Chromatography - A Century of Discovery 1900-2000 (Journal of Chromatography Library)

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JOURNAL OF CHROMATOGRAPHY LIBRARY – volume 64

chromatography a century of discovery 1900–2000 the bridge to the sciences=technology

The Evolution of Chromatography. The Bridge to the Sciences=Technology. Some of the early scientists who invented, rediscovered, and=or advanced chromatography include: M.S. Tswett, L.S. Palmer, R. Kuhn, A.W.K. Tiselius, A.J.P. Martin, R.L.M. Synge, F. Sanger, S. Moore and W.B. Stein, and the Awardees in Chapters 2, 4, 5 and S-9, S-10, and S-11. Who is the next farsighted scientist?

JOURNAL OF CHROMATOGRAPHY LIBRARY – volume 64

chromatography a century of discovery 1900–2000 the bridge to the sciences=technology edited by

Charles W. Gehrke Professor Emeritus of Biochemistry, University of Missouri, Columbia, Missouri, USA

Robert L. Wixom Professor Emeritus of Biochemistry, University of Missouri, Columbia, Missouri, USA

Ernst Bayer Professor of Organic Chemistry, University of Tu¨bingen, Tu¨bingen, Germany

2001

ELSEVIER Amsterdam – London – New York – Oxford – Paris – Shannon – Tokyo

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands  2001 Elsevier Science B.V. All rights reserved. This work is protected under copyright by Elsevier Science, and the following terms and conditions apply to its use: Photocopying: Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. Permissions may be sought directly from Elsevier Science Global Rights Department, PO Box 800, Oxford OX5 1DX, UK; phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also contact Global Rights directly through Elsevier’s home page (http://www.elsevier.com), by selecting ‘Obtaining Permissions’. In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: (978) 7508400, fax: (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London W1P 0LP, UK; phone: (+44) 171 631 5555, fax: (+44) 171 631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works: Tables of contents may be reproduced for internal circulation, but permission of Elsevier Science is required for resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage: Permission of the Publisher is required to store or use electronically any material contained in this work, including any chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier Science Global Rights Department, at the mail, fax and e-mail addresses noted above. Notice: No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drugs dosages should be made. First edition 2001 Library of Congress Cataloging in Publication Data Chromatography: a century of discovery 1900-2000: the bridge to the sciences=technology = edited by Charles W. Gehrke, Robert L. Wixom and Ernst Bayer.– 1st ed. p. ; cm – (Journal of chromatography library ; v. 64) Includes bibliographic references and indexes. ISBN 0-444-50114-2 (hc) 1. Chromatographic analysis–History–20th century. I. Gehrke, Charles W. II. Wixom, Robert L. III. Bayer, Ernst. IV. Series. QD79.C4 C4837 2001 543’.089’09–dc21 2001053230 British Library Cataloguing in Publication Data Chromatography: a century of discovery 1900-2000: the bridge to the sciences=technology. – (Journal of Chromatography library; v. 64) 1. Chromatographic analysis – History 2. Chemists I. Gehrke, Charles W. (Charles William), 1917- II. Wixom, Robert L. III. Bayer, Ernst. IV. Journal of Chromatography 5430 .089 ISBN 0444501142

ISBN:

0 444 50114 2

1 The paper used in this publication meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper). Printed in The Netherlands.

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Preface “Chromatography — A Century of Discovery 1900–2000 — The Bridge to the Sciences=Technology”, is a documentary of seminal events, developments, discovery, and history of chromatography in the 20th century that presents the beginnings and story of chromatography which revolutionized the sciences for students, researchers, and science for the 21st century. Its central theme is the impact of chromatography as a branch of science on different facets of science — analytical chemistry, instrumentation, biomedical-, environmental-, pharmaceutical-, and space sciences, etc. Who are the Chromatography Award winners? They were nominated by their colleagues, evaluated by their peers and recognized for outstanding contributions. Each Awardee was recognized as a recipient of one or more international or national Awards for their investigations in the separation sciences. The 13 most important Awards and their respective sponsoring professional societies are listed in the outline of Chapter 2. We invited over 100 Awardees in Chromatography to write about their unique activities and careers in their own words and to share how their advances have impacted the science disciplines. The book represents the combined thinking and contributions of many chromatographers and colleagues. E. Bayer (Professor of Chemistry, Director of the Research Center for Nucleic Acid and Peptide Chemistry, of the University of Tu¨bingen) is Editor in Europe. Chapter 2 is a compilation of international and national award winners in chronological order (most since 1975) from 13 professional societies and associations for 1900–2000. Among the very early group (1900s–1920s) were M.S. Tswett, L.S. Palmer and C. Dhe´re´, who contributed fundamentally to liquid adsorption chromatography. From E. Lederer on (1931), classical column chromatography was widely used across Europe. R. Kuhn and L. Zechmeister of Europe and H.H. Strain of the USA were widely recognized as pioneers in this field. The 1940s saw the seminal work of A.J.P. Martin and R.L.M. Synge on liquid–liquid partition chromatography, which then was utilized by them in the development of paper chromatography. At that time, A. Tiselius systematized the various chromatographic processes. Ion-exchange chromatography also started in the 1940s, with the Manhattan Project group utilizing the new synthetic polymeric resins for the separation of rare earths. Soon after, S. Moore and W.R. Stein (in the 1950s) extended IEC to the separation of amino acids and E. Glueckauf, in England, provided the interpretations of the basis of IEC separation. W.E. Cohn at Oak Ridge used IEC for the analysis of nucleic acids, and S. Moore and W.R. Stein, together with D.H. Spackman developed the first instrument — the amino acid analyzer. E. Cremer in Austria, J. Jana´k in Czechoslovakia, C.S.G. Phillips in England and A.A. Zhukhovitskii in the Soviet Union carried out pioneering work on gas adsorption chromatography, but the real

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breakthrough was represented by the adaptation of the principles of partition to gas chromatography by A.T. James and A.J.P. Martin in 1952. At least a score of prominent scientists made fundamental contributions to the development of chromatography. The reader is referred to “75 Years of Chromatography – A Historical Dialogue” by L.S. Ettre and A. Zlatkis, which tells the stories of 65 pioneers. It is true, and most important, that chromatography in the 20th century revolutionized analytical chemistry and research problems across the sciences. The discoveries of chromatography fundamentally changed investigation of naturally occurring substances. Before chromatography the key word was ‘isolation’ of one or only a few components of a substance; thus to obtain it large amounts of materials had to be prepared. Chromatography allowed separation of all the components present and brought in the world of ‘microchemistry’ in analytical chemistry. The changes in the direction of the Sciences, introduced by GC, PC, TLC, IEC, HPLC, and AC=SEC in the 20th century are paradigm shifts leading to many new discoveries. Unique aspects: This centennial documentary’s purpose is fulfilled by the Pioneers and Builders of Chromatography in their ‘Seminal Concepts’ in chromatography. Each Awardee was asked to write about their scientific discovery(s), and activities in chromatography. Their presentations (Chapter 5) are preceded by a short biography and in some cases picture(s) of the Awardees, their research years, and the place of their laboratory accomplishments to fully reflect the advances they have made in the sciences. Thus, this book is dedicated to the Chromatography Awardees and is written by them. This is their Book! Several in-depth feature chapters cover the Beginnings of Chromatography. M.S. Tswett, the inventor of chromatography, and several other early pioneers (Chapter 1) are highlighted; a discussion of the contributions of several Nobel laureates is included (Chapter 2). An extensive bibliography on the History of the Evolution of Chromatography (Chapter S-8); a presentation of Major International Symposia supporting chromatography as a Bridge to selected sciences is included (Chapter 3). Also, well-known chromatographers have written special chapters on Supports, and Stationary Phases and Separations (Chapter S-11), followed by a chapter on Paradigm Shifts in Science (Chapter S-9) and one on the early Evolution of Scientific Instrumentation (Chapter S-10). Chapters 1–7 are in the bound volume, and Chapters S-7 to S-15 are on the internet at http://chemweb.com/preprint/. Chapter 6 is a unique presentation of Chromatography Around the World detailing the chromatography research in Japan, Russia, China, Latin America, The Netherlands and other countries. Prominent chromatographers wrote these chapters from their respective countries. Chapter S-14 presents 24 Future Chromatographers of the 21st century who received their PhD’s recently with their abstracts and selected references. This is followed by a projection and perspectives of 41 of the Award Winners into the 21st century on the impact of Chromatography — the advances made in the many science disciplines — and the development of emerging technologies in the new millennium. New discoveries in the biosciences and medicine, agriculture, the environment and separations technology in the 21st century will rely immeasurably on the 20th century research tools in chromatography and those yet to be developed.

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This book is recommended for Students in the Sciences and Research, Chromatographers at all levels: professional scientists; research chromatographers in academia, government, and industry; science libraries in academia, industry and professional societies, historians and philosophers of science; and educators and students at both high school and university levels. This book describes ‘Chromatography as the Bridge’ — a key foundation built in the 20th century for major advances and discoveries yet to come across the sciences of the 21st century. C HARLES W. G EHRKE ROBERT L. W IXOM and E RNST BAYER (Editors)

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Dedication To our scientific colleagues: Who are the pioneers and builders of chromatography. Who have contributed to this historical compendium. Who have provided scientific thought, experiments, evaluation and communications to the world at large, and who interact with leaders in communities, states and international realms. Our sincere thanks are extended to the many accomplished scientists from around the world who have graciously and diligently presented their research findings in their contributed papers. Their efforts have allowed this treatise to present an international, and a comprehensive perspective of the field of chromatography. In this fast-developing science and technology, their research findings have and will, play important roles in the advancement of science in many disciplines. To our respective research institutions: Whether academe, government, corporate, institutes, and foundations that have supported this and other scientific research enterprises. To our respective family members: Who have given freely of their tangible support and valuable suggestions. Our thanks are also extended to the editors of Elsevier Science.

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Acknowledgements The Editors of this book had the benefit of many discussions and advice from other chromatographers. However, one scientist stands out, Leslie S. Ettre, who not only contributed four chapters, but has served valiantly on many policy issues, a variety of specifics and relationships, and as a friendly critic. With his multiple experiences as editor and his wealth of earlier chapters, reviews, historical explorations and several books, the knowledgeable reader will not be surprised that we, the Editors, have deeply appreciated the above role. Chapters 5 and 6, in recognition of achievements in chromatography are a logical extension of the 1979 book by Ettre and Zlatkis titled “75 Years of Chromatography — A Historical Dialogue”. We, the Editors, have had many helpful conversations with and advice from D.W. Armstrong, V.G. Berezkin, P.R. Brown, T.L. Chester, C.A. Cramers, P. Flodin, G. Guiochon, S. Hjerte´n, Cs. Horva´th, W.G. Jennings, R.E. Kaiser, K. Jinno, B.L. Karger, J.J. Kirkland, F.M. Lanc¸as, K. Macek, P. Sandra, L.R. Snyder, R. Tijssen, K.K. Unger, C. Welch and Y. Zhang. We have appreciated the persistence and thoroughness of Nicole Hininger, a University of Missouri student, who served as a Library Research Assistant. Many valuable suggestions and some difficult online searches were provided by the excellent Reference Librarians at the University of Missouri: Brenda Graves-Blevins, Janice Dysart, Rebecca S. Graves, E. Diane Johnson, Paula Roper and Caryn Scoville. The Editors have received helpful input from the librarians of the Chemical Heritage Foundation initiated by the (American Chemical Society and other sponsors, Philadelphia, Pennsylvania) and Chemical Abstracts (American Chemical Society, Columbus, OH). Preparation of copy for this book is in large part due to the excellent secretarial skills plus accuracy and patience of Crista B. Chappell, Nancy Harrison, Cinda Hudlow, Cynthia Mercado, Kara Seidel, Valerie Wedel, and Kelly Willcut at the University of Missouri, Columbia, MO and University of Tu¨bingen, Germany. The Editors have warmly appreciated the graphic artwork by Sammae Heard, MU Graphic Artist, and the pen and ink drawings by Corrine Barbour, MU Graduate Art Student. Research, preparation, writing and editing this book was supported financially by the University of Missouri–Columbia, Missouri, USA: ž Chancellor, Richard L. Wallace, Vice Provost Jack O. Burns, and their Office of Research. ž School of Medicine, and Dean Robert Churchill. ž College of Agriculture, Food and Natural Resources, and Dean Thomas Payne. ž Department of Biochemistry, and Chair William Folk.

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ž Experiment Station Chemical Laboratories (Agriculture), and Director Thomas P. Mawhinney. ž University of Tu¨bingen, Tu¨bingen, Germany. ž Analytical Biochemistry Laboratory (ABC Labs), Columbia, MO, USA and CEO Jake Halliday.

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Editors Charles William Gehrke was born on July 18, 1917 in New York City. He studied at The Ohio State University, receiving a B.A. in 1939, a B.Sc. in Education (1941) and an M.S. in Bacteriology in (1941). From 1941 to 1945, he was Professor and Chairman of the Department of Chemistry at Missouri Valley College, Marshall, Missouri teaching chemistry and physics to World War II Navy midshipmen (from destroyers, battleships, and aircraft carriers in the South Pacific) for officer training. These young men returned to the war as deck and flight officers. In 1946, he returned as instructor in agricultural biochemistry to The Ohio State University in 1967 receiving his Ph.D. in 1947. In 1949, he joined the College of Agriculture at the University of Missouri–Columbia (UMC), retiring in Fall 1987 from positions as Professor of Biochemistry, Manager of the Experiment Station Chemical Laboratories, and Director of the University Interdisciplinary Chromatography Mass-Spectrometry facility. His duties also included those of State Chemist for the Missouri Fertilizer and Limestone Control laws. He was Scientific Coordinator at the Cancer Research Center in Columbia until 1997. Gehrke is the author of over 260 scientific publications in analytical and biochemistry. His research interests include the development of quantitative, high-resolution gas- and liquid chromatographic methods for amino acids, purines, pyrimidines, major and modified nucleosides in RNA, DNA, and methylated ‘CAP’ structures in mRNA; fatty acids; biological markers in the detection of cancer; characterization and interaction of proteins, chromatography of biologically important molecules, structural characterization of carcinogen–RNA=DNA adducts; and automation of analytical methods for nitrogen, phosphorus, and potassium in fertilizers. He developed automated spectrophotometric methods for lysine, methionine, and cystine. He has lectured on gas–liquid chromatography of amino acids in Japan, China, and at many universities and institutes in the United States and Europe. Gehrke analyzed lunar samples returned by Apollo flights 11, 12 and 14–17 for amino acids and extractable organic compounds as a co-investigator with Cyril Ponnamperuma, University of Maryland, and with a consortium of scientists at the National Aeronautics and Space Administration (NASA), Ames Research Center, California, and the University of Maryland, College Park, MD. Awards and honors In 1971, he received the annual Association of Official Analytical Chemists’ (AOAC) Harvey W. Wiley Award in Analytical Chemistry. He was recipient of the Senior Faculty Member Award, UMC College of Agriculture, in 1973. Invited by the Soviet Academy of Sciences, he gave a summary presentation on organic substances in lunar fines at

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Charles W. Gehrke and Robert L. Wixom. Photograph taken on the University of Missouri Campus, Columbia, MO, USA, July 2000.

Ernst Bayer. Photograph taken at the Symposium on Environmental Technologies in the Research Center of PETROBRAS, Brazilian Oil Company, September 2000.

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the August 1974 Oparin International Symposium on the ‘Origin of Life’. In 1975, he was selected as a member of the American Chemical Society Charter Review Board for Chemical Abstracts. Sponsored by five Central American Governments, he taught chromatographic analysis of amino acids at the Central American Research Institute for Industry in Guatemala, 1975. Gehrke was elected to Who’s Who in Missouri Education, recipient of the UMC Faculty-Alumni Gold Medal Award in 1975 and the Kenneth A. Spencer Award from the Kansas City Section of the American Chemical Society for meritorious achievement in agricultural and food chemistry, 1979–1980. He received the Tswett Chromatography Memorial Medal from the Scientific Council on Chromatography, Academy of Sciences of the USSR, Moscow, 1978 and the Sigma Xi Senior Research Award by the UMC Chapter, 1980. In 1986, Gehrke was given the American Chemical Society Midwest Chemist Award. He was an invited speaker on ‘Modified Nucleosides and Cancer’ in Freiburg, German Federal Republic, 1982, and gave presentations as an invited scientist throughout Japan, People’s Republic of China, Taiwan, Philippines, and Hong Kong (1982 and 1987). He was elected to the Board of Directors and Editorial Board of the AOAC, 1979–1980; President-Elect of the Association of Official Analytical Chemists International Organization, 1982–1983; and was honored by election as the Centennial President in 1983–1984. He developed ‘Libraries of Instruments’, an interdisciplinary research programs on strengthening research in American Universities. Gehrke is founder, board member, and former Chairman of the Board of Directors (1968–1992) of the Analytical Biochemistry Laboratories, Inc., a private corporation of 250 scientists, engineers, biologists, and chemists specializing in chromatographic instrumentation, and addressing world-wide problems on pharmaceutical and environmental issues to the corporate sector. He is a member of the board of SPIRAL Corporation, Dijon, France. Over 60 masters and doctoral students have received their advanced degrees in analytical biochemistry under his direction. In addition to his extensive contributions to amino acid analysis by gas chromatography, Gehrke and colleagues have pioneered in the development of sensitive, high-resolution, quantitative high-performance liquid chromatographic methods for over 100 major and modified nucleosides in RNA, DNA, tRNAs and mRNA, and then applied these methods in collaborative research with scientists in molecular biology across the world. At the 1982 International Symposium on Cancer Markers, Freiburg, German Federal Republic, E. Borek stated that “Professor Gehrke’s chromatographic methods are being used successfully by more than half of the scientists in attendance at these meetings.” His involvement in chromatography began in the early 1960s with investigations on improved GC methods for fatty acid analysis. Gehrke is widely known for developing a comprehensive quantitative gas chromatographic method for the analysis of amino acids in biological samples and ultra-micro methods for life molecules in moon samples. This method was used and advanced in the analysis of lunar samples when he was co-investigator with NASA. In the 1970s, his major interests shifted towards the development of quantitative HPLC methods for the analysis of various important substances in biological samples, especially the modified nucleosides in tRNA as biomarkers in cancer research.

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Major research contributions: ž Developed eight methods adopted as Official Methods by AOAC International (formerly Association of Analytical Chemists). Sampling, Ca, Mg, K(2), P(2), N(2). ž First to develop and automate AOAC official chemical methods for fertilizers (1950s– 1980s) ž First to discover quantitative GLC of total protein amino acids (1960s–1970s), 45 publications. ž First to develop quantitative HPLC of total nucleosides in tRNA, mRNA, DNAs and rRNAs (1970s–1990s), 31 publications. ž First to use HPLC–MS nucleoside chromatography in molecular biology (1987– 1994), 23 publications. ž First to use GLC and HPLC methods for metabolites in body fluids as potential biological markers (1971–1994), 54 publications. ž First to use GLC in analysis of Apollo 11–17 moon samples at ultra high sensitivity levels (1969–1974), 10 publications. ž First to propose a Lunar=Mars-Based Analytical Laboratory (1989–1999). Books: (Author–Editor) 1979

1987

1990

1993

1997

1954–1995

Author, in: L.S. Ettre and A. Zlatkis (Eds.) ‘75 Years of Chromatography — A Historical Dialogue’. Elsevier Science Publishers, Amsterdam, The Netherlands, pp. 75–86. C.W. Gehrke, K.C. Kuo and R.L. Zumwalt (Eds.) — ‘Amino Acid Analysis by Gas Chromatography’, three volumes, CRC Press, Boca Raton, FL; 19 Chapters by 29 authors (5 chapters by C.W.G.) 543 pp. C.W. Gehrke and K. Kuo (authors=editors) ‘Chromatography and Modification of Nucleosides,’ a three volume treatise published by Elsevier in the Journal of Chromatography Library Series addressing ‘Analytical Methods for Major and Modified Nucleosides’, ‘Biochemical Roles and Function of Modification’, ‘Modified Nucleosides in Cancer and Normal Metabolism’, and ‘A Comprehensive Database of Structural Information on tRNAs and Nucleosides by HPLC, GC, MS, NMR, UV, and FT-IR combined techniques,’ 1206 pp. C. Ponnamperuma and C.W. Gehrke (Eds.), Proceedings of the Ninth College Park Colloquium — A Lunar-Based Chemical Analysis Laboratory, A. Deepak Publishing, Hampton, VA, 282 pp. C.W. Gehrke, Mitchell K. Hobish, Robert W. Zumwalt, Michel Prost and Jean Degre´s, ‘A Lunar-Based Analytical Laboratory’ (C. Ponnamperuma memorial Volume, A. Deepak Publishing, Hampton, VA, 329 pp. Nine additional chapters and reviews in other scientific journals and books.

In 1989 and 1993, C.W. Gehrke and C. Ponnamperuma of the University of Maryland were named co-principal investigators on a proposal to address the scientific technical

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concerns of placing on the moon a chemical laboratory which would be automated, miniaturized, computer robotic-operated and would support NASA programs in the study of five aspects of the exploration of space: (a) astronaut health; (b) closed environment life support; (c) lunar resources; (d) exobiology; and (e) planetology. Gehrke received the American Chemical Society National Award in Separations Science and Technology in 1999 and the American Chemical Society National Award in Chromatography in 2000 and the Ohio State University Alumni Achievement Award in 2001. Robert L. Wixom, Co-Editor of this book, was born on July 6, 1924 in Philadelphia, PA. In 1947, he graduated with a B.Sc. in Chemistry from Earlham College, Richmond, IN. He conducted his graduate studies and thesis at the University of Illinois under the guidance of William C. Rose, receiving his Ph.D. in Biochemistry in 1952. Wixom held teaching=research faculty appointments in the Department of Biochemistry, School of Medicine, University of Arkansas (1952–1964) and the Department of Biochemistry, School of Medicine=College of Agriculture, UMC (1964–1992). He took year-long sabbatical=research leaves at Oxford University (1961–1962), University of Wisconsin (1970–1971), Massachusetts Institute of Technology (1978–1979), and the Fox Chase Institute for Cancer Research (1985–1986). His 40 years of research (45 peer-reviewed papers, two reviews) and graduate teaching focused on amino acid and protein metabolism. He taught intermediate and advanced biochemistry to medical students, graduate students in diverse departments and undergraduate students with a variety of majors. Wixom guided the Advanced Biochemistry Laboratory course at UMC for 20 years, which covered several experiments in chromatography and 15 years teaching a course on Biochemical Information Retrieval. He has received three teaching awards. He served as a Departmental Representative to the Graduate Faculty Senate (1980–1993) and its Chair (1989–1992); this included a key role in three major new university programs. He officially retired in 1992 as Emeritus of Biochemistry, but continues many similar activities. Reflecting other earlier interests, Wixom was the co-initiator of the UMC Environmental Affairs Council, served as their first chair for three years (1990–1994), and continues as a member during retirement. He initiated and served as senior Editor of the 1997 book, “Environmental Challenges for Higher Education: Integration of Sustainability into Academic Programs”. The preceding experiences served as the educational background for his present role as Co-Editor of the book “Chromatography — A Century of Discovery 1900–2000”. Ernst Bayer was born on March 24, 1927 in Ludwigshafen=Rhein, Germany, where he also visited primary and secondary schools from 1934 to 1947. From 1947 to 1952, he studied chemistry at the University of Heidelberg, and made his master thesis in Physical Chemistry. From 1952 to 1954, he completed his Ph.D. thesis under the advice of Nobel Laureate R. Kuhn, Max-Planck-Institute for Medical Research, Heidelberg University on the structure of hemovanadin, a vanadium compound occurring in marine tunicates. In March 1955, after a short postdoctoral fellowship in Kuhn’s laboratory, Bayer was

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appointed as Director of the Department of Biochemistry of the Government Research Institute at Geilweilerhof. At this Institute, Bayer had also the task to study the quality of wine and new cultivated sorts of wine. He developed in 1956, gas chromatographic (GC methods for flavoring substances in wines and other beverages, and demonstrated the use of GC in many other areas of natural compound chemistry derivatives. Also in this period, preparative GC with large diameter columns up to 10 cm was demonstrated to be a useful tool, and in 1959 he published the separation of the pheromones of silk moth using the insects as a specific detector. At Geilweilerhof, Bayer started his investigations on metal proteins and biomimetic selective sequestering of metal ions, which led to the development of polymers for the selective enrichment of gold, uranium and copper from seawater. From 1958 to 1962, Bayer was appointed as lecturer at the Institute for Organic Chemistry, University of Karlsruhe. He continued his work on various aspects of GC, metal proteins, selective enrichment of metal ions, recognized the importance of metal chelation for the color of flowers and fruits (blue color of cornflower versus red color of roses), and isolated flavor components of various beverages. In 1957, his monograph, “Gas Chromatography” was published in German, and soon translated into English and Russian. This book became for many years a guide for users of GC. In 1962, he was appointed Professor and Head of the Department of Organic Chemistry, University of Tu¨bingen, a position he held until his retirement in 1996. At present, he is Head of the Research Center for Nucleic Acid and Peptide Chemistry of the University of Tu¨bingen. From 1967 to 1971, he held also the position of a distinguished R.A. Welch Professorship at the University of Houston. Bayer has pioneered in HPLC, hyphenation of separation methods with MS and NMR. He published in 1972 the analysis of complex peptide mixtures, and detected the inherent failure sequences in solid phase peptide synthesis (SPPS), which led to optimization and acceptance of SPPS, and made HPLC as the standard method for control. In 1976, he reported the first HPLC of dansyl-amino acids with fluorescence detection and reported detection limits down to the lower femtomoles. This performance has not been much improved since then. In 1974, Bayer reported the method known as template chromatography, using specific interactions of oligonucleotides and peptides. He made some of the first studies on the structure of the stationary reversed phases using solid state NMR (Cross Polarization and Magic Angle Spinning techniques). He used pulse field gradients to derive values for the local axial and radial dispersion coefficients and determined the amount of mobile phase percolating through the bed and of stagnant fluid. Recently he has concentrated on miniaturized separation methods like capillary HPLC, and capillary electrochromatography and their on-line coupling to MS and NMR. He designed an instrument for the alternative use for all capillary separation methods (HPLC, CE, CEC). In 1998, he published a new MS detection method, called Coordination Ion Spray (CIS–MS), which is based on the on-line formation of charged coordination compounds of the analytes. Non-polar compounds like sugars, lipids, terpenes, and vitamins can be detected as silver, palladium or boron complexes, which are not sensitively detected with electrospray MS. It is obvious that not all of the contributions of Bayer, which are reported in his 550 original publications, can be summarized here.

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Bayer is internationally recognized with many honors for his research in different areas: 1978 A.J.P. Martin Award and the Tswett Medal of the USSR Academy of Sciences, for outstanding contributions to the development of chromatography. 1981 British Petroleum Energy Research Prize and the Max-Bergmann Medal for Peptide Chemistry for the development of Chirasil-Val and its application to study racemization of peptides and amino acids. 1985 Philip Morris Research Prize, ‘Men and Environment’ for the development of a thermocatalytic, biomimetic process to convert biomass and sludge to a petroleum like oil. 1986 Tswett Chromatography Award for research in chromatography. 1989 First Class Merit Cross of the Federal Republic of Germany; in 1990, the International Rheinland-Prize for Environmental Protection for development of analytical methods relevant for the environment, and the Richard Kuhn Medal of the German Chemical Society for his research in structure elucidation of metal proteins and antibiotics. 1993 Fritz Pregl Medal of the Analytical Chemistry Society of Austria. 1994 Fresenius Prize of the German Chemical Society for his contributions to analytical chemistry and the Grand Merit Cross of the Federal Republic of Germany. 1997 Maria Sklodowska Curie Medal of the Polish Chemical Society for his contributions to natural compound chemistry, and in 2000, the MTE. Golay Award for his contributions to capillary HPLC, capillary electrochromatography and hyphenated techniques. 2000 Hala´sz Medal of the Hungarian Society. 2001 American Chemical Society National Award in Chromatography. Bayer has served in many positions in the German Chemical Society, as a member of the editorial staff in many scientific journals, in government and science commissions, as a member of the IUPAC analytical chemistry nomenclature commission and member of committees of scientific congresses.

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Chapter Outline

Chromatography a Century of Discovery 1900–2000 The Bridge to the Sciences and Technology

The Pioneers and Builders of Chromatography

1.

A. B. C. D. E. F. G. H. I. J.

2.

A. B. C.

The Beginnings of Chromatography — The Pioneers (1900–1960) — Robert L. Wixom Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The initiation in Switzerland and Russia . . . . . . . . . . . . . . . . . Adsorption chromatography (1900–1950s) . . . . . . . . . . . . . . . . Partition chromatography (1940s–1950s) . . . . . . . . . . . . . . . . Paper- and thin-layer chromatography (two forms of planar chromatography) Ion-exchange chromatography (IEC) (1930s–1960s) . . . . . . . . . . . Chromatography of petroleum . . . . . . . . . . . . . . . . . . . . . The literature of chromatography . . . . . . . . . . . . . . . . . . . . Integration of seminal concepts with chromatography leaders . . . . . . . From the inventors to the builders of chromatography . . . . . . . . . . . What is required to be one of the award winners? . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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The Builders of Chromatography — Major Chromatography Awards and the Award Winners — Leslie S. Ettre Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nobel Prize in Chemistry by the Nobel Foundation (1948–1999) . . . . . . . . . National Award in Chromatography of the American Chemical Society (1961–2001) National Award in Separations Science and Technology of the American Chemical (1984–2001) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . Society . . . .

1 2 4 7 11 15 17 21 23 26 27 29 30

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Chapter Outline D. E. F. G. H. I. J. K. L. M.

3.

A. B. C. D. E. F. G. H. I.

4.

XIX

A.J.P. Martin Award of the Chromatographic Society (1978–2000) . . . . . . . . . . . . M.S. Tswett Chromatography Award of the International Symposia on Advances in Chromatography (1974–1988) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M.J.E. Golay Award in Capillary Chromatography of the International Symposia on Capillary Chromatography (1989–2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stephen Dal Nogare Award in Chromatography of the Chromatographic Forum of the Delaware Valley (1972–2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Keene P. Dimick Award in Chromatography by the Society for Analytical Chemists of Pittsburg (1988–2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Silver Jubilee Award of the Chromatographic Society (1982–2000) . . . . . . . . . . . . Award for Achievements in Separation Science of the Eastern Analytical Symposium (1986– 2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . COLACRO Medal (1986–2000) of the Congresso Latino Americano de Cromatografia . . . Leroy S. Palmer Award of the Minnesota Chromatography Forum (1980–2000) . . . . . . M.S. Tswett Chromatography Memorial Medal of the All-Union Scientific Council on Chromatography, Academy of Sciences of the U.S.S.R. (1978–1979) . . . . . . . . . . .

Major International Symposia Supporting Chromatography — Leslie S. Ettre

43 44 44 47 48 49 49 50 51 52

55

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . International symposia on (gas) chromatography by the (British) Chromatographic Society (1956–2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Symposia on gas chromatography organized by the Instrument Society of America (1957– 1963) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . International symposia by the French Society G.A.M.S. (1961–1969) . . . . . . . . . . . International symposia on advances in chromatography (1963–1988) . . . . . . . . . . . International symposia on high-performance liquid chromatography (HPLC) (1973–2000) . . International symposia on capillary (gas) chromatography (1975–2000) . . . . . . . . . . Danube symposia on chromatography (1976–1993) . . . . . . . . . . . . . . . . . . . COLACRO Latin American congresses on chromatography and related techniques (1986– 2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (PITTCON) (1950–2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

55 58 59 60 60 61 62 63 64 65

Chromatography — The Bridge to Environmental, Space and Biological Sciences — Charles W. Gehrke (continued in Chapter S-12)

69

A. B. C. D.

Early years of automated chemistry . . . . . . . . . . . . . . . . . . . . . Chromatography in environmental analysis over the last 30 years . . . . . . . . Amino acid analysis — gas–liquid and ion-exchange chromatography — 30 years Chromatography in space sciences — GLC and IEC of Apollo moon samples . .

72 74 76 83

5.

Prominent Chromatographers and their Research – Seminal Concepts in Chromatography=Separation Sciences — Charles W. Gehrke, Robert L. Wixom and Ernst Bayer

99

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Seminal concepts and new discoveries . . . . . . . . . . . . . . . . . . . . . . . . Relation of seminal concepts and awardees . . . . . . . . . . . . . . . . . . . . . .

107 108 108

A. B. C.

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Chapter Outline

D. E.

Prominent chromatographers (76 awardees) . . . . . . . . . . . . . . . . . . . . . . Summary: if Mikhail Tswett were alive today . . . . . . . . . . . . . . . . . . . . .

6.

Chromatography around the World — Charles W. Gehrke, Robert L. Wixom and Ernst Bayer

A. B. C. D. E.

7. A. B. C.

Chromatography in Japan — Kiyokatsu Jinno . . . . . . . . . . . . . . . . . Chromatography in Russia in the 20th century — Viktor G. Berezkin . . . . . . . Chromatography in China — Yukui Zhang and Guowang Xu . . . . . . . . . . Development of chromatography in Latin America — Fernando Mauro Lanc¸as . . Chromatography in The Netherlands (University of Amsterdam) — Hans Poppe, Schoenmakers and Robert Tijssen (see their References in Chapter S-13) . . . . .

109 598

601 . . . . . . . . . . . . Peter . . .

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Overview: Chromatography — A New Discipline of Science — Charles W. Gehrke, Robert L. Wixom and Ernst Bayer Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Attributes of modern chromatography . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography in the near future . . . . . . . . . . . . . . . . . . . . . . . . . .

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603 621 644 659 670

687 687 688 689

691

Author and Scientist Index (see separate complementary author=subject index for the Supplement on the Internet) 695 Subject Index

705

A New Discipline of Science Chromatography Chapters S-7 To S-15 are on the Internet at Chem Web Preprint Server (http:==www.chemweb.com=preprint=).

S-7. Overview: Chromatography – A New Discipline of Science — Charles W. Gehrke, Robert W. Wixom and Ernst Bayer S-8. Bibliography of Publications – The History of the Evolution of Chromatography — Leslie S. Ettre A. B. C.

Introduction Books and Booklets Journal Papers and Book Chapters General

Chapter Outline D. E. F. G. H. I. J. K. L.

The Precursors of Chromatography (Pre-Tswett) M.S. Tswett. His Life, Activities, and Correspondence Nobel Prize Lectures Evolution of Liquid Chromatography Evolution of Gas Chromatography Petroleum Chromatography Key Contributors to the Evolution of Chromatography Evolution of Chromatographic Instrumentation Meetings, Associations, and Personal Recollections

S-9. Milestones and Paradigm Shifts in Chromatography — Robert L. Wixom A. B. C. D.

E.

F.

Introduction Nobel Awardees Who Advanced Chromatography Nobel Awardees Who Used Chromatography Natureof Paradigm Shifts Paradigm Shifts in Chromatography: a. Carotenoids b. Other Natural Products c. Chromatography of Amino Acids, Peptides and Proteins d. Affinity Chromatography e. Chiral Chromatography f. Supercritical-Fluid Chromatography g. Instruments for Chromatography Further Developments in Chromatography a. Size-Exclusion Chromatography b. High-Performance Liquid Chromatography c. Detectors in Chromatography d. Hyphenated /Coupled /Tandem Techniques in Chromatography e. Women Scientists in Chromatography Summary (see Chapter 1 for Pioneers in Chromatography)

S-10. Evolution and Instrumentation in Chromatography — Leslie S. Ettre A. B.

Introduction Gas Chromatography – Instrumentation, Detectors and Columns Liquid Chromatography – Pumps, Detectors and Columns

S-11. Advances in Chromatographic Column Technology — Ernst Bayer, Walter G. Jennings, Ron E. Majors, J. Jack Kirkland, Klaus K. Unger, Heinz Engelhardt, Gerhard Schomburg, William H. Pirkle, Christopher J. Welch, Daniel W. Armstrong, Jerker O. Porath, Jan B. Sjo¨vall and Charles W. Gehrke A.

B.

Introduction Supports, Stationary and Bonded Phases a. Column Development – An Abbreviated History — Walter G. Jennings b. Future Advances in Column Technology — Ronald E. Majors Contributions by Other Chromatographers

XXI

XXII

Chapter Outline

S-12. Chromatography – The Bridge to the Environmental, Space and Biological Sciences — Charles W. Gehrke (a continuation of Chapter 4) S-13. Chromatography around the World – References for Chapter 6 are given in Chapter S-13; the printed volume has – Japan (Kiyokatsu Jinno), Russia (Victor G. Berezkin), China (Yukui Zhang and Guowang Xu), Latin America (Fernando M. Lanc¸as) and The Netherlands (Hans Poppe, Peter J. Schoenmakers and Robert Tijssen) S-14. Future Chromatographers of the 21st Century — Contributions by 24 Younger Scientists S-15. Chromatography for the Next Millennium: Continuing Discovery and Emerging Technology — Perspectives by 41 Chromatography Awardees

Appendices 1–7 — Robert L. Wixom and Charles W. Gehrke 1. 2. 3. 4.

5.

Glossary of Common Chromatography Terms Deceased Chromatographers – Recognition and References Main Current Periodicals Covering Chromatography – Serial Books, Review Journals, Research Journals Selected Earlier Chromatography Books (Pre-1980) A. General, Earlier Chromatography Books B. Planar Chromatography (Paper-TLC) Books C. Liquid Chromatography Books – Early HPLC D. Ion-Exchange Chromatography Books E. Gas Chromatography Books F. Size-Exclusion Chromatography Books G. Affinity Chromatography Books H. Chromatography Handbooks Chromatography Books by Series (Post-1990) A. Journal of Chromatography Library Series of Books B. Chromatographic Science Series of Books C. Separation Science Series of Books D. Books on Chromatography by the American Chemical Society E. Books on Chromatography by the Royal Society of Chemistry F. Books by the Chromatographic Society (UK) G. Chromatography Books by Subject Areas (Post-1990) a. General Chromatography Books b. Planar Chromatography Books c. Gas Chromatography Books d. Ion-Exchange Chromatography Books e. Size-Exclusion Chromatography Books f. High-Performance Liquid Chromatography Books g. Affinity Chromatography Books h. Electrophoresis=Capillary Electrophoresis, Etc. Books i. Supercritical-Fluid Chromatography=Extraction Books j. Chiral Chromatography Books

Chapter Outline 6.

7.

XXIII

Published Chromatography Symposia (1994–1999) A. Symposia in Journal of Chromatography A B. Symposia in Journal of Chromatography B C. Symposia in Chromatographia D. Recent and Future Chromatography Symposia (2000 and 2001) Books on Methods Related to Chromatography A. Laboratory Techniques in Biochemistry and Molecular Biology B. Methods of Biochemical Analysis C. CRC Series in Analytical Biotechnology D. Methods in Molecular Biology E. Methods in Biotechnology F. Methods in Enzymology G. Methods in Molecular Medicine H. Other Book References on Methods

Author and Scientist Index for the Supplement – see the Internet Chem Web Preprint Server (http:==www.chemweb.com=preprint=) Subject Index for the Supplement (Chapters S-7 to S-15)

XXIV

List of Contributors The Editors are pleased that 125 living Awardees and contributors have responded to our request for their concise research presentations. Their presentations and institutional addresses may be found in Chapters 5 and 6. The contributors in Chapter 5 are presented in alphabetical order. Similarly, biographical information and a thesis abstract for 24, mostly 1999 Ph.D. investigators, may be found in Chapter S-14. Both are listed in Author Index (d=deceased). For complete addresses see the contribution in Chapters 5 and 6 of each scientist=contributor. Daniel W. Armstrong Iowa State University Ernst Bayer Universita¨t Tubingen Viktor G. Berezkin, Corresponding author, Institute of Petrochemical Synthesis Prominent chromatographers from Russia: Vadim A. Davankov Laboratory at the Institute of Element-Organic Compounds Boris V. Ioffe d State University, Leningrad (St. Petersburg), Russia Andrei V. Kiselev d State University of Moscow Karl I. Sakodynskii d Karpov Institute of Physical Chemistry, Moscow M.S. Vigdergauz d Institute of Organic and Physical Chemistry Aleksander A. Zhukhovitskii d All-Union Research Institute for Geological Prospecting of Petroleum (VNI GNI)

M.I. Yanovskii d Morton Beroza USA Gu¨nter Blobel Rockefeller University Jerald S. Bradshaw Brigham Young University Phyllis R. Brown University of Rhode Island Tom L. Chester Miami Valley Laboratories, Proctor and Gamble Corporation Carel A. Cramers Lab. of Instrumental Analysis John V. Dawkins Loughborough University Heinz Engelhardt Universita¨t Des Saarlandes Leslie S. Ettre Yale University Michael B. Evans United Kingdom Per G.M. Flodin Artimplant AB

List of Contributors

XXV

James S. Fritz Iowa State University

Hiroyuki Hatano d Kanagawa Dental College

Charles W. Gehrke University of Missouri

Nobuo Ikekawa d Niigata College of Pharmacy

J. Calvin Giddings d University of Utah

Daido Ishii Kumamoto Institute of Techology

Robert Grob Anal. Chem. Consultant

Hiroshi Miyazaki Niigata College of Pharmacy

Georges Guiochon University of Tennessee

Tsuneo Okuyama Tokyo Dental College

Andra´s Guttman Novartis Agricultural Discovery Institute

Shigeru Terabe Himeji Institute of Technology

Steven B. Hawthorne University of North Dakota

James W. Jorgenson Univ. of North Carolina

Frederich G. Helfferich The Pennsylvania State University

Olga Kaiser and Rudolf E. Kaiser Institut fu¨r Chromatogrophie

Jo¨rgen Hermansson Chrom. Tech. AB., Stockholm

Barry L. Karger Northeastern University

Herbert H. Hill Washington State University

Jerry W. King National Center for Agricultural Utilization Research

Stellan Hjerte´n Biomedical Center Csaba Horva´th Yale University Daido Ishii Kumamoto Institute of Technology

J. Jack Kirkland Agilent Technologies Ernst G. Klesper University of Technology John H. Knox University of Edinburgh

Reed M. Izatt Brigham Young University

Fernando M. Lanc¸as, Corresponding author, Universidade de Sa˜o Paulo

Jaroslav Janik Academy of Sciences of the Czech Republic

Other chromatographers from South America:

Egil Jellum Institute of Clinical Biochemistry

Clyde N. Carducci University of Buenos Aires

Walter G. Jennings J&W Scientific Company

Remolo Ciola Refinery Research Center

Kiyokatsu Jinno, Corresponding author, Toyohashi University of Technology

Armando M. Moreno Universidad Nacional Autonoma de Mexico

Shoji Hara Tokyo College of Pharmacy

Joaquim Lubkowitz Separation Systems, Inc.

XXVI

List of Contributors

Milton L. Lee Brigham Young University

Jacques Rijks The Netherlands

Hendrik Lingeman Vrije Universiteit

Pat J.F. Sandra Research Institute for Chromatography

Charles H. Lochmu¨ller Duke University

Frederick Sanger Cambridge University

James E. Lovelock Combe Mill, UK

Raymond P.W. Scott Consultant, USA

Karel Macek Czech Academy of Sciences

Gerhard Schomburg Max-Planck Institut

Ronald E. Majors Agilent Technologies

Robert E. Sievers University of Colorado at Boulder

Karin Markides Uppsala University

Colin F. Simpson United Kingdom

Archer J.P. Martin National Inst. Med. Res. (Ex)

Jan B. Sjo¨vall Karolinska Institutet

Michel Martin Ecole Supe´rieure de Physique et de Chimie Industrielles

Hamish Small Dow Chemical Co. (Retired)

Daniel E. Martire Georgetown University Robert B. Merrifield The Rockefeller University Hiroshi Miyazaki Kawasaki, Japan E. David Morgan Keele University Milos V. Novotny Indiana University Janusz Pawliszyn University of Waterloo

Roger M. Smith Loughborough University Lloyd R. Snyder LC Resources Inc. Jun Suzuki Soda Aromatic Co., Ltd. Shigeru Terabe Himeji Institute of Technology Toyohide Takeuchi Gifu University Robert Tijssen, Corresponding author, Universiteit van Amsterdam Other co-authors from the Netherlands:

William H. Pirkle University of Illinois

Hans Poppe University of Amsterdam

Colin F. Poole Wayne State University

Peter J. Schoenmakers University of Amsterdam

Jerker O. Porath Uppsala University

Klaus K. Unger Johannes Gutenberg-Universita¨t

Michel Prost Spiral Corp., Dijon, France

Irving W. Wainer Georgetown University Medical Center

List of Contributors

XXVII

Harold F. Walton Retired, USA

Robert L. Wixom University of Missouri-Columbia

Phillip C. Wankat Purdue University

Edward S. Yeung Iowa State University of Science and Technology

Christopher J. Welch Merck & Co., Inc. Ian D. Wilson Drugs Kinetics Group - Zeneca

Yukui Zhang and Guowang Xu and Peichang Lu Chinese Academy of Sciences

XXVIII

Contents Preface . . . . . Dedication . . . . Acknowledgements Editors . . . . . Chapter Outline . List of Contributors

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THE PIONEERS AND BUILDERS OF CHROMATOGRAPHY 1. 2.

The Beginnings of Chromatography — The Pioneers (1900–1960) — Robert L. Wixom . . The Builders of Chromatography — Major Chromatography Awards and the Award Winners — Leslie S. Ettre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Major International Symposia Supporting Chromatography — Leslie S. Ettre . . . . . . . Chromatography — The Bridge to Environmental, Space and Biological Sciences — Charles W. Gehrke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prominent Chromatographers and their Research – Seminal Concepts in Chromatography= Separation Sciences — Charles W. Gehrke, Robert L. Wixom and Ernst Bayer . . . . . . . Chromatography around the World — Charles W. Gehrke, Robert L. Wixom and Ernst Bayer Overview: Chromatography — A New Discipline of Science — Charles W. Gehrke, Robert L. Wixom and Ernst Bayer . . . . . . . . . . . . . . . . .

687

Permissions for Copyright Items . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Author and Scientist Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

691 695 705

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OVERVIEW: CHROMATOGRAPHY – A NEW DISCIPLINE OF SCIENCE

See on the internet at Chem Web Preprint Server (http:==www.chemweb.com=preprint=) S-7.

Overview: Chromatography – A New Discipline of Science — Charles W. Gehrke, Robert W. Wixom and Ernst Bayer S-8. Bibliography of Publications – The History of the Evolution of Chromatography — Leslie S. Ettre S-9. Milestones and Paradigm Shifts in Chromatography — Robert L. Wixom S-10. Evolution and Instrumentation in Chromatography — Leslie S. Ettre S-11. Advances in Chromatographic Column Technology — Ernst Bayer, Walter G. Jennings, Ron E. Majors, J. Jack Kirkland, Klaus K. Unger, Heinz Engelhardt, Gerhard Schomburg, William H. Pirkle, Christopher J. Welch, Daniel W. Armstrong, Jerker O. Porath, Jan B. Sjo¨vall and Charles W. Gehrke S-12. Chromatography – The Bridge to the Environmental, Space and Biological Sciences — Charles W. Gehrke

1 39 55 69 99 601

Contents S-13. Chromatography Around the World – References for Chapter 6: in Part A – Japan (Kiyokatsu Jinno), Russia (Victor G. Berezkin), China (Yukui Zhang and Guowang Xu), Latin America (Fernando M. Lanc¸as) and The Netherlands (Hans Poppe, Peter J. Schoenmakers and Robert Tijssen) S-14. Future Chromatographers of the 21st Century — Contributions by 24 Younger Scientists S-15. Chromatography for the Next Millennium: Continuing Discovery and Emerging Technology — Perspectives by 41 Chromatography Awardees Appendices 1–7 — Robert L. Wixom and Charles W. Gehrke Author and Scientist Index for the Supplement Subject Index for the Supplement

XXIX

1

CHAPTER 1

The Beginnings of Chromatography — The Pioneers (1900–1960) Robert L. Wixom University of Missouri, Columbia, MO 65212, USA

CONTENTS

A. B. C.

D.

E. F. G. H. I. J.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Possible approaches . . . . . . . . . . . . . . . . . . . . . . . Nature of this chapter . . . . . . . . . . . . . . . . . . . . . . . The initiation in Switzerland and Russia . . . . . . . . . . . . . . . . . . . Adsorption chromatography (1900–1950s) . . . . . . . . . . . . . . . . . Partition chromatography (1940s–1950s) . . . . . . . . . . . . . . . . . . C.1. Liquid–liquid partition chromatography (LLC) . . . . . . . . . . . . C.2. Gas–liquid partition chromatography (GLC) . . . . . . . . . . . . . Paper- and thin-layer chromatography (two forms of planar chromatography) . . D.1. Paper chromatography (PC) . . . . . . . . . . . . . . . . . . . . D.2. Thin-layer chromatography (TLC) . . . . . . . . . . . . . . . . . Ion-exchange chromatography (IEC) (1930s–1960s) . . . . . . . . . . . . . Chromatography of petroleum . . . . . . . . . . . . . . . . . . . . . . . The literature of chromatography . . . . . . . . . . . . . . . . . . . . . . Integration of seminal concepts with chromatography leaders . . . . . . . . . From the inventors to the builders of chromatography . . . . . . . . . . . . . I.1. Other early chromatography leaders . . . . . . . . . . . . . . . . What is required to be one of the award winners? . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References for introduction . . . . . . . . . . . . . . . . . . . . References for Mikhail S. Tswett . . . . . . . . . . . . . . . . . . References for predecessors of Mikhail S. Tswett . . . . . . . . . . References on Leroy S. Palmer . . . . . . . . . . . . . . . . . . . References on other early followers of M.S. Tswett . . . . . . . . . . References on partition chromatography (LLC) . . . . . . . . . . . References for gas–liquid chromatography (GLC) . . . . . . . . . . References on paper- and thin-layer chromatography (TLC) . . . . . . References on ion-exchange chromatography (mainly early investigators) References for petroleum chromatography . . . . . . . . . . . . . . References on the literature of chromatography . . . . . . . . . . .

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Chapter 1 References on ‘From the inventors to the builders of chromatography’ . . . . . . References for summary . . . . . . . . . . . . . . . . . . . . . . . . . .

37 38

“Separation is as old as the earth : : : ” J. Calvin Giddings Unified Separation Science, 1991

INTRODUCTION This chapter presents chromatography as a branch of science that bridges a century of science and discovery. Chromatography represents the premier analytical method of the 20th century for the advancement of a variety of disciplines of science. The evolution of chromatography is depicted in the frontispiece as a bridge and shows some of the inventors — the builders who advanced chromatography and who constructed the foundation of chromatography for later sciences and technology. To continue with J. Calvin Giddings’ thoughts [1], the cloud of dust and gases gathered and coalesced to form our planet Earth. Metal ions separated forming crystals and ores — iron in brassy-yellow pyrite (FeS2 ) or hematite ore (Fe2 O3 ), silicon in clear, hexagonal cross-section of crystallized quartz (SiO2 ), multi-colored wavelite with a radial fibrous structure (Al3 (OH)3 (PO4 )2 Ð 5 H2 O, red cinnabar (HgS) and lead in the shiny, gray, cubes of galena (PbS). The sun’s energy, captured in photosynthesis led to the separation of a carbon-prevalent biosphere and an oxygen-rich atmosphere (21%); nitrogen was concentrated in the atmosphere (70%), but is also abundant in the proteins and nucleic acids of plant-, microbial-, and animal cells. Much more recently, Homo sapiens has learned to distinguish protein- vs. carbohydrate-rich foods, isolated natural products for medical use (e.g., quinine for malaria, etc.), and extracted natural dyes from concentrated plant sources (blue indigo from Indigofera genus; yellow quercitin from the black oak, Quercus velutina; red carmine from the female cochineal insect, etc.). After the Big Bang and evolution, mankind has traveled in time through the late stages: Agricultural Revolution

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Industrial Revolution (1700s–1800s)

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Scientific=Technology Revolution (1800s–1900s)

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Information Explosion (late 1900s–2000s)

Present-day culture and science rests upon the scientific traditions and cultures of Egypt, Mesopotamia, Greece, Rome, Islam, China and Western Europe; however, we have spiraled far beyond these roots [2]. Within this context and in the past century, chromatography and separation science have incubated and grown with a flourish. But how do we scientists place a handle — a meaningful organizational structure on chromatography — a body of knowledge that is now too large for many scientists to fully grasp?

The Beginnings of Chromatography — The Pioneers (1900–1960)

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Possible approaches As stated in the preface, the goal of this book is to recognize the pioneers and the builders of chromatography, their discoveries and their personal recollections. Interwoven in their contributions and in other chapters will be seminal concepts, and the developmental events during the past century. By undertaking the above goal, we are also embarking on writing a history of this branch of science. Several approaches may be found for organizing a presentation in the field of history of science: ž Portrayal of the sequence of scientific discoveries and their interrelationships. (Example: M. Florkin and E.H. Stotz (Eds.), Comprehensive Biochemistry, Elsevier Publishing Co., Vols. 1–34, 1972–1986.) ž Scientific biographies of the leading members of the branch of science under review. (Examples: L.S. Ettre and A. Zlatkis (Eds.), 75 Years of Chromatography — A Historical Dialogue, Elsevier, 1979; or M. Florkin and E.H. Stotz (Eds.), Comprehensive Biochemistry — Sections on Personal Recollections, Elsevier, Vols. 35–40, 1983–1997.) ž Flow of major concepts and hypotheses in a branch of science, the evidence for the same, and sometimes or even frequently, their modification or even disavowal. (Example: T.S. Kuhn, The Structure of Scientific Revolutions, University of Chicago Press, Chicago, IL, 1962, 1970 and 1996.) ž Schools of scientific discipline, usually led by a distinguished scientist guiding his=her colleagues, postdoctoral associates and graduate students. The above examples suggest the merits of each approach. Indeed some treatises include more than one approach. The Frontispiece Bridge emphasizes the crucial role of the earlier scientists=chromatographers to recognize the distinctive merits of chromatography, to build the scientific structure of chromatography and to enhance the connection — the bridge — to other scientific disciplines. Last, but not least, these scientists inspired others to continue to construct bridges. Since chromatography is a relatively young branch of science, this book emphasizes the scientific biographies of international and national awardees and contributors in chromatography and separation science. Some authors of other chromatography books have a brief historical description for their specific chromatography area that is being described. V.G. Berzekin has written to us that this book is also “the Bridge from the XIX to the XXI Century”. Thus, let us begin.

Nature of this chapter This chapter is a brief sketch — a beginning for the rest of the book, as each of the following subsections, A to F, has many references that are also presented by L.S. Ettre in Chapter S-8. Furthermore, the history of these six subject areas has an extensive number of books that are cited in Appendices 4, 5 and 7, whose subdivisions are partially parallel to the outline of this chapter. For Chapter 1, the reference numbers for journal articles or books are in regular type, and cross-references to other chapters (‘C’) or the Appendices

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(‘A’) are presented at the end of each subsection in italics along with the chapter number and subsection, i.e., ‘C-4B’ refers to Chapter 4-B, S-7D refers to a chapter in the Supplement (http:==www.chembweb.com=preprint) and ‘A-5G’ to Appendix 5-G. Each main chromatographer is followed by a ‘See Chapter 5B’ plus a letter code that defines their area of chromatography as described in 5B.

A. THE INITIATION IN SWITZERLAND AND RUSSIA No discussion of the evolution of chromatography in the 20th century can be complete without dealing with the life and activities of its inventor. Mikhail Semenovich Tswett was born on May 14, 1872, in the small northern Italian town of Asti. His parents were on an extended holiday in Italy and were traveling by train toward the Lago di Maggiore, the beautiful lake in northern Italy. The trip had to be interrupted because of the condition of the mother who then died soon after the birth of her son. His father took the infant to Switzerland and Tswett grew up there, first in Lausanne and M.S. Tswett, circa later in Geneva where he studied botany at the University. By the 1910. beginning of 1896, he finished his doctorate thesis dealing with the structure of plant cells and chloroplasts and the movement of the protoplasma [1]. Late spring of that year, he moved to Russia, joining his father who at that time was

Location of towns important in Tswett’s life.

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a high government official in the Crimea. Tswett had high hopes for an early academic appointment, but soon found that without a Russian advanced university degree this was not possible. Therefore, he moved to St. Petersburg and while having a temporary position in a laboratory, he carried out scientific research so that he could submit a thesis and receive an academic degree. This was accomplished in the fall of 1901 at the University of Kazan’ where he duly received a Russian Magister’s degree. Soon afterward he moved to Warsaw, in Russian-occupied Poland, and in the next 14 years, he was active there, first at the University, then in 1907 at the Veterinary Institute, and finally, from 1908 on at the Polytechnic Institute, although always in relatively minor positions. It should be noted that the controversy concerning the non-acceptance of his Swiss academic degree and then, for the next 15 years, the impossibility for him to obtain a senior university appointment made Tswett very bitter. He considered this discrimination because of his foreign background; as a conclusion, he became a loner and reacted very harshly even to the mildest criticism. His strong polemic nature became a serious handicap in his professional life and was justly criticized when, in 1918, he was nominated for the Chemistry Nobel Prize [2]. Tswett’s thesis work for the Magister’s degree dealt with the physico-chemical structure of plant chlorophyll [3] and represented the start of his research which eventually led to the development of chromatography. It is very intuitive to follow the successive steps of Tswett’s investigations because these demonstrate his logical thinking. Tswett’s aim was to isolate chlorophyll as close as possible to its natural state. During this work he found that while polar solvents (e.g., ethanol) can be used to extract chlorophyll from leaves, non-polar solvents (e.g., petroleum ether) are unable to do this. However, after chlorophyll was isolated from plants, it could be easily dissolved in these solvents. Tswett correctly concluded that this behavior is not due to simple solubility problems or to a chemical change of chlorophyll in ethanol to a ‘soluble’ form, but is “rather due to the interference of the molecular forces of the tissue, that is to say, to adsorption”, and to the relative strength of the solvents compared to the adsorption forces of the plant tissues. The next step in his work was the study of the interaction of plant pigments with over 100 different powdered organic and inorganic materials which may act as adsorbents, aiming to establish the general adsorption behavior of these substances. Eventually, these investigations led to a method permitting the separation of chlorophylls and some carotenoids by stepwise selective adsorption and extraction. The results of these studies were summarized by Tswett in a major lecture presented on March 21, 1903, in Warsaw, which was published two years later in a local scientific periodical [4]. From this method it was not far to realize that separation by adsorption–desorption can also be carried out in a continuous way [5,6]. From 1903 to 1905, Tswett developed this method and first referred to its existence (calling it only a “new, reliable method”, without giving any details) in two polemic publications criticizing the results of Hans Molisch (1856–1937), then Professor at the University of Prague, on the pigments of brown algae. After being hard-pressed to present actual data and disclose the way his investigations were carried out, Tswett finally submitted in June

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and July 1906 his fundamental two papers to the Berichte der deutschen botanischen Gesellschaft, the journal of the German Botanical Society, published in the Fall. The first [7] dealt with his investigations of plant chlorophylls while the second [8] discussed in detail the new separation method developed by him, which he for the first time called “chromatography”. These two papers contain all the important aspects of chromatography, including proper selection of the adsorbent, proper utilization of the solvents and the possibility of using columns with different diameters (from 2–3 mm up to 10–20 mm) and lengths. These papers created considerable controversy and in the following years Tswett carried out a lot of polemics with scientists in different countries who have worked in the same field [9]. (For a detailed discussion of this controversy, see the papers by T. Robinson [10] and L.S. Ettre [11,12].) Meanwhile Tswett summarized the knowledge he gathered on plant pigments, particularly chlorophylls, in a book published in 1910 in Russian [13]. A separate section of his book dealt with adsorption and chromatography and in it, Tswett further expanded the description of the technique and its use, also emphasizing the possibility of preparative separations on columns having a diameter of 30 mm and packing length of 80 mm. Between 1906 and 1911, Tswett further expanded his research on plant pigments, including the carotenoids. It is practically unknown that this name for the polyene pigments — used universally since the 1930s — was first proposed by him, in a paper published in 1911 [14]. However, from 1912 on, he published almost nothing; his health started to deteriorate and then came World War I with the interruption of normal life. Warsaw was occupied by German troops in the summer of 1915 and the Polytechnic Institute was evacuated to Nizhnii Novgorod, but there was no possibility to carry out any research there. Finally, in 1917, Tswett was appointed a full Professor of Botany and the Director of the Botanical Gardens at the University of Yure’ev (today: Tartu, in Estonia). He moved there in September 1917, but within a few months, German troops also occupied the Baltic area. A few months later, the Russian professors moved to Voronezh, in Russia, to start a new State University. Tswett was already very ill at that time, and he died in Voronezh on June 26, 1919, only 47 years old. Thus, the life of one of the most original thinkers of this period ended prematurely. During his lifetime, Tswett’s work on chromatography was not appreciated and was belittled by his peers. However, within a decade after his death, its importance was finally recognized and applied in almost every branch of science. Today, 80 years after his death, we consider chromatography as one of the most important inventions of the 20th century. By Leslie S. Ettre See Chapter 5B, a, b Many other references on M.S. Tswett are available [15]. Though Tswett is generally regarded as the father of chromatography, several other scientists have been identified as making brief entries in the 1800s into the general area of chromatography. The names and contributions of the predecessors of M.S. Tswett follow with appropriate reference(s):

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ž Friedlieb F. Runge (1794–1867) was a German chemist who studied the spread of coal tar dyes on paper and made ‘self-grown’ pictures for the ‘Friends of Beauty’; this area might be considered a precursor of the later (1940s) paper chromatography [1]. ž Friedrich Goppelsroeder (1837–1919) was a student of Friedrich Schoenbeim (1799–1867) and improved the latter’s approach for separation of dyes on hanging strips of unsized paper by capillary migration. Though he published a 1906 monograph, and again in 1909, his work was ignored and then lost until the 1940s [2].

B. ADSORPTION CHROMATOGRAPHY (1900–1950s) In adsorption chromatography, the molecules are separated on the basis of their adsorptive properties, where the stationary phase is a solid adsorbent usually in a column or on a plate and eluted by the mobile phase that may be aqueous or organic solvent(s). The adsorption chromatography research of M.S. Tswett was scoffed at by some, but followed by others that are now presented. The individual whose research had the most impact on later investigators was Leroy S. Palmer. Leroy Sheldon Palmer (1887–1944) was a pioneer in chromatography research shortly after M.S. Tswett. Palmer was a student at the University of Missouri-Columbia, Missouri, USA, earning a B.Sc. in chemical engineering in 1909 (a new department that began in 1903), and his M.A. in chemistry in 1910 [1]. His Master’s thesis was primarily an outline of his doctoral problem and a detailed literature survey. Palmer’s investigations leading to his Ph.D. degree began in October 1909 and were completed in the spring of 1913 in the College of Agriculture [2]. The full text of his thesis was published in four issues of the Bulletins of the University of Missouri Agricultural Experiment Station [1], and then in five successive research papers [3,4]. Upon entering the Graduate School in 1909, he joined the Cooperative Government Dairy Research Laboratory with Clarence H. Eckles (1875–1933), Professor and Chair of the Department of Dairy Husbandry (1901–1918). His thesis problem was to investigate the observations made by many dairy farmers, namely that butter from cows on summer pasture with fresh grass or green alfalfa hay has a deep yellow color, whereas cows consuming stored foodstuffs in winter produce a butter that has usually a very light color. Just a few years earlier, M.S. Tswett had invented adsorption chromatography and had separated the chlorophylls, carotenoids and xanthophylls from plant leaves (described more fully in the preceding paragraphs). R. Willsta¨tter and W. Mieg [5] had just established the elementary composition of ‘carotin’ and ‘xanthophyll’ in 1907, but their actual structures and various isomers were not deciphered until the late 1920s. Thus Palmer had to make a choice in 1910 to isolate individual pigments utilizing their selective solubility and purify them in a multi-step process, ending in

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crystallization, or follow Tswett’s method of adsorption chromatography; he chose the latter. Palmer found Tswett’s 1906 paper in the Berichte der deutschen botanischen Gesellschaft, at a time when European scientists tended to ignore Tswett’s observations. Relying on Tswett’s style of adsorption chromatography, Palmer found carotenoids in his extracts of butterfat and thus explained the change in color from summer to winter being due to dietary intake. Palmer, like Tswett, found that inulin and sucrose were suitable alternative adsorbents to calcium carbonate and used elution to remove the colored solutions, rather than extrusion as in C. Dhe´re´’s laboratory. He also used prefractionation of the pigments before chromatography, differential solvent extraction and a crude spectrometric examination. His research represented probably the first use of chromatography after Tswett’s basic 1906 publications and introduced chromatography to scientists in the USA [3]. Palmer, after completion of his thesis research on carotene in butter by chromatography, stayed on at the University of Missouri with additional chromatographic studies of nutritional problems, and examined the pigments in other biological tissues: body fat, corpeus luteum and skin secretions of the cow, the yellow pigments in blood serum, the fate of carotenes during digestion and in human milk fat. Palmer extended his investigations to other animals: hen (and eggs), sheep, goat, swine, and horse [1,3], and found that sheep, swine and rabbit differ from the others in the absence of carotenoids in their fatty tissue (5 papers in 1915–1916) [1]. Thus Palmer was the first to introduce adsorption chromatography into the study of animal systems, animal nutrition and biochemistry. In 1918, C.H. Eckles moved to the University of Minnesota and invited L.S. Palmer to join him. With this transfer, Palmer switched his research area to minerals and vitamins in animal nutrition and over the subsequent years developed a strong leadership role in this area — regionally (Head of the Division of Agricultural Chemistry, 1942–1944) and internationally [1]. However, before leaving carotenoids and chromatography, he wrote a thorough 1922 book, “Carotinoids and Related Pigments — The Chromolipids” [6] in which he elaborately detailed the chromatography method and its applications, listed Tswett’s 13 earlier papers, including a 17 page bibliography, and a detailed discussion of chromatography for laboratory investigations. The Palmer 1922 book was part of a new monograph series of the American Chemical Society and hence received worldwide attention. Bearing in mind, the criticisms of and the then somewhat obscurity of Tswett’s work, Palmer’s book brought chromatography, carotenoids and Tswett’s contribution’s to scientists elsewhere. Thus Palmer’s research and writings serve as the bridge (connection) between Tswett and the resurgence of chromatography in the early 1930s by R. Kuhn, E. Lederer, P. Karrer and others. (continued in Chapter S-9B, see R. Kuhn.) See Chapter 5B, b Other early followers of M.S. Tswett: ž Gottfried Kra¨nzlin (1882), the first follower of M.S. Tswett, was a botany graduate student at the University of Berlin (1906–1907). He followed Tswett’s 1906 description of chromatography, and used a CaCO3 adsorption column to purify

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chlorophylls and xanthophylls. His 1908 thesis was published in a specialized journal and was soon forgotten. Soon, thereafter, he went to Africa and left the area of chromatography [1]. ž Theodor Lippmaa (1892–1944), an Estonian scientist, received his Ph.D. in botany in 1926 from Tartu University and published six research papers in 1926 on ‘thuyorhodine’, later renamed ‘rhodoxanthin’, a plant pigment. For two of these papers, he followed Tswett’s chromatographic procedures despite the criticism of Tswett’s work in the 1920s. His subsequent research was unrelated botanical studies in Africa and Europe that were a precursor to present-day environmental protection [2]. ž Charles Dhe´re´ (1876–1955) was the first scientist in Europe to recognize the overall importance of chromatography [3]. Dhe´re´ studied medicine in Paris (M.D. in 1898), but never practiced as a physician. After several years at the Sorbonne, he joined the University of Fribourg, Switzerland in 1900 as an Associate Professor of physiology, biological chemistry and microbiology (such joint appointments were common in this period), and then in 1908 as a full Professor until his 1938 retirement. His service at Fribourg included two periods as Dean of the Faculty of Science (1916–1917 and 1933–1934). His primary interest was the investigation of biological substances, mainly by ultraviolet and fluorescence spectroscopy. One of Dhe´re´’s students, Wladyslaw Fr¯anciszek de Rogowski (1886–1945) from Poland, received his doctorate in 1912, examined the chromatography on CaCO3 columns of chlorophylls and their UV absorption and confirmed the earlier findings of M.S. Tswett. Another Dhe´re´ student, Guglielmo Vegezzi (1890–1955) from Switzerland, started his thesis work in 1913, and after military service, completed his doctoral work in 1916. He extended Rogowski’s methods with minor modifications to study invertebrate pigments, such as those from bile and liver of escargot and the eggs of the spider crab. His research with Dhe´re´ is summarized in six papers (1916–1917). Then he joined the Swiss Federal Administration of Alcohol. Dhe´re´ prepared the first thorough summary of M.S. Tswett’s life and scientific work — a 50 page paper in the journal, Candollea. Apparently, Dhe´re´, Rogowski and Vegezzi did not undertake further investigations in chromatography after the period described. Thus to evaluate, L.S. Palmer conducted chromatography research from 1910 to 1918, wrote nine papers on carotenoids, and most importantly, wrote a rather thorough 1922 chromatography book [6]. Palmer cited Goppelsroeder’s research, but not that of Kra¨nzlin, Dhe´re´, Rogowski and Vegezzi in his 16 pages of references in 2 columns in his book. (Scientific communication was more limited at this period in the absence of airplanes for travel to research conferences, radio, TV, the Web and the Internet.) To summarize, L.S. Palmer in the United States and Charles Dhe´re´ in Europe were the middle men, the conservers of Tswett’s insights and the bridge to the research of Edgar Lederer and Richard Kuhn — some 20 years later. For adsorption chromatography, the next major event came in the laboratory of Richard Kuhn who had a long, distinguished record of research. One of his research assistants, Edgar Lederer, had read the above book by Leroy S. Palmer (about 1930) and soon thereafter found the book and papers by Michael Tswett. The other part of this story, described under R. Kuhn, is that Kuhn, Winterstein and Lederer published

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a key 1931 paper [4] on the purification of xanthophylls on a CaCO3 adsorption chromatography column. Later, after many other discoveries, R. Kuhn won the Nobel Prize in 1938 “for his work on carotenoids and vitamins”, which will be continued later. Most readers know that Edgar Lederer (1908–1988) moved in 1933 to France with his French wife due to political reasons and to the rise of anti-semitism and began an active research career, mainly at the Institut de Chimie des Substances Naturelles du C.N.R.S. [5,6], and received recognition by being one of the five 1976 M.S. Tswett Awardees. Lederer’s research continued with contributions on carotenoids (astracene from red boiled lobster shells, astoxanthine from the skin of goldfish), vitamin A2 , perfumes, the pheromone from the queen bee, lysopine from crown gall, ascaryl alcohol from a parasitic nematode; microbial lipids, glycolipids and a peptidolipid and muramyl peptides — usually with reliance on chromatography. Perhaps more important in the long run are the many significant chromatography books that he wrote (1934, 1949, 1952, 1954–1957, 1960). This editor read the latter books as a graduate student and remembers the clarity in his writing; these books were probably read by many other young investigators in the 1950s and 1960s. Appendix 3 has several short biographical articles about E. Lederer, but his longer, historical biography is lucid and gripping to read for his discoveries, associations and the unfolding of chromatography [6]. Lederer’s shadow is thus long in its present-day influence. The many transformations of chromatography led some to overlook its quite modest origins. Indeed, when Kuhn, Winterstein and Lederer presented their chromatographic progress in the early 1930s at a colloquium at the Chemical Institute of the University of Munich, whose Institute Director was the then well-known Henrich Wieland (1928 Nobel Prize in Chemistry for his investigations on bile acids) remarked, “Up to now, we have learned with much effort to distil, crystallize and recrystallize. Now they come along and just pour the stuff through a little tube.” See Chapter 5B, b During the early and mid-1930s, Paul Karrer (1889–1971) was very active in natural products research at the University of Zurich, Switzerland. After learning the Kuhn=Lederer results, he used adsorption chromatography in his investigations and published the results in his many research papers in the 1930s. He was recognized as the 1937 recipient of the Nobel Prize in Chemistry “for his investigations on carotenoids, flavins and vitamins A and B2 ”. La´szlo´ Zechmeister (1889–1972) was another early leader in chromatography. He was born in Hungary and was a graduate student of Richard Willsta¨tter before World War I. After the war ended, he returned to his home in Budapest, Hungary and became in 1923 a Professor at the University of Pe´cs, Hungary. Being interested in carotenoids, he followed the Kuhn=Lederer group in carotenoid chromatography. In 1934, he wrote a book on carotenoids and then in 1937 wrote the first textbook on chromatography, which had three later editions plus his 1950 book, Progress in Chromatography (see A-4A). With the political instability in Europe in the 1930s, he moved to the California Institute of Technology as a Professor in 1940, and organized a vigorous laboratory to examine

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natural products by chromatography. Though he retired in 1959, his lucid books were very effective in the subsequent development and spread of chromatography [7,8]. See Chapter 5B, b Most scientists remember A.W.K. Tiselius and associate him with free-flow electrophoresis, but his fertile mind led also to significant developments in chromatography in the 1940s and 1950s as described by one of his students, Per Flodin in S-9A. Aloysius I.M. Keulemans (1908–1977) was another prominent chromatographer. Most of his career was at the University of Technology, Eindhoven, The Netherlands, where he conducted research on gas chromatography and developed a significant program to guide the next generation of chromatographers; this included over 200 research papers plus 150 M.S. and 20 Ph.D. degrees for students from his institute. The 1957 and 1959 editions of his textbook on gas chromatography described reports from chromatographers (J.J. Van Deemter, F.J. Zuiderweg, A. Klinkenberg and H. Boer at Shell Development, Amsterdam, The Netherlands) and E. Glueckauf and N.H. Ray (England) and other early investigators [9,10]. The Keulemans’ books were translated to a number of different languages and served as a major source of information to practical chromatographers (cited in A-4E). See Chapter 5B, b Adsorption chromatography may be extended to become a preparative liquid chromatography [11,12]. This introduction for adsorption chromatography, whether liquid or gas chromatography, may be supplemented by other sources [13,14]. At this point, an overall look on the developing patterns for the varieties of chromatography is presented (Fig. 1.1). Note: Additional comments and references may be found later in Chapter S-9B (R. Kuhn, P. Karrer) and A-4A, A-4E and A-5Ga.

C. PARTITION CHROMATOGRAPHY (1940s–1950s) This subject area has been divided into liquid–liquid chromatography and gas–liquid chromatography. In an earlier decade, these groups based on the physical state of the mobile phase were thought to be distinct; now we know that while one mechanism may be dominant, other factors are definitely involved.

C.1. Liquid–liquid partition chromatography (LLC) The research of the 1930s, as just described, was dominated by the emphasis on adsorption chromatography. A major turning point came in 1941 with the research by Archer J.P. Martin and Richard L.M. Synge at Cambridge University, United Kingdom [1–4] on partition chromatography. They used a silica gel column with water as the

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Fig. 1.1. Outline of the historical flow of scientific thought in chromatography (1900-1960s). This figure will serve as an outline for subsequent sections. Partition chromatography and its sequential development occurred over the 1940s to 1960s period. Note: Additional comments and references may be found in later Chapters S-9A, S-9B (R. Kuhn, R. Karrer), A-4A, A-4E and A-5G.

stationary phase plus a mobile phase of water-saturated butanol to separate acetyl amino acids to initiate partition chromatography. Column partition chromatography [5] led soon to paper chromatography and thin-layer chromatography, gas–liquid partition chromatography and in the mid-1960s to high-performance liquid chromatography (HPLC). Note: Further development of TLC is described in Chapters 1-D2, 2, 5 and S-11.

C.2. Gas–liquid partition chromatography (GLC) Gas chromatography is a long standing method with ample research to demonstrate its effectiveness in separating the components of mixtures of volatile compounds. Over the decades, it has become the principal method of analysis for volatile, heat-stable, organic compounds. Martin and Synge [1] conceived the possibility of GLC, but a decade passed until examined experimentally by James and Martin [2]. Their simple system relied on a GLC column to separate volatile fatty acids and identify them by titration. This report then became a key turning point for the 1950s decade, during which many investigators built their own GC instruments (i.e., E. Cremer, G.E. Hesse, J. Jana´k

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and others). Since the development of the GC method, the needed instruments and detectors required major considerable capital investment, blocks of time, and organized effort, it is not surprising that scientists with the petroleum companies having major analytical problems moved rapidly ahead. The observation that many components of petroleum are volatile at room temperatures (C2 –C5 ), mild temperatures (C6 –C10 ), or the elevated temperatures usually used in GC (up to 300ºC) had a key role in the above advance. Subsequently, elevated temperatures have been regularly used and may be at a fixed temperature, or a programmed temperature increase during the GC. GLC has advanced rapidly on many, more or less, simultaneous developments with respect to the support and stationary phase, column selection — an elongated tube, flow controllers, carrier gas sample inlet devices, column ovens, power supplies, detection systems, and troubleshooting. The original qualitative method developed in two directions: a rigorous quantitative method and a semi-preparative approach. Initially, columns were ‘packed’ with the desired stationary phase, but then gave way, under M.J.E. Golay’s invention and investigations in the late 1950s and 1960s, to ‘open tubular’ columns [3–6]; the latter may be capillary columns (usually less than 0.35 mm) or larger diameter (up to 2.5 mm). Initial columns were made from stainless steel tubing, which was replaced by glass capillaries when techniques were developed to prepare a stable internal coating. Later glass capillaries were replaced by fused-silica columns. These columns are usually used as a coil and strengthened by a polyimide outside coating. A thin, uniform film (¾0.25 µm) of the desired stationary phase is frequently coated on the inside wall of the capillary column. Carrier gases for GLC must be pure and inert; they have usually included hydrogen, helium, or most frequently nitrogen. Since the sample must be delivered to the head of the GC column with a minimum initial bandwidth, sample inlets, also called injectors or injection ports, may be a vaporization injector (high temperatures to vaporize the sample rapidly), or on-column injectors (deliver sample directly into the column). Special microsyringes are used to introduce the sample into the injector. The carrier gases (H2 , He, N2 ) undergo a straight line increase in viscosity with the temperature rise (0–350ºC), but a pressure rise (up to 5 atm) had a negligible influence on viscosity [7]. Multiple detectors are now available and are discussed in Chapter S-10. Another wave of research in the 1960s was to modify the non-volatile compounds by preparing volatile derivatives, such as: methyl esters for organic acids, reaction with trialkylsilyl (R3 Si) groups to form TMS ethers, use of N-methyl-trifluoroacetamide for steroids; anhydrides (acetic-, trifluoroacetic- or pentafluoropropionic anhydride, etc.) employed for alcohols and phenols; use of acetone to form ketals, etc. The reagent, bis trimethylsilyl trifluoroacetamide (BSFTA, a C.W. Gehrke patent), has been widely used for amino acids, nucleosides, proton donors, etc. The list of derivatives is much longer; their selection requires insight and knowledge of the substrate, the derivatizing reagent, the column and the instruments for detection. The more detailed earlier history of GC has been described [5,6,8]. Though gas chromatography is usually for an analytical objective, several modifications have led to making it a preparative GC [9]. The lively interaction between ideas, gas chromatographers, and international symposia (1963–1988) has been carefully reviewed [10,11]. Many of the GC pioneers (about 21 scientists=awardees) of the 1950s have

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departed from us; their names and some references to describe their research and lives may be found in Appendix 2 and=or the Author=Scientist Index. Note: To be brief, the potential realm for GC in the 1960s has undergone a marked outreach by many new investigators. Many made contributions to the L.S. Ettre and A. Zlatkis book [5]; their research, contributions and short biographies are described briefly in Tables 2 to 4 of Section G of this chapter and later in Chapters 4, 5, S-9 and S-11. Readers will find valuable additional information and the subsequent flow of historical thought in E. Bayer’s contribution [8]. Eighty-four gas chromatographers, who made notable contributions for this subarea, are listed in the Author=Scientist Index with the code letter ‘d’. (Our code letters for seminal concepts and new discoveries by chromatographers are described fully at the end of this chapter, Table 1.2.) Further information on gas–liquid chromatography may be found in the Appendices [12] and recent reviews [13–17]. Others have stated that chemistry has permeated throughout modern human life. The use of chromatography is so much around us and yet is hidden from most people’s attention; year after year, chemical analyses are performed to protect our food, water, air, medicines, etc. The lead organization in this area since the 1900s publishes the book, Official Methods of Analysis of AOAC International (formerly the Association of Official Analytical Chemists), edited by William Horwitz [18]. Their 15th edition, 1990, lists 229 refereed chromatographic methods, of which 130 are GC, 82 are LC, and 17 are general. These methods are associated with classes of compounds: organic acids, alcohols, aldehydes and ketones; monosaccharides; lipids, fatty acids and their derivatives; sterols, steroids and pseudohormones; antibiotics and other drugs, vitamins and vitamin antagonists, flavoring agents, mold metabolites, pesticides, PCBs, nitrosoamines, methyl mercury, dyes, TNT and other nitro derivatives, plus other miscellaneous compounds. Of course, chromatographic procedures have been developed and studied collaboratively to measure many other substances. The 17th edition of AOAC Methods (2000) has 2700 chemical and microbiological methods, 85 new methods, and 110 newly modified methods, and is now available [18]. The American Society of Testing Materials (ASTM) has 15 sections and 73 reference volumes for the 1999 Annual Book of ASTM Standards (coal, pipes, wood, air pollution, etc.), in which Vol. 14.02 has 18 chromatographic methods, covering GC, LC, SFC, ion chromatography, detectors and sampling [19]. Another important source of GC abstracts was the ‘Gas Chromatography Literature, Abstracts and Index’ [20], published by the Preston Technical Abstracts Co. These examples illustrate the penetration of chromatography into the food-, beverage-, pharmaceutical- and other consumer industries. Modern chromatography may not be recognized by the lay citizen, but its pervasive nature surrounds him=her and society. Note: To supplement these notes on early years, see C-1F, C-2, C-4, C-5, C-6A, C-6E, C-6E, S-11 and A-4E.

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D. PAPER- AND THIN-LAYER CHROMATOGRAPHY (TWO FORMS OF PLANAR CHROMATOGRAPHY) D.1. Paper chromatography (PC) One of the simplest forms of chromatography evolved from Liquid–Liquid Partition Chromatography (LLC). Noting that partition chromatography has a support phase of silica gel and a mobile phase in a column, Consden, Gordon and Martin [1] transferred the partition concept in 1944 to cellulose-bound water, i.e., a sheet or strips of filter paper in a closed glass tank. The paper sheet may be run in an ascending mode due to capillary flow, or with an overhead trough, run in a descending solvent flow. The solvent systems employed can be varied with the nature of the solutes; many solvents have been examined and lengthy tables have been published [2,3]. The separated solutes, if not colored, are detected by spray reagents, such as ninhydrin for amino acids; again long descriptions are available. The mechanism of separation in paper chromatography is that of partition of the solutes between the bound-water in cellulose and the ascending solvent. Resolution in paper chromatography is enhanced by drying the sheet after ‘one-dimensional chromatography’, rotating it 90º and placing it in a second solvent system to give ‘two-dimensional chromatography’. The hundreds of publications on paper chromatography have been distilled into many books and handbooks [2]. Paper chromatography [2] had an earlier key (pre-1961) role in the recognition of many, then new, amino acids in: ž Mammalian fluids — butyrine, β-aminoisobutyric acid, 1-methyl histidine, felinine, γ-aminobutyric acid and lanthionine. ž Microorganisms — α-aminoheptylic acid, α,ε-diaminopimelic acid, and γ-aminobutyric acid. ž Plants — γ-methyleneglutamine, γ-methyleneglutamic acid, α-aminopimelic acid, γ-aminobutyric acid, γ-hydroxyglutamic acid, pipecolic acid, 5-hydroxypipecolic acid and L-allohydroxyproline. ž Antibiotics — N-methyl-L-isoleucine, N-methyl-L-valine, N-methylglycine (sarcosine) and lanthionine [3]. Note: Paper chromatography has a considerable volume of research publications (see the papers in S-8H, the books in A-4AB, A-5G, and A-7BH), or the Author=Scientist Index for those scientists with a code letter of ‘b’.

D.2. Thin-layer chromatography (TLC) The transfer of chromatography from a column to a rigid plate, usually glass, to support the sorbent was initiated by two Russian scientists (N.A. Izamilov and M.S. Shraiber, 1938–1939) [4], who called their method ‘spot chromatography’. Next, two Americans (J.E. Meinhard and N.F. Hall, 1949) [5] investigated this area of chromatography and named their approach ‘surface chromatography’. The name, thin-layer chromatography or TLC, came later. However, J.G. Kirchner and his associates (papers in 1951, 1952 and 1954) [6] were the first to undertake systematic studies on TLC,

16

Chapter 1

but with the then lack of available spreading equipment, achieving a uniform layer of sorbent was difficult. Egon Stahl made improvements in sorbents and spreading apparatus (1956 and 1958 papers). By hindsight, the turning point, or the breakthrough, seems to be the subsequent books on TLC by E. Stahl (1962, 1967) and J.G. Kirchner (1967, 1978) [7–9]. TLC is also partition (or sometimes adsorption) chromatography using plates to hold the sorbent; plates are usually 20 ð 20 cm glass sheets (or 5 ð 20, 10 ð 20 and 20 ð 40 cm), but may be also plastic sheets or aluminum foil. Sorbents for partition chromatography include silica gel, cellulose, polyamide powders, Florisil, kieselguhr, etc. The silica gel may be substituted to be a less polar form, i.e., silica > amino silica > cyano silica > octadecyl (C18 ) silica. The particle size of TLC sorbents is between 10 µm and 50 µm. Alumina (Al2 O3 ) as a sorbent provides the conditions of adsorption chromatography. Sometimes a binding agent (usually plaster of Paris, calcium sulfate monohydrate) is needed to hold the silica gel to the plate. Pulverized ion-exchange resins or Sephadex may be used to mimic IEC or SEC. The performance of a TLC sorbent depends on the specific surface area, pore volume, mean pore diameter and pore size distribution, plus particle distribution, and size. The test sample is applied near the bottom edge of the plate, which is then dried and placed into a closed rectangular tank. Many solvent systems have been tested, but the choice depends on the nature of the mixture to be separated and the character of the sorbent. Other variations of TLC include ‘forced-flow planar chromatography’ (FFPC), ‘overpressured-layer planar chromatography’ (OLPC) and ‘centrifugal force planar chromatography’ (CPC) [10]. Thus, thin-layer chromatography has become a widely used separation method due to its rapidity of separation, simplicity in its use, low volume of solvent needed and rather low cost, plus a variety of now commercially available prepared plates and apparatus. Like paper chromatography, TLC may be one-dimensional or two-dimensional. One-dimensional TLC allows application of multiple spots of unknown solutes to be tested for purity. Earlier TLC was mainly a qualitative identification and separation tool in the 1950s–1960s (usually a 10–200 µg sample size and a 250 µm thickness layer). With improved commercially available sorbents came uniform particle size and a variety of sorbents compatible with detection instruments; then TLC became more quantitative (usually 5–500 ng of sample and 150 µm thickness). When smaller particle size (6 µm) and a narrow particle range are utilized, the conditions for ‘high performance TLC’ (HPTLC) are met. When the layer thickness is increased to 250–2000 µm, TLC becomes a preparative tool (5–500 mg of sample in 50–1000 µl of solution). TLC has many other variations, such as reversed phase TLC and modified silicas (added magnesium acetate for phospholipids, added potassium oxalate for polyphosphoinositides, added ammonium sulfate for heat charring and detection of solutes, or added silver nitrate to detect compounds with –C C, double bonds, etc. Note: The number of articles in the research journals proceeded rapidly in the 1960s and 1970s, leading to additional books [7], and eight more books in the 1990s decade [8]. Recent excellent reviews are more detailed than appropriate for this introduction [9–12]. Thus TLC as a qualitative research tool, has had considerable amplification in uses, and has made inroads to serve also as a quantitative procedure. The preceding

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changes in the direction of chromatographic science (Fig. 1.1) in the past century are major milestones that have led to many other new discoveries (see Table 1.3 of this chapter and S-9).

E. ION-EXCHANGE CHROMATOGRAPHY (IEC) (1930s–1960s) The phenomenon of ion-exchange has been going on for a long time in the natural environment of inanimate soils, sands and rocks, or more specifically clays, glauconites, zeolites and humic acids. They are insoluble solids and carry exchangeable cations or anions [1–4]. The humic acids are complex colloids formed from decaying plant tissues, probably from pectins and gums [2]. Whenever salt-containing solutions percolate through the ground cover, an exchange of ions may occur. “Ion-exchangers consist of a framework carrying a positive or negative electric surplus charge, which is compensated by mobile counter ions of opposite sign. A simple model for the ion-exchanger is a sponge carrying an electric charge which must be compensated by charged particles within its pores” [4]. The zeolites are aluminum silicates and include analite, chabazite, harmotone, heulandite and natrolite; they have a three-dimensional framework with channels and cavities in the overall lattice. With trivalent aluminum in the lattice, sodium-, potassium-, calcium- and barium ions move freely within this lattice; one major use of the zeolites was for water softening in areas of hard water (called then ‘permutits’). Based on the above observations, Adams and Holmes in the 1930s [5] made a deliberate invention to synthesize ion-exchange resins, i.e., to introduce sulfonic acid groups into a phenol-formaldehyde resin (‘Bakelite resins’), or m-phenylenediamine into phenol-formaldehyde polymers [3,5]. Soon thereafter, the former I.G. Farbenindustrie in Germany developed synthetic resins and were followed by companies in the United States and United Kingdom. Rohm and Haas Co., Philadelphia, PA, called their cation series of sulfonated polystyrene resins, ‘Amberlites’, such as Amberlite IR-1, IR-100, IRC-120, etc.; Dow Chemical Co., Midland, MI, developed the ‘Dowex’ resins, such as Dowex 50, a sulfonated styrene divinyl benzene copolymer; Permutit Ltd., United Kingdom prepared the ‘Zeo-Karbs’ and many others [3,4]. Vinyl-addition polymers with substituted carboxyl groups were Amberlites (IRC-50, XE-89, etc.), Permutit’s Zeo-Karbs, and many other trade names [3]. Tertiary alkyl amines were widely used to produce anion-exchangers (Dowex-1, Amberlite-IRA-400, etc.) along with incorporation of dimethylethanolamine (Dowex-2, Amberlite-IRA-410, etc.) [3,4]. The next stage in the history of IEC involved the merger of two areas of investigation: the isolation and study of properties of the rare earths [4], and the fission products of radioactive decay. The rare earths are 15 elements in the Periodic Chart (elements 58–71 plus yttrium-39); they are metals having three electrons in their outer orbit (therefore trivalent). Only cerium and lanthanum were examined in detail prior to World War II; they were isolated by fractional crystallization, fractional precipitation, amalgam extraction and=or liquid–liquid extraction. Ion-exchange chromatography became the fifth and main method for their isolation during and after World War II. For the elution of the rare earths, buffers of citrate, malate, glycolate, lactate, α-hydroxyisobutyrate, and ethylenediamine tetraacetate (EDTA) were used for the analytical objective, and later

18

Chapter 1

for the preparative scale [2–4]. A major issue of the J. Am. Chem. Soc. published 15 papers on wartime IEC research in 1947 [6a]: ž Oak Ridge National Laboratory (ORNL) (papers by E.R. Tompkins, J.-X. Khym, and W.E. Cohn) — Ion exchange as separations method. ž ORNL (papers by G.E. Boyd) — The exchange adsorption of ions by organic zeolites. ž Iowa State College (papers by F.H. Spedding and co-workers — Separation of rare earths by IEC. ž Miscellaneous papers by J.A. Ayers, J.A. Marinsky, W.C. Bauman, R. Kunin and R.J. Myers. ž Other early reports on IEC during World War II (F.H. Spedding and E.R. Tompkins; T.R.E. Kressman and J.A. Kitchener; R. Kunin and R.J. Myers; E. Glueckauf and associates; and S.M. Partridge) may be found in the 1949 conference report of the Faraday Society, UK [6b], a collection of early IEC papers [7], and followed by other books in the 1950s and 1960s [8]. The 1940–1941 advent of World War II led many governments and scientists to develop new weapons and defenses: explosives, land mines, war gases, particularly the nerve gases, radar, and last, but not least, the atomic bombs with their escalation of destruction, along with advances to deliver these weapons. The then secret Manhattan Project undertook major research to develop the atomic bomb and to identify both the metallic ions and the radiations emitted during the stepwise radioactive decay. The available, synthetic ion-exchangers — the substituted polystyrene resins (the Dowex series) and the substituted acrylic resins (the Amberlite series) — were utilized for separation of these nuclides. The key investigators of IEC at the Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA were Waldo E. Cohn, Kurt A. Kraus and Edward H. Tompkins [9]. W.E. Cohn (1910–1999) earned his B.Sc., M.Sc. and Ph.D. degrees (1938, with the senior biochemist, David M. Greenberg) at the University of California, taught at Harvard Medical School (1939–1942), and then became a group leader of the Plutonium Project, University of Chicago and Manhattan Project, Oak Ridge National Laboratory (1942–1947). During these years, he worked on the ion-exchange separation of rare earth elements and fission products [10]. In post-World War II years, he did research to develop ion-exchange methods for the isolation (and analysis) of the purine and pyrimidine bases and the mononucleotides of the nucleic acids (DNA and RNA) (about 40 research papers and 5 reviews in the 1948 to 1967 period (from his resume by ORNL)) [cf. 11–13]. He was an early scientist in the 1950s to organize and promote the distribution of radioactive isotopes for use in medical research and treatment. He was the initial editor for 8 volumes of the annual series, Progress in Nucleic Acid Research (1963–1968), chair of the Oak Ridge Town Advisory Council (1953–1955), and an amateur cellist; he retired in 1975. Not surprisingly, Cohn was the third recipient of the National Chromatography Award of the American Chemical Society (1963). See Chapter 5B, c and A-2 To again step back in time, Kurt A. Kraus (1914–1995) was born in Windsheim, Germany, came to the United States in 1935 and was naturalized as a citizen in 1941. He

The Beginnings of Chromatography — The Pioneers (1900–1960)

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received his B.Sc. at Harvard University (1938) and his Ph.D. degree at Johns Hopkins University (1941). After two years at Tulane University, he joined the Metallurgical Laboratory, Chicago, became a group leader at Oak Ridge National Laboratory (1945– 1948), Tennessee [9], and then their Director of the Water Research Program of ORNL (1962–1971). His early research focused on inorganic chemistry and ion-exchange of alkali metal ions [14–16]. Later he conducted research on hydrous oxides, properties of solutions, adsorbent studies, inorganic polymers, desalination, hyperfiltration studies, and pollution control, that were described in many papers in the open literature, contract reports and 9 patents (1965–1971). Kraus was the 1966 recipient of the National Chromatography Award of the American Chemical Society. See Chapter 5B, c and A-2 Information on Edward R. Tompkins (1998†) — an ORNL scientist — is difficult to find. Four of his significant papers are cited [9,17–19]. Other investigators and related IEC findings are described in the review by L.S. Ettre [9]. See Chapter 5B, c Frank H. Spedding (1902–1984) was the chemist who made distinct early contributions for IEC. He was born in Ontario, Canada, earned his B.Sc. (1925) and M.Sc. (1926) at the University of Michigan and his Ph.D. in chemistry at the University of California (1929). After several short appointments elsewhere, he joined Iowa State College (1937) (now Iowa State University), serving there as Professor (1941–1973), Director of their Atomic Project (part of the Manhattan Project) (1942–1948), and then became Director of the Ames Laboratory, United States Department. of Energy (1947–1968). He was a member of the National Academy of Sciences and has several awards other than in chromatography. His ‘Ames group’, as a part of the Metallurgical Project within the overall Manhattan Project (World War II), focused on the pilot plant production of high grade uranium; 1000 tons of pure uranium metal were isolated at the Ames pilot plant with a dozen large IEC columns (40 inch in diameter and 10 ft. in height), 12 to 18 day runs and collecting 45 liters per 12 hours [20–23]. This role of IEC in World War II has been thoroughly reviewed by L.S. Ettre [9]. Spedding’s et al. research examined the isolation and properties of the rare earth elements (particularly the lanthanide series by IEC), atomic and molecular spectra, metallurgy of uranium, thorium and other rare metals, plutonium chemistry, atomic energy chemistry, and absorption spectra of solids at low temperatures. His research papers may be identified in several books [20–22]. The references for the early investigation and properties of the rare earths are available [9,21–26a]. See Chapter 5B, c and A-2 Another investigator, Olof Samuelson (1914–2000) conducted research on ionexchange chromatography during the above early time period. He published about 27

20

Chapter 1

research papers in the 1939 to 1960 period in Scandinavian journals and completed his thesis in 1944. Subsequently he has served as Professor of Engineering Chemistry, Chalmers University of Technology, Go¨teborg, Sweden. His early research focused on the IEC separation of organic acids, sugars, aldehydes and ketones as their bisulfite addition products, Fe3C and AI3C from other metal ions. His two books (1st ed. in 1953, 2nd ed. in 1963) have a thorough discussion on the concepts and applications of IEC and the cited references prior to 1963 [26b]. His post-1963 research (about 240 papers) relied on anion-exchange chromatography to examine uronic acids, aldonic acids, aldouronic acids and other organic acids, bleaching of kraft pulp without chlorine and cellulose degradation in bleaching by oxygen or nitrogen dioxide. See Chapter 5B, c Eugen Glueckauf (1906–1981) also made contributions to IEC. He was born in Germany with parents from Jewish origin, and owners of a textile manufacturing business. His studies began at the University of Berlin, but soon changed to the Technische Hochschule at Charlottenburg (Dipl. Ing., 1930), and went on then to study surface adsorption and surface reactions (Dr. Ing., 1932). With the political developments then in Germany, he migrated to England in 1933 and accepted an assistant position with F.A. Paneth (F.R.S.) at the Royal College of Science (Imperial College), South Kensington. His research focused on helium analysis to determine the age of meteorites; since neon interfered with the helium analysis, he developed a 12-stage adsorption–desorption cascade with charcoal cooled in liquid nitrogen as adsorbent that separated He and Ne. For several years, he examined the production of He after bombardment of boron with neutrons, and beryllium with γ-rays. In 1937, he was appointed Professor of Chemistry at Durham College (later the University of Durham). With the outbreak of World War II, he was interned for 5 months in 1940, but released by the efforts of F.A. Paneth. In 1944, he was invited to join the extramural work of the Department on ‘Tube Alloys’ (the code name for atomic energy research). Early research dealt with separation of LiC isotopes by IEC and Ne isotopes by low temperature (196ºC) adsorption columns [27]. In 1947, he transferred to the newly created Harwell Laboratories, and was stepwise promoted to Deputy Chief Scientist in 1952. In the mid-1950s, he achieved separation of the hydrogen isotopes on a Pd-asbestos column. He developed nine patents and retired in 1971. During this later 19 years, he focused on radioisotope chemistry, solvent extraction, concentrated electrolyte theory, membrane science and chromatography. Several of Glueckauf’s papers dealt with the principles of IEC, though the equations apply to all forms of chromatography. A memorial biography provides many additional details and his IEC references [28]. See Chapter 5B, a, c, d, s and A-2 Once the above basic knowledge for the ion-exchange resins was developed, use of this method spread rapidly to the separation of carbohydrates as their borate derivatives, amino acids, nucleic acid derivatives, antibiotics, alkaloids and more complex biological fluids [2–4,6–8].

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Furthermore this research base led to the examination of related approaches to make phospho-ion-exchangers, electron exchange resins (hydroquinone condensed with phenol and formaldehyde, or vinyl hydroquinone polymerized with divinylbenzene) and chelating resins (containing EDTA-like functional groups). The team, Stanford Moore (1913–1983) and William B. Stein (1911–1980), Rockefeller University, New York, NY, USA, achieved the next major advance in ion-exchange chromatography. This pair and their colleagues conducted research in the early 1950s on quantitative amino acid analysis by starch partition chromatography on starch columns, and in the late 1950s by ion-exchange chromatography. Key features of sensitivity, precision, automation and quantitation were developed for the first time. Their extensive basic knowledge led them in the early 1960s to determine the amino acid composition of the enzyme, ribonuclease, and then to ascertain the amino acid sequence of ribonuclease. This research led to their 1972 Nobel Prize in Chemistry. The ion-exchange principle has been extended to modified cellulose derivatives [29,30], altered polydextrans [31], and cross-linked agarose [32], along with the development of many commercial grades of polymers and refinements [33]. Note: Subsequent developments in ion-exchange chromatography for analytical and preparative objectives for many classes of compounds have been reviewed [1,2,25,33]. At this point in the introduction to chromatography, an Outline of Major Variants of Chromatography (common terms for the forms of chromatography) so far discussed should be presented. The 1991 outline by J. Calvin Giddings, presented in Table 1.1 below, serves this purpose.

F. CHROMATOGRAPHY OF PETROLEUM As a consequence of the exploration, fractionation, processing and utilization of oil, gasoline, kerosene, organic gases and related products, the analysis and chemistry of these hydrocarbon mixtures occurred more recently — more or less parallel to the rise of the internal combustion engine and the post-1900 rise of the modern automobile. Since these developments are described elsewhere, they are briefly mentioned here to place in perspective the rapid developments of the chromatography of petroleum in the 1940s to 1950s. Hydrocarbons in gas form may arise from (1) anaerobic bacterial fermentation producing methane or marsh gas (about 109 tons per year for the earth), (2) coal mining leading to a methane-rich mine drainage gas, or (3) landfill gas from human waste. Natural gas, like fossil-based oil and coal from deep, rich accumulations, is mainly methane with low concentrations of He, N2 , CO2 , ethane and higher alkanes [1]. Gases from petroleum refineries are more complex. Regardless of source, hydrogen sulfide is removed due to its toxicity and corrosive nature in an amine scrubbing plant; carbon dioxide is separated by an alkali scrubbing process; water is deleted by a glycol scrubbing tower and possible hydrocarbon liquids are eliminated by chilling or adsorption. Such natural gases are widely used as fuels in residences and industries, and as feedstock in the chemical industries [1,2]. The many components and biogenesis of petroleum is highly complex [3].

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Chapter 1

TABLE 1.1 MAJOR VARIANTS OF CHROMATOGRAPHY

From J.C. Giddings, Unified Separation Science, 1991, Chapter 10, p. 232.

Building on the earlier research of M.S. Tswett, L.S. Palmer, C. Dhe´re´, A.J.P. Martin and A.T. James, the merits of gas chromatography for separation and analysis of petroleum products were very clear, due to its volatile, heat-stable, organic compounds found in petroleum. After the 1859 drilling of the first oil well at Titusville, PA, USA, by Edwin Drake, oil production increased in the United States and many other countries to meet the demands of industrial growth, plus the later needs of automobiles, trucks and diesel engines. The United States Geological Survey, that commenced in 1879, began a major survey of petroleum components under the leadership of David Talbot Day (1859–1915); he did not know the complexity of petroleum components [4]. During and after his graduate studies at the University of Maryland, he worked at the United States Geological Survey, starting full time in 1895 and held a variety of administrative positions [4]. In several lectures (1897, 1900), Day proposed that primary oil migrated through rock formations by diffusion, not the earlier distillation mechanism; his theory called ‘filtration hypothesis’ was never followed with written data nor methods. (His later emphases focused on utilization of oil shales, cracking

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heavier oils to gasoline and private consultation.) Day’s work was continued by Joseph E. Gilpin (1866–1924), a Professor of Chemistry at John Hopkins University, and his students who tested the ‘filtration hypothesis’ by allowing oil to diffuse down, and later upwards through a column of fuller’s earth [4]. Gilpin’s four papers (1909–1913 — after Tswett’s earlier papers) reported some fractionation of oil, but not complete separations. Carl Engler (1842–1925), a German organic chemist at the University of Halle and later the Technical University at Karlsruhe, heard Day’s 1902 lecture, set out to test the ‘filtration hypothesis’ and relied mainly on Day and Gilpin’s conditions; they did not show separation. L.S. Ettre concluded that the methodology of Day, Gilpin and Engler “can be considered as precursors of the chromatographic technique; however, it should not be envisaged as the beginnings of chromatography” [4]. (L.S. Ettre’s article [4] has many other details and references on these three scientists.) Fractional distillation of the petroleum volatiles was the main earlier method of analytical approach, but slowly in the 1940s, “fractional desorption from an adsorbent” developed in the early investigations of S. Claesson [5] and C.S.G. Phillips [6], who used columns (2, 8 or 15 mm ID, 30 cm long) of activated coal or coconut charcoal and 4 levels of temperature between 7º and 127ºC to separate low molecular weight hydrocarbons. Briefly, another early report by W.M. Smit explored what he called “adsorptive percolation” of petroleum hydrocarbons on silica gel [7]. A.S.C. Lawrence and D. Barby used powdered coke or alumina and successive solvent elutriation of fuel oil [8]. A turning of direction, or inflection point, was the pioneering work of A.T. James and A.J.P. Martin in 1952 [9]. It is interesting to note that some of the GC research by E. Cremer [10], G. Hesse, and J. Jana´k [11] preceded that of A.T. James and A.J.P. Martin [12,13]. Note: Most readers are aware of the overlap in the leading GC investigators and the petroleum chromatographers. Deceased chromatographers who explored hydrocarbon mixtures include D.H. Desty, A. Zlatkis and others; the living chromatographers who have been active contributors to petroleum chromatography are identified in the Author=Scientist Index (with the code letter ‘j’ — see Table 1.2 for description of these code letters). Since this chapter is an introduction, the reader is referred to the many, more detailed books=reviews (see earlier C-1C, S-9A and Appendices 2, 4-A, 4-E, 5ABCDE, and several in 7). The outstanding recent book on petroleum chromatography, edited by E.R. Adlard [2] reviews the quantitative analytical procedures, waxes, hydrodynamic chromatography of polymers, petroleum geochemistry, several detectors, multi-column GC systems, SFC, HPLC, data handling and capillary electrophoresis.

G. THE LITERATURE OF CHROMATOGRAPHY To read and understand an article, review or book, one usually reads in sequence — paragraph by paragraph, or chapter by chapter — a linear pattern. To integrate the whole, book writers include a table of contents, cross-references in the text, author index and subject index. To supplement these literary devices, the following Fig. 1.2 on Driving Forces in Modern Chromatography is presented to emphasize the robust

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Chapter 1

DRIVING FORCES IN MODERN CHROMATOGRAPHY AND CONNECTIONS WITHIN THIS BOOK Scientists (See Chapters 1, 2, 5, 7, S-9, S-10 and S-11)

Seminal Concepts/ Subject areas (See Chapter 5, 6 and S-9)

Experimental Investigations and Instrumentation/ Automation

Research Publications (See Chapters 5, 6 and S-9, Appendices 4, 5 and 6)

(See References cited in later Chapters)

Scientific Organizations

Emerging Technologies

(See Chapters 2 and 3, Appendices 5 and 6)

(See Chapters 4, 5, 6, S-9, S-10, S-11, S-12 and S-15)

Chromatography Applications and New Scientific Industries

Fig. 1.2. Driving forces in modern chromatography. This overall figure summarizes the known relationships of chromatography (or science in general) and will be expressed in greater detail in the stated chapters or appendices. The arrows highlight the connections, or the flow of thought, experiments, and the needed process of communication that leads to emerging applications in new scientific industries.

nature of and the interactions with chromatography at the turn of this millennium. Only the partially linear and main pattern of driving forces in chromatography is presented in Fig. 1.2, in the connections to the chapters and appendices of this book. Other cross-connections exist and are more subtle and variable. This chapter — a sketch of the history of chromatography — is an introduction for the chapters to follow. Chapter S-8 by L.S. Ettre provides a bibliography of references for books, chapters, journal articles relevant to this chapter and this book; the interested reader is encouraged to examine further these sources. As stated earlier, one goal of this book is to highlight the significant research contributions, discoveries and personal recollections of over 100 chromatography awardees [1]. Another goal is to emphasize the role of the scientists, their seminal concepts, the science of chromatography, the research publications, scientific societies, institutions and companies, the developing technologies and new scientific industries and their interactions as shown in Fig. 1.2. Notes: The seven appendices provide supplementary information on mainly books and also overlap with many chapters. An excellent early reference to peruse is the 1949 Faraday Society Discussions [1], which provides a dramatic contrast of the state of chromatography knowledge in 1949 with that of 2000 [2]. However, a recognized imbalance of limited references to journal articles is corrected by the references cited

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in other books. For instance, I.M. Hais and K. Macek made valiant efforts to collate and publish the accumulating papers on paper and thin-layer chromatography in the 1950s to 1960s [3]. E. Lederer and M. Lederer cited 1879 references in their 1954 book on Chromatography [4]. Similarly, Z. Deyl (Ed.) in his 1984 book, Separation Methods [5], has 512 journal references for gas chromatography, 145 references for adsorption and partition chromatography, 169 for gel chromatography, 230 for affinity chromatography, 176 for planar chromatography, and 301 references for electromigration techniques. L.S. Ettre in his 1980 review, Evolution of Liquid Chromatography — A Historical Overview [6], has 296 references on adsorption chromatography, partition chromatography (paper- and TLC), IEC, gel chromatography, affinity chromatography and HPLC. Cs. Horva´th, editor of his 5-volume series, 1980–1988 book, High-Performance Liquid Chromatography [7] has hundreds of references on HPLC. J.C. Giddings in his book, Unified Separation Science [8], and many other authors show a similar thoroughness for references of their stated subject area. Another excellent source to find reviews and journal articles is Chemical Abstracts, Vol. 1, 1907 to Vol. 132, 2000, published by the American Chemical Society, Washington, DC, USA, which covers the world’s chemical literature with a very broad scope; its earlier Collective Decennial Indexes and now Collective Quinquennial Year Indexes enable a scientist to make a faster search through the thousands of citations. Chemical Abstracts has some 241 CA SELECTS as a biweekly, current awareness bulletins, that includes GC, GPE, HPLC, IEC, ion chromatography, paper chromatography and TLC, as well as specific chemical groups, such as amino acids, peptides and proteins, enzymes, prostaglandins, natural products, steroids, etc. However, when one’s intent is focused on chromatography, the frequently prepared Bibliography Section of the Journal of Chromatography A (edited by Z. Deyl, J. Jana´k, V. Schwarz and K. Macek and published by the Elsevier Science Publishers, Amsterdam, The Netherlands) is valuable in content and organization [9]. This Bibliography has major sections on liquid column chromatography, gas chromatography, planar chromatography, gel electrophoresis and capillary electrophoresis=electrokinetic chromatography with each having some 38 subsections plus other subdivisions; this structure of literature organization facilitates quick searches. Chromatography Abstracts, 1986–1999 under the current guidance of the (British) Chromatographic Society and the Royal Society of Chemistry is another reference source [10]; it started in 1958 under another name, Gas Chromatography Abstracts. However, in this day of computers, many scientists may prefer seeking the desired reference information by the ‘on-line’ approach. Professional societies have had considerable input into the advance of chromatography through their journals and research conferences. The latter provide an opportunity to present research papers, overall or review papers, presentation of research awards and frequently a related symposium. Furthermore, such conferences provide the desired training and retraining. Two older societies, Society for Analytical Chemists of Pittsburgh (SACP) and the Spectroscopy Society of Pittsburgh (SSP), combined strengths in 1949 to plan the Pittsburgh Conference on Analytical Chemistry (hereafter PITTCON). PITTCON celebrated its 50th birthday in 1999 [11]. Leslie S. Ettre presents a longer discussion of PITTCON and other chromatography societies (see C-3 and C-8).

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Chapter 1

TABLE 1.2 INTEGRATION OF SEMINAL CONCEPTS WITH CHROMATOGRAPHY LEADERS Code

Seminal concepts and research areas

Chapter locations

a. b.1 b.2 b.3 b.4 c. d.

Theoretical contributions to chromatography Early adsorption chromatography Early partition chromatography Paper chromatography Thin-layer chromatography Ion-exchange chromatography Gas chromatography=capillary gas chromatography

e. f. g. h. i.

Supports, stationary-, and bonded-phases chromatography Detectors in chromatography Size-exclusion chromatography High-performance liquid chromatography Affinity chromatography=bioaffinity chromatography=biosensors Petroleum chromatography Instrumentation in chromatography Electrophoresis=capillary electrophoresis=capillary electrochromatography Ion chromatography Synthetic and biological membrane separations and other techniques Supercritical-fluid chromatography=extraction Hyphenated=coupled=tandem techniques in chromatography Chiral chromatography and pharmaceutical separations Biomedical sciences and chromatography Environmental sciences and chromatography Space sciences and chromatography

6-ABCE 1-B, 6-BE, 8-E, 9-AB, 1-C, 5, 6-BE, 8-G, 9-AB 1-D, 5 1-D, 5 1-E, 3-CD, 5, 9-A 1-3, 3-CD, 5, 6-ABCDE, 8-H, 9-AB, 10-A 5, 6-AB, 11, 14 5, 6-ADE, 9-E 5, 6-AE, 9-E, 14 4-E, 5, 6-ABCDE, 9-B, 9-E, 11 5, 6-A, 9-D

j. k. l. m. n. o. p. q. r. s. t.

1-F, 5, 6-BD 5, 6-ADE, 9-D, 10-A 5, 6-ACDE, 9-A, 14 5, 6-A, 14 5 5, 6-A, 6-ADE, 9-D, 14 5, 6-ADE, 9-E, 14 5, 6-A, 9-D, 11-C, 14 5, 6-ACD, 11, 12-E, 14 4-B, 5, 6-ACD, 14 4-D, 5, 14

Chapter locations are indicated by a number (1–15) and subsections by a letter; chapters with a number higher than 7 are in the Supplement.

H. INTEGRATION OF SEMINAL CONCEPTS WITH CHROMATOGRAPHY LEADERS The emphasis in this book is on the scientific biographies and the accomplishments of the chromatography leaders=awardees. However to integrate seminal concepts and research areas with the chromatography leaders, the editors have devised the following letter code ‘a’ to ‘t’ in the first column of Table 1.2. These letter codes are also used later in other chapters. Note: For Table 1.2 and elsewhere, the editors grouped together in ‘l’ the three electrodriven separations; strictly speaking the first two are not chromatography. However, these widely utilized methods shade into electrochromatography and are thus listed together. Since this book concentrates on chromatography, these three separation methods will be handled only briefly. Recent developments suggest that they will be dominant ultramicro techniques in the 21st century.

The Beginnings of Chromatography — The Pioneers (1900–1960)

27

The scientific literature has a generally recognized pattern: research investigation ! oral presentations ! written scientific papers ! comprehensive reviews (or book chapters) ! scientific books ! major treatise=handbooks. The literature process is usually additive, selective in later stages, questioning and yet confirming, postulating and also leading to new approaches=experiments. Since the original research literature may be identified as stated above, the editors have developed appendices for references of books that enhance=extend the subject areas of the subsequent chapters and for the awardee’s contributions.

I. FROM THE INVENTORS TO THE BUILDERS OF CHROMATOGRAPHY The brilliant beginnings of chromatography, just presented in this chapter, were followed by the rapid and widespread dissemination of the concepts and practices of chromatography by the recognized builders of chromatography; these awardees are listed in the 13 tables in Chapter 2, and their interactions with many professional societies. These international, national, and regional societies have supported the enhancement of chromatography in the 1930 to 2000 period, particularly the post-1960 decades by: ž Publishing research papers. ž Organizing symposia, research conferences and oral papers (see also C-3). ž Holding informal discussions, scientific forums, displays and interactions with the scientific industries (see also C-3, C-4, C-6, S-8 and S-15). ž Training and retraining of scientists with advanced knowledge (see also S-14). ž Recognizing scientific leadership and creativity in chromatography by identifying awardees who have received these prestigious awards (see also C-2 and C-5). Using a different approach than ‘from the Inventors to the Builders of Chromatography’, Leslie S. Ettre has prepared an excellent review, entitled ‘Chromatography: The separation technique of the 20th century’ and describes many of the same phenomena, but adds other directions and interpretations [1]. The flexibility of chromatography is influenced by flow of the mobile phase (gravity pressure, capillary action and electroosmosis), wide temperature range in GLC and HPLC, sample size, column length and diameter (or dimensions of flat plates), all of which in turn have changed chromatography from a technique (extrusion adsorption chromatography) to a sensitive, automated, instrumental method [1]. Another historical essay emphasizes similar concepts, scientists and the development of chromatographic processes [2]. Thus chromatography, as a new branch of science, has had a series of significant changes — the chromatographic milestones presented next in Table 1.3. They should be considered as ‘paradigm shifts’ (a subject to be described later in S-9C).

I.1. Other early chromatography leaders The present book, Chromatography — A Century of Discovery, with its scientific biographies is thus similar to and advances to 2000 the book by L.S. Ettre and A. Zlatkis

28

Chapter 1

TABLE 1.3 CHROMATOGRAPHIC MILESTONES OR PARADIGM SHIFTS Date

Major events in chromatography – the milestones

Some early investigators in each area of chromatography

Chapter locations

1901–1903

M.S. Tswett

1-A

1903–1906 1912–1922 1931–1938

The beginning of adsorption chromatography First publications Confirmation and extension Rediscovery and recognition

1-A 1-B 1-A, 9-B

1938–1960s

Thin-layer chromatography

1941

A.W.K. Tiselius

9-A

1940s

Partition chromatography concept and plate theory Liquid–liquid chromatography Adsorption chromatography – 3 modes of development Ion-exchange chromatography

M.S. Tswett L.S. Palmer and C. Dhe´re´ R. Kuhn, E. Lederer, P. Karrer, H.H. Strain and L. Zechmeister N.A. Izamilov, J.G. Kirchner, M.S. Shraiber, and E. Stahl A.J.P. Martin and R.L.M. Synge

1-E, 5

1944

Paper chromatography

1952 Mid-1940s–1960s

Gas–liquid chromatography Gas–solid chromatography

Mid-1950s

Automated IEC of amino acids

W.E. Cohn, K.A. Kraus, F.H. Spedding, O. Samuelson and H.F. Walton R. Consden, A.H. Gordon and A.J.P. Martin; F. Sanger A.T. James and A.J.P. Martin E. Cremer, E. Glueckauf, G.E. Hesse, J. Jana´k, A.V. Kiselev, P.C. Lu, and A.A. Zhukhovitskii S. Moore and W.H. Stein

1959–1960s

Gel filtration=molecular sieve=size-exclusion chromatography HPLC developed and established

1940s

1960s–1970s

1960s–1970s

Large elution columns for chromatography

1966–1980s

Chiral chromatography

1968 1990s

Affinity chromatography Instrument development, process design and process chromatography Chromatography-on-a-chip

1990s

P.G.M. Flodin and J. Porath

1-D 9-A

1-D

1-C

1-E, 5, 9-A 5, 9-E 5, 9-E

K.A. Cramers, J.C. Giddings, C.W. Gehrke, I. Hala`sz, Cs. Horva´th, J.F.K. Huber, J.J. Kirkland, L.R. Synder and others E. Bayer, K.P. Hupe, G. Guiochon, Cs. Horva´th, J. Porath and K.I. Sakodynskii E. Gil-Av, D. Armstrong, V.A. Davankov, W. Pirkle, C. Welch and others C.B. Anfinsen Many investigators and companies

9-D 9-D, 10

A. Guttman and others

5, 9-F

5

5, 9-D

Chapter locations are indicated by a number and subsections by a letter, as earlier.

(Eds.), 75 Years of Chromatography — A Historical Dialogue, Elsevier Scientific Publishing Co., 1979. The objective of Table 1.4 is to integrate and to cross-refer these two books for the benefit of the reader.

The Beginnings of Chromatography — The Pioneers (1900–1960)

29

TABLE 1.4 OTHER EARLIER CHROMATOGRAPHY LEADERS 1,2 Name

Institution 3 , country 4

Subject areas(s) 5

Ettre=Zlatkis book (see pages below)

Adlard, Edward R. 6 Boer, Hendrik Dijkstra, Greult Grant, David W. Heftmann, Erich Horning, Marjorie G. James, Anthony T. 6 Kaiser, Rudolph E. 7 Karmen, Arthur 6,8 Kirchner, Justus G. Kova´ts, Ervin sz. 6 Lederer, Michael Liberti, Arnoldo 6 Patton, Hugh W. Phillips, Courtenay S.G. 6 Ray, Neil H. Rohrschneider, Lutz Schwartz, Robert D. Scott, Charles D. Stross, Fred H. Teranishi, Roy Van Deemter, Jan J.

Shell Res. Ltd., UK Shell Lab., NL Utrecht Univ., NL Brit. Carb. Res. Assoc., UK WRRC, USDA, USA Baylor Col. Med., USA Unilever Res., UK Inst. for Chromatogr., D A. Einstein Col. Med., USA USDA, Coca-Cola Co., USA Ecole Polytech., L., Fed., CH Lab. Chromatogr., I Univ. of Rome, I Eastman Chem. Prod., USA Oxford Univ., UK ICI Corp. Lab., UK Chemische Werke Hu¨ls, D Pennzoil Co., USA Oak Ridge Natl. Lab., USA Shell Dev. Co., CA, USA; Univ. Wash., USA WRRC, USDA, USA Shell Lab., NL

d, f, s d, k d, k d, j b, r d, r a, d, l, r a, b, d, s d, f b a, d, e a d, f d, f a, d, f d, f, j d d, f, g, j h, r d, f, j d a, d

1–10 11–19 43–51 115–123 125–130 142–150 167–172 187–192 193–200 201–208 231–236 247–253 255–263 309–313 315–322 345–350 351–360 381–390 391–395 443–446 453–460 461–465

1

For deceased, earlier Chromatography Leaders and their page reference in the Ettre and Zlatkis 1979 book, see Appendix 2. 2 An exception for Table 1.4 should be noted. Some of the 1979 living and deceased contributors are omitted here, as they are mentioned in subsequent chapters and located in the Author=Scientist Index. Thus, Table 1.4 complements these chapters and Appendix 2 that covers deceased chromatographers. These chromatographers were active during the 1930s to 1980s; many have retired; those who have passed away may be found in A-2. Most of the chromatography awards had not been established for these earlier decades. 3 The institutional and country affiliations correspond to their 1979 listings; some may have moved. 4 For country abbreviations, see the Author and Scientist Index. 5 The Subject Area(s) for their research are described in Table 1.2. 6 These investigators have received one or two of the awards. 7 W. Bertsch, R.E. Kaiser decorated by German president, J. High Resolut. Chromatogr., 19 (1996) 710. 8 L.S. Ettre and G. Malikin, Editorial on A. Karmen, Chromatographia, 51 (5=6) (2000) 260–261.

To summarize, these earlier chromatographers made distinctive contributions, mainly in gas chromatography, supports and stationary phases, along with the needed detectors and instrumentation.

J. WHAT IS REQUIRED TO BE ONE OF THE AWARD WINNERS? Most award winners in science have the following common requirements: ž Conducted outstanding research as recognized in peer-reviewed, scientific journals.

30

Chapter 1

ž Made significant laboratory discoveries and seminal ideas that have led to an original concept or new experimental approaches. ž Published a trail of research papers that documented and extended these approaches or concepts. ž Received recognition by peer review of their colleagues in the appropriate scientific society, forum, or symposia. Though most scientists are familiar with ‘peer-review’, others in different disciplines and the lay community are not fully aware of the term. This communication gap between scientists in their ‘laboratories’ or ‘ivory tower’(?), with citizens at large and policy makers in key positions is a major consideration and requires additional comment. While there are many exceptions, the same comment applies also to the limited interaction of scientific societies, the media and officials in national and international governments. ‘Peer review’ in the scientific community applies to the careful evaluation and detailed consideration of: (a) research grant applications to government agencies or private foundations; (b) original research papers for professional scientific journals, whether sponsored by scientific societies or private publishers; (c) or a review of nominations for scientific awards. ‘Peer review’ refers to the deliberate, studied, in-depth evaluation by one’s colleagues to accept, reject, or recommend modifications of a grant application or a scientific paper. ‘Peer review’ is conducted on an anonymous basis and has frequently led to improvement in the grant application or scientific paper. With some modifications, the same qualities are included in the ‘behind the scenes steps’ for the ‘peer review’ of nominations for the chromatography awards. These awards and their ‘peer-reviewed’ awardees represent the collective best in the chromatographic sciences. The social=intellectual organization of ‘peer review’ is performed by scientific societies, which, of course, have other responsibilities in the communication of science. Note: Further information on the awardees is described in C-2, 5, and S-9A and 9B and the related scientific societies in Chapters 1G, 2, and 3.

SUMMARY Chromatography has grown as a branch of science, and is now a powerful research tool ranging across the sciences. The genius of Mikhail S. Tswett, the Father of Chromatography, in the 1900s has had a profound subsequent impact in the sciences; he was a true inventor, whose ideas have grown after his death to become the most widely used laboratory separation method of all time. Chromatography has grown over the last century to be a vigorous enterprise of scientists; plus societies with the background of their patrons (or support) from government, academia, and research institutions; and the corporate sector and private foundations. The scientific community and public at large has benefited from these interactions, as presented by the awardees in other chapters by: ž Professional interactions of scientists among universities, industry, and across the semi-artificial boundary lines of cultures and nations. ž Personal knowledge and frequent long-standing friendships of scientists, despite such possible barriers as race, religion, patriotism and language.

The Beginnings of Chromatography — The Pioneers (1900–1960)

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Fig. 1.3. Passing the Baton of Chromatography.

ž Shared or common language among scientists with respect to their experiments, hypotheses, small and large molecules, reactions and their governing conditions, controls and signals for these processes. ž Resolution of key problems in medicine and human health, pharmacology and new drugs, agriculture and food production (including herbicides and pesticides, nutrients and food composition), scientific industry and technical advances, petroleum processing, environmental conservation and avoidance of pollution, education and the rising generation of future leaders. Chromatography as one of the separation sciences has become a major ‘bridge’, or ‘common denominator’ for analytical methods and biological=medical sciences research. They have caused many changes in other sciences, but particularly in analytical chemistry — a change from the determination of one or several components in a sample to the separation, detection and the quantitative measurement of all the components in a sample. The chromatography bridge (see the frontispiece) depicts the spanning from the foundation into the 21st century, by those on the bridge, and by the thousands of other chromatographers; they have provided insights and research tools to make new discoveries possible for the advancement of society. For instance, chiral chromatography has resulted in immediate and profound changes in the pharmaceutical industry, medical practice and government regulations. What is the discovery process? C.S.G. Phillips in a thoughtful essay on this question provides the examples in chromatography; to state his guides — “to solve a problem, cross-fertilization, analogy, simplicity, observation, serendipity, rejection” [see 1]. This discovery process is leading to the new areas of genomics, proteomics, and bioinformatics [2] as described in Chapters 5 and S-9F. Already a new journal, Proteomics, will be started in 2001 by Wiley VCH and the editor, Michael J. Dunn. Science, including chromatography, is a human adventure from the unknown to the known as depicted in earlier chapters and then projected into the future in Chapter 15 (in the supplement) (Fig. 1.3).

32

Chapter 1

REFERENCES Note: Journal references use the journal title abbreviations of Chemical Abstracts Service Source Index (CASSI), which also provides other essential journal information, including library holdings.

References for introduction 1. 2.

J.C. Giddings, Unified Separation Science, Wiley Interscience Publisher, New York, NY, 1991, 320 pp. D.C. Lindberg, The Beginnings of Western Science, 600 BC to 1450 AD, The University of Chicago Press, Chicago, IL, 1992, 455 pp.

References for Mikhail S. Tswett 1.

2. 3.

4.

5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15.

M.S. Tswett, E´tudes de physiologie cellulaire: Contribution a` la connaissance des mouvements du protoplasme, des membranes plasmiques et des chloroplastes, Bulletin de Laboratoire de Botanique Ge´ne´rale de l’Universite´ de Gene`ve, 1 (3) (1896) 125–206. L.S. Ettre, M.S. Tswett and the 1918 Nobel Prize in Chemistry, Chromatographia, 43 (1996) 343–351. M.S. Tswett, Fiziko-khimicheskoe stroenie khlorofil’nogo zerna. Eksperimental’noe i kriticheskoe izsledovanie (The physico-chemical structure of the chlorophyll particle. Experimental and critical study), Trudy Obshchestva Estestvoispytatelei Pri Imperatorski Kazanskom Universitet 35 (3) (1901) 1–268. A summary of this paper was published under the title: Recherches sur la constitution physico-chimique du grain de chlorophylle, in: Botanisches Centralblatt, 89 (1902) 120–123. M.S. Tswett, O novoi kategorii adsorbtsionnykh yavlenii i o primenenii ikh k biokhimicheskomu analizu (On a new category of adsorption phenomena and their application to biochemical analysis), Trudy Varshavskogo Obshchestva Estestvoispytatelei, Otdelenie Biologii, 14 (1905) 20–39; for English translation, see Refs. 5, 6a. G. Hesse and H. Weil (Eds.), Michael Tswett’s First Paper on Chromatography, M. Woelm, Eschwege, 1954. V.G. Berezkin (Compiler), Chromatographic Adsorption Analysis: Selected Works of M.S. Tswett, Ellis Horwood, New York, NY, 1990; a: pp. 9–19; b: pp. 21–26; c: pp. 27–34; d: pp. 35–79. M. Tswett, Physikalisch–Chemische Studien u¨ber das Chlorophyll. Die Adsorption, Ber. Dtsch. Bot. Ges., 24 (1906) 316–326; for English translation, see Ref. 6b. M. Tswett, Adsorptionsanalyse und chromatographische methode. Anwendung auf die Chemie des Chlorophylls, Ber. Dtsch. Bot. Ges., 24 (1906) 384–393; for English translation, see Refs. 6c and 9. H.H. Strain and J. Sherma, Michael Tswett’s contributions to sixty years of chromatography, J. Chem. Educ., 44 (1967) 235–242. T. Robinson and M. Tswett, Chymia, 6 (1960) 146–161. L.S. Ettre, Evolution of liquid chromatography: A historical overview, in: Cs. Horva´th (Ed.), High-Performance Liquid Chromatography — Advances and Perspectives, Vol. I. Academic Press, New York, NY, 1980, pp. 1–74. L.S. Ettre, Those who are no longer with us, in: L.S. Ettre and A. Zlatkis (Eds.), 75 Years of Chromatography — A Historical Dialogue, Elsevier, Amsterdam, 1979, pp. 483–490. M.S. Tswett, Khromofilly v Rastitel’nom i Zhivotnom Mire (Chromophylls in the Plant and Animal World). Karbasnikov Publishers, Warsaw, 1910; for partial English translation, see Ref. (6d). ¨ ber den makro- und mikrochemischen Nachweis des Carotins, Ber. Dtsch. Bot. Ges., 29 M. Tswett, U (1911) 630–636. Additional references on M.S. Tswett may be found in C-8AEF, particularly that of K.I. Sakodynskii, J. Chromatogr., 73 (1972) 303–360, with many unique photographs.

The Beginnings of Chromatography — The Pioneers (1900–1960)

33

References for predecessors of Mikhail S. Tswett (Titles of articles are omitted here, since the full citation is found in Chapter S-8.) 1. 2.

H.H. Bussemas, G. Harsch and L.S. Ettre, F.E. Runge, Chromatographia, 38 (3=4) (1994) 243–254. H. Newesly, F. Goppelsroeder, Chromatographia, 30 (9=10) (1990) 595–596.

References on Leroy S. Palmer 1. 2. 3. 4. 5. 6.

L.S. Ettre and R.L. Wixom, Leroy S. Palmer and the beginnings of chromatography in the USA, Chromatographia, 37 (1993) 659–668. L.S. Palmer, A study of the natural pigment of the fat of cow’s milk, Ph.D. thesis in Dairy Husbandry, College of Agriculture, University of Missouri, Columbia, MO, 1913, 205 pp. L.S. Palmer and C.H. Eckles, Carotin — The principal natural yellow pigment of milk fat : : : etc., J. Biol. Chem., 17 (1914) 191–210, 211–221, 223–236, 237–243 and 245–249. L.S. Ettre, Those who are no longer with us — L.S. Palmer and others, in: L.S. Ettre and A. Zlatkis (Eds.), 75 Years of Chromatography: A Historical Dialogue, Elsevier, Amsterdam, 1979, pp. 483–491. ¨ ber die gelben Begleiter des Chlorophylls, Liebigs Ann., 355 (1907) R. Willsta¨tter and W. Mieg, U 1–28. L.S. Palmer, Carotinoids and Related Pigments — The Chromolipids, American Chemical Society Monograph Series, Chemical Catalog Co., New York, NY, 1922, 316 pp.

References on other early followers of M.S. Tswett 1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13.

H.H. Bussemas and L.S. Ettre, G. Kra¨nzlin, Chromatographia, 39 (5=6) (1994) 369–374. L.S. Ettre, T. Lippmaa, Chromatographia, 20 (7) (1985) 399–402. V.R. Meyer and L.S. Ettre, Early evolution of chromatography: The activities of Charles Dhe´re´, J. Chromatogr., 600 (1992) 3–15. R. Kuhn, A. Winterstein and E. Lederer, The xanthophylls, Hoppe-Seyler’s Z. Physiol. Chem., 197 (1931) 141–160; R. Kuhn and E. Lederer, Fractioniering und isomerisierung des Carotins, Naturwissenschaften, 19 (1931) 306. E. Lederer, in: L.S. Ettre and A. Zlatkis (Eds.), 75 Years of Chromatography — A Historical Dialogue, Elsevier, Amsterdam, 1979, pp. 237–245. E. Lederer, Adventures and research, Chapter 9, in: A. Neuberger, L.L.M. Van Deenen and G. Semenza (Eds.), Comprehensive Biochemistry, Vol. 36, Elsevier, Amsterdam, 1986, pp. 437–490. L. Zechmeister, in: L.S. Ettre and A. Zlatkis (Eds.), 75 Years of Chromatography — A Historical Dialogue, Elsevier, Amsterdam, 1979, pp. 491–494. L.S. Ettre, La´szlo´ Zechmeister — A pioneer of chromatography, Anal. Chem., 61 (1989) 1315A– 1322A and 62 (1990) 71A. L.S. Ettre and A. Zlatkis (Eds.), 75 Years of Chromatography — A Historical Dialogue, Elsevier, Amsterdam, 1979, 502 pp. L.S. Ettre, Chromatography: The separation technique of the 20th century, Chromatographia, 51 (1) (2000) 7–17. E. Geeraert and M. Verzele, Preparative liquid chromatography: History and trends, Chromatographia, 11 (1978) 640–644. L.S. Ettre, Preparative liquid chromatography: History and trends, Supplemental remarks, Chromatographia, 12 (5) (1979) 302–304. E. Lederer and M. Lederer, Chromatography: A Review of Principles and Applications, Elsevier,

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Chapter 1

Amsterdam, 1953, 460 pp.; see Chapters 1 to 6 for a more detailed survey of early adsorption chromatography. 14. See Appendices 4-AH and 5-G.

References on partition chromatography (LLC) 1. 2. 3. 4. 5.

A.J.P. Martin and R.L.M. Synge, Separation of the higher monoaminoacids by counter-current liquid– liquid extraction: The amino acid composition of wool, Biochem. J., 35 (1941) 91–121. E.R. Adlard, 90th Birthday of A.J.P. Martin, Chromatographia, 51 (5=6) (2000) 255. A.J.P. Martin, Future possibilities in micro-analysis, Chromatographia, 51 (5=6) (2000) 256–259. A.J.P. Martin, in: L.S. Ettre and A. Zlatkis (Eds.), 75 Years of Chromatography — A Historical Dialogue, Elsevier, Amsterdam, 1979, pp. 285–296. See Appendices 4-ABC, 5-ABCDE, 5-G, 6-AB and 7-AH.

References for gas–liquid chromatography (GLC) 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

A.J.P. Martin and R.L.M. Synge, A new form of chromatogram employing two liquid phases. 1. A theory of chromatography, 2. Application to the micro-determination of the higher monoamino-acids in proteins, Biochem. J., 35 (1941) 1358–1368. A.T. James and A.J.P. Martin, Gas–liquid partition chromatography: The separation and micro-estimation of volatile fatty acids from formic acid to dodecanoic acid, Biochem. J., 50 (1952) 679–690. L.S. Ettre, Capillary columns — From London to London in 25 years, Chromatographia, 16 (1982) 18–25. L.S. Ettre, Open-tubular columns: Past, present and future, Chromatographia, 34 (1992) 513–528. M.J.E. Golay, in: L.S. Ettre and A. Zlatkis (Eds.) 75 Years of Chromatography — A Historical Dialogue, Elsevier, Amsterdam, 1979, pp. 104–114. W. Jennings, E. Mittlefehldt and P. Stremple, Analytical Gas Chromatography, Academic Press, San Diego, CA, 1st ed. in 1987, 2nd ed. in 1997, 389 pp. J.V. Hinshaw and L.S. Ettre, The variation of carrier gas velocities with temperature, J. High Resolut. Chromatogr., 20 (1997) 471–481. E. Bayer, Gas Chromatography, Elsevier, Amsterdam, 1961, 240 pp.; preceded by Gaschromatographie, 1959, 163 pp. Note: Has 420 early GC references. L.S. Ettre, 25 Years of international symposia on advances in chromatography, J. Chromatogr., 468 (1989) 1–34. A. Zlatkis and V. Pretorius (Eds.), Preparative Gas Chromatography, Wiley Interscience, New York, NY, 1971, 402 pp. L.S. Ettre, Gas chromatography — Past, present and future, LC ž GC Europe, 14 (2) (2001) 72–74. See Appendices 4-A, 4-E and 4-H; 5-ABCDEFG, 5-G, 7-BE and 7-H. C.F. Poole and H.-G. Janssen, Contemporary capillary gas chromatography, Part I, Chromatogr. A, 842 (1999) 1–426 and Part II, 843 (1999) 1–433. C.A. Bruckner, B.J. Prazen and R.E. Synovec, Comprehensive two-dimensional high speed gas chromatography with chemometric analysis, Anal. Chem., 70 (1998) 2796–2804. J.W. Elling and 8 co-authors, Hybrid artificial intelligence tools for assessing GC data, Anal. Chem., 69 (13) (1997) 409A–415A. G.A. Eiceman, H.H. Hill Jr. and J. Gardea-Torresdey, Gas chromatography — Part I, Anal. Chem., 70 (12) (1998) 312R–339R; ibid. Anal. Chem., 72 (12) (2000) 137R–144R. L.S. Ettre, Evolution of capillary gas chromatography, in: H. Issaq (Ed.), Century of Chromatography, in press, 2001. W. Horwitz (Ed.), Official Methods of Analysis of AOAC International, AOAC Int., 15th ed. in 1990, 17th ed. in 2000.

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19. American Society for Testing and Materials (ASTM), Annual Book of ASTM Standards, ASTM, West Conshohocken, PA; 15 Sections, 73 Volumes, 1999; see particularly Vol. 14.02 for 18 chromatography methods. 20. Editors, Gas Chromatography Literature, Abstracts and Index, Preston Technical Abstracts Co., Niles, IL, Vol. 1 in 1968 to Vol. 24 in 1991.

References on paper- and thin-layer chromatography (TLC) 1.

R. Consden, A.H. Gordon and A.J.P. Martin, Qualitative analysis of proteins: A partition chromatographic method using paper, Biochem. J., 38 (1944) 224–232. 2. For books on paper chromatography, see Appendices 4-AB, 5-G and 7-BH. 3. J.P. Greenstein and M. Winitz, Chemistry of the Amino Acids, Vol. 2, Chapter 15, Wiley, New York, NY, 1961, 3 Vols., pp. 1366–1500. 4. M.S. Shraiber, The beginnings of thin-layer chromatography, J. Chromatogr. Sci., 73 (1972) 367–370; M.S. Shraiber and N.A. Izmailov, Spot chromatographic adsorption analysis and its application in pharmacy communication, J. Planar Chromatogr., 8 (1995) 402–405; transl. from original Russian Journal, Farmatsiya, 3 (1936) 1–7. 5. J.E. Meinhard and N.E. Hall, Surface chromatography, Anal. Chem., 21 (1949) 185–188. 6. J.G. Kirchner, Thin-layer chromatography — Yesterday, today and tomorrow, J. Chromatogr. Sci., 11 (1973) 180–183. 7. See Appendix 4-B for full citation to early books on TLC by K. Randerath (1962, 1966), E. Stahl (1962–1967), G. Pataki (1966, 1971), J.G. Kirchner (1967, 1978), G.K. Macek (1972), A. Zlatkis and R.E. Kaiser (1977). 8. See Appendices 5-BG and 7-BH for planar chromatography books in the 1990s. 9. V.G. Berezkin, The discovery of thin-layer chromatography, J. Planar Chromatogr., 8 (1995) 401–402. 10. J. Sherma, Planar chromatography, Anal. Chem., 70 (12) (1998) 7R–26R; ibid., Anal. Chem., 72 (2000) 9R–25R. 11. S. Nyiredy, Planar chromatography — Chapter 2, in: E. Heftmann (Ed.), Chromatography, Elsevier, Amsterdam, 5th ed., 1992, pp. A110–A150. 12. B. Fried and J. Sherma, Thin-Layer Chromatography, M. Dekker, New York, NY, 4th ed., 1999, 499 pp.

References on ion-exchange chromatography (mainly early investigators) 1.

R. Kunin and R.J. Myers, Ion-Exchange Resins, Wiley, New York, NY, 1st ed. in 1950, 2nd ed. in 1958, 466 pp. 2. J.A. Kitchener, Ion-Exchange Resins, Methuen, London; or Wiley, New York, NY, 1957, 109 pp. 3. C. Calmon and T.R.E. Kressman (Eds.), Ion-Exchangers in Organic and Biochemistry, Interscience Publishers, New York, NY, 1957, 761 pp. 4. F. Helfferich, Ion-Exchange, McGraw Hill, New York, NY, 1st ed. in 1959, 2nd ed. in 1962, 624 pp. 5. B.A. Adams and E.L. Holmes, Adsorptive properties of synthetic resins, J. Soc. Chem. Ind., London, 54 (1938) 1 (T). 6a. E.R. Tompkins and 14 other authors, Ion-exchange as a separations method and other topics, J. Am. Chem. Soc., 69 (1947) 2769–2881. 6b. A. Tiselius and others, Chromatographic analysis — 42 papers presented, of which 11 concerned ion-exchange chromatography, Discuss. Faraday Soc., 7 (1949) 1–336. 7. H.F. Walton (Ed.), Ion-Exchange Chromatography, Benchmark Papers in Analytical Chemistry (early papers reprinted), Dowden, Hutchinson Ross, Stroudsburg, PA, 1976, 440 pp. 8. See Appendix 4-D for additional early IEC books by O. Samuelson, S. Blasius, R. Kunin, W. Riema´n and H.F. Walton, J.-X. Khym, and J.A. Marinsky and Y. Mana.

36 9. 10. 11. 12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26a. 26b.

27.

28. 29. 30. 31. 32.

33.

Chapter 1 L.S. Ettre, Preparative liquid chromatography and the Manhattan project, LC ž GC North America, 17 (12) (1999) 1104–1108. W.E. Cohn, The nature and production of artificial radioactivity, Adv. Biol. Med. Phys., 1 (1948) 118–149. W.E. Cohn, Nucleic acid derivatives, in: C. Calmon and T.R.E. Kressman (Eds.), Ion-exchangers in Organic and Biochemistry, Interscience Publications, New York, NY, 1957, pp. 345–359. W.E. Cohn, Column chromatography of nucleic acid derivatives and related substances, in: E. Heftmann (Ed.), Chromatography — A Laboratory Handbook of Chromatographical and Electrophoretic Methods, Reinhold, New York, NY, 2nd ed., 1967, pp. 627–660. W.E. Cohn, Paper 32, 1950, in: H.F. Walton (Ed.), see above Reference 7. M. Lederer, Kurt A. Kraus — Obituary, J. Chromatogr., 738 (1996) 155–156. F. Nelson, D.C. Michelson, H.O. Phillips and K.A. Kraus, Ion-exchange procedures. VII. Separation of alkali metal ions, J. Chromatogr., 20 (1965) 107–121. K.A. Kraus, Papers 16 to 19 and 28, 1951–1958, in: H.F. Walton (Ed.); see above Reference 7. E.R. Tompkins, Discuss. Faraday Soc., 7 (1949) 232–237; see Reference 6b. S.W. Mayer and E.R. Tompkins, J. Am. Chem. Soc., 69 (1947) 2866–2874. E.R. Tompkins, Paper 2, 1947, in: H.F. Walton (Ed.), see above Reference 7. F.H. Spedding, Metallurgy of Uranium and its Alloys, U.S. Atomic Energy Commission, National Nuclear Energy Series, Vol. 12A, Washington, DC, 1963, 1977, 208 pp. F.H. Spedding and A.H. Daane (Eds.), The Rare Earths, Wiley, New York, NY, 1961, 641 pp.; R.E. Kreiger Publisher, 1971 reprint. F.H. Spedding, Papers 5, 8, 13, 1947 to 1955; in: H.F. Walton (Ed.); see above Reference 7. T. Moeller, The Chemistry of the Lanthanides, Reinhold Publishing, New York, NY, 1963, 117 pp. N.E. Topp, The Chemistry of the Rare Earth Elements, Elsevier, Amsterdam, 1965, 164 pp. H.F. Walton, Ion-exchange chromatography, Chapter 5, in: E. Heftmann (Ed.), Chromatography, Part A: Fundamentals and Techniques, Elsevier, Amsterdam, 5th ed., 1992, pp. A227–A265. See also the eight recent IEC books cited in the Appendices 5-A, 5-E, and 5-G. O. Samuelson, Ion Exchangers in Analytical Chemistry, Almqvist and Wiksell, Stockholm; Wiley, New York, NY, 1st ed., 1953; ibid, Ion Exchange Separations in Analytical Chemistry, same publishers, 2nd ed., 1963, 474 pp. E. Glueckauf, K.H. Barker and G.P. Kitt, Theory of chromatography VIII. The separation of lithium isotopes by ion exchange and of neon isotopes by low temperature adsorption columns, Discuss. Faraday Soc., 7 (1979) 199–213. D.H. Everett, Eugen Glueckauf, Biogr. Mem. Fellows R. Soc., 30 (1982) 193–274. H.A. Sober and E.A. Peterson, Chromatography of proteins on cellulose ion-exchangers, J. Am. Chem. Soc., 76 (1954) 1711–1712. E.A. Peterson and H.A. Sober, Chromatography of proteins. I. Cellulose ion-exchangers, J. Am. Chem. Soc., 78 (1956) 751–755. J. Porath and E.B. Lindner, Separation methods based on sieving and ion exclusion, Nature, 191 (1961) 69–70. J. Porath, T. La˚a˚s and J.-Ch. Janson, Agar derivatives for chromatography, electrophoresis and gel-bound enzymes. III. Rigid agarose gels cross-linked with divinylsulfone, J. Chromatogr., 103 (1975) 49–62. O. Mikesˇ, Ion-exchange chromatography, Chapter 45, in: Z. Deyl (Ed.), Separation Methods, Vol. 8, 1984, pp. 205–270 with 422 references; in: A. Neuberger and L.L.M. van Deenen (General Editors), New Comprehensive Biochemistry, Elsevier, Amsterdam, Vol. 1 in 1981 to Vol. 38 in 1999.

References for petroleum chromatography 1.

D.H. Desty and A. Goldup, Chromatography of hydrocarbons, in: E. Heftmann (Ed.), Chromatography — A Laboratory Handbook of Chromatographic and Electrophoretic Methods, 3rd ed., Van Nostrand Reinhold, New York, NY, 3rd ed., 1975, pp. 915–955.

The Beginnings of Chromatography — The Pioneers (1900–1960) 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13.

37

E.R. Adlard (Ed.), Chromatography in the Petroleum Industry, Vol. 56 in the Journal of Chromatography Library Series, Elsevier, Amsterdam, 1994, 430 pp. R.P. Philip, Geochemistry in the search for oil, Chem. Eng. News, 64 (6) (1986) 28–43. L.S. Ettre, Early petroleum chemists and the beginnings of chromatography, Chromatographia, 40 (314) (1995) 207–216. S. Claesson, Studies on adsorption and adsorption analysis with special reference to homologous series, Arkiv. Kemi, Min. Geol. A, 23 (1) (1946) 123. C.S.G. Phillips, The chromatography of gases and vapors, Discuss. Faraday Soc., 7 (1949) 241–248. W.M. Smit, Chromatography of petroleum hydrocarbons, Discuss. Faraday Soc., 7 (1949) 248–255. A.S.C. Lawrence and D. Barby, Chromatographic fractionation of black oils, Discuss. Faraday Soc., 7 (1949) 255–258. A.T. James and A.J.P. Martin, Gas–liquid partition chromatography; The separation and microestimation of volatile fatty acids from formic acid to dodecanoic acid. Biochem. J., 50 (1952) 679– 690. For References on Erika Cremer’s early GC research, see Appendix 2. For References on Gas Chromatography and Petroleum Chromatography, see L.S. Ettre in S-8IJ. E. Lederer and M. Lederer, Chromatography — A Review of Principles and Applications, Elsevier, Amsterdam, 2nd ed., 1955, 460 pp. D.H. Desty and A. Goldup, Chromatography of hydrocarbons, in: E. Heftmann (Ed.) Chromatography, Reinhold Publishing, New York, NY, 1961, 753 pp.

References on the literature of chromatography 1. 2.

See also preface and Chapters 2, 4, 5, 6 and S-9. Faraday Society (A. Tiselius, A.J.P. Martin and others), Chromatographic analysis, Discuss. Faraday Soc., 7 (1949) 7–336. 3. I.M. Hais and K. Macek (Eds.), See several references in Appendix 4B. 4. E. Lederer and M. Lederer, Chromatography — A Review of Principles and Applications, Elsevier, Amsterdam, 1st ed. in 1954, 2nd ed. in 1957, 711 pp. 5. Z. Deyl (Ed.), Separation Methods, in: A. Neuberger and L.L.L. van Deenen (Eds.), New Comprehensive Biochemistry, Vol. 8, Elsevier, Amsterdam, 1984, 526 pp. 6. L.S. Ettre, Evolution of liquid chromatography — A historical overview, Chapter 1, in: Cs. Horva´th (Ed.) HPLC — Advances and Perspectives, Academic Press, New York, NY, 1980, Vol. 1, pp. 1–74. 7. Cs. Horva´th (Ed.), High Performance Liquid Chromatography — Advances and Perspectives, Academic Press, New York, NY, 5 Vols., 1980–1988. 8. J.C. Giddings, Unified Separation Science, Wiley–Interscience Publishers, New York, NY, 1991, 320 pp. 9. Z. Deyl, J. Jana´k, V. Schwarz and K.M. Macek (Eds.), Bibliography Section of the Journal of Chromatography A, Elsevier, Amsterdam. 10. Chromatography Abstracts, published currently by the (British) Chromatographic Society and the Royal Society; started 1958 as Gas Chromatography Abstracts; see Appendix 3 for its several predecessors and their dates. 11. J. Wright, Vision, Venture and Volunteers: 50 Years of History of the Pittsburgh Conferences on Analytical Chemistry and Applied Spectroscopy, Pittsburgh Conference and Chemical Heritage Foundation, Pittsburgh, PA, 1999, 186 pp.

References on ‘From the inventors to the builders of chromatography’ 1.

L.S. Ettre, Chromatography: The separation technique of the 20th century, Chromatographia, 51 (2000) 7–17.

38 2.

Chapter 1 E. Smolkova¨-Keulemansova¨, A few milestones on the journey of chromatography, J. High Resolut. Chromatogr., 23 (2000) 497–501.

References for summary 1. 2.

C.S.G. Phillips, Chromatography and the discovery process, J. Chromatogr., 468 (1989) 35–42. A.D. Baxevanis and B.F.F. Ouellette (Eds.), Bioinformatics — A Practical Guide to the Analysis of Genes and Protein, Methods of Biochemical Analysis, Vol. 39, Wiley–Interscience, New York, NY, 1998, 370 pp.

39

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The Builders of Chromatography — Major Chromatography Awards and the Award Winners Leslie S. Ettre Department of Chemical Engineering, Yale University, New Haven, CT, USA *

CONTENTS A. B. C. D. E. F. G. H. I. J. K. L. M.

*

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nobel prize in Chemistry by the Nobel Foundation (1948–1999) . . . . . . . . . . . . . National Award in Chromatography of the American Chemical Society (1961–2001) . . . . National Award in Separations Science and Technology of the American Chemical Society (1984–2001) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.J.P. Martin Award of the Chromatographic Society (1978–2000) . . . . . . . . . . . . M.S. Tswett Chromatography Award of the International Symposia on Advances in Chromatography (1974–1988) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M.J.E. Golay Award in Capillary Chromatography of the International Symposia on Capillary Chromatography (1989–2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stephen Dal Nogare Award in Chromatography of the Chromatographic Forum of the Delaware Valley (1972–2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Keene P. Dimick Award in Chromatography by the Society for Analytical Chemists of Pittsburg (1988–2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Silver Jubilee Award of the Chromatographic Society (1982–2000) . . . . . . . . . . . . Award for Achievements in Separation Science of the Eastern Analytical Symposium (1986– 2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . COLACRO Medal (1986–2000) of the Congresso Latino Americano de Cromatografia . . . Leroy S. Palmer Award of the Minnesota Chromatography Forum (1980–2000) . . . . . . M.S. Tswett Chromatography Memorial Medal of the All-Union Scientific Council on Chromatography, Academy of Sciences of the U.S.S.R. (1978–1979) . . . . . . . . . . .

Mailing address: P.O. Box 6274, Beardsley Station, Bridgeport, CT 06606-0274, USA

40 41 43 43 43 44 44 47 48 49 49 50 51 52

40

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Their names and countries are given in the following 13 Tables.

“We all stand on the shoulders of our predecessors; thus is it not conspicuous that we can see further than they could?” Friedrich A. Kekule´ (1829–1890)

INTRODUCTION A number of national and international awards honor scientists active in the field of chromatography. Obviously, we should start with the Nobel prizes in this listing: in the past three prizes were awarded specifically for achievements in chromatography. The two principal chromatography awards are the National Award in Chromatography of the American Chemical Society, and the A.J.P. Martin Award of the (British) Chromatographic Society. A further major award is the National Award in Separations Science and Technology of the American Chemical Society; although many of its recipients received it for achievements other than chromatography, some were honored specifically for activities related to the various chromatographic techniques. Other major national and international recognitions in chromatography are the M.S. Tswett Chromatography Award which has been presented between 1974 and 1988 by the International Symposia on Advances in Chromatography, the M.J.E. Golay Award in Capillary Chromatography presented by the International Symposia on Capillary Chromatography, the Stephen Dal Nogare Award of the Chromatography Forum of the Delaware Valley and the Keene P. Dimick Award administered by the Society for Analytical Chemists of Pittsburgh; both are presented at the yearly Pittsburgh Conferences on Analytical Chemistry and Applied Spectroscopy. Further major awards are the Silver Jubilee Award of the Chromatographic Society, the Award for Achievements in Separations Science of the Eastern Analytical Symposium, the COLACRO Medal presented by the biannual chromatography symposia held in Latin America, and, finally, the Leroy Sheldon Palmer Award of the Minnesota Chromatography Forum.

The Builders of Chromatography — Major Chromatography Awards and the Award Winners 41

The Chromatography Memorial Medal was presented to a number of scientists, associations and companies by the Scientific Council on Chromatography of the Academy of Sciences of the USSR in 1978 to 1979, on the occasion of the 75th anniversary of the discovery of chromatography. Although it is not an award in the same sense as those mentioned previously, we list its recipients here for completeness. Below the history of the individual awards is given briefly and the scientists who have received the awards listed. The institutional affiliation of the recipients and their geographical location are given for the time of receiving the award and thus recognizes the institution where the awardees’ research had been carried out. The address given may not be satisfactory for mail contact, since the awardee may have moved, retired or died, or it contains insufficient detail for postal delivery. The listing of the awardees corresponds to the known status as of December 2000. All living awardees were invited to present their significant research and brief biography. Almost all responded and hence the reader is referred to Chapter 5 and=or 6. The biography and summary of activities of many of the scientists have also been included in the 1979 book by L.S. Ettre and A. Zlatkis, “75 Years of Chromatography — A Historical Dialogue”; they are designated here with the superscript letter ‘e’. Deceased scientists are indicated by the superscript letter ‘d’. The abbreviations used in listing the awardees’ affiliation are self-explanatory, i.e., Univ., Inst., Corp., Inc., U.K., U.S.A. and the U.S. Postal Service abbreviations for States in the U.S.A. We are aware that there are also other international and national awards related to achievements in chromatography. However, in the interest of conciseness, these are reluctantly omitted.

A. NOBEL PRIZE IN CHEMISTRY BY THE NOBEL FOUNDATION (1948–1999) Three Nobel prizes were awarded for major achievements which also specifically included further development of the chromatographic techniques; two of these were shared awards (Table 2.1). In many cases, the achievements honored by other Nobel prizes in Chemistry or Medicine=Physiology would have been difficult without the use of chromatography. We do not consider these here, because the awardees did not contribute to the TABLE 2.1 NOBEL LAUREATES IN CHEMISTRY RELATED TO CHROMATOGRAPHY Year

Awardee=Affiliation=Country

1948 1952

Arne W.K. Tiselius d,e , Uppsala Univ., Uppsala, Sweden Archer J.P. Martin e , Medical Research Council, London, U.K. Richard L.M. Synge d,e , Rowett Research Inst., Aberdeen, Scotland, U.K. Stanford Moore d,e , Rockefeller Inst., New York, NY, U.S.A. William H. Stein d,e , Rockefeller Inst., New York, NY, U.S.A.

1972

42

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advancement of chromatography; they only used the methods. These Nobelists relied on the chromatographic procedures along with other research methods, but in so doing, showed the versatility, selectivity and sensitivity of the chromatography approach; hence, their contributions are described in Chapters 1 and=or S-9.

TABLE 2.2 RECIPIENTS OF THE NATIONAL AWARD IN CHROMATOGRAPHY OF THE A.C.S. Year

Awardee=Affiliation=Country

1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001

Harold H. Strain d,e , Argonne National Labs, Argonne, IL, U.S.A La´szlo´ Zechmeister d,e , California Inst. of Technology, Pasadena, CA, U.S.A Waldo E. Cohn d , Oak Ridge National Lab, Oak Ridge, TN, U.S.A. Stanford Moore d,e and William H. Stein d,e , Rockefeller Inst., New York, NY, U.S.A Stephen Dal Nogare d,e , E.I. duPont de Nemours & Co., Wilmington, DE, U.S.A Kurt A. Kraus d , Oak Ridge National Lab, Oak Ridge, TN, U.S.A. J. Calvin Giddings d,e , Univ. of Utah, Salt Lake City, UT, U.S.A. Lewis G. Longsworth d , Rockefeller Univ., New York, NY, U.S.A. Morton Beroza, U.S. Dept. of Agriculture, Research Services, Beltsville, MD, U.S.A. Julian F. Johnson, Univ. of Connecticut, Storrs, CT, U.S.A. No award was presented. J. Jack Kirkland e , E.I. duPont de Nemours & Co., Wilmington, DE, U.S.A. Albert Zlatkis d,e , Univ. of Houston, Houston, TX, U.S.A. Lockhard B. Rogers d , Purdue Univ., Lafayette, IN, U.S.A. Egon Stahl d,e , Univ. des Saarlandes, Saarbru¨cken, Germany James S. Fritz, Iowa State Univ., Ames, IA, U.S.A. Raymond P. W. Scott e , Hoffman-LaRoche Co., Nutley, NJ, U.S.A. Archer J.P. Martin e , Univ. of Houston, TX, U.S.A. Evan C. Horning d,e , Baylor Coll. Medicine, Houston, TX, U.S.A. James E. Lovelock e , Univ. of Reading, Bowerchalke, U.K. Marcel J.E. Golay d,e , Perkin-Elmer Corp., Norwalk, CT, U.S.A. Barry L. Karger, Northwestern Univ., Boston, MA, U.S.A. Csaba G. Horva´th e , Yale Univ., New Haven, CT, U.S.A. Lloyd R. Snyder e , L.C. Resources, Inc., Orinda, CA, U.S.A. Leslie S. Ettre e , The Perkin-Elmer Corp., Norwalk, CT, U.S.A. Milos V. Novotny, Indiana Univ., Bloomington, IN, U.S.A. Charles H. Lochmu¨ller, Duke Univ., Durham, NC, U.S.A. Milton L. Lee, Brigham Young Univ., Provo, UT, U.S.A. Fred E. Regnier, Purdue Univ., Lafayette, IN, U.S.A. John H. Knox, Univ. of Edinburgh, Edinburgh, U.K. Hamish Small, Dow Chemical Co., Midland, MI, U.S.A. Josef F.K. Huber d,e , Univ. of Vienna, Vienna, Austria James W. Jorgenson, Univ. of North Carolina, Chapel Hill, NC, U.S.A. Willliam H. Pirkle, Univ. of Illinois, Urbana, IL, U.S.A. Klaus K. Unger, Johannes Gutenberg Univ., Mainz, Germany Stellan Hjerte´n, Uppsala Univ., Uppsala, Sweden Peter W. Carr, Univ. of Minnesota, Minneapolis, MN, U.S.A. Georges Guiochon, Univ. of Tennessee, Knoxville, TN, U.S.A. Daniel W. Armstrong, Univ. of Missouri, Rolla, MO, U.S.A. Charles W. Gehrke e , Univ. of Missouri, Columbia, MO, U.S.A. Ernst Bayer, Univ. of Tu¨bingen, Germany

The Builders of Chromatography — Major Chromatography Awards and the Award Winners 43

B. NATIONAL AWARD IN CHROMATOGRAPHY OF THE AMERICAN CHEMICAL SOCIETY (1961–2001) This award was established in 1959, originally as the American Chemical Society (ACS) Award in Chromatography and Electrophoresis; the first award was presented in 1961. In 1971 the title of the award was narrowed to only chromatography (Table 2.2). The aim of the award is to recognize outstanding contributions to the field of chromatography, with particular consideration given to the development of new methods. The award is announced at the Fall National Meeting of the Society and is then presented at the following Spring National Meeting. Usually, there is a special session at the meeting, with contributed lectures to honor the award winner who also presents an award address. In general, the award is given every year to a single person only; only once (in 1964) was the award presented to two persons. The award is not restricted to American scientists. However, most of the time, it had been given to scientists active in the U.S.A.; from the 40 awards, only seven were presented to scientists working outside the United States. Between 1961 and 1970 the award was sponsored by Lab-Line Instruments, Inc. Since 1972, the award has been sponsored by SUPELCO, Inc. and consists of a honorarium (presently $5000) and a certificate. C. NATIONAL AWARD IN SEPARATIONS SCIENCE AND TECHNOLOGY OF THE AMERICAN CHEMICAL SOCIETY (1984–2001) This award was established in 1982, with the aim to recognize outstanding accomplishments of scientists and engineers in fundamental or applied research directed to separations science and technology (Table 2.3). According to the rules, the scope of the award is to be as broad as possible, covering all fields where separations science and technology is practiced, including (but not limited to) biology, chemistry, engineering, geology and medicine. In other words, the field of the award is much broader than chromatography. However, this award is included in this compilation because some of the award winners received it for their contribution to chromatography or related techniques. The award is announced at the Fall National Meeting of the Society and is then presented at the following Spring National Meeting. Usually a special session within the program of the ACS Division of Industrial and Engineering Chemistry honors the awardee who also is invited to present an award address during the session. Between 1982 and 1996 the award was sponsored by Rohm and Haas Company. In 1996, IBC Advanced Technologies, Inc. and Millipore Corporation assumed sponsorship. The award consists of a honorarium (presently $5000) and a plaque. D. A.J.P. MARTIN AWARD OF THE CHROMATOGRAPHIC SOCIETY (1978–2000) This award was established in 1978 by the then Chromatography Discussion Group, which later changed its name to The Chromatographic Society (Table 2.4). The

44

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TABLE 2.3 RECIPIENTS OF THE AWARD IN SEPARATIONS SCIENCE AND TECHNOLOGY OF THE A.C.S. Year

Awardee=Affiliation=Country

1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001

P.B. Broughton, U.O.P., Inc., Alan S. Michaels, A.S. Michaels, Inc., Chestnut Hill, MA, U.S.A. J. Calvin Giddings d,e , Univ. of Utah, Salt Lake City, UT, U.S.A. Friedrich G. Helfferich, Pennsylvania State Univ., University Park, PA, U.S.A. Norman N. Li, Catholic Univ. of America, Washington, DC, U.S.A. Jay M.S. Henis, Monsanto Co., St. Louis, MO, U.S.A. Henry Freiser, Univ. of Arizona, Tucson, AZ, U.S.A. Georges Guiochon, Univ. of Tennessee, Knoxville, TN, U.S.A. Milos V. Novotny, Indiana Univ., Bloomington, IN, U.S.A. James R. Fair, Univ. of Texas, Austin, TX, U.S.A. Phillip C. Wankat, Purdue Univ., Lafayette, IN, U.S.A. Georges Belfort, Rensselaer Polytechnic Inst., Troy, NY, U.S.A. Reed M. Izatt and Jerald S. Bradshaw, Brigham Young Univ., Provo, UT, U.S.A. C. Judson King, Univ. of California, Berkeley, CA, U.S.A. Barry L. Karger, Northeastern Univ., Boston, MA, U.S.A. Charles W. Gehrke e , Univ. of Missouri, Columbia, MO, U.S.A. Earl P. Horwitz, Eichron Industries Inc., Darien, IL, U.S.A. Csaba Horva´th e , Yale Univ., New Haven, CT., USA

The individuals in italics made distinctive chromatography contributions.

aim of the award is to recognize some special contribution to the advancement of chromatography, not necessarily limited to the purely scientific aspects of the technique. The award consists of a gold medal depicting A.J.P. Martin, the inventor of partition chromatography.

E. M.S. TSWETT CHROMATOGRAPHY AWARD OF THE INTERNATIONAL SYMPOSIA ON ADVANCES IN CHROMATOGRAPHY (1974–1988) The organizers of the International Symposia on Advances in Chromatography instituted the M.S. Tswett Chromatography Award (certificate and medal) to honor scientists who significantly contributed to the advancement of chromatography (Table 2.5). The awards were presented each year starting in 1974 during the opening session of the symposium.

F. M.J.E. GOLAY AWARD IN CAPILLARY CHROMATOGRAPHY OF THE INTERNATIONAL SYMPOSIA ON CAPILLARY CHROMATOGRAPHY (1989–2000) The Golay Award in Capillary Chromatography was originally instituted in 1989 by the organizers of the Symposium on Capillary Chromatography (Table 2.6). From

The Builders of Chromatography — Major Chromatography Awards and the Award Winners 45 TABLE 2.4 RECIPIENTS OF THE A.J.P. MARTIN AWARD Year

Awardee=Affiliation=Country

1978

Ernst Bayer, Tu¨bingen Univ., Tu¨bingen, Germany C.H.E. Knapman, U.K. Atomic Energy Authority, British Nuclear Fuel Ltd., Capenhurst, U.K. Georges Guiochon, Ecole Polytechnique, Palaiseau, France G.A.P. Tuey, May & Baker, Ltd., Dagenham, U.K. Edward R. Adlard e , Shell Research Ltd., Thorton Research Centre, Chester, U.K. Leslie S. Ettre e , The Perkin Elmer Corp., Norwalk, CT, U.S.A Courtenay S.G. Phillips e , Oxford Univ., Oxford, U.K. Raymond P.W. Scott e , The Perkin-Elmer Corp., Norwalk, CT, U.S.A. Gerhard Schomburg e , Max Planck Inst. fu¨r Kohlenforschung, Mu¨lheim, Germany Ralph Stock, Trent Polytechnic, Nottingham, U.K. C.E. Roland Jones, Chromsultants Ltd., Redhill, Surrey, U.K. Arnoldo Liberti d,e , Univ. di Roma`, Rome, Italy John H. Knox, Edinburgh Univ., Edinburgh, U.K. Ervin sz. Kova´ts e , Ecole Polytechnique Fe´de´rale, Lausanne, Switzerland J. Calvin Giddings d,e , Univ. of Utah, Salt Lake City, UT, U.S.A. Udo A. Th. Brinkmann, Free Univ., Amsterdam, The Netherlands Josef F. K. Huber d,e , Univ. of Vienna, Austria Lloyd R. Snyder e , LC Resources, Orinda, CA, U.S.A. Rudolf E. Kaiser e , Inst. fu¨r Chromatographie, Bad Du¨rkheim, Germany Karel A.M.G. Cramers, Univ. of Technology, Eindhoven, The Netherlands Egil Jellum, Inst. of Clinical Biochemistry, Oslo, Norway William H. Pirkle, Univ. of Illinois, Urbana, IL, U.S.A. Daniel W. Amstrong, Univ. of Missouri, Rolla, MO, U.S.A. Denis H. Desty d,e , Walton-on-Thames, U.K. David E. Games, Univ. of Wales, Swansea, U.K. Barry L. Karger, Northeastern Univ., Boston, MA, U.S.A. James W. Jorgenson, Univ. of North Carolina, Chapel Hill, NC, U.S.A. Irving W. Wainer, McGill Univ., Montreal, Canada Heinz Engelhardt, Univ. des Saarlandes, Saarbru¨cken, Germany Fred E. Regnier, Purdue Univ., Lafayette, IN, U.S.A. Klaus K. Unger, Johannes Gutenberg Univ., Mainz, Germany Csaba Horva´th e , Yale Univ., New Haven, CT, U.S.A. Pat J. Sandra, Univ. of Ghent, Ghent, Belgium Milos V. Novotny, Indiana Univ., Bloomington, IN, U.S.A. Shigeru Terabe, Himeji Inst. of Technology, Himeji, Japan Milton L. Lee, Brigham Young Univ., Provo, UT, U.S.A. J. Jack Kirkland e , Agilent, Zorbax R&D, Newport, DE, U.S.A. Walter G. Jennings, J&W Corp., Folsom, CA, U.S.A. Albert Zlatkis d,e , Univ. of Houston, TX, U.S.A. Geoffrey Eglinton, Univ. of Bristol, Bristol, U.K. Hans Poppe, Univ. of Amsterdam, Amsterdam, The Netherlands C.H. Mosbach, University of Lund, Sweden W.S. Hancock, Agilent Technologies, Palo Alto, CA, USA

1980 1982

1984 1985 1986 1988

1989 1990

1991

1992 1993

1994 1995 1996 1997 1998 1999 2000

1990 on, the financial sponsorship of the award was taken over by the Perkin-Elmer Corporation. The award consists of a honorarium (presently $5000 per year) and a medal.

46

Chapter 2

TABLE 2.5 RECIPIENTS OF THE M.S. TSWETT CHROMATOGRAPHY AWARD Year

Awardee=Affiliation=Country

1974

Erika Cremer d,e , Leopold-Franzens Univ., Innsbruck, Austria Denis H. Desty d,e , British Petroleum, Sunbury-on-Thames, U.K. Aloysius I.M. Keulemans d,e , Univ. of Technology, Eindhoven, The Netherlands Andrei V. Kiselev d,e , State Univ., Moscow, U.S.S.R. Archer J.P. Martin e , Univ. of Sussex, Brighton, U.K. Gerhard Hesse d,e , Univ. of Erlangen-Nu¨rnberg, Erlangen, Germany Evan C. Horning d,e , Baylor Univ. of Medicine, Houston, TX, U.S.A. Jaroslav Jana´k e , Inst. of Instrumental Analytical Chemistry, Brno, Czechoslovakia James E. Lovelock e , Bowerchalke, U.K. Courtenay S.G. Phillips e , Oxford Univ., Oxford, U.K. Marcel J.E. Golay d,e , The Perkin-Elmer Corp., Norwalk, CT, U.S.A. Georges Guiochon, Ecole Polytechnique, Palaiseau, France Anthony T. James e , Unilever, Sharnbrook, U.K. Edgar Lederer d,e , Inst. de Chimie des Substances Naturelles, Gif-sur-Yvette, France Victor Pretorius d,e , Univ. of Pretoria, Pretoria, Republic of South Africa Ervin sz. Kova´ts e , E´cole Polytechnique Fe´de´rale, Lausanne, Switzerland John H. Purnell d , Univ. of Wales, Swansea, U.K. Aleksandr A. Zhukhovitskii d,e , Steel & Alloys Inst., Moscow, U.S.S.R. Leslie S. Ettre e , The Perkin-Elmer Corp., Norwalk, CT, U.S.A. J. Calvin Giddings d,e , Univ. of Utah, Salt Lake City, UT, U.S.A. Raymond P.W. Scott e , Hoffmann-La Roche, Nutley, NJ, U.S.A. Per Flodin e , Chalmers Univ. of Technology, Go¨teborg, Sweden Jerker O. Porath e , Uppsala Univ., Uppsala, Sweden Istva´n Hala´sz d , Univ. des Saarlandes, Saarbru¨cken, Germany Csaba Horva´th e , Yale Univ., New Haven, CT, U.S.A. Arnoldo Liberti d,e , Univ. of Rome, Rome, Italy Karl I. Sakodynskii d,e , Karpov Inst. of Physical Chemistry, Moscow, U.S.S.R. Robert E. Sievers, Univ. of Colorado, Boulder, CO, U.S.A Hiroyuki Hatano d , Kyoto Univ., Kyoto, Japan Nobuo lkekawa d , Inst. of Technology, Tokyo, Japan Arthur Karmen e , Albert Einstein Univ. of Medicine, Bronx, New York, NY, U.S.A. Seymour R. Lipsky d,e , Yale Univ. Medical School, New Haven, CT, U.S.A. Egil Jellum, Inst. of Clinical Biochemistry, Oslo, Norway Gerhard Schomburg e , Max Planck Inst. fu¨r Kohlenforschung, Mu¨lheim, Germany Albert Zlatkis d,e , Univ. of Houston, Houston, TX, U.S.A. Milton L. Lee, Brigham Young Univ., Provo, UT, U.S.A. Milos V. Novotny, Indiana Univ., Bloomington, IN, U.S.A. John H. Knox, Edinburgh Univ., Edinburgh, U.K. Karel Macek, Inst. of Physiology, Charles Univ., Prague, Czechoslovakia Colin F. Poole, Wayne State Univ., Detroit, MI, U.S.A. Ernst Bayer, Univ. of Tu¨bingen, Tu¨bingen, Germany Karel A. Cramers, Univ. of Technology, Eindhoven, The Netherlands Shoji Hara, College of Pharmacy, Tokyo, Japan Barry L. Karger, Northeastern Univ., Boston, MA, U.S.A. Hiroshi Miyazaki, Nippon Kayaku Co., Tokyo, Japan Marjorie G. Horning e , Baylor Univ. of Medicine, Houston, TX, U.S.A. Daido Ishii, Nagoya Univ., Nagoya, Japan J.B. Sjo¨vall, Karolinska Inst., Stockholm, Sweden

1975

1976

1977

1978

1979 1980 1981

1982

1983

1984 1985

1986

1987

The Builders of Chromatography — Major Chromatography Awards and the Award Winners 47 TABLE 2.5 (continued) Year

Awardee=Affiliation=Country

1988

Phyllis R. Brown, Univ. of Rhode Island, Kingston, RI, U.S.A. Fabrizio Bruner d , Univ. of Urbino, Urbino, Italy Tsuneo Okuyama, Metropolitan Univ., Tokyo, Japan

TABLE 2.6 RECIPIENTS OF THE M.J.E. GOLAY AWARD Year

Awardee=Affiliation=Country

1989

Rudolf E. Kaiser e , Inst. fu¨r Chromatographie, Bad Du¨rkheim, Germany Raymond D. Dandeneau, Hewlett-Packard, Inc., Avondale, PA, U.S.A. Ernest H. Zerenner, Hewlett-Packard, Inc., Avondale, PA, U.S.A. Gerhard Schomburg e , Max Planck Inst. fu¨r Kohlenforschung, Mu¨lheim, Germany Daido Ishii, Nagoya Univ., Nagoya, Japan Milos V. Novotny, Indiana Univ., Bloomington, IN, U.S.A. Leslie S. Ettre e , Yale Univ., New Haven, CT, U.S.A. Konrad Grob, Kantonales Labor, Zu¨rich, Switzerland James W. Jorgenson, Univ. of North Carolina, Chapel Hill, NC, U.S.A. Pat J. Sandra, Univ. of Ghent, Belgium Walter G. Jennings, J&W Corp., Folsom, CA, U.S.A. Fabrizio Bruner d , Univ. of Urbino, Urbino, Italy Karel A. Cramers, Eindhoven Inst. of Technology, Eindhoven, The Netherlands Milton L. Lee, Brigham Young Univ., Provo, UT, U.S.A. Hans Poppe, Univ. of Amsterdam, Amsterdam, The Netherlands Csaba Horva´th e , Yale Univ., New Haven, CT, U.S.A. Kiyukatsu Jinno, Toyohashi Univ. of Tech., Toyohashi, Japan Shigeru Terabe, Himeji Inst. of Tech., Himeji, Japan John H. Knox, Univ. of Edinburgh, U.K. Ernst Bayer, Univ. of Tu¨bingen, Tu¨bingen, Germany

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999

2000

G. STEPHEN DAL NOGARE AWARD IN CHROMATOGRAPHY OF THE CHROMATOGRAPHIC FORUM OF THE DELAWARE VALLEY (1972–2000) The Chromatography Forum of the Delaware Valley, formed in 1966, is one of the most active scientific organizations on chromatography in the U.S.A. “Delaware Valley” refers to the area on the two sides of the lower part of the Delaware River and of Delaware Bay, encompassing the State of Delaware, the eastern part of Pennsylvania and the southwestern part of New Jersey, where some of the most important chemical industries of the U.S.A. are located. Stephen Dal Nogare was one of the pioneers of gas chromatography in the U.S.A. He was associated with E.I. DuPont de Nemours and Co. (the world’s largest chemical company), at its Experimental Station, located in Wilmington, Delaware, and served as the second president of the group; he died suddenly in 1968. In 1972 the group

48

Chapter 2

TABLE 2.7 RECIPIENTS OF THE STEPHEN DAL NOGARE AWARD Year

Awardee=Affiliation=Country

1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000

Lockhard B. Rogers d , Purdue Univ., Lafayette, IN, U.S.A. Stuart P. Cram, National Bureau of Standards, Washington, DC, U.S.A. J. Jack Kirkland e , E.I. DuPont de Nemours & Co. Research Center, Wilmington,DE, U.S.A. Barry L. Karger, Northeastern Univ., Boston, MA, U.S.A. Lloyd R. Snyder e , Technicon, Inc., Tarrytown, NY, U.S.A. Georges Guiochon, Ecole Polytechnique, Palaiseau, France Csaba Horva´th e , Yale, Univ., New Haven, CT, U.S.A. J. Calvin Giddings d,e , Univ. of Utah, Salt Lake City, UT, U.S.A. Evan C. Horning d,e , Baylor Univ. of Medicine, Houston, TX, U.S.A. Josef F.K. Huber d,e , Univ. of Vienna, Vienna, Austria Marcel J.E. Golay d,e , The Perkin-Elmer Corp., Norwalk, CT, U.S.A. John H. Knox, Edinburgh Univ., Edinburgh, U.K. Hamish Small, Dow Chemical Co., Midland, MI, U.S.A. James E. Lovelock e , Bowerchalke, U.K. Gerhard Schomburg e , Max Planck Inst. Fu¨r Kohlenforschung, Mu¨lheim, Germany Fred E. Regnier, Purdue Univ., Lafayette, IN, U.S.A. Harold F. Walton, Univ. of Colorado, Boulder, CO, U.S.A. Phillis R. Brown, Univ. of Rhode Island, Kingston, RI, U.S.A. Robert L. Grob, Villanova Univ., Villanova, PA, U.S.A. James S. Fritz, Iowa State Univ., Ames, IA, U.S.A. Heinz Engelhardt, Univ. des Saarlandes, Saarbru¨cken, Germany Jack A. Rijks, Univ. of Technology, Eindhoven, The Netherlands Pat J. Sandra, Univ. of Ghent, Ghent, Belgium Charles W. Gehrke e , Univ. of Missouri, Columbia, MO, U.S.A. Peter W. Carr, Univ. of Minnesota, Minneapolis, MN, U.S.A. Daniel E. Martire, Georgetown Univ., Washington, DC, U.S.A. James W. Jorgenson, Univ. of North Carolina, Chapel Hill, NC, U.S.A. Milton L. Lee, Brigham Young Univ., Provo, UT, U.S.A William F. Pirkle, Univ. of Illinois, Urbana, IL, U.S.A.

established in his memory this annual award to be presented to an outstanding scientist in the field of chromatography (Table 2.7). The award is presented every year, usually at the Pittsburgh Conference on Analytical Chemistry, which is one of the world’s most important conventions on scientific instruments including chromatography (see Chapter 4 for further description). The award consists of a honorarium and a plaque.

H. THE KEENE P. DIMICK AWARD IN CHROMATOGRAPHY BY THE SOCIETY FOR ANALYTICAL CHEMISTS OF PITTSBURG (1988–2000) The Keene P. Dimick Award in Chromatography was originally instituted in 1987 by the Keene P. Dimick Foundation and the Dimick Family, in memory of K.P. Dimick, the founder of Wilkens Instruments (Aerograph), the predecessor of the present Chromatography Division of Varian Associates (Table 2.8). The award is to honor scientists

The Builders of Chromatography — Major Chromatography Awards and the Award Winners 49 TABLE 2.8 RECIPIENTS OF THE KEENE P. DIMICK AWARD Year

Awardee=Affiliation=Country

1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000

Milton L. Lee, Brigham Young Univ., Provo, UT, U.S.A. Herbert H. Hill, Washington State Univ., Pullman, WA, U.S.A. Milos V. Novotny, Indiana Univ., Bloomington, IN, U.S.A. Harold M. McNair, Virginia Polytechnic Inst. and State Univ., Blacksburg, VA, U.S.A. Robert E. Sievers, Univ. of Colorado, Boulder, CO, U.S.A. Egil J. Jellum, Biochemical Inst., Oslo, Norway Thomas L. Chester, Procter & Gamble Corp., Miami Valley Labs., Cincinnati, OH, U.S.A. Steven B. Hawthorne, Univ. of North Dakota, Grand Forks, ND, U.S.A. Gerhard Schomburg e , Max Planck Inst. fu¨r Kohlenforschung, Mu¨lheim=Ruhr, Germany Walter G. Jennings, J&W Scientific, Folsom, CA, U.S.A. Leslie S. Ettre e , Yale Univ., New Haven, CT, U.S.A. Karel A. Cramers, Eindhoven Univ. of Technology, Eindhoven, The Netherlands Jerry W. King, Natl. Ctr. for Agric. Utilization Research, Peoria, IL, USDA, U.S.A.

with notable achievement in gas chromatography, which has resulted in significant applications of broad utility. They appointed the Society for Analytical Chemists of Pittsburgh, one of the organizers of The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, to administer the award (see Chapter 3 for further information). The first award was presented in 1988; the awards are presented each year at the Pittsburgh Conference. The award consists of a honorarium (presently $5000) and a plaque. This award is now closed, with the 2000 award being the last.

I. SILVER JUBILEE AWARD OF THE CHROMATOGRAPHIC SOCIETY (1982–2000) The Chromatography Discussion Group (today: the Chromatographic Society) established this silver medal award in 1982 on the occasion of its Silver Jubilee, to be awarded mainly to scientists in the middle of their career to acknowledge their contribution to chromatography (Table 2.9).

J. AWARD FOR ACHIEVEMENTS IN SEPARATION SCIENCE OF THE EASTERN ANALYTICAL SYMPOSIUM (1986–2000) The Eastern Analytical Symposium (EAS) is a major American scientific meeting held annually since 1959 in the New York City area, consisting of various scientific sessions of all branches of analytical chemistry and an exposition. A number of awards are presented during the meeting, among them the award for Achievements in Separation Science (Table 2.10). This award was established in 1986 to honor scientists who significantly contributed to the advancement of separation science. It is presented

50

Chapter 2

TABLE 2.9 RECIPIENTS OF THE SILVER JUBILEE AWARD Year

Awardee=Affiliation=Country

1982

Konrad Grob, Kantonales Labor, Zu¨rich, Switzerland Robert Tyssen, Shell Research, Amsterdam, The Netherlands Peter Simmonds, Ringwood, Hampshire, U.K. Henri Colin, Varex Corp., Rockville, MD, U.S.A. Jo¨rgen Hermansson, Uppsala Univ., Uppsala, Sweden John C. Berridge, Pfizer Research Centre, Sandwich, U.K. Eric D. Morgan, Univ. of Keele, Straffordshire, U.K. Peter J. Schoenmakers, Philips Research, Eindhoven, The Netherlands Joan Albaige´s, Spanish Centre for R&D, Barcelona, Spain Keith D. Bartle, Leeds Univ., Leeds, U.K. Hendrik Lingeman, Free Univ. Amsterdam, The Netherlands David M. Goodall, Univ. of York, York, U.K. Wolfgang Lindner, Karl-Franzens Univ., Graz, Austria Colin F. Poole, Wayne State Univ., Detroit, MI, U.S.A. Christopher M. Riley, Univ. of Kansas, Lawrence, KS, U.S.A. Karin E. Markides, Uppsala Univ., Uppsala, Sweden Brian J. Clark, Dept. of Pharmacy, Bradford Univ., Bradford, U.K. Werner G. Kuhr, Univ. of California, Riverside, CA, U.S.A. Ian D. Wilson, Zeneca Plc., Macclesfield, Cheshire, U.K. Jeremy K. Nicholson, Univ. of London, London, U.K. Patrick Camilleri, Smith Kline Beecham Pharmaceuticals, Harlow, U.K. Norman Smith, Glaxo Research Centre, Stevenage, U.K. Colin F. Simpson, Birbeck College, Univ. of London, London, U.K. Michel Martin, Ecole Supe´rieure de Physique et de Chimie Industrielles, Paris, France Roger M. Smith, Loughborough Univ. of Technology, Loughborough, U.K. Janusz Pawliszyn, Univ. of Waterloo, Waterloo, Canada Graham Nickless, Univ. of Bristol, Bristol, U.K. Claus Albert, University of Tu¨bingen, Germany Phillip Marriott, Royal Melbourne Institute of Technology, Melbourne, Australia

1984 1986 1988 1989

1990

1991

1992 1993 1994 1995 1996 1997 1998 1999 2000

at the annual EAS during a special session honoring the award winner who is also presenting an award address. Originally, ABC Corporation sponsored the award. In 1995 sponsorship was taken over by the Waters Corporation. The award consists of a honorarium (presently $1000) and a plaque.

K. COLACRO MEDAL (1986–2000) OF THE CONGRESSO LATINO AMERICANO DE CROMATOGRAFIA This award is presented at the biannual COLACRO (Congresso Latino Americano de Cromatografia) meetings to scientists who have made a significant contribution to the promotion of chromatography in Latin America (Table 2.11). The award consists of a medal and a certificate.

The Builders of Chromatography — Major Chromatography Awards and the Award Winners 51 TABLE 2.10 RECIPIENTS OF THE AWARD FOR ACHIEVEMENTS IN SEPARATION SCIENCE OF THE EASTERN ANALYTICAL SYMPOSIUM Year

Awardee=Affiliation=Country

1986 1987 1986 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000

Csaba Horva´th e , Yale Univ., New Haven, CT, U.S.A. Haleem J. Issaq, National Cancer Inst., Frederick Cancer R&D Center, Frederick, MD, U.S.A. Milos V. Novotny, Indiana Univ., Bloomington, IN, U.S.A. Harold M. McNair, Virginia Polytechnic Inst. and State Univ., Blacksburg, VA, U.S.A. Daniel W. Armstrong, Univ. of Missouri, Rolla, MO, U.S.A. Robert L. Grob, Villanova Univ., Villanova, PA, U.S.A. Daniel E. Martire, Georgetown Univ., Washington, DC, U.S.A. J. Jack Kirkland e , Rockland Technologies, Newport, DE, U.S.A. Lloyd R. Snyder e , LC Resources, Inc., Orinda, CA, U.S.A. James W. Jorgenson, Univ. of North Carolina, Chapel Hill, NC, U.S.A. Fred E. Regnier, Purdue Univ., Lafayette, IN, U.S.A. Barry L. Karger, Northeastern Univ., Boston, MA, U.S.A. William H. Pirkle, Univ. of Illinois, Urbana, IL, U.S.A. Milton L. Lee, Brigham Young Univ., Provo, UT, U.S.A. Peter W. Carr, Institute of Technology, Minneapolis, MN, U.S.A.

TABLE 2.11 RECIPIENTS OF THE COLACRO MEDAL Year

Awardee=Affiliation=Country

1986 1988 1990 1992 1994 1996

Harold M. McNair, Virginia Polytechnic Inst. and State Univ., Blacksburg, VA, U.S.A. Fernando Lanc¸as, Univ. of Sa˜o Paulo, Sa˜o Paulo, Brazil Remolo Ciola, Univ. of Sa˜o Paulo, Sa˜o Paulo, Brazil Armando Mangarrez, Univ. of Mexico, Mexico City, Mexico Karel A. Cramers, Univ. of Technology, Eindhoven, The Netherlands Pat J. Sandra, Univ. of Ghent, Ghent, Belgium Joaquin Lubkowitz, Separation Sciences, Gulf Breeze, FL, U.S.A. Milton L. Lee, Brigham Young Univ., Provo, UT, U.S.A. Clyde Carducci, Univ. of Buenos Aires, Argentina Milos V. Novotny, Indiana Univ. Bloomington, IN, U.S.A R. Saelzer and M. Vega, Univ. of Conceptio´n, Chile

1998 2000

L. LEROY S. PALMER AWARD OF THE MINNESOTA CHROMATOGRAPHY FORUM (1980–2000) The Minnesota Chromatography Forum (MCF), formed in 1978, is one of the largest regional scientific organizations in the field of chromatography. The objectives of the group are to maintain and promote education, discussion and exchange of information with respect to all fields of chromatography. Besides periodic evening meetings, the MCF organizes each year a three-day Symposium consisting of posters and papers presented by chromatographers from the region as well as invited speakers. Short courses are also held during the Spring Symposium and an instrument exhibition helps the participants to learn about the newest instrumentation.

52

Chapter 2

TABLE 2.12 RECIPIENTS OF THE LEROY S. PALMER AWARD Year

Awardee=Affiliation=Country

1980 1981 1982 1983 1984 1985

Leslie S. Ettre e , The Perkin-Elmer Corp., Norwalk, CT, U.S.A. Larry Bell, Larry Bell & Assoc., Hopkins, MN, U.S.A. Donald F. Hagen, 3M Company, St. Paul, MN, U.S.A. Walter G. Jennings, Univ. of California, Davis, CA, U.S.A. Peter W. Carr, Univ. of Minnesota, Minneapolis, MN, U.S.A. Lloyd R. Snyder e , LC Resources, Inc., Orinda, CA, U.S.A. Larry D. Bowers, Univ. of Minnesota, Minneapolis, MN, U.S.A. Susan M. Price, Twin City Testing Corp., St. Paul, MN, U.S.A. Mark L. Brenner, Univ. of Minnesota, Minneaplis, MN, U.S.A. James S. Fritz, Iowa State Univ., Ames, IA, U.S.A. Jonathan W. DeVries, General Mills, Minneapolis, MN, U.S.A. Shoukry K. W. Kahlil, North Dakota State Univ., Fargo, ND, U.S.A. Kay N. Olson, Univ. of Minnesota, Minneapolis, MN, U.S.A. Craig G. Markell, 3M Company, St. Paul, MN, U.S.A. Gary A. Reineccius, Univ. of Minnesota, Minneapolis, MN, U.S.A. Dennis C. Johnson, Iowa State Univ., Ames, IA, U.S.A. Edward S. Yeung, Iowa State Univ., Ames, IA, U.S.A. John A. Freeburg, Hewlett-Packard Co., St. Paul, MN, U.S.A. James M. Broge, Sandoz Nutrition, St. Louis Park, MN, U.S.A. Wils B. Bergstrom, St. Paul Technical Inst., St. Paul, MN, U.S.A. Patricia H. Sackett, 3M Pharmaceuticals, St. Paul, MN, U.S.A. Steve Pierson, Chrom. Tech., Inc. Apple Valley, MN, U.S.A. Peter Johnson, 3M Pharmaceutical, Maplewood, MN, U.S.A.

1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000

The group has about 600 active members, primarily affiliated with universities, research institutions and industrial companies in the State of Minnesota; however, chromatographers from the neighboring States of Iowa, North and South Dakota and Wisconsin also participate in the meetings of the MCF. The Palmer Award was instituted in 1980 (Table 2.12) in the memory of Leroy Sheldon Palmer (1887–1944), former professor and head of the Division of Agricultural Biochemistry at the University of Minnesota, an early American pioneer in the use of chromatography for the investigation of carotenoids and related pigments (see also Chapter 1). The purpose of the award is to honor contributions by chromatographers in the region to both the science of chromatography and to the activities of the Minnesota Chromatography Forum; however, some scientists outside the region are also honored. The award is presented during the annual Spring Symposium of the MCF and consists of a plaque. M. M.S. TSWETT CHROMATOGRAPHY MEMORIAL MEDAL OF THE ALL-UNION SCIENTIFIC COUNCIL ON CHROMATOGRAPHY, ACADEMY OF SCIENCES OF THE U.S.S.R. (1978–1979) This medal, originally issued in 1978 on the occasion of the 75th anniversary of the discovery of chromatography by M.S. Tswett, was to recognize the services of

The Builders of Chromatography — Major Chromatography Awards and the Award Winners 53 TABLE 2.13 U.S.S.R., M.S. TSWETT CHROMATOGRAPHY MEMORIAL MEDAL RECIPIENTS Individuals Australia Austria Bulgaria Czechoslovakia France German Democratic Republic German Federal Republic Hungary Italy Israel Netherlands Poland Republic of South Africa Romania Spain Sweden Switzerland United Kingdom

U.S.A.

U.S.S.R.

E. Dawes E. Cremer d,e , J.F.K. Huber e N. Kotzev J. Franc, I. Hais d , J. Jana´k e , E. Keulemansova-Smolkova, K. Macek C.L. Guilemin, G. Guiochon, E. Lederer d,e M. Mohnke, H.G. Struppe

E. Bayer, I. Hala´sz d , G. Hesse d,e , R.E. Kaiser e , H. Kelker, L. Rohrschneider e , G. Schomburg e , E. Stahl d,e G. Schay d , L. Szepesy F. Bruner d , M. Lederer e , A. Liberti d,e E. Gil-Av d K.A. Cramers, J.J. Van Deemter e , G. Dijkstra e W. Kemula d , A. Wakmundski d V. Pretorius d,e M. Felipescu M. Gassiot Matas G. Widmark Kurt Grob d , E. sz. Kova´ts e E.R. Adlard e , D.R. Deans, D.H. Desty d,e , E. Glueckauf d,e , A.T. James e , J.H. Knox, J.E. Lovelock e , A.J.P. Martin e , C.S.G. Phillips e , J.H. Purnell d , R. Stock, R.L.M. Synge d,e C.W. Gehrke e , J.C. Giddings d,e , M.J.E. Golay d,e , E. Grushka, L.S. Ettre e , C. Hamilton d , E.C. Horning d,e , M.G. Horning e , C. Horva´th e , J.H. Purnell d , R. Stock A.A. Akhrem, V.B. Aleskovskii, T.G. Andronikashvili, B.G. Belenk’ii, V.G. Berezkin, V.V. Brazhnikov, P.I. Brouthsek, T.I. Bulenkov, K.V. Chmutov d , V.A. Davankov, A.A. Dazkevitsh, M.I. Dement’eva, O.E. Eisen, N.P. Gnusin, R.V. Golovnya, V.I. Gorshkov, V.D. Grebenjuk, B.V. Ioffe d , Ya. I. Yashin, V.I. Kalmanovskii, E.I. Kazanzev, Yu I. Khol’kin, O.G. Kirret, A.V. Kiselev d,e , A.N. Korol, Uy A. Kovan’ko, E.A. Ku¨llik d , B.N. Laskorin, E.T. Lippmaa, N.N. Matorina, V.P. Meleshko, P.P. Nazarov, Yu. S. Nikitin, B.N. Nikolskii, B.P. Okhotnikov, C.M. Ol’shanova, A.B. Pashkov, D.P. Poshkus, V.V. Ratshinksii, R.N. Rubinstein, B.A. Rudenko, K.I. Sakodynskii d,e , C.M. Saldadze, G.V. Samsonov, E.M. Savitzkaya, M.M. Ssenyavin, Z.M. Shapiro, F.M. Shemiakin, K.D. Shcherbakova, M.S. Shraiber d , Yu. V. Shostenko, V.S., Soldatov, G.L. Starobinez, V.L. Tal’rose, A.S. Teviina, G.A. Tsikin, N.N., Tunizkii, E.V. Vagin, M.M. Vigdergauz d , D.A. Vyakhirev d , S.A. Volkov, S.P. Zhdanov, A.A. Zhukhovitskii d,e , I.C. Zitovich, P.P. Zolotarev

Associations and Journals Chromatographia Chromatography Discussion Group Elsevier Scientific Publishing Co. Journal of Chromatographic Science Journal of Chromatography

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TABLE 2.13 (continued) Instrument Companies and Supply Houses Australia Scientific Glass Engineering Ltd.Pty Czechoslovakia Laboratori Pristrojne, Lachema France Jobin and Ivon German Chromatron Democratic Republic German Federal E. Merck Republic United Kingdom Pye-Unicam Italy Carlo Erba Strumentazione Japan Jasco, Shimadzu Netherlands Chrompack, Packard-Becker Sweden Optilab U.S.A. E.I. du Pont de Nemours, Inc., Hamilton, Inc., Hewlett-Packard Co., Perkin-Elmer Corp., Supelco, Inc., Varian Inc. U.S.S.R. Experimental Factory of Diatomites, Experimental Factory of Oil Treatment, Factory of Reagents Institutes and Construction Bureaus in the U.S.S.R Construction Bureau of Automation Construction Bureau of Automation in Petrochemistry Construction Bureau of Chromatography Construction Bureau of the Estonian Academy of Sciences Construction Bureau of the Institute of Organic Chemistry Construction Bureau of the Institute of Petroleum Synthesis Institute of Luminofors Institute of Microcrystals

scientists who gave their thoughts, energy, strength and time to the development of chromatography and its application for solving the many problems facing our society (Table 2.13). The medal was first presented at the Memorial Symposium held in 1978 in Tallin, Estonian S.S.R., to the leading scientists present at the meeting. However, it was immediately recognized that many significant scientists, with major contributions to the evolution of chromatography, were not present at the meeting. Therefore, the Council decided to also present the medal to a number of scientists in various countries who had a significant role in the evolution of chromatography. In addition, the Scientific Council also presented the medal to instrument companies, supply houses, construction bureaus, as well as to journals and institutions which significantly contributed to the evolution of chromatography. This medal is not an award in the usual sense; it is included here for completeness. The full list of the recipients of the U.S.S.R. Chromatography Memorial Medal recipients are listed in Table 2.13.

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Major International Symposia Supporting Chromatography Leslie S. Ettre Department of Chemical Engineering, Yale University, New Haven, CT, USA *

CONTENTS A. B. C. D. E. F. G. H. I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . International symposia on (gas) chromatography by the (British) Chromatographic Society (1956–2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Symposia on gas chromatography organized by the Instrument Society of America (1957– 1963) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . International symposia by the French Society G.A.M.S. (1961–1969) . . . . . . . . . . . International symposia on advances in chromatography (1963–1988) . . . . . . . . . . . International symposia on high-performance liquid chromatography (HPLC) (1973–2000) . . International symposia on capillary (gas) chromatography (1975–2000) . . . . . . . . . . Danube symposia on chromatography (1976–1993) . . . . . . . . . . . . . . . . . . . COLACRO Latin American congresses on chromatography and related techniques (1986– 2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (PITTCON) (1950–2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

55 58 59 60 60 61 62 63 64 65

INTRODUCTION In a rapidly growing scientific discipline, the continuous exchange of information among scientists is of vital importance. Publication of new results is one way to achieve this; however, frequent personal contact is even more important because it facilitates informal discussions. Scientific meetings, symposia and congresses represent the best forum where scientists from different geographical areas can meet, present their newest results and discuss them with their peers. Such meetings also provide the possibility for * Mail address: P.O. Box 6274, Beardsley Station, Bridgeport, CT 06606-0274, USA

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younger scientists to become acquainted with the internationally recognized authorities in their field. Modern chromatography evolved in the middle of the 20th century and its rapid growth was unparalleled in the history of science; within two decades chromatography became the most widely used analytical separation technique in the world. The evolution of modern chromatography coincided with improvements in transportation. While before the World War II, a week or more was needed to travel from one continent to the other, this could now be done in less than one day. Also, this period represented the beginning of the second industrial revolution and the internationalization of economy, commerce and science. All these facilitated participation at various meetings on the international level. In this way chromatography has served as a kind of bridge between countries, continents and ideological boundaries. This situation was best characterized by one of the early pioneers who said that “chromatography has a dual face: it is the best method to separate compounds, but it is also the best method to unite people”. Some of these meetings were organized by existing, traditional scientific associations; however, in a number of cases new organizations were set up to deal specifically with chromatography. In addition ad hoc committees, without any formal permanent structure, organize some of the important symposium series. In these cases the chairman of the meeting always plays a particularly important role. In each case, the listing is preceded by a brief summary, explaining the origin of the series and indicating the organization, which had been responsible for the meetings. The listings give the date and location of the meetings and, whenever possible, the name of the person or persons who had prime responsibility for their success. We are considering here only those international meeting series which have dealt with chromatography in general or with one of the principal chromatographic techniques (gas or liquid). These are listed in Table 3.1. We realize that there are a number of other regular symposium series, which, however, cover only a specialized, narrower field (e.g., preparative chromatography, protein chromatography) or serve only a smaller geographical area. It would have been impossible to include all of these. Naturally, papers on chromatography and its applications have always been presented during the regular meetings of the national chemical societies. However, we disregarded TABLE 3.1 MAJOR INTERNATIONAL SYMPOSIUM SERIES IN THE FIELD OF CHROMATOGRAPHY A. International Symposia on (Gas) Chromatography (1956–2000) B. Symposia on Gas Chromatography by the Instrument Society of America (1957–1963) C. Journe´es Internationales d’E´tude des Me´thodes de Se´paration Imme´diate et de Chromatographie (1961–1969) D. International Symposia on Advances in Chromatography (1963–1988) E. International Symposia on High-Performance Liquid Chromatography (1973–2000) F. International Symposia on Capillary (Gas) Chromatography (1975–2000) G. Danube Symposia on Chromatography (1976–1993) H. COLACRO Latin American Congress on Chromatography and Related Techniques (1986–2000) I. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (PITTCON) (1950–2000)

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these because the main profile of these meetings was chemistry in general and chromatography only had a subordinate role. There were, however, two such sessions held during the National Meetings of the American Chemical Society (ACS), which deserve mentioning because they represent important milestones in the evolution of chromatography. The first is a special Symposium on Ion-Exchange Separations, held in 1947, during the 112th National ACS Meeting, in New York City. During the Second World War, as part of the Manhattan Project, two major groups — at Oak Ridge, TN, and at Iowa State University, in Ames — carried out research on the possibility of separating rare earths by ion-exchange chromatography. Their work was successful and even made possible the separation of rare earths on a preparative scale. During the war this work was classified; finally, in 1947, permission was granted to the researchers participating in this project to publicly report on their activities. This took place at this special Symposium where members of the two groups presented a total of 13 papers; these were then subsequently published as a separate issue of the Journal of the American Chemical Society. The second symposium held in conjunction with a National ACS Meeting which had a particular importance in the evolution of modern chromatography, was the one-day Symposium on Gas Chromatography held during the 129th National ACS Meeting, in Dallas, Texas, April 8–13, 1956. This was the first major meeting on gas chromatography in the United States; in fact, it even preceded the First International Symposium on Gas Chromatography held in London, May 30–June 1, 1956. In the United States the annual Pittsburgh Conferences on Analytical Chemistry had an important role in the evolution of laboratory instrumentation, in the introduction of new instruments including, naturally, also gas — and later, liquid — chromatographs. Papers presented at these meetings have dealt primary with the design and performance of new instruments which were shown the first time at the exhibition. Because of their importance in the evolution of modern chromatography, these conferences are also included in our discussion. It should be noted, however, that similar periodic meetings, combined with exhibitions, were also held in other countries, serving the same purpose: e.g., the ACHEMA and ANALYTICA meetings in Germany, the Salon de Chimie in France, or the yearly meetings of the Chemical Society of Japan, and the biannual conferences of BECIA in China. Before we discuss the individual symposium series, we should specifically mention two international meetings held in the second part of the 1940s which were very important in providing an excellent summary of the status of chromatography at that time. The Conference on Chromatography was organized on November 29–30, 1946, by the New York Academy of Sciences. Although just one year after the end of the War, two key participants could already attend the meeting from Europe: S. Claesson, from the University of Uppsala, Sweden, an associate of Arne Tiselius who, two years later, received the Nobel Prize for his investigations on electrophoresis and adsorption chromatography, and A.J.P. Martin, from England, the co-inventor of partition chromatography, receiving in 1952 the Nobel prize together with R.L.M. Synge, honoring this achievement. Claesson’s lecture dealt with displacement and frontal techniques on which he had worked together with Tiselius, while Martin summarized their work on partition chromatography. The lectures of this meeting were later published as a separate issue of the Annals of the New York Academy of Sciences.

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The second important international symposium of this period entitled Chromatographic Analysis was organized by the Faraday Society on September 22–24, 1949, at Reading University, in England. A total of 42 lectures were presented and the participants included scientists from ten countries. Major presentations have dealt with the theory and practice of column and paper chromatography using adsorption, partition and ion-exchange techniques. It is particularly important to mention that at this meeting, C.S.G. Phillips of Oxford University already described the possibilities of separation by gas adsorption chromatography in the elution mode.

A. INTERNATIONAL SYMPOSIA ON (GAS) CHROMATOGRAPHY BY THE (BRITISH) CHROMATOGRAPHIC SOCIETY (1956–2000) This is the oldest symposium series originated in 1956 and held biannually since then, in even-numbered years, in various European cities. The first symposium in 1956 was organized mainly by British petroleum chemists (although the subject of the overwhelming part of the lectures was outside the petroleum field) and was held under the auspices of the British Institute of Petroleum. After this symposium prominent chromatographers formed the Gas Chromatography Discussion Group, which took over the responsibility for the organization of these biannual symposia. Until 1972 this group was legally part of the Institute of Petroleum; from then on, it became an independent body with a large number of foreign members, particularly from Scandinavia forming a separate section within the association. In 1970 the attribute ‘gas’ was dropped from the name of the group. Finally, on May 3, 1984, the group was reorganized as The Chromatographic Society. In 1991 a separate Irish Section was also formed. The name of the first meeting in 1956 was Vapor Phase Chromatography; starting with the 1958 meeting this was changed to Gas Chromatography. At the 1966 meeting its scope was enlarged to also include ‘associated techniques’. Finally, in 1970 the name of the symposia was changed to Chromatography in general, encompassing all chromatographic techniques. In 1969, an agreement was made between the group, as well as two other national organizations, the French G.A.M.S. (Groupement pour l’Analyse des Me´thodes Spectrographiques et des Me´thodes Physiques d’analyse) and the Arbeitskreis Chromatographie of the Gesellschaft Deutscher Chemiker, the German Chemical Society, that from then on the biannual symposia will be organized on a tripartite basis. Until 1972, the proceedings of the symposia were published in book form by Butterworths Publishers and the Institute of Petroleum. From 1974 on, the papers have always been published as a separate issue of a journal. From 1978 to the present, the Chromatographic Society has sponsored the A.J.P. Martin Award, which, along with the Awardees, is presented in Chapter 2D. In the listing below, the chairmen of the individual symposia responsible for the organization are also indicated. 1. 2.

May 30–June 1, 1956 May 19–23, 1958

London, U.K. Amsterdam, The Netherlands

S.F. Birch J. Boldingh

Major International Symposia Supporting Chromatography 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

June 8–10, 1960 June 13–16, 1962 September 8–10, 1964 September 20–23, 1966 June 25–28, 1968 Sept. 28–Oct. 1, 1970 October 9–13, 1972 Sept. 30–Oct. 4, 1974 July 5–9, 1976 September 25–29, 1978 June 30–July 4, 1980 September 13–17, 1982 October 1–5, 1984 September 21–26, 1986 September 25–30, 1988 September 23–28, 1990 September 13–18, 1992 June 19–24, 1994 September 15–20, 1996 September 13–18, 1998 October 1–5, 2000

Edinburgh, U.K. Hamburg, Germany Brighton, U.K. Rome, Italy Copenhagen, Denmark Dublin, Ireland Montreux, Switzerland Barcelona, Spain Birmingham, U.K. Baden-Baden, Germany Cannes, France London, U.K. Nu¨rnberg, Germany Paris, France Vienna, Austria Amsterdam, The Netherlands Aix en Province, France Bournemouth, U.K. Stuttgart, Germany Rome, Italy London, U.K.

59 R.C. Chirnside C.S.G. Phillips and H. Kienitz D.H. Desty G.B. Marini-Bettolo C.G. Scott C.L.A. Harbourn R. Stock G. Guiochon D.R. Deans K.P. Hupe G. Guiochon C.E.R. Jones E. Bayer M. Martin and P. Devaux J.F.K. Huber U.A.Th. Brinkmann A.M. Siuffi and M. Martin M.B. Evans H. Engelhardt F. Dondi K. Bartle

B. SYMPOSIA ON GAS CHROMATOGRAPHY ORGANIZED BY THE INSTRUMENT SOCIETY OF AMERICA (1957–1963) After the success of the 1956 London Symposium organized by the British group (see above), chromatographers in the United States decided to organize a similar symposium series in the odd-numbered years. For convenience the symposia were organized under the auspices of the Instrument Society of America, and held at the facilities of Michigan State University, in East Lansing, MI. The first three symposia — held in 1957, 1959 and 1961 — were a success, with a number of major papers presented there. However, mainly due to organizational problems the planning of future symposia faced increasing difficulties. A further symposium was still held in 1963, but with significantly reduced participation and with it, the symposium series ceased to exist. The papers presented at these symposia, including also the discussions, were published with a delay of one year or more (in the case of the second symposium, only two years later) by Academic Press under the title Gas Chromatography, with the organizers serving as the editors. Meetings in East Lansing, Michigan and organizers are listed below for this symposium, 1. 2. 3. 4.

August 28–30, 1957 June 10–13, 1959 June 13–16, 1961 June 18–21, 1963

V.J. Coates, H.J. Noebels, I.S. Fagerson N. Brenner, H.J. Noebels, R.F. Wall N. Brenner, J.E. Callen, M.D. Weis L. Fowler

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C. INTERNATIONAL SYMPOSIA BY THE FRENCH SOCIETY G.A.M.S. (1961–1969) This association was originally founded in 1944 as the Groupement pour l’Analyse des Me´thodes Spectrographiques (G.A.M.S.) to help the exchange of information among scientists involved in emission spectrometry and metal analysis. Its scope was progressively extended to include other spectroscopic methods and, from 1957 on, also other physico-chemical methods of analysis, particularly (gas) chromatography. At that time the name of the society was changed to Groupement pour l’Avancement des Me´thodes Spectroscopiques et des Me´thodes Physiques d’Analyse, and in 1989 to Groupe pour l’Avancement des Sciences Analytiques; however, the acronym G.A.M.S. has continued to indicate the association. Finally, in 1997, G.A.M.S. was dissolved and a new organization, the Association Franc¸aise des Sciences Se´paratives created. Starting in 1961, the G.A.M.S organized biannual international symposia called Journees Internationales d’E´tude des Me´thodes de Se´paration Imme´diate et de Chromatographie (J.I.S.I.C.) in various major European cities. The principal theme of these symposia was chromatography, but papers on other separation methods were also presented. In 1969 an agreement was made between the G.A.M.S., the Arbeitskreis Chromatographie of the German Chemical Society, and the British Chromatography Discussion Group, that from then on the biannual international symposia organized since 1956 by the British group will be organized on a tripartite basis. As a conclusion of this agreement G.A.M.S. suspended its J.I.S.I.C. series of meetings. The five international J.I.S.I.C. symposia organized by G.A.M.S. are listed below, giving also the names of the principal organizers: 1. 2. 3. 4. 5.

June 13–15, 1961 June 14–16, 1963 September 19–25, 1965 October 10–13, 1967 October 7–10, 1969

Paris, France Milano, Italy Athens, Greece Heidelberg, Germany Lausanne, Switzerland

P. Chovin G. Parissakis H. Kienitz E. sz. Kova´ts

D. INTERNATIONAL SYMPOSIA ON ADVANCES IN CHROMATOGRAPHY (1963–1988) This symposium series originated in 1963 by a number of prominent American chromatographers as a forum where both established chromatographers and younger representatives of the field participate, present reports on their work, and have the opportunity to exchange their ideas in an informal setting. A special emphasis was on the rapid publication of the presented papers in a journal, with broad distribution. A. Zlatkis, Professor at the University of Houston, Texas, accepted the responsibility of organizing the symposia; for this reason, this symposium series is often called the ‘Zlatkis meetings’. The first three symposia were held in Houston, Texas; from 1967 on they were also held at other locations in the United States and Canada; from 1975 on, the symposia alternated between the United States and Europe and two

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meetings were also held in Japan. In 1988 it was decided to discontinue this symposium series. Originally the scope of the meetings was restricted to gas chromatography; however, starting with the fourth symposium, it was extended to encompass all chromatographic techniques. According to the original concept, the proceedings of the symposia were published within a short time in widely distributed journals: Analytical Chemistry, the Journal of Gas Chromatography, and since 1974 as separate issues of the Journal of Chromatography. (See Appendices 3 and 6 for references) Starting in 1974, the M.S. Tswett Chromatography Award was presented during the opening session of the symposium, see Chapter 2E. The individual meetings of this series are listed below: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

January 21–24, 1963 March 23–26, 1964 October 18–21, 1965 April 3–6, 1967 January 20–23, 1969 June 2–5, 1970 November 29–December 3, 1971 April 16–19, 1973 November 4–7, 1974 November 3–6, 1975 November 1–5, 1976 November 7–10, 1977 October 16–19, 1978 September 24–28, 1979 October 6–9, 1980 September 28–October 1, 1981 April 5–8, 1982 April 15–17, 1982 October 3–6, 1983 April 16–18, 1984 June 3–6, 1985 September 15–17, 1986 October 7–9, 1986 September 8–10, 1987 August 28–September 1, 1988

Houston, TX, USA Houston, TX, USA Houston, TX, USA New York, NY, USA Las Vegas, NV, USA Miami Beach, FL, USA Las Vegas, NV, USA Toronto, Ontario, Canada Houston, TX, USA Mu¨nchen, Germany Houston, TX, USA Amsterdam, The Netherlands St. Louis, MO, USA Lausanne, Switzerland Houston, TX, USA Barcelona, Spain Las Vegas, NV, USA Tokyo, Japan Amsterdam, The Netherlands New York, NY, USA Oslo, Norway Houston, TX, USA Chiba (Tokyo), Japan Berlin, Germany Minneapolis, MN, USA

E. INTERNATIONAL SYMPOSIA ON HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) (1973–2000) With the growing importance of modern, high-performance liquid chromatography, it was soon considered as important that a special symposium devoted only to this technique should be organized, particularly since at that time liquid chromatography still occupied a relatively minor place at the major chromatography symposia. Thus, a three-day symposium called Symposium on Column Liquid Chromatography was

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held in May 1973, at Interlaken, Switzerland. Encouraged by its success, a similar symposium was held three years later, in the United States. There, it was decided to organize similar symposia on a biannual basis, alternately in Europe and the United States. Due to the exponential growth of liquid chromatography, from 1981 on the symposia, which by then were named the International Symposia on High-Performance Liquid Chromatography (HPLC), were held on an annual basis. Recently, due to the expansion of the field, the name of the symposia was again changed, to International Symposia on High-Performance Liquid Phase Separations and Related Techniques. A permanent international committee organizes these yearly HPLC symposia. Each symposium has a general chairman who is responsible for the local arrangements and organization. In general most of the papers presented at these symposia were subsequently published in separate issues of the Journal of Chromatography. (See Appendix 6 for post-1990 symposia references.) The individual symposia of this series are listed below, giving also the general chairmen who were principally responsible for their organization. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

May 2–4, 1973 May 17–19, 1976 September 27–30, 1977 May 7–10, 1979 May 10–15, 1981 June 7–11, 1982 May 2–6, 1983 May 20–25, 1984 July 1–5, 1985 May 18–23, 1986 June 28–July 3, 1987 June 19–24, 1988 June 25–30, 1989 May 20–25, 1990 June 3–7, 1991 June 14–19, 1992 May 9–14, 1993 May 8–13, 1994 May 28–June 2, 1995 June 16–21, 1996 June 22–27, 1997 May 3–8, 1998 May 30–June 4, 1999

Interlaken, Switzerland Wilmington, DE, USA Salzburg, Austria Boston, MA, USA Avignon, France Cherry Hill, NJ, USA Baden-Baden, Germany New York, NY, USA Edinburgh, U.K. San Francisco, CA, USA Amsterdam, Netherlands Washington, DC, USA Stockholm, Sweden Boston, MA, USA Basel, Switzerland Baltimore, MD, USA Hamburg, Germany Minneapolis, MN, USA Innsbruck, Austria San Francisco, CA, USA Birmingham, U.K St. Louis, MO, USA Granada, Spain

W. Simon J.J. Kirkland J.F.K. Huber B.L. Karger G. Guiochon R.A. Barford K.P. Hupe Cs. Horva´th J.H. Knox R.E. Majors H. Poppe G. Guiochon D. Westerlund B.L. Karger F. Erni F.E. Regnier K.K. Unger P.W. Carr and L.D. Bowers W. Lindner W.S. Hancock A.F. Fell D.W. Armstrong E. Gelpi

F. INTERNATIONAL SYMPOSIA ON CAPILLARY (GAS) CHROMATOGRAPHY (1975–2000) This symposium series started in 1975 by R.E. Kaiser (Institut fu¨r Chromatographie, Bad Du¨rkheim, Germany) as an informal meeting discussing advances in capillary gas chromatography. The first four symposia were held in Hindelang, in the Bavarian Mountains, as biannual meetings. In 1983, the organization was taken over by a group

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headed by P. Sandra (University of Ghent, Belgium) and moved to Riva del Garda, in northern Italy. From 1985 on, the symposia became a yearly event and their scope was extended to include all chromatography techniques dealing with microcolumn separations; however, the emphasis still remained on gas chromatography. From 1986 on, the symposia alternated between Europe and the United States, and periodically symposia were also held in Japan. Since 1989 the M.J.E. Golay Award in Capillary Chromatography has been presented at the yearly meetings to scientists with major achievements in the theory and practice of capillary chromatography; please refer to Chapter 2. In order to facilitate discussion, the text of the presentations or at least an extended abstract has always been available at the meeting. Subsequently, most of the papers found publication in the chromatography journals. The M.J.E. Golay awardees are presented in Chapter 2F. Below the individual meetings of this series are listed. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

May 4–7, 1975 May 2–6, 1977 April 28–May 3 1979 May 3–7, 1981 April 26–28, 1983 May 14–16, 1985 May 11–14, 1986 May 19–21, 1987 May 16–19, 1988 May 22–25, 1989 May 14–17, 1990 September 11–14, 1990 May 13–16, 1991 May 25–29, 1992 May 24–28, 1993 September 26–30, 1994 May 7–11, 1995 May 20–24, 1996 May 18–22, 1997 May 26–29, 1998 June 20–24, 1999 November 8–12, 1999

Hindelang, Germany Hindelang, Germany Hindelang, Germany Hindelang, Germany Riva del Garda, Italy Riva del Garda, Italy Gifu, Japan Riva del Garda, Italy Monterey, CA, USA Riva del Garda, Italy Monterey, CA, USA Kobe, Japan Riva del Garda, Italy Baltimore, MD, USA Riva del Garda, Italy Riva del Garda, Italy Wintergreen, VA, USA Riva del Garda, Italy Wintergreen, VA, USA Riva del Garda, Italy Park City, UT, USA Gifu, Japan

G. DANUBE SYMPOSIA ON CHROMATOGRAPHY (1976–1993) In the 1970s contact between scientists in the west and in the so-called socialist countries intensified; however, scientists living in the Soviet block still had only limited possibilities to participate at international symposia held in the west. Therefore, a proposal had been made to establish a symposium series similar to the biannual International Symposia on Chromatography (ISC) organized by the British Chromatography Discussion Group (The Chromatographic Society), but the meetings should be held

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in cities located in countries of the Soviet block. According to the plans prominent western scientists would also be invited to participate at the meetings. In this way a large number of chromatographers from the socialist countries could have contact with their western colleagues which otherwise would be impossible. The new symposium series was named after the river Danube flowing through both the West and East and thus, serving as a link between the various countries. The first Danube Symposium held in 1976 in Hungary was a success; therefore, it was decided to continue it on a biannual basis, but in the odd-numbered years in order not to compete with the ISC series, which is always held in even-numbered years. After the collapse of the Soviet Union and the elimination of the so-called iron curtain dividing Europe, the rationale behind this symposium series became obsolete. Therefore, it was decided in 1991 to discontinue it after the 1993 meeting, the planning of which was then already under way. The individual meetings of the Danube Symposium series are listed below, giving also the names of the principal organizers. 1. 2. 3. 4. 5. 6. 7. 8. 9.

September 28–30, 1976 April 18–20, 1979 August 31–September 3, 1981 August 29–September 2, 1983 November 11–16, 1985 October 12–17, 1987 August 21–25, 1989 September 2–6, 1991 August 23–27, 1993

Szeged, Hungary Karlovy Vary, Czechoslovakia Sio´fok, Hungary Bratislava, Czechoslovakia Yalta, U.S.S.R. Varna, Bulgaria Leipzig, East Germany Warsaw, Poland Budapest, Hungary

L. Szepesy J. Jana´k L. Szepesy J. Garaj K.I. Sakodynskii D. Shopov W. Engewald E. Szoczewinski L. Szepesy

H. COLACRO LATIN AMERICAN CONGRESSES ON CHROMATOGRAPHY AND RELATED TECHNIQUES (1986–2000) COLACRO is the abbreviation for Congresso Latino–Americano de Cromatografia e Te´cnicas Afin (Latin American Congress on Chromatography and Related Techniques). The proposal for this symposium series originated in 1984 during the First Brazilian Symposium on Chromatography held in conjunction with the annual meeting of the Brazilian Chemical Society. In 1985, F. Lanc¸as of the University of Sa˜o Paulo established a local group to organize the first meeting planned for 1986. At that time an International Scientific Committee, consisting of members from Latin America, the United States and Europe, was established to guarantee the international character of the meeting and its scientific quality. Based on the success of the first meeting, it was decided to hold the series on a biannual basis, in various Latin American countries. For this reason a permanent Latin American Committee on Chromatography was set up, consisting of representatives of each country, appointed by the national chemical associations, which has the overall responsibility for the organization of the individual meetings. The original aim of these symposia was to promote the application of chromato-

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graphic techniques in Latin America. Soon, however, these meetings exceeded this goal and became a major international event where, in addition to chromatographers from Latin America, many scientists from North America and Europe, and even Asia, participate, and the number of participants approaches the level of the other, better known international symposia. The scientific program of the meetings includes plenary lectures by invited international guests, posters of contributed papers, technical seminars, discussion sessions and workshops; an exhibition of scientific instruments is always held in conjunction with the meeting. In addition a number of short courses are also organized in the days before the symposium. At the opening session, the COLACRO Medal is presented to scientists who have directly or indirectly helped to promote chromatography in Latin America; refer to Chapter 2K for these Awardees. The date, location and principal organizers of the COLACRO symposia are listed below: 1. 2. 3. 4. 5. 6. 7. 8.

March 17–19, 1986 October 18–20, 1988 March 14–16, 1990 April 21–23, 1992 January 11–15, 1994 January 23–25, 1996 March 25–27, 1998 April 12–14, 2000

Rio de Janeiro, Brazil Buenos Aires, Argentina ´ guas de Sa˜o Pedro, Brazil A Mexico City, Mexico Conception, Chile Caracas, Venezuela ´ guas de Sa˜o Pedro, Brazil A Buenos Aires, Argentina

F.M. Lanc¸as D. Escatl F.M. Lanc¸as H. Gomez D. von Baer I. Romero F.M. Lanc¸as C. Carducci

I. PITTSBURGH CONFERENCE ON ANALYTICAL CHEMISTRY AND APPLIED SPECTROSCOPY (PITTCON) (1950–2000) The Pittsburgh Conferences on Analytical Chemistry and Applied Spectroscopy (PITTCON) originated from the activities of two professional societies in the greater Pittsburgh area: the Spectroscopy Society of Pittsburgh (SSP), and the Society for Analytical Chemistry of Pittsburgh (SACP), established in 1946 and 1942, respectively. After holding separate annual meetings for a few years, the two societies decided to combine their individual meetings, adding also an exposition of modern laboratory equipment. The first such meeting was held in February 1950, in Pittsburgh. Due to the great interest in this convention, the two societies decided to make the Conference and Exposition a jointly organized annual event. The start of the Pittsburgh Conferences and Expositions coincided with the rapid growth of the American scientific instrument industry. In such a rapidly growing field there is particularly a great need to periodically demonstrate the newest products, exchanging ideas with the leaders in the field as well as with the prospective users of these instruments. The Pittsburgh Conferences and Exhibitions, called generally by the acronym PITTCON, represented an ideal forum for such an activity. As a conclusion, these annual meetings soon became one of the most important places where new laboratory instruments were displayed at the exhibition and described in lectures during the conference. These activities also brought more and more participants from

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outside the Pittsburgh area. Naturally, the attendees from the many laboratories, both industrial and academic, also wanted to participate in the program of the conferences and workshops, by presenting papers on their newest results and organizing symposia and short courses on the newest directions in analytical chemistry. As a conclusion of these activities, the PITTCON meetings and exhibitions rapidly became very important national and, within about two decades, international events. Today, these meetings represent worldwide the most important annual conventions in the field of chemical sciences, with the total number of attendees around 30,000. Numerical data illustrating this growth is presented in Table 3.2 below. The annual PITTCON meetings cover more than just chromatography; they encompass the whole field of analytical chemistry. However, they were particularly important in the evolution of modern chromatography. After all, chromatography is now an instrument-based analytical method and thus a possibility for the periodic display and description of the newest instruments is indispensable for the growth of the field. This was provided by PITTCON; thus the evolution of modern gas and, later, liquid chromatography went step by step with the evolution of these meetings. This is the reason to include the PITTCON conferences in this compilation. In the first two years the meetings were co-chaired by representatives of the two societies. From 1952 on, the conference was organized jointly by the two societies which elect the president of the convention who, in turn, is assisted by many volunteers from the membership of the societies. Until 1967 the yearly meetings were held in Pittsburgh, in the William Penn (Penn-Sheraton) Hotel, but they simply outgrew the local possibilities. Therefore, in 1968, PITTCON moved to Cleveland (Ohio), occupying the large convention hall of the city. After over a decade even this became too small and so, in 1980, PITTCON moved to Atlantic City (New Jersey). From 1985 on, it was decided to embark in a multi-city rotation that has included New York City, Chicago (Illinois), Atlanta (Georgia) and New Orleans (Louisiana), cities in which very large convention and exhibition halls exist. The 1999 meeting, representing the fiftieth anniversary of PITTCON, was held in Orlando, Florida. However, the meetings and expositions are still organized by the two Pittsburgh societies and their many volunteers. The dates and locations of the 50 years of annual PITTCON conferences are listed in Table 3.2, with the names of the presidents, and provides numerical data demonstrating their evolution, giving the total number of attendees, (A), exhibiting companies (B) and presented papers (C). TABLE 3.2 50 YEARS OF EVOLUTION OF PITTCON Serial No.

Date (Year, M=DD)

Location

President

A

B

C

1

1950, 2=15–17

Pittsburgh, PA

800

14

56

2

1951, 3=5–7

Pittsburgh, PA

Mary E. Warga C. Manning Davis Robert A. Friedel John J. McGovern Henry Freiser

1,006

17

87

Major International Symposia Supporting Chromatography

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TABLE 3.2 (continued) Serial No.

Date (Year, M=DD)

Location

President

A

B

C

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

1952, 3=5–7 1953, 3=2–6 1954, 3=1–5 1955, 2=28–3=4 1956, 2=27–3=2 1957, 3=4–8 1958, 3=3–7 1959, 3=2–6 1960, 2=29–3=4 1961, 2=27–3=3 1962, 3=5–9 1963,3=4–8 1964, 3=2–6 1965, 3=1–5 1966, 2=21–25 1967, 3=6–10 1968, 3=4–8 1969, 3=3–7 1970, 2=2–6 1971, 3=1–5 1972, 3=6–10 1973, 3=5–9 1974, 3=4–8 1975, 3=3–7 1976, 3=1–5 1977, 2=28–3=4 1978, 2=27–3=3 1979, 3=5–9 1980, 3=10–14 1981, 3=9–13 1982, 3=8–12 1983, 3=7–11 1984, 3=5–9 1985, 2=25–3=1 1986, 3=10–14 1987, 3=9–13 1988, 2=22–26 1989, 3=6–10 1990, 3=5–9 1991, 3=4–8 1992, 3=9–13 1993, 3=8–12 1994, 2=27–3=4 1995, 3=5–10 1996, 3=3–8 1997, 3=16–21 1998, 3=1–6 1999, 3=7–12 2000, 3=12–17 2001, 3=4–9

Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Cleveland, OH Cleveland, OH Cleveland, OH Cleveland, OH Cleveland, OH Cleveland, OH Cleveland, OH Cleveland, OH Cleveland, OH Cleveland, OH Cleveland, OH Cleveland, OH Atlantic City, NJ Atlantic City, NJ Atlantic City, NJ Atlantic City, NJ Atlantic City, NJ New Orleans, LA Atlantic City, NJ Atlantic City, NJ New Orleans, LA Atlanta, GA New York, NY Chicago, IL New Orleans, LA Atlanta, GA Chicago, IL New Orleans, LA Chicago, IL Atlanta, GA New Orleans, LA Orlando, FL New Orleans, LA New Orleans, LA

Hugh F. Beeghly C. Burton Clark Melvin L. Moss Robert K. Scott Raymond G. Russel Neil E. Gordon, Jr. James F. Miller Edwin S. Hodge Laben M. Melnick Andrew G. Sharkey Fritz Will, III James E. Paterson Francis P. Byrne Bruce M. LaRue James P. McKaveney Frank E. Dickson Richard T. Oliver Gerald L. Carlson Robert Mainier William G. Fateley Harry W. Fracek Joseph R. Ryan Joseph A. Feldman Charles McCafferty, Jr. Alex J. Kavoulakis John F. Jackovitz Jane H. Judd Herbert L. Retcofsky Harold A. Sweeney John E. Graham Robert Badoux, Sr. Richard Obrycki Ralph M. Raybeck Allen J. Sharkins Richard S. Danchik John A. Queiser George L. Vassilaros Paul E. Bauer Ann C. Cibulas Ernest F. Tretow John P. Auses Victor C. Zadnik W. Richard Howe Herald A. Barnett John D. Sember Joanne H. Smith Sarah L. Shockey Thomas J. Conti Hyman Schultz Michael N. Carmosino

1,300 1,360 1,653 2,050 2,800 2,839 2,856 3,182 3,366 3,550 3,734 3,918 4,103 4,585 4,808 5,393 5,405 6,232 6,539 6,384 6,803 7,486 8,369 9,318 10,959 12,051 13,489 15,838 16,032 17,270 19,884 21,728 24,648 20,733 29,146 31,555 25,264 26,741 34,048 29,947 27,987 28,941 30,922 31,089 34,079 31,411 28,118 29,893 15,741 13,903

24 26 36 37 50 50 75 75 102 108 136 153 154 178 181 189 211 220 236 246 247 253 275 301 308 319 362 369 451 498 560 560 630 730 767 790 830 850 860 988 1,011 1,083 1,083 1,163 1,116 1,157 1,217 1,276 2,222 2,295

121 129 122 183 187 131 152 160 169 222 185 226 226 292 260 298 266 291 314 317 385 318 434 532 492 483 648 700 820 884 849 946 962 1,213 1,086 1,097 1,134 1,324 1,059 1,373 1,862 1,864 1,812 1,828 1,909 1,992 1,931 1,960 27,670 24,970

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Two National Chromatography Awards, the Stephen Dal Nogare Award and the Keene P. Dimick Award, are presented at the PITTCON meetings; lists of these Awardees are presented in Chapters 2G and 2H. Further information on PITTCON may be found in their 50th year history: J. Wright (Ed.), Vision, Venture, and Volunteers, The Pittsburgh Conference, Pittsburgh, PA and Chemical Heritage Foundation, Philadelphia, PA, 1999.

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Chromatography — The Bridge to Environmental, Space and Biological Sciences Charles W. Gehrke Department of Biochemistry and the Experiment Station Chemical Laboratories, College of Agriculture, University of Missouri, Columbia, Missouri 65212, USA

CONTENTS A. B.

C.

D.

Charles W. Gehrke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Early years of automated chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography in environmental analysis over the last 30 years . . . . . . . . . . . . . B.1. Charles W. Gehrke and Lyle D. Johnson — Analytical Biochemistry Laboratories (ABC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.2. The Belgian dioxin crisis . . . . . . . . . . . . . . . . . . . . . . . . . . Amino acid analysis — gas–liquid and ion-exchange chromatography — 30 years . . . . . C.1. Teaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C.2. Summation on early GC research on fatty acids and amino acids . . . . . . . . . C.3. In conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography in space sciences — GLC and IEC of Apollo moon samples . . . . . . . D.1. Experimental — methods and results . . . . . . . . . . . . . . . . . . . . . D.2. The controversy of contamination — The National Academy of Sciences experiment References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

69 72 74 74 76 76 77 78 82 83 83 94 96

CHARLES W. GEHRKE Charles William Gehrke was born July 18, 1917 in New York City. He studied at The Ohio State University receiving a B.A. degree in 1939, a B.Sc. degree in education in 1941 and a M.S. degree in bacteriology in 1941. From 1941 to 1945 he was Professor and Chairman of the Department of Chemistry at Missouri Valley College, Marshall, Missouri. In 1946, he returned to Ohio State University as an instructor in agricultural biochemistry and received his Ph.D. degree in 1947. In 1949 he joined the College of Agriculture of the University of Missouri in Columbia, Missouri, retiring in 1987.

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He is now Professor Emeritus of Biochemistry and Manager of the Experiment Station Chemical Laboratories (ESCL). His duties also included those of State Chemist for the Missouri Fertilizer and Limestone Control Laws. From 1987 to 1997, after retirement from the university, he was Scientific Coordinator at the Cancer Research Center, Columbia, Missouri. Gehrke is the author of over 260 scientific publications in analytical- and biochemistry. His research interests included the automation of analytical methods for nitrogen, phosphorus, Charles W. Gehrke potassium and sampling in fertilizer and for other biologically important molecules, e.g., spectrophotometric methods for lysine, methionine and cystine; the development of automated quantitative gas and liquid chromatographic methods for fatty acids, amino acids, purines, pyrimidines, biogenic amines, nucleosides and biological markers in cancer detection, and the characterization and interaction of proteins. Gehrke has been an invited scientist on GLC analysis of amino acids at many universities and institutes in the United States, Europe, Russia, China and Japan. As an invited teacher under the sponsorship of five Central American governments, he taught chromatographic analysis of amino acids at the Central American Research Institute for Industry in Guatemala, 1975. He participated in the analysis of all of the lunar samples brought back by the Apollo 11–17 (1969 to 1974) missions for amino acids and extractable organic compounds with C. Ponnamperuma (University of Maryland) and a consortium of scientists with the National Aeronautics and Space Administration. In 1974, he was invited by the Soviet Academy of Sciences to make the summary presentation on organic substances in lunar fines at the 50th International Seminar, The Origin of Life, Moscow State University, Moscow. In 1987, C.W. Gehrke, R.W. Zumwalt, and K. Kuo were the authors=editors of a three volume, 519 page treatise, ‘Amino Acid Analysis by Gas Chromatography’, published by CRC Press, Boca Raton, Florida. These volumes address sample preparation, derivatization of amino acids as N-TFA-n-butyl esters, trimethylsilyl, heptafluorobutyryl, and N-TFA-n-propyl esters. Applications are presented by 29 chromatographers on all aspects of amino acid analysis. In 1990, Gehrke and K. Kuo were the authors=editors of a three-volume treatise entitled, Chromatography and Modification of Nucleosides, published by Elsevier in the Journal of Chromatography Library Series. These volumes address ‘Analytical Methods for Major and Modified Nucleosides’, ‘Biochemical Roles and Function of Modification’, ‘Modified Nucleosides in Cancer and Normal Metabolism,’ and ‘A Comprehensive Database of Structural Information on tRNAs and Nucleosides by HPLC, GC, MS, NMR, UV, and FT–IR combined techniques’ (1206 pages total). He initiated and is the continuing Chair of the Advisory Council for the University of Missouri Diabetes Center and Cosmopolitan International (1976 to present). A complete biosketch on Gehrke is given in the Editors’ section. Information on his research activities, awards, and honors, major research contributions, author=editor of (8) books, teaching and involvement in the corporate sector is presented there. Cyril Ponnamperuma, Director of the Laboratory of Chemical Evolution, Uni-

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A.I. Oparin and C. Ponnamperuma (right) at the International Conference on Origin of Life in Cortina d’Ampezzo, Italy (1966).

versity of Maryland, College Park, MD ((UMCP), and NASA Principal Investigator, and General Chairman (USA) of the Second Lunar Analysis Laboratory Workshop (LAL-II), died suddenly on Tuesday, December 20, 1994, after suffering a cardiac arrest while working at his office at the University of Maryland, College Park, Maryland. Ponnamperuma was a central figure in the organization of the LAL-II Workshop, from which these Proceedings emerged, as well as the earlier First Lunar-Based Chemical Analysis Laboratory (LBCAL-I) Workshop, which he hosted at the University of Maryland, College Park, in 1989 [1a]. Author Arthur C. Clarke said that Ponnamperuma was the “world’s leading authority on the origins of life, and extended his deepest sympathy to Cyril’s family, and to let them know that thousands of people of many nations — by no means all of them scientists — will miss his warm and compassionate personality.” C. Ponnamperuma wrote over 400 publications on chemical evolution and the origin of life. In October 1994, he was named by Pope John Paul II to the Pontifical Academy of Sciences, a prestigious body of international scientists. One of the University of Maryland’s most renowned scientists, C. Ponnamperuma joined the faculty in 1971, coming from the position of Chief, Chemical Evolution Branch, Exobiology Division of NASA–Ames Research Center at Moffatt Field, California. Upon his arrival at University of Maryland, College Park, MD, he founded

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the Laboratory of Chemical Evolution and served as its Director until his death. He also had recently been named to head the UMCP new North–South Center for Sustainable Development, to study and support the development of third-world countries. In 1984, C. Ponnamperuma was appointed Science and Technology Advisor to the President of Sri Lanka, and served as chairman of that country’s National Science Policy Planning Commission from 1985 to 1987. In 1990 he was awarded the ‘Vidya Jothi’ (Luminary of Science) Medal for his services to science and to Sri Lanka. C. Ponnamperuma’s contributions to science have been recognized by other nations in recent years. In 1991, the government of France conferred on him the title of ‘Chevalier des Arts et Lettres’ for promoting international understanding. In 1991, the University of Maryland celebrated his international accomplishments by awarding him its first Distinguished International Service Award. In 1993 the Russian Academy of Creative Arts awarded him the first Harold Urey Prize in recognition of his outstanding contributions to the study of the origins of life. In 1993, C. Ponnamperuma served as a General Chairman (USA) of the International Conference on Space Exploration and the Future of Humans in Space in Dijon, France, which was immediately followed by the Second Lunar Analytical Laboratory Workshop (LAL-II). The ‘Dijon Declaration’ on the exploration of space emerged from those meetings [1b]. His life, his science, his accomplishments will serve as a legacy for many young scientists. A.I. Oparin, shown with C. Ponnamperuma in Italy, is considered the ‘father of chemical evolution’. A.I. Oparin studied plant physiology at Moscow State University where he later served as professor. He helped found, with the botanist A.N. Bakh, the Bakh Institute of Biochemistry, which the government established in 1935. Oparin became director of the institute in 1946. As early as 1922, Oparin was speculating on how life first originated and made the then controversial suggestion that the first organisms must have been heterotrophic — that is, they could not make their own food from inorganic starting materials, but relied on organic substances. This questioned the prevailing view that life originated with autotrophic organisms, which, like present-day plants, could synthesize their nutrients from simple inorganic materials. A.I. Oparin’s view has gradually gained acceptance in many circles. Oparin did much to stimulate research on the origin of life and organized the first international meeting to discuss the problem, held in Moscow in 1957.

A. EARLY YEARS OF AUTOMATED CHEMISTRY Analytical chemistry and biochemistry are changing disciplines; the 20th century marks a period where revolutions have occurred in analytical chemistry that will have a dramatic impact through the coming years of the 21st century. These changes have been brought about to a large extent by new analytical methods in chromatography. We are now in a decade of chromatographies and hyphenated techniques, interfaced with high- and low-resolution mass spectrometry and computers for fast data reduction. Some of our most important environmental problems have been solved with this array of instrumentation combined with sensitive and selective analytical chromatographic methods and detectors.

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The world of analytical chemistry has changed immeasurably since the 1950s. The analytical laboratory of today is far different from that in which most of us were trained; the diverse types of chromatographic methods available to us today have greatly altered our approaches to the problems of analysis and constitute important successes in every discipline of the analytical and biological sciences. See Chapter 5B a, c, d, e, h, k, r, s As Manager of the Experiment Station Chemical Laboratories (ESCL) (from 1950 to 1987), starting in 1950, my mission was to help professors and graduate students in the college on the use of chemistry in their research programs. I had just completed 7 years of teaching all aspects of chemistry at Missouri Valley College, Marshall, Missouri, and thus looked forward to my new position as Associate Professor and the challenges of research support in chemistry to agriculture. Deans John Longwell and Sam Shirkey of the College of Agriculture, University of Missouri, Columbia, Missouri stated in 1950 that my responsibilities as State Chemist were to streamline and update the analysis programs for N, P, K, Ca, Mg, and sampling in fertilizers and limestones; of course, this program brought substantial funding of $500,000 to 1 million dollars per year to the College. I also had the responsibility of teaching analytical chemistry and biochemistry to the graduate students in the College of Agriculture. In a few years with a broad knowledge in analytical chemistry and the help of 12 graduate students and 20 staff members, the analyses for these elements were quickly changed from manual to automated methods using the equipment of the Technicon Corporation. As a result, 8 automated methods, called ‘Missouri Methods’ were adopted by AOAC International

Fig. 4.1. Photo of Dr. Charles W. Gehrke (left) 1984 Centennial President of the International Association of Official Analytical Chemists (AOAC), Dr. Linus Pauling (keynote speaker), and Walter Bontoyan, right, past president, Washington, D.C.

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(formerly Association of Analytical Chemists) as ‘Official Methods’. We could now quantitatively analyze 30 to 60 samples per hour, whereas formerly this same work required days. Fig. 4.1 shows a photo of Gehrke as centennial President of the AOAC in 1984 with keynote speaker, Nobel Laureate Linus Pauling, and past president, Walter Bontoyan.

B. CHROMATOGRAPHY IN ENVIRONMENTAL ANALYSIS OVER THE LAST 30 YEARS B.1. Charles W. Gehrke and Lyle D. Johnson — Analytical Biochemistry Laboratories (ABC) In 1968, I founded the Analytical Biochemistry Laboratories (ABC), a for profit corporation, with two of my graduate students — Jim Ussary of my staff and David Stalling of the National Fishery Laboratory in Columbia, Missouri. The University was only mildly receptive. Technicon Corporation of Tarrytown, NY wanted me to set up an automated laboratory for testing of agricultural feeds, fertilizers and amino acids. This was a natural, as my job in the Experiment Station Chemical Laboratories (ESCL) was the same for the College of Agriculture and its staff. The Graduate School offered help and a site in the University Research Park near the Reactor facility. I said I would think about it. After due consideration I declined, as I could not accept the work and avoid a conflict of interest. We then raised $200,000 of private money, bought 70 acres of land on the outskirts of Columbia and started the ABC Co. In 2000, we occupy three additional large laboratories in the University Reactor for pharmaceutical studies and the distribution of short-lived radioactive isotopes to pharmaceutical companies. Jim Ussary was our first CEO of ABC from 1968 to 1980. The three founders are still members of the Board of Directors in 2001; however, the work of our company is now 80% directed to pharmaceutical analysis and bio-analytical chemistry. Over 200 scientists are now employed in Columbia, Missouri. In the early years, our work was centered on the analysis of pesticides and aquatic toxicology services. Lyle Johnson, one of my first graduate students, directs the analytical environmental and field studies programs of ABC with 35 employees. Introduction Over the past three decades, developments in analytical chemistry have increased our understanding of life and environmental effects on the basis of molecular chemistry. We have been able to detect and quantitate trace amounts of compounds affecting the very basics of life processes. The existence and persistence of some industrial and agricultural chemicals since 1960 had caused concern in the reproduction and longevity of higher food chain animals. The discovery and innovation of progressive analytical procedures allowed scientists and consequently government regulators to develop chemistries and to implement enforcement guidance, harmonious with nature and beneficial to mankind and wildlife environment.

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Detector and column developments Two significant developments in analytical separation techniques had a large impact on the environmental regulatory process. J.E. Lovelock, in 1958, observed a physical characterization of noble gases ionized by radioactive beta emitters [1c]. This characteristic was quenched by electrophilic compounds passing through the gaseous plasma, which led to the development of the argon-ionization detector, followed by the electroncapture detector. The latter is highly sensitive to halogen-containing compounds and many other organic species (see also Lovelock in Chapter 5). Although chromatographic separations of chlorophyll pigments were reported by Tswett decades earlier, gas–solid chromatography was reported using a common detergent as the solid phase, with an inert gas passed through as the mobile phase, was used for the separation of pesticides (Chapter 1-C). Early detectors of gas–solid chromatography used biological detectors and other awkward devices; the science was desperately in need of an electronic means of monitoring the elution of the analyte. The development of the Lovelock electron capture detector [1c] was such a significant event, which through its capacity to detect pesticides in just about everything, in all kinds of matrices, set the scene for Rachel Carson in 1962 and the environmental movement. It detected picomolar quantities of halogenated compounds, such as for industrial control releases in the 40s, and a couple of decades following. These chlorinated bullets were ‘knocking holes’ in the reproductive function of higher food chain birds, most notably our national avian hero, the American Bald Eagle. The process of eggshell thinning, and hatchling mortality caused a significant decline in the population of the eagle. The GC analyses showed that the use of polychlorinated biphenyls, an excellent electrical insulator in transformers, though resistant to biodegradation, found its way into the environmental food chain. Other agro-chemicals, such as DDT, chlordane, heptachlor, aldrin, dieldrin, endrin, BHC, lindane, methoxychlor and toxaphene, were suspected of also contributing to adverse environmental effects, that was complicated by their long biological half life. The Lovelock detector made it practical to detect these chlorinated species selectively over many other organic molecules. This capability led to screening and monitoring programs sponsored by governmental agencies, academic research groups, and industrial organizations for wildlife, soil, water, air and foodstuff, giving a total bio-system perspective of chemical exposure. These early years of cause and effect led government regulators to understand the long-term effects of persistent chemicals to body functions from chronic exposure. Significance During the course of the last 30 years, there have been many monitoring programs for food by USDA and FDA to determine the safety of food items. Super Fund Cleanup Sites have required a tremendous amount of effort and accountability. More recently, in August 1996, Congress entered into legislation the ‘Food Quality and Protection Act’, which required a more holistic approach to agricultural chemical exposure. It required consideration for chemicals with ‘common mechanistic’ modes, a 10-fold safety factor for children, screening of compounds for possible endocrine disruption, and a total perspective evaluation for all exposures, whether the source be food, home, lawn, work-

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place, water, and=or air. In order to measure the actual levels of exposure, instead of perceived or estimated exposure, refinements in chromatographic techniques for quantitation and confirmation of compounds were developed and effectively implemented. Conclusions A powerful separation tool, high-performance liquid chromatography has been in need of a selective and sensitive detection system for environmental analysis. Recently major improvements have been made in the transportation of analytes from the HPLC column effluent to the mass spectrometer analyzer. Future developments in protein sequencing through the use of LC–MS=MS systems will greatly enhance our understanding of metabolic systems and forecast their malfunctions as a diagnostic tool. Similarly, understanding and identifying gene traits via analytical protein mapping have been, and will continue to be, important techniques for developing and producing transgenic plants capable of resisting disease and insects. Herbicidal resistance allowing selective plant control, and engineering of plants for selected food or energy traits are common goals of many agricultural industries. Continued development of advanced analytical technology will allow our 21st century civilization to enjoy higher standards of living, with curtailed impact on our environment.

B.2. The Belgian dioxin crisis Thirty-eight years after the start of the environmental movement, 1999, in Belgium, will be remembered as the year of the ‘dioxin crisis’. This is a very important example of an environmental problem and its solution involving contamination of chicken feed with PCB’s, PCDD’s and PCDF’s. At the Research Institute for Chromatography (RIC), Pat Sandra relates how he used matrix solid phase dispersion clean up and CGC–µECD and CGC–MS for fast screening of samples and the solution of the dioxin crisis in Belgium. The complete story of this 1999 crisis is presented in Chapter 5 by P. Sandra.

C. AMINO ACID ANALYSIS — GAS–LIQUID AND ION-EXCHANGE CHROMATOGRAPHY — 30 YEARS In 1959, William Albrecht, Chair of the Soils Department at the University of Missouri, called me to his office and said, “Gehrke, there is a great need and there must be a better way to measure amino acids than by bacterial turbidometric assay.” I responded, “Yes, by gas–liquid chromatography”, although at this time my experience was limited. A few laboratories were just starting some studies for GC of amino acids. In 1959, we purchased the first gas chromatograph at the university, a Perkin Elmer 154C. This was the challenge and the start of my work on new methods by GC for amino acids. Our accomplishments have been most rewarding. Our goals in the Experiment Station Chemical Laboratories were the development of automated analytical, and chromatographic methods as ‘research tools’ — useful for advancing investigations in biochemistry, agriculture, space sciences, and medicine.

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Our studies in this field started mid-1959 with the gas chromatographic analysis of amino acids in proteins. At that time amino acids were analyzed by the tedious bacteriological, paper chromatographic, and manual ion-exchange methods. A method was needed to determine, rapidly and accurately, the amino acids in agricultural and other biological samples. Our investigations have resolved a number of questions, whose results can be summarized as follows: (a) direct esterification of amino acids; (b) development of special chromatographic columns, esterification and acylation organic reaction conditions; (c) investigation on the hydrolysis of proteins as a function of time and temperature; (d) the successful routine analysis of amino acids in biological substances (blood plasma, corn and soybean grain hydrolysates, urine, etc.); and (e) the analysis of nanogram amounts of amino acids using a ‘solvent venting’ system. From our publications, I would like to quote a few examples from the early period dealing with fundamentals [2–6] and then later papers discussing newer developments in the GLC analysis of protein amino acids [7,8]. The use of gas–liquid chromatography (GLC) has become an important method for the analysis of amino acids. The classical ion-exchange method of Nobel laureates Stein and Moore (see Chapter S-9A) developed in the late 1950s is now complemented and supplemented by these excellent GLC methods. Several types of derivatives have been used, but the most reliable and common are the N-trifluoroacetyl (N-TFA) n-butyl esters that we developed in the 1960s [16,17,18]. We also invented a special device, a (‘Sol-Vent’) device to the injection port, which allows injection of large amounts of solvents and samples [9]. In the 1970s, we were extracting 100 mg of lunar samples, and with a final derivatization volume of 100 µl, using this Sol-Vent device, we were able to inject 75 µl of the derivatized sample (75 mg) [10]. This results in greater sensitivity, accuracy, and precision, especially for very small samples. These GLC methods opened doors to researchers because of their rapidity, sensitivity, simplicity, accuracy, and economics and have been adopted widely throughout the world. In our research, attention was also directed to sample preparation methods, as these are as vital in amino acid analysis as the methods of measurement. We conducted experiments to obtain rapid, accurate, and precise procedures for protein hydrolysis and sample cleanup with subsequent gas–liquid chromatographic analysis. The use of ultrasonication and reduced pressure to remove dissolved air from the sample solution prior to hydrolysis assured a good recovery for methionine and cystine. These techniques combined with a 4-h hydrolysis at 145ºC using a 6 N HCl gave results in good agreement with the common hydrolysis conditions of 18 to 24 h at 110ºC. We prepared physiological fluids for free amino acid analysis by precipitating the protein with saturated picric acid followed by cation exchange clean up. These techniques for sample preparation and chromatographic analysis of amino acids provided chemists with valuable tools for the analysis of biological samples by gas–liquid chromatography. C.1. Teaching In 1968, I proposed a series of mini-courses to the Graduate Faculty. Three of these were very successful: (1) Chromatography, (2) Automation in Analytical Chemistry, and

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Fig. 4.2. University of Missouri Chromatography Team. Dr. Robert W. Zumwalt, Mr. Kenneth C. Kuo, Dr. Charles W. Gehrke and Dr. David L. Stalling in Woodland Floral Gardens adjacent to the Experiment Station Chemical Laboratories (left to right).

(3) Mass Spectrometry. These were 2 semester hour courses directed to the needs of graduate students in agriculture, chemistry, and biochemistry to advance their research. The 2-hour lectures were in the evening and the 4-hour laboratory during the day. About 20 students enrolled each semester and applied these tools later in their research problems. The advanced instrumentation in the ESCL was used and staff assisted in the courses. Over 5000 requests for reprints for our papers on the GLC analysis of amino acids were received. Norway set up a central laboratory at its Agricultural Research Station in Aas for this determination. In the early 1970s and 1980s, at least 50 scientists each year from laboratories in England, Europe, Sweden, South Africa, Japan, Central and South America, and others visited our laboratories at the University of Missouri and ABC, to learn directly of these methods.

C.2. Summation on early GC research on fatty acids and amino acids Some of our earliest work in the late 1950s was on gas chromatography of the volatile fatty acids in rumen fluids and, in 1977, we published a definitive summary report on a ‘Rapid microdetermination of fatty acids in biological materials by gas–liquid chromatography’ [11] using an internal standard method and ‘on-column’ methylation of the acids with trimethyl (α,α,α-trifluoro-m-tolyl) ammonium hydroxide (TMTFTH) (see references for later articles). The earlier research was done by my graduate students Lamkin and Goerlitz [12,13]. During the period of the 1950s to the 1980s, I received substantial grants from

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the NIH, NSF, NASA, and DOE, the University and corporate sector to develop and advance analytical and chromatographic methods as ‘research tools’. Automation of these methods was also of great importance. Thus, a concerted effort was placed on the development of quantitative GLC methods for amino acids, genetic bases, nucleosides, and nucleotides, sugars, biogenic amines and polyamines, etc., from the macro to nanogram levels, for non-protein amino acids (about 80 different molecules), and as an investigator with NASA in the search for amino acids in the Apollo 11 to 17 lunar sample fines (1965 to 1974). Applications of these methods led to solving important biological problems. In 1982, I put together a group of faculty research scientists and proposals to the National Institutes of Health (NIH) and the National Science Foundation (NSF) for $250,000 each for MS and NMR facilities, which would be matched by the Graduate School, thus totaling $1,000,000. Paul Agris, Professor in Biological Sciences and Richard Loeppky, Professor of Chemistry, helped line up the supporting proposals. We were successful and approved, but when we went to the Graduate Dean, he laughed, and said, “I never once thought you could do it”, he came across with $500,000. Thus, our facilities were top rate for research with our courses in chromatography. After 1975, we directed our efforts toward the development of quantitative high performance liquid chromatography (HPLC) methods for major and modified nucleosides in biological materials (plasma, tissue, urine), and hydrolysates of RNA and DNA with the measurement of more than 67 major and modified nucleosides, useful as indicators of cancer or ‘tumor markers’. Also, simple, sensitive, quantitative, high-performance liquid chromatographic methods were developed at the nanogram level for measuring neurotransmitters, such as histamine, norepinephrine, octopamine, normetanephrine, dopamine, serotonin, and tyramine in plasma, tissue and other biological fluids. I will now discuss in more detail three areas of activities involving our development and use of chromatographic techniques in: ž GC research on amino acids (see section C, also see Chapter S-12); ž the search for life molecules in lunar soil (see section D); ž chromatography of modified nucleosides as biologic markers in cancer and structural characterization (see section E in Chapter S-12, on the Chem Web Preprint Server (http:==www.chemweb.com=preprint=)). The central role of proteins (French prote´ine, ‘primary substance [of the body]’, from Greek, pr¯otos, first) and their building blocks, the amino acids, in biology has evoked intense and continued interest in protein and amino acid chemistry by scientists representing a wide spectrum of disciplines. The array of substances subjected to examination for their amino acid content is therefore extraordinarily broad, ranging from exotic meteorites and lunar samples, to newly synthesized or isolated peptides and proteins, or the exudate of a leaf. Over the past 5 decades, chromatographic techniques have emerged as the dominant means of amino acid determination. Some milestones of that development are apparent: Nobel laureates A.J.P. Martin and R.L.M. Synge (1941), working in the laboratories of the Wool Industries Research Association in Leeds, England performed partition chromatography of the N-acyl amino acids. They addressed the problem of analyzing the amino acids in protein hydrolysates and laid the theoretical foundation on which partition chromatography is based. Nobel laureates

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S. Moore and W. Stein, along with D.H. Spackman at the Rockefeller University, New York, pioneered the elegant automated ion-exchange amino acid analysis (1958) which has had a profound impact on amino acid and protein research. In 1952 the Nobel laureate A.J.P. Martin together with A.T. James described the fundamental parameters of gas–liquid chromatography (GLC), including a theory of its operation in terms of the theoretical plate concept which he initially elaborated in 1941, and laid the foundation for further development of the technique (see also Chapter S-9A and Chapter 1-C for a fuller description and references). The earliest gas chromatographic (GC) method for analysis of amino acids was described by Hunter et al. [14] in 1956, and involved separation of the aldehydes which resulted after decarboxylation and deamination with ninhydrin. In 1958 [15], Bayer reported the GC separation of N-trifluoroacetyl (N-TFA) methyl esters, and N-acylated amino acid esters have subsequently emerged as by far the most widely used class of derivatives. In the early 1960s, Gehrke and his doctoral students William Lamkin, David Stalling, Dan Roach and Frank Shahrokhi at the University of Missouri–Columbia, laid the foundations that resulted in the synthesis of the 20 reference standard compounds, and established the organic reaction and chromatographic separation conditions for the first quantitative amino acids analysis by GLC of the 20 N-TFA n-butyl esters [16,17]. In 1963, our research on GLC methods for amino acids led to intensive research in more than 100 laboratories across the world directed to studies on the merit of different derivatives, chromatographic columns, detectors, and applications to research in medicine, agriculture and the environment. Some of these studies are briefly described below. Numerous reports of GLC techniques for amino acid determination began to emerge in the late 1960s, mainly spurred by the result of improved resolution and speed of analysis as compared to the ion-exchange techniques of the day. Advances in GC detectors, column, materials, and quantitative derivatization methods during the 1960s and 1970s encouraged further research; and continued improvements in all phases of GC instrumentation and column technology into the 1980s have enhanced the capabilities of GC for amino acid analysis. Development of the GC methodology was followed by interfaced GC=mass spectrometric (MS) analysis and characterization of unknowns and analysis of amino acids enantiomers, and then by the more recent reversed-phase liquid chromatography approaches. This story on accomplishments continues with the excellent contributions by the 28 pioneering scientists in the 20 chapters of a three-volume treatise by C.W. Gehrke, K. Kuo and R.W. Zumwalt (1987) [18]. Analytical and chromatographic strategies for separating, identifying, and quantitating amino acids in the array of matrices have been varied, dictated by both the methodology available and the demands presented by the specific analytical problem whether in a research setting or for compositional information. Ion-exchange, gas– liquid, and reversed-phase chromatographic techniques continue to evolve to meet the ever-increasing demands for improved resolution, sensitivity, speed, and versatility [18]. The methodology of choice depends on the analytical requirements and problems at hand. For a protein chemist involved in structural analysis of a particular protein, the analytical demands are not the same as for a nutritional chemist involved in determining the nutritional quality of foods and feeds, the clinical chemist engaged in determining

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Fig. 4.5. GLC analysis of corn hydrolysate. Cation-exchange cleaned N-TFA n-butyl esters.

amino acids in physiological fluids to aid in diagnosis of disease, or to the biogeochemist interested in the extent of racemization of amino acids in fossils. Earlier and now, the complexity and diversity of the sample matrices that are encountered require a methodology providing high resolution, selectivity, and a wide linear response range of 106 . GC=FID (flame ionization detection) is the method of choice and in these situations GC will provide more reliable data. The inherent strengths of GC methods (resolution, sensitivity, versatility, cost) to a wide range of amino acid analytical problems and applications are presented in these three volumes [18]. Some highlights of this wide ranging research follows. In biomedical research, the problem is a general one, the need for new techniques, and their application to solve old problems, and to probe new ideas of approach to solve intractable new problems. Whatever the disease or biochemical research objective, a research tool is required that will provide a reliable measurement of the molecules under study. GLC of amino acids in all of its ramifications provides the research scientist with powerful new tools and approaches to help in obtaining the needed answers to advance science. In 1965, after we developed the quantitative derivatization and separation in a two column system of the 20 protein amino acids as the N-TFA-n-butyl esters by GC, the Associate Dean of the College called a press conference to announce this important scientific advance at the Experiment Station. We were the first to develop this technology; 100 laboratories across the world were working on this subject. In my position as manager of the ESCL of the college, I reported directly to the Dean. However, as others have probably experienced, the department chair, asserted that these

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Fig. 4.7. GLC analysis of bovine blood plasma. Deproteinized with picric acid, cation-exchange cleaned.

developments were poor science, unworthy of a press release, an unfortunate example of administrative shortsightedness and jealousy. Peer review and time tells another story that a paradigm shift in advancement of amino acid gas chromatography had occurred. In Chicago, of that same year, at the National ACS Meeting, future Nobel laureate Stanford Moore invited me to his suite in the Hilton Hotel for breakfast for a private discussion on gas chromatography (GC) of amino acids. At the end of breakfast, he told me that he was going to buy a GC for amino acid analysis as a complement to ion-exchange.

C.3. In conclusion The chromatographies and separation sciences, are a major ‘bridge’ or ‘common denominator’ for analytical methods in the biological sciences research. The importance of research and new methods of measurement to the advancement of our society and the developing world depends upon expanding and new knowledge from every source for continued growth. Problems in nutrition, pollution, drugs, environment, and biotechnology are now being solved by chromatography and interfaced MS in weeks and months; formerly years of study were involved. The genius of Mikhail Tswett, the father of chromatography, in the early 1900s has had a profound impact to this point in history.

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To illustrate the significance of Tswett’s work, 56 world-leading chromatographers paid tribute to his accomplishments by contributing chapters to 75 Years of Chromatography — A Historical Dialogue, which was published in 1979 to commemorate the 75th anniversary of Tswett’s invention of chromatography. That volume, edited by L.S. Ettre and A. Zlatkis [19], provides a unique historical perspective as it relates developments and applications of chromatography by scientists from disciplines that range from petroleum chemistry to medicine. Their accomplishments promise to open even wider doors as chemistry and biology are brought more closely together to more effectively serve mankind.

D. CHROMATOGRAPHY IN SPACE SCIENCES — GLC AND IEC OF APOLLO MOON SAMPLES The lunar samples from Apollo flights 11 through 17 provided the students of chemical evolution with an opportunity of examining extraterrestrial materials for evidence of early prebiological chemistry in the solar system [10]. Our search was directed to water-extractable compounds with emphasis on amino acids. Gas chromatography, ionexchange chromatography and gas chromatography combined with mass spectrometry were used for the analysis. The characterization of carbon compounds indigenous to the lunar surface is of particular interest as these investigations could result in findings which would advance our knowledge of the processes of chemical evolution. The Apollo missions have provided us with the requisite extraterrestrial material for study. The paper [10] presents both our search for water-extractable organic compounds, with emphasis on amino acids, in Apollo 17 fines, and a summary of the analysis for amino acids in samples from Apollo flights 11 through 17. The chromatography and procedural techniques that are described on the following pages, and developed and used in investigations on the lunar regolith samples, are directly applicable to the search for life molecules (amino acids and genetic code) in Mars meteorites, and the Mars samples planned for return to Earth in 2007.

D.1. Experimental — methods and results Apparatus and reagents A gas chromatograph (GLC), with ethylene glycol adipate column and Sol-Vent system (described by Zumwalt et al. [52]) was used for the analyses of extracts of the lunar fines. The derivatization reagents, n-butanol Ð 3 N HCl, dichloromethane, and trifluoroacetic anhydride (TFAA) were of the quality described by Gehrke et al. [53]. Derivatizations of the water extracts of the lunar material were conducted in Pyrex micro-reaction vials with all-Teflon screw caps. A classical ion-exchange (CIE) analysis, by a Durrum D-500 computer-controlled amino acid analyzer, was used for amino acids; the CIE analyses do not require the prior derivatization steps involved in GLC analyses. A detailed description of the apparatus and reagents is given by Gehrke et al. [10,54].

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Fig. 4.10. University of Missouri Research Team at NASA Ames Research Center, Moffatt Field, CA, July 1969. Dr. Robert W. Zumwalt, Dr. Charles W. Gehrke, holding sample of Apollo 11, and Dr. David L. Stalling (left to right).

A summary follows of our experiences and techniques used in the analysis of samples from Apollo missions 11 to 17. The studies were conducted at the Ames Research Center, Moffett Field, CA, the University of Missouri–Columbia, Missouri, and the University of Maryland, College Park, MD, 1969 to 1974. Our search was directed to water-extractable compounds with emphasis on amino acids, using GC, IEC, and GC–MS for the analysis. It is our conclusion that amino acids are not present in the lunar regolith above the background levels of our investigation (ca. 1 to 3 ng=g). Preparation of extracts and hydrolysates Apollo 17 samples 72501.62 (4 g) and 70011.37 SECS (4 g) were used in these studies. Sample 72501.62 was a soil sample from material sampled to a depth of 5 cm on a steep uphill slope. The unsieved material was less than 1 mm in size. Sample 70011.37 was specially provided to those interested in organic carbon analysis. This sample was presumed and reported to have been exposed to some rocket exhaust. The lunar fines were transported from the moon to the laboratory in especially solvent cleaned Teflon bags that showed a background level response of less that 1 ng=g. All reagents (TFAA, n-butanol, CH2 Cl2 , and double distilled (dd) ultra pure H2 O) were specially purified to give a procedural blank response of <1 ng=g. Micro derivatization techniques were developed; all work was done in a 100 clean room. In general, more time was spent on analyzing total performance samples of 5–20 ng

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of each amino acid, total procedural blanks, and sensitivity studies than in analyzing the lunar fines. The sensitivity of our GC method was at <500 pg of each amino acid. The extracts were prepared by hydrothermal extraction at 165ºC for 1 h as described by Cheng and Ponnamperuma [55]. Aliquots of the extract were analyzed for free amino acids (F), and a corresponding aliquot hydrolyzed with 50 µl of 6 N HCl (H). Heating at 500ºC via furnace for 4 h cleaned all glassware. The lunar sample sizes analyzed ranged from 100 mg to 1 g. The glass reaction vials were provided with Teflon liners; also, all Teflon micro vials were used and the reaction volume was kept at <100 µl. Otherwise the amino acid derivatives would oil out on the walls of the vial if the reaction volume were less than 65 µl. A two-gram portion of lunar fines was placed in two separate Pyrex glass extraction tubes (4 g total); ca. 6 ml of water were added to each and the tubes were sealed with a hydrogen–oxygen torch. The selected tubes were shaken well, then placed in a 165 to 170ºC oven for 1 h. At the end of this period, the tubes were centrifuged for 10 min at 3000 rpm and the supernatants were removed by pipettes connected to all-glass syringes with Teflon tubing. The supernatants from each sample were divided into four equal parts, two parts being analyzed directly by GLC and CIE for water-extractable free amino acids. The other two fractions were hydrolyzed with 50 µl 6 N HCl for 16 h under vacuum at 105ºC prior to analysis by GLC and CIE. A second (water) extract of the sample was prepared as described above after removal of the initial water extracts. The total procedural blanks for the analysis of all reagents and pyrolyzed sand blanks showed no amino acid contamination above the 1 ng=g level by GLC and CIE. The analysis of a clean fingerprint showed amino acids present at 5 to 200 ng=g response for each. Thus, careful handling of samples and use of laboratory procedures was crucial. Glycine was definitely observed in the (F) and (H) extracts of Apollo sample 70011.37 SECS at levels of 19 ng=g (F) and 11 ng=g (H) by CIE. The glycine found is unlikely to be indigenous to the lunar fines as the sample was taken near the lander and shown to be contributed by synthesis of glycine on the moon by the rocket fuel and oxidizer. Special GC chromatographic techniques and columns employing a Sol-Vent venting system (Gehrke patent US 3,881,892) using a cold spot to trap the volatile amino acid derivatives and to vent solvent and reaction products was employed. In this way we were able to inject 75 µl of the derivatization volume into the GC. Our clean up, chromatography, and the above procedural techniques that we used on the lunar regolith samples are directly applicable to the later search for life molecules in Mars meteorites and returned Mars samples. Analyses were also conducted on two Teflon lunar sample collection bags. The Teflon flight bags Nos. 113 and 114 were the backup for Apollo 17. These bags were handled and cleaned like all of the flight equipment with one exception. The Teflon bags and dispenser were transferred to the SNAP line, then opened and the bags removed and repackaged in Teflon bags. Handling was done with organic solvent-cleaned stainless steel forceps. Five ml of doubly distilled water were placed in each bag, the bags were agitated manually, then the water removed for evaporation, derivatization, and GLC analysis. For the last three years, the search for extraterrestrial life is again being pursued intensely in the Mars meteorite ALH-84001 from Antarctica. A final decision has not

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Fig. 4.11. Recovery of amino acids from water.

yet been reached; the scientific evidence is still circumstantial. In the 1970s our team made an equally intense search for amino acids in the lunar regolith. The question: Are there life molecules on the moon? Yes or no? Our answer was no! Amino acids were not present above experimental background at the 1–2 ng=g level in lunar fines. The scientific debate has become heated when primitive life was reported to exist on Mars 3.6 billion years ago by a NASA–Stanford team led by David McKay and Richard Zare (1997). Mars is destined to receive humans early in the 21st century, preceded by many international missions to space station Freedom and robotic missions to the Moon and Mars. First, we must ‘learn to live in space’. The Moon represents a base that provides the opportunities and challenges to assemble the international, interdisciplinary intellectual scientific teams to make the next step before human exploration of Mars and the search for evidence in Martian soil and samples returned to Earth laboratories. Our experiences learned in moon analysis will be useful in Mars exploration and returned sample study. Standards, reagents and blanks Initial experiments were conducted to establish the purity of the water used for extraction, and the integrity of the derivatization reagents. A complete procedural blank was prepared by subjecting a 4 g sample of pyrolyzed Ottawa sand (1000ºC, 24 h) to the manipulative extraction, hydrolysis and analysis regime to be used for the lunar fines. Figs. 4.11–4.14 represent the GLC analysis of the water used for the extraction of the lunar fines and the analysis of the hydrolyzed water extract of the pyrolyzed sand blank.

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Fig. 4.12. GLC of contaminated water.

Fig. 4.13. GLC analysis of unhydrolyzed water blank and hydrolyzed H2 O extract of sand blank. (NASA Apollo 14 experiment at Berkeley.)

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Fig. 4.14. GLC analysis of a single finger print.

The GLC results showed no significant amino acid contamination at the 1 ng g1 level. Analyses of the hydrolyzed procedural blank by CIE indicated no significant amino acid contamination was present at the 0.5 ng g1 level, confirming the integrity of the analytical procedure (Fig. 4.15). The GLC analysis of a standard mixture containing 6 ng of each amino acid is presented in Fig. 4.16. Corresponding to Fig. 4.16 for GLC, standards of 6 amino acids at the 2 ng and the 10 ng level were analyzed by CIE. In Fig. 4.15 we show the responses for the 6 amino acids of major concern, aspartic acid, threonine, serine, glutamic acid, glycine and alanine. GLC of Apollo 17 sample No. 72501.62 Fig. 4.17 presents the chromatograms obtained from the unhydrolyzed and hydrolyzed extracts of Apollo 17 sample No. 72501.62. Three significant peaks were observed in the unhydrolyzed extract with the largest identified tentatively as oxalic acid (ca. 25 ng), eluted at ca. 145ºC. This peak, along with the two similar peaks eluted near threonine and serine, have been observed in the analyses of lunar samples returned by earlier Apollo flights (Zumwalt et al., 1971; Gehrke et al. [53,54]). These peaks are significantly reduced in size after hydrolyzing with 6 N HCl, with the largest also being eluted ca. 1 min earlier than the largest peak in the unhydrolyzed sample. The three large peaks were not observed on the CIE chromatogram and thus were ninhydrin negative. The amino acids of greatest interest were alanine, glycine, threonine, serine, aspartic and glutamic acids. These amino acids, in nearly all cases, except glycine, were not

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Fig. 4.15. Ion-exchange analysis of amino acids — Apollo 17 lunar fines.

observed by GLC to be present above the 1 ng g1 level in Apollo 17 extracts, both free (F) and hydrolyzed (H), although the elution positions of glycine and threonine, in part, were obscured by non-amino acid peaks. However, analyses of the hydrolyzed fraction of the same Apollo 17 water extract showed glycine to be less than 1 ng g1 (Fig. 4.17). Less than 2 ng g1 of threonine were observed, as the threonine elution was partially obscured. In separate analysis the amino acids were not found to be present above the

Fig. 4.16. Derivatization — GLC of chromatography standard, 6 ng of each.

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Fig. 4.17. GLC analysis of water extract of Apollo 17 lunar fines.

1 ng g1 level. The relative sizes of the three peaks eluted in the glycine, threonine, and serine regions of the chromatogram were somewhat different than in Fig. 4.17, but similar reductions in peak size after hydrolysis were observed. GLC of Apollo 17 sample No. 70011.37 SECS The GLC data for the free amino acids and 6 N HCl hydrolyzed aqueous extracts are given in Table 4.1 [see 10]. Glycine was found at a level of 12 ng g1 (F) and 5 ng g1 (H). However, the other amino acids (named above) were not found in either the free or hydrolyzed extracts at concentrations greater than 1 ng g1 . CIE analysis of Apollo 17 sample No. 72501.62 Fig. 4.15 presents the chromatograms obtained by the Durrum D-500 amino acid analyzer of the unhydrolyzed water extracts of the Apollo 17 lunar samples, and the results of these analyses are presented in Table 4.1 [10] along with the GLC data. Glycine was found at 1.5 ng g1 (F) and 4 ng g1 (H). The others were at a level less than 0.5 ng g1 in this sample. CIE analysis of Apollo 17 sample No. 70011.37 SECS Glycine was definitely observed in the (F) and (H) extracts at levels of 19 ng g1 (F) and 11 ng g1 (H). Alanine, threonine, serine, aspartic and glutamic acids were at a level of 1 ng g1 or less. It was interesting to note that glycine was present in a larger amount in the unhydrolyzed aqueous extract; this was also observed for the GLC

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Fig. 4.18. GLC analysis of water extract of lunar sample Teflon collection bags.

analysis. Of importance is the observation that the other amino acids were at one or sub-nanogram g1 level, thus ruling out contamination; otherwise a pattern for these common amino acids would have been observed. The CIE chromatogram tracing (Fig. 4.15) for Apollo sample No. 70011.37 SECS shows glycine present at ca. 10 ng=g. We found glycine in the soil samples taken at the rocket landing. No other amino acids were seen, thus excluding earthly contamination. The glycine was synthesized from the rocket fuel (unsymmetrical dimethyl-hydrazine, NH2 –NH2 ) and oxidant (N2 O4 ) of the moon lander, and placed on the moon below the rocket during landing. Teflon bags The GLC analyses of water washes of the two Teflon sample collection bags are shown in Fig. 4.18. No evidence for amino acids was found in the water wash of Teflon bag No. 113, and only a small peak at the elution position of alanine (ca. 2 ng) was observed in the wash of bag No. 114. These experiments, along with the analyses of the water and sand blank (Fig. 4.13), gave a relative thorough study of the low levels of organic background peaks resulting from the analytical procedure and aided in the interpretation of the lunar sample results. Aminoacetonitrile precursors experiment CIE analysis showed that one microgram of aminoacetonitrile contained 1.2 ng of glycine as impurity. In a procedural blank, glycine was detected at 1.6 ng µg1 of its added precursor (F) and at 4 ng µg1 (H). When 84 µg of amino acetonitrile in 3 ml of H2 O was added to one gram of the Apollo 17 fines (72501.62), then the mixture

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Fig. 4.19. Flow diagram of precolumn venting system (Sol Vent, Gehrke patent, USA 3881892).

Fig. 4.20. Photo in clean room for analysis of Apollo 12 samples, Moffatt Field, CA (1970). Drs. Charles W. Gehrke and Robert W. Zumwalt.

subjected to extraction, evaporation, hydrolysis and CIE analysis, glycine was found at a level of 7 ng µg1 (F) and 13 ng µg1 (H). No significant amount of amino acids other than glycine was detected. A discussion is presented on the fundamental aspects of biological chirality and its necessity for life [64].

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Throughout these investigations of the returned lunar samples, our primary aim has been the search for, and identification of, water-extractable, derivatizable-organic compounds by the GLC method and ninhydrin-active organic compounds by the CIE method with particular emphasis on the amino acids. Analyses by both GLC and CIE on the same sample extracts produced independent yet supporting analytical results. Also, the glycine found is unlikely to be indigenous to the lunar fines, particularly in light of the work by Sagan et al. [56], regarding amino acid destruction under simulated lunar conditions. He reported that the half life of amino acids to a depth of several cm was found to be 4000 yr under simulated lunar conditions of proton flux (108 p cm2 s1 ), vacuum (105 Torr), and temperatures of 120 to 140ºC, thus negating the reported existence of amino acids on the Moon for ¾109 yr. Sagan concludes that similar remarks apply to other organic compounds, which are not particularly stable to heating and proton irradiation. The generation of amino acids from precursors in the solar wind has been suggested to occur during the extraction and=or HCl hydrolysis of an aqueous extract of lunar materials by Sidney W. Fox [57]. Indeed, glycine can be formed from simpler molecules by the Strecker synthesis, for example: Reaction .1/

HCHO C NH3 C HCN ! NH2 CH2 CN C H2 O

Reaction .2/

NH2 CH2 CN C 2H2 O ! NH2 CH2 COOH C NH3

The results of our investigation indicate that the Apollo 17 fines catalyze the generation of glycine from aminoacetonitrile. Nonetheless, the presence of 10 to 20 ng of glycine in the aqueous extract would require that aminoacetonitrile would have to be present at a level of approximately one hundred micrograms in one gram of the

Fig. 4.21. Scene at Laboratory of Chemical Evolution, University of Maryland, College Park, MD in the clean room for analysis of Apollo 14–17 samples (1971). Drs. Chou N. Cheng (left), Charles W. Gehrke (center) and Akira Shimoyama.

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lunar fines. Obviously, if we start with hydrogen cyanide, it would have to be more abundant than aminoacetonitrile in the lunar fines even though reaction (1) is known to proceed easily, and its yield of aminoacetonitrile is more than 90% [58]. Inasmuch as such abundance of aminoacetonitrile or hydrogen cyanide in the lunar fines cannot be rationalized, we attributed the presence of glycine in the Apollo sample 70011.37 to contamination by the rocket exhaust. The differences in amino acid levels, in particular, glycine, that have been found between the Apollo 17 sample known to have been contaminated by rocket exhaust (70011.37) and the samples taken from a slight depth or distance, could implicate hypergolic rocket fuels as the source of the glycine found. Emanuel [59] reported that rocket fuels employing hydrazine or its substituted derivatives give positive ninhydrin reactions, or aminonitriles from fuel combustion. Unsymmetrical dimethyl NH2 NH2 was used as the fuel and N2 O4 as the oxidant. This is convincing evidence that the most likely source for amino acids in returned lunar samples is contamination, either in acquisition or return of samples, or during preparation and analysis in laboratories on earth. The sources, nature, and levels of contaminants have been the subject of several studies [57,60,61,62,63]. It is our conclusion that the presence and survival of amino acids in the environment of the lunar regolith appears to be highly unlikely.

D.2. The controversy of contamination — The National Academy of Sciences experiment In 1969, our team from Missouri reported that the Apollo 11 lunar fines contained no detectable amounts (less that 2 to 3 ng=g) of amino acids [52a]. While Fox et al. [52b], using ion-exchange chromatographic methods reported hydrolysates of aqueous extracts of Apollo 11 fines contained in nanomoles=gram: aspartic acid 0.03, glutamic acid 0.05, glycine 0.28, and alanine 0.14, representing about 60 ng=g of total amino acids in Apollo 11 fines. For Apollo 12, in 1970, we again reported that if amino acids were present in lunar fines, the level is 2 to 3 ng=g or less; Fox and Hare stated that the level was 30 ng=g. This created quite a controversy in the scientific community that had to be resolved. The story of Apollo 13 is well known. The National Academy of Sciences on recommendations of Nobel laureate H. Urey, requested that NASA set up a special experiment on Apollo 14 to settle this inconsistency of results between the GC and ion exchange methods as given by Gehrke and Fox, respectively. An ‘environmental container’ sample was obtained of Apollo 14 soil from the moon; the sample container was not to be opened in Houston but to be taken directly to the University of California, Berkeley Space Sciences Laboratory and opened there. This was only to be done after Gehrke, Kuo and Zumwalt assured NASA that the Berkeley Laboratory was clean. This laboratory was just opened as a new 100 clean room NASA facility. Our team used Ottawa sea sand, pyrolized it in a furnace for 24 h at 1000ºC to destroy organic matter, then placed the open sand in the hoods for 24 h subjecting it to air flow, after which it was extracted with hot water and the extract derivatized for amino acids, then analyzed at the Ames Research Center, near San Francisco. The laboratories were found to be clean (see Fig. 4.13). We were then to open, at Ames, the Apollo 14 container for the

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Fig. 4.22. Mass spectrum (EI) of TMS cytosine in the Murchison meteorite.

sample, place it in a reflux apparatus, make the extract together by the Gehrke and Fox teams as a joint team, then divide the sample extract, and analyze one half by GLC (Gehrke group) and one-half by (IEC) (the Fox group). The results by the Gehrke group were less than 1 ng=g of amino acids in the Apollo 14 sample; however, the Fox group was unable to analyze their half of the extract because of technical difficulties. This answered the controversy on the presence or absence of amino acids in lunar soil. The Gehrke reports were confirmed as less than 2ng=g or no detectable amount. Fig. 4.15 shows the results that we obtained on an Apollo 17 sample by ion-exchange chromatography. Data are presented for a chromatographic procedural blank, a 2 ng and a 10 ng standard and, an Apollo 17 sample picked up under the lander on the moon. Glycine was found at 11 ng=g for the sample taken from under the rocket ship. If there were earthly contamination, six other amino acid would have been seen as shown for the 2 ng and 10 ng standards. In this case, glycine was synthesized from N2 O4 and unsymmetrical dimethyl hydrazine and placed on the moon, and we found it. We are deeply indebted to the following scientists for their valuable advice and contributions to the research on analyses for organic materials in samples of Apollo 11 to 17: Walter Aue, David Stalling, Don Roach, and Jay Rash, formerly of the University of Missouri, for their work on gas–liquid chromatography; Roy Rice for his contributions to the mass spectrometric studies; Cyril Ponnamperuma, Keith

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Kvenvolden, James Lawless and Sherwood Chang, of the NASA Ames Research Center for their combined help throughout the program, also Chao-Nang Cheng, Martha Gay, Pat Buhl and Ramsay Pal of the Laboratory of Chemical Evolution of the University of Maryland for many aspects of their work. The mass spectrum (EI) of TMS cytosine in the Murchison meteorite is show in Fig. 4.22. Note: For Figs. 4.3, 4.4, 4.6, 4.8, 4.9, 4.23–4.37 and Tables 4.1–4.3 see Chapter S-12 on the ChemWeb Preprint server (http:==www.chemweb.com=preprint=).

REFERENCES (See also Chapter S-12 on the chem web preprint server (http:==www.chemweb.com= preprint=) 1a. 1b. 1c. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16.

C. Ponnamperuma and C.W. Gehrke (Eds.), Proceedings of the Ninth College Park Colloquium — A Lunar-Based Chemical Analysis Laboratory, A. Deepak Publishing, Hampton, VA, 1993, 282 pp. C.W. Gehrke, M.K. Hobish, R.W. Zumwalt, M. Prost and J. Degre´s, A Lunar-Based Analytical Laboratory (Cyril Ponnamperuma Memorial Volume), A. Deepak Publishing, Hampton, VA, 329 pp. J.E. Lovelock, A sensitive detector for gas chromatography, J. Chromatogr., 1 (1958) 35–46. W.M. Lamkin and C.W. Gehrke, Quantitative gas chromatography of amino acids: preparation of n-butyl N-trifluoracetyl esters, Anal. Chem., 37 (1965) 383–389. D.L. Stalling and C.W. Gehrke, Quantitative analysis of amino acids by gas chromatography: acylation of arginine, Biochem. Biophys. Res. Commun., 22 (1966) 329–335. D. Roach and C.W. Gehrke, Direct esterification of the protein amino acids: gas–liquid chromatography of the N-TFA n-butyl esters, J. Chromatogr., 44 (1969) 269–278. R.W. Zumwalt, D. Roach and C.W. Gehrke, Gas–liquid chromatography of amino acids in biological substances, J. Chromatogr., 53 (1970) 171–193. D. Roach and C.W. Gehrke, The gas–liquid chromatography of amino acids, J. Chromatogr., 43 (1969) 303–310. C.W. Gehrke, K. Kuo and R.W. Zumwalt, The complete gas liquid chromatographic separation of the twenty protein amino acids, J. Chromatogr., 57 (1971) 209–217. C.W. Gehrke, D.R. Younker, K.O. Gerhardt and K.C. Kuo, Gas–liquid chromatography of histidine, arginine, and cystine. Interaction with liquid and solid support phases, J. Chromatogr. Sci., 17 (1979) 301–307. C.W. Gehrke, K.C. Kuo, R.W. Zumwalt and D.L. Stalling, A Sol-Vent chromatographic system, USA Patent No. 3,881,892 (1975). C.W. Gehrke, R.W. Zumwalt, K.C. Kuo, C. Ponnamperuma and A. Shimoyama, Search for amino acids in Apollo returned lunar soil, Origins of Life, 6 (1975) 541–550. K.O. Gerhardt and C.W. Gehrke, Rapid microdetermination of fatty acids in biological materials by gas–liquid chromatography, J. Chromatogr. Biomed. Appl., 143 (1977) 335–344. C.W. Gehrke and W.M. Lamkin, Determination of steam volatile fatty acids by gas–liquid chromatography, J. Agric. Food Chem., 9 (1961) 85–87. C.W. Gehrke and D.F. Goerlitz, Quantitative preparation of methyl esters of fatty acids for gas chromatography, Anal. Chem., 35 (1963) 76–80. I.R. Hunter, K.P. Dimick and J.W. Corse, Determination of amino acids by ninhydrin oxidation and gas chromatography, Chem. Industry, (1956) 294–295. E. Bayer, Separation of derivatives of amino acids using gas–liquid chromatography, in: D.D. Desty (Ed.), Gas Chromatography 1958, Butterworths, London, 1958, 333 pp. W.M. Lankin and C.W. Gehrke, Quantitative gas chromatography of amino acids: preparation of n-butyl N-trifluoroacetyl esters, Anal. Chem., 37 (1965) 383–389.

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17. C.W. Gehrke, W.M. Lamkin, D.L. Stalling and F. Shahrokhi, Quantitative gas chromatography of amino acids, Biochem. Biophys. Res. Commun., 19 (1965) 328–334. 18. C.W. Gehrke, K.C. Kuo and R.W. Zumwalt, Amino Acid Analysis, Vols. I, II, III, CRC Press, Boca Raton, FL, 1987, 163 pp., 172 pp., 208 pp. 19. L.S. Ettre and A. Zlatkis (Eds.), 75 years of Chromatography — A Historical Dialogue, Elsevier, Amsterdam, 1979, 502 pp. 20. F.E. Kaiser, C.W. Gehrke, R.W. Zumwalt and K.C. Kuo, Amino acid analysis: hydrolysis, ion-exchange cleanup, derivatization and quantitation by gas–liquid chromatography, J. Chromatogr., 94 (1974) 113– 133. 21. C.W. Gehrke, L.L. Wall Sr., J.S. Absheer, F.E. Kaiser and R.W. Zumwalt, Focus: amino acid analysis. Sample preparation for chromatography of amino acids: acid hydrolysis of proteins, J. Assoc. Off. Anal. Chem., 68 (1985) 811–821. 22. C.W. Gehrke and D.L. Stalling, Quantitative analysis of the twenty natural protein amino acids by gas–liquid chromatography, Sep. Sci., 2 (1967) 101–138. 23. F. Shahrokhi and C.W. Gehrke, Gas–liquid chromatography of iodine-containing amino acids, Anal. Biochem., 24 (1968) 281–291. References 24 to 50: see Chapter S-12, Chem Web Preprint server (http:==www.chemweb.com=preprint= 51. C.W. Gehrke, L.L. Wall, J.S. Absheer Sr., F.E. Kaiser and R.W. Zumwalt, Focus: amino acid analysis. Sample preparation for chromatography of amino acids: acid hydrolysis of proteins, J. Assoc. Off. Anal. Chem. 68 (1985) 811–821. 52. C.W. Gehrke, R.W. Zumwalt, D.L. Stalling, D. Roach, W.A. Aue, C. Ponnamperuma and K.A. Kvenvolden, A search for amino acids in Apollo 11 and 12 lunar fines, J. Chromatogr., 59 (1971) 305–319. 52a. C. Ponnamperuma and C.W. Gehrke et al., Search for organic compounds in the lunar dust from the sea of tranquility, Science, 167 (1970) 760–762. 52b. S.W. Fox et al., Bio-organic compounds and glassy microparticles in lunar fines and other materials, Science, 167 (1970) 767–770. 53. C.W. Gehrke, R.W. Zumwalt, W.A. Aue, D.L. Stalling and J.J. Rash, A search for organics in hydrolysates of lunar fines, J. Chromatogr., 54 (1971) 169–183. 54. C.W. Gehrke, R.W. Zumwalt, K.C. Kuo, C. Ponnamperuma, C.-N. Cheng and A. Shimoyama, Extractable organic compounds in Apollo 15 and 16 lunar fines, Geochim. Cosmochim. Acta, 37 (1973) 2249–2259. 55. C.N. Cheng and C. Ponnamperuma, Extraction if amino acids from soils and sediments with superheated water, Geochim. Cosmochim. Acta, 38(12) (1974) 1843–1848. 56. C. Sagan, E. Bilson, F. Raulin and P. Shapshak, Search for indigenous lunar organic matter, Space Life Sci., 3(4) (1972) 484–489. 57. S.W. Fox, K. Harada and P.E. Hare, Accumulated analyses of amino acid precursors in returned lunar samples, Proc. Fourth Lunar Sci. Conf., 2 (1973) 2241–2248. 58. G.A. Menge, New technique for the preparation of amino nitriles, J. Am. Chem. Soc. 56 (1934) 2197–2198. 59. C.F. Emanuel, Ninhydrin, Chem. Eng. News, 50 (July 1, 1972) p. 38. 60. B. Hamilton and B. Nagy, Problems in the search for amino acids in lunar fines, Space Life Sci., 3 (1972) 432–438. 61. J.J. Rash, R.W. Zumwalt, C.W. Gehrke, K.C. Kuo, K.A. Kvenvolden and D.L. Stalling, GLC of amino acids: a survey of contamination, J. Chromatogr. Sci., 10 (1972) 444–450. 62. P.E. Hare, in: G. Eglington and M.T.J. Murphy (Eds.), Organic Geochemistry, Springer, New York, NY, 1969, p. 45. 63. C.W. Gehrke, R.W. Zumwalt, K.C. Kuo, W.A. Aue, D.L. Stalling, K.A. Kvenvolden and C. Ponnamperuma, Amino acid analyses of Apollo 14 samples, Geochim. Cosmochim. Acta, 36 (1972) 2119–2129. 64. C.J. Welch and J.I. Lunine, The Chirons of Titan: A search for extra terrestrial enantioenrichment, Enantiomer, 6 (2–3) (2001) 67.

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CHAPTER 5

Prominent Chromatographers and their Research Seminal Concepts in Chromatography/Separation Sciences Charles W. Gehrke a , Robert L. Wixom b and Ernst Bayer c a Department

of Biochemistry and the Experiment Station Chemical Laboratories, College of Agriculture, University of Missouri, Columbia, MO 65212, USA b Department of Biochemistry, University of Missouri, Columbia, MO 65212, USA c Institut fu ¨ r Organische Chemie, Universita¨t Tu¨bingen Research Center, Auf der Morgenstelle 18, 72076 Tu¨bingen, Germany

CONTENTS A. B. C. D.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Seminal concepts and new discoveries . . . . . . . . . . . . . . . . . . . . . . Relation of seminal concepts and Awardees . . . . . . . . . . . . . . . . . . . . Prominent chromatographers (76 Awardees) . . . . . . . . . . . . . . . . . . . . D.1. Daniel W. Armstrong . . . . . . . . . . . . . . . . . . . . . . . . . . 1.I. Unconventional approaches for understanding and developing separations . . . 1.I.1. In the beginning, pseudophase theory and use . . . . . . . . . . 1.I.2. The enantiomeric separations revolution . . . . . . . . . . . . 1.I.3. Other areas of endeavor . . . . . . . . . . . . . . . . . . . 1.I.4. Looking back . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . 1.II. The advent of broadly applicable, high efficiency enantiomeric separations by A review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.II.5. Ligand exchange chromatography . . . . . . . . . . . . . . . 1.II.6. Macrocyclic stationary phases . . . . . . . . . . . . . . . . . 1.II.7. π-Complex stationary phases . . . . . . . . . . . . . . . . . 1.II.8. Polymeric stationary phases . . . . . . . . . . . . . . . . . . 1.II.9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . LC: . . . . . . . . . . . . . .

107 108 108 109 109 110 110 111 113 114 114 116 117 118 119 120 121 121

100

Chapter 5 D.2. 2.I.

Ernst Bayer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Personal views on the development of chromatography . . . . . . . . . . . . . 2.I.1. GC at the Wine Research Institute-Geilweilerhof . . . . . . . . . . 2.I.2. GC research at Karlsruhe . . . . . . . . . . . . . . . . . . . . . 2.I.3. GC in Tu¨bingen and the wider scientific community . . . . . . . . . 2.I.4. Chromatography research at the University of Houston . . . . . . . . 2.I.5. Chromatography research during retirement . . . . . . . . . . . . . 2.I.6. Environmental chemistry and ecology . . . . . . . . . . . . . . . 2.I.7. Conversion of biomass to oil . . . . . . . . . . . . . . . . . . . 2.I.8. My role in teaching and administration . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.3. Morton Beroza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.I. Insect sex pheromones: some contributions from chromatography . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.4. Gu¨nter Blobel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.5. Phyllis R. Brown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.I. Breaking the gender barrier: a woman in chromatography . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.6. Thomas L. Chester . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.I. Supercritical-fluid chromatography — on the road to unification . . . . . . . . . 6.I.1. Our role in SFC . . . . . . . . . . . . . . . . . . . . . . . . . 6.I.2. A few words on safety . . . . . . . . . . . . . . . . . . . . . . 6.I.3. Looking at the future . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.7. Karel A. Cramers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.I. Reflections on research in gas chromatography in the Eindhoven Laboratory of Instrumental Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.8. John Vernon Dawkins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.I. Characterization of polymers by gel permeation chromatography . . . . . . . . . 8.I.1. Discovery, contribution, event(s), recollections . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.9. Heinz Engelhardt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.I. From TLC to high performance instrumental analytical techniques . . . . . . . . 9.I.1. Preparation and characterization of stationary phases . . . . . . . . . 9.I.2. Post-column derivatization . . . . . . . . . . . . . . . . . . . . 9.I.3. Chromatography and extraction with supercritical fluids . . . . . . . 9.I.4. Capillary electrophoresis . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.10. Leslie S. Ettre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.I. The remembrances of a chromatographer . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.11. Michael Bryan Evans . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.I. Inverse gas chromatography in the study of the oxidative degradation of unsaturated elastomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.I.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 11.I.2. Initial experiments . . . . . . . . . . . . . . . . . . . . . . . . 11.I.3. Study of the oxidation of squalene as a model for the oxidative degradation of natural rubber . . . . . . . . . . . . . . . . . . . . . . 11.I.4. Study of the oxidation of sulphurated squalene as a model for the oxidative degradation of natural rubber vulcanizates . . . . . . . . .

123 123 125 126 128 129 131 132 133 133 134 135 136 141 142 143 144 145 154 154 155 155 159 159 161 162 162 168 168 169 169 170 171 172 173 175 175 175 176 178 180 182 183 185 185 185 186 187

Prominent Chromatographers and their Research 11.I.5.

D.12. D.13. D.14.

D.15. 15.I.

D.16. 16.I.

D.17. 17.I. D.18. 18.I. D.19. 19.I.

D.20. 20.I.

D.21. 21.I.

D.22.

D.23.

Study of the inhibited oxidation of squalene as a model for antioxidant protected natural rubber vulcanizates . . . . . . . . . . . . . . . . 11.I.6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . J. Calvin Giddings (1930–1996) . . . . . . . . . . . . . . . . . . . . . . . Selected references . . . . . . . . . . . . . . . . . . . . . . . Robert L. Grob . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Georges Guiochon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coworkers and research interests . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Andra´s Guttman (invited paper) . . . . . . . . . . . . . . . . . . . . . . . Integrated microfabricated device technologies . . . . . . . . . . . . . . . . . 15.I.1. Future prospectives . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Steven B. Hawthorne . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development of analytical SFE: trying to put science into sample preparation . . . 16.I.1. Why study extraction? . . . . . . . . . . . . . . . . . . . . . . 16.I.2. First attempts . . . . . . . . . . . . . . . . . . . . . . . . . . 16.I.3. Conventional wisdom versus scientific reality . . . . . . . . . . . . 16.I.4. Present and future studies: SFE as a tool to investigate analyte=matrix interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.I.5. ‘Subcritical’ (hot=liquid) water extractions . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Friedrich G. Helfferich . . . . . . . . . . . . . . . . . . . . . . . . . . . Half a century as a kibitz . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Jo¨rgen Hermansson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Direct resolution of enantiomers using immobilized α1 -acid glycoprotein . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Herbert H. Hill, Jr. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ambient pressure ionization and ion mobility separation in chromatography . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Vilhelm Einar Stellan Hjerte´n . . . . . . . . . . . . . . . . . . . . . . . . Scientific contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.I.1. Early studies of separation methods . . . . . . . . . . . . . . . . 20.I.2. Hjerte´n’s present main interests . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Csaba Horva´th . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . My focus on chromatography over 40 years . . . . . . . . . . . . . . . . . . 21.I.1. My acquaintanceship with chromatography . . . . . . . . . . . . . 21.I.2. Growing up . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.I.3. New life in America . . . . . . . . . . . . . . . . . . . . . . . 21.I.4. Waiting for moon rocks begets HPLC . . . . . . . . . . . . . . . 21.I.5. Reversed-phase chromatography wears the crown . . . . . . . . . . 21.I.6. Dawn of biotechnology . . . . . . . . . . . . . . . . . . . . . . 21.I.7. The expanding scope of electrochromatography . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Josef Franz Karl Huber . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Daido Ishii and Toyohide Takeuchi . . . . . . . . . . . . . . . . . . . . . .

101

188 189 189 190 191 192 193 194 199 200 201 203 204 205 206 206 207 208 210 211 211 212 213 217 218 218 225 226 227 230 230 231 232 233 234 235 237 238 238 239 239 240 242 243 245 247 247 248 250 250 250

102

Chapter 5 23.I. Development of microcolumns for LC . . . . . . . . . . . . . . . . . . . . 23.I.1. Open-tubular capillary LC . . . . . . . . . . . . . . . . . . . . 23.I.2. Direct coupling of microcolumn LC with fast-atom bombardment mass spectrometry (FABMS) . . . . . . . . . . . . . . . . . . . . . . 23.I.3. Unified chromatography . . . . . . . . . . . . . . . . . . . . . 23.I.4. Future prospects of microcolumn LC . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.24. Reed M. Izatt and Jerald S. Bradshaw . . . . . . . . . . . . . . . . . . . . . 24.I. Selective ion separations using solid-phase extraction procedures . . . . . . . . . 24.I.1. Separation of nuclear waste from storage solutions [5,6a] . . . . . . . 24.I.2. Separation and purification of mine drainage solutions [6a] . . . . . . 24.I.3. Removal of unwanted Bi3C impurity from a copper concentrate solution [5,6] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.I.4. Separation and purification of precious metals [6,7] . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.25. Jaroslav Jana´k . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.I. Personal recollections, achievements and opinions . . . . . . . . . . . . . . . 25.I.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 25.I.2. The origin of the gas chromatograph . . . . . . . . . . . . . . . . 25.I.3. Opinions . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.26. Egil Jellum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.I. Chromatography, mass spectrometry and electrophoresis for diagnosis of human disease, particularly metabolic disorders . . . . . . . . . . . . . . . . . . . . 26.I.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.I.2. GC–MS, HPLC, 2D-PAGE and CE for diagnosis and studies of human disease, particularly metabolic disorders . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.27. Walter G. Jennings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27.I. Conversion of the industrial analyst to capillary GC . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.28. James W. Jorgenson . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.I. Scientific biography of James W. Jorgenson . . . . . . . . . . . . . . . . . . 28.I.1. My early formative years . . . . . . . . . . . . . . . . . . . . . 28.I.2. Separation and analysis of complex mixtures . . . . . . . . . . . . 28.I.3. Separations of the new millennium . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.29. Rudolf E. Kaiser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.I. My occupation and my hobby — chromatography . . . . . . . . . . . . . . . 29.I.1. Update: 2000 . . . . . . . . . . . . . . . . . . . . . . . . . . 29.I.2. First example . . . . . . . . . . . . . . . . . . . . . . . . . . 29.I.3. Second example . . . . . . . . . . . . . . . . . . . . . . . . . 29.I.4. Third example . . . . . . . . . . . . . . . . . . . . . . . . . 29.I.5. Lessons learned . . . . . . . . . . . . . . . . . . . . . . . . . 29.I.6. Satisfaction . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.30. Barry L. Karger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30.I. Reminiscenses of 40 years in separation science . . . . . . . . . . . . . . . . 30.I.1. A look at separation: science in the 21st century . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.31. Jerry W. King . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.I. The use of supercritical fluids in analytical chemistry . . . . . . . . . . . . . .

251 252 253 255 257 257 257 259 260 260 261 261 262 262 263 264 264 265 269 270 270 271 271 272 277 277 280 282 283 285 285 287 289 290 290 292 295 296 296 297 298 299 300 300 301 307 308 309 310

Prominent Chromatographers and their Research 31.I.1.

D.32. 32.I.

D.33. 33.I. D.34. 34.I.

D.35. 35.I.

D.36. 36.I.

D.37. 37.I.

D.38. 38.I.

In summary . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . J.J. Kirkland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Search for new measurement technologies . . . . . . . . . . . . . . . . . . . 32.I.1. Separation potpourri . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Ernst Klesper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Progress during the first 20 years and some later developments . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . John H. Knox . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 years exploring chromatography . . . . . . . . . . . . . . . . . . . . . . 34.I.1. Early days . . . . . . . . . . . . . . . . . . . . . . . . . . . 34.I.2. Mainly GC in Edinburgh . . . . . . . . . . . . . . . . . . . . . 34.I.3. Liquid chromatography – band dispersion studies . . . . . . . . . . 34.I.4. Column packings and the Wolfson Liquid Chromatography Unit . . . 34.I.5. Mechanisms in chromatography . . . . . . . . . . . . . . . . . . 34.I.6. Preparative HPLC . . . . . . . . . . . . . . . . . . . . . . . . 34.I.7. Size-exclusion chromatography (SEC) . . . . . . . . . . . . . . . 34.I.8. Dead volumes and solvent peaks . . . . . . . . . . . . . . . . . 34.I.9. Miniaturization of HPLC and electrochromatography . . . . . . . . 34.I.10. Postscript . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Milton L. Lee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quarter of a century of research in capillary column separations at Brigham Young University . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35.I.1. Capillary column technology . . . . . . . . . . . . . . . . . . . 35.I.2. High resolution chromatographic analysis of polycyclic aromatic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35.I.3. Capillary column supercritical-fluid chromatography . . . . . . . . . 35.I.4. Time-of-flight mass spectrometry . . . . . . . . . . . . . . . . . References: key papers selected . . . . . . . . . . . . . . . . . . Hendrik Lingeman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Automation of all steps in chromatography . . . . . . . . . . . . . . . . . . References for original papers (11 cited of 110 published) . . . . . . References for review papers (4 cited of ca. 35 published) . . . . . . References for book chapters (ca. 20) or books (5) (partial list) . . . . Per G.M. Flodin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sephadex and gel filtration . . . . . . . . . . . . . . . . . . . . . . . . . 37.I.1. The Institute of Biochemistry (1950–1953) . . . . . . . . . . . . . 37.I.2. AB Pharmacia (1954–1962) . . . . . . . . . . . . . . . . . . . 37.I.3. Sephadex . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37.I.4. Gel filtration . . . . . . . . . . . . . . . . . . . . . . . . . . 37.I.5. Some characteristic features of SEC . . . . . . . . . . . . . . . . 37.I.6. Final remarks . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . James S. Fritz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advances on several fronts . . . . . . . . . . . . . . . . . . . . . . . . . 38.I.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 38.I.2. Solid-phase extraction . . . . . . . . . . . . . . . . . . . . . . 38.I.3. Ion chromatography . . . . . . . . . . . . . . . . . . . . . . . 38.I.4. Mobile-phase modifiers in HPLC: when bigger is better . . . . . . . 38.I.5. Ion chromatography–capillary electrophoresis (IC–CE) . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

103 315 316 316 317 325 328 329 330 343 346 347 347 348 349 351 353 354 354 355 355 356 357 357 359 359 361 363 363 364 364 365 366 367 367 367 368 368 370 370 372 373 374 375 375 376 376 376 377 378 379 380

104

Chapter 5 D.39. Charles H. Lochmu¨ller . . . . . . . . . . . . . . . . . . . . . . . . . . . 39.I. Discoveries, contributions, events and recollections . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.40. James E. Lovelock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40.I. Some historical comments . . . . . . . . . . . . . . . . . . . . . . . . . . 40.I.1. Significant scientific contributions . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.41. Karel Macek . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.I. Research and editorial activity in chromatography . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.42. Karin E. Markides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References: selected publications . . . . . . . . . . . . . . . . . D.43. Michel Martin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.I. Contribution to the field of separation science . . . . . . . . . . . . . . . . . 43.I.1. High-performance liquid chromatography . . . . . . . . . . . . . . 43.I.2. Field-flow fractionation . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.44. Daniel E. Martire . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44.I. Thermodynamics and theory of chromatography: development of new methods and a unified approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.45. Robert B. Merrifield . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45.I. The role of chromatography in solid phase peptide synthesis . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.46. Hiroshi Miyazaki . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46.I. Microanalyses of biologically important substances and drugs in biological specimens by gas chromatography, mass spectrometry, and isotachophoresis . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.47. E. David Morgan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47.I. Isolation, structure and quantification of insect substances . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.48. Milos V. Novotny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48.I. Chromatography, a journey from central Europe to America . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.49. Janusz Pawliszyn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49.I. Solid phase microextraction . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.50. William H. Pirkle and Christopher J. Welch . . . . . . . . . . . . . . . . . . 50.I. Chiral HPLC and enantioseparation of pharmaceuticals . . . . . . . . . . . . . 50.I.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 50.I.2. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . 50.I.3. Pirkle-type CSPs . . . . . . . . . . . . . . . . . . . . . . . . 50.I.4. Advantages of Pirkle-type CSPs . . . . . . . . . . . . . . . . . . 50.I.5. Preparative enantioseparation by HPLC . . . . . . . . . . . . . . 50.I.6. Discovery of new CSPs . . . . . . . . . . . . . . . . . . . . . 50.I.7. A new approach to CSP development . . . . . . . . . . . . . . . 50.I.8. Summary and conclusion . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.51. Colin F. Poole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.I. Reflections of a polychromatographer . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

381 382 385 385 386 388 389 389 390 395 396 398 399 400 400 403 405 405 406 407 411 412 412 413 413 414 419 419 421 424 424 426 434 434 435 440 440 440 441 441 442 443 444 444 445 448 450 451 453 454 459

Prominent Chromatographers and their Research D.52. Jerker Porath . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.I. Half a century of struggling with molecular sieving and affinity chromatography . . 52.I.1. Preliminaries . . . . . . . . . . . . . . . . . . . . . . . . . . 52.I.2. Discovery of Sephadex . . . . . . . . . . . . . . . . . . . . . . 52.I.3. From ion exchange — to adsorption — to affinity chromatography . . 52.I.4. Cross-linked agarose . . . . . . . . . . . . . . . . . . . . . . . 52.I.5. EDAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.I.6. IMAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.I.7. Strategies and future prospects . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.53. Michel Prost (invited paper) . . . . . . . . . . . . . . . . . . . . . . . . . 53.I. Capillary–gas chromatography coupled-mass spectrometric analysis of steroids, biogenic amines and amino acids in biological materials . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.54. Jacques Rijks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.55. Pat J. Sandra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55.I. Thirty years in separation sciences (1969–1999) . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . 55.II. Solving the Belgian dioxin crisis by analyzing PCBs in fatty matrices . . . . . . . 55.II.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 55.II.2. Methodologies for the analysis of PCBs in fatty matrices . . . . . . . 55.II.3. Sample preparation method for PCB analysis developed at RIC . . . . 55.II.4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.56. Gerhard Schomburg . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56.I. Surface modification in separation systems of chromatography and electrophoresis: 30 years of progress . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56.I.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 56.I.2. Surface modification in GC, SFC, HPLC, CZE and CEC . . . . . . . 56.I.3. Capillary-gas chromatography (CGC) . . . . . . . . . . . . . . . 56.I.4. High-performance liquid chromatography (HPLC) . . . . . . . . . . 56.I.5. Capillary zone electrophoresis (CZE), and micellar electrokinetic chromatography (MECC) . . . . . . . . . . . . . . . . . . . . . . . 56.I.6. Dynamic surface modification . . . . . . . . . . . . . . . . . . . 56.I.7. Permanent coatings . . . . . . . . . . . . . . . . . . . . . . . 56.I.8. Capillary electrochromatography (CEC) . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.57. Raymond P.W. Scott . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57.I. Forty-five years of chromatography research and development . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.58. Robert E. Sievers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58.I. The reach and stimulus of chromatography in diverse realms . . . . . . . . . . . 58.I.1. Early experiments . . . . . . . . . . . . . . . . . . . . . . . . 58.I.2. The reach and influence of chromatography . . . . . . . . . . . . . 58.I.3. Environmental studies and needs . . . . . . . . . . . . . . . . . 58.I.4. Gas chromatography and NMR of metal chelates . . . . . . . . . . 58.I.5. Thin films and coatings . . . . . . . . . . . . . . . . . . . . . 58.I.6. Selective detectors and sorbents . . . . . . . . . . . . . . . . . . 58.I.7. Aerosolization and fine powder synthesis . . . . . . . . . . . . . . 58.I.8. Waste clean-up . . . . . . . . . . . . . . . . . . . . . . . . . 58.I.9. The influence of chromatography in broadly diverse publications . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . .

105 459 460 460 461 463 464 465 466 468 470 470 472 474 475 477 477 478 487 488 488 490 490 495 495 495 498 498 498 499 500 501 503 504 504 505 506 507 511 511 513 513 513 515 515 516 517 517 518 518 518

106

Chapter 5 58.I.10.

D.59. 59.I.

D.60. 60.I.

D.61. 61.I.

D.62. 62.I.

D.63. 63.I.

D.64. 64.I.

D.65. 65.I.

Kudos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Colin F. Simpson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Research and development of interest in chromatography and electrophoresis . . . 59.I.1. Teaching of separation science . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Jan Bertil Sjo¨vall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Non-polar neutral and ion-exchanging derivatives of Sephadex for extraction and sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Hamish Small . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ion chromatography and hydrodynamic chromatography . . . . . . . . . . . . 61.I.1. Ion chromatography . . . . . . . . . . . . . . . . . . . . . . . 61.I.2. Hydrodynamic chromatography . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Roger M. Smith . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Retention and selectivity in liquid and supercritical=superheated separations . . . . 62.I.1. Introduction and application of retention indices in liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.I.2. Effects of mobile phase additives in liquid chromatography . . . . . . 62.I.3. Separation of basic drugs on silica . . . . . . . . . . . . . . . . . 62.I.4. Packed column supercritical-fluid chromatography . . . . . . . . . . 62.I.5. Supercritical-fluid extraction of natural products . . . . . . . . . . . 62.I.6. Retention prediction in capillary electrophoresis and electrochromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.I.7. Superheated-water chromatography . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Lloyd R. Snyder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A search for simple LC models . . . . . . . . . . . . . . . . . . . . . . . 63.I.1. Retention vs. structure . . . . . . . . . . . . . . . . . . . . . . 63.I.2. High-performance liquid chromatography . . . . . . . . . . . . . . 63.I.3. Gradient elution theory . . . . . . . . . . . . . . . . . . . . . . 63.I.4. Selectivity optimization . . . . . . . . . . . . . . . . . . . . . 63.I.5. Computer simulation . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Shigeru Terabe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Micellar electrokinetic chromatography (MEKC) . . . . . . . . . . . . . . . . 64.I.1. Prelude to MEKC . . . . . . . . . . . . . . . . . . . . . . . . 64.I.2. Success in MEKC experiments . . . . . . . . . . . . . . . . . . 64.I.3. Applications of MEKC . . . . . . . . . . . . . . . . . . . . . . 64.I.4. Future prospects of MEKC . . . . . . . . . . . . . . . . . . . . 64.I.5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Klaus K. Unger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The impact of silica chemistry on separation science and technology: a personal view spanning three decades . . . . . . . . . . . . . . . . . . . . . . . . . 65.I.1. Introduction to the field . . . . . . . . . . . . . . . . . . . . . 65.I.2. Breakthrough studies in silica bead manufacturing and surface modification of silica . . . . . . . . . . . . . . . . . . . . . . . . . . 65.I.3. The switch to high-resolution biopolymer separations . . . . . . . . 65.I.4. Novel ordered mesoporous silicas: the gateway to new adventures in separation science and technology . . . . . . . . . . . . . . . . .

519 519 521 523 530 533 533 534 540 541 543 543 545 545 546 546 547 548 549 549 549 550 550 551 551 552 553 553 554 556 557 559 560 560 562 562 562 564 565 565 565 566 567 567 567 569 570

Prominent Chromatographers and their Research 65.I.5.

E.

Capillary electro-chromatography (CEC): challenges and opportunities for high throughout separations . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.66. Irving W. Wainer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66.I. Enantioselective chromatography in the pharmaceutical and pharmacological sciences References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.67. Harold F. Walton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.68. Phillip C. Wankat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68.I. Interactions in research on large-scale chromatography and adsorption . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.69. Ian David Wilson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69.I. The fascination of separations . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . D.70. Edward S. Yeung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70.I. Laser-based detection for chromatography . . . . . . . . . . . . . . . . . . . 70.I.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 70.I.2. Laser-induced fluorescence (LIF) detection . . . . . . . . . . . . . 70.I.3. Optical rotation detector . . . . . . . . . . . . . . . . . . . . . 70.I.4. Indirect detection . . . . . . . . . . . . . . . . . . . . . . . . 70.I.5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary: if Mikhail Tswett were alive today – additional Awardees are found in Chapters 4 and 6 and in the Supplement Chapters S-8 to S-15 . . . . . . . . . . . . . . . . . . .

107

571 572 572 573 577 578 579 583 584 587 587 588 589 594 594 595 595 596 596 597 598 598 598

A. INTRODUCTION This chapter — the cardinal chapter of the book — presents each of the chromatography Awardees=scientists including a brief biography of each of them. The Awardees were encouraged to write about some unique activities, their research experiences, and to share how their advances have impacted the scientific disciplines. A description of their discovery(s) and contributions to chromatography, closely related sciences and recollections is given in their following written personal accounts. The contributors portray some unique associations, some humorous events or personal connections that are omitted in the standard style of research journals. Chapter 5 is, thus, an extension of the earlier Chapter 2 on the Builders of Chromatography and the chromatography Awardees in 13 Tables, which gives only their award, the date of award, and their institutional affiliation. Thus, scientific biography and discovery become part of the history and evolution of science. Our goal as Editors was to identify the research areas of the chromatography Awardees and then relate their contributions — their pioneering ideas and their scientific validation to seminal concepts. Since the living Awardees are very versatile individuals, they are listed in an alphabetical order and followed by a brief footnote stating the one or more seminal concepts that they addressed. Chapter 2 lists several Awardees in 2000; however, our publication timeline prevented the Editors from including some of their written personal stories in this chapter.

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Chapter 5

B. SEMINAL CONCEPTS AND NEW DISCOVERIES The Editors, upon review of almost 100 years of chromatographic books, symposia, reviews and literature, present the following 20 seminal concepts or subject areas. Some areas are judgment calls beyond the realm of science and subject to later modification by others; similarly, the cited contributions of the Awardees may have a different expression of views. The seminal concepts, listed below, are also partially related to the historical sequence of the development of chromatography, as described in the earlier Chapters 1, 4, 5, 6, S-8, S-9, S-12, S-13 and S-14. In this way, the original objective to integrate the seminal concepts with chromatography leaders is partially fulfilled in the following outline: Seminal concept or subject areas and code letters:

Chapters or subject index:

a. b.

Subject index 1-BCD

c. d. e. f. g. h. i. j. k. l. m. n. o. p. q. r. s. t.

Theoretical contributions to chromatography Early adsorption chromatography=partition chromatography (plus PC and TLC) Ion-exchange chromatography Gas chromatography Supports-, stationary- and bonded-phase chemistry Detectors in chromatography Size-exclusion chromatography High-performance liquid chromatography Affinity chromatography=bioaffinity chromatography=biosensors Petroleum chromatography Instrumentation in chromatography Electrophoresis=capillary electrophoresis=electrochromatography Ion chromatography Synthetic and biological membrane separations and their techniques Supercritical-fluid chromatography=extraction Hyphenated=coupled=tandem techniques in chromatography Chiral chromatography and pharmaceutical separations Biomedical sciences and chromatography Environmental sciences and chromatography Space sciences and chromatography

1-E 1-C 11 9-Ec 9-Ea 9-Eb 9-Dd 1-F 9-Dg, 10 Subject index 1-E Index 9-Df 9-Ed 9-De Subject index Subject index 4, 12

Chapters with a number higher than 7 are in the Supplement.

Before taking a running plunge into the current discussion of the branches of chromatography, the editors suggest exploring a comprehensive review of some of the above 20 seminal concepts or areas. Several of the pertinent earlier references in Appendices 4, 5 and 6 may provide the needed background preparation in reading the events and discoveries described by the 112 living Awardees and contributors in this chapter and Chapter S-14.

C. RELATION OF SEMINAL CONCEPTS AND AWARDEES The above letters (a to t) appear with the following Awardee contributions as related to the research expertise of the contributors and later in the author and scientist index.

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Some pertinent notes follow: ž The Nobel Awardees are described in Chapters 1, 2, S-9 and two in this chapter. ž Deceased chromatographers are presented in Chapters 1, 2 and S-9 with further information and selected references in Appendix 2. ž Some of the living Awardees listed in the tables of Chapter 2 have not provided their invited contribution to this chapter. The above 20 seminal concepts, investigated by the chromatography Awardees, have led to a succession of new discoveries, which have had major impact in the many growing areas of science and the related emerging scientific technologies. In Chapter S-15, the Editors present the perspectives of 41 chromatographers on past chromatographic science with a view of the near future for the advancement of the sciences and technologies and the continuing discovery in the 21st century.

D. PROMINENT CHROMATOGRAPHERS (76 AWARDEES) D.1. Daniel W. Armstrong Daniel W. Armstrong was born on November 2, 1949, Ft. Wayne, IN. He received his B.Sc. in Science and Math from Washington and Lee University (1968–1972), a M.Sc. in Chemical Oceanography from Texas A&M University (1974– 1975) and a Ph.D. in Chemistry from Texas A&M University (1975–1977). Curators’ Professor of Chemistry, University of Missouri, Rolla, MO, from 1988 to 2000. His present position is Caldwell Chair of Chemistry at Iowa State University, Ames, IA. Armstrong has 260 papers, 16 chapters and one book (W.L. Hinze and D.W. Armstrong (Eds.), “Use of Ordered Media and Chemical Separations”, American Chemical Society, Washington, DC, 1981). His eight patents include: Bonded Phase Material for Chromatographic Separations, September 3, 1985; Fused Silica Capillary Coated with Permethylated-S-Hydroxypropyl Cyclodextrins and Analogues for Separations, August 14, 1990; Chiral Stationary Phase for Gas Chromatography, August 12, 1991; Chiral Stationary Phase for Chromatographic Separations, October 13, 1992; Stereoselective Adsorption Bubble Process, May 13, 1997; Macrocyclic Antibiotics as Separation Agents, May 6, 1997; and Activated Carbon Produced from Agricultural Materials, March 16, 1999. He currently has two patents pending. His work has been recognized through a number of citations: Teaching Excellence Award from the ‘Arts and Sciences Council’ of Texas Tech University, 1985; Faculty Excellence Award, 1988, Teaching Excellence Award 1988–1989, 1992, 1994; Curator’s Distinguished Professorship, 1989, all three of them from the University of Missouri at Rolla; 1990 EAS Award in Chromatography; 1991 Chromatography Society’s Martin Medal; 1992 ISCO Award; 1993 President’s Award for Research and Creativity; 1993 49th ACS Midwest Regional Award; R&D 100 Award 1995; AAPS Research Fellow Award 1995; American Microchemical Societies’ Benedetti-Pichler Award 1996; 1997

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Karen Morehouse Best Paper Award presented by the 12th Annual Conference on Hazardous Waste Research; 1998 American Chemical Society Helen M. Free Award for Public Outreach; and the 1999 American Chemical Society Award in Chromatography; and the 1999 Hope College Distinguished Scholar Award. He was elected as a member of the Chemistry Honor Society in 1975, Phi Lamba Upsilon (PLU); Who’s Who, American Men and Women of Science, Directory of World Researchers, and others. He is a member of the Editorial Boards of the Journal of Chromatography, Journal of Liquid Chromatography, Journal of Pharmaceutical and Biomedical Analysis, Chirality, Journal of Planar Chromatography, Journal of Microcolumn Separations, LCžGC Magazine, Separation Science and Technology. He is a member of the Instrumentation Advisory Board of Analytical Chemistry, Section Editor for Amino Acids, Editor of Chirality, separations associate Editor for Analytical Chemistry, and on the Editorial Advisory Board of Chromatographia. He belongs to the following professional societies: the American Association for the Advances of Science, American Chemical Society, Smithsonian Associate, Sigma XI. He has participated in over 230 invited seminars and oral presentations. Approximately 7.4 million dollars in grants and contracts have been awarded for his work. Three ‘Classic Most Cited Papers’ (one in 1984 and two in 1985) are listed in the Scientific Citation Index Review (SCI). See Chapter 5B, a, b, d, e, h, q, r

1.I. UNCONVENTIONAL APPROACHES FOR UNDERSTANDING AND DEVELOPING SEPARATIONS Daniel W. Armstrong Caldwell Chair of Chemistry, Iowa State University, Ames, IA, USA

1.I.1. In the beginning, pseudophase theory and use Modern separation methods, particularly chromatography and electrophoresis, have revolutionized many branches of science and technology. My initial research introduction to chromatography (¾mid 1970s) did not involve separations per se, but rather its use to make physicochemical measurements. Specifically, we were involved in using and developing liquid chromatography (and later capillary electrophoresis) as an effective means to measure binding constants of molecules to such things as micelles, cyclodextrins, etc. [1,2]. Today, separation-based methods are among the most useful ways to measure association constants [3]. In some cases, where molecules do not undergo any spectroscopic changes upon binding, they are the most effective methods for evaluating binding behavior. At about the same time as the above, I used both classic LC and HPLC in an attempt to separate products from a micelle catalyzed reaction [4]. It was clear that the micelles

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in the reaction mixture were affecting the separation. At that point it was obvious to me that solutions of micelles could be used in place of organic solvents or hydro-organic solvent mixtures as the mobile phase in liquid chromatography. Partitioning to the micellar pseudophase would offer unique selectivities for the separation of neutral and charged analytes [5,6]. Not long afterwards we first used cyclodextrins in a pseudophase-type separation system [7,8]. Prior to these studies, the deliberate and widespread use of micelles and cyclodextrins in analytical chemistry was virtually unknown. The term pseudophase separations was first coined in 1980 [9]. More importantly the theory and mechanism of pseudophase separations (or separations based on ordered media) was thoroughly outlined and then supported experimentally [10–12]. Today micelles and cyclodextrins are well-known, important elements in a variety of modern, high performance analytical procedures including chromatography, capillary electrophoresis, not to mention a variety of spectroscopic methods.

1.I.2. The enantiomeric separations revolution Our early work with cyclodextrin [7–9] led me into the area of chiral molecular recognition and enantiomeric separations [13–15]. My main interest was to understand, in a fundamental manner, the ultimate in molecular recognition (i.e., the ability of one molecule to distinguish between enantiomers of another molecule). Developing a highly successful class of chiral stationary phases that would be used worldwide (as they are today) was never a prime consideration. Prior to the early 1980s, enantiomeric separations tended to be avoided or ignored, as they were thought to be tedious, difficult or impossible to do. I developed the original stable cyclodextrin-bonded phase for HPLC because: (a) using cyclodextrins in the mobile phase was very expensive at that time, unless one recycled the cyclodextrin additive; (b) the cyclodextrin mobile phase additive approach produced relatively inefficient separations; and (c) I was curious as to the reversed-phase selectivity of cyclodextrin columns (which separated on the basis of inclusion complexation) versus that of the more common alkyl-type reversed-phase stationary phases. Thus, our early work on cyclodextrins consisted of roughly equal parts of theory =mechanism, achiral retention=separations and enantiomeric separations. While all of these studies received a good deal of attention from academia, industry and government scientists, it was the enantiomeric separations that seem to have garnered the most attention (at the time I felt a disproportionate share). I did not fully appreciate the practical consequences of our work or the rapidity with which it (and our later work on enantiomeric separations) would be adapted. Our work, along with that of only a few others, fundamentally changed the way the pharmaceutical industry developed, tested, assayed and produced drugs products. It provided the main impetus for the Food and Drug Administration (FDA), issuing a 1992 policy statement on the development of new stereoisomeric drugs [16]. Our work gave rise to the development of new industries that made and marketed commercial chiral stationary phases, produced bulk enantiomeric separations, and analyzed chiral molecules for a variety of clinical, pharmaceutical, environmental and agrochemical concerns. The impact in science, technology and industry has been profound and continues unabated today.

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The mechanistic aspect of our cyclodextrin (and other chiral selector research) work overlapped with the practical aspects that were so popular with others. By understanding how and why chiral recognition occurs, one can use it more effectively to achieve the desired separation. By the mid-1980s, we had a basic understanding of the chiral recognition mechanism for cyclodextrins in aqueous solutions [13–15]. Our early modeling and mechanistic work gave rise to a set of rules for separation. Often we could predict what type of molecule would or would not separate. This work also led us to synthesize specific derivatives of cyclodextrin that had different interactions and produced separations that could not be obtained by native cyclodextrin [17–19]. Most intriguing was our discovery that host–guest inclusion complexation was not always necessary or even preferable for chiral recognition with cyclodextrins. In the gas phase and in nonhydrogen-bonding solvents, such as acetonitrile (i.e., the polar organic mode), external adsorption and association produced enantioseparations that could not be obtained under conditions that favored inclusion complexation [19,20]. This greatly enhanced the understanding of these systems and expanded their utility as well. By the early 1990s, the use of cyclodextrins had become well established and popular in analytical chemistry. We turned our attention to a new class of macrocyclic molecules, the glycopeptides [21–24]. These compounds (vancomycin, teicoplanin, ristocetin A, avoparcin, etc.) also happened to be a very important class of antibiotics. My interest, however, stemmed from their structure. They were moderate in size, chiral molecules that had a diversity of functionality only approached by that of glycoproteins, which are very much larger. In addition, the functionalities of these chiral selectors were in relatively close proximity to one another. This could provide opportunities for multiple, simultaneous interactions [21–24]. In addition, the structures of the macrocyclic glycopeptides are known and they were of a size that they could be effectively modeled and used in a variety of physicochemical studies. The teicoplanin chiral selector phase (CSP) rapidly became the column of choice for the LC resolution of most amino acids [23]. Its enantioselective retention mechanism also was elucidated [24]. The macrocyclic glycopeptides quickly became one of the most broadly applicable classes of chiral selectors in the world (even outstripping cyclodextrins in some areas). They have been widely utilized in HPLC, CE and TLC. One particularly novel aspect of our research was the first separation of enantiomers using bubbles [25,26]. Adsorptive bubble separation methods are known to be useful for processing large amounts of material at a relatively low cost (such as sulfide minerals in the mining industry). We demonstrated that a variety of surface-active chiral collectors could be used to enrich enantiomers in conjunction with an inexpensive glass countercurrent foam column [25]. In a significant extension of this work, molecularly imprinted polymers (MIPs) and heterogeneous solutions were used for the bubble fractionation of enantiomers from solution [26]. The powdered MIPs served both as the chiral collector and the foaming agent. As solid particles, the MIPs were easily recycled which is important in process-scale separations. The widespread publicity regarding high efficiency enantioselective separations, and its direct effect on the pharmaceutical industry and FDA policy, gave a significant boost to asymmetric synthesis. This was because of the heightened interest in testing and producing single enantiomer drugs. In addition, these new analytical approaches were

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the most effective means to measure enantiomeric excesses (e.e.s.) and to prepare chiral intermediates. Later it was shown that hundreds of the most commonly used commercial chiral catalysts, auxiliaries and synthons had significant levels of enantiomeric impurities [27,28]. The reader is also referred to Chapter S-9D, on paradigm shifts in chromatography, for some of the early publications on enantiomeric separations by chromatography.

1.I.3. Other areas of endeavor 1.I.3.1. Increasing chromatographic efficiency via turbulent flow In 1988, we published a series of papers on a variation of countercurrent chromatography (CCC). CCC has a liquid mobile phase and a liquid stationary phase. One of the papers involved the theory and mechanism of band broadening in this form of chromatography [29]. We found that at higher flow rates, chromatographic efficiency increased rather than decreased as predicted by the van Deemter equation. Indeed the van Deemter plots had maxima rather than the minima found for most forms of chromatography. The reason was essentially due to the turbulent mixing that enhanced mass transfer. This may have been the first practical example and theoretical treatment of this phenomenon in chromatography [29]. It now appears that turbulent mixing might be useful in enhancing other forms of chromatography. Columns and media are being developed which can be used at the flow rates necessary to achieve turbulent mixing and high efficiencies. 1.I.3.2. Gas and hydrocarbon separations Several years ago I was approached by an industrial scientist who wanted to know why someone could not develop a rugged gas chromatography (GC) column, for separating gases and light hydrocarbons, that was not adversely affected by water. We worked on several variations of cyclodextrins bonded to fused silica and silica gel. Eventually we came up with a gas-solid chromatography (GSC) column that could separate most gases, hydrocarbons and halocarbons and showed no deleterious effects from water [30,31]. It could be used to directly analyze automobile exhaust and a variety of gaseous and volatile pollutants [30,31]. It was commercialized as the Gas-Pro column first by Advanced Separation Technologies in New Jersey and later by J&W Scientific Co. in California. Among its many uses, it is now being adapted for a NASA mission probe to Venus where it will be used to measure the atmospheric gases on that planet. 1.I.3.3. Gradient fractionation of polymers Polymers can be chromatographically separated by molecular weight or by composition (for copolymers) by gradient LC. Early on I was involved in a small controversy with those who claimed polymers behaved the same (and thus could be treated) as small molecules in liquid chromatography. Now it is well known (and was amply

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demonstrated then) that this is not the case. In the early 1980s, we developed the theory, mechanism and several practical examples of the gradient LC fractionation of polymers [32]. The theory and mechanism for this separation was somewhat vague and controversial until we used the Flory–Huggins theory of dilute polymer solutions to provide the technique with a firm theoretical background. As a result of this treatment the concept of the critical mobile phase composition (Xc) was introduced to liquid chromatography [32]. Macromolecular solutes below Xc are completely retained, while above Xc they are rapidly eluted. Because the retention factor of a solute is related to Xc, traditional chromatographic parameters such as theoretical plate numbers have little meaning. This approach forms the basis for the chromatographic separation of both synthetic and biological polymers (although biological polymers can be even more complex).

1.I.4. Looking back Research has been one of the great joys of my life. It helps to satisfy my curiosity and gives one a sense of accomplishment. It requires imagination, a good deal of hard work and sufficient scientific background combined with common sense to recognize what to do and how to do it. Some research is analogous to ‘detective-work’, where you attempt to ferret out unknown answers (i.e., solve mysteries). Some research is analogous to ‘architecture= construction work’, where you design and build something interesting and useful. Research has many faces which is one of its attractions. I always felt that there were nearly unlimited options and directions in which to go. What better way to avoid boredom and do something useful or important. An integral part of my research was working with students and colleagues. These were people who enjoyed what they were doing. Watching them ‘light up’ when they solved a problem or understood something for the first time (often after weeks of struggle) is a wonderful experience. They cannot hide their sense of accomplishment when their first scientific paper is published or when they come back from their first job interview. I have seen 60-year-old colleagues with the same sense of excitement when they discover an answer, solve a problem or publish a particularly elegant paper. My research in separations has been fun and rewarding. I cannot imagine having done anything else for this long a period of time.

References 1. 2. 3.

4.

D.W. Armstrong, R. Seguin and J.H. Fendler, Partitioning of amino acids and nucleotides between water and micellar hexadecyltrimethyl-ammonium halides, J. Mol. Evol., 10 (1977) 241–250. D.W. Armstrong and G.Y. Stine, Evaluation of partition coefficients to micelles and cyclodextrins via planar chromatography, J. Am. Chem. Soc., 105 (1983) 2962–2964. D.W. Armstrong, Determination of association constants by chromatography and electrophoresis, in: P.R. Brown and E. Grushka (Eds.), Advances in Chromatography, Vol. 39, Dekker, New York, NY 1998, pp. 239–262. D.W. Armstrong, R. Seguin, C.J. McNeal, R.D. Macfarlane and J.H. Fendler, Spontaneous polypeptide

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6. 7. 8.

9. 10. 11. 12.

13. 14. 15.

16. 17. 18. 19. 20.

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formation from amino acyl adenylates in surfactant aggregates, J. Am. Chem. Soc., 100 (1978) 4605– 4606. D.W. Armstrong and J.H. Fender, Differential partitioning of tRNAs between micellar and aqueous phases: a convenient gel filtration method for separation of tRNAs, Biochim. Biophys. Acta, 478 (2) (1977) 75–80. D.W. Armstrong and S.J. Henry, Use of an aqueous micellar mobile phase for separation of phenols and polynuclear aromatic hydrocarbons via HPLC, J. Liq. Chromatogr., 3 (5) (1980) 657–662. D.W. Armstrong, Application of pseudophase liquid chromatography (PLC): highly selective mobile phase for present and future separations, Am. Lab., 13 (8) (1981) 14–20. D.W. Armstrong, Use of micellar and cyclodextrin solutions in liquid chromatographic separations, pp. 1273–1282, in: K.L. Mittel and E.J. Fendler (Eds.), Proc. Int. Symp. Soln. Behavior Surfact., Plenum Press, New York, NY, 1982. D.W. Armstrong, Pseudophase liquid chromatography: applications to TLC, J. Liq. Chromatogr., 3 (6) (1980) 895–900. D.W. Armstrong and F. Nome, Partitioning behavior of solutes eluted with micellar mobile phases in liquid chromatography, Anal. Chem., 53 (11) (1981) 1662–1666. D.W. Armstrong, Micelles in separations: application and theory, Sep. Purif. Methods, 14 (1985) 213–304. L.A. Spino and D.W. Armstrong, Least-squares iterations: nonlinear evaluation of cyclodextrin multiple complex formation with static and ionizable solutes, in: W.L. Hinze and D.W. Armstrong (Eds.), Ordered Media in Chemical Separations, ACS Symposium Series 342, American Chemical Society, Washington, DC, 1987, Chapt. 11, pp. 235–246. D.W. Armstrong and W. DeMond, Cyclodextrin bonded phases for the liquid chromatographic separation of optical, geometrical and structural isomers, J. Chromatogr. Sci., 22 (1984) 411–415. D.W. Armstrong, T.J. Ward, R.D. Armstrong and T.E. Beesley, Separation of drug stereoisomers by the formation of β-cyclodextrin inclusion complexes, Science, 232 (1986) 1132–1135. S.M. Han and D.W. Armstrong, HPLC separation of enantiomers and other isomers with cyclodextrinbonded phases: Rules for chiral recognition, in: A.M. Krustolvic (Ed.), Chiral Separations by HPLC: Applications to Pharmaceutical Compounds, Ellis Horwood Ltd: Chichester, West Sussex, 1989, Chapt. 10, pp. 208–284. FDA’s policy statement for the development of new stereoisomeric drugs, Chirality, 4 (1992) 338–340. D.W. Armstrong, A.M. Stalcup, M.L. Hilton, J.D. Duncan, J. Faulkner, Jr. and S.C. Chang, Derivatized cyclodextrin for normal phase LC separation of enantiomers, Anal. Chem., 62 (1990) 1610–1615. A.M. Stalcup, S.C. Chang, D.W. Armstrong and J. Pitha, (S)-2-hydroxypropyl β-CD: a new chiral stationary phase for reversed-phase liquid chromatography, J. Chromatogr., 513 (1990) 181–194. A. Berthod, W. Li and D.W. Armstrong, Multiple enantioselective retention mechanisms on derivatized cyclodextrin gas chromatographic chiral stationary phases, Anal. Chem., 64 (1992) 873–879. S.C. Chang, G.L. Reid, S. Chen, C.D. Chang and D.W. Armstrong, Evaluation of a new polar-organic HPLC mobile phase for cyclodextrin bonded chiral stationary phases, Trends Anal. Chem., 12 (1993) 144–153. D.W. Armstrong, Y. Tang, S. Chen, Y. Zhou, C. Bagwill and J.R. Chen, Macrocyclic antibiotics as a new class of chiral selectors for liquid chromatography, Anal. Chem., 66 (1994) 1473–1484. M.P. Gasper, A. Berthod, U.B. Nair and D.W. Armstrong, Comparison and modeling study of vancomycin, ristocetin A and teicoplanin for CE enantioseparations, Anal. Chem., 68 (1996) 2501– 2514. A. Berthod, Y. Liu, C. Bagwill and D.W. Armstrong, Facile liquid chromatographic enantioresolution of native amino acids and peptides using a teicoplanin chiral stationary phase, J. Chromatogr. A, 731 (1996) 123–137. U.B. Nair, S.S.C. Chang, D.W. Armstrong, Y.Y. Rawjee, D.S. Eggleston and J.M. McArdle, Elucidation of vancomycin’s enantioselective binding site using its copper complex, Chirality, 8 (1996) 590–595. D.W. Armstrong, E.Y. Zhou, S. Chen, K. Le and Y. Tang, Foam flotation enrichment of enantiomers, Anal. Chem., 66 (1994) 4278–4282. D.W. Armstrong, J.M. Schneiderheinze, Y.S. Hwang and B. Sellergren, Bubble fractionation of

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30.

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Chapter 5 enantiomers from solution using molecularly imprinted polymers as collectors, Anal. Chem., 70 (1998) 3717–3719. D.W. Armstrong, J.T. Lee and L.W. Chang, Enantiomeric impurities in chiral catalysts, auxiliaries and synthons used in enantioselective synthesis, Tetrahedron Asymmetry, 9 (1998) 2043–2064. D.W. Armstrong, J.T. Lee, B. He and T. Yu, Enantiomeric impurities in reagents for asymmetric synthesis, II, Tetrahedron Asymmetry, 10 (1999) 37–60. D.W. Armstrong, G.L. Bertrand and A. Berthod, Study of the origin and mechanism of band broadening and pressure drop in centrifugal counter current chromatography, Anal. Chem., 60 (1988) 2513–2519. D.W. Armstrong, K. Le, G.L. Reid, III, S.C. Lee, K.K. Beutelmann, M. Horak and P. Tran, Gas-solid chromatographic analysis of automobile tailpipe emissions as a function of different engine and exhaust system modifications, J. Chromatogr. A, 688 (1994) 201–209. D.W. Armstrong, G.L. Reid and J. Luong, Gas separations: A comparison of GasPro and aluminum oxide PLOT columns for the separation of highly volatile compounds, Curr. Separations, 15 (1) (1996) 5–17. D.W. Armstrong and R.E. Boehm, Gradient liquid chromatographic separation of macromolecules: Theory and mechanism, J. Chromatogr. Sci., 22 (1984) 378–385 and references therein.

1.II. THE ADVENT OF BROADLY APPLICABLE, HIGH EFFICIENCY ENANTIOMERIC SEPARATIONS BY LC: A REVIEW Daniel W. Armstrong Caldwell Chair of Chemistry, Iowa State University, Ames, IA, USA

Throughout most of the 20th century, the resolution of enantiomeric compounds was considered one of the most difficult problems in separation science. Prior to the early 1980s, enantiomeric separations tended to be avoided or ignored as they were often thought to be tedious, difficult or impossible to do. At that time, limited large-scale resolution of racemic mixtures could sometimes be done by techniques, such as fractional recrystallization of diastereomeric salts, enzymatic resolution, or microbiologic digestions, for example. There were no widely applicable and efficient analytical (or semipreparative) methods that permitted quick resolution and accurate quantitation of enantiomers. Some of the early research for chromatographic separation of enantiomers is described in Chapter S-9D, paradigm shifts in chromatography. In a 10-year period (from the early 1980s to the early 1990s), the rapid and routine separation of enantiomers went from practically nonexistent, to commonplace. This in itself is a tribute to the relatively few scientists who were instrumental in this revolution, as well as to the first companies (most of which were small, innovative enterprises) that first commercialized this technology. The expansion of enantiomeric separations was fueled by: (a) the commercialization of useful liquid chromatography (LC) chiral stationary phases; (b) a tremendous increase in publications in this area; and (c) an increased understanding of the mechanism of chiral recognition and its importance in many scientific disciplines. The development of new and more useful approaches for the analyses of enantiomers continues today in LC, GC, and CE. The earliest review on chiral stationary phases (CSPs) for LC was published in 1984 [1]. This was followed by many subsequent reviews [2–5].

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By the beginning of the 21st century, enantiomeric separations and analyses have become essential in a wide variety of areas. These include the pharmaceutical and medicinal sciences (including pharmacokinetics and pharmacodynamics), since it is known that different enantiomers can have different physiological effects and biological disposition [6,7,8]. The tremendous advances in the LC analysis of enantiomers provided the impetus for the Food and Drug Administration (FDA) to develop and issue guidelines for the development of new stereoisomeric drugs in 1992 [9]. In the environmental area, many chiral pesticides and herbicides have enantioselective effects and biodegradation rates [4,10]. The food and beverage industry is becoming increasingly concerned with the analysis of enantiomers which can affect flavor, fragrance, nutrition and can be used to monitor fermentation, age and even adulteration of products [11,12]. Enantiomeric separations are frequently the most useful way to determine the enantiomeric purity of newly synthesized chiral compounds and in elucidating reaction mechanisms [13,14]. Stereochemical analysis also can be of importance in geochemistry [15–18], geochronology [19], biochemistry, and in some areas of materials science [20]. Many elegant studies on chiral recognition have been reported. These studies have led to a better understanding of the molecular recognition and the enantioselective separation process. These are outlined in some of the aforementioned reviews. Although the number of publications involving enantiomeric separations and the number of commercially available CSP has expanded substantially, there are relatively few basic types of CSPs. Indeed, of the total number of enantiomeric separations that can be done by LC, the vast majority can be done on perhaps 6 or 7 different columns. In the next few paragraphs, the five basic classes of chiral stationary phases for HPLC are outlined. Types of CSPs for LC: (1) ligand exchange; (2) macrocyclic; (3) π-complex; (4) polymeric; and (5) synthetic. These are outlined below. It should be noted that there can occasionally be some overlap between these classes.

1.II.5. Ligand exchange chromatography Ligand exchange types of CSPs were developed by Davankov and were the first practical approach for the LC resolution of amino acids [21]. A chiral bidentate ligand (such as proline, hydroxyproline, etc.) was attached to a support. Copper (II) was added to the mobile phase and coordinated with both the chiral selector on the stationary phases and the free amino acids in solution, to form transient diastereomeric complexes. This method also can be used to resolve other racemates that can act as bidentate ligands, such as α-hydroxycarboxylic acids. A variety of different bidentate chiral selectors have been attached to different supports (silica gel, polystrene-divinylbenzene, etc.). Researchers have used different transition metals in the mobile phase, although copper(II) seems to be the most generally applicable [21]. This ‘ligand-exchange’ approach is limited to the resolution of specific chiral molecules that can selectively coordinate to the CSP. The copper salt-containing mobile phase gives some background absorbance when using UV-detection. Also, it can present a problem when using electrochemical detection. When doing preparative or semipreparative separations, the copper must be removed from the isolated product. It should be noted that the ligand

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exchange approach is the basis of the only commercially successful CSP for thin layer chromatography [22].

1.II.6. Macrocyclic stationary phases There are three main classes of CSPs that utilize macrocyclic compounds as chiral selectors. There are: (a) cyclodextrins; (b) antibiotics; and (c) crown ethers. Altogether, this particular group of chiral selectors is responsible for >95% of all GC enantiomeric separations (i.e., mainly on derivatized cyclodextrins), >95% of all CE enantiomeric separations (i.e., cyclodextrins plus glycopeptide antibiotics), and ¾50% of all LC separations reported. 1.II.6.1. Cyclodextrins Cyclodextrins were first used by Armstrong et al., as mobile phase additives in TLC to separate isomeric compounds [23,24]. Later they were immobilized to form a highly effective CSP [25–27]. The β-cyclodextrin bonded phase was the first commercially successful reversed phase CSP. At the time of this early work, the concept of an inclusion complex and its role in retention and selectivity were foreign to most chromatographers and separation scientists. By the mid-1980s the chiral recognition mechanism of cyclodextrin in aqueous solutions was fairly well understood [25–28]. The utility of cyclodextrin-based CSPs was enhanced in two different ways. First, a variety of different cyclodextrin derivatives were made and immobilized on a chromatographic support [29,30]. The various moieties used to functionalize cyclodextrins can greatly alter their enantioselectivity, thereby expanding their overall usefulness. For example, aromatic functionalized cyclodextrins can be used in the normal phase mode as a π-complex CSP, or in the reversed phase mode where inclusion complexation dominates. Completely different types of chiral molecules are resolved in each mode. Hydroxypropyl-functionalized cyclodextrins provide the most facile means of resolving tertiary butyl oxycarbonyl (t-BOC) amino acids and a variety of other chiral molecules [29,31]. A somewhat different experimental approach was found to enhance the usefulness of cyclodextrin-CSPs and produce unusually enantioselectivities. In this approach, inclusion complexation is suppressed by using a nonhydrogen bonding, polar-organic solvent (such as acetonitrile) as the main component of the mobile phase [32,33]. Although this is sometimes referred to as ‘the polar-organic mode’, it is most closely related to normal phase separations. The acetonitrile tends to occupy the cyclodextrin cavity. It also accentuates hydrogen bonding between the hydroxyl groups on the cyclodextrin and any hydrogen bonding groups on the chiral analyte [32,33]. A hydrogen bonding solvent (such as methanol) can be added to decrease the retention of highly retained compounds. Very small amounts of glacial acetic acid and triethylamine are added to control the protonation of the analyte and to enhance enantioselectivity. In this mode the analyte is thought to reside on top of the cyclodextrin selector (somewhat like a ‘lid’) in such a way that hydrogen bonding is maximized. Chiral compounds containing two hydrogen bonding groups (one of which should be α or β to the chiral

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center) and a bulky group (such as an aromatic ring) are often resolved via this approach. Also, this is an excellent technique for preparative separations. 1.II.6.2. Macrocyclic antibiotics Macrocyclic antibiotics are the newest and fastest growing class of chiral selectors [34–36]. Six types of glycopeptide based CSPs are available commercially. Among these are vancomycin, teicoplanin, ristocetin A, avoparcin, and the aglycone of teicoplanin. The glycopeptide macrocyclic chiral selectors appear to have broad applicability. They are multimodal CSPs as they can be used effectively in the reversed phase mode, normal phase mode, or ‘polar-organic’ mode. The enantioselectivity is usually different in each mode [34–36]. These CSPs are useful for preparative separations as well. The ‘principle of complementary separations’ is a very useful concept when doing method development work with glycopeptide-based CSPs. Vancomycin and teicoplanin are similar, closely related chiral selectors. They can have similar, but not identical enantioselectivities. Consequently, if a partial separation is obtained on one CSP, in many cases, one can go directly to a related column and obtain a baseline separation (using identical or very similar separation conditions). 1.II.6.3. Crown ethers The first real alternative to ligand exchange LC resolution of amino acids was provided by chiral crown ethers. Chiral crown ether stationary phases for LC were first developed by Cram and co-workers [37–39]. Cations the size of KC and NHC 4 tend to form inclusion complexes with [18]-crown-6-polyethers. Cram et al., demonstrated the chromatographic resolution of primary amino-containing chiral compounds on a CSP consisting of a chiral crown ether covalently linked to a polymeric support. The mobile phase must be acidic so that the analyte’s primary amine functional group is in the ammonium ion form. Ions that compete for inclusion in the crown ether (such as KC ) must be excluded from the mobile phase and separation solutions. The crown ether-type of chiral selector works only for primary amine containing compounds. It will not work for secondary amines and amino acids (such as proline, hydroxyproline, etc.) or other types of compounds. The commercial version of this column uses a ¾102 M solution of HClO4 as the mobile phase [40–42]. The use of other acids either greatly diminish or negate the separation on this column. This creates a problem when doing preparative separations, since evaporating the solvent can leave an explosive mixture of perchlorate plus organic material. 1.II.7. π-Complex stationary phases CSPs that interact with racemic analytes through one or more π–π interactions have been used since at least the 1960s in chromatography [43]. Early versions of these columns were sometimes referred to as ‘charge transfer’ stationary phases. The chiral selector in these types of stationary phases contained either a π-donor or π-acceptor

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moiety [43–46]. If the CSP was a π-donor, then the analyte to be resolved must contain a π-acceptor group or vice versa. Other simultaneous interactions must be present as well, if enantioselective separations are to occur. Generally these other interactions consist of hydrogen bonding, steric repulsion and=or dipolar interactions. Separations on the π-complex type of CSPs are usually done in the normal phase mode. This is because π–π interactions, hydrogen bonding, and dipolar interactions are more pronounced in nonpolar solvents. Mikesˇ and co-workers first developed both π-donor and π-acceptor CSP for HPLC [44–46]. Later Pirkle and co-workers [47–49] and Oi et al. [50–52] developed analogous CSPs, some of which were commercialized. The ionically attached dinitrobenzoylphenylglycine CSP was the first column commercialized for the chromatographic resolution of enantiomers [47]. Subsequently, several different research groups (and some companies) developed entirely analogous types of CSPs. One shortcoming of most of these CSPs is that the analyte to be resolved must have a complementary π-donor or π-acceptor group. Since most compounds of industrial interest did not have these groups, derivatization of one’s analyte often was required. The most recent versions of the π-complex CSPs contain both π-donor and π-acceptor groups [53,54]. 1.II.8. Polymeric stationary phases Polymeric chiral selectors can be classified in several different ways. There are naturally occurring chiral polymers, synthetic chiral polymers and hybrid varieties. Although interesting from an academic standpoint, the totally synthetic chiral polymers have not had a great impact on the LC separation of enantiomers, to date. The principal synthetic polymers used as CSPs are poly(triphenylmethylmethacrylate) and poly(2-pyridyl diphenylmethyl methacrylate) types [55]. These exist in right- and left-hand helical forms depending on the configuration of the chiral catalyst used in the polymerization reaction [55]. Most compounds resolved on these CSPs are better resolved on one or more of the other CSPs described in this monograph. Recently a hybrid polymeric CSP has been introduced [56]. It is based on the N ,N 0 -diallyl-L-tartardiamide monomer, which has been derivatized with any of several aromatic acid chloride moieties. This chiral moiety is then attached and crosslinked on silica gel that was previously silanized with an unsaturated organosilane. These CSPs are used in the normal phase mode [56]. Up to the present time, the natural occurring chiral polymers have had a far greater impact on LC enantioseparations than the synthetic or hybrid polymer CSPs. The natural polymers can be divided into two main types: (a) proteins; and (b) carbohydrates. 1.II.8.1. Proteins Bonded protein CSPs have played an important role in the analytical separations of enantiomers [57–59]. They are used in the reversed-phase mode with aqueous buffers or hydro-organic solvent systems. Early versions of these columns suffered from a lack of hardiness and longevity. However, the second generation of protein-based CSPs had

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much improved stability and efficiency. The α1 -acid glycoprotein column has proven to be particularly useful to the pharmaceutical industry for quickly resolving a variety of amine containing compounds. Because of the large size of the chiral selector, protein LC columns have the least capacity of any chiral stationary phases. Also, they still tend to be more labile than most other CSPs. For these reasons protein-based CSPs appear to be decreasing in importance (as a class) relative to other classes of CSPs. 1.II.8.2. Carbohydrates Cellulose and amylose are among the most common of naturally occurring chiral polymers. They are very poor chiral selectors in their native state. However, when their hydroxy-functional groups are derivatized (particularly with aromatic moieties via ester or carbamate linkages) and they are properly immobilized on a silica support, they become highly effective chiral stationary phases. The initial work on functionalized cellulose-based CSPs was done in Europe [60,61]. Later, Daicel Ltd. of Japan commercialized a series of different derivatized cellulose CSPs and eventually, derivatized amylose [62,63]. The most widely useful of these derivatized carbohydrate CSPs are those that contain 3,5-dimethylphenylcarbamate functional groups. Also these chiral selectors are adsorbed onto wide pore silica gel and not covalently attached as are most of the other CSPs. These chiral polymer CSPs can be used with supercritical fluids as can most of the other classes of CSPs.

1.II.9. Conclusions While LC has played a leading role in enantioselective separations, it is the combination of all of the often complementary separation techniques (LC, CE, GC, TLC), that has allowed today’s scientists to successfully address and solve most problems involving the resolution and analysis of enantiomeric compounds. The other important aspect of the enantiomeric separations work described herein, is that it has greatly enhanced our understanding of molecular recognition. CSPs also were beneficial in the study of chromatographic theory and mechanisms. For example, one can isolate and study chiral vs. nonchiral retention mechanisms. Enantiomers can be useful probe molecules since they have the same size, shape, solvent solubility, etc. Mechanistic studies involving chiral molecules and=or CSPs will undoubtedly increase in the foreseeable future.

References 1. 2. 3. 4. 5. 6. 7.

D.W. Armstrong, J. Liq. Chromatogr., 7 (S-2) (1984) 353. D.W. Armstrong, Anal. Chem., 59 (1987) 84A. D.W. Armstrong and S.M. Han, CRC Crit. Rev. Anal. Chem., 19 (1988) 175. A.M. Krstulovı´c (Ed.), Chiral Separations by HPLC, Ellis Horwood, Chichester, 1989. S. Ahuja (Ed.), Chiral Separations, Am. Chem. Soc., Washington, DC, 1997. E.J. Ariens, Clin. Pharmacol. Ther., 42 (1987) 361. E.J. Ariens, E.W. Wuis and E.F. Veringa, Biochem. Pharmacol. 37 (1988) 9.

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Chapter 5 Anonymous, Chirality, 4 (1992) 338. D.W. Armstrong, G.L. Reid, III, M.L. Hilton and C.-D. Chang, Environ. Pollut., 79 (1993) 51. D.W. Armstrong, C.-D. Chang and W.Y. Li, J. Agric. Food Chem., 38 (1990) 1674. K.H. Ekborg-Ott and D.W. Armstrong, Stereochemical Analyses of Food Components, in: S. Ahuja (Ed.), Chiral Separations, Am. Chem. Soc., Washington, DC, 1997, Chapter 9, p. 201. D.W. Armstrong, T.J. Lee and L.W. Chang, Tetrahedron Asymmetry, 9 (1998) 2043–2064. D.W. Armstrong, L. He, T. Yu, J.-T. Lee and Y.-S. Liu, Tetrahedron Asymmetry, 10 (1999) 37–60. G. Eglinton and M. Calvin, Sci. Am., 216 (1967) pp. 32–43. R.P. Philip, Chem. Eng. News, 64 (6) (1986) 28–43. D.W. Armstrong, Y. Tang and J. Zukowski, Anal. Chem., 63 (1991) 2858. D.W. Armstrong, E.Y. Zhou, J. Zukowski and B. Kosmowska-Ceranowicz, Chirality, 8 (1996) 39. J.L. Bada, in: G.C. Barrett (Ed.), Chem. Biochem. Amino Acids, Chapman and Hall, New York, NY, 1985, p. 6. K. Robbie, M.J. Brett and A. Lakhtakia, Nature, 384 (1996) 616. V.A. Davankov and S.V. Rogozhin, J. Chromatogr., 60 (1971) 280. V.A. Davankov, A.A. Kurganov and A.S. Bocklov, in: J.C. Giddings, E. Grushka, J. Cazes and P.R. Brown (Eds.), Advances in Chromatography, Vol. 22, Marcel Dekker, New York, NY, 1983. S.M. Han and D.W. Armstrong, Enantiomeric Separation by TLC, in: J.C. Touchstone (Ed.), Planar Chromatogr. Life Sci., Vol. 108, J. Wiley and Sons, New York, NY, 1990, p. 8. D.W. Armstrong, J. Liq. Chromatogr., 3 (1980) 895. W.L. Hinze and D.W. Armstrong, Anal. Lett., 13 (1980) 1093. D.W. Armstrong and W. DeMond, J. Chromatogr. Sci., 22 (1984) 411. D.W. Armstrong, W. DeMond and B.P. Czech, Anal. Chem., 57 (1985) 481. D.W. Armstrong, T.J. Ward, R.D. Armstrong and T.E. Beesley, Science, 232 (1986) 1132. J.A. Hamilton and L. Chen, J. Am. Chem. Soc., 110 (1988) 4379. A.M. Stalcup, S.C. Chang, D.W. Armstrong and J. Pitha, J. Chromatogr., 513 (1990) 181. D.W. Armstrong, A.M. Stalcup, M.L. Hilton, J.D. Duncan, J.R. Faulkner, Jr., and S.C. Chang, Anal. Chem., 62 (1990) 1610. S.C. Chang, L.R. Wang and D.W. Armstrong, J. Liq. Chromatogr., 15 (1992) 1411. D.W. Armstrong, S. Chen, C. Chang and S. Chang, J. Liq. Chromatogr., 15 (1992) 545. S.C. Chang, G.L. Reid III, S. Chen, C.D. Chang and D.W. Armstrong, Trends Anal. Chem. 12 (1993) 144. D.W. Armstrong, Y. Tang, S. Chen, Y. Zhou, C. Bagwill and J.-R. Chen, Anal. Chem., 66 (1994) 1473. D.W. Armstrong, Y. Liu and K.H. Ekborg-Ott, Chirality, 7 (1995) 474. A. Berthod, Y. Liu, C. Bagwill and D.W. Armstrong, J. Chromatogr. A, 731 (1996) 123. R. Helgeson, J. Timko, P. Moreau, S. Peacock, J. Mayer and D.J. Cram, J. Am. Chem. Soc., 96 (1974) 6762. G.D.Y. Sogah and D.J. Cram, J. Am. Chem. Soc., 98 (1976) 3038. M. Newcomb, J. Toner, R. Helgeson and D.J. Cram, J. Am. Chem. Soc., 101 (1979) 4941. T. Shinbo, T. Yamaguchi, K. Nishimura and M. Sugiura, J. Chromatogr., 405 (1987) 145. M. Hilton and D.W. Armstrong, J. Liq. Chromatogr., 14 (1991) 9. M. Hilton and D.W. Armstrong, J. Liq. Chromatogr., 14 (1991) 3673. L.H. Klemm and D. Reed, J. Chromatogr., 3 (1960) 364. F. Mikesˇ, G. Boshart and E. Gil-Av, J. Chromatogr., 122 (1976) 205. F. Mikesˇ and G. Boshart, J. Chromatogr., 149 (1978) 455. F. Mikesˇ and G. Boshart, Chem. Commun., 173 (1978). W.H. Pirkle and J.M. Finn, J. Org. Chem., 46 (1981) 2935. W.H. Pirkle and M.H. Hyun, J. Chromatogr., 322 (1985) 309. W.H. Pirkle, K.C. Deming and J.A. Burke, Chirality, 3 (1991) 183. N. Oi, M. Nagase and T. Doi, J. Chromatogr., 257 (1983) 111. N. Oi and H. Kitahara, J. Chromatogr., 265 (1983) 117. N. Oi and H. Kitahara, J. Liq. Chromatogr., 9 (1986) 443. N. Oi, J. Kitahara and T. Doi, European Patent # EP029793 (1988).

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54. W.H. Pirkle and C.J. Welch, J. Liq. Chromatogr., 15 (1992) 1947. 55. Y. Okamoto, S. Honda, I. Okamoto, H. Yuki, S. Murata, R. Noyori and H. Takaya, J. Am. Chem. Soc., 103 (1981) 6971. 56. S.G. Allenmark, S. Anderson, P. Moller and D. Sanchez, Chirality, 7 (1995) 248. 57. J. Hermansson, J. Chromatogr., 269 (1983) 71. 58. S. Allenmark, B. Bomgren and H. Boren, J. Chromatogr., 269 (1983) 63. 59. J. Haginaka, C. Seyama and N. Kanasugi, Anal. Chem., 67 (1995) 2579. 60. H. Hakli, M. Mintas and A. Mannschreck, Chem. Ber., 112 (1979) 2028. 61. K.R. Lindner and A. Mannschreck, J. Chromatogr., 193 (1980) 308. 62. Y. Okamoto, M. Kawashima, K. Yamamoto and K. Hatada, Chem. Lett., 739 (1984). 63. Y. Okamoto, R. Aburatani, T. Kukumoto and K. Hatada, Chem. Lett., 1857 (1987).

D.2. Ernst Bayer Ernst Bayer was born on March 24, 1927 in Ludwigshafen=Rhein, Germany, where he also visited primary and secondary schools from 1934 to 1947. From 1947 to 1952, he studied chemistry at the University of Heidelberg, and made his master thesis in physical chemistry. From 1952 to 1954, he completed his Ph.D. thesis under the advice of Nobel Laureate Professor Richard Kuhn, Max-Planck-Institut for Medical Research, Heidelberg University on the structure of hemovanadin, a vanadium compound occurring in marine tunicates. A complete presentation on the professional activities of Professor Ernst Bayer is detailed under the Editor’s section giving information on his education, books, professional positions; his pioneering research in HPLC and the hyphenation of separation methods with MS and NMR, his work on template chromatography using specific interactions of oligonucleotides and peptides, his research on miniaturized separation methods and a new MS detection method called coordination ion spray (CIS–MS). Also, a documentation is given of the many honors and awards that he has received, and the many positions in the German Chemical Society that he has held, and as a member of the editorial staff of several scientific journals and commissions. See Chapter 5B, a, b, d, e, f, h, i, k, l, p, r, s, t

2.I. PERSONAL VIEWS ON THE DEVELOPMENT OF CHROMATOGRAPHY Ernst Bayer Universita¨t Tu¨bingen, Research Center for Nucleic Acid, and Peptide Chemistry, Institut fu¨r Organische Chemie, 72076 Tu¨bingen, Auf der Morgenstelle 18, Germany

My first experience with chromatography dates back to 1952 to 1954 when I was doing research for my Ph.D. degree in the laboratory of Richard Kuhn, Heidelberg. Kuhn

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received the Nobel Prize in 1938 for his work on isolation and structure elucidation of carotinoids and vitamins and the success of his work was based mainly on his rediscovery of Tswett chromatography, together with his coworkers Edgar Lederer and A. Winterstein. He told us young students the early story of Tswett chromatography, and discussed the reasons for the lack of acceptance of the method. Richard Kuhn was a student of Willsta¨tter, and Willsta¨tter, one of the early pioneers in chlorophyll chemistry, was skeptical about Tswett’s chromatography after his lack of success in reproducing Tswett’s results in the years of 1912–1925. Nonetheless, Willsta¨tter translated the thesis of Tswett, written in 1910, into German and later passed this translation on to his student Richard Kuhn. It was Kuhn’s opinion that Willsta¨tter’s assistants had not selected suitable adsorbents from those originally described by Tswett and as a result the chlorophyll had decomposed on the column. The rather skeptical report of Willsta¨tter, who received the Nobel prize in 1915 for his investigations of chlorophyll and anthocyanines and who was the most famous chemist in the field of plant pigments between 1900 and 1930, discouraged most scientists from pursuing the method further. After Kuhn’s successful use of Tswett Chromatography, Willsta¨tter told Kuhn that his assistants had obviously failed to follow exactly Tswett’s recommendations. Kuhn told us that Willsta¨tter himself never had doubts about the potential of chromatography and in 1928 in a publication about the selective adsorption of enzymes, Willsta¨tter [1] wrote that, “one should not forget the success of the chromatographic adsorption analysis of Tswett.” Fortunately some scientists were courageous enough to apply chromatography e.g., Palmer and Eckles [2] in 1914 for the separation of carotene and xanthophyll. When the young E. Lederer joined Kuhn’s laboratory in Heidelberg in 1930 to investigate the isolation of pure carotinoids and elucidate their structure, he decided to try again Tswett’s adsorbents. Fortunately Richard Kuhn was still in possession of the translation of Tswett’s thesis given to him by Willsta¨tter, and remembered which adsorbents Willsta¨tter’s assistants unsuccessfully tried. With calcium carbonate, the separation of carotinoids was readily achieved and a series of papers about the chromatography of natural compounds was published by Kuhn and his coworkers, E. Lederer, A. Winterstein and H. Brockmann [3]. This was the renaissance of column liquid chromatography and now other laboratories such as those of P. Karrer in Zu¨rich and L. Zechmeister also took up the method. Besides history, we discussed and followed in Heidelberg closely the development of chromatography techniques. During this period between 1951 to 1954 several important chromatographic publications appeared and Kuhn invited the young scientist Egon Stahl to give a lecture about the rapidly growing field of thin-layer chromatography while Kuhn’s former coworker Theodor Wieland reported on his research on carrier-free electrophoresis. Also the introduction of gas chromatography by A.T. James and A.J.P. Martin in 1952 and the analysis of amino acids with ion exchangers by W.H. Stein and S. Moore in 1951 were subjects of our discussions. My scientific career was thus connected with separation science right from the very beginning and in the course of my Ph.D. thesis, I used both amino acid separation and electrophoresis.

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2.I.1. GC at the Wine Research Institute-Geilweilerhof In the spring of 1955, I left the stimulating atmosphere of Kuhn’s laboratory and accepted a position as Head of the Department of Biochemistry of the Government Institute for Wine Research, Geilweilerhof. One of our tasks there was to correlate the components of grapes before and after fermentation with the quality of wine. Especially the flavoring components and their development during ripening of the grapes, fermentation and aging were of interest. At this time no suitable analytical methods were available for volatile compounds. For this reason we first developed a method for the quantitative determination of esters and aldehydes by paper chromatography and were able to establish some interesting correlations with the taste of alcoholic beverages. Despite these results, the broad variety of volatile flavoring compounds were not revealed since no general method for the analysis of volatile compounds was available. At this point I remembered our discussions in Kuhn’s laboratory about the publication on gas chromatography by A.T. James and A.J.P. Martin. Between 1952 and 1955 only a handful of publications about the use of gas–liquid chromatography had appeared. Prior to 1955, the development of instrumentation and applications took place predominantly in a geographically relatively restricted area in the United Kingdom and in The Netherlands. However, in 1955, some commercial instruments were available in the USA, and by 1958 in Germany. The proximity of researchers in this region led to extremely fast exchange of information among the very early pioneers of gas–liquid chromatography, especially A.T. James and A.J.P. Martin (who invented the method), E.R. Adlard, T. Ambrose, D.H. Desty, J. Lovelock, C.S.G. Phillips, J.H. Purnell, R.P.W. Scott in the UK and J.J. van Deemter, A.J. Klinkenberg, A.I.M. Keulemans and G. Dijkstra in The Netherlands. The British Gas Chromatography Discussion Group was the exchange place, and the early meetings they organized, in the UK and Amsterdam in particular the great symposia of 1956 in London and 1958 in Amsterdam, were the highlights. As with many scientists in the rest of the world, I had to make contact with these early pioneers to participate in their experience and to build my own GC equipment. In 1955, I became acquainted with A.I.M. Keulemans through a Dutch company producing essential oils for which we both consulted. We arranged to visit several laboratories in the UK and the Netherlands and to see their home-built instruments for GC. Most of these we found to be unsuitable for our problems because temperatures up to 220ºC were necessary for the elution of volatile derivatives of amino acids and many essential oils. This demanded temperature-stable stationary phases and thermal conductivity cells. We therefore constructed a GC instrument capable of operation at temperatures up to 220ºC and demonstrated this instrument in late 1955 in a seminar held at the wine research institute at Geilweilerhof where Keulemans and I both gave a lecture. This was an important event for Germany, with numerous scientists and students from the universities of Karlsruhe, Heidelberg and even Aachen, and from companies such as BASF and Siemens attending. Siemens delivered the thermal conductivity cells trimmed for our use at elevated temperatures. How rudimentary the knowledge of GC was still at this time was shown by the fact that Kienitz, the director of analytical research of BASF, discussed with Keulemans and me whether it would also be

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worthwhile for BASF to construct and use a gas chromatograph. In the spring of 1956, Kuhn invited me and Keulemans to give our lectures and to demonstrate our instrument at the seminar of the Max-Planck-Institute in Heidelberg. This was a big success in my young career. In 1956 and 1957, our research concentrated on the separation of volatile amino acid derivatives and flavoring compounds of wine, as well as the first GC separation of a pheromone, that of the silk-worm moth, using the male moth as detector. I was very proud to be invited to present these results at the great symposium on GC in Amsterdam in 1958 [5], this symposium was probably the most important one in the history of GC, with M.J.E. Golay and G. Dijksta reporting on capillary gas chromatography, Lovelock on his ionization detectors and McWilliam on the flame ionization detector. The proceedings of the Amsterdam symposium certainly give an excellent impression of the dawning of gas chromatography. In January 1958, I made my ‘habilitation’ at the Technical University of Karlsruhe, and was appointed lecturer for organic chemistry. In 1962, I was offered the Chair of Organic Chemistry at the University of Tu¨bingen, taking up the position of Professor of Organic Chemistry and Director of the Organic Chemistry Department in October 1962. I started work in the field of preparative GC as early as 1957 at Geilweilerhof. Isolation and separation of small amounts of natural compounds was easy with GC. However for structural elucidation, it was necessary to isolate sufficient quantities for analysis by spectroscopic methods, such as IR, NMR, and MS since on-line coupling had not yet been invented. We thus started work to scale up GC to larger column diameters, which at this time was against the accepted theory in that separation efficiency decreases with the square root of the diameter of the column. We were able to show that this theory, in which the column is treated as a bundle of capillaries, is wrong on account of its neglect of radial diffusion, and we constructed and used a preparative gas chromatograph with columns of up to 10 cm diameter. Several engineering problems, especially associated with heat transfer, had to be overcome and I was fortunate to be able to engage an engineer, K.P. Hupe, with whom I have had a very fruitful cooperation since 1959. At this time no apparatus for large scale separation was commercially available. Using our home-built preparative gas chromatograph, we separated and identified the components of many essential oils, such as the flavor components from Bartlett pears, rum, cherries, cheddar cheese and mushrooms and investigated the equilibrium between furanose-, pyranose- and aldehyde-forms of monosaccharides.

2.I.2. GC research at Karlsruhe I should also mention a situation arising from my change of position from Geilweilerhof to the University of Karlsruhe. At the governmental research institute I had an excellent staff of technicians but was only able to take one technician with me when I moved to the Technical University of Karlsruhe. In addition it was 3 months before the first Ph.D. students joined my group. In this situation I found time to write my monograph “Gas Chromatography”, a practically oriented handbook published in German in 1958, with Russian and English translations appearing in 1960 and 1961, respectively [6]. The book proved very popular. The first edition 1958 had only to

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consider 300 references. When I wrote the second edition in 1962, there were already 3500 publications on GC, from which I selected 1300 citations. This shows the exceptional growth of GC within a few years, made possible especially by the availability of commercial instruments. Besides GC, I had already concentrated on other fields during my time at Geilweilerhof and I continued to follow these other interests in Karlsruhe. We designed extremely metal-specific polymers for the enrichment of metals. Their use became very popular later when we reported on the selective enrichment of gold, uranium, and copper from seawater from the Gulf of Naples. We were also successful in isolating the substance responsible for the blue color of cornflowers by means of preparative, carrier-free electrophoresis. The coloring principle was found to be a chelate of the anhydrobase of cyanin. This was in contrast to the publications of Willsta¨tter and Karrer, who considered the blue color of cornflowers to be an alkaline salt of cyanin whereas the red color of roses is due to cyanin in an acidic cell medium. It could be very easily shown that the cell medium of cornflowers is slightly acid, and that the blue color is stable even at pH 3, where an alkaline salt of phenols is not existent. These publications of a young scientist just 30 years of age challenging the views of very established scientists were noted with interest. Although our work on preparative GC together with Hupe made good progress, no instrument company was prepared to risk commercializing it. I still remember a discussion with an instrument company, where E. Kova´ts and myself tried to convince them to build a preparative gas chromatograph. Most companies just recommended repeated separations on an analytical column while Beckman, under the impression of the invalid ‘bundle of capillaries’ theory, constructed the preparative GC ‘Megachrome’, in which the mobile phase was split after injection onto 8 columns in parallel and reunited before entering the detector. This system was very difficult to regulate, and was very soon taken off the market. Our approach of larger diameter columns still proves sound. When I moved to Tu¨bingen, Hupe decided to stay in Karlsruhe and in 1962, together with an electronic engineer, he set up the company Hupe-Busch. Working at the beginning in a kitchen, they successfully commercialized our preparative GC instrument. In addition Hupe bought the license to commercialize the glass capillary drawing machine designed by Desty. This was very important for the survival of capillary GC in the scientific community, because Perkin Elmer, the exclusive holder of the Golay patent, distributed solely metal capillaries that were not suitable for labile compounds. At least in research laboratories, including our laboratory in Tu¨bingen, capillary GC could be used. It was only later, when the mechanically more stable fused silica capillary columns were introduced and the Golay patents also expired, that capillary GC found the wide distribution it now enjoys. By 1958, the development of GC was no longer a privilege of northern Europe, but spread out over the whole scientific community and to the benefit of humanity. It entered industry and even changed the entire face of analytical chemistry as a result of increasing automation and miniaturization. This in turn had a tremendous impact on life sciences and earth sciences where e.g., it enabled for the first time analysis of the world-wide distribution and transport of halogenated toxic substances such as herbicides. Before the advent of GC, there was no method available for analyzing traces of herbicides and insecticides, such as dieldrin or DDT, in the ice of the Antarctic or

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in seawater. With the halogen-specific detectors of Lovelock, this was possible and, together with the observations of environmentally oriented observers, these analyses sharpened our perception of the dangers of the uncontrolled use of toxic chemicals. It was luck for humanity that at the right time these trace analysis detectors were invented and halogenated compounds could be monitored in concentrations just still below dangerous toxic activity. Regulation then could be established. Globalization of GC after 1958 and the availability of commercial instruments led to the establishment of chromatographic discussion groups in most countries, and symposia were organized. My personal contacts extended especially to France, where the GAMS organized several meetings, and I became acquainted with G. Guiochon. Also contacts were established beyond the iron curtain to colleagues of the former German Democratic Republic, especially R. Kaiser, and Russian colleagues such as K.I. Sakodynskii and others. The relations to Keulemans were maintained. He moved from the Shell Laboratories to occupy a newly created chair of instrumental analysis at the University of Eindhoven. Among his coworkers were the young scientists C. Cramers, E.B. Everaerts and J.F.K. Huber, a former student of E. Cremer at the University of Innsbruck, Austria. My first contacts to the United States were established to A. Zlatkis in the University of Houston, Texas.

2.I.3. GC in Tu¨bingen and the wider scientific community In Tu¨bingen, I was able to build up a strong group for natural compound chemistry. The research on flavor compounds, sugars and preparative GC continued. In the meantime, as in France and the United Kingdom, a working group on GC was also established in Germany (Arbeitskreis Chromatographie) and G.E. Hesse was elected as the first chairman. Several young scientists were very active in this group: G. Schomburg, important for development of capillary gas chromatography, I. Hala´sz, who came to Germany in 1956 from Hungary and, after some years in industry, moved to Frankfurt as a lecturer and later to Saarbru¨cken to occupy the chair of applied physical chemistry. It was Hala´sz who, among other important contributions, introduced chemically modified silica for gas chromatography and was later the father of reversed phases for HPLC. Many important scientists in the field of separation methods such as C. Horva´th and H. Engelhardt were his coworkers. Also, Henneberg, who reported as early as 1959 on the on-line coupling of gas chromatography to mass spectrometry, belonged to our group. It took another five years before the first commercial GC–MS (produced by LKB and using the Becker-Ryhage jet interface to separate the analyte from the excess of carrier gas) became available. Other members were E. Stahl, active in the development of thin-layer chromatography, L. Rohrschneider, working on the selectivity of stationary phases, H. Kelker and R. Kaiser, who moved in 1961 from the German Democratic Republic to West Germany. Considering the now global distribution of GC, the British Gas Chromatography Discussion Group decided for the first time to hold their 1962 meeting outside of the UK and Holland. In Hamburg and, under the auspices of the German Chemical Society, our German Arbeitskreis was the local organizer. Later on the Chromatography Discussion Group, the French GAMS and the

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German Arbeitskreis collaborated to biannually organize these meetings, which became the most important chromatographic events in Europe. In the USA, A. Zlatkis organized a very important series of meetings since 1963 (see Chapter 3D). From Germany, I. Hala´sz, G. Schomburg and I participated at the first meeting in Houston and in many other consecutive meetings. It was in 1965, at one of the early meetings in Houston that the discussions started as to whether conventional column liquid chromatography should not be replaced by an approach, which took into account the theory of gas chromatography, especially the van Deemter equation. C. Giddings, A.I.M. Keulemans, C. Horva´th, J.F.K. Huber, G. Guiochon, I. Hala´sz, R.P.W. Scott and V. Pretorius were particularly active in these discussions between 1965 and 1968. It was quite obvious what was required for optimization of liquid chromatography — the particle size of the stationary phase had to be reduced for better separation efficiency. This in turn required a higher pressure to drive the mobile phase through the column. In addition, the question of detection had to be resolved. Probably the first international symposium at which HPLC was a broader issue was the Zlatkis meeting in New York in 1967. It took still more than five years until 1973, in Interlaken, that the series of excellent meetings on column liquid chromatography was inaugurated by G. Guiochon, J.F.K. Huber, K.P. Hupe, B.L. Karger, J.J. Kirkland, J.H. Knox and W. Simon. At the meeting in 1967 different groups already reported on HPLC, predominantly under isocratic conditions, and with normal phase stationary phases. At this meeting I reported about the Micro Adsorption Detector (MAD), a generally applicable detector for normal phase HPLC using organic solvents as mobile phases. It was based on monitoring the heat of adsorption=desorption of the analyte. If there is any interaction of the analyte with the stationary phase, the heat of adsorption associated with this interaction can be measured. Together with K. Hupe we designed an HPLC instrument using this MAD detector which was commercialized by the Hupe-Busch Company in 1968. The MAD was excellent for normal phase HPLC with organic solvents, however less sensitive for the polar solvents used in reversed-phase HPLC which became popular since the early 1970s. Therefore Hupe also used UV-detection in his instrument. Around 1970 to 1971, the company of Hupe had a considerable share of the European HPLC market, and his company had to move to new buildings to keep pace with the exponential growth of HPLC. At this point the larger instrument companies decided to step with full power into the HPLC market, and finally Hewlett-Packard acquired the company Hupe-Busch, later moving to Waldbronn, where they still manufacture HPLC instruments. This is a good example of technology transfer from the university to a small company and finally to large scale manufacturing. A small company would not have had the financial capacity to stay competitive in this fast growing field of HPLC. This was an early example of the development chain from science to start-up company and finally to larger companies.

2.I.4. Chromatography research at the University of Houston In January 1967, a considerable proportion of my activities was transferred to the United States, where I accepted the Robert A. Welch Chair at the Chemistry Department,

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University of Houston, Texas, occupying it until 1970. Besides HPLC investigations, the continuing research on metal sequestering polymers and our work in the field of metal proteins, I started investigations on solid phase peptide synthesis, an ingenious method invented by R. Merrifield. In 1968, we synthesized some hormones such as oxytocin, and the larger polypeptide apoferrodoxin with 55 amino acids. Subsequently we had to investigate the purity of the synthesized peptides using liquid chromatography and GC– MS. Also racemization was a big issue. Fortunately E. Gil-Av from Israel, the pioneer of GC enantiomer separation, was a visiting professor at the University of Houston from 1967 to 1969, and was involved in investigations of the moon samples from the APOLLO-projects together with Oro´. The investigation of racemization of amino acids was a crucial point. If amino acids were found in moon rocks as an indication of extraterrestrial life, they should be racemized, whereas contaminations from earth should not be racemized. Gil-Av and Oro´ did not find any amino acids at all in the moon samples. However in a joint paper with these authors we investigated the racemization in peptide synthesis. Unfortunately the chiral phases available at that time lacked thermal stability. We synthesized many chiral phases for GC, and finally in 1976, together with H. Frank and G.J. Nicholson we were successful in coupling L- or alternatively D-valine to a copolymer of dimethylsiloxane and carboxyalkylmethylsiloxane to form a chiral polysiloxane which was sufficiently temperature-stable to allow separation of the enantiomers of all proteinogenic amino acids. Since both enantiomers of these chiral polysiloxanes are available, inversion of the order of elution of peaks can be used to identify enantiomers. This is an advantage of ‘Dand L-Chirasil-Val,’ as we subsequently named these phases. Even today, Chirasil-Val is still in use. It was the first example of immobilized chiral groups on polysiloxanes, an approach taken up later by numerous authors for many chiral groups. Our continued interest in polymer-supported peptide synthesis led to further engagement in peptide and amino acid analysis. From 1974, we investigated selective peptide–nucleotide interactions and their use for separation, which we called Template Chromatography. In 1976, we separated the dansyl derivatives of amino acids and peptides by HPLC using fluorescence detection, and reported limits of detection of only a few femtomoles. We isolated and elucidated the structure of some peptide antibiotics such as phosphinotricin and of the vanadium compound of the mushroom Amanita muscaria, which we named amavadin. However, probably the most important contribution over the years after 1967 was the very careful analysis of the peptides synthesized by the solid phase method using GC, MS and HPLC [7]. Very early we were able to detect the so-called ‘failure sequences’. Since the reaction steps in a polymer-supported synthesis do not proceed with 100% yield even with large excesses of coupling components truncated and also deleted sequences result, and these can only be separated at the end of the synthesis. Even if every coupling and deprotection step proceeds with 95% yield and a separation of all failure sequences is possible, the theoretical yield of a 30-mer polypeptide such as the insulin B chain is only 4.7%, and for a 124-mer such as ribonuclease only 0.0002%. Thus, in addition to the main product, n-1 failure sequences with one amino acid less can be expected and even today no separation method can reliably separate these contaminants if n exceeds 20–30. In 1967, this limit lay much lower, below

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n D 10–15, and the solid phase peptide synthesis (SPPS) technique had not yet been optimized. In 1973, we introduced HPLC for the separation of synthetic peptides, and this subsequently became the standard method for SPPS. We optimized the polymer carriers to improve coupling yields and in 1971, together with M. Mutter and H. Hagenmaier, we introduced the liquid phase polymer supported peptide synthesis using polyethylene glycol as soluble polymer, later (1985), together with W. Rapp, using as a hybrid the graft–copolymers of polystyrene–polyethyleneglycol [7]. There is no doubt that the parallel development of analytical methods contributed in a decisive manner to the success of polymer supported synthesis. Without the development of HPLC, SPPS probably would have died out in 1970 to 1975. Our analytical investigations on the failure sequences were sometimes a thorn in the flesh of uncritical users of the solid phase peptide synthesis, but they finally helped to establish this fine method while also explaining the restriction of the method to synthesis of proteins with a molar mass of less than 12,000 Da because of the limits imposed by the separation techniques available. Using soluble polymers as a support for metal-containing active centers, we also developed a new class of homogeneous catalysts which can be separated from products by ultrafiltration. This solves one of the basic problems of the lower molecular-mass homogeneous catalysts. Such soluble polymers with chelating groups can also be utilized to separate polymer-bound metals from non-bound metals by ultrafiltration after metal binding, this representing the only metal-sequestering method in homogeneous solution. Since 1977, we have investigated continuous flow NMR spectroscopy and their on-line coupling with HPLC together with K. Albert and Bruker Physik. When we started this work, many obstacles had to be overcome, and it was a long-term, high-risk project. We were however successful and HPLC-NMR on-line coupling is nowadays commercially available and has found application, especially for problems where coupling with MS does not yield sufficient structural information. Very detailed investigations of the structures of reversed-phases were performed since 1983 using 29 Si and 13 C cross-polarization and magic angle spinning NMR. Together with K. Unger, in Mainz, and M. Hearn, in Australia, correlations of the conformation of peptides with the dynamic behavior of the n-alkyl ligands of the reversed-phases could be established. Fruitful cooperation in this project with the group of C. Cramers in Eindhoven should also be mentioned.

2.I.5. Chromatography research during retirement A series of recent publications deals with the use of pulsed field gradient NMR of the spatial and time resolved axial and radial dispersion coefficients. The cooperation with G. Guiochon and the enthusiasm of my former coworker U. Tallarek was important for the progress in this area. This method affords ready determination of some of the characteristics of the kinetics of mass transfer in chromatography. Thus it is possible to determine the amount of mobile phase exchanged between the stream of fluid percolating through the bed and the pools of stagnant fluid in the particle pores. In the last five years since my retirement, I have concentrated on the miniaturized

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separation methods of capillary HPLC, capillary electrochromatography and capillary electrophoresis, together with their on-line coupling with MS and NMR. In 1994, we described an instrument that can be used alternatively for all three separation methods even under conditions of gradient elution. This instrumentation can be used without interface for coupling to electrospray MS, e.g., capillary electrochromatography–MS (1999). For on-line coupling to NMR, nanoscale NMR with detection cells of 80–240 nl has been developed and a routine interface constructed with high resolution and limit of detection (LOD) down to lower ng. 2D NMR techniques such as correlation spectroscopy (COSY) and total correlation spectroscopy (TOCSY) are also possible with this setup. Applications for peptides, oligonucleotides, drug metabolites and beer bitter compounds demonstrated the progress achieved with this method [8]. In 1998, we published a new mass spectrometric detection method for on-line coupling with separation methods, which we called Coordination Ionspray MS (CIS–MS). CIS–MS is based on the on-line post-column formation of charged coordination complexes which then can be sprayed and transferred to an MS detector, without requiring a high voltage electric field or other physical impact. Non-polar compounds which normally are not sensitively detected by electrospray MS such as sugars, fully protected peptides, olefines, terpenes, saccharides, lipids etc., can be detected by forming positively charged silver or palladium complexes or negatively charged boron complexes. In MS–MS experiments, new fragmentation patterns of coordinated product ions facilitate structural elucidation, e.g., the position or stereochemistry of double bonds in lipids. Another field of common interest is the design of a series of charged nanoparticles to selectively extract oligonucleotides from biological matrices like antisense-oligonucleotides and PCR products, and submit these to electrospray ionization (ESI–MS) and tandem mass spectrometry. A series of publications starting from 1997 describes this development for the analysis of nucleotide drugs. Since my retirement I have had the privilege of being able to continue my research in the Center of Peptide and Nucleotide Chemistry of the University of Tu¨bingen where the contact with my young coworkers keeps me active, and I can still discuss scientific questions with my friends at meetings.

2.I.6. Environmental chemistry and ecology Environmental chemistry and analysis was never a principal facet of my research. However, I encouraged my former coworkers H. Hagenmaier and P. Krauss to enter this field, where especially Hagenmaier made significant contributions to the analysis and the mechanism of formation of chlorinated dibenzodioxins and dibenzofurans. One field in my research with ecological implications was the invention of a process to convert biomass rich in lipids and proteins to an oil which is very similar to diesel fuel. We were interested in the origin of petroleum, and it is well established that oil originated by degradation of biomass under relatively mild temperature conditions (<300ºC); however, on the other hand lipids are stable at these temperatures, thus we postulated that a catalyst must have participated in the conversion and that such catalysts should be present in oil shales.

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Using oil shales obtained from the south of Tu¨bingen, we extracted the oil, mixed the ground inorganic residue with lipids, proteins and sewage sludge and subjected this material to temperatures between 280 and 400ºC under exclusion of oxygen. With such catalysts, an oil rich in aliphatic hydrocarbons was produced. With sewage sludge from industrial and domestic sources, a kind of diesel oil could be obtained in yields of 20–30%. The products were subjected to very careful analysis. It could be shown that no condensed toxic aromatic hydrocarbons or dioxins are formed. Even if dioxins were present, they are destroyed by this catalytic low temperature conversion. Together with Canadian and Australian colleagues, especially T. Bridle, we developed low temperature conversion to a technical scale, with plants planned for operation in 2000 in Germany and Australia.

2.I.7. Conversion of biomass to oil This process is also excellent for treatment of renewable biomass such as agricultural waste. Thus we began activities in Brazil treating different kinds of biomass, e.g., also waste from sugar cane production. In contrast to developed countries, 70% of Brazil’s primary energy still comes from renewable resources. For sustainable development of the Southern Hemisphere, it is important that they rely on their special resources, which must be supported. In the frame of this work, I was asked to give my consent to an Institute for Sustainable Development of the Southern Hemisphere, associated with my name. However since climate change affects one increasingly with increasing age, I abandoned this plan and now I prefer to travel once or twice a year to visit the very active group working in Brazil on low temperature conversion. For the development and optimization of the process, the analysis was the most important part and new methods for the quantitative analysis of unsaturated hydrocarbons were developed. The opportunity to travel in countries with different cultures is certainly very rewarding for a scientist. Besides the scientific discussions, gaining an understanding of foreign cultures, their history and their present development has always been an important aspect for me. Very often it corrects one’s views which tend to be limited by one’s own surroundings and culture. Similarly, visits of scientists from foreign countries, from western countries, but also from the near and far east, from Africa and China, contribute in this respect. I had approximately 35 Ph.D. students, 35 postdoctoral fellows, and 31 visiting professors from foreign countries during my career, not to mention the numerous visiting guests for a few days. It is rewarding for me that many look back with pleasure on their time in Tu¨bingen and keep in contact. My German coworkers have also always taken advantage of these contacts with foreign scientists.

2.I.8. My role in teaching and administration Teaching students is one of the important tasks of a university professor, especially when it comes to the highest rank, the graduate students. I had approximately 220 German Ph.D. students, and quite a number of German postdoctoral fellows, most of

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who found their employment in industry. However a considerable proportion also took up an academic career at universities. From my coworkers, 22 received professorships in various fields, from organic chemistry, biochemistry to environmental chemistry. Many also have contributed to the field of separation techniques: K.P. Hupe, K. Albert, H. Frank, H. Hagenmaier, G. Jung, W. Ko¨nig, P. Krauss, J. Metzger and V. Schurig. One cannot avoid that besides research and teaching, duties in administration and organization must be done. I served as Dean of our faculty, as Vice-President of the University from 1976 to 1979, and as President of the Senate. In the German Chemical Society I served from 1981 to 1997 on the Board of Directors, from 1993 to 1996 as Vice-President, and from 1972 to 1988 as Head of the Arbeitskreis Chromatography. Maybe more important, however, was my activity as head of the ‘GDCh-Advisory Committee on Existing Chemicals’ from 1982 to 1998. To advise the German government on the regulation of existing chemicals, the Federal Ministry of Environment of Germany and the Chemical Industry agreed in 1992 to set up a commission with members from all relevant public environmental agencies, the chemical industry, and scientists. They should identify chemicals with environmentally dangerous potential, write comprehensive reports and, if necessary, propose regulations. The agreement was that both government and industry would follow the recommendations of the advisory committee. We scrutinized about 1200 substances, published reports on approximately 300 substances and proposed regulations ranging from total banning to restrictions in production and use. This was an excellent model for cooperation between industry and government for the benefit of the environment. When I retired from this important commission, the responsibility for existing chemicals was transferred to the control of the European Community. This advising committee was very time-consuming. However I always thought that I have to give back some of my time in exchange for the freedom a university professor enjoys. We have the privilege of being free to select the topic we want to study, while a considerable proportion of the research funding comes from public organizations.

References 1. 2.

3. 4. 5. 6. 7. 8.

R. Willsta¨tter, Untersuchungen u¨ber Enzyme I, Berlin, Julius Springer, 1928, p. 67. L.S. Palmer and C.H. Eckles, Carotin — The principal natural yellow pigment of milk fat: its relations to plant carotin and the carotin of body fat, corpus luteum and blood serum. I: The chemical and physiological relation of the pigments of milk fat to the carotin and xanthophylls of green plants, J. Biol. Chem., 17 (1914) 191–210. R. Kuhn, A. Winterstein and E. Lederer, The xanthophylls, Z. Physiol. Chem., 197 (1931) 141–160. S. Moore, W.H. Stein, Chromatography of amino acids on sulfonated polystyrene resins, J. Biol. Chem., 192 (1951) 663–681. E. Bayer, in: D.H. Desty (Ed.), Gas Chromatography 1958, Butterworths, London, 1958, 333 pp. E. Bayer, Gaschromatographie, Springer-Verlag, Berlin, Germany, 1st edn., 1958; 2nd edn., 1961. E. Bayer, Gas Chromatography, Elsevier, Amsterdam, The Netherlands, 1961. E. Bayer, Protein synthesis, Angew. Chem. Int. Ed. Engl., 30 (2) (1991) 113–129. P. Gfro¨rer, J. Schewitz, K. Pusecker and E. Bayer, On-line coupling of capillary separation techniques with 1H NMR, Anal. Chem., 71 (9) (1999) 315A– 321A.

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D.3. Morton Beroza Morton Beroza was born on March 7, 1917 in New Haven, Connecticut, USA. His advanced education was at George Washington University, Washington DC, B.Sc. (1943), and at Georgetown University, Washington, DC, M.Sc. (1946) and Ph.D. (1950). After active duty with the US Navy (1943–1946), he served from 1948 to 1974 as a chemist with the US Department of Agriculture (USDA). He was a Group Leader for several projects in the Entomology Research Division, Agricultural Research Service, USDA, and finally served as Chief, Organic Chemicals Synthesis, Agricultural Environmental Quality Institute, USDA, Beltsville, MD, USA. He organized and served as Editor or Co-editor of key symposia of the American Chemical Society on juvenile insect hormones and insect pheromones (attractants) [1] and has written a comprehensive book on controlled release pheromone systems [2]. He was the Scientific Coordinator for the Association of Official Analytical Chemists from 1975 to 1981 [3]. He was an adjunct professor of the American University, Washington, DC and served as mentor for six graduate students. He was an Advisory Board member of seven national=international journals, served on many professional organizations and has lectured widely in the United States and many foreign countries. He has received many professional awards — some of which are: the American Chemical Society Award in Chromatography and Electrophoresis-1969, Harvey W. Wiley Award in Analytical Chemistry of the Association of Official Analytical Chemists (AOAC)-1970, ACS International Award for Research in Pesticide Chemistry-1977. Within the USDA, he has received five Superior Service Awards and seven Certificates of Merit. However, the most significant two are the USDA-Agricultural Research Service (ARS) Hall of Fame Award in 1997 and the Sterling B. Hendricks Memorial Lectureship of ARS, which was presented at the August 22–26, 1998 meeting of the American Chemical Society’s Division of Agrochemicals. Since his retirement from the USDA (January 1974), Dr. Beroza has served as a consultant for a number of organizations including: ž Health-Chem. Corp, NJ — insecticides, pheromones, etc. ž Association of Official Analytical Chemists (AOAC), VA — revised seven manuals for the Environmental Protection Agency (EPA), VA, on pesticides, quality control and gas chromatography; ž Campbell Soup Co., NJ — analytical chemistry, particularly pesticide residues; ž Whitaker Corp., PA — synthesis of insect pheromones; ž Clement Associates, VA — pesticides and other environmental chemicals; ž Midwest Research Institute, MO — research on 2,4,5-T herbicide; ž Bristol Myers Squibb, NJ — pharmaceutical problems. See Chapter 5B, d, k, s

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3.I. INSECT SEX PHEROMONES: SOME CONTRIBUTIONS FROM CHROMATOGRAPHY Morton Beroza * Agricultural Environmental Quality Institute, US Department of Agriculture, Beltsville, MD, USA (retired)

The investigations of Morton Beroza have been expressed in 315 research publications; chapters in books, authored books and 25 patents. The subjects covered include: synthesis and analysis of insecticides, five alkaloids, spectroscopy, isolation and determination of structure of constituents of plants and insects, analysis of pesticide residues (methods for over 100 compounds were developed), instrumental analysis, chromatography methods to determine the structure of compounds (see below), the identification of insect sex pheromones and the technology to use them effectively in pest management [1–3]. Early studies concerned the Mediterranean fruit fly (Ceratis capitata), which was a major pest in Florida fruit orchards; the sex attractant was identified as sec-butyl ester of 6-methyl-3-cydohexene-1-carboxylic acid — now known as siglure. Later the commercial product was found to be a less effective lure in the traps set out in infested areas than the laboratory-prepared compound [4]. When the synthetic method was modified to prepare the cis-isomer, partition column chromatography (silicic acid) showed separation of the cis- and trans-isomers of the commercial product. Further research showed that the trans-product was the potent isomer and that the commercial product had only 70% of the trans-isomer; this accounted for its lower activity as an attractant [4]. He was responsible in 1965 for a key concept to identify pesticides by ‘p-values’ [5]. Since positive identification of trace components in the nanogram range is crucial, and the then conventional analytical procedures were inadequate, he proposed confirmation by partition values (‘p-values’) — the ratio of distribution of the component between two immiscible phases in a binary solvent system. Beroza and Bowman developed ‘p-values’ for over 100 pesticides and related substances [5]. Subsequently AOAC verified and adopted the ‘p-value’ procedure. As a teenager, he worked on machinery in his father’s shop (a skilled tool and die maker). As a result, he was later able to build equipment for his laboratory research; some of the instrumentation he developed are: ž Gas chromatographic apparatus to help determine the carbon skeleton of a compound by instantaneous catalytic removal (with a hydrogen carrier gas) of oxygen, nitrogen, halogens, sulfur; and reduction of multiple bonds prior to GC analysis of the hydrocarbon products with flame-ionization or thermal-conductivity detectors [6]. ž A micro-ozonizer coupled with a gas chromatograph to detect double bonds in organic compounds (only 1–5 µg needed) [7] (available through Supelco Co., PA). ž A compact tube heater to concentrate dilute solutions of pesticide residues, or other compounds [8]. The heater concentrates 10 ml of solvent to less than 1.0 ml in *

Present address: 821 Malta Lane, Silver Spring, MD 20901, USA

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Fig. 1. These leafless forests seem to be late fall or winter, but the photograph, taken in July, shows Dr. Beroza and staff inspecting the severely defoliated trees due to the gypsy moth (Cape Cod, MA).

20–30 min, automatically turns off and requires no manual attention (manufactured by Kontes Glass Co., NJ). ž A TLC plate densitometer to provide quantitative scans at selected wavelengths of micrograms of solute separated on the plate. It is based on a sealed, unitized fiber-optic head that detects light reflected from the plate [9]. Electrophoretic scans, as used in genome chemistry, may also be run. The apparatus is one of the first to apply fiber optics in a scientific instrument. To return to insect sex attractants (pheromones), the gypsy moth was brought to Medford, MA in 1869 to start a silk-producing industry. About 20 years later, their caterpillars “were all over the streets and homes of Medford. The leaves of virtually every tree and bush were stripped clean” [10,11]. An early estimate observed that the insect stripped the leaves of 400,000 acres of timber per year. After further spread, the moth threatened the eastern seaboard hardwood forests, but also could defoliate shade trees, fruit trees and ornamentals (Figs. 1 and 2). The virgin female does not fly, but

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Fig. 2. A single gypsy moth caterpillar (about 2 in. long) may eat several leaves daily. Three successive years (1970s) of gypsy moth infestation killed more than 1.0 million oaks, 39,000 Eastern hemlocks, and 98,000 white pines near Newark, NJ.

emits an odor to which the male, a strong flier, responds. Traps contained a female moth, or an extract of the female moth, or the pure attractant (only 2 pg showed activity) as bait, lured the male moth, which became entangled in a sticky substance (Fig. 3). Since a female gypsy moth after mating lays about 400 eggs, the attractant could be used for both detection and for control. Beroza and colleagues used 78,000 female tips (the last two abdominal segments that contain the attractant) to isolate a few µg of the pure attractant (Fig. 4) by extraction steps, GC chromatography on Florisil and TLC on silica gel; bioassays with live male moths and GC–MS were relied on to detect the attractant [11]. Degradation of the 10–15 µg pheromone precursor and the few µg of the isolated natural sex lure and then subsequent synthesis (Fig. 5) confirmed the structure as cis-7,8-epoxy-2-methyloctadecane [11–15]. At least 21 other insect sex attractants are known and are usually long chain alcohols, acids, acetate esters or terpene-like compounds [13]. Developments in GC and TLC, plus MS, IR, UV and NMR spectrometers have facilitated this research on only mg amounts of the attractants [13–15].

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Fig. 3. Gypsy moth trap that contains 0.1 µg disparlure per trap which lasted at least 3 months in the field [11].

His major research contributions are best summarized in the words presented for his USDA-Agricultural Research Service Award in 1997: “Morton Beroza has an international reputation for discovering ingenious and inventive tools for controlling insect pests safely within their ecological domain. He developed pest-control strategies that used chemicals that do not linger in the environment. His scientific legacy influences insect-pest-management programs of Federal, state, local, and international agencies. Beroza revolutionized and inspired research worldwide with his discovery of the gypsy moth pheromone, or sex attractant. Disparlure is now used as the bait to trap male gypsy moths in more than 400,000 traps each year in the United States. The traps detect the presence of the chewing pest that defoliates an average of 3.3 million acres of hardwood trees per year. Recognizing that multiplying in massive numbers can be an insect species’ best survival strategy, Beroza was the first to propose using sex pheromones to confuse and disrupt

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Fig. 4. Fractionation of gypsy moth sex attractant [11].

Fig. 5. Synthesis of the gypsy moth sex attractant [11].

mating. Farmers now use this confusion tactic to protect apples, cotton, corn, and other crops from some insect pests. He also developed trimedlure, TML, for detecting the destructive Mediterranean fruit

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fly. Today, TML is used to bait medfly detection traps wherever fruit is grown. In the US, such traps help protect over $4 billion of agricultural commodities. To help fight the latest outbreak of medfly in Florida, officials distributed 17,000 traps baited with TML. Beroza also discovered or contributed to the development of other attractants used to trap melon flies, Japanese beetles, house flies, fire ants, corn earworm, and many other pests.”

Dr. Beroza’s most recent award is the Sterling B. Hendricks Memorial Lectureship, which was presented at the August 22–26, 1998 meeting of the Division of Agrochemicals of the American Chemical Society. Reflecting his earlier described four instruments, this lectureship recognized that he: “ : : : invented numerous microanalytical techniques including ‘carbon skeleton chromatography’ for rapid identification of the carbon backbone of microgram quantities of a variety of unknown compounds. This technique and others he devised greatly facilitated the elucidation of the chemical structure of biologically active compounds from insects and other natural sources. All these techniques have been, and continue to be, used by analytical chemists all over the world : : : ”

The above 1950–1970s research continues to the present with advances in microencapsulation and controlled release of pesticides for cockroaches, termites and other insects [16] — a summary of 18 papers presented at the Division of Agrochemicals, American Chemical Society’s meeting, August 20–24, 2000. Prepared by Dr. Robert L. Wixom

References 1.

2. 3.

4. 5. 6.

M. Beroza (Editor or Co-Editor), Symposia of the American Chemical Society, Division of Pesticide Chemistry, Washington, DC, USA: Chemicals Controlling Insect Behavior, Academic Press, 1970, 170 pp; Chemistry and Action of Juvenile Insect Hormones, Academic Press, 1972, 341 pp; Pest Management with Insect Sex Attractants and Other Behavior-Controlling Chemicals, Am. Chem. Soc., Symp. #23, 1976, 192 pp; and Insect Pheromone Technology: Chemistry and Applications, Am. Chem. Soc., Symp. #190, 1982, 260 pp. A.F. Kydonieus and M. Beroza (Eds.), Insect Suppression with Controlled Release Pheromone Systems, CRC Press, Boca Raton, FL, USA, 1982, 2 Vols., 586 pp. R.R. Watts (Ed.), M. Beroza and R.L. Caswell for revisions, Analytical Reference Standards and Supplemental Data for Pesticides and other Organic Compounds, Office of Research and Development, U.S. Environmental Protection Agency and Environmental Toxicology Division, Association of Official Analytical Chemists, 1980 revision, 182 pp. N. Green and M. Beroza, Cis–trans isomers of 6-methyl-3-cyclohexene-1-carboxylic acid and their sec-butyl esters, J. Org. Chem., 24 (1959) 761–764. M. Beroza and M.C. Bowman, Identification of pesticides at nanogram levels by extraction p-values, Anal. Chem., 37 (2) (1965) 291–292. M. Beroza, Determination of the chemical structure of microgram amounts by gas chromatography, Anal. Chem., 34 (1962) 1801–1811; M. Beroza and R. Sarmiento, Determination of the carbon skeleton and other structural features of organic compounds by gas chromatography, Anal. Chem., 35 (1963) 1353–1357; M. Beroza and R. Sarmiento, Carbon skeleton chromatography using hot-wire thermal conductivity detection, Anal. Chem., 36 (1964) 1744–1750; M. Beroza and R. Sarmiento, New catalyst and technique for analyzing fatty acids, their esters and long-chain compounds by carbon-skeleton chromatography, Anal. Chem., 37 (1965) 1040–1041. M. Beroza, Determination of

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Chapter 5 the chemical structure of organic compounds at the microgram level by gas chromatography, Acc. Chem. Res., 3 (1970) 33–40. M. Beroza, Microanalytical methodology relating to the identification of insect sex pheromones and related behavior-control chemicals, J. Chromatogr. Sci., 13 (1975) 314–321. M. Beroza and B.A. Bierl, Apparatus for ozonolysis of milligram amounts of compounds, Anal. Chem., 38 (1966) 1976–1977; ibid, Rapid determination of olefin positions in organic compounds in microgram range by ozonolysis and gas chromatography, Anal. Chem., 39 (1967) 1131–1135. M. Beroza and M.C. Bowman, Device and procedure for concentrating solutions to a small volume with minimum attention, Anal. Chem., 39 (1967) 1200–1203. M. Beroza and K.R. Hill, Determination of reflectance of pesticide spots on thin-layer chromatograms using fiber optics, Anal. Chem., 40 (11) (1968) 1608–1613; M. Beroza, Optical Fibers, McGraw Hill Yearbook of Science and Technology, McGraw Hill, 1971, pp. 308–310. M. Beroza and E.F. Knipling, Gypsy moth control with the sex attractant pheromone, Science, 177 (4042) (1972) 19–27; re. disparlure. M. Beroza, Microanalytical methodology relating to the identification of insect sex pheromones and related behavior-control chemicals, J. Chromatogr. Sci., 13 (7) (1975) 314–321. M. Beroza, Identification of trace amounts of organic compounds, J. Assoc. Off. Anal. Chem., 54 (1971) 251–258. B.A. Bierl, M. Beroza and C.W. Collier, Potent sex attractant of the gypsy moth: Its isolation, identification, and synthesis, Science, 170 (1970) 87–89. M. Beroza, Insect sex attractants, Am. Scientist, 59 (3) (1971) 320–325. Anonymous, Sex attractant curbs gypsy moth, Agricult. Res., 24 (5) (1975) 8–11. S.L. Wilkinson, Pest control by controlled release, Chem. Eng. News, 78 (37) (2000) 25–27.

D.4. Gu¨nter Blobel Rockefeller University, New York, NY, USA

Nobel awardee in physiology=medicine, 1999 Gu¨nter Blobel was born on May 21, 1936 in Waltersdorf, East Germany. He remembers the fire bombing of nearby Dresden during World War II. He earned a medical degree at the University of Tu¨bingen and learned his main interest was research. He moved to Madison, Wisconsin to be with his brother, while working on his Ph.D. (oncology) at the University of Wisconsin (1967). He then moved to Rockefeller University, New York as a post-doctoral fellow with George E. Palade, an innovative cell biologist, pioneer in electron microscopy of cells, and a 1974 Nobel Awardee. He joined the staff of Rockefeller University becoming a Professor in 1976, and has become a USA citizen. Blobel was the 1999 Nobel Awardee in Physiology and Medicine [1–3]. By the early 1970s, considerable knowledge had accumulated on the structure of subcellular organelles and their respective functions: the nucleus for repository of genetic information, the mitochondria as the powerhouses of the cell, the lysosomes for breakdown of cellular waste, the Golgi apparatus for glycoprotein biosynthesis, the ribosomes and endoplasmic reticulum for protein biosynthesis and other entities unique to specific organs. Each organelle was surrounded by a lipid self-sealing bilayer

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membrane that provides compartmentalization of regions with different functions and chemical compositions. However the interactions of the organelles and the trafficking of proteins among them were primarily a mystery at that time. These are the areas in which Gu¨nter Blobel has made significant and steady advances over the subsequent 28 years, based on the elegant use of chromatography and other separation procedures. In 1971, Blobel demonstrated that an amino acid sequence at the end of a newly synthesized protein chain has the information that guides the proteins for secretion and enables them to pass through the membranes of the ribosome where they are synthesized; this sequence became known as the ‘signal peptide’ [4]. His key 1975 experiments [5,6] showed that pancreatic membranes (or enzymes) had cleaved the initially synthesized light chain of an immunoglobulin to the mature form of the light chain. He also proposed that translocation takes place through a protein-conducting channel and used electrophysiology experiments to demonstrate the existence of this channel for protein transport [7]. Hence the signal sequence functions as a key to open this normally closed channel through the lipid membrane [1–7]. For the readers who are chemists, an analogy follows. Since the human cell contains about one billion protein molecules, anarchy might prevail in the absence of the above signal sequence; similarly an automobile factory needs the guide of an assembly line to produce a uniform pattern for car models. Thus the sequence of 10–30 amino acids on the new protein serve as ‘address tags,’ or ‘zip codes’ to direct the protein to other intracellular organelles or with some, for secretion outside the cell. Subsequent reports from his laboratory showed that similar mechanisms operate in yeast and plant cells in a similar manner as animal cells. So far we have only alluded to chromatography. Since Blobel works daily with proteins, searching his many papers shows that the Blobel team has frequently relied on hydrophobic chromatography, adsorption chromatography, affinity chromatography, IEC, SEC, HPLC and sucrose-density gradient ultracentrifugation [7–12]. Blobel has made brilliant insights into cell biology [13], namely, in the words of the Nobel Awards Committee, for the discovery that “proteins have intrinsic signals that govern their transport and localization in the cell.” These accomplishments were achieved by elegant chromatography and several other approaches. Such research may lead to insights on some genetic transport diseases, such as hereditary hyperoxaluria, cystic fibrosis and others. By Robert L. Wixom See Chapter 5B, b, c, g, h, i, r

References 1. 2. 3. 4.

New York Times, October 12, 1999; Science section — Rockefeller biologist wins Nobel prize for protein cell research. M. Hageman, Protein zipcode makes Nobel journey, Science, 286 (1999) 666. M.T. Heemels, Nobel goes to pioneer of protein guidance mechanisms, Nature, 401 (1999) 625–626. G. Blobel and D.D. Sabatini, Ribosome membrane interaction in eukaryotic cells, Biomembranes, 2 (1971) 193–195.

144 5. 6. 7. 8. 9. 10. 11. 12. 13.

Chapter 5 G. Blobel and B. Dobberstein, Transfer of proteins across membranes. I. Presence of immunoglobulin light-chains on membrane-bound ribosomes of murine myeloma, J. Cell Biol., 67 (1975) 835–851. G. Blobel and D. Dobberstein, Transfer of proteins across membranes II. Reconstitution of functional rough microsomes from heterologous components, J. Cell Biol., 67 (1975) 852–862. S.M. Simon and G. Blobel, A protein-conducting channel in the endoplasmic reticulum, Cell, 65 (1991) 371–380. P. Walter and G. Blobel, Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum, Proc. Natl. Acad. Sci. USA, 77 (1988) 7112–7116. E.A. Evans, R. Gilmore and G. Blobel, Purification of microsomal signal peptides as a complex, Proc. Natl. Acad. Sci. USA, 83 (1986) 581–585. M.P. Rout and G. Blobel, Isolation of the yeast nuclear pore complex, J. Cell Biol., 123 (1993) 771–783. M.S. Moore and G. Blobel, Purification of a Ran-interacting protein that is required for protein import into the nucleus, Proc. Natl. Acad. Sci. USA, 91 (1994) 10212–10216. G. Blobel, Intracelluar protein traffic, HHMI Research in Progresss, 1999; http:==www.hhmi.org=science=cellbio=biobel.htm. P. Pugsley, Protein Targeting, Academic Press, San Diego, CA, 1989, 279 pp.

D.5. Phyllis R. Brown Phyllis R. Brown, a pioneer in the application of HPLC to biomedical research, was born in Providence, Rhode Island on March 16, 1924. She attended Simmons College and received her B.Sc. degree in chemistry from George Washington University. After an educational hiatus of 18 years, she returned to school and received her Ph.D. in chemistry in 1968 from Brown University under the supervision of John O. Edwards. After postdoctoral work in the Pharmacology Section at Brown, she became an Assistant Professor in the Department of Chemistry at the University of Rhode Island and was promoted to Associate Professor in 1977, and Full Professor in 1980. She was a visiting professor at the Hebrew University in Israel in 1979, and 1983, and received a Fulbright Fellowship for Israel in 1987 and a Lady Davis Fellowship in 1994. She is a member of Phi Beta Kappa, Sigma Xi, and Phi Kappa Phi and received the Dal Nogare Award in Chromatography in 1989, The Tswett Chromatography Medal, The Governor’s Science and Technology Award, The Excellence in Research Award from the Graduate School at Brown University, and The Scholarly Achievement Award from the University of Rhode Island. Brown has published 200 scientific articles. She wrote the first book on the Biomedical and Biochemical Applications of HPLC (Academic Press, 1973), and with a co-author, wrote the first book on Reversed-Phase HPLC (Wiley, 1982). Both books have been translated into Japanese. She edited a book on the HPLC of nucleic acids (Dekker, 1984), and was Co-editor of another book on HPLC (Wiley, 1989). She also is the senior author of a book, “HPLC and CE: Principles and Practise” (Academic Press, 1997). She was an Editor with J. Calvin Giddings and Eli Grushka of Volumes 14–33 of the “Advances in Chromatography”: series (Dekker), and with Eli Grushka is currently editing the series which is published annually. Brown has been on the Editorial Boards of Analytical Chemistry, Journal of Chromatography, and The University Press of New England. Currently she is on the Boards

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The author with one of her first graduate students, Richard A. Hartwick, in front of a Water’s liquid chromatograph in 1978. Most of the original work done on the separation of nucleotides, nucleosides and their bases was done by Rick for his Master’s and Doctoral theses.

of the Journal of Liquid Chromatography, Journal of Chromatographic Science, and LCžGC. She is best known for her research on the analysis of nucleic acid constituents in biological matrices, both by HPLC and CE. The HPLC methods she developed in the early 1970s for the separation of nucleotides in cells and of nucleosides and their bases in physiological fluids are still being used to study metabolism in normal subjects and patients with various diseases. She was the first to use reversed-phase HPLC methods to determine the concentrations of nucleosides and bases in biological matrices and established a range of normal values of these compounds in blood of humans, mammals and marsupials. Brown has an active research group at URI and her current interests involve the development and optimization of HPLC, CE and MS analyses for the pharmaceutical industry, biotechnology, medical research and environmental monitoring. She has also investigated mechanisms of separation, especially of nucleotides, nucleosides, and their bases by HPLC, CE and more recently by MS. See Chapter 5B, a, h, l, r

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The participants in the Dal Nogare Symposium at the Pittsburgh Conference held in Atlanta, 1989. From left to right: Georges Guiochon, Eli Grushka, Mathew McKee, Csaba Horva´th, Mary Ellen McNally, and seated J. Calvin Giddings and the author.

5.I. BREAKING THE GENDER BARRIER: A WOMAN IN CHROMATOGRAPHY Phyllis R. Brown Department of Chemistry, Pastore Hall, 51 Lower College Road, University of Rhode Island, Kingston, RI 02881-0809, USA

I never expected to have a career as a chemist, especially as an analytical chemist. Although I had worked for a year in Washington, DC after receiving my B.Sc. in chemistry from George Washington University, my husband and I then returned to Providence, RI. I applied to Brown University to work for a master’s degree but the chemistry department was not interested in admitting a woman and an ‘older’ married woman at that. There were no jobs in the Rhode Island area at that time in chemistry so I put away my books and thoughts of a career in chemistry and settled down to being a wife, mother and community volunteer (e.g., PTA, scouts etc.). When my youngest child (of 4) was in school all day, I started to think of what I would do for the rest of my life. With the support and encouragement of my husband I decided to try my hand at graduate school. Joseph Bunnett, Chairman of the Chemistry Department at Brown University took me on as a challenge; to see if someone could come back to school after

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a hiatus of 18 years. I started school part time, then after three years became a full time student and in 1968 received my Ph.D. My work in chromatography had an inauspicious but maybe prophetic beginning. As a graduate student I wrote my first paper with my major professor, John O. Edwards, on a quick TLC test for mercaptan groups. As a physical organic major, becoming an analytical chemist never occurred to me. However, when I was doing postdoctoral work in the pharmacology section at Brown, the head of the section came back from a meeting and announced that he saw a new instrument ‘A Nucleic Acid Analyzer’. The analyzer had been developed by Csaba Horva´th and Sandy Lipsky and was being manufactured by the Picker Nuclear Corp. Horva´th and Lipsky had published two elegant papers, one on the HPLC separation of nucleotides and the other on the analysis of nucleosides and their bases. Since we were analyzing nucleotides in cell extracts by open column chromatography, the section head thought that this instrument might be useful in our work. However, he did not want ‘a white elephant’ in the laboratory, therefore he only leased it for three months. The analyzer was promptly nicknamed ‘The White Elephant’. As I was the newest person in the group and the only one with a chemistry background, the instrument was assigned to me; no one else wanted anything to do with it. The ‘analyzer’ which, in reality was a high performance liquid chromatograph dedicated to the analysis of nucleotides and nucleosides came with one sheet of instructions on its operation and three sheets of instructions on how to purify the phosphate buffer. Because I had experience with both TLC and GC, I was convinced that the analyzer had to be better than the method we were using for our analyses. Luckily, I succeeded within the three months [1]. I found that HPLC could be applied to all kinds of pharmacology and biochemical investigations and my career as a chromatographer was launched. As I often tell my graduate students, chromatography is one-third science, one-third art and one-third black magic. In the early days of HPLC, the black magic played a very significant role and I had more than my share of problems in producing results in our pharmacology studies efficiently and rapidly. Part of my time was spent in advising ‘would be users’ how to use this new temperamental technique. In 1970, when I was laid up with a back problem, I decided to put together a simple, easy to read book on HPLC. After I wrote a table of contents and the first chapter, I contacted six science publishers. Only Academic Press was interested and I signed a contract with Academic Press to write a book on “High Pressure Liquid Chromatography” as the technique was then called. When I completed the manuscript, I sent it to a number of friends in the field who critiqued it and gave me constructive suggestions. However my good friend Dennis Gere did not respond to my request. Dennis who was indispensable to me in helping me set up the instrument and keep it running in those critical early months and years, later confessed that he had dropped the manuscript and could not put it back together since I had not numbered the pages. The book was finally published in 1973 [2], and in 1997, was rewritten with the inclusion of capillary electrophoresis (CE) by Andrea Weston and myself [3]. My work at Brown resulted in 21 papers, mainly on the HPLC analysis of nucleotides and nucleosides and the use of HPLC in pharmacology studies. The group was working on the metabolism of nucleoside cancer chemotherapeutic drugs and how they affected the metabolism of naturally occurring nucleotides. Our work on the nucleotide profiles

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of primitive and vertebrate species attracted the attention of Australian scientists Eric Guiler and John Sallis at the University of Tasmania. They asked me to collaborate with them to investigate the unusual metabolism of the Tasmanian devil. The devil had a very high level of the enzyme, acid phosphatase; this high a level was present only in human males with prostate cancer. During the great blizzard of 1978 in RI, I wrote my part of a joint grant proposal, which was funded by NSF and we collaborated on this project for about 10 years. We were able to show blood nucleotide profiles as well as nucleoside profiles were unique for various marsupial species and each species could be placed on the family tree by its erythrocytic nucleotide and plasma nucleoside profiles. One of my earliest collaborative studies was with Anne Fausto-Sterling of Brown. We showed that HPLC could be successfully applied to studies of DNA and RNA synthesis [4]. Later collaboration included work with Sloane Kettering Hospital, NIH, RI Hospital, NY University Hospital, Roger Williams Hospital, MIT, Hebrew University, Waters Assoc., Perkin Elmer, Gillette and Pfizer, Inc. In the early 1970s, I was having problems with discrimination at Brown and in August of 1973 I heard that the University of Rhode Island (URI) was looking for someone with experience in chromatography to take Douglas Rosie’s position for a ‘one-year only’ appointment in the Department of Chemistry. I had to leave Brown on two weeks notice, close my laboratory, prepare notes to teach a freshman class (which I had never taught before) and start a laboratory at URI. I had no equipment, no start-up money, no grants and only one year in which to be productive or it would indeed be a ‘one-year only’ position. I appealed to my friends in the companies that manufactured liquid chromatographs and within a few months I had an old Varian (called ‘the urine analyzer’), an old DuPont (which soon went out of the HPLC business) and a brand new Waters, thanks to Jim Little. When I approached Jim about the use of an instrument, his only question was “when do you want it?” Of course I answered “immediately” and then told him I did not know when or if I could ever pay for it. Luckily, he had enough faith in my work to ‘loan’ me a new HPLC. Within three years, I was finally able to pay for that instrument as well as two others from grant money. Parts of these instruments are still running in my laboratory. I am very grateful to Jim and Waters for giving me the chance to show how powerful this technique was for the studies in the life sciences. I was also fortunate that I was assigned a graduate course, which attracted a half a dozen bright young graduate students. We all worked setting up the instruments so that at the next Pittsburgh Conference in Cleveland we were all able to give presentations on our work. At URI one of my first graduate students, Richard A. Hartwick, investigated the potential use of nucleotide profiles as biochemical markers for the diagnosis and monitoring of cancer and other diseases. Charles Gehrke and his group at the University of Missouri were doing similar work on nucleosides at that time. However, no normal levels of nucleotides had been established; thus we first had to determine the concentrations of nucleotides in blood cells of a normal population without disease [5]. Fred Rabel, who was then at Whatman, introduced me to microparticle chemically bonded C18 packings [6]. He gave me some prototype C18 columns to try out for the analysis of the nucleosides and bases. The new packings worked like a charm; much better than the ion exchange stationary phases previously used (Fig. 1) [7]. We then

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Fig. 1. Separation of 0.1–0.5 nmol of nucleosides, bases, nucleotides, aromatic amino acids and some of their metabolites. Injection volume, 40 µl of a solution 1:0 ð 105 M in each standard; Column, µBondapak=C18 (10 µm particle size); eluents: low strength, 0.02 M KH2 PO4 , pH 5.6; high strength, 60% methanol; gradient slope, 0.69%=min, linear; temperature, ambient; flow, 1.5 ml=min. Peak identity: Cyt, cytosine; Ord. orotidine; Ura, uracil; Tyr, tyrosine; Cyd, cytidine; Hyp, hypoxanthine; Urd, uridine; 5-AICAR, 5-aminoamidazole carboxamide riboside; 7-m-Ino, 7-methyl inosine; 7-m-Xao, 7-methyl xanthosine; 7-m-Guo, 7-methyl guanosine; β-NAD; Ino, inosine, Guo, guanosine; 20 -dIno, 20 -deoxyinosine; dThd, deoxythymidine; 1-m-Ino, 1-methylinosine; N1 -m-Guo, N1 -methylguanosine; N2 -m-Guo, N2 -methylaguanosine; Kyn A., kynurenic acid; Ado, adenosine; Thb, theobromine; N22 -m-Guo, N2 -dimethylguanosine; Thp, theophylline; Dyp, dyphylline; 6-m-Ado, 6-methyladenosine; I-3-Prp A., indole-3-propionic acid; Caf, caffeine [7].

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Fig. 2. Chromatogram of a serum filtrate from a normal donor using both UV (254 nm) and fluorometric detection (285 nm, excitation, 320 nm emission cut-off filter); injection volume: 80 µl; chromatographic conditions same as in Fig. 1 [8].

determined the concentrations of these compounds in blood fluids of normal and cancer populations (Fig. 2) [8]. We were the first to use the microparticle chemically bonded ion exchange packings for nucleotides [5] and the microparticle chemically bonded reverse phase packing for the nucleosides, bases, and UV absorbing amino acids [6]. We then started using these columns for the analysis of other classes of biomolecules and for polynuclear aromatic hydrocarbons. We also developed a fast selective assay for adenosine, which was later used in cardiac studies. In 1982, together with Ante Krstulovic, we wrote the first book on “Reversed-Phase Liquid Chromatography” [9]. A student in one of my analytical classes, Mona Zakaria, asked me if we could separate α and β carotenes by HPLC. Since I had no experience with the carotenes, I gave her my standard answer: “If they are soluble in some solvent, we should be able to separate these compounds provided you can find the appropriate mobile and stationary phases”. With the help of Rick and Ante, she worked out a separation that is still being used (Fig. 4) [10]. An elegant kinetic study of the acid catalyzed break-down of NADH [11] was done by Joy Miksic, who was also one of my first graduate students. We were able to show which of two pathways proposed by Oppenheimer and Kaplan was correct but most importantly that HPLC could be used for kinetic studies. For the first decade at URI,

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Fig. 3. Serum profile of a post-operative breast cancer patient with metastasis to the bone. Injection volume: 80 µl; chromatographic conditions same as in Fig. 1 [8].

the majority of our work was on the development and optimization of RPLC assays for compounds in the purine and pyrimidine metabolic pathways and the application of these assays to medical, biochemical and pharmaceutical investigations. We did considerable work on the preparation of biological matrices and on the evaluation of detectors for these assays. A total analysis is as good as the weakest step and we learned that sample preparation and detection are vitally important in a reliable HPLC assay. We also found that peak identification was important and before MS was available as a detector for HPLC, peak identification was a problem in physiological fluids. I developed the enzyme peak-shift method for identification of biologically active molecules, especially those in the purine and pyrimidine metabolic pathways [1]. An interesting application of HPLC was in the assay of enzymes such as adenosine deaminase and purine nucleoside phosphorylase. Using a simplex optimization technique, another graduate student Anne Halfpenny was able to determine the activity of two enzymes simultaneously. In the fall of 1979, I spent a sabbatical semester in Jerusalem working with Eli

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Fig. 4. Resolution of α- (#2) and β-carotene (#3), (Rs D 1:2). Chromatographic conditions: column, Partisil-5=ODS, 5 µm; eluent, 8.5% chloroform in acetonitrile; flow rate, 2.0 µl=min; temperature, ambient; detection, visible, 470 nm; sensitivity, 0.02 A.U.F.S. [10].

Grushka at the Hebrew University. During that period and in subsequent sabbaticals in Israel, we investigated the structure–retention relationships in RPLC of purine and pyrimidine compounds. We formulated general rules by which we could predict the retention order of nucleosides and their bases [11]. Later, together with some of my graduate students, we investigated the mechanisms of retention, and stationary phase effects in these separations. For his graduate research, Sebastian Assenza quantified the structure–retention relations and Pamela Perrone investigated the use of ion pairing for not only the separation of nucleotides but also for nucleosides and their bases. With this solid background on the analysis of purine and pyrimidine compounds, we were able to classify RPLC profiles of serum nucleosides and their bases from patients with acute and chronic leukemia. Using chemometrics Hubert Scoble, another of my graduate students, separated cleanly normal populations from those with the leukemias (Fig. 5) [13]. The calculated ‘disease index’ data is represented as continuous distribution functions. The

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Fig. 5. The calculated disease index data is represented as continuous distribution functions. The curve on the right is a normal population, and that on the left, is of a population with chronic lymphocytic leukemia, and included individuals receiving no medications as well as those receiving chemotherapy [13].

curve on the right is a normal population, and that on the left is that of a population with chronic lymphocytic leukemia, and included individuals receiving no medications as well as those receiving chemotherapy. Our work continued and now includes the development and optimization of HPLC analyses for many different groups of biomolecules: amino acids, peptides, enantiomers, vitamins, biogenic amines, triglycerides and many types of drugs including anti-aids drugs. We have scaled up equipment for preparative work and scaled down for microbore and short column assays. One regret I have is that we have not yet gotten into the ‘separations on a chip’ which I see as a way of the future. Recently we did work on HPLC=ESI=MS which is an exciting technique in biotechnology and the pharmaceutical industry and we are currently finding HPLC–FT-IR very valuable for pharmaceutical work. We were among the first to see the potential of capillary electrophoresis (CE) for the separation of cations and we investigated the use of CE for nucleic acid constituents, amino acids, peptides and enantiomers. Currently, an exciting area of research is the use of conducting polymers as coatings for the capillaries in CE. My wonderful group of graduate students has led me into new projects and into attacking analytical problems with fresh eyes and enthusiasm. I was fortunate to have been active in research when the great potential of HPLC for use in the life sciences was realized. In my first book I predicted in the early 1970s that “HPLC would open new horizons for the analysis

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of non-volatile, thermally labile molecules that are so important in biochemistry and medical research” [2]. HPLC and other separation techniques developed in the past three decades have indeed paved the way for the exciting break-throughs in the human genome project, biotechnology, bioengineering and medical research. Most important, it is from the analytical laboratories that the rapid, sensitive, selective and reliable analyses, which are vital to the life sciences, have emerged. I cannot close without paying tribute to J. Calvin Giddings who was my role model as a scientist, writer and Editor. He was also my mentor and my friend. An outstanding teacher, I learned a great deal from him during the twenty years we were associated as Editors of the ‘Advances in Chromatography’ series. His high professional standards and his great personal integrity influenced me as a scientist and enriched my life personally. References 1. 2. 3. 4. 5. 6.

7.

8.

9. 10.

11. 12. 13.

P.R. Brown, The rapid separation of nucleotides in cell extracts using high pressure liquid chromatography, J. Chromatogr., 52 (1970) 257–272. P.R. Brown, High Pressure Liquid Chromatography: Biochemical and Biomedical Applications, Academic Press, New York, 1973, 202 pp. A. Weston and P.R. Brown, HPLC and CE: Principles and Practise, Academic Press, New York, 1997, 280 pp. A. Fausto-Sterling, L.M. Zhentlin and P.R. Brown, Rates of RNA synthesis during embryogeneses in Drosophila melanogaster, Dev. Biol., 40 (1974) 78–83. R.A. Hartwick and P.R. Brown, Performance of microparticle chemically-bonded anion exchange packings in the analysis of nucleotides, J. Chromatogr., 112 (1975) 651–661. R.A. Hartwick and P.R. Brown, Evaluation of microparticle chemically-bonded reverse phase packings in the high pressure liquid chromatographic analysis of nucleosides and their bases, J. Chromatogr., 126 (1976) 679–691. R.A. Hartwick, S.P. Assenza and P.R. Brown, Identification and quantitation of nucleosides, their bases and other UV-absorbing compounds in serum using Reversed-Phase High Performance Liquid Chromatography: I. Chromatographic methodology, J. Chromatogr., 186 (1979) 725–736. R.A. Hartwick, A.M. Krstulovic and P.R. Brown, Identification and quantitation of nucleosides, their bases and other UV-absorbing compounds in serum using Reversed-Phase High performance Liquid Chromatography: II. Evolution of Hurman sera, J. Chromatogr., 186 (1979) 737–754. A.M. Krstulovic and P.R. Brown, Reverse Phase Liquid Chromatography: Theory, Practise and Biomedical Applications, Wiley Interscience, New York, 1982, 296 pp. M. Zakaria, K. Simpson and A.M. Krstulovic, Use of Reversed-Phase High Performance Liquid Chromatographic analysis for the determination of provitamin A carotene in tomatoes, J. Chromatogr., 176 (1979) 109–117. J.M. Miksic and P.R. Brown, The reactions of reduced nicotinamide adenine dinucleotide in acid: Studies by reversed phase high pressure liquid chromatography, Biochemistry, 17 (1978) 2234–2238. P.R. Brown and E. Grushka, Structure retention relations in the reversed-phase high performance chromatography of purine and pyrimidine compounds, Anal. Chem., 52 (1980) 1210–1215. H. Scoble, M. Zakaria, P.R. Brown and H.F. Martin, Liquid chromatographic profile classification of acute and chronic leukemias, Computers Biomed. Res., 16 (1983) 300–310.

D.6. Thomas L. Chester Thomas L. Chester was born on September 28, 1949, in Jacksonville, FL. He received his B.Sc. degree in chemistry from the Florida State University in 1971.

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He then moved to Charleston, SC, where he worked for the Verona Division of the Baychem Corporation (now Bayer) at their plant in Bushy Park. In the fall of 1972, Tom enrolled in the graduate program at the University of Florida where he earned the Ph.D. degree in 1976 under the direction of J.D. Winefordner. He then joined the Procter and Gamble Company, Cincinnati, OH, where he is Section Head in the Corporate Research Division. Tom is on the Editorial Advisory Board of the Journal of High Resolution Chromatography, the Journal of Microcolumn Separations, the Journal of Supercritical-fluids, and Instrumentation Science and Technology. He served on the Instrumentation Advisory Panel for Analytical Chemistry. He was chair of the American Chemical Society Subdivision of Chromatography and Separations Chemistry. He is the founder and President of Supercritical Conferences, the organization producing the International Symposia on Supercritical-fluid Chromatography and Extraction. He is also Treasurer of the TriState Supercritical-fluids Discussion Group located in Cincinnati. Tom has authored over 50 publications and co-edited a book, “Unified Chromatography”. His recent research interests include unified chromatography and chromatography optimization. The Cincinnati Section of the American Chemical Society named him the 1993 Chemist of the Year. He was the recipient of the Keene P. Dimick Award in 1994. He is an Adjunct Professor of Chemistry at the University of Cincinnati. See Chapter 5B, a, o 6.I. SUPERCRITICAL-FLUID CHROMATOGRAPHY — ON THE ROAD TO UNIFICATION Thomas L. Chester The Procter and Gamble Company, Miami Valley Laboratories, P.O. Box 538707, Cincinnati, OH 45253-8707, USA

6.I.1. Our role in SFC We became interested in supercritical-fluid chromatography (SFC) for one overwhelming reason. As late as the 1980s we had a particular problem analyzing samples that were complicated mixtures: solutes had to contain a chromophore or fluorophore for really good detection limits in HPLC. Refractive index detection was a poor substitute and, since it is not compatible with gradient elution, was of little value when the samples contained solutes with a wide range of retention. We really wanted a sensitive, universal detector for HPLC. GC had the advantage of the flame ionization detector (FID), essentially a universal detector for carbon-containing solutes. However, the FID was limited to solutes with enough volatility and thermal stability for successful GC.

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This left a hole into which a large fraction of our applications fell — samples where not all the solutes could be detected adequately in HPLC, and where at least some of the solutes were not volatile or stable enough for successful GC. Since we were working in an upstream corporate laboratory instead of one belonging to a business unit, we only got involved in the most difficult analysis problems, and a great many of them fell into this hole. It was not really SFC that captured our interest, but the prospect of using CO2 as the mobile phase, as CO2 promised to be a solvating mobile phase able to dissolve and transport low-volatility solutes at temperatures much lower than GC, yet was completely compatible with the FID. Furthermore, the critical temperature of CO2 is near ambient, so with only a small temperature increase it can be made supercritical. In this state there is only one fluid phase which shares the properties of both the liquid and vapor states. This allows us to continuously vary the solvating strength using pressure, and cover a strength range from gas (with essentially zero solvent strength) to something close to liquid hexane without ever experiencing a phase change from vapor to liquid or vice versa. We could program the mobile-phase strength using pressure (or density), in analogy with temperature programming in GC and gradient elution in HPLC, all the while using the FID! This combination of a solvating mobile phase with continuously programmable strength plus universal detection went a long way toward filling the hole in our analysis capabilities. Of course, a great deal of work was necessary to make this practical, particularly for open-tubular columns because of the small diameters they required for reasonable analysis times. First, the interface to the FID was tricky. It was necessary to use a flow restrictor to couple the column exit to the FID flame. The amount of restriction has to be correct to keep the mobile-phase velocity near the appropriate rate on the column when the column inlet is pressurized to several hundred atmospheres. There is very little pressure drop (less than one atmosphere) over the length of an open-tubular column under these conditions, so the column outlet pressure is almost the same as at the inlet, yet the FID operates at ambient pressure. The restrictor must maintain the pressure at the column outlet and keep the solutes dissolved as long as possible before jetting them into the flame. In the early FID trials, cooling due to the expansion of the CO2 would often cause the less-volatile solutes to condense in the restrictor. Spikes would occur in the signal whenever a condensed solute particle broke loose from the restrictor and entered the flame. Several groups came up with independent solutions. We were all using capillary tubes of various sorts for restrictors, but Claudia Smith in our group found that tapering the capillary outlet by drawing it out in a flame was a very effective way of postponing the decompression to the last instance [1]. Drawing these by hand was never reproducible, so we later programmed a laboratory robot for the job [2]. These restrictors served us well for many years until we switched to a fiber-optic splicer for narrowing the restrictor outlets. Of course, CO2 is a good mobile phase only when it can dissolve the solutes of interest. The dipole moment of CO2 is zero, so it is not a very good solvent for very polar solutes. However, another co-worker, David Innis, convinced us that silylating such solutes was worth a try. It worked amazingly well [3]. We do not necessarily

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Fig. 1. Separation of silylated Maltrin M100, a malto-oligosaccharide. The numbers indicate the number of glucose units in each peak. The molecular weight of the DP18 compound is 2934 before derivatization, and 6966 when silylated. (Adapted from Chester et al. [3] and used with permission.)

produce volatile derivatives, as is the object in GC, but are aiming instead at reducing the polarity of polar solutes and improving their solubilities in CO2 . Thus we were able to make and elute some surprisingly heavy derivatives and detect them with the FID. Fig. 1 shows an example of this capability. Open-tubular SFC continued to be plagued, however, by the relatively crude, splitting injection methods in use at the time. No one had paid much attention to phase behavior and mass transfer of sample solutions when injected into a stream of CO2 . Reproducibility of open-tubular SFC results was terrible. The effective injection volumes were measured in nanoliters. Even with the sub-nanogram detection capabilities of the FID, trace analysis is impossible when the sample volumes are so small. So, we undertook a fundamental study to learn about the transport of things injected into streams of CO2 [4]. We began by injecting neat liquids of various sorts into CO2 flowing through an otherwise empty fused-silica tube in a GC oven and connected with a restrictor to an FID. This work provided the key knowledge for us to develop a retention gap injection procedure for open-tubular SFC [5]. The simple arrangement is shown schematically in Fig. 2. We accomplished about a 100-fold increase in the effective injection volumes and completely eliminated all sources of splitting in the system, all for the price of one union and a few meters of uncoated fused silica. This, plus fixing losses that had been occurring in the injection valve, solved the reproducibility problem as demonstrated in Fig. 3. We also developed a rapid flow-injection technique for determining the critical loci of CO2 -solvent mixtures, and published many of these (for example [4,6]). These plots provide the guidance necessary for setting the initial temperature and pressure when using the retention gap technique: conditions must be selected to ensure that liquid– vapor phase separation occurs in the retention gap. The plots are also useful for selecting temperature and pressure to avoid liquid–vapor phase separation when using binary

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Fig. 2. The retention-gap injection set-up for open-tubular SFC. The inlet tube and column are typically 50 µm-inside-diameter and, although shown separate here, are wound together in the oven on the same column support. (Adapted from Ziegler et al. [5] and used by permission of the American Chemical Society).

Fig. 3. Ten superimposed chromatograms of Neodol 23-6.5, a nonionic surfactant made by ethoxylating a mixture of C12 and C13 alcohols. Injection was performed using the retention gap method for otSFC [5]. 10 m ð 50 µm SB-biphenyl column, 120ºC, pressure programmed from 70 to 80 atm at 1 atm=min, then 80–415 atm at 3 atm=min. (From the 7th International Symposium on Supercritical-fluid Chromatography and Extraction, Indianapolis, IN, 1996, used here by permission of Supercritical Conferences, Cincinnati, OH.)

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mobile phases of CO2 plus a liquid modifier in techniques like packed-column SFC, and for understanding phase behavior and mass transfer through detector interfaces. We explored the limits of the open-tubular SFC technique, and pushed into new areas for the FID [7]. We found (not unexpectedly) that temperatures much higher than those originally anticipated for CO2 had benefits of improving diffusion rates and shortening analysis times. We also found surprising ability to adjust the selectivity between dissimilar solutes by temperature and pressure tuning. The disadvantage of using higher temperatures is that much more pressure is necessary to reach useful CO2 densities than when using lower temperatures. Above about 200ºC, the upper half of the usable density range is out of the pressure limits of the commercial instruments. The technology to use much higher pressures safely, particularly with the small volumes encountered in open-tubular and microcolumn SFC, is very ordinary, but simply not commercially available on turn-key, analytical SFC instruments. Commercial open-tubular SFC, at the time of this writing, is aimed almost exclusively at petroleum applications where very high pressures are not necessary. Additional pressure remains a major, untapped opportunity when unmodified CO2 is used in SFC.

6.I.2. A few words on safety Safety is often a concern for people first encountering the pressures used in SFC. While caution is necessary in everything we do in a laboratory, SFC requires only a little more attention than GC and HPLC. The main taboo is to never isolate a liquid in a closed system with no means for pressure relief. For example, never put two valves in series without a rupture disk or relief valve in the same volume. This caution is necessary because whenever any fixed volume is completely filled with a liquid, enormous pressure can be generated if the temperature goes up just a few degrees. This is why glass-bottled drinks always have a gas headspace. More care is necessary with supply lines and tubes in SFC than in GC or HPLC, not because of the pressure but because the tubes can be propelled by their own contents if a fitting is not made properly or is disassembled while under pressure. This is not a serious problem in GC (because the pressures are much lower), or in LC (because the fluids, although pressurized, do not expand significantly upon decompression). The pressure range of SFC is the same as that of HPLC, and no other special precautions not already practiced in HPLC have been necessary for analytical-scale columns.

6.I.3. Looking at the future [8–10] One of the most important things holding back the development of chromatography has been its partitioning into specific, stand-alone techniques like GC, HPLC, SFC, etc. Promoters continue to spend much effort building the image of their particular technique of choice. Unfortunately, some of this energy also goes into discrediting rival techniques. J. Calvin Giddings, Daido Ishii, Daniel Martire, and others realized that chromatog-

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Fig. 4. These are generalized pressure–temperature–composition phase diagrams for a type I binary mobile phase. Type I means the two mobile-phase components are miscible in all proportions. The shaded region in a is the region of liquid–vapor phase separation. Only one continuous phase exists in the volume surrounding the shaded region. The temperature, pressure, and composition range where GC and LC are practiced are indicated in a. The pressure–temperature–composition regions of several other named techniques are plotted in b–e. The boundaries between these techniques are only arbitrary definitions. Unified chromatography results when the arbitrary definitions are ignored and chromatography is considered in the entire, continuous region of the phase diagram, f. Thus, once the mobile-phase components are chosen, the chromatographer is limited only by liquid–vapor phase separation which must usually be avoided, and by the temperature and pressure safety limits of the system. The operator is free to find the global optimum for the particular separation under consideration wherever it might exist within the continuum. (Adapted from Chester [8] and used by permission of the American Chemical Society.)

raphy is universal. This universality is easily appreciated once we realize that ambient temperature and pressure, while very convenient for us to use, are not necessarily the best choices for our separations. Only fairly recently have researchers considered temperatures much above ambient in HPLC. Yet they are still limited by the normal boiling points of their mobile phases when the column outlet is vented to atmosphere. Few people have realized that boiling can be avoided and new realms of mobile phase behavior can be made available by controlling the column outlet pressure. I am not advocating just raising the outlet pressure a few bars to suppress cavitation, but instead controlling it over the entire range where benefits can be derived. These include the ability of using temperatures greatly exceeding the normal boiling point of the mobile phase, or using mobile phase components that are not liquids at ambient conditions. It amuses me to think what chromatography would be like if developed on another planet where the ambient conditions are different than ours. What would the first

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interplanetary meeting be like if our counterparts attended and defended their parameter choices (their ambient conditions) and their mobile phase choices as strongly as we seem to embrace and defend ours? The obvious conclusion is that no particular temperature and outlet pressure will be best for every chromatogram. Unified chromatography results if we simply include both temperature and column outlet pressure in the parameters we vary when optimizing a separation. We must have maps to tell us the range of parameters over which the mobile phase is well behaved [8–10]. These maps are simply phase diagrams for the mobile phase. All of the various, individually named chromatographic techniques appear in these diagrams, as shown in Fig. 4. When the mobile phase components are miscible in all proportions, there is no discontinuity between techniques whatsoever. The similar techniques, separated only by arbitrary definitions, merge together to make the unified picture. Optimization in unified chromatography is simply a matter of including both temperature and outlet pressure among the variables, and then finding the global optimum for the separation according to the constraints of importance to the method user. In many cases this will lead to conditions that are much different from those we consider normal today, but who can argue if these conditions produce the global optimum for the problem at hand? We simply need to expand our thinking and push ourselves to use the full range of fluid properties to more fully realize the capabilities of chromatography. References 1.

T.L. Chester, Capillary supercritical-fluid chromatography with flame ionization detection, reduction of detection artifacts and extension of detectable molecular weight range, J. Chromatogr., 299 (1984) 424–431. 2. T.L. Chester, D.P. Innis and G.D. Owens, Separation of sucrose polyesters by capillary supercriticalfluid chromatography=flame ionization detection with robot-pulled capillary restrictors, Anal. Chem., 57 (1985) 2243–2247. 3. T.L. Chester and D.P. Innis, Separation of oligo- and polysaccharides by capillary supercritical-fluid chromatography, J. High Resolut. Chromatogr. Commun., 9 (1986) 209–212. 4. T.L. Chester and D.P. Innis, Dynamic film formation and the use of retention gaps with direct injection in open-tubular supercritical-fluid chromatography, J. Microcol. Sep., 5 (1993) 261–273. 5. T.L. Chester and D.P. Innis, Quantitative open-tubular supercritical-fluid chromatography using direct injection onto a retention gap, Anal. Chem., 67 (1995) 3057–3063. 6. J.W. Ziegler, J.G. Dorsey, T.L. Chester and D.P. Innis, Estimation of liquid-vapor critical loci for CO2–solvent mixtures using a peak-shape method, Anal. Chem., 67 (1995) 456–461. 7. T.L. Chester and P. Innis, Investigation of retention and selectivity in high-temperature, high-pressure, open-tubular supercritical-fluid chromatography with CO2 mobile phase, J. Microcol. Sep., 5 (1993) 441–449. 8. T.L. Chester, Chromatography from the mobile phase perspective, Anal. Chem., 69 (1997) 165A– 169A. 9. T.L. Chester, The road to unified chromatography: The importance of phase behavior knowledge in supercritical-fluid chromatography and related techniques, and a look at unification, Microchem. J., 61 (1999) 12–24. 10. T.L. Chester. Unified chromatography from the mobile phase perspective, in: J.F. Parcher and T.L. Chester (Eds.), Unified Chromatography, ACS Symposium Series, American Chemical Society, Washington, DC, USA, in press.

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D.7. Karel A. Cramers Karel A. Cramers was born in Breda, The Netherlands, on September 4, 1935. He received the M.Sc. degree in Chemical Engineering from Eindhoven University of Technology in The Netherlands in 1963. He earned his Ph.D. in Analytical Chemistry with a thesis on capillary gas chromatography in 1967, also in Eindhoven. In 1968 he spent one year in California, USA, on a sabbatical leave with the Hamilton Company in Whittier and the University of California, Los Angeles, USA. Since 1978, he has been full Professor and Chairman of the Laboratory of Instrumental Analysis at Eindhoven University of Technology. During his education, he has worked with pioneers in chromatography as A.I.M. Keulemans, A.J.P. Martin and M.J.E. Golay. K.A. Cramers is an Editor of the Journal of High Resolution Chromatography and is on the Editorial Advisory Board of Chromatographia and the Journal of Microcolumn Separations. He has authored or co-authored about 300 publications in separation sciences and hyphenated techniques. Under his direction, more than 30 Ph.D. theses were defended in Eindhoven. Research interest involve all high-resolution separations: Fast GC, HPLC, MECC and CZE. Much attention is given to sample preparation, sample introduction and to coupling to mass spectrometry. He received the M.J.E. Golay Award during the 19th International Symposium on Capillary Chromatography in May 1997, Wintergreen, VA, USA. In March 1999, he received the Keene P. Dimick Award during the Pittsburgh Conference in Orlando, FL, USA for his accomplishments in chromatography. Since 1978, he totally received eight international awards. He is also a permanent member of the scientific committees of a number of International Symposium series: Capillary Chromatography and Electrophoresis (Europe, USA, Japan) Hyphenated Methods (Belgium) and the Latin American Congress on Chromatography, COLACRO (Latin America). See Chapter 5B, a, d, e, i, l, m, p

7.I. REFLECTIONS ON RESEARCH IN GAS CHROMATOGRAPHY IN THE EINDHOVEN LABORATORY OF INSTRUMENTAL ANALYSIS Karel A. Cramers Technische Universiteit Eindhoven, P.O. Box 513 (STO 3.33), 5600 MB Eindhoven, The Netherlands

I have been with the University of Technology of Eindhoven, The Netherlands for more than 40 years. I am currently Professor of Analytical Chemistry there. In 1958 I entered the Laboratory of Instrumental Analysis. The first chair in Europe with this subject was funded in Eindhoven. I started working under the guidance of the late

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Professor Cramers with a donated GC–MSg system on the occasion of the 25th anniversary of the Laboratory of Instrumental Analysis in Eindhoven.

A.I.M. Keulemans, one of the pioneers laying the foundations of gas chromatography in the late fifties. During the first ten years, I had the opportunity to work and discuss with Archer Martin, Marcel Golay and Josef Huber, who all spent periods in the laboratory. Looking back at this long period in Eindhoven, it is clear that many research subjects have been tackled. The core activities, however, were in the field of separation sciences, especially on capillary and miniaturized systems. Already from our start in 1958, we concentrated on capillary columns, introduced by Marcel Golay in 1956. During the following years in capillary gas chromatography, much attention was paid to the study of the effect of experimental factors on the repeatability and accuracy of retention indices. Together with Jos Curvers and Jacques Rijks, the latter now retired, a series of papers dealing with programmed and isothermal retention data and their interrelations were published ([1,2] among others). Another important subject worked at in Eindhoven during the whole period by several post-doctoral fellows and Ph.D. students is large volume sample introduction for trace analysis with capillary GC. Programmed temperature vaporizing (PTV) injection systems were developed and optimized (Jacques Rijks, Hans Mol, Jacek Staniewski) by studying the underlying physical principles [3]. PTV injectors were later successfully used for both fast capillary gas chromatography and for large volume sample introduction. Other research efforts included the understanding of the basic factors of supercritical fluid chromatography and extraction (Hans-Gerd Janssen), preand post-column hyphenation especially with MS (Piet Leclercq), characterization of

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silica surfaces by solid state NMR (Jan de Haan, Martin Hetem, Alex Scholten), micellar electrokinetic chromatography (Pim Muijselaar) and miniaturization in liquid chromatography (Henk Claessens). In Eindhoven we always tried to base our research on theoretical modelling going back to the underlying physical or engineering principles. This often allowed us to understand the analytical problem and to carry out optimization. Looking back on my career and to the impact it has had on the advancement of chromatographic science, I think my major contribution was on the subject ‘Fast gas chromatography’. A large part of my invited key note and plenary lectures were on this subject. Also in the award addresses explaining why I was selected, much attention was paid to my contributions to the study of increased speed of analysis by gas chromatography: therefore, I feel it appropriate to go in some detail on this subject rather than give an extensive overview of all my research activities in the past 40 years. We commenced research in the area of high-speed gas chromatography in 1978 after my appointment as Professor and Director of the Laboratory of Instrumental Analysis as successor of A.I.M. (Lou) Keulemans. It is clear that many scientists have been working since the early sixties on the same subject, most notably Dennis Desty using glass capillary columns. Our start in fast capillary GC was greatly enhanced by the introduction of fused silica columns by Dandeneau and Zerenner in 1979. We quickly figured out that the main road to fast GC was to enhance radial mass equilibrium inside the column. The various routes to this goal were studied during many years. With Kees Schutjes as Ph.D. student, we started an extensive theoretical study. Using the Golay plate height equation for capillary columns describing the dependence of the experimental plate height upon carrier gas velocity and column parameters, and the Poiseuille flow equation for capillary columns, Schutjes and associates [4] derived theoretical models for fast capillary GC. It was shown that the relationship of the analysis time with the column inside diameter is dependent on the pressure gradient, more exactly on the ratio of the inlet to outlet pressure of the column. Assuming the average carrier gas velocity to be maintained at the optimum value u opt , the analysis time is predicted to decrease with the second power of the column diameter, when the pressure gradient is small. For a large gradient, a linear relationship is expected, since the required carrier gas velocity is now predicted to be independent of the column diameter. The theoretical model is shown to be valid for isothermal and for temperature programmed conditions. The temperature programming rate must, however, be increased proportionally to the gain in analysis speed which is obtained. Only recently instruments allowing very high programming rates became available. In this and following research projects, it could be demonstrated that reduction of the column inner diameter allows the speed of analysis in capillary gas chromatography to be greatly increased. Importantly, the quality of the separation as judged from the resolution of the compounds, is thereby maintained. By consequence, this approach appears particularly suited for decreasing the analysis times of complex mixtures, which is a subject of great economic importance. In a joint publication with Georges Guiochon and Vidal Madjar [5], we addressed the consequences of operating a column under conditions of carrier gas velocities much larger than u opt . In order to restore the accompanying loss in peak resolution a longer column has to be selected, thus re-establishing the original plate number, it

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was concluded and proven by experiments that high-speed open tubular columns under conditions of large pressure gradients should be operated very close to the optimum conditions, contrary to the ideas of many colleague chromatographers. In Schutjes thesis [6], it is also demonstrated that reduction of the inner diameter enhances the applicability of columns with very high plate numbers. A full chromatogram showing the separation of some 400 components of a condensate of natural gas on a 95 m ð 65 µm column having one million theoretical plates was presented. In the eighties, we extensively studied the gain in separation speed, that can be obtained by operating the column under vacuum outlet conditions [7]. The principle had already been shown by Locke and Brandt in 1963. It will be clear that the detector must be able to operate at reduced pressures. Detectors of choice proved to be the thermal conductivity detector and of course the mass spectrometer. It appeared that the increase of speed of analysis at vacuum outlet conditions is most pronounced for wide bore and=or short columns. For example a wide bore column, with dc 530 µm, L D 40 cm, yielding 1000 theoretical plates will operate a factor of 6 faster under reduced outlet pressures. This can be explained from the effect of the reduced outlet pressure on the average column pressure. At a smaller average column pressure the average diffusion coefficient will be larger and this implies shorter analysis times. The same principle explains the higher speeds of analysis obtainable with hydrogen as the carrier gas. A reduction in the column diameter as described above lowers the contribution of the velocity profile .CM -term in the Golay plate height equation) to the chromatographic dispersion. The chromatographic dispersion can also be lowered by changing the velocity profile. A possible way to change the velocity profile is to create turbulent flow (in open tubular column with Reynolds numbers >2300). With turbulent flow the velocity profile is largely flattened, thus decreasing the flow inequalities; further, the effective diffusion coefficient of the component is considerably increased by convective contributions. As a consequence, peak broadening in the mobile phase due to the velocity profile is expected to be largely reduced. Only a few experimental results on turbulent flow in GC have been reported dating back to the sixties. In 1988, my Ph.D. student Andrew van Es started an investigation, in this work he used the instrumentation we developed for narrow bore columns, instrumentation compatible with peak widths of a few milliseconds. Stationary phase effects were minimized by selecting a suitable thin film column (length ranging from 25 to 5 m, internal diameter 320 µm, and a film thickness of 0.12 µm OV-1). Experiments showed that low plate heights equal or even smaller than the inside column diameter could be obtained under very high-speed conditions (inlet pressure D 50 bar, u Ł 15 m=s; Re 104 / [8]. At these very high flow rates the flame ionization detector could not be used, the flame was extinguished. We had to turn to a low volume photoionization detector. Unfortunately, the dependence of the plate height on the retention factor is significantly higher than under laminar flow conditions, limiting the profitable use of turbulence to solutes with a low retention factor. This effect is clearly demonstrated in Fig 1 [8]. Also the instrumental requirements with respect to time constants of sample introduction and detection are very severe in turbulent gas chromatography. Taking into account the high-pressure drop required for turbulent flow, a reduction of the column diameter appeared to be a better approach to increase the

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Fig. 1. Representative chromatogram of a hydrocarbon headspace sample under turbulent flow conditions. Column: L D 5 m, i.d.D 0.32 mm; df D 0:12 µm; pi D 50 bar [8].

analysis speed in capillary GC. During these experiments, we asked Marcel Golay, former Extraordinary Professor in our laboratory, for expert advice concerning the several contradictory theories on turbulent chromatography. He immediately became extremely interested in the subject. During the last months of his life, he visited our laboratory several times and he derived a theory for turbulent dispersion in capillary GC. His manuscript “Calculations Relative to Turbulent Capillary Gas Chromatography” was meant to constitute his opening lecture at the 10th International Meeting on Capillary Chromatography in Riva del Garda, in May 1989. We were shocked by the message of his sudden death in his native country of Switzerland during the night of April 28–29, 1989. As a tribute to the great scientist Marcel Golay, inventor of capillary chromatography, we evaluated his postulates in the paper [9]. In parallel and essential for the theoretical treatment of the various concepts leading to ‘fast gas chromatography’ compatible hardware had to be developed. Lack of commercially available instrumentation has hindered the wide-spread acceptance of narrow-bore columns until recently. In our laboratory various injection systems and detectors were evaluated for coupling to high-speed GC-columns. To mention a few highlights, Andrew van Es focused on the problem of sample introduction onto narrow bore columns. The sample introduction system must be able to deliver an extremely narrow input band width at elevated pressures. Several specially developed systems were described, capable of generating input band widths as low as 1 ms. This allowed column diameters at least down to 10 µm i.d. One of these sample introduction systems is part of a commercially available miniaturized gas chromatograph with extremely low dead volumes. An etching technique called ‘silica on micromachining’ allows chromatographic parts to be integrated on a silicon wafer. With a micro cold trap=thermodesorption system both preconcentration of the sample as well as an extremely narrow input band width (1 ms) could be obtained [10], enabling analysis times below 1 s as demonstrated in Fig. 2 [12]. Column properties such as diameter and film thickness have a large influence on the sample capacity of the column. The working range, i.e., the ratio between sample capacity

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Fig. 2. Fastest narrow-bore capillary-column separation to date. Hydrocarbons (C6–C9) at 72ºC. Column 30 cm ð 50 µm i.d., OV-1, inlet pressure 4.5 bar, carrier gas helium. Re-injection by rapid heating (4000ºC=s during 50 ms) after cold-trapping at 75ºC [12].

and minimum detectable amount appears to decrease strongly with column inside diameter. Experiments showed that a microchip thermal conductivity detector, as well as conventional thermal conductivity detectors at reduced pressure were highly compatible with narrow-bore columns. Also flame ionization detectors and small volume electron capture detectors could be used in combination with fast GC. My Ph.D. student, Peter Van Ysacker, studied different types of mass analyzers, including the ion trap, sector instrument, reflection time-of-flight and the orthogonal acceleration time-of-flight mass spectrometer, as mass spectrometric detectors in combination with narrow-bore capillaries. The potentials and limitations of all these mass spectrometers were discussed in detail. Special emphasis was paid to the maximum scan speed, detection limits, mass spectrometric resolution and the quality of the mass spectra obtained. With mass-scanning instruments including the quadrupole, the ion trap and the sector machine, the acquisition was limited to a maximum of some 20 scans per second. This maximum scan rate is sufficient for chromatographic separations in the minute range. With non mass-scanning techniques, such as the time-of-flight mass spectrometer and a prototype of a microchannel plate mass spectrometer, a complete spectrum could be recorded in 100 µs or less enabling coupling to fast 50 µm narrow-bore columns and analyses in the seconds range. Ref. [11] is a recent review on high-speed GC–MS from our laboratory. A comprehensive overview of the various concepts leading to fast gas chromatography is presented in a review article [12]. Finally, I hope and believe that after the ‘usual’ delay time of some ten years, our and our colleagues’ activities in the field of fast gas chromatography will be accepted in daily life chromatography. In the near future, open tubular columns of 100 µm inside diameter will be used; a compromise between analysis time and instrument capability. I think more research has to be devoted to the important role of selectivity

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(stationary phase selectivities, selectivity tuning, etc.) on the attainable speed of analysis. Non-scanning mass spectrometers (e.g. time-of-flight machines) will become essential tools in separation sciences.

References 1.

J. Curvers, J. Rijks, K.A. Cramers, K. Knauss and P. Larson, Temperature programmed retention indices: Calculation from isothermal data. Part I: Theory, J. High Resolut. Chromatogr. Chromatogr. Commun., 8 (1985) 607–611. 2. J. Curvers, J. Rijks, K.A. Cramers, K. Knauss and P. Larson, Temperature programmed retention indices: calculation from isothermal data. Part II: Results with non polar columns, J. High Res. Chromatogr. Chromatogr. Commun., 8 (1985) 611–618. 3. J. Staniewski and J.A. Rijks, Solvent evaporation rate in temperature-programmed injection of large sample volumes in capillary gas chromatography, J. Chromatogr., 623 (1992) 105–113. 4. C.P.M. Schutjes, E.A. Vermeer, J.A. Rijks and K.A. Cramers, Increased speed of analysis in isothermal and temperature-programmed capillary gas chromatography by reduction of the column inner diameter, J. Chromatogr., 253 (1982) 1–16. 5. C.P.M. Schutjes, P.A. Leclercq, J.A. Rijks, K.A. Cramers, C. Vidal Madjar and G. Guiochon, Model describing the role of the pressure gradient on efficiency and speed of analysis in capillary gas chromatography, J. Chromatogr., 289 (1984) 163–170. 6. C.P.M. Schutjes, Ph.D. Thesis, High Speed, High Resolution Capillary Gas Chromatography, Eindhoven University of Technology, 1983. 7. K.A. Cramers and P.A. Leclercq, Considerations on speed of separation, detection and identification limits in capillary GC and MS, CRC Crit. Rev. Anal. Chem., 20 (1988) 117–147. 8. A.J.J. van Es, J.A. Rijks and K.A. Cramers, Turbulent flow in capillary gas chromatography, J. Chromatogr., 477 (1989) 39–47. 9. A.J.J. van Es, J.A. Rijks, K.A. Cramers and M.J.E. Golay, Turbulent flow in capillary gas chromatography, J. Chromatogr., 517 (1990) 143–159. 10. A.J.J. van Es, H.G.J. Janssen, K.A. Cramers and J.A. Rijks, Sample enrichment in high speed narrow bore capillary gas chromatography. J. High Resolut. Chromatogr. Chromatogr. Commun., 11 (1988) 852–857. 11. P.A. Leclercq and K.A. Cramers, High speed GC–MS, Mass Spectrom. Rev., 19 (1998) 37–49. 12. K.A. Cramers, H.G. Janssen, M.M. van Deursen and P.A. Leclercq, High-speed gas chromatography: an overview of various concepts, J. Chromatogr. A, 856 (1999) 315–329.

D.8. John Vernon Dawkins John Dawkins was born on September 2, 1939. He received his education at the University of Birmingham, UK (1958–1964) with a B.Sc. in Chemistry, 1st Class Honours (1961), Ph.D. (1964), D.Sc. (1983), respectively. Earlier appointments include Postdoctoral at Duke University, USA (1964–1966) and University of Bristol, UK (1966–1967). He held a Senior Research Scientist position at ICI Petrochemical and Polymer Laboratory, UK from 1967–1972). From 1972 to the present, Dawkins has been associated with Loughborough University, UK, as Lecturer,

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Department of Chemistry (1972), Senior Lecturer (1980–1984), Reader (1984–1987) and Professor of Polymer Chemistry (1987 to date). He was the recipient of the 1985 Royal Society of Chemistry Award for Chromatography and Separation Chemistry, comprising of a bronze medal and a prize of £100; made contributions to the developments of products and equipment for polymer characterization — an infrared gel permeation chromatograph (1973), polystyrene gels (1978) and polyacrylamide gels (1981). Some of his external appointments include as Visiting Professor, Polymer Science Program, Institute of Materials Science, University of Connecticut, USA (1981–1982); Editor, “Developments in Polymer Characterization”, Volumes 1–5, published by Elsevier Applied Science Publishers Ltd. (1977–1986); Member of the Editorial Board, Journal of Liquid Chromatography, published by Marcel Dekker, Inc. (1979–1992); Member of the Permanent Scientific Committee for the International Symposium on Polymer Analysis and Characterization (1986–date); Member of the Editorial Board of the International Journal of Polymer Analysis and Characterization, published by Gordon and Breach Science Publishers (1995–date); and Joint Editor-in-Chief (1987– 1992) and Editor-in-Chief (from 1993) of the European Polymer Journal, published by Elsevier Science Ltd. Dawkins’ award citation from the Royal Society of Chemistry reads for ‘Distinguished outstanding contributions to the theory and practice of high performance gel permeation chromatography with special reference to polymer chemistry’. See Chapter 5B, a, e, g

8.I. CHARACTERIZATION OF POLYMERS BY GEL PERMEATION CHROMATOGRAPHY John V. Dawkins Department of Chemistry, Loughborough University, Loughborough, Leicestershire LE11 3TU, UK

8.I.1. Discovery, contribution, event(s), recollections My research on gel-permeation chromatography (GPC), also known as size-exclusion chromatography (SEC), commenced in 1967. At that time there was a need to produce accurate data for molecular weight distribution and average molecular weights for synthetic polymers by GPC. Reliable instrumentation was developed, and a chromatograph based on this work was marketed by Applied Research Laboratories (UK) in 1973. Calibration methods as a function of polymer structure, polymer conformation (random coil and rod-like chains), and polymer-solvent interactions were studied in detail. The experimental GPC studies of polystyrene with poor and theta solvents with columns containing cross-linked polystyrene gels merited special consideration [1]. From these studies it was proposed that GPC functioned by a network-limited separation involving

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a primary size exclusion mechanism and a secondary mechanism arising from polymer– gel interactions, e.g., adsorption, partition or secondary exclusion. Comprehensive treatments for retention in polymer separations by GPC were formulated and involved interpretations of distribution coefficients for primary and secondary mechanisms in terms of entropy, enthalpy and free energy contributions. These retention treatments initially reported in 1975, were presented at an international symposium held at Birmingham, UK (1976), at an American Chemical Society meeting in Chicago (1977), and at an IUPAC conference in Prague (1978) [2]. Molecular weight distributions of polymers determined by GPC are too broad. On moving to Loughborough in 1972, a programme aimed at improving the efficiency of GPC columns was initiated, so that errors in neglecting corrections for chromatogram broadening when calculating molecular weight data would not be significant. Initial experiments demonstrated that column efficiency for GPC separations of low polymers with porous silica and alumina increased as the particle size of the packing decreased [3]. Polymerization procedures were developed for the preparation of porous microspheres of cross-linked polystyrene and cross-linked polyacrylamide having sufficient rigidity for use in high performance GPC. High efficiency polymer packings (with particle diameters of 10 µm) based on this work have been marketed by Polymer Laboratories from 1978. Studies of column efficiency with these packings and appropriate operation of improved instrumentation were performed as a function of the flow velocity of the eluent, and of the diffusion coefficient of the polymer. Experimental conditions for particle size of the column packing and eluent flow rate for polydisperse polystyrene were identified for high speed separations and for precise determinations of molecular weight distribution [4]. High performance separations were extended to porous silica microspheres, with and without a bonded phase, for synthetic polymers and biopolymers in organic and aqueous eluents [5]. Column efficiency data were interpreted in terms of dispersion mechanisms for non-permeating and permeating polymers in the mobile and stationary phases. From theoretical considerations, an equation was derived for column efficiency, represented by plate height, to include three terms for eddy diffusion (mobile phase), mass transfer and true polydispersity for the molecular weight distribution. Experimental results were interpreted with this equation, and diffusion coefficients for polymers in a porous gel and polydispersities were calculated for polystyrenes and proteins. The first presentations of this theoretical approach were made at the international symposium held in Strasbourg (1979), at an American Chemical Society meeting in Las Vegas (1980), and at the Faraday Symposium of the Royal Society of Chemistry at Sussex, UK (1980) [6,7].

References 1.

J.V. Dawkins and M. Hemming, Gel permeation chromatography with cross-linked polystyrene gels and poor and theta solvents for polystyrene, 2. Separation mechanism, Makromol. Chem., 176 (1975) 1795–1813.

2.

J.V. Dawkins, Thermodynamic interpretation of polystyrene retention on cross-linked polystyrene gels

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in GPC with poor and theta solvents, J. Polymer Sci., Polymer Phys. Edn., 14 (1976) 569–571. J.V. Dawkins and G. Yeadon, High performance gel permeation chromatography of polystyrene, Polymer, 20 (1979) 981–989. J.V. Dawkins, T. Stone, G. Yeadon and F. P. Warner, High performance GPC with cross-linked polystyrene gels: Influence of particle size on the polydispersity of high polymers, Polymer, 20 (1979) 1164–1166. J.V. Dawkins and G. Yeadon, High performance gel permeation chromatography of polystyrene with silica microspheres, J. Chromatogr., 188 (1980) 333–345. J.V. Dawkins and G. Yeadon, Macromolecular separations by liquid exclusion chromatography, Faraday Symposia, 15 (1980) 127–138. J.V. Dawkins and N. P. Gabbott, Macroreticular polyacrylamide gel particles for aqueous high performance gel permeation chromatography of poly(ethylene oxide), Polymer, 22 (1981) 291–292.

D.9. Heinz Engelhardt Heinz Engelhardt was born on August 31, 1936 in Nu¨rnberg. He received his Diploma in Organic Chemistry in 1962 at the University of Erlangen-Nu¨rnberg. He continued research for a doctoral degree (Dr. Rer. Nat.) at the Institute of Organic Chemistry under the supervision of G. Hesse at the same university, which he finished in 1965. In the period of 1962 to 1971, he worked as teaching assistant in the organic chemistry laboratory, only interrupted for 13 months (1969–1970) for a stay as research associate at Northeastern University, Boston, MA in the laboratory of B.L. Karger. After returning to Erlangen, he finished his Habilitation (Dr. Habil.), and became Privat-Dozent in 1971. In the same year he became Professor at the Department of Applied Physical Chemistry at the University of the Saarland at Saarbru¨cken. In 1993, a new Institute of Instrumental and Environmental Analysis was founded at the same University by him, which Engelhardt is leading presently. In 1979, he was Visiting Professor at Northeastern University, Boston, MA, and in 1998 at the Universite´ de Marseille, France. The work of over 90 Master and Ph.D. students has been published in more than 230 papers related to separation sciences (thin layer, and classical column chromatography, HPLC, SFC, SFE, CE, and CEC). In 1975, Engelhardt wrote a book on HPLC (Hochdruck-Flu¨ssigkeits-Chromatographie) with a second German edition in 1977 and an English edition in 1979, which was subsequently translated into Russian and Chinese (both 1980). He also edited “Practice of HPLC” in 1993. Together with two of his students he wrote a book on ‘Kapillarelektrophorese’ in German in 1994, and in English in 1996. Engelhardt is on the Editorial Board of the J. of Chromatography, and Editor of Chromatographia since 1984. In 1992, he received the Dal Nogare Award in Chromatography from the Chromatography Forum of the Delaware Valley; in 1993 the A.J.P. Martin Medal from the Chromatographic Society of Great Britain, and in 1997 the Hala´sz Medal in Chroma-

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Professor Engelhardt in the laboratory at the Universita¨t des Saarlandes.

tography from the Hungarian Society for Separation Sciences. In 1999 he was a visiting Professor at the University of Innsbruck, Austria. In February 2000 he received a Dr. honoris causa from the University of Aix-Marseille, France. See Chapter 5B, a, e, h, k, l, o, p, s

9.I. FROM TLC TO HIGH PERFORMANCE INSTRUMENTAL ANALYTICAL TECHNIQUES Heinz Engelhardt Universita¨t des Saarlandes, Fachrichtung 12.6 Instrumentelle Analytik=Umweltanalytik, Im Stadtwald, Bau 12, D-66123 Saarbru¨cken, Germany

At Erlangen University chromatography was already an intrinsic part of chemical education around 1960. This was the reason I started with paper chromatography as an analytical tool, and with classical column chromatography in large dimensions (5 cm column diameter) in my master thesis to isolate heart glycosides from plant tissue. Chromatography was one of the favorite techniques of my teacher G. Hesse in the organic chemistry laboratory. During my master thesis I had the first success; I convinced my advisor that TLC is a much better tool than paper chromatography because of the ease of universal detection, higher speed of analysis and better sensitivity. With the precious sample of his ‘pure’ heart glycoside, I could demonstrate with

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TLC that it contained at least three constituents compared to one component in paper chromatography. Therefore, the decision was: chromatography is the field I should pursue. While running the big columns, I was always surprised that colored zones already emerged from the column while they were still way up in the column at the glass wall. I believed that it was due to heat of adsorption as the cause and thus started to measure zone profiles in big columns by inserting thermocouples in the column. This was not so easy and my colleagues were always joking about my ‘bleeding’ columns. I used Sudan red as a cheap and marker indicator. The temperature in the middle of the column was several degrees higher than at the wall. Temperature programming was used then as a technique to accelerate classical column chromatography. An old photometer was used as UV-detector in gravity driven LC. TLC was still the main research work at this time, like standardized water control for TLC plates, but I was looking for other stationary phases like titanium salts and polyphosphates. After some aberration and embarrassment with organic synthesis, I returned to chromatography when starting as a post-doc in Barry Karger’s laboratory at Northeastern University in 1969. The year I spent there has been one of the most important years in my career. Not only with the research in HPLC I did there on liquid chromatography, heavily loaded columns, etc., but also the persistent friendship with Barry started, and last but not least, I met I. Hala´sz. Returning to Erlangen, HPLC was introduced there, and I became Privat-Dozent for organic-analytical chemistry in 1971. In the meantime, a research institute had been established by the German Science Foundation at the University of the Saarland at Saarbru¨cken. I. Hala´sz was appointed as Head and I received a call in the same year as Professor to this Institute which was associated with the Institute of Physical Chemistry. After the retirement of I. Hala´sz in 1987, the Institute remained in existence and was renamed in 1993 as the Institute of Instrumental and Environmental Analysis. It is difficult to select only some of our achievements from the work of over 80 students who received their Ph.D.s with me. They all did their research with great endeavor and skill and everybody contributed to our overall knowledge and experience in chromatography. The results of their work have been summarized in monographs on HPLC [1–3], on capillary electrophoresis [4,5], and in more than 250 papers in scientific journals.

9.I.1. Preparation and characterization of stationary phases A significant part of our research has been the preparation and characterization of bonded stationary phases for HPLC. Starting with studies on retention mechanisms [6], and the properties of polar phases in protein analysis [7,8], a new technique for the preparation of polymer encapsulated stationary phases was developed [9,19]. By this technique, not only RP columns with excellent chromatographic properties can be prepared for the separation of basic analytes and better stability (up to pH 8.5) in aqueous solutions; it is also possible to prepare by this method ion exchangers [11] and stationary phases with a high selectivity and efficiency for enantiomeric separations.

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Fig. 1. Comparison of enantioselectivity between monomeric and polymeric chiral stationary phases. Upper chiral selector: L-phenylalanine- D-menthylesteramide. Sample: D,L-oxazolidinon. Upper chromatogram: Chiral silane bonded to silica; surface concentration, 2.37 µmol=m2 ; mobile phase, n-heptane–isopropanol (95 : 5); flow, 1 ml=min; k D 0:87; Þ D 1:00. Lower chromatogram: Chiral acrylamide bonded on silica, modified with vinylsilane; surface concentration, 2.44 µmol=m2 ; mobile phase, n-heptane–tetrahydrofuran (90 : 10); flow, 1 ml=min; k1 D 0:76; Þ D 2:93.

This is demonstrated in Fig. 1, where the same selector has been bonded to the silica surface by a bristle type reaction and by polyencapsulation. Only in the latter case could enantioselectivity be achieved. The characterization of packed columns for their efficiency and separation performance paralleled the work in phase synthesis. A commonly applicable test procedure has been developed [12] which allows one not only to determine the hydrophobic retention and selectivity of packed columns, but also to select columns for their potential to separate basic solutes with symmetrical peaks and high efficiency [13]. With the retention behavior of at least six solutes, the columns can be characterized unequivocally [14]. Meanwhile, a test also has been developed that permits the measurement of the surface contamination of stationary phases with metal ions. It could be demonstrated, that the columns are collecting metal ions on their surface originating from the chromatographic equipment, column hardware, and eluents. These test procedures can also be used to validate chromatographic columns. The validation of chromatographic instruments, e.g., flow accuracy measurements of pumps, or the linearity of detectors [15] is a useful extension of the work on stationary phase characterization.

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9.I.2. Post-column derivatization The limited sensitivity of chromatographic photometric detectors has initiated our work with post-column reaction detection, open tubes are used for the storage of eluent and reagent after the column during reaction time. However, due to the restricted mass transfer in open tubes with liquids, radial mixing has to be enforced. This can be achieved by continuously changing the geometrical direction of the flow of the capillaries. Such devices can easily be prepared by knitting or stitching organic polymeric capillary tubes [16]. The principle of reaction detection has been applied in the determination of free formaldehyde in cosmetics [17], nail varnishes and wall paints. For the analysis of carbamate residues in plants, a two step reaction detector gave similar detection sensitivity as gas chromatography, but sample preparation time could be reduced from 12 h to 30 min, due to the extremely selective detection of methylamine, a common hydrolysis product of all carbamates [18]. The detection sensitivity for proteins could be enhanced by post-column hydrolysis in a micro-wave oven and subsequent derivatization of the formed primary amino groups with OPA [19]. By applying knitted tubes to flow injection analysis, detection sensitivity could also be improved there [20]. Post-column derivatization is a generally applicable technique for enhancement of detection sensitivity and selectivity when the reaction times can be kept below 2 min. The pressure drop is much lower than with packed bed reactors. There are no limitations for reagent composition; the only prerequisite is that reaction product and reagent should have different absorption and=or emission wave-lengths.

9.I.3. Chromatography and extraction with supercritical fluids The renaissance of SFC started in the 1980s with the use of capillaries. In our opinion, this was the wrong direction, because of the low diffusion coefficients of the analytes in the mobile phase. Under the pressure conditions required for solute elution, their diffusion coefficients resemble more those in liquids than in gases. Narrower capillaries would be required than those applied in capillary SFC. Therefore, we started very early in using packed HPLC columns in SFC [21]. At the same time fluid systems were used for the extraction of pesticides from soil [22], toxic contaminants from food [23], and the total fat content of food [24]. Our studies on the retention mechanisms in SFC showed that carbon dioxide is a less polar eluent than pentane in the predominantly normal phase retention mechanism in SFC. This technique is superior to GC and LC in the separation of unsaturated fatty acids up to a preparative scale [25]. Packed columns SFC has a distinct place under the chromatographic techniques as a normal phase system, where the influence of the water of the mobile phases is negligible.

9.I.4. Capillary electrophoresis While working in separation science, capillary electrophoresis and capillary electrochromatography are challenging new areas in research. Coating of capillaries to

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Fig. 2. Fast CE with PEEK capillaries: separation of alkali ions. Capillary: PEEK i.d. D 75 µm; L D 35=26:5 cm; U D 30 kV; UV detection: 230 nm (through coupled HP high sensitivity cell). Buffer: 9 mM pyridine, 12 mM glycolic acid, 5 mM crown-6; pH D 3.6. Injection: 5 s at 35 mbar.

diminish wall adsorption and to modify the electro-osmotic flow has been a consequence from our work in modifying surfaces of silica to the transfer to fused silica capillary surfaces [26,27]. To circumvent some of the intrinsic problems of fused silica capillaries the potential of organic polymeric capillaries in CE has been studied [28]. The potential of PEEK (poly-ether-ether ketone) for fast analysis is demonstrated in Fig. 2. Our main effort in CE was in the separation of small molecules, the separation of enantiomers, but lately also in the analysis of synthetic polyelectrolytes [29]. Parallel with our work on fundamental studies of analytical separation techniques, the application of the methods to solve analytical problems was one of the main aims in our laboratory. This has only been possible through the intensive contact with the users of chromatography in pharmaceutical and chemical industries. This leads to the main work at the university: teaching young chemists in the fundamentals of separation science and guiding them to perform research at a high quality level. Furthermore, the transfer of acquired knowledge to the people in industry by giving seminars or teaching practical courses in the laboratory is of similar importance for the further application of separation techniques.

References 1. 2. 3. 4. 5.

H. Engelhardt, Hochdruck-Flu¨ssigkeits-Chromatographie, Springer, Heidelberg, 1975; 2. Auflage 1977. H. Engelhardt, High Performance Liquid Chromatography, Springer, Heidelberg, 1979; Russian translation, 1980; Chinese translation, 1980. H. Engelhardt (Ed.), Practice of High Performance Liquid Chromatography, Springer, Heidelberg, 1986. H. Engelhardt, W. Beck and Th. Schmitt, Kapillarelektrophorese, Vieweg, Braunschweig, 1994. H. Engelhardt, W. Beck and Th. Schmitt, Capillary Electrophoresis, Vieweg, Braunschweig, 1996.

Prominent Chromatographers and their Research 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

26. 27. 28. 29.

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K. Karch, I. Sebestian, I. Hala´sz and H. Engelhardt, Optimization of reversed-phase-separation, J. Chromatogr., 122 (1976) 171–184. H. Engelhardt and D. Mathes, High-performance exclusion chromatography of water-soluble polymers with chemically bonded stationary phases, J. Chromatogr., 185 (1979) 305–319. H. Engelhardt and P. Orth, Alkoxy silanes for the preparation of silica based stationary phases with bonded polar functional groups, J. Liq. Chromatogr., 10 (1987) 1999–2022. H. Engelhardt, H. Lo¨w, W. Eberhardt and M. Maub, Polymer encapsulated stationary phases: advantages, properties and selectivities, Chromatographia, 27 (11=12) (1989) 535–543. H. Engelhardt and M.A. Cunat-Walter, Polymer encapsulated stationary phases with improved efficiency, Chromatographia, 40 (11=12) (1996) 657–661. F. Steiner, C. Niederla¨nder and H. Engelhardt, Optimization of alkali-alkaline earth cation separation on weak cation-exchangers, Chromatographia, 43 (3=4) (1996) 117–123. H. Engelhardt and M. Jungheim, Comparison and characterization of reversed phases, Chromatographia, 29 (1=2) (1990) 59–68. H. Engelhardt, H. Lo¨w and W. Go¨tzinger, Chromatographic characterization of silica based reversed phases, J. Chromatogr., 544 (1991) 371–379. H. Engelhardt, M. Arangio and T. Lobert, A chromatographic test procedure for reversed-phase HPLC column evaluation, LC-GC, 15 (9) (1997) 856–866. H. Engelhardt and Ch. Siffrin, Means of validation of HPLC systems and components, Chromatographia, 45 (1997) 35–43. H. Engelhardt and U.D. Neue, Reaction detector with three dimensional coiled open tubes in HPLC, Chromatographia, 15 (1982) 403–408. H. Engelhardt and R. Klinkner, Determination of free formaldehyde in the presence of donators in cosmetics by HPLC and post column derivation, Chromatographia, 20 (1985) 559–565. H. Engelhardt and B. Lillig, Optimization of a reaction detector for analysis at the femtomol level of carbamate insecticides, Chromatographia, 21 (1986) 136–142. H. Engelhardt, M. Kra¨mer and H. Waldhoff, Enhancement of protein detection and flow injection analysis, J. Chromatogr., 535 (1990) 41–53. H. Engelhardt, R. Klinkner and G. Scho¨ndorf, Post-column reaction detection and flow injection analysis, J. Chromatogr., 535 (1990) 41–53. H. Engelhardt and A. Grob, On-line extraction and separation by supercritical fluid chromatography with packed columns, J. High Resolut. Chromatogr., 11 (1988) 38–42. H. Engelhardt and A. Grob, Extraction of pesticides from soil with supercritical CO2 , J. High Resolut. Chromatogr., 11 (1988) 726. H. Engelhardt and P. Haas, Possibilities and limitations of SFE in the extraction of aflatoxin B1 from food matrices, J. Chromatogr. Sci., 31 (1993) 13–19. P. Lembke and H. Engelhardt, Development of a new SFE method for rapid determination of total fat content of food, Chromatographia, 35 (9–12) (1993) 509–516. H. Engelhardt and R. Krumbholz, P. Lembke, Patent No. EP 558,774 (1993) Manufacture of polyunsaturated fatty acids and their derivatives from animal and vegetable fatty acids and fatty acid derivatives. J. Kohr and H. Engelhardt, Capillary electrophoresis with surface coated capillaries, J. Microcolumn Sep., 3 (1991) 491–495. H. Engelhardt and M.A. Cunat-Walter, Preparation and stability tests for polyacrylamide-coated capillary electrophoresis, J. Chromatogr. A, 716 (1–2) (1995) 27–33. H. Bayer and H. Engelhardt, Capillary electrophoresis in organic polymer capillaries, J. Microcolumn. Sep., 8 (7) (1996) 479–484. H.N. Clos and H. Engelhardt, Separations of anionic and cationic synthetic polyelectrolytes by capillary gel electrophoresis, J. Chromatogr. A, 802 (1) (1998) 149–157.

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D.10. Leslie S. Ettre Leslie Stephen Ettre was born on September 16, 1922, in Szombathely (Hungary), but grew up in Szentgottha´rd, a small town on the border of Hungary with Austria. After finishing his primary and secondary education in the local schools, he studied at the Technical University Budapest, graduating in 1945 with a degree in chemical engineering. Later he also received a technical doctorate (Dr. Tech.) from the same school. Between 1946 and 1949, he was associated with the Gedeon Richter Pharmaceutical Co., in Budapest, as a chemical engineer. In 1949, he joined the Research Institute for the Heavy Chemical Industries, in Veszpre´m (Hungary) as a research associate, advancing to the Head of the Technical Office of the Institute. He also taught at the local University of Chemical Engineering. In 1953, after a short period as the head of the department in charge of the industrial chemical research institutes in the Ministry for the Chemical Industry, he was appointed as the Head of the Industrial Department at the Hungarian Research Institute on Plastics, in Budapest. In September 1956, he accepted a position at BUNA, one of the largest chemical plants in Eastern Germany, but his stay there was cut short by the Hungarian revolution. After moving to West Germany by the year’s end, he became a chemist in the R&D laboratories of the LURGI Companies, in Frankfurt am Main, in charge of the gas chromatography laboratory. He immigrated to the United States in the fall of 1958 when he joined the Perkin-Elmer Corporation, in Norwalk, CT, as an applications engineer. During his 32-year association with the company, he had various appointments with increasing responsibilities. His last position was that of a senior scientist, the highest nonmanagerial position at the company. He retired from Perkin-Elmer at the end of 1990. Since 1988, he has also been affiliated with the Department of Chemical Engineering of Yale University (New Haven, CT), first as a senior lecturer; starting with the 1990=1991 school year he was appointed an adjunct professor to the department. He retired from teaching in 1994, but continues his affiliation with the Chemical Engineering Department as a research affiliate. He is the author and co-author of over 200 publications and 15 books, and the editor of 41 books. Between 1968 and 1974, he has served as the Executive Editor of the Encyclopedia of Industrial Chemical Analysis, a 20-volume set published by John Wiley and Sons. He has been an editor of Chromatographia from 1970 until the end of 1994 when he became a member of the journal’s advisory board. Presently he is also on the Editorial Advisory Boards of LC-GC Magazine, both its American and European (international) editions, and of the Magyar Kemikusok Lapja (Hungarian Chemical Journal), the publication of the Hungarian Chemical Society. He has also served on the Editorial Advisory Boards of the Journal of Chromatographic Science (1963–1994) and the Journal of Liquid Chromatography (1984–1993). Between 1966 and 1973, he has served as the chairman of the Subcommittees on Research (1966– 1970) and Nomenclature (1970–1973) of Committee E-19 of the American Society for Testing and Materials (ASTM); between 1981 and 1991, he has been a member

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of the Commission of Analytical Nomenclature, International Union of Pure and Applied Chemistry (I.U.P.A.C.), where he was responsible for the development of the new Unified Nomenclature for Chromatography issued in April 1993. Between 1982 and 1987, he has served as a member of the Executive Committee of the (British) Chromatographic Society, and between 1988 and 1993 as a member of the Executive Committee of the Chromatography Subdivision, Division of Analytical Chemistry of the American Chemical Society. He has lectured widely in the world on the invitation of universities, scientific associations and academies, and has been active in the organization of several international symposia on chromatography. He has received the National Award in Chromatography of the American Chemical Society (1985) and the A.J.P. Martin Award of the Chromatographic Society (1982), the highest American and European honors in this field, respectively — he is also the recipient of the M.S. Tswett Award in Chromatography of the International Symposium on Advances in Chromatography (1978), the Anniversary M.S. Tswett Chromatography Medal of the All-Union Scientific Council on Chromatography of the USSR Academy of Sciences (1979), the L.S. Palmer Award of the Minnesota Chromatography Forum (1980), the Anniversary Medal of Tartu University (Estonia, 1981), the Merit Award of the Western Carolinas Chromatography Discussion Group (1987), the M.S. Tswett Medal of the Russian Chromatographic Society (1991), the M.J.E. Golay Award (1992) and the Jubilee Award (1998) of the International Symposium on Capillary Chromatography, and the Keene P. Dimick Award in Chromatography of the Society for Analytical Chemists of Pittsburgh (1998). In 1992, he was elected an honorary member of the Hungarian Chemical Society and in 1995 he received the Golden Diploma of Budapest Technical University. In the past 40 years, his major area of research has been chromatography. His activities covered a wide variety of fields including trace analysis, studies on detector response, headspace sampling, reaction-gas chromatography, the retention index system and particularly the theory and practice of open-tubular columns; his first pioneering book on this subject was published in 1965. In the last two decades his interest has focused on the history of chromatography, particularly on the early evolution of the various techniques, looking at them in the proper historical and political context and investigating their interaction with other scientific disciplines. See Chapter 5B, a, d, e, h, k

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10.I. THE REMEMBRANCES OF A CHROMATOGRAPHER Leslie S. Ettre * Department of Chemical Engineering, Yale University, New Haven CT, USA

I started to be involved in chromatography in the spring of 1957. I was a penniless refugee in Frankfurt am Main, Germany, waiting for an immigration visa to the United States. However, after some delay I was told that it may take over a year; the accelerated process for Hungarian refugees was being applied only to those who were registered in Austria which I bypassed. Therefore I was advised to settle in Germany, at least temporarily. I applied for a position at LURGI, one of the principal European companies engaged in developing chemical processes and building factories for these processes. Upon checking my background they saw in my resume the titles of a couple of my publications from Hungary that dealt with pilot plant separations of various tar components. Because of this, my interviewers felt that I must be familiar with separation methods and they offered me a job as an analytical chemist, with the main task to bring in operation a recently purchased gas chromatograph (a Perkin-Elmer Model 154B): in fact, this was one of the first instruments sold in Germany. Their problem was that nobody was in charge of the instrument and thus, it never worked properly although they had great expectations of its use. I did not know anything about gas chromatography at that point, but — as the Hungarian proverb says — need is the best teacher, and within a few months I managed to establish a well-functioning laboratory with three technicians, adding two more instruments to the original one and analyzing daily a fairly large number of a wide variety of samples. When I say ‘analyzing’, I do not mean just obtaining a chromatogram with nice peaks, but to establish the qualitative and quantitative composition of these samples. I was continuously reminded that optimization of pilot plant operation for the development of chemical processes and the design of new plants depended on the answers I supplied. Each sample was a ‘first’: there were no standard methods, no publications on similar problems, and nobody who could advise me about the conditions or the columns to be used: I had to figure them out myself. In retrospect I can say that the 17 months spent at LURGI represented an excellent schooling. I would strongly advise my young colleagues to serve for a couple of years in a service laboratory which has to provide answers in a short time to other parts of an organization whose operation depends on these results. At that time Perkin-Elmer just opened an office in Frankfurt and I was continuously bugging their people for information, but they were of little help since they were also going through the same learning period. However, this continuous contact with them had an unexpected and very pleasant result. When finally, I was able to immigrate to the United States in September 1958, I was almost immediately hired by Perkin-Elmer on their recommendation. I found out only later that their ‘recommendation’ was actually not very flattering: “we had so much trouble with this guy as a customer that you better hire him, so that he won’t be a customer anymore” was the advice of the Frankfurt *

Correspondence address: P.O. Box 6274, Beardsley Station, Bridgeport, CT 06606-0274, USA

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office manager to the people in Norwalk. This is how my 32-year association with Perkin-Elmer started. At Perkin-Elmer I became a member of their Applications Engineering Group and two years later the head of the GC group within it. It was an exciting time: gas chromatography had just started its exponential growth and we were in the frontline. Somebody once remarked jokingly that I was present at the creation; my answer was that I was there a few seconds after the start. At that time, everything we did was new: new developments, new applications, and new instrumental modifications, developing and building various gadgets permitting to enter a new field. It was our duty to help our customers in the use of their instruments, to open new fields for GC and also to advise our own people how the existing instruments could be improved. During this crucial period of instrument development — not only in GC but in other fields as well — the Application Engineering Groups of the instrument companies served a vital role. In fact our activities were considered so important that in 1961 Analytical Chemistry devoted one of its Report for Analytical Chemists to the key role of these groups in instrumental analysis. This report characterized our role in the most fitting way, saying that “the Applications Engineer represents the customer’s viewpoint within the corporation and the corporation’s technology to the customer.” From the mid-1960s on, the field matured and this pioneering role of the Applications Engineering Groups diminished; in fact they were incorporated in the general R&D and marketing functions of the companies. Still, when reminiscing on my activities in the past 40 years, I want to emphasize their role — our role — in the early development of the technique. This decade was probably the most exciting period of my professional life. It is almost impossible to summarize one’s professional activities in a few pages; it would look like a listing of titles of publications and could easily be interpreted as an attempt of self-aggrandizement, over-emphasizing one’s own role. Therefore, I would rather deal with three particular aspects. It is often emphasized that the frequent meetings, symposia, the cooperation of scientists and the almost continuous contact had characterized the growth of chromatography across borders and oceans. I was part of this and, when looking back to the past 40 years, the opportunity to meet so many interesting people represents one of the most exciting aspects of my activities. My connection with them usually went over a professional relationship, becoming a true friendship, not only here in the US, but also in many different parts of the world. Unfortunately, many of them are no longer with us; however, their achievements form a permanent part of the history of chromatography. One of the great pleasures of my life was the possibility to travel, mainly on chromatography business: participating at meetings, lecturing or visiting laboratories. Recently I made a mental inventory of the many places I visited and I was surprised to realize that they were located in almost three scores of countries in the five continents. This was probably the most exciting part of my career. I enjoyed traveling, and it was always more than a business trip: I always tried not to be a simple tourist but observe and understand the different cultures and customs and try to become a part of them. When I was in high school, I learned a Latin proverb (at that time in Hungary we still had Latin for six school years) according to which peregrinare necesse est, and this

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proverb expresses perfectly my feeling. It means ‘it is necessary to travel,’ but the verb peregrinare means more than just traveling: it implies to stay at places and live there according to the local habits. If I would be asked to give a single advice to young people, it would be to visit as many places in the world as possible, learn their customs and habitat, and appreciate the multitude of cultures you will encounter. Finally I would like to return to chromatography. Soon after joining Perkin-Elmer, I had the good fortune to meet M.J.E. Golay and from then on until practically the last days of his life — he died in 1989, a few weeks before his 87th birthday — I had the privilege of being able to cooperate with this great scientist. Of course, Marcel is known as the inventor of open-tubular (capillary) columns and this field has also occupied a significant part of my activities over the past 40 years; the citation of my American Chemical Society National Award in Chromatography in 1985 referred to my contributions “to the theory and practice of open-tubular columns.” However, I am not shy to say that everything I did in this field, I learned from him, Marcel was not a teacher in the common sense: he never taught anybody how to do something but rather how to approach a problem and how to think. He conveyed an attitude, a philosophy, and this was the most important legacy he could give to others. He was also a polyhistor in the classical sense of this term: his interest ranged from physics and mathematics to literature and art, from the creation of the Universe to the Rubik cube. He could explain with authority not only the theory of chromatography, but also the best way to cook a lobster or to test the ‘dryness’ of a martini cocktail. Looking back on a lifetime of activities, one remembers not only the achievements and successes but also the failures and missed opportunities. However, most important is the overall assessment, and for me it is definitely positive. It was an exciting time to participate in the evolution of one of the most important methods of instrumental analysis and to be able to cooperate with a large number of prominent scientists across the whole world. But most importantly, it certainly was a great fun. References Selected books 1. 2. 3.

L.S. Ettre, Open-Tubular Columns in Gas Chromatography, Plenum Press, New York, 1965. 164 pp. L.S. Ettre and A. Zlatkis (Ed.), 75 Years of Chromatography — A Historical Dialogue, Elsevier, Amsterdam, 1979, 502 pp. B. Kolb and L.S. Ettre, Static Headspace — Gas Chromatography — Theory and Practice. Wiley-VCH, New York, 1997, 298 pp. Selected Research Publications.

Selected research publications 1. 2. 3.

L.S. Ettre and W. Averill, Investigation of the linearity of a stream splitter for capillary gas chromatography, Anal. Chem., 33 (1961) 680–684. A. Zlatkis, D.C. Fenimore, L.S. Ettre and J.E. Purcell, Flow programming: a new technique in gas chromatography, J. Gas Chromatogr., 3 (1965) 75–81; (12) 16A–17A. L.S. Ettre and E.W. March, Efficiency, resolution and speed of open-tubular columns as compared to packed columns, J. Chromatogr., 91 (1974) 5–24.

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4.

L.S. Ettre and J.E. Purcell, Porous-Layer Open-Tubular Columns: Theory, Practice and Applications. J.C. Giddings and R.E. Keller (Eds.): Advances in Chromatography, Vol. 10, Dekker, New York, NY, 1974, pp. 1–97. 5. L.S. Ettre, J.E. Purcell, J. Widomski, B. Kolb and P. Pospisil: Investigations of equilibrium headspace open-tubular column gas chromatography, J. Chromatogr. Sci., 18 (1980) 116–124. 6. L.S. Ettre, Open-tubular columns prepared with very-thick liquid phase film. I. Theoretical basis, Chromatographia, 17 (1983) 553–559. 7. L.S. Ettre, G.L. McLure and J.D. Walters: Open-tubular columns prepared with very thick liquid phase film 11. Investigations on column efficiency, Chromatographia, 17 (1983) 569–706. 8. L.S. Ettre, Performance of open-tubular columns as function of tube diameter and liquid phase film thickness, Chromatographia, 18 (1984) 477–488. 9. J.V. Hinshaw and L.S, Ettre, Selectivity tuning in serially connected open-tubular (capillary) columns in gas chromatography, Chromatographia, 21 (1986) 561–572, 669–680. 10. J.V. Hinshaw and L.S. Ettre, Aspects of high-temperature capillary gas chromatography. J. High Resolut. Chromatogr., 12 (1989) 251–254. 11. B. Kolb and L.S. Ettre, Theory and practice of multiple headspace extraction, Chromatographia, 32 (1991) 505–513.

Selected publications on history of chromatography 1. 2.

3.

L.S. Ettre and A. Zlatkis (Eds.), 75 Years of Chromatography — A Historical Dialogue, Elsevier, Amsterdam, 1979, 502 pp. L.S. Ettre, Evolution of liquid chromatography: A historical overview, pp. 2–74, Vol. 1–1980, in: C. Horva´th (Ed.), High Performance Liquid Chromatography, Academic Press, New York, NY, 5 Vols., 1980–1988. Please refer to Chapter S-8 on the ChemWeb Preprint server (http:==www.chemweb.com=preprint=) for additional publications.

D.11. Michael Bryan Evans Michael Bryan Evans was born in Sible Hedingham, Essex, United Kingdom, on September 7, 1931. He was educated at Kings School, Macclesfield, where he completed his School Certificate at age 16. Mike’s first job was as a technician in the analytical laboratory at the Dunlop Rubber Company in Manchester, where he was encouraged to continue his education leading to an Ordinary National Certificate (ONC) in Chemistry and the award of a Technical State Scholarship, which enabled a return to full-time education. He graduated in 1954 with an External London B.Sc. and the Associateship of the Royal Institute of Chemistry. In 1956, he joined the British (now Malaysian) Natural Rubber Producers’ Research Association (NRPRA), in Welwyn Garden City, UK, as an instrumental analyst. Here he was introduced, in 1959, to gas chromatography, a topic that was to occupy his interest for the next 40 years. At BRPRA research was carried out which was to lead to the award of a London University Ph.D., as an external student, in 1968. A chance meeting with a former teacher from Salford resulted in a part-time lectureship in physical chemistry at the then Hatfield Technical College, later to become Hatfield Polytechnic and today is the University of Hertfordshire. Mike was appointed

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Michael B. Evans at University Degree Ceremony, November 1998, at which he received his D.Sc. degree.

Senior Lecturer in Analytical Chemistry in 1966, with promotion to Principal Lecturer in 1972, Head of Department in 1982, followed by a Professorship in 1986. Today he is an Emeritus Professor of Analytical Chemistry. His research has concentrated on GLC, HPLC and more recently CEC, topics in which he has gained an international reputation, with medal awards in 1984 and 1994. In particular, he is noted for his collaborative studies with scientists in industry. Mike has published 87 papers in refereed journals, presented 48 conference papers and has supervised 20 Ph.D. students. In 1997 he was awarded a D.Sc. by the University of Hertfordshire for a submission entitled “Studies in analytical chemistry with special emphasis on separation methods”. He has been an active member of the Chromatographic Society for 40 years, occupying with distinction the offices of Secretary, Treasurer, Deputy Chairman and for three years, President. Recently he was made an honorary life member in recognition of his services to the Society. In 1994, Mike was the Chairman of the 19th International Symposium on Chromatography held in Bournemouth, UK.

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In 1984, he received the Royal Society of Chemistry Award in Analytical Separation Methods the citation read as follows: “Distinguished for his continuous record of research in chromatography from 1961 to date. ‘His work has encompassed both gas and liquid chromatographic analysis and he has made a significant contribution to the theoretical aspects of the subject as well as to its application in the polymer and pharmaceutical fields. Michael Evans has played an important role in the training of young scientists and research workers throughout his career.’”

See Chapter 5B, a, d, e, i, m, r

11.I. INVERSE GAS CHROMATOGRAPHY IN THE STUDY OF THE OXIDATIVE DEGRADATION OF UNSATURATED ELASTOMERS Michael Bryan Evans University of Hertfordshire, 20 Hertford Road, Digswell, Welwyn, Hertfordshire AL6 0DE, UK

11.I.1. Introduction The oxidative degradation of polymers is a common cause of the failure of manufactured articles under service conditions. This is particularly the case with unsaturated elastomers, such as natural rubber, which are prone to aerobic oxidation. Much research has been directed towards the solution of these problems, through studies involving the base polymers, formulated products and structurally related model compounds. Generally these have involved measurements of physico-chemical properties and uptake of oxygen to monitor the effects of oxidation. The purpose of the work described in our research was to test the feasibility of using inverse gas chromatography as an alternative method for studying the oxidative degradation of rubber. A technique, that is a variant of gas–liquid chromatography, in which retention measurements are used to monitor chemical changes occurring within a substance used as the liquid phase.

11.I.2. Initial experiments Squalene, a naturally occurring acyclic dihydrotriterpene consisting of six isoprene units, is commonly used as a model for natural rubber in oxidation studies. However, as squalene is rapidly oxidized, even at moderate temperatures, it was decided to use its hydrogenated analogue squalane for the preliminary studies. Particularly as squalane is an established stationary phase and readily available in a pure state. A squalane=Celite column was prepared, conditioned in a stream of oxygen free nitrogen, at 100ºC and chromatograms were recorded for calibration solutions, containing n-alkanes as internal standards, as soon as a stable base-line was observed. Six

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compounds of increasing polarity were used as the test solutes for the measurement of Kova´ts retention indices. The carrier gas was switched to air and the column oxidized at 100ºC for a period of 4 h, at the end of which, the retention characteristics of the stationary phase were checked by chromatography of the calibration solutions with nitrogen as carrier gas. The cycle of oxidation and retention measurement was continued over a period of a week. Overnight the column was left at room temperature after flushing with nitrogen. The retention indices of all the test solutes were found to decrease with oxidation time, pass through a minimum value and then increase. In each case, the decreases in retention were accompanied by improvements of peak symmetry, the peaks being fully symmetrical at the minimum and beyond. These observations were consistent with oxidation of squalane to form products, which are selectively adsorbed on the support surface, thereby precluding the adsorption of solute molecules leading to peak tailing. Once a coherent protective film had been formed, further oxidation would be expected to lead to increases of retention due to the presence in the stationary phase of the excess of oxygenated groups, as observed. On this evidence, the minimum of the curve may be regarded to be the retention index of a particular compound corresponding to gas–liquid partition between the mobile gas phase and pure squalane. Support for this hypothesis was provided by the results of a further oxidation experiment in which the support was deliberately contaminated with silica gel, to increase its effective adsorptivity, and a study of the effect of adding diglycerol, as a support deactivator, to squalane=C-22 firebrick columns. The minimum retention index values observed for the individual test solutes, which covered the range 800–1000, for the different columns were in good agreement to within š5 index units. The above experiments not only demonstrated that the retention index measurements could be used to monitor stationary phase oxidation, but also provided evidence of the occurrence of liquid–solid adsorption during the gas–liquid chromatography of polar solutes on apolar columns and its prevention by hydroxylic surfactants.

11.I.3. Study of the oxidation of squalene as a model for the oxidative degradation of natural rubber The modern theories of the oxidation of unsaturated hydrocarbons are substantially based upon the studies of Bateman and his co-workers at NRPRA, who established the free-radical nature of the process and the involvement of alkyl radicals arising from initially formed allylic hydroperoxides. With 1,5-dienes, such as cis-1,4-polyisoprene (natural rubber) and squalene, hydroperoxide formation is accompanied by the introduction of peroxide groups to yield products which undergo main-chain scission, with the elimination of leavulinaldehyde. Thus the oxidation of a squalene column would be expected to give rise to shifts of retention index accompanied by increases of the detector standing current. Provided oxygen was rigorously excluded during their preparation, and subsequent use, it proved possible to produce columns with reproducible retention characteristics. Solutes with hydrogen donor and acceptor functional groups generally were found to give asymmetric tailing peaks, typical of apolar stationary phases. When deliberately

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oxidised, using air as carrier gas, characteristic shifts of retention index were observed, similar to those that occurred with squalane, but in a considerably reduced time span. In order to monitor the oxidation of squalene by inverse gas chromatography alkyl bromides were chosen as physico-chemical probes, insofar as they were sufficiently sensitive to the changes of liquid phase selectivity, but not affected by adsorption at the liquid–solid interface. Plots of retention index versus volumetric airflow were obtained, which were reproducible from column to column, similar to those obtained by oxygen absorption measurements. Furthermore, oxidation curves obtained at temperatures between 60 and 100ºC gave rise to a linear Arrhenius plot of log dI=dT vs. 1=T and activation energy of 94 kJ=mol, typical of olefin oxidation. The rate of oxidation of the squalene columns also was found to be affected by contamination with transition metal salts, such as Co(II), Fe(III) and Mn(II). Again the GC oxidation curves were found to be consistent with the results of previous investigations into the effect of metal ions on the oxidation behavior of olefins. In each of the above experiments, the changes of retention were accompanied by displacement of the baseline due to the evolution of leavulinaldehyde, the presence of which in the exit gas was confirmed by smell and formation of the 2,4-dinitro-phenylhydrazone. The above changes were as anticipated; on the other hand, the concomitant decrease of peak efficiency was unexpected. In order to investigate this effect, a fresh column was prepared and oxidized, as previously, and values for retention index, capacity factor and plate number determined. After a short induction period, autocatalysis occurred as reflected by rapid changes of retention and a catastrophic fall in column efficiency from an initial 2280 plates=m to a final value of 30 plates=m. Furthermore, a repeat experiment revealed a marked increase of the C-term of the van Deemter equation possibly due to cross-linking leading to a highly viscous product in which analyte diffusion would be reduced. Polyisoprenes may be cross-linked by a variety of reactions of which peroxide vulcanization yields well defined products involving the formation of carbon–carbon bonds leading to a three dimensional network. Reaction of squalene with dicumyl peroxide gave rise to highly viscous products, which proved to be unsuitable for the preparation of column packings by the slurry process. Accordingly vulcanized squalene column packings were produced by carrying out the cross-linking reaction in situ. The resulting columns were found to yield plate values which decreased as the molar proportion of dicumyl peroxide increased, an observation which strongly supports the oxidative cross-linking hypothesis.

11.I.4. Study of the oxidation of sulphurated squalene as a model for the oxidative degradation of natural rubber vulcanizates Vulcanization is the key process whereby the elastic, strength and load-bearing qualities, which are innate in raw rubber, are fully realized. In rubber technology, sulphur vulcanization, leading to the introduction of sulphidic cross-links, is the most commonly used process. Vulcanization with sulphur alone is a relatively inefficient process and today has been largely replaced by systems involving the addition of accelerators or sulphur donors. Such reactions give rise to structurally simpler networks,

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in which dialkenyl mono- and disulphidic cross-links predominate, and products with superior physical properties. In order to extend the applicability of inverse gas chromatography to technologically important systems the technique was evaluated by a study of the oxidation of squalene vulcanized by unaccelerated and accelerated sulphuration reactions. Mixtures of squalene with sulphur, sulphur=mercaptobenzthiazole=zinc oxide and tetramethylthiuram disulphide=zinc oxide, respectively, were heated in Carius tubes at 140ºC and the products used to prepare column packings by the slurry technique. Individual columns were conditioned in a stream of nitrogen and then oxidized at 100ºC with air as carrier gas. The reaction was monitored by chromatography of 1-bromopentane together with n-alkane standards and plots of retention index versus reaction time constructed. For each system autoxidation was preceded by an induction period where retention index values increased gradually, due to the formation of polar oxygenated sulphur groups within the cross-links. Consistent with the results of previous studies on the oxidation of dialkenyl sulphides the induction periods were found to increase with the chain length of the sulphur cross-links, with the exception of the sulphur donor reaction product. Here an extended induction period was observed that was shown to be due the formation, as a by-product of the reaction, of zinc dimethyldithiocarbamate, a known oxidation inhibitor.

11.I.5. Study of the inhibited oxidation of squalene as a model for antioxidant protected natural rubber vulcanizates Rubber articles are normally protected from the deleterious effects of atmospheric oxidation by the addition of small quantities of antioxidant. Materials that either interrupt the propagation reaction or decompose the initially formed hydroperoxides into compounds that are unable to promote the radical chain reaction. Amine and phenolic antioxidants function by removal of the chain carrying species, on the other hand, metal dithiocarbamates act as hydroperoxide decomposers converting them into the corresponding alcohols. In order to demonstrate the applicability of inverse gas chromatography to the testing of antioxidant efficiency, a series of squalene columns were prepared containing 0.05% of 2,6-di-tert-butyl-4-methylphenol (BHT), N -iso-propyl N 0 -phenyl-p-phenylenediamine (IPPD) and cadmium di-n-butyldithiocarbamate (CdDBC), respectively. Each was oxidised in a stream of air at 100ºC, whilst the polarity of the column was monitored by measurement of the retention index of 1-bromopentane. The oxidation curves obtained for the antioxidant protected systems all displayed definite induction periods, during which the retention index of 1-bromopentane remained constant. The results indicated a wide variation in the protection afforded by the three antioxidants. Whereas the column containing BHT reached the autoxidative stage in 5 h, those protected by CdDBC and IPPD took 20 and 80 h, respectively. The inferior performance of the phenolic antioxidants was unexpected because when tested by the oxygen absorption method BHT and IPPD were found to be equally efficient. However, whereas the test procedures used in rubber technology involve enclosed systems, inverse gas chromatog-

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raphy is a dynamic method. Clearly loss of BHT due to its vaporization during inverse gas chromatography would explain the apparently anomalous result. Experiments with pre-heated squalene=BHT columns and with a novel antioxidant prepared by interaction of PEG 400 and 3,5-di-tert-butyl-4-hydroxybenzoyl chloride provided confirmatory evidence. In service, car and truck tyres are subject to heat build up in a stream of air; thus inverse gas chromatography would appear to have distinct advantages over the methods generally used in rubber technology.

11.I.6. Conclusions Inverse gas chromatography has been shown to be a useful technique for study of the oxidation behavior of gum rubbers and vulcanizates that is complementary to oxygen absorption methods. The technique also provides a means of testing the efficiency of antioxidants under service conditions.

References Inverse gas chromatography in the study of polymer degradation 1. 2. 3. 4.

M.B. Evans and R. Newton, Part 1: Oxidation of squalene as a model for the oxidative degradation of natural rubber, Chromatographia, 9 (1976) 561–566. M.B. Evans and R. Newton, Part 2: Oxidation of sulphurated squalene as a model for the oxidative degradation of natural rubber vulcanizates, Chromatographia, 11 (1978) 311–315. M.B. Evans and R. Newton, Part 3: Study of the inhibited oxidation of squalene as a model for antioxidant protected natural rubber vulcanizates, Chromatographia, 12 (1979) 83–88. M.B. Evans, Part 4: A study of model systems for reinforced vulcanizates, Chromatographia, 36 (1993) 241–245.

Gas–liquid chromatography in qualitative analysis 1. 2. 3. 4. 5.

M.B. Evans and J.F. Smith, Part 6: An investigation of the changes in relative retention data accompanying the oxidation of apolar liquid phases, J. Chromatogr., 28 (1967) 277–284. M.B. Evans, Part 11: A study of the retention characteristics of diglycerol deactivated gas chromatographic columns, Chromatographia, 4 (1971) 441–447. J.D. Carmi, M.B. Evans and R. Newton, Part 15: Deactivation of diatomaceous supports by amine antioxidants, J. Chromatogr., 166 (1978) 101–109. A.D. Dale and M.B. Evans, Part 19: The use of antioxidants to delay the oxidation of polyoxyethylene glycol stationary phases, J. Chromatogr., 552 (1991) 161–167. M.B. Evans, M.I. Kawar and R. Newton, An investigation of the changes of column efficiency accompanying the oxidation of apolar gas chromatographic stationary phases, Chromatographia, 14 (1981) 398–402.

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D.12. J. Calvin Giddings (1930–1996) J. Calvin Giddings was born in Utah in 1930 and received the majority of his education in Utah. He was a graduate of American Fork High School and Brigham Young University and did his graduate research under the renowned physical chemist, Henry Eyring, at the University of Utah where he received his Ph.D. in 1954. He did postdoctoral work under Joseph Herschfelder at the University of Wisconsin and then in 1957, he joined the faculty of the Department of Chemistry at the University of Utah. He became an Associate Professor in 1959 and Professor in 1966. Although his training was mainly in physical chemistry, he became one of the top theoreticians in the field of separation science and a giant in this area. He was a brilliant pioneer who blazed the trail with his precise mathematical treatment of the mechanisms involved in chemical separations and made outstanding contributions to the development of chromatographic theory. He was able to picture a complex process in his mind, describe it mathematically and then clearly paint a word picture. In 1958, he developed the random walk theory of chromatography [1], and later the quasi-equilibrium model that elegantly simplified the mathematical description of the complex processes that occurred in a chromatographic column during a separation [2]. He proposed that the optimal velocity of a separation was based on a balance between rate and diffusion processes. He realized that both liquid and gas chromatography could be described using the same theoretical models [3]. He used the models he developed not only to describe the processes in the separations, but also to optimize separations. Giddings felt that theory was important because it provided “the power of prediction, control, correlation and calculation.” He found that the mechanisms of physical and chemical phenomena could be elucidated and simplified by theory, and he showed how theory could guide the development and optimization of a separation system. According to him, theoretical work was “like laying the bricks of a satisfying edifice, tying diverse chromatographic phenomena to dynamical roots which lead to predictions of efficiency.” In his classic book “Dynamics of Chromatography”, Volume I, published by M. Dekker in 1965 [4], he laid the foundation that made possible the developments in liquid chromatography as well as anticipating many of the advances that were made after the book was published. His more recent book “Unified Separation Science” [5] is a basic text relating various separation techniques. In addition, he was the Founder and Executive Editor for many years of the ‘Advances in Chromatography’ series and the journal “Separation Science”, long before the importance of chromatography and related techniques for the biological sciences and other scientific disciplines was recognized. One of his most important contributions to separations was his ability to connect real life phenomena and the theoretical equations that describe these phenomena. His theoretical approach to band broadening in chromatography was the foundation of developments in the field. His early work was done with gas and paper chromatography and later included liquid chromatography. His paper chromatography work is still

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relevant to modern TLC [6] and his GC work is the basis of modern HPLC. He then developed a group of techniques he called ‘field-flow fractionation’ (FFF) [7]. These techniques extended the range of chromatographic separations from small molecules to macromolecules, and particles of every shape and size. Giddings was a superb but demanding teacher whose lectures were crystalline clear. He was also a meticulous writer, whose manuscripts were models of clarity, both in thought and presentation. He published over 400 articles, wrote four and edited 32 books. Most of his articles were on separations; however he also wrote articles on such diverse topics as flame kinetics, nuclear kinetics, snow and avalanche physics, as well as articles on his outdoor activities and exploration expeditions. He was an avid environmentalist and in 1973 he wrote a textbook for undergraduates “Chemistry, Man and Environmental Change” [8], and edited a book with B.M. Monroe in 1972 “Our Chemical Environment”. He loved nature and was an outstanding sportsman, excelling in kayaking, skiing and mountain climbing. Giddings received many awards for his pioneering work including the American Chemical Society Awards in Chromatography and Electrophoresis (1967), in Analytical Chemistry and in Separation Science and Technology (1986), M.S. Tswett Medal in Chromatography, the Nichols Award from the New York Section of the ACS, the Dal Nogare Award in Chromatography, a Fulbright Fellowship and the Roscoe Award for Outstanding Environmental Achievement in Education. He was nominated for the Nobel Prize in 1984 and 1992. J. Calvin Giddings died at the age of 66 on October 24, 1996 in his home in Utah after a valiant fight with cancer. He was not only a brilliant scientist and writer, a superb teacher, a renown explorer, and a dedicated environmentalist, but most importantly, a wonderful human being who cared deeply about his students, colleagues, friends, family and the world he lived in. By Phyllis R. Brown See Chapter 5B, a, b, d, h

Selected references 1. 2. 3. 4. 5. 6. 7. 8.

J.C. Giddings, The random downstream migration of molecules in chromatography, J. Chem. Ed., 35 (1958) 588–591. J.C. Giddings, Nonequilibrium and diffusion. A common basis for theories of chromatograhy, J. Chromatogr., 2 (1959) 44–52. J.C. Giddings, Evidence on the nature of eddy diffusion in gas chromatography from inert (nonsorbing) column data, Anal. Chem., 35 (1963) 1338–1341. J.C. Giddings, Dynamics of Chromatography, Vol. I, Marcel Dekker, New York, NY, 1965, 323 pp. J.C. Giddings, Unified Separation Science, Marcel Dekker, New York, NY 1992. A. Ruoff and J.C. Giddings, Paper geometry and flow velocity in paper chromatography, J. Chromatogr., 3 (1960) 438–442. J.C. Giddings, Extending the molecular weight range of liquid chromatography to one trillion, J. Chromatogr., 125 (1976) 3–16. J.C. Giddings, Chemistry, Man and the Environment, Canfield Press, San Francisco, CA, 1973, 472 pp.

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D.13. Robert L. Grob Robert L. Grob was born in Wheeling, WV, on February 13, 1927. He is Emeritus Professor of Analytical Chemistry at Villanova University. He received his B.Sc. in Chemistry from the Franciscan University of Steubenville in Ohio and a M.Sc. and Ph.D. in Analytical Chemistry from the University of Virginia. After completing his Ph.D. (1955), he joined the Analytical Research Group of Esso Research and Engineering Co. in Linden, NJ. From 1957 to 1963, he was Associate Professor of Chemistry at the Jesuit University of Wheeling, WV. He joined the Faculty of Villanova University in 1963 and was promoted to full Professor in 1967. He directed a very active research program and is credited with mentoring 28 M.Sc. and 23 Ph.D. students. His research interests are in chemical separations and environmental analysis. He was visiting Professor of Chemistry at the University of Hawaii at Manoa (Spring– 1980, Fall–1984). In 1983, he received the Outstanding Research Scholar Award from Villanova University and the Chromatography Award for his contributions to the theory, instrumentation and applications of chromatography from the Chromatography Forum of the Delaware Valley (CFDV). During the fall of 1987 he delivered the Plenary Address at the Analytical Symposium in Hat Yai, Thailand. He then spent three months lecturing at Thai Universities; a second lecture tour of Thai Universities was made in 1990. In March 1990, R.L. Grob was awarded the Stephen Dal Nogare Award in chromatography for his significant contributions to the field of separations science at the 41st Pittsburgh Conference in New York City. In November 1991, he was awarded the Eastern Analytical Symposium Award for achievements in chromatography. In March 1992, he was presented the Fr Daniel Egan Award from his Undergraduate Alma Mater in recognition of a distinguished career in the fields of science and teaching. R.L. Grob is author of “Modern Practice of Gas Chromatography” (3rd Ed.); “Chromatographic Analysis of the Environmental” (2nd Ed.); co-author of “Environmental Problem Solving Using Gas and Liquid Chromatography”; contributing author for the “Treatise on Analytical Chemistry, Contemporary Topics in Analytical Chemistry and Clinical Chemistry”, and “Progress in Analytical Chemistry”; as well as more than two hundred research papers. He is a founder of the CFDV and has been awarded life membership in the Forum. He has been given Emeritus Status in the ACS, is a member of the Analytical Chemistry and Chromatography Sub-Sections of the ACS, Society of Sigma XI and Phi Kappa Phi National Honor Society. He is a Consultant in Analytical and Environmental Chemistry to several Chemical and Environmental Companies in the Philadelphia area. Dr. Grob’s research is decribed in his books. See Chapter 5B, b, d, h, s

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D.14. Georges Guiochon Georges Guiochon was born in Nantes, France on September 6, 1931. His education and academic grades are a M.Sc. degree in engineering from Ecole Polytechnique, Paris, France, with a Ph.D. thesis obtained under L. Jacque´ at Ecole Polytechnique, Paris and defended in chemistry at the University of Paris. He has held the following academic positions: until 1986, Maıˆtre de Conferences then Professor of Chemistry, Ecole Polytechnique, Paris, France; until 1984, Professor, University of Paris, VI (Universite´ Pierre et Marie Curie); until 1984, Director, Laboratoire de Chimie Analytique Physique (CAP); 1984–1987, Professor, Georgetown University, Washington, DC; 1987–present, UTK=ORNL Distinguished Scientist. He is a member of the following professional societies: American Chemical Society, Washington Chromatography Discussion Group, American Institute of Chemical Engineers, and is a member of the Editorial Advisory Boards of the: Journal of Chromatographic Sciences, Journal of Chromatography, Analytical Chemistry (1980– 1983; Associate Editor, 1986–1993), Journal of Liquid Chromatography, and Chromatographia. G. Guiochon is an internationally known scientist in chromatography and holds the present position of Professor of Chemistry as Distinguished Scientist, University of Tennessee, Knoxville, and Oak Ridge National Laboratory. He has been honored with many prizes and awards including: Charles Bihoreau Award (French Association of Petroleum Chemists and Engineers); Chevalier de L’Ordre National du Me´rite; Honorary member of the Chromatographic Society (UK); Fellow of the Chromatographic Society; Tswett Medal (Advances in Chromatography); Dal Nogare Award (Chromatography Forum of the Delaware Valley); A.J.P. Martin Award (Chromatography Discussion Group, UK); Kirkpatrick Chemical Engineering Achievement Honor Award (this latter award was given to Societe´ National Elf-Aquitaine and Societe´ de Recherches Techniques et Industrielles for the development of preparative gas chromatography, an effort in which he played a critical role as scientific consultant); ACS National Award in Separations Science and Technology, 1991; ACS National Award in Chromatography, 1998; Istva´n Hala´sz Memorial Award, 1999, Doctor Honoris Causa from the Technical University, Budapest, Hungary and the University of Pardubice (Czech Republic). His research interests cover a broad perspective of subjects in chromatography such as adsorption and gas chromatography, thermodynamics of interfaces, adsorption and liquid chromatography, mass transfer in chromatography, preparative chromatography, column packing and homogeneity of the column bed, quantitative analysis, coupling between mass spectrometry and chromatography, two dimensional chromatography, and analytical applications. See Chapter 5B, a, b, e, h, p

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Coworkers and research interests The work described below would not have been possible without the cooperation of 335 co-authors, graduate students, post-doctoral fellows or other associates with whom I worked intensely during the years. It is, unfortunately, impossible to name them all here and would be unfair to only identify a few of them. Still, I wish to acknowledge my mentor, L. Jacque´, who taught me more than science, an attitude toward science in the making. Also, it is difficult to account properly for the achievements of a career spanning a number of years, spent on two continents, working on several dozens of projects, big or small, with more than a hundred co-workers and publishing more than seven hundred papers and a half-dozen books. Some projects, which are dear to my heart, may have had few results; the results of others, which were important in their time, have become so trivial that they tend to be forgotten. The senior author is poorly placed to make a balanced synthesis. My research interests include: ž Adsorption and gas chromatography [1,2] Chromatography is an excellent method for the measurement of thermodynamic data related to adsorption. We have shown how to make it very accurate (see [13]). Besides adsorption energies, free energies and entropies, we have measured the heat capacity of sorbed molecules and the virial coefficient of interaction between sorbed molecules. These data cannot be measured at low surface coverages by the more conventional methods used in adsorption studies (manometry, calorimetry and thermogravimetry) which are inaccurate at low concentrations. The use of the conventional, semi-empirical Lennard-Jones potential permits the direct calculation of the interaction potential between two atoms. Summing up those potentials for all the atoms of a graphite crystal (assumed to be perfect and infinite) and for the atoms of small molecules, permits a calculation of the potential adsorption energy of the molecule on graphitized carbon black. The agreement between this potential energy and the measured adsorption energy is only fair, however. We have shown that the agreement between calculated and measured adsorption energy becomes excellent if the calculations are made using the methods of statistical mechanics without any approximation, and taking into account the fact that the polarizability of graphite is not homogeneous, using the known components of the polarizability tensor of graphite, and the best values of the magnetic susceptibility, the polarizability and the Van der Waals radius for the atoms involved, those of graphite and of the adsorbent molecule. Calculations were made for benzene, naphthalene, anthracene, phenanthrene, and their polymethyl and polychloro derivatives. These molecules have no internal rotation degree of freedom. The comparison between the values measured for 1-butene and n-butane, and those calculated for the different conformations of these molecules gave an estimate of the potential energy barrier to internal rotation, in good agreement with independent results. ž Thermodynamics of other interfaces [3,4] The relationship between chromatographic retention data and solution thermodynamics is known since the early beginning of chromatography. Martire and Purnell among

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others have shown and explained the reasons for the frequent disagreement between activity coefficients measured by gas chromatography and by conventional methods. Because of the rather large specific surface area of the support used, adsorption at the gas–liquid, gas–solid and liquid–solid interfaces are important. Their contribution to retention varies widely in relative importance with the nature of the liquid phase and the solute. We have carried out many studies involving the thermodynamics of adsorption at the gas–liquid interface and later the influence of complexation equilibria. ž Adsorption and liquid chromatography [5,6] Systematic investigations of the properties of the interface between a solution and the surface of silica chemically bonded to alkyl chains with 8–18 carbon atoms were carried out. The structure of this interface is most unusual since it is, by some respects, analogous to an immobilized expanded liquid and since the solute molecule may actually penetrate inside the interface. The study of the variation of retention of homologous compounds with the length of the alkyl chain has led to the discovery of several new phenomena. Liquid–solid equilibria are fundamental to the understanding of liquid chromatography, particularly under nonlinear conditions. We have studied them actively these last fifteen years. The equilibrium isotherms of numerous compounds, of many binary mixtures and of two ternary systems have been determined, many on RPLC systems, a few on enantioselective stationary phases or on more conventional adsorbents. In all cases, the isotherms were fit to appropriate models. Several antilangmuirian isotherms were found. Out of approximately a dozen pairs of enantiomers studied, the equilibrium data of ten were found to be well accounted for by a bilangmuir model. One of the two terms of this model is identical for the two enantiomers and accounts for the contribution of the nonselective interactions which take place between the molecules of the analyte and the stationary phase. The second term differs for the two enantiomers, some times greatly. It accounts for the enantioselective interactions which permit the chiral separation. The equilibrium data of one of the ternary systems fit well the Langmuir model. This behavior was explained by determining the activity coefficients of the solutes in the mobile phase: they are nearly independent of the concentration. The methods used for the determination of equilibrium isotherms were also actively studied, and results obtained with different methods were compared systematically. Two new methods were developed, the simple wave method which relies on the particular solution of the equilibrium dispersive model for wide injection pulse; and the numerical solution of the inverse problem of chromatography which is now possible because of the considerable progress made these last fifteen years in computer hardware and software. This last method has great potential. ž Mass transfer in chromatography [7,8] Mass transfer phenomena control the efficiency of chromatographic columns. They have been thoroughly investigated in the past and we have contributed to these studies in gas and in liquid chromatography. At the analytical level, work on the theory of optimization of experimental conditions showed us that very narrow bore open tubular columns could permit the achievement of extremely fast analyses and of very efficient

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separations in gas chromatography. We demonstrated these possibilities by performing very early some extremely fast separations (in a few seconds or less). This work paved the way for modern fast GC. Unfortunately, a variety of practical problems do not allow its convenient extension to liquid chromatography. We developed methods for the rapid determination of the rate coefficient of mass transfer in nonlinear chromatography. The same breakthrough curves, which are used for the determination of equilibrium isotherms, can also be used for these kinetic measurements. The slope of a breakthrough curve in the inflection region increases with increasing rate coefficient in a lumped kinetic model. Parameter identification allows an easy determination of these coefficients. The interpretation of the results is more difficult. In many cases, we have found a positive dependence of the rate coefficient on the concentration. This could be explained in part by the important role often played by surface diffusion in mass transfer in HPLC. ž Preparative chromatography [9,10] In preparative applications, the chemist does not look for information, as in analytical applications, but for pure compounds prepared in amounts as large as possible in a given time. We have demonstrated that optimization of experimental conditions is simple through the solution of the general equations of chromatography at finite concentration. Work in preparative chromatography was pursued over thirty years, in GC as well as LC. The main thrust of my current research is the theory and applications of preparative high performance liquid chromatography. The theoretical work deals with the simulation of the migration of large concentration bands through chromatographic columns, using a general model based on basic principles of physical chemistry. The model is the set of partial differential equations obtained by writing a mass balance equation and a proper kinetic equation for each compound involved (including the retained components of the mobile phase). These equations are nonlinear. We have generated computer programs that permit the numerical calculations of solutions of these equations under all kinds of experimental conditions. Depending on the boundary conditions selected, the solution of this system can predict accurately the elution profiles of large bands (with or without volume overload), the breakthrough curves, the displacement profiles, or the system peaks. We are presently studying the properties of the column response to changes in the input conditions, when the concentration of the input band is large and=or the kinetics of mass transfer between phases is fast. This approach works even when the kinetics of the mass transfer is slow and the column efficiency is very low. Our experiments allowed a detailed investigation of the optimization of the design and operating conditions in various modes of chromatography. These results are of great practical interest for industrial applications because they were all validated by numerous experimental results. For close to twenty different component pairs and a few ternary mixtures, we have determined the equilibrium isotherms and the rate constant coefficients, calculated the band profiles under various sets of experimental conditions, and sometimes determined the production rate, recovery yield, and purity. Then, we compared the results with experimental data obtained on the same systems. In all cases, there was an excellent

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agreement between experimental results and the prediction of the model. Work is continuing to extend these results to simulated moving bed, an implementation of preparative chromatography, which is nearly continuous but separates only two fractions. A simulated moving bed seems to be well suited for the industrial preparation of pure enantiomers, which gives it great importance in the pharmaceutical industry. ž Column packing and homogeneity of the column bed [11,12] Chromatographers have long suspected that column beds are not homogeneous. The column bed has usually a cylindrical symmetry, with a radial distribution of packing density, porosity, and permeability, hence of local mobile phase velocity, dispersion coefficient, and concentration. This causes the elution of a band to begin in the column center and to take place later at the column wall. This causes a broadening of the bands recorded by bulk detectors and a loss of efficiency, resolution and production rate at a given purity. The phenomenon is explained by the peculiar properties of particulate materials. There is friction between the particles in the powder and friction between the packed bed and the wall. Because it permits a leak-proof contact of the bed at the wall, this friction is not entirely detrimental. It cannot be eliminated. Work is continuing on further attempts at understanding better the behavior of the column bed and improving the column performance. ž Quantitative analysis [13,14] A detailed study of the various phenomena involved in gas-chromatographic analysis permitted the identification and the control of most of the sources of error in the measurement of peak height, peak area, retention times and the higher moments of a peak profile. We derived relationships between precision and the fluctuation of the experimental parameters. This work permitted the design, construction and operation of a gas chromatograph giving a relative standard deviation of 5 to 8 ð 103 % for the retention times on graphitized carbon black (only this material is compatible with obtaining such a precision). With this instrument, we could measure the heat capacity of sorbed molecules and carry out various studies on the peak profiles. This work was useful for the design of process-control analyzers and of various industrial equipments for routine analysis, since it allowed the calculation of specifications for the different components of a gas chromatograph as a function of the required precision. Although many investigations on the performance of instrument parts of HPLC were carried out, no systematic studies of the entire system were ever undertaken. Recently, we measured the reproducibility and repeatability of the retention data, peak area, peak asymmetry, and column efficiency obtained with different popular brands of columns for reversed phase liquid chromatography. This work demonstrated the great improvements made in the reliability of the commercially available HPLC instruments and the high quality of the available columns. As a matter of fact, for all practical purposes, the accuracy of routine analyses is now determined by the skills of the analyst. ž Coupling between mass spectrometry and chromatography [15] Chromatography separates substances and does it well, but it supplies no useful data for their identification, it only gives the free energy of transfer of the analyte between

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a mobile phase and an often ill-defined stationary phase. Mass spectrometry is an ideal complement to chromatography as it does supply a large amount of information on pure compounds, either allowing the identification or giving important clues on the nature of the separated compounds. This is exemplified by the success of GC=MS, which we pioneered in the 1960s. To develop the coupling between the LC column and a mass spectrometer, we chose the direct, straightforward solution: the LC effluent is nebulized through a diaphragm. The positively charged droplets drift through a convergent then divergent heated chamber where the solvent vaporizes. Ions are formed by ejection from the droplets or reaction with the solvent plasma in the source. These ions are formed with little internal energy, so the mass spectrometer records mainly quasi-molecular ions or aggregates with one or several solvent molecules. Excellent results were obtained with complex compounds, such as vitamin B12 for which the base peak is a quasi-molecular ion, and little fragmentation is observed. ž Two dimensional chromatography [16,17] The separation power of a chromatographic column is often too small to permit the separation of the components of a complex mixture. Successive analyses on different columns using different retention mechanism offer a solution, but it is long and tedious. We tried to combine two chromatographic separations, using two different retention mechanisms along two perpendicular directions, on a thin square column .10 ð 10 ð 0:1 cm) replacing the conventional cylindrical column. The separated components were eluted and detected using a diode array UV detector. This method was similar to two-dimensional thin layer chromatography, except that the flow rate of the eluent was kept constant and the detection was on-line. Preliminary results were excellent. A theoretical study predicted a separation power more than one order of magnitude larger than for a conventional LC column. This work was interrupted in 1984 due to my move from France to the USA. ž Analytical applications [18–20] Over the years, we have made major contributions to the development of analytical applications by GC or LC and of chromatographic methodology, for example in the analysis of essential oils, of halocarbons in the atmosphere, organic pollutants in the river Oise, and the tap water plant in the northern suburb of Paris; in the analysis of crude oil cuts and of crude oil heavy fractions. In these fractions we studied and identified new organic acids (steranic, and hopanic acids), several dozens of new aza-arenes (alkyl benzoquinolines and dibenzoquinolines), many alkylcarbazoles, benzo- and dibenzocarbazoles and alkyl-dienzothiofenes. Selective extraction methods were developed which are still in use; those developed for acidic and basic components were so highly selective as to be practically specific for those classes, with yields in the 98–100% range.

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References 1.

2.

3. 4.

5. 6.

7.

8. 9. 10.

11.

12. 13. 14.

15.

16.

17. 18. 19. 20.

C. Vidal-Madjar, M.F. Gonnord and G. Guiochon, Determination of enthalphy, entropy and free energy of adsorption of vapors on graphitized carbon black. Problems of calculation by statistical thermodynamics and of measurements, Advanc. Chromatogr., 13 (1975) 177. C. Vidal-Madjar, M.F. Gonnord, M. Goedert and G. Guiochon, Gas solid chromatography measurements of the change in the heat capacity during adsorption and graphitized thermal carbon black, J. Phys. Chem., 79 (1975) 732. C. Devillez, C. Eon and G. Guiochon, The Kelvin capillary phenomena in chromatography and its influence upon the determination of adsorption coefficients, J. Colloid Interface Sci., 49 (1974) 232. H. Colin, A.M., Krstulovic, M.F. Gonnord, Z. Yun, P. Jandera and G. Guiochon, Investigation of selectivity in reversed-phase liquid chromatography. IV. Effects of stationary and mobile phases on retention of homologous series, Chromatographia, 17 (1983) 9. T. Fornstedt, P. Sajonz and G. Guiochon, A closer study of chiral retention mechanisms, Chirality, 10 (1998) 375–381. F. James, M. Sepu´lveda, F. Charton, I. QuiZones and G. Guiochon, Determination of binary competitive equilibrium isotherms from the individual chromatographic band profiles, Chem. Eng. Sci., 54 (1999) 1677–1696. P. Sajonz, H.G.-Sajonz, G. Zhong and G. Guiochon, Application of the shock layer theory to the determination of the mass transfer rate coefficient and its concentration dependence for proteins on anion exchange columns, Biotech. Progr., 13 (1997) 170. K. Miyabe and G. Guiochon, Analysis of surface diffusion phenomena in reversed-phase liquid chromatography, Anal. Chem., 71 (1999) 889–896. G. Guiochon, S.G. Shirazi and A.M. Katti, Fundamentals of Preparative and Nonlinear Chromatography, Academic Press, Boston, MA, 1994. G. Guiochon, A.M. Katti, M. Diack, M. Zoubair El Fallah, S. Golshan-Shirazi, S.C. Jacobson and A. Seidel-Morgenstern, Prediction of high concentration band profiles in liquid chromatography, Acc. Chem. Res., 25 (1992) 366–374. M. Sarker, A.M. Katti and G. Guiochon, Consolidation of the packing material in chromatographic columns under dynamic axial compression. II — Consolidation and breakage of several packing materials, J. Chromatogr. A, 719 (1996) 275–289. G. Guiochon, E. Drumm and D. Cherrak, Evidence of a wall friction effect in the consolidation of beds of packing materials in chromatographic columns, J. Chromatogr. A, 835 (1999) 41–58. M. Goedert and G. Guiochon, High precision measurements in gas chromatography. IX. A study of systematic errors on the determination of retention times, Anal. Chem., 45 (1972) 1188. M. Kele and G. Guiochon, Repeatability and reproducibility of retention data and band profiles on RPLC columns. II — Results obtained with symmetry C18 columns, J. Chromatogr., 830 (1999) 55–79. P. Arpino, J.P. Bounine, M. Dedieu and G. Guiochon, Optimization of the instrumental parameters of a combined liquid chromatograph-mass spectrometer, coupled by an interface for direct liquid introduction. IV. A new desolvation chamber for droplet focusing or Townsend discharge ionization, J. Chromatogr., 271 (1983) 43. L.A. Beaver, M.F. Gonnord, A.M. Siouffi, M. Zakaria and G. Guiochon, A theoretical investigation of the potentialities of the use of a multidimensional column in chromatography, J. Chromatogr., 255 (1983) 415. M. Zakaria, M.F. Gonnord and G. Guiochon, Applications of two-dimensional thin layer chromatography, J. Chromatogr., 271 (1983) 127. H. Colin, A. Siouffi and G. Guiochon, Separation of free sterols by high-performance liquid chromatography, Anal. Chem., 52 (1979) 1661. H. Colin, J.M. Schmitter and G. Guiochon, Liquid chromatography of aza-arenes, Anal. Chem., 53 (1981) 625. A. Stolyhwo, M. Martin and G. Guiochon, Analysis of lipid classes by HPLC with the evaporative light scattering detector, J. Liq. Chromatogr., 10 (1987) 1237.

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D.15. Andra´s Guttman (invited paper) Andra´s Guttman was born on August 16, 1954 in Pe´cs, Hungary. He graduated with a M.Sc. in chemical engineering at the University of Veszpre´m, Hungary, where his Ph.D. was awarded in 1981. He started working as an Assistant Professor in the Semmelweis University of Medicine in Budapest, Hungary, first on enzyme kinetics using electrophoretic methods in the Department of Biochemistry and later he conducted drug metabolite studies by combined chromatographic methods in the Institute of Pharmacodynamics. He moved to the USA in 1987 to engage in further scientific work at the Barnett Institute, Northeastern University, Boston. Under the guidance of Barry L. Karger he became involved in the early development of capillary gel electrophoresis for the analysis of biologically important molecules. In 1990, he joined the capillary electrophoresis development team of Beckman Instruments, Palo Alto, CA, to solve basic separation-related problems. Here, he worked on fundamental principles of capillary gel electrophoresis and developed numerous novel separation methods for the analysis of DNA, protein, and complex carbohydrate molecules, as well as, small chiral drugs. In 1996, he got involved in the development of an agarose gel-based, microfabricated DNA fragment analyzer at Genetic BioSystems, San Diego, CA, as chief scientific officer. Since 1999, he is a Principal Scientist and the leader of the Microfluidics Development Group in the Novartis Agricultural Discovery Institute in La Jolla, CA. He is responsible for the implementation of an integrated microfabricated ‘Lab-on-a-Chip’ device for high throughput genomic and proteomic applications. Andra´s Guttman is one of the pioneers of capillary gel electrophoresis. He was the first to attain single base resolution of large DNA molecules on gel-filled capillary columns. Since the foundation of modern gel electrophoresis-based DNA sequencing, this advance finally enabled the recent completion of the sequencing of the entire human genome. Andra´s Guttman made significant contributions to capillary gel electrophoresis based carbohydrate analysis and sequencing, as well. His present interest is in the development of integrated microfabricated analytical devices for ultra-fast and large scale DNA and protein analysis. He has authored and co-authored more than a hundred scientific papers and book chapters and has numerous patents, is Co-editor of the book “Integrated Microfabricated Device Technologies”, in press, Publisher Marcel Dekker; has organized several special symposiums on multi-dimensional separation techniques in the Pittsburgh conference series. He is the elected co-chair of the High Performance Capillary Electrophoresis and Related Microseparation Techniques Conference, which will be held in San Diego in 2003. He is an Associate Director of the California Separation Science Society. Andra´s Guttman was awarded the degree of Doctor of Chemical Sciences by the Hungarian Academy of Sciences in 1996 and received the Annual Award of the Hungarian Chemical Society in 2000. See Chapter 5B, a, f, l, q, r

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15.I. INTEGRATED MICROFABRICATED DEVICE TECHNOLOGIES Andra´s Guttman Novartis Agricultural Discovery Institute, 3115 Merryfield Row, La Jolla, CA 92121, USA

Interdisciplinary science and technologies have converged in the past few years to create exciting challenges and opportunities, which involve novel, integrated microfabricated separation systems, facilitating high throughput applications for the biotechnology and the pharmaceutical industry. These new devices are referred to as ‘Lab-on-a-Chip’ or ‘Micro Total Analysis Systems’ (µTAS) and their development involves both established and evolving technologies including microlithography, micromachining, microelectromechanical systems (MEMS) technology, microfluidics and nanotechnology [1]. The development of these new devices and systems requires a high level of interaction and cooperation among engineers, computer scientists, materials scientists, chemists, molecular biologists, geneticists and clinical scientists. Some of the main applications for this novel ‘synergized’ technology will include high-throughput microscale bioseparations (genetic and proteomic analysis) for the biotechnology industry; clinical chemistry (particularly DNA and immuno diagnostics) for the medical field; drug discovery, combinatorial chemistry and industrial process control for the pharmaceutical industry; and portable=hand-held analytical instrumentation for the environmental and defense sector. Microdevices are advantageous in several aspects, such as the use of low volumes of reagent, nano=pico-liter sample requirement, as well as, readiness of serial and=or parallel processing, consequently leading to revolutionary gain in overall analysis time. Miniaturization in the form of silica microchip is an elegant solution to reduce the size of analytical devices, while increasing their capacity and functionality. Microfabricated channels in silica wafers act like capillaries providing similar means of both electric field-mediated separations (zone electrophoresis, micellar electrokinetic chromatography, gel electrophoresis, isoelectric focusing, isotachophoresis and electrochromatography), and pressure-mediated (capillary liquid chromatography) separations. Since these microfabricated channels are significantly shorter than regular capillaries, e.g., in electric field-mediated separations high field strengths can be applied, resulting in extremely rapid (seconds) analysis times. Parallel setup in the way of using arrays of microchannels, etched into a glass wafer ensures high throughput processing. Earlier efforts to miniaturize automated separation devices, such as gas chromatography [2], and slab gel electrophoresis [3] suggested that separation science can leverage from miniaturization in a way similar to what microchips did in the electronics=computer industry, especially, since microfabrication employs the same techniques developed for the silicon microchips in the electronics=computer industry. Microchannels fabricated in glass wafers (similar to fused silica capillaries) could be effectively utilized for rapid and highly reproducible separations [4]. A modest ten-fold miniaturization can result in a 100-fold increase in separation speed and resolution, and a 1000-fold decrease in necessary sample and buffer volumes. In addition, the microchip format highly favors parallel processing, i.e., arrays of microfabricated channels are as easily etched into glass wafers as one channel with marginal additional cost, concomitantly leading to a suitable solution to parallel processing.

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Microfabricated electric field mediated separation devices were first introduced by Manz et al. [5]. These devices not only provided improved separation performance, but also increased sample throughput by orders of magnitude that represents a significant advantage particularly in analytical biotechnology and pharmaceutical applications where large libraries of samples need to be analyzed. Some of the most important features of microfabricated separation devices are the implementation of precisely controlled injection volumes, extremely short separation distances and instant readiness of multiplexing and system integration. The improved analytical capability of microfabricated device technology [6–10] to advance electric field and pressure mediated separations is already well established. One of the most genuine benefits of microfabricated devices over such conventional techniques as slab gel or capillary electrophoresis based systems is the fact that in addition to easy multiplication of the separation channels, other functional and structural elements can be integrated into a single microdevice. These additional functionalities include such structures as reaction chambers (e.g., polymerase chain reaction, PCR or restriction digestion for restriction fragment length polymorphism, RFLP), pre- or postseparation labeling, sample purification (e.g., SFE, solid phase extraction) and fraction collection. It is important to note here that miniaturization not only enables the use of significantly smaller sample volumes and reagent consumption (up to 2–3 orders of magnitudes), but also has the capability of enhancing reaction speed and greatly reducing the time requirement of on-the-chip chemical=biochemical reactions such as PCR [11] due to micro-miniaturization. Integration of these additional functional elements is indeed the real implementation of the well-publicized ‘Lab-on-a-Chip’ concept. Fig. 1 exhibits such an integrated microfabricated device including reagent=sample reservoirs, mixing compartments, reaction chambers, separation channels, pre- and post-separation labeling elements and fraction collection assembly. Until recently, almost all microfabricated electric field mediated separation devices have been made of glass or fused-silica wafers, since the fabrication process of these materials is well understood [11]. The numerous surface modification chemistries (coating of the inner glass surface) had been developed for silica material in liquid chromatography and capillary electrophoresis and used since, thus were readily applicable to these substrates. The typical fabrication process of glass and fused silica microdevices [12] starts with the deposition of a thin metal film onto the relevant surface substrate, followed by the deposition of a thin film of spincoated photoresist. The channel design is then simply transferred to the substrate using a photomask. Ultraviolet (UV) exposure of the photoresist is followed by chemical removal of the resist and the metal layer wherever they were exposed to UV radiation. The channels and other structural elements are then isotropically wet-etched into the substrate by means of immersing the wafer into an NH4 =HF bath. Note that this etching process results in channel widths of approximately twice the channel depths. To obtain deeper and wider channels longer etching times should be used. After drilling access holes (e.g., laser drilling) into the appropriate parts of the wafer, a monolithic closed channel network is formed by bonding a cover plate over the substrate using thermal fusion. The resulting microchip is ideally suited to create novel complex analytical devices, with no added methodological complexity or time investment, compared to a single channel device. Fluid transport by both pressure

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Fig. 1. Integrated microchip device and components.

and electrokinetic means can support controlled parallel and serial processing of fluids in the microchannel structure. Utilization of microfluidic valves and vents allows sample routing to any of the channel structures. Sample injection onto the microfabricated separation device is usually accomplished by the so-called cross-channel injecting method. Such a cross-channel injector is shown in Fig. 1. Application of electric potential between sample and sample=waste reservoirs electrokinetically pumps sample solution into the cross-section at the beginning of the separation channel. This picoliter sample injector is solely controlled by the electric field distribution in the relevant functional elements. Due to the minute sample volumes, extremely sensitive detection methods are required, like laser-induced fluorescence (LIF); however, other methods, such as electrochemical [13] and more recently mass spectrometric [14] detectors have also been reported. Typical applications include separation of biopolymers like DNA and protein molecules, as well as, analysis of small molecules such as combinatorial libraries. Fig. 2 depicts an extremely rapid separation of a 100 base pair (bp) ladder on a gel filled microfabricated device.

15.I.1. Future prospectives The advent of integrated microfabricated device technology, this powerful and rapid analysis method, opens up new opportunities in large scale micro bioseparations for the biotechnology industry, capable of revealing global changes in gene expression both in the messenger ribonucleic acid (mRNA) and protein level (proteome). This is

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Fig. 2. Rapid analysis of a 100–1000 bp dsDNA ladder on a microfabricated device. Separation matrix, 0.5ð POP-6 for DNA sequencing gel; labeling, 1ð Sybr Gold; Injection, 100 µm cross; separation conditions, L D 30 mm, E D 200 V=cm, LIF detection, 532 nm=585 nm.

bringing about revolutionary transition in our capabilities to examine living systems on a molecular basis, which is crucial for the modern analytical biotechnology and pharmaceutical industry. Entering the age of genomics and proteomics, we expect to see a paradigm shift towards miniaturized, high resolution, micro-separation techniques used in integrated and automated fashion to solve formidable separation problems and provide means for ultra-high throughput micro analysis. Note that most current separation protocols for DNA and protein analysis, already in use in molecular biology and biotechnology laboratories, can be readily transferred to microfabricated devices. A multiplexed 96-channel device can readily analyze 13,824 samples in a 24-h time period considering 10 min cycling time and fully automated sample handling and reagent replenishment. However, the real strength of this technique, besides the potential of miniaturization, is the possibility to integrate existing methods and functionalities in a way that allows for sample preparation, reactions, analysis and even fraction collections carried out on a single microchip (‘Lab-on-a-Chip’). Microfabricated devices are intrinsically acquiescent to full automation enabling large scale micro-analyses requiring considerably less human intervention than conventional techniques, resulting in significant savings in time, labor and cost. References 1. 2. 3.

R.F. Service, Microchip arrays put DNA on the spot, Science, 282 (1998) 396–401. S.C. Terry, J.H. Jerman and J.B. Angell, Gas-chromatographic analyzer fabricated on a silicon wafer, IEEE Trans Electron Devices, 26 (1979) 1880–1886. A. Guttman, High performance ultrathin layer agarose gel electrophoresis, Trends Anal. Chem., 18 (1999) 694–702.

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L.C. Waters, S.C. Jacobson, N. Kroutchinina, J. Khandurina, R.S. Foote and J.M. Ramsey, Multiple sample PCR amplification and electrophoretic analysis on a microchip, Anal Chem., 70 (1998) 5172– 5176. A. Manz, D.J. Harrison, E.M.J. Verpoorte, J.C. Fettinger, A. Paulus, H. Luedi and H.M. Widmer, Planar chips technology for miniaturization and integration of separation techniques into monitoring systems — capillary electrophoresis on a chip, J. Chromatogr., 593 (1992) 253–258. F. von Heeren, E. Verpoorte, A. Manz and W. Thormann, Micellar electrokinetic chromatography separations and analyses of biological samples on a cyclic planar microstructure, Anal. Chem., 68 (1996) 2044–2053. A. Ewing, P.F. Gavin, P.B. Hietpas and K.M. Bullard, Continuous separations in microfabricated channels for monitoring ultrasmall biological environments, Nature Med., 3 (1997) 97–99. P.A. Walker, M.D. Morris, M.A. Burns and B.N. Johnson, Isotachophoretic separations on a microchip. Normal Raman spectroscopy detection, Anal. Chem., 70 (1998) 3766–3769. B. He, N. Tait and F. Regnier, Fabrication of nanocolumns for liquid chromatography, Anal. Chem., 70 (1998) 3790–3797. B. Zhang, F. Foret and B.L. Karger, A microdevice with integrated liquid junction for facile peptide and protein analysis by capillary electrophoresis=electrospray mass spectrometry, Anal. Chem., 72 (2000) 1015–1022. R.P. Oda, M.A. Strausbauch, A.F.R. Huhmer, N. Borson, S.R. Jurrens, J. Craighead, P.J. Wettstein, B. Eckloff, B. Kline and J.P. Landers, Infrared-mediated thermocycling for ultrafast polymerase chain reaction amplification of DNA, Anal. Chem., 70 (1998) 4361–4368. C.S. Effenhauser, G.J.M. Bruin and A. Paulus, Integrated chip-based capillary electrophoresis, Electrophoresis, 18 (1997) 2203–2213. A.T. Woolley, K. Lao, A.N. Glazer and R.A. Mathies, Capillary electrophoresis chips with integrated electrochemical detection, Anal. Chem., 70 (1998) 684–688. D. Figeys and R. Aebersold, Nanoflow solvent gradient delivery from a microfabricated device for protein identifications by electrospray ionization mass spectrometry, Anal. Chem., 70 (1998) 3721– 3727.

D.16. Steven B. Hawthorne Steven Hawthorne was born in Rapid City, SD, on April 8, 1954. After receiving both B.Sc. and M.Sc. degrees in chemistry from South Dakota State University in 1976 and 1978, respectively, Steve Hawthorne worked for the U.S. Air Force at Brooks Air Force Base, Texas until 1980. He left to earn a Ph.D. in Analytical=Environmental Chemistry at the University of Colorado (Boulder) which was awarded in 1984. He was then employed at the Energy and Environmental Research Center at the University of North Dakota in Grand Forks, ND, where he is a Senior Research Scientist in environmental chemistry. Hawthorne serves on the advisory board for several international meetings, serves as peer-reviewer for several government and private funding agencies, as well as for several professional journals. He has published over 100 major scientific articles and has contributed to and=or edited several books and special journal issues, mostly in the areas of collection, extraction, and analysis of environmental samples. He was a Co-Professor of the American Chemical Society (ACS) short course in sample preparation, and has trained many students in his capacity of Adjunct Professor at the University of North Dakota and the University of Waterloo. Recent research interests include using selective supercritical-fluid extraction methods to study the sequestration of organic pollutants in

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soils and sediments, and the development of subcritical water (hot=liquid water) as an extraction and chromatographic fluid. Hawthorne has received several awards including the Pittsburgh Conference’s Keene P. Dimick award for ‘outstanding accomplishments in chromatography’ (1995); ‘The Award of Excellence’ (1994) from the International Symposium on Supercritical-Fluid Chromatography and Extraction; the ISCO Award for ‘significant contributions to instrumentation for separations’ (1993); and the U.S. Department of Energy Distinguished Lectureship award. See Chapter 5B, a, o, p, s 16.I. DEVELOPMENT OF ANALYTICAL SFE: TRYING TO PUT SCIENCE INTO SAMPLE PREPARATION Steven B. Hawthorne Energy and Environmental Research Center, University of North Dakota, Box 9018, Grand Forks, ND 58202-9018, USA

16.I.1. Why study extraction? Analytical methods for determining organic constituents in samples ranging from soils and sediments to biological tissues generally require two basic steps: extraction= preparation (in which the target analytes are removed from the sample matrix to a

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convenient solvent), and chromatographic determination of the extracted constituents. This volume celebrates the development of chromatography since Tswett’s first report in 1906, and all would agree that this development has been extraordinary. In contrast, there has been almost no development of extraction methods until the late 1980s, and the Soxhlet apparatus (virtually unchanged from Soxhlet’s original report in 1879) was, and still is, required for many methods mandated by regulatory agencies. Few, if any, other articles in this volume address the extraction=preparation step, despite its importance to chromatographic analyses. I am aware of no major awards which are given for sample preparation, and in fact, am only aware of two other researchers who have received awards for research, at least in part, into extraction processes: Jerry King (U.S. Department of Agriculture) who received the Wiley Award for his work with SFE and SFC on food and related samples, and Janusz Pawliszyn (University of Waterloo), who received the Jubilee Award for his work on solid-phase micro extraction (SPME). It is not hard to understand this, given the fact that very, very few universities offer course work in any form of sample extraction. This shows a lack of interest in the science of extraction and related sample preparation techniques either at the undergraduate or graduate level. Hopefully, the next 100 years of development will see more education, research, and developments in the extraction process. In an attempt to share some of my laboratory’s fascination with the extraction of organic pollutants, the contributions we feel are most significant from our SFE research are briefly described below. The best studies, of course, are not performed in isolation and the references at the end list some of the collaborators who contributed greatly to our progress.

16.I.2. First attempts As a new Ph.D. in 1984, I had started my first ‘real’ job, and was faced with obtaining research grants. Through the suggestion of Doug Raynie (now of Proctor and Gamble, USA), my new colleague Dave Miller and I became interested in the developing field of supercritical-fluid chromatography (SFC), particularly coupled with mass spectrometry. Initial attempts to write a proposal to obtain funding from the US Environmental Protection Agency for developing SFC=MS convinced me that we really had no new ideas in the field, and could never compete with the experts (e.g., Milton Lee). However, Dave and I concluded that the EPA may be interested in using supercritical fluids for extracting organic pollutants, and perhaps we could hide a little SFC=MS research in such a proposal. Funding was obtained, and our first paper describing SFE of environmental samples was published in 1986 [1]. This paper also described our first, and reasonably successful, attempt to directly couple SFE with capillary GC by inserting the outlet restrictor of the SFE cell directly into the GC column through an on-column injection port. Publications in analytical SFE were dominated by Jerry King’s research group for food and related samples, and by Dave’s and my findings for environmental samples, until 1989. At that time, the need to reduce organic solvent use in analytical laboratories

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caused a huge increase in the number of papers published describing the development and use of analytical SFE for a variety of sample matrices. Interest and activity in the technique grew sufficiently to induce Analytical Chemistry to request a review for their ‘A’ pages section which appeared in 1990 [2].

16.I.3. Conventional wisdom versus scientific reality In many ways, the scientific and commercial development of SFE was very different from other areas of chromatographic separations. The normal progression of scientific understanding to automated instrumentation capable of operating in a routine laboratory typically has required a number of years to achieve, e.g., gas chromatography was first described in 1952 and the first commercial instrument was introduced in 1955. The time from our first publication in 1986 (performed with home-built equipment modified from crude SFC instrumentation) to the availability of automated commercial instruments from several companies also occurred in a few years. Not surprisingly, such rapid commercialization of a process that was very poorly understood led to less-than-ideal instruments. A more serious impediment to the development of SFE was the nearly total lack of understanding of the physicochemical processes governing the extraction process, at least for environmental matrices. Several ‘conventional wisdom’ assumptions became popular that misguided the actual practice of SFE, and greatly reduced its initial effectiveness. Our laboratory was intensely interested in understanding the SFE process for environmental matrices, and believe some of our most important contributions to the field came in challenging some of the ‘conventional wisdom’ guiding SFE. Some of the more important examples which were investigated in our laboratory (and with our collaborators) are described below. Initially, nearly all SFE development focused on the analyte to be extracted. For very polar compounds which had very poor solubilities in carbon dioxide, the normal approach was to add organic modifiers such as methanol to the carbon dioxide in an attempt to increase the solubility of the desired analyte. In 1992, we reasoned that, since many such analytes were derivatized after extraction to facilitate GC analysis, we may be able to derivatize such organics during the SFE process, and thus make the analytes more soluble in the supercritical carbon dioxide, as well as provide a derivatized analyte ready for GC analysis. Our initial studies demonstrated the use of the in-situ derivatization approach for organics ranging from acid herbicides to phospholipid-derived fatty acids [3]. As SFE developed in several laboratories, it became increasingly clear that for most samples, the sample matrix limited the extraction much more than the characteristics of the target analyte. Likely the worst assumption ever made by most workers in the field and, for that matter, by investigators in any extraction research, was that spiked analytes added to a solid sample matrix could be used to determine the extraction behavior of the real analytes on a sample. Although the determination of extraction efficiencies based on spike recoveries was not only accepted, but required by regulatory agencies, its validity had essentially never been tested. Many SFE methods were developed

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(and, unfortunately still are) based on spike recoveries, and then fail when applied to real analytes. This is a particular problem with environmental samples (e.g., soils and sediments) where exposure over long periods of time causes the analytes to be more closely associated with the sample matrix. This problem was most clearly demonstrated by extracting PAHs from historically-contaminated samples (soils, sediments, and air particulate matter) along with recently spiked deuterated PAHs. In some cases, the spiked deuterated PAHs could be completely extracted when only a few percent of the ‘real’ PAHs were extracted, clearly demonstrating that recently spiked analytes do not experience the same matrix interactions as the ‘native’ analytes, and should, therefore, not be used for developing any quantitative extraction method [4]. Regardless of the SFE conditions used, nearly all samples in our laboratory showed a rapid extraction of the target analytes at the beginning of the extraction (this fraction includes recently spiked analytes), followed by a much slower ‘kinetically-limited’ fraction of the same analyte molecules. It quickly became clear that samples which were easy to extract had most of the analyte molecules associated with ‘loose’ sites, and samples which were difficult to extract had the majority of analyte molecules which were associated with ‘tight’ or ‘kinetically-limited’ sites. Armed with many data sets demonstrating these observations, a one-month visit with Tony Clifford and Keith Bartle (University of Leeds, UK) resulted in the ‘hot-ball’ model which successfully describes SFE behavior of real samples ranging from contaminated soils to polymers to plant material [5]. This model teaches that diffusion of the analyte in the sample matrix limits the extraction of ‘tightly-bound’ analytes, and did much to teach investigators to focus SFE development on the sample matrix, rather than only on the target analyte. The new (but now obvious) information that overcoming the interactions of target analytes with the sample matrix was the key to obtaining high recoveries led to the testing of fluids with higher dipole moments than carbon dioxide [6]. Studies on the mechanisms of organic modifiers added to carbon dioxide clearly showed that the modifier’s action was by disrupting matrix binding sites, and demonstrated that modifiers should be selected based on matrix characteristics rather than analyte characteristics [7]. Finally, and most successfully, the realization that the extraction of ‘tight’ analytes was limited by kinetic (not thermodynamic) problems suggested that higher temperature extractions may be useful. At this period of development in SFE, one of the most accepted rules was that extractions were controlled by the fluid density, and that high density was always best. Since raising temperature (at a constant pressure) lowered the fluid density, conventional wisdom dictated that raising the extraction temperature should not be done. In direct contrast to this conventional wisdom, we demonstrated that extraction temperatures up to 200ºC (or even higher) with pure carbon dioxide were extremely effective for increasing SFE recoveries of typical environmental pollutants [8]. However, it was unclear whether this increase in SFE rates and recoveries were a result of better extraction kinetics, or increased solubilities. Again, at this time, conventional wisdom said that raising temperature (at a constant pressure) should reduce solubilities because of the lower carbon dioxide density. Since virtually no solubility data was available at temperatures above 80ºC, we developed a simple method to determine the solubility of typical organic pollutants over a wide range of temperatures and pressures.

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These studies provided some of the first high temperature solubility data, and clearly demonstrated that raising temperature was much more effective than raising pressure for increasing solubilities (although raising both temperature and pressure is best), as long as the analytes had any significant vapor pressure (i.e., if the analyte can be analyzed by gas chromatography). The collaboration with Leeds also resulted in a simple predictive model for organic solubilities in carbon dioxide over wide temperature ranges [9]. A final contribution to the understanding of analytical SFE processes was developed when we realized that most investigators looked at extractions like chromatography — i.e., that controlling the thermodynamics of the elution was most important, and that the kinetics of analyte desorption were not important (or were relatively fast). In fact, as chromatographers we are trained to set carrier fluid flow rates so that analyte sorption=desorption kinetics are not a problem. However, in extraction, the analytes start on the stationary phase (i.e., the sample matrix), and the kinetics of analyte desorption from the matrix active sites can be the most significant problem limiting the extraction rate, as discussed above. The simplest way to determine if an extraction is limited by the thermodynamics of the process (i.e., the sample=fluid distribution which depends on analyte solubility, called the ‘elution’ step) or the kinetics of analyte release from the sample matrix (the ‘desorption’ step) is to simply determine the effect of flow rate on the extraction rate. Simply put, if doubling the carbon dioxide flow rate doubles the rate of extraction, the extraction is controlled by the elution step (the thermodynamics of the process). If doubling the carbon dioxide flow rate has little or no effect on the extraction rate, the extraction is controlled by the rate of desorption from the matrix active sites [10]. This simple approach to differentiating the mechanisms of extraction seems to be easier to communicate to chromatographers than some of the more complex models discussed above.

16.I.4. Present and future studies: SFE as a tool to investigate analyte/matrix interactions Many users of SFE were frustrated by the fact that different matrices could show varying extraction characteristics for the same analytes. However, we have found this ‘problem’ to be a powerful tool to investigate how organic pollutants are associated with soils, sediments, and other environmental matrices. The ‘hot-ball’ models developed with the Leeds group show essentially identical mathematical form to models attempting to predict transport and fate of organic pollutants in the environment. We are presently investigating the use of SFE to predict environmentally important processes such as bioavailability of pollutants, their treatability, and their environmental mobility. Initial results are very promising that the ability of SFE to find ‘loosely-bound’ and ‘tightly-bound’ pollutants and to determine their kinetics of release will result in a short-term (hours) laboratory test to predict long-term (years) behavior of pollutants in the environment. Our first efforts on using SFE to study PCB binding to aged sediments demonstrated the potential for measuring pollutant ‘availability’ rather than the traditional approach of only measuring a pollutant’s total quantity [11].

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16.I.5. ‘Subcritical’ (hot/liquid) water extractions Finally, our recent efforts have focused on the use of ‘subcritical’ water (water above 100 and below 374ºC, with enough pressure to maintain the liquid state) as an extraction fluid for both polar and non-polar organics. Our initial experiments were performed in an effort to understand the effect of water on conventional SFE. (In truth, the initial experiments were performed to settle a bet with Mary Ellen McNally of DuPont — neither of us correctly predicted the results!) When we heated the water during a soil extraction to observe the effect on the soil, we were amazed to see very high amounts of PAHs in the extract. Further work showed that liquid water at high temperatures (e.g., 150–250ºC) is an extremely effective extraction fluid for both polar and non-polar analytes, and led to the development of a new approach for organic extractions [12]. Since virtually no data on solubility changes with temperature were available for ‘subcritical’ water, we developed a method to measure solubilities of ‘insoluble’ organics, and found that increases in solubilities were typically five orders-of-magnitude (or more) when water is heated from ambient to 200–250ºC [13]. Clearly, such a wide range of control over solubility should be useful for performing a range of different separations. As we learn more about using subcritical water for extractions, it is ironic to note that the story of extraction and chromatography has now come full circle in our laboratory. Our initial work with SFE was based on the then-popular field of supercritical fluid chromatography (SFC). In contrast, our work with subcritical water extraction has led to its first use as a chromatographic carrier fluid, which can be used as pure water to perform reverse-phase separations (even with FID detection) by simply using temperature programming instead of conventional solvent mixing [14]. I believe that this is the first example of extraction research leading to chromatographic research, and hope that these developments demonstrate how the fields of extraction and chromatographic separations could benefit from better interactions and parallel investigations.

References 1. 2. 3. 4.

5. 6.

7.

S.B. Hawthorne and D.J. Miller, Extraction and recovery of organic pollutants from environmental solids and Tenax-GC using supercritical CO2 , J. Chromatogr. Sci., 24 (1986) 258–264. S.B. Hawthorne, Analytical-scale supercritical fluid extraction, Anal. Chem., 62 (1990) 633A. S.B. Hawthorne, D.J. Miller, D.E. Nivens and D.C. White, Supercritical fluid extraction of polar analytes using in situ chemical derivatization, Anal. Chem., 64 (1992) 405–412. M.D. Burford, S.B. Hawthorne and D.J. Miller, Extraction rates of spiked versus native PAHs from heterogeneous environmental samples using supercritical fluid extraction and sonication in methylene chloride, Anal. Chem., 65 (1993) 1497–1505. K.D. Bartle, A.A. Clifford, S.B. Hawthorne, J.J. Langenfeld, D.J. Miller and R. Robinson, A model for dynamic extraction using a supercritical fluid, J. Supercrit. Fluids, 3 (1990) 143–149. S.B. Hawthorne, J.J. Langenfeld, D.J. Miller and M.D. Burford, Comparison of supercritical CHClF2 , N2 O, and CO2 for the extraction of polychlorinated biphenyls and polycyclic aromatic hydrocarbons, Anal. Chem., 64 (1992) 1614–1622. J.J. Langenfeld, S.B. Hawthorne, D.J. Miller and J. Pawliszyn, Role of modifiers for analytical-scale supercritical fluid extraction of environmental samples, Anal. Chem., 66 (1994) 909–916.

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Chapter 5 J.J. Langenfeld, S.B. Hawthorne, D.J. Miller and J. Pawliszyn, Effects of temperature and pressure on supercritical fluid extraction efficiencies of polycyclic aromatic hydrocarbons and polychlorinated biphenyls, Anal. Chem., 65 (1993) 338–344. D.J. Miller, S.B. Hawthorne, A.A. Clifford and S. Zhu, Solubility of polycyclic aromatic hydrocarbons in supercritical carbon dioxide from 313K to 523K and pressures from 100 bar to 450 bar, J. Chem. Eng. Data, 41 (1996) 779–786. S.B. Hawthorne, A.B. Galy, V.O. Schmitt and D.J. Miller, Effect of SFE flow rate on extraction rates: Classifying sample extraction behavior, Anal. Chem., 67 (1995) 2723–2732. E. Bjo¨rklund, S. Brwadt, L. Mathiasson and S.B. Hawthorne, Determining PCB sorption=desorption behavior on sediments using selective supercritical fluid extraction. 1. Desorption from historically contaminated samples, Environ. Sci. Technol., 33 (1999) 2193–2203. S.B. Hawthorne, Y. Yang and D.J. Miller, Extraction of organic pollutants from environmental solids with sub- and supercritical water, Anal. Chem., 66 (1994) 2912–2920. D.J. Miller, S.B. Hawthorne, A.M. Gizir and A.A. Clifford, Solubility of polycyclic aromatic hydrocarbons in subcritical water from 298K to 498K, J. Chem. Eng. Data, 43 (1998) 1043–1047. D.J. Miller and S.B. Hawthorne, Subcritical water chromatography with flame ionization detection, Anal. Chem., 69 (1997) 623–627.

D.17. Friedrich G. Helfferich Friedrich G. Helfferich was born in 1922 in Berlin Germany. He received his Vordiplom and Diplom degrees at the University of Hamburg, Germany, 1949, 1952 and his Dr. Rer. Nat. at the University of Go¨ttingen, Germany, 1955. His academic positions are: Research Assistant, Max-Planck-Institut, Go¨ttingen, Germany; 1951–1956, Research Assistant, MIT, Cambridge, MA; 1954, Research Assistant, California Institute of Technology, Pasadena, CA; 1956–1958, Shell Development Company, Emeryville, CA, and Houston, TX, 1958–1979 (chemist, engineer, supervisor, acting department head, senior staff); Professor of Chemical Engineering, The Pennsylvania State University, University Park, PA, 1980–1990; and as a Visiting Professor and Lecturer, University of California at Berkeley, CA, 1962; University of Houston, Rice University, University of Texas at Austin, TX, 1980; East China Institute of Chemical Technology, Shanghai, 1987. Activities:

Honors:

Editor-in-Chief and Founder, Reactive Polymers (Elsevier), 1981 to 1991. Lecturer, AIChE Life Long Learning Series, since 1982. Consultant: du Pont, Duracell, Exxon Chemical, Raychem, Sepracor, Union Carbide. Fulbright Travel Scholar, 1954; Chairman, Gordon Research Conferences on Ion Exchange, 1967; Separation and Purification, 1994; American Society of Engineering Education, Award for Excellence in Instruction, 1985; American Chemical Society, Award in Separation Science, 1987; Co-Director, NATO Advanced Study Institute on Migration and Fate of Pollutants in Soils, Maratea, Italy, 1992.

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Over 160 technical publications including 5 books, 8 patents. American Institute of Chemists, Fellow Emeritus. American Institute of Chemical Engineers, Fellow. Reaction kinetics, chemical process development, enhanced oil recovery, dynamics of multi-component systems, ion exchange, chromatography. Racewalking, windsurfing, travel, languages, writing children’s stories, military history. See Chapter 5B, a

17.I. HALF A CENTURY AS A KIBITZ Friedrich G. Helfferich Department of Chemical Engineering, The Pennsylvania State University, 158 Fenske Laboratory, University Park, PA 16802-4400, USA

My first encounter with chromatography dates back a bit over half a century, to a time when that science was still true to its name, when conducting a chromatographic separation meant watching bands of different colors develop in a glass column. It was the beauty of these patterns that got me hooked. Yet, life had other things in store for me, and when I returned to chromatography many years later, the attraction and challenge were those of its mathematical, rather than visual patterns. My first constructive involvement in chromatography arose from a practical need: To recover a diamine from a large excess of ammonia in dilute aqueous solution. This led me to formulate the principle of ligand exchange chromatography, in which the stationary phase is an ion exchanger carrying a coordinating transition-metal ion [1]. Separation is by virtue of differences in the strengths of complexes formed with the metal ion. Foremost among later applications of this principle are its use for excellent chiral separations [2], and as IMAC (immobilized metal affinity chromatography, renamed from MCAC, Metal Chelate Affinity Chromatography), an adaptation to large biomolecules [3]. My next innovative contribution stemmed from the realization that there are two kinds of velocities of motion in a column to consider and that, at concentrations high enough for sorption isotherms to be nonlinear, the two differ in magnitude [4]. In the language of physics, one is a particle velocity, indicating how fast (on the average) a respective molecule or ion progresses down the column; the other is a wave velocity, telling how fast a variation of a physical quantity such as a concentration is propagated. An easily perceived analogy is with a tornado; its winds of, say, 120 mph are a particle velocity, whereas its eye moves with a wave velocity of maybe 10 mph. This recognition led me to develop, with Don Peterson, the tracer pulse technique for chromatographic determination of isotherms, especially in multi-component systems [5]. That technique measures the wave velocity of the labeled species, from which the latter’s distribution coefficient can be calculated directly. Moreover, within any one coherent pulse, all

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species have the same wave velocity (that is the coherence condition!!). In contrast, concentration pulses on a non-zero background travel at concentration velocities, whose relationship with isotherms is more complex and much harder to evaluate. To wit, injection of a single-component pulse into a multi-component stream at the column entry produces as many composition peaks and dips as there are sorbable components, and these travel at different wave velocities, each involving concentration variations of all participants, and none with a retention time directly related to any of the distribution coefficients. The recognition of these intriguing and counterintuitive facets of nature sparked in me a deep interest in multi-component nonlinear chromatography and its physics and mathematics. In a year of wonder, work, frustration, and glimpses of a greater truth, it led me to formulate the general concept of ‘coherence’ [6,7] and then to develop, in cooperation with Gerhard Klein, the tools for its practical application, among them the h-transformation (for hyperplane transformation) for rough-and-ready, fast mathematics [7,8]. Coherence in multi-component wave propagation essentially denotes propagational stability. However, since stability in systems with fluid flow has all kinds of connotations not intended here, I decided to coin a different term based on a loose analogy with optics. On the most fundamental level, coherence can be viewed as a generalization of the ideas of equilibrium and steady state as ‘stable’ states to which things settle down if not disturbed any further. As we have known since our high-school days, a closed system left alone comes to equilibrium (with exceptions). If we allow flow through the system, but impose fixed boundaries and boundary values, the system comes to a steady state (with more exceptions). If we drop the restriction to fixed boundaries and boundary values, as we must for traveling waves, the system still shakes itself down to a distinct ‘stable’ state in which the concentration profiles of its components no longer shift relative to one another: it becomes coherent (with still more exceptions). If solute concentrations were people, one might say they look for congenial companions, and once they have found them, they want to keep traveling in their company. On a more practical level, a very simple sufficient and necessary condition for a wave (i.e., concentration variation) to be coherent can be stated: The wave velocities of all particles must be the same at any point in space and time within the wave. This allows only certain distinct composition variations for different particles. Coming back to our example of a concentration pulse injected at the column entry into a multi-component stream: The injected pulse is noncoherent (i.e., it does not meet the coherence condition); therefore it cannot travel as such. Instead, it is resolved into a set of coherent response pulses of different wave velocities for each of the different particles, much like a multi-component sample injected in conventional chromatography is resolved into single-component peaks. Likewise, any composition variation at the column entry, be it a pulse or step, abrupt or gradual, is resolved into a set of coherent waves, provided no new variations are imposed or encountered and the column is long enough for an entirely coherent pattern to develop. Composition variations that meet the coherence condition can be plotted as curves in the hodograph space (i.e., space with concentrations as coordinates). Once behavior has become coherent, the composition profile in the column (and the composition history of

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the effluent) runs exclusively along such curves, called composition paths. The grid of composition paths is given by sorption equilibrium of each respective component, and is independent of the compositions of the entering fluid and those existing initially in the column. This makes it possible to construct a path grid once and for all for a given sorbent and then find the response under any initial and entry conditions much as one would plot a trip by car on a road map. The overwhelming majority of work on mathematics of nonlinear chromatography has been on systems with uniform initial composition in the column and constant composition of the entering fluid (called Riemann conditions by mathematicians). In this case, behavior is essentially coherent from the start, and the coherence concept has little to offer. The concept comes in its own when successive or gradual input variations and columns with nonuniform initial compositions are considered. Here, faster waves generated by later input variations may catch up with slower ones generated earlier or contained in the column at start. Coherence and its tools provide an immediate understanding of such wave interferences and allows predictions to be made without further calculation. In essence, the two (or more) interfering coherent waves merge into a noncoherent wave that is again resolved into new coherent ones: Noncoherence does not know and does not care whether it came into being at the column entry when injection was started, or the composition of the entering fluid was changed, or farther downstream and later through interference of coherent waves; in both cases it is resolved into a new coherent pattern by exactly the same rules. It is in its applications to noncoherent situations that the concept is most valuable as it shows us what will happen during such more complex transients and why. Like equilibrium and steady state, coherence is a very general concept, at least in multi-component nonlinear wave propagation, and is by no means confined to chromatography. The most fruitful among the other applications have been to multiphase, multi-component flow in enhanced oil recovery [9,10], dynamics of multi-component continuous countercurrent mass-transfer operations including distillation [11], and precipitation-dissolution waves in aquifers [12]. Other interesting ones, such as the dynamics of disturbances in catalytic fixed-bed and trickle-phase reactors, are challenges still waiting for takers. The differences within the scientific community in perception of and attitude toward coherence have been psychologically interesting and a little ironic. Theoreticians of nonlinear chromatography, preoccupied with behavior under uniform initial and constant entry conditions, long took for granted what amounts to entirely coherent behavior, oblivious to the fact that more complex conditions give rise to temporary noncoherence. In contrast, engineers concerned with fixed-bed adsorption at first questioned the validity of the concept, while mathematicians familiar with nonlinear wave theory, but unaware of the concept’s practical implications and predictive power, declared it trivial. The greatest obstacle to accepting the coherence concept appears to be the belief that it must be very hard to understand — if not, why has it not been in our books all along? But a search for hidden complexity is a wild-goose chase. Coherence is as simple an idea as is equilibrium and steady state (and as mysterious, if you will), both of which we have taken for granted since our academic infancy, so much so that we have long

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forgotten to ask for proof. I remember my reaction, at age five, when my mother tried to explain equilibrium to me. I did not believe her; the world I knew was push and pull, never at rest, and even if she was right, at equilibrium nothing happened, so what was the use talking about it? A point well taken, yet equilibrium is the basis of much of our scientific and technical thinking. Reaction to coherence has often been similar. I have been asked, “Why should I learn it?” There is nothing to learn, but much to apply to, and from successful application springs acceptance and appreciation. One of science’s greats (I forget whether it was Dirac or de Broglie) once said, “You don’t learn a theory, you get used to it.” This is as true for coherence as it is for equilibrium and steady state. While coherence is a law of nature, a facet of the world we live in, the h-transformation is merely a mathematical tool. Much like matrix algebra, the LaPlace transformation, or, closer to home, the method of characteristics, it makes mathematical problems more tractable, but does not convey better understanding. In contrast to the coherence concept, the (nonlinear) h-transformation is restricted to systems with multicomponent Langmuir isotherms, or ion exchange with constant separation factors (under these conditions the simultaneous first-order differential equations are quasi-linear). The transformation replaces the solute concentrations with new, abstract variables hi that are the roots of a simple polynomial. They have no physical significance. The advantage gained is that all quantities of interest, such as wave velocities, sharpening or spreading of waves, and compositions of zones between waves in a coherent pattern, can be calculated with very simple algebraic equations from just the hi of the compositions of the entering fluid and those existing initially in the column. As an example, in twenty-five years of displacement development, the mathematics for calculating the times and column lengths required for given separations had not progressed beyond three-component mixtures [13] and, even for so few components, required quite lengthy algebra. With the h-transformation it became possible to calculate the requirements for separation of mixtures of fifty or more components [14], and that with equations so simple that even an old-fashioned hand-held programmable calculator such as the HP-41C could manage cases of that complexity with ease. (The h-transformation is equivalent to the ω-transformation introduced later by Rhee et al. [15,16].) My work on nonlinear waves in chromatography has been summarized recently in a series of tutorial articles in the Journal of Chromatography [17]. If I have one regret, it is that the concepts I developed and the practical tools based on them did not become available earlier. Today, our computers are so powerful, fast, and cheap to use that even very complex chromatographic problems can be calculated just by programming ‘brute-force’ forward integration of the simultaneous differential material balances (continuity conditions) of all participants over space and time. My work would have been of much greater utility in an earlier age when that was still unthinkable. Nevertheless, I take consolation from the fact that a better conceptual understanding, the ability to see what will happen, to know without need for calculation, is apt to lead to creative ideas and novel solutions. Progress tends to result from a grasp of the how and why, something wave theory can provide, but computer calculation does not, because each run gives an answer only for the exact conditions specified. In any grass-roots problem, in chromatography as elsewhere, there are more potential approaches than could be calculated in a lifetime even on a supercomputer, and a sound knowledge of

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basic concepts can at the very least help to decide what to calculate. This conviction, the friends I made, and the enjoyment of the abstract beauty of wave mathematics that rivals the visual one of the colorful patterns in primeval chromatography, these have been my reward. I am conscious of the high honor of being included in this volume, of traveling in the company of the greatest minds in chromatography. If I am found wanting, my excuse is to have been a kibitz, an amateur among professionals, that chromatography has been a mere hobby of mine, and that my contributions were those of a Charter Member of the Club of Weekend Theoreticians.

References 1. 2. 3.

4. 5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15.

16. 17.

F. Helfferich, Ligand exchange, a novel separation technique, Nature, 189 (1961) 1001–1002. S.V. Rogozhin and V.A. Davankov, Chromatography of ligands on dissymmetrical complexing adsorbents; new principle of racemate splitting, Dokl. Akad. Nauk. SSSR, 192 (1970) 1288; V.A. Davankov, Ligand-exchange chromatography of chiral compounds; in: D. Cagniant, (Ed.), Complexation Chromatography, (Vol. 57 of Chromatographic Science series), M. Dekker, New York, NY, 1992, Chapter 5. J. Porath, J. Carlsson, I. Olsson and G. Belfrage, Metal chelate affinity chromatography: A new approach to protein separation, Nature, 258 (1975) 598–599. F. Helfferich, Travel of molecules and disturbances in chromatographic columns: A paradox and its resolution, J. Chem. Ed., 41 (1964) 410–413. F. Helfferich and D.L. Peterson, An accurate chromatographic method for sorption isotherms and phase equilibria, Science, 142 (1963) 661–667. F.G. Helfferich, Theory of chromatography: Basis of a generalized theory for multi-component systems with interdependent nonlinear isotherms, Shell Development Co., Technical Report 315– 64 (1964). F.G. Helfferich, Multi-component ion exchange in fixed beds. Generalized equilibrium theory for systems with constant separation factors, I&EC Fundam., 6 (1967) 362–364; Chromatographic Behavior of Interfering Solutes, in: R.F. Gould (Ed.), Adsorption from Aqueous Solution, ACS Advances in Chemistry Series #79, 1968, Chapter 5. F.G. Helfferich and G. Klein, Multi-component Chromatography: Theory of Interference, M. Dekker, New York, NY, 1970; Reprint University Microfilms International, Ann Arbor, No. 2050382. F.G. Helfferich, Theory of multi-component, multiphase displacement in porous media, SPI J., 21 (1981) 51–62. L.W. Lake, Enhanced Oil Recovery, Prentice-Hall, Englewood Cliffs, NJ, 1989, Sections 5.6 and 5.7. Y.-L. Hwang and F.G. Helfferich, Dynamics of continuous countercurrent mass-transfer processes, Parts III and IV, Chem. Eng. Sci., 44 (1989) 1547–1568; 45 (1990) 2907–2915. F.G. Helfferich, The theory of precipitation=dissolution waves, Am. Inst. Chem. Eng. J., 35 (1989) 78–87. J.E. Powell, H.R. Burkholder and D.B. James, Elution requirements for the resolution of ternary rare-earth mixtures, J. Chromatogr., 32 (1968) 559–566. F. Helfferich and D.B. James, An equilibrium theory for rare-earth separation by displacement development, J. Chromatogr., 46 (1970) 1–28. H.K. Rhee, R. Aris and N.R. Amundson, On the theory of multi-component chromatography, Phil. Trans. Roy. Soc., A 267 (1970) 419–455; First-order Partial Differential Equations, Vol. II: Theory and Applications of Hyperbolic Systems of Quasi-linear Equations, Prentice-Hall, Englewood Cliffs, 1986, p. 257. F.G. Helfferich, The h- and ω-transformations in multi-component fixed-bed adsorption: Equivalent mathematics, different scope, Chem. Eng. Sci., 46 (1991) 3320–3323. F.G. Helfferich et al., Non-linear waves in chromatography, Parts I, II and III, J. Chromatogr. A, 629 (1993) 97–122; 734 (1996) 7–48; 768 (1997) 169–205.

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D.18. Jo¨rgen Hermansson Jo¨rgen Hermansson was born in 1948. He studied at the University of Uppsala where he received his University Diploma in Pharmacy 1973. In 1973, he was employed by the National Board of Health and Welfare where he started the work on his Ph.D. in 1976. He received his Ph.D. in Analytical Pharmaceutical Chemistry in 1981 and was employed until 1983 by the National Board of Health and Welfare. Between 1983 and 1989 he was the Head of the Department of Biomedicine at Apoteksbolaget AB. Since 1986, he holds the post of Associate Professor at the Department of Analytical Pharmaceutical Chemistry, University of Uppsala. In 1985, he founded ChromTech AB where he is the President. J. Hermansson is the author of numerous scientific papers in various fields associated with liquid chromatography and is active in Editorial Advisory Boards of scientific journals. He is one of the pioneers in chiral chromatography, with more than twenty years in the field and is the inventor of chiral columns of which the most well known is the column based on α1 -acid glycoprotein (AGP) as the chiral selector. Today, the AGP range of columns are among the most widely used chiral separation tools worldwide. In 1988, he received the Jubilee Medal from the British Chromatographic Society for his contribution to the field of chiral chromatography. Besides chiral chromatography he is very active in HPLC bioanalysis, where his research interest is focused on the development of restricted access media columns, enabling determination of drugs and metabolites by direct injection of protein rich samples, like serum=plasma, on the HPLC system. See Chapter 5B, i, q, r, s

18.I. DIRECT RESOLUTION OF ENANTIOMERS USING IMMOBILIZED α1 -ACID GLYCOPROTEIN Jo¨rgen Hermansson ChromTech Ltd., No. 2 The Courtyard, Greenfield Farm Trading Estate, Congleton, Cheshire CW12 4TR, UK

In 1971–1972, I performed my first chromatographic experiments. As a young student at the University of Uppsala I was asked by Finn Sandberg at the Department of Pharmacognosy if I was interested in working on a project where the goal was to isolate and characterize alkaloids from different African herbs. My contribution was to develop preparative chromatographic methods and elucidate the structure of the isolated compounds using NMR, MS and IR. I accepted the offer and in my spare time I was occupied by this work. The chromatographic separations were performed using a huge handpacked 2-m-long glass column. The column was fed with mobile phase by gravity.

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The herbs were ground and the alkaloids were extracted by acidified methanol. The solvent was evaporated and the residue was applied onto the column. In order to see the migrating zones on the column, I used UV-lamps of different wavelength. Working with preparative chromatography in a glass column was a very good way to get an insight in the secrets of chromatography. I could see the moving zones in different colours and how they broadened during the migration. I remember that I was extremely fascinated and since that time I have been really hooked on chromatography. After this period I was determined to continue in some way to study chromatography to get a deeper insight in the chromatographic processes. After receiving my university degree, I started a research position at the National Board of Health and Welfare, Department of Drugs, which gave me the possibility to start working on my Ph.D. at University of Uppsala, in the Department of Analytical Pharmaceutical Chemistry supervised by Go¨ran Schill. Ion-pair liquid chromatography was one of the most important research areas in the department. My first project was focused on chromatography of glucuronide-conjugated drugs and conjugates of endogenous compounds like steroids. We started with the antiepileptic drug 5,5-diphenylhydantoin (DPH). This drug has a prochiral center and when hydroxylated enantiomers are formed, the hydroxylated metabolite is conjugated with D-glucuronic acid and excreted in urine as diastereomers. I utilized the fact that the body formed diastereomeric derivatives of the enantiomers of the 4-OH-DPH metabolite. I developed a reversed-phase method that gave a separation factor of 1.06 that allowed partial resolution of the diastereomers of the 4-OH-DPH-glucuronides. We found a preponderance of the (S)-4-OH-DPH glucuronide of approximately 20 : 1 in urine of patients treated with DPH. This is most likely the result of a stereoselective hydroxylation of DPH. I was also involved in another project started together with Christer von Bahr, Department of Clinical Pharmacology, Karolinska Institute, concerning stereoselective metabolism of drugs, preferentially the β-blockers propranolol, alprenolol and metoprolol. I was focused on developing chromatographic methods enabeling the resolution of enantiomers present in biological matrixes at a very low concentration. Both in vitro studies using human and dog liver microsomes as well as human liver cells and in vivo studies in man were performed. Concentration–effect relationship studies were also carried out since the concentration ratio may vary under different conditions and since only the S-enantiomer contributes to the clinical effect. At this time no chromatographic method was available for the determination of enantiomers in human plasma at low concentration (pg=ml–ng=ml). It was a great challenge for a young scientist to start working with this kind of project. I decided to synthesize chiral reagents, namely symmetrical anhydrides of Boc-amino acids. They were prepared using phosgene. These reagents were very reactive and could easily form diastereomeric derivatives with the β-blockers without racemization. The diastereomeric derivatives were resolved on non-chiral reversed-phase columns. Using these methods, human kinetic data of the enantiomers of many β-blockers were studied. Enantiomer concentration–effect relationship studies were also performed. These studies in the late seventies and the early eighties were pioneering in this area and the research field has rapidly expanded following our initial studies. After developing the initial indirect methods for the separation of enantiomers, I

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decided to put in a major effort to elucidate the possibilities to create a chiral phase with a very broad applicability and the ability of direct resolution of enantiomers without derivatization. Several advantages could be obtained with the direct methods; they are faster since no diastereomeric derivatives have to be prepared and the risk of racemization during the formation of the diastereomeric derivative is eliminated. Since my research activities were focused around drugs and the majority of the drugs are amines, I concentrated the studies on basic drugs. This was a great challenge since amines at that time were not even very well chromatographed on ordinary non-chiral reversed-phase columns, due to the lack of good silica surface chemistry. A direct separation was reported by Hendersson and Rule in 1939, who demonstrated the separation of the enantiomers of p-phenylenediiminocamphor on D-lactose. However, chemically bonded chiral phases were prepared much later. In 1960, Klemm and Reed utilized the properties of nitroaromatic compounds to form complexes with other aromatic hydrocarbons via π–π interactions. They obtained a partial resolution of both 1-naphthyl-2-butyl ether and 2,4,5,6-dibenzo-9,10-dihydrophenanthrene. Fifteen to twenty years later other groups (Davankov, Pirkle, etc.) contributed with many interesting chiral bonded phases using low molecular weight chiral selectors. The phases that had been developed so far could not be applied to the direct resolution of basic drugs, i.e., the majority of the drugs, without derivatization and the phases had a quite narrow applicability. At this time I also synthesized many of chiral phases based on low molecular weight compounds immobilized on silica (unpublished results). However, the results obtained were not good enough. Since separations with low resolution were obtained in many cases, and the peak symmetry was poor, I decided to concentrate my efforts on developing a chiral phase that could resolve underivatized basic drugs and had the ability to resolve a very broad range of compounds from different compound classes (basic, acidic and non-protolytes). From the results we obtained in the metabolic studies with human and animal liver microsomes and liver cells, it was obvious that enzymes generate stereoselectivity for an extremely broad range of drugs. These results together with early indications from the literature (Willsta¨tter pointed out in 1904 that enantiomers might be adsorbed by proteins with different strengths) gave me the idea that a protein might be the ideal chiral stationary phase. This was also supported by the fact that McMenamy and Oncley in 1958 observed that the enantiomers of tryptophan were bound to albumin with different strength. This finding was utilized by Stewart and Doherty who immobilized albumin on a soft gel for the separation of tryptophan isomers. I thought that the ‘right’ proteins might have the ability to resolve a broad range of chiral compounds due to the many possibilities for stereoselective interactions in the binding site(s) of the proteins. In the beginning of the eighties we started working with human serum albumin which was immobilized onto silica particles. We succeeded in resolving both the enantiomers of tryptophan as well as the enantiomers of warfarin. However, the separation efficiency was low and we did not succeed in resolving basic compounds. I was not satisfied with the results and they were only presented at a local scientific meeting. During that time I was testing a number of proteins as chiral selectors in an attempt to find a selector which could be used for the resolution of a broad range of compounds from different compound classes beside the basic drugs. One of the proteins

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Fig. 1. Determination of the enantiomeric purity of (C)-naproxen. Column: CHIRAL-AGP .100 ð 4:0 mm). Mobile phase: 10 mM sodium phosphate buffer pH 7.0.

I isolated from human plasma was α1 -acid glycoprotein (AGP). I had a great interest in this protein due to the extremely high stability. I was aware of the contribution of AGP in binding of drugs in plasma. This protein is an acidic protein with a low isoelectric point which I thought could favor ionic bonding between the positively charged drug and the negatively charged groups in the protein binding site. In late 1981, I prepared the first AGP column by immobilizing the protein on wide pore silica. When I saw the first results from the evaluation, I understood that this chiral selector would open a new field for direct resolution of enantiomers of drugs. For the first time it was possible to dissolve a racemic basic drug (underivatized) and inject it on the column and obtain a high separation factor and a base-line resolution. The first paper was published 1983 [1]. The peaks showed tailing and the chromatographic performance was not good with this premature version of the AGP column. However, I saw that the principles worked, which was the most important thing at this stage. In fact I received a tremendous interest for this separation technique, phone calls, letters and faxes came from all over the world. I was now convinced that I had a very good basic concept but I had to focus on how to optimize the surface chemistry and how to immobilize the protein in an optimal way in order to obtain a good chromatographic performance. We also focused on

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Fig. 2. Effect of pH on diperodon. Column: As in Fig. 1. Mobile phases: 0.5% 2-propanol in ammonium acetate buffer pH 4.1 (acetate concentration 96 mM) and 6% 2-propanol in 10 mM sodium phosphate buffer pH 7.0.

studies designed for elucidating the mechanisms behind the chiral recognition process [2–5]. The graduate student Ma¨rit Eriksson and I performed a lot of these experiments. Adsorption studies were performed in order to characterize the number and nature of the adsorption sites involved in the chiral recognition process. From these studies we concluded that one high affinity site and a site with lower affinity was involved in the binding of the analytes. All analytes, independently of their nature, were bound to and compete for the same site(s). In order to study the influence of the immobilization procedure on the conformation of AGP, we performed fluorescence studies, utilizing the tryptophan residues of native and immobilized AGP. A 20-nm red shift was obtained for immobilized AGP compared with the emission maximum of 338 nm obtained for native AGP. This demonstrated that one or more tryptophan residues are exposed on the protein surface after immobilization, indicating that the immobilized form of AGP is unfolded to some extent compared to native AGP. However, chromatographic experiments with native AGP as a complexing agent in the mobile phase and experiments with immobilized AGP showed strong similarities. This demonstrates that even though the immobilization procedure affects the conformation of AGP, there still exist large similarities between the immobilized and native AGP concerning the chiral recognition ability. One of the most fascinating properties of AGP is that it is possible to induce enantioselectivity using charged or uncharged modifiers in the mobile phase [5]. Many of these studies were performed together with my wife, Inger Hermansson. For example the enantioselectivity of the acidic anti-inflammatory drug naproxen could be increased from 1.75 to 13 by adding 5 mM N,N-dimethyloctylamine (DMOA) to the mobile phase [5]. Fig. 1 demonstrates the test of the enantiomeric purity of (C)-naproxen produced by fractional crystallization. Naproxen is, in this example, chromatographed using a mobile phase of 10 mM phosphate buffer pH 7.0. As can be seen naproxen is easily

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Fig. 3. Separation of the enantiomers of the basic drugs clenbuterol (a) and cyamemazine (b). Column: As in Fig. 1. Mobile phases: (a) 1% 2-propanol in sodium acetate buffer pH 5.0 (acetate concentration 15 mM). (b) 1% 2-propanol in sodium acetate buffer pH 4.0 (acetate concentration 60 mM).

resolved without DMOA in the mobile phase. Also uncharged modifiers could induce chiral selectivity. For example, the anti-inflammatory acidic drug tiaprofen, showed no enantioselectivity when chromatographed in a pure phosphate buffer pH 6.5. However, adding 4–5% (v=v) of 2-propanol resulted in a separation factor of about 1.4. All NSAIDs of the arylpropionic acid type could easily be resolved on the AGP column. By circular dichroism studies (CD), we have demonstrated that the induction of the enantioselectivity is most likely obtained by a reversible change of the secondary structure induced by the organic solvent. Our initial interpretation of the CD data led us to the conclusion that no changes of the secondary structure were obtained. The changes were very small due to the low molar absorptivity of the amide group. However, a re-investigation clearly indicated changes of the secondary structure. Transformation of parts of the peptide chain with an unordered structure or β-conformation to helical form is most likely the reason for the induction of enantioselectivity using uncharged modifiers. This might also be the reason for the increasing enantioselectivity obtained using the charged modifiers reported above [5]. AGP is a protein with a low isoelectric point (pI), about 2.7. This means that in the ‘normal’ pH range, 4–7, the chiral selector has a net negative charge. Chromatography of hydrophobic basic analytes at pH 7, where these analytes are positively charged, gives strong ionic bonding to anionic groups in the binding site. Thus, very high retention is obtained and uncharged organic modifiers have to be added to the mobile phase in order to reduce the retention. The addition of high concentrations of uncharged modifiers reduces the enantioselectivity. A reduction

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Fig. 4. Separation of the enantiomers of the antiulcer drug omeprazole. Column: As in Fig. 1. Mobile phase: 10% acetonitrile in 10 mM sodium phosphate buffer pH 6.5.

of the pH towards the isoelectric point, pH 4-6, reduces the retention and since only a low concentration of organic modifier has to be added, normally high enantioselectivity can be obtained. The chromatograms in Fig. 2 demonstrate the resolution of the enantiomers of diperodon at pH 4.1 and 7. As can be seen, the retention at pH 7 is very high compared to what was obtained at pH 4.1. At pH 4.1 base-line resolution was obtained within a very short time. A general rule for hydrophobic basic analytes is that the chromatographic performance is also much better at lower pH. Fig. 3a and b demonstrate the separation of the enantiomers of the basic drugs clenbuterol and cyamemazine using pH 5 and 4, respectively. A very large number of basic drugs of different character have been resolved on the AGP column as reported in a number of publications [2–4, 6–8]. Fig. 4 demonstrates the resolution of the world number one drug at the moment, the antiulcer drug omeprazole, with a chiral sulphur atom. It is a weak basic compound. The pH is also an important tool for optimizing the enantioselectivity and the reten-

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tion of acidic compounds. Normally the enantioselectivity and the retention increases by reduction of pH. This is most likely caused by a reduction of the net negative charge of the chiral selector by reducing the pH towards the isoelectric point, giving the negatively charged analyte the prerequisites for stereoselective binding to the protein. A large number of nonprotolytes of different types (alcohols, esters, sulphoxides, amides, ketones etc.) have also been resolved on the AGP column illustrating the extremely broad applicability of the AGP column. Studies have also been performed in order to elucidate the relationship between molecular structure and the enantioselectivity. One of the conclusions that can be drawn is that the steric bulk on a basic nitrogen is important for the chiral recognition. Normally higher enantioselectivity is obtained with an increasing bulk on a basic nitrogen. We have also observed that the distance between the stereogenic center and a basic nitrogen affects the enantioselectivity. The shorter the distance the higher the enantioselectivity. However, if there is more than one hydrogen bonding group in the vicinity of the stereogenic center beside the basic nitrogen, high separation factors can be obtained. Even with a distance of up to seven atoms between the stereogenic center and the basic nitrogen, separation factors of about 1.6 could be obtained [6]. The AGP column is today one of the most widely used chiral columns worldwide. The column is used by scientists at pharmaceutical companies, universities and hospitals. One reason for the widespread use of the column is the extremely broad applicability. Basic compounds (primary-, secondary-, tertiary amines and quaternary ammonium compounds), acidic compounds (carboxylic acids and weaker acids) and non-protolytes (alcohols, esters, sulphoxides etc.) can be resolved.

References 1. 2.

3.

4.

5.

6. 7. 8.

J. Hermansson, Direct liquid chromatographic resolution of racemic drugs using α1-acid glycoprotein as the chiral stationary phase, J. Chromatogr., 269 (1983) 71–80. M. Enquist and J. Hermansson, Influence of uncharged mobile phase additives on retention and enantioselectivity of chiral drugs using an α1-acid glycoprotein column, J. Chromatogr., 519 (1990) 271–283. M. Enquist and J. Hermansson, Separation of enantiomers of beta-receptor blocking agents and other cationic drugs using a CHIRAL-AGP column. Binding properties and characterization of immobilized α1-acid glycoprotein, J. Chromatogr., 519 (1990) 285–298. J. Hermansson and A. Grahn, Optimization of the separation of enantiomers of basic drugs. Retention mechanisms and dynamic modification of the chiral bonding properties on an α1-acid glycoprotein column, J. Chromatogr. A, 694 (1995) 57–69. J. Hermansson and I. Hermansson, Dynamic modification of the chiral bonding properties of a CHIRAL-AGP column by organic and inorganic additives. Separation of enantiomers of antiinflammatory drugs, J. Chromatogr. A, 666 (1994) 181–191. J. Hermansson, K. Stro¨m and R. Sandberg, Relationship between enantioselectivity and solute structure on a chiral α1- acid glycoprotein column, Chromatographia, 24 (1987) 520–526. J. Hermansson, Enantiomeric separation of drugs and related compounds based on their interaction with α1- acid glycoprotein, Trends Analyt. Chem., 8 (7) (1989) 251–259. J. Hermansson, Immobilized α1-acid glycoprotein and cellobiohydrolaze as chiral selectors. Basic characteristics and unique modifier effects, Proceedings of the Chiral Europe ’95 symposium.

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D.19. Herbert H. Hill, Jr. Herbert H. Hill was born on November 25, 1945, in Helena, AR. He earned his B.Sc. degree from Rhodes College, Memphis, TN in 1970, his M.Sc. from the University of Missouri, Columbia, MO in 1973 and his Ph.D. in Chemistry from Dalhousie University, Halifax, NS, Canada in 1975. He received both his M.Sc. and Ph.D. degrees under the direction of Walter A. Aue. Before joining the faculty at Washington State University, he served as a postdoctoral fellow, sponsored by the Ontario Ministry of the Environment, in the laboratory of F.W. Karasek at the University of Waterloo, Waterloo, ON, Canada from 1975 to 1976. At Washington State University he has been an Assistant Professor of Chemistry from 1976 to 1980, an Associate Professor of Chemistry from 1980 to 1985 and a Full Professor of Chemistry since 1985. He has also been an Adjunct Professor of Pharmacology and Toxicology at Washington State University since 1985, Guest Professor in the Department of Chemistry, Kyoto University, Kyoto, Japan in 1983 to 1984 and Visiting Professor at the University of Bayreuth, Bayreuth, Germany in 1994. From 1985 to 1987 he served as Director of the Office of Grant and Research Development at Washington State University. He is on the Editorial Advisory Board of the Journal of Microcolumn Separations (JMS), Field Analytical Chemistry and Technology (FACT), and the International Journal of Ion Mobility Spectrometry (IJIMS). He is a founding member of the International Society of Ion Mobility Spectrometry (ISIMS) and serves on the steering committee for the International Ion Mobility Spectrometry Workshop and Conference. He co-edited the books “Instrumentation for Trace Organic Monitoring” (1992) and “Detectors for Capillary Chromatography” (1992). He has authored over 140 major scientific publications, mainly in the area of separation sciences and ambient pressure ionization processes. He received, in 1989, the Keene P. Dimick Award for Chromatography administered by the Pittsburgh Society for Analytical Chemistry. He was also awarded an Izaak Walton Killam Memorial Scholarship in 1974 for advanced study at Dalhousie University, Halifax, NS, Canada and a fellowship from the Japan Society for the Promotion of Science in 1983. “Professor Hill has made significant contributions to the development of detectors for gas, supercritical fluid and liquid chromatography. Focussing especially on detectors based on ambient pressure ionization and ion mobility, Professor Hill has developed novel chromatographic detectors based on flame ionization, photon ionization, surface ionization, coronaspray ionization and electrospray ionization. Most recently, his work in ambient pressure ion-mobility spectrometry has enabled the high-resolution separation of isomeric peptides and protein conformers.”

See Chapter 5B, d, f, k, h, m, o, p, r, s

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19.I. AMBIENT PRESSURE IONIZATION AND ION MOBILITY SEPARATION IN CHROMATOGRAPHY Herbert H. Hill, Jr. Department of Chemistry, Washington State University, Pullman, WA 99164-4630, USA

Since 1957, when the first ionization detectors for gas chromatography were developed, chromatography and gas-phase ambient pressure ionization processes have been intrinsically linked. Not only does the high efficiency of ambient pressure ionization processes enable the detection of trace quantities of components separated by chromatography, but the ion selectivity possible with these processes enhances the qualitative nature of the chromatographic separation. Over the years I have focussed my attention on the detection and selection process which occurs after chromatography, taking advantage of gas-phase ion mobilities to provide a second dimension to chromatographic separation. In the early 1970s, working under the guidance of Professor W.A. Aue, I developed a novel ionization detector, which we named the hydrogen atmosphere flame ionization detector (HAFID) [1]. Developed for the sensitive and selective determination of trace quantities of metal and silicon containing compounds after gas chromatography, the HAFID proved to be a non-traditional example of how differences in gas-phase ion mobilities can provide sensitive and selective detection after chromatographic separation. Used for the determination of organolead in gasoline, organotin in fish, and silylated pesticides in soils, the response ions of this detector were identified as large cluster ions containing SiO and SiO2 with mobilities considerably lower than normal flame ionization background ions [2]. This difference in gas-phase ion mobility between the background ions and the response ions permits the selective detection of the large response ions at an electrode positioned several centimeters above the flame. In the electric field free region of the detector near the flame, the more mobile background ions diffuse more rapidly to the walls of the detector or to negative ions and are neutralized. The heavier, less mobile ions remain in the gas plume and are carried to the electrode, providing a sensitive and selective response. The more traditional method for using gas-phase ion mobility with chromatography is to couple an ion mobility spectrometer with a chromatograph. When I first began working with ion mobility spectrometry (IMS), in the mid-1970s, under the guidance of F.W. Karasek, it was known as plasma chromatography (PC). This direct analogy with chromatography was due to the mechanism of ion separation in which gas-phase ions are separated by their interaction with neutral buffer gas molecules or atoms as they move through an electric field. Also referred to as gas-phase electrophoresis or ion chromatography, the term ion-mobility spectrometry has gained the widest acceptance since its introduction in the early 1980s. This analogy to mass spectrometry has been unfortunate; the separation mechanism, especially for large biomolecules (as will be discussed later), is more similar to chromatography than mass spectrometry. Early efforts to couple IMS with chromatography defined several modes of operation [3], but were essentially unsuccessful due to excessive column bleed, complex ion-molecule reactions, and long clearance times. In the early 1980s, I completely

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redesigned the ion mobility spectrometer to efficiently interface it to capillary-gas chromatography (CGC) [4]. The success of this CGC–IMS hyphenated system and the new IMS instrument design was evident by the selection of our CGC–IMS separation for the cover of the first monograph published in ion mobility spectrometry [5]. The novel design had a number of salient features: First, the ion mobility drift tube was completely sealed. Prior to this innovation, ion mobility tubes were constructed using an open tubular design similar to those in vacuum systems. Under ambient pressure conditions, however, analytes and contaminants were not pumped away and could remain in the instrument for minutes and sometimes days after introduction. The lack of efficient clearance times prevented the detection of a series of compounds introduced into the spectrometer after chromatographic separation. A second design change, also focused on decreasing clearance times, took advantage of the rapid rates of ion-molecule reaction under ambient pressure conditions. Because equilibrium can be established rapidly at atmospheric pressure, the size of the ion-molecule reaction chamber could be significantly reduced. The rapid ionization rate coupled with the reduced ionization region permitted the sensitive detection of each compound as it eluted from the capillary column. Third, the standard ion mobility spectrometer in those days used a bi-directional flow pattern in which the sample and carrier gas were introduced at the ionization end of the IMS tube and swept toward the center of the instrument. Simultaneously, the IMS buffer gas was introduced at the collector end of the spectrometer and swept toward the center of the tube. Both gases exited in the center of the IMS tube. Unfortunately this configuration allowed the contamination of the ion separation region of the spectrometer with neutral compounds which then underwent ion-molecule reactions as the ions separated in the tube. In our design we introduced a unidirectional flow pattern which eliminated complex ion-molecule reactions in the ion separation region of the spectrometer. Today, all commercial, ion mobility spectrometers have adopted this unidirectional flow pattern. Finally, in order to reduce the complexity of the ion-molecule reactions in the instrument the temperature of the spectrometer was raised significantly over that which had been used previously. At high temperatures, the presence of water cluster ions and dimer ions are reduced to a minimum. With high-temperature IMS, the thermal energy of the buffer gas is not sufficient to break covalent bonds in the ion but is sufficient to break non-covalent bonds such as those which lead to water clusters and dimer formation. In addition, the high temperature helps to keep the instrument clean. With the introduction of this IMS design, IMS could be interfaced to a variety of chromatographic separation techniques. During the 1980s, we successfully interfaced IMS to capillary gas chromatography, supercritical-fluid chromatography, capillary electrophoresis, and liquid chromatography. A review of this work along with other detectors for capillary chromatography can be found in the literature [6]. The development of Fourier transform IMS offered increased sensitivity and resolution [7]. In addition, we reported several novel ionization sources for IMS. In the early 1980s we demonstrated that the linear dynamic range for IMS could be extended several orders of magnitude by using a photo-ionization source. Surface ionization using a MoO source was introduced for the selective detection of drugs and other nitrogen containing compounds.

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Fig. 1. Ion mobility separation of isomeric peptides demonstrating that IMS is an ion size separation technique. Reprinted with permission from C. Wu; W.F. Siems.; J. Klasmeier and H.H. Hill, Jr. Anal. Chem., 72 (2) (2000) 391–395. Copyright American Chemical Society.

Secondary ionization from an electrospray source was demonstrated for the ionization of gas-phase molecules. This secondary ionization source produces spectra similar to that obtained with the positive mode of the 63 Ni radioactive source. Direct coronaspray and electrospray ionization sources were developed for the ionization and introduction of liquids into the IMS after liquid chromatographic separation [8]. Recently, the most significant developments with which we have been involved are the separation and detection of biomolecules by IMS. By developing a cooled electrospray ionization source [9], we were the first to ionize and separate protein conformers under ambient pressure conditions. Because IMS separates on the basis of the size=charge ratio rather than the mass=charge ratio as in mass spectrometry, or the distribution coefficient as in chromatography, separation by IMS offers an orthogonal method to these traditional methods of separation. For example, Fig. 1 shows the separation of two isomeric peptides in which the amino acid order is reversed. In the case of Gly–Arg–Gly–Asp–Ser, the Gly is the N-terminal end of the peptide and the Ser is the C-terminal end. From modeling studies we know that the charge on the peptide is located on the Gly and Arg amino acids and the C-terminal end of the peptide folds up to stabilize the charge at the N-terminal end. Conversely, in the case of the Ser–Asp–Gly–Arg–Gly peptide, the charge was located on the N-terminal Ser group and the basic Arg amino acid. Thus the peptide is stretched apart by the coulombic repulsion of the two charge sites and the molecule is elongated. Until recently, IMS was considered a low-resolution technique. However, we have

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developed a design in which the maximum theoretical resolving power possible by IMS has been achieved under ambient pressure conditions [10]. We have now achieved resolving powers as high as 230. The resolving power in IMS is calculated as the peak arrival time over the peak width at half maximum. Thus, an IMS resolving power of 230 is equivalent to a chromatographic efficiency of over 300,000 theoretical plates. In addition to developing high-resolution IMS, we have now demonstrated that the type of buffer gas can effect ion separation. For example, when helium is used as the buffer gas, the chloroaniline ion is eluted before the iodoaniline ion. When nitrogen is the buffer gas the two ions co-elute and when carbon dioxide is the buffer gas, iodoaniline elutes first. This separation difference as a function of buffer gas composition has led us to develop a separation factor, Þ, for IMS similar to that used in chromatography. By maximizing N and adjusting Þ, ion mobility spectrometry has developed into a powerful separation method, which is more analogous to chromatography than to mass spectrometry. Because its separation occurs in milliseconds rather than seconds as in chromatography and microseconds as in mass spectrometry, IMS is ideally suited to provide orthogonal separation information when located between chromatography and mass spectrometry.

Acknowledgments While my name appears on the papers cited and others we have published, it is really the students who deserve the credit for the growth of IMS as a chromatographic detector and separation device. To date, 18 Ph.D. and 6 M.Sc. students have received their degrees in my laboratory working on projects related to IMS and chromatography. References 1.

H.H. Hill, Jr. and W.A. Aue, Performance of silicon-doped hydrogen atmosphere flame ionization detector for gas chromatography, J. Chromatogr., 122 (1976) 515–526. 2. C.H. Lillie, D.G. McMinn and H.H. Hill, Jr., Mass spectral identification of response ions in a metal selective flame ionization detector, Intern. J. Mass Spectro. Ion Processes, 103 (1991) 219–230. 3. F.W. Karasek, H.H. Hill, Jr. and S.H. Kim, Gas chromatographic detection modes for the plasma chromatograph, J. Chromatogr., 135 (2) (1977) 329. 4. M.A. Baim and H.H. Hill, Jr., Tunable selective detection for capillary-gas chromatography by ion mobility monitoring, Anal. Chem., 54 (1982) 38. 5. H.H. Hill, Jr. and M.A. Baim, Plasma chromatography as a gas-chromatographic detection method; in: T.W. Carr (Ed.), Plasma Chromatography, Plenum Press, New York (1984). 6. H.H. Hill, Jr. and D.G. McMinn, Detectors for Capillary Chromatography, John Wiley and Sons, New York, NY, 1992. 7. F.J. Knorr, R.L. Eatherton, W.F. Siems and H.H. Hill, Jr., Fourier transform ion-mobility spectrometry, Anal. Chem., 57 (1985) 402. 8. C.B. Shumate and H.H. Hill, Jr., Coronaspray nebulization and ionization of liquid samples for ion-mobility spectrometry, Anal. Chem., 61 (1989) 601. 9. D.P. Wittmer, Y.H. Chen, B.K. Luckenbill and H.H. Hill, Jr., Electrospray-ionization ion mobility spectrometry, Anal. Chem., 66 (1994) 2348–2355. 10. C. Wu, W.F. Siems, G.R. Asbury and H.H. Hill, Jr., Electrospray-ionization high-resolution ion-mobility spectrometry, Anal. Chem., 70 (1998) 4929–4938.

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D.20. Vilhelm Einar Stellan Hjerte´n Stellan Hjerte´n was born on April 2, 1928 in Forshem, Sweden. He married Laila Woxtro¨m in 1965; they have one daughter, Marie-Christine. He received the Studentexamen (Gymnasium diploma) in 1948 and was awarded Count Karl’s Scholarship for the highest grade, and his B.Sc. in mathematics, physics, and chemistry in 1954, and his M.Sc. in chemistry, in 1958. He received his Ph.D. degree in biochemistry at Uppsala University in 1967. The title of his thesis was “Free Zone Electrophoresis”. His academic appointments include an Assistant Professorship in Biochemistry at Uppsala University in 1967. He was promoted to Professor in 1969. S. Hjerte´n has received a number of prestigious awards. The most important awards during recent years are: in 1985, the Bjo¨rke´n Prize (the highest award of Uppsala University) for Development of novel electrophoretic and chromatographic separation methods; in 1988, the Electrophoresis Society Founders’ Award for outstanding contributions to the field of electrophoresis, both in practice and theory; in 1993, the Frederick Conference Award: in recognition of outstanding contributions to the field of capillary electrophoresis; in October 1994, the Hirai Prize (Japan) for new approaches in the design of capillary electrophoresis experiments; in January 1996, at HPCE 1996, he was recognized for distinguished advancements to the field of capillary electrophoresis; in March 1996, he received the American Chemical Society National Award in Chromatography; in June 1996, the Torbern Bergman Medal; and in April 2001 the Pierce Award in affinity chromatography and biological recognition. In 1999, he became Doctor Honoris Causa at the University Medical School, Pe´cs, Hungary and in 2001 at Vytautas Magnus University, Kaunas, Lithuania. Hjerte´n’s present research interests are centered on the following projects: ž Chromatography (HPLC) of macromolecules on compressed continuous polymer beds. ž High-performance capillary electrophoresis (HPCE), particularly of macromolecules. ž Methods for the purification of hydrophobic membrane proteins in the presence of SDS, followed by renaturation of the proteins. ž Synthesis of gels mimicking antibodies in their selective recognition of proteins. ž Crystallization of membrane proteins. ž Development of a pump for microchromatography. ž Capillary and microchip chromatography and electrochromatography on non-compressible continuous beds (monoliths) and homogenous gels. See Chapter 5B, a, e, h, i, l, m, n, s

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Professor Hjerte´n at the bench in the laboratory of Arne Tiselius, a Nobel Laureate. Hydroxyapatite columns were being developed for chromatography of proteins and nucleic acids. (Hydroxyapatite is now a classical adsorbent).

20.I. SCIENTIFIC CONTRIBUTIONS Vilhelm Einar Stellan Hjerte´n Department of Biochemistry, University of Uppsala, Biomedical Center, P.O. Box 576, S-75123 Uppsala, Sweden

Stellan Hjerte´n was born on April 2, 1928 and started his scientific career in 1954. He took a Ph.D. in 1967 in biochemistry and was appointed Professor at Uppsala University in 1969. Characteristic of Hjerte´n’s way to develop new methods is that he often tries to combine experimental investigations with theoretical studies and complemented with practical applications. As a pupil of Arne Tiselius (who received the Nobel Prize in Chemistry in 1948), Hjerte´n was early influenced by Tiselius’s great interest in the development of new methods for the separation of biopolymers and realized the immense importance of such research for the progress of biochemistry and related disciplines. For space limitations, only the separation methods that he has introduced and now used world-wide are mentioned below, although many of the other methods are of great interest both from a scientific and practical point of view (the studies of membrane proteins are not treated in this review). For a more exhaustive account see S. Hjerte´n, Uppsala School in Separation Science: My Contributions and Some Personal Reflections and Comments; in H.J. Issaq (Ed.), A Century of Separation Science, Marcel Dekker, Inc., New York, in press.

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20.I.1. Early studies of separation methods Chromatography on hydroxyapatite, first trade name ‘Bio-Gel HT’ [1,2], later several others. Hjerte´n’s role in the introduction of hydroxyapatite as an adsorbent for chromatography is described in Current Contents; this week’s citation classic [3]. Gel filtration (size-exclusion chromatography, molecular-sieve chromatography) on cross-linked polyacrylamide, trade name ‘Bio-Gel P’ [4]. Hjerte´n was the first to observe the molecular-sieving properties of cross-linked dextran gels; for a review, see [5]. It may also be of interest to note that Hjerte´n separated macromolecules (proteins) on polyacrylamide gels before dextran gels (Sephadex) were introduced for such separations as pointed out by A. Tiselius [6], and at a time when many, including Tiselius, had doubts whether the slow diffusion of macromolecules into and out of a gel particle would permit separation of molecules as large as proteins. Gel filtration (size-exclusion chromatography, molecular-sieve chromatography) on agarose, trade name ‘Sepharose’, ‘Bio-Gel A’ [7]. ‘Superose’ is the trade name for agarose-based HPLC beds, similar to those prepared in Hjerte´n’s Laboratory [8]. Gel filtration (size-exclusion chromatography, molecular-sieve chromatography) on agarose beads covered with dextran [8]. ‘Superdex’ is the trade name for columns, similar to those prepared in Hjerte´n’s Laboratory. Different modes of high-performance liquid chromatography (HPLC) on compressed beds of agarose beads, and on columns of coated silica beads; (see for instance [9]). These techniques have the attractive feature that the resolution is independent of or increases with an increase in flow rate, which is contradictory to classical chromatographic theory. These silica beads were probably the first reported to be stable at high pH. Electrophoresis and immunoelectrophoresis in agarose gels [10,11]. These gels are used particularly for analysis of proteins and nucleic acids. Electrophoresis in polyacrylamide gels. Two groups in the USA (Raymond and Weintraub; Ornstein and Davis) and Hjerte´n in Sweden independently studied the usefulness of polyacrylamide gels for analytical electrophoresis of proteins; for a review, see [5]. An apparatus for preparative separations was also designed which permits fractionation of proteins in the range 1 mg to 1 g with a resolution comparable to that obtained in analytical polyacrylamide gel electrophoresis. Hydrophobic-interaction chromatography. Hjerte´n introduced this term, which now is generally accepted, in connection with synthesis and studies of beds with noncharged, nonpolar ligands; for a review, see [12]. Capillary electrophoresis. In his thesis on capillary electrophoresis [13], Hjerte´n stated that there are two methods to perform electrophoresis in capillaries — one being based on rotation of the horizontal capillary around its long axis, and the other on the use of a stationary capillary with a very narrow bore; the latter method is nowadays called high-performance capillary electrophoresis (HPCE). Hjerte´n’s work was focused on the first method particularly since at this time (in 1967), no UV-detectors sensitive enough to detect zones in a tube with diameters much below 1 mm were available, nor were fused silica capillaries. In this article, Hjerte´n: (1) showed, starting from a theoretical discussion, how one could proceed in practice in order to eliminate

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electroendosmosis, including adsorption (by permanent or dynamic coating); (2) derived an equation for thermal zone deformation; (3) showed that a constant current gives a migration velocity which is independent of the temperature variations in the buffer and is therefore preferable to constant voltage in most electrophoresis experiments in order to get reproducible results; (4) traced the light when it transverses a capillary tube to derive the relationship between the transmission of the sample in a capillary and the concentration of the sample; (5) introduced indirect detection; (6) discussed the influence of the sample concentration and the mobility of the buffer ion on peak asymmetry; (7) separated inorganic and organic ions, proteins, nucleic acids, virus and whole cells (bacteria) by capillary zone electrophoresis (CZE), both on an analytical and micropreparative scale; and (8) described isoelectric focusing of proteins (the first focusing experiments performed in the absence of an anti-convection medium). All these points are, of course, relevant to all electrophoresis experiments in capillaries, independent of the diameter of the capillary, i.e., also when it is in the range of 0.025– 0.2 mm, which are common dimensions for the stationary electrophoresis tube used in high-performance capillary electrophoresis. Many of the problems in modern HPCE were, accordingly, solved as early as 1967. Therefore, it is not surprising that colleagues call him ‘the father’ of capillary electrophoresis. Electrophoretic sieving in gel-free media with dissolved polymers (M.D. Zhu, J.-C. Chen and S. Hjerte´n, United States Patent No. 5,089,111. Filed January 27, 1989 and September 27, 1990, approved February 18, 1992). These media are replaceable and thus permit automated HPCE analyses of DNA and proteins. Capillary electrophoresis, polyacrylamide and agarose gel electrophoresis, as well as electrophoresis in polymer solutions, i.e., fields where Hjerte´n did pioneering work, were prerequisites for the success of the Human Genome Project and will probably become — along with several of the chromatographic methods mentioned herein, including continuous beds (monoliths) — equally important in studies of proteomics. 20.I.2. Hjerte´n’s present main interests (1) Chromatography (HPLC) of macromolecules on compressed continuous polymer beds, trade name ‘UNO’ (monoliths). No preparation of beads is required. These beds have the unique property that the resolution is not affected by the flow rate or even increases with an increase in flow rate, which is impossible to achieve according to classical chromatographic theory. The continuous bed in the non-compressed format is very attractive for capillary chromatography (i.d. 5–300 µm) and easy to prepare: an appropriate monomer solution is sucked into a fused silica capillary and polymerized under such conditions that a polymer rod forms with channels, through which the mobile phase can pass. Frits are not required since the polymer rod (the bed) is linked covalently to the capillary wall. These beds are, no doubt, more appropriate also for capillary electrochromatography than are conventional packed beds [14]. (2) High-performance capillary electrophoresis (HPCE), particularly of macromolecules. Hjerte´n was the first to introduce a polymer coating to suppress adsorption, capillary gel electrophoresis, capillary isoelectric focusing, the partial filling technique, replaceable polymer solutions and low-conductivity buffers permitting field strengths as

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high as 2000 V=cm, which means very short analysis times. He has also made important contributions to the development of the theory of capillary electrophoresis. See for instance [15]. (3) Methods for the purification of hydrophobic membrane proteins in the presence of SDS, followed by renaturation of the proteins. This approach facilitates very much the separation of proteins insoluble in water; for an introduction to this technique, see [16]. (4) Synthesis of gels mimicking antibodies in their selective recognition of proteins. A gel prepared for recognition of myoglobin from horse is so highly selective that it adsorbs this protein, but not myoglobin from whale although these two proteins have very similar structures [17,18]. (5) Crystallization of membrane proteins. Capillary electrophoresis is used for analysis of proteins before, after, and during crystallization as well as prior to and following exposure to X-rays [19]. (6) Development of a pump for microchromatography. The standard HPLC pumps give a strong pulsation with attendant variations in the base line when used at the very low flow rates required in microchromatography. Therefore Hjerte´n has designed a thermal pump, based on the expansion of a liquid upon an increase in temperature [20]. The expanded liquid displaces the mobile phase through the column. The lack of such a micro-pump has impeded the development of microchromatography. (7) Capillary and microchip chromatography and electrochromatography on non-compressible continuous beds and homogeneous gels [21–23]. Electrochromatography is often performed in packed beds, which means that the zone broadening is substantial. By replacing this bed by a continuous bed or a homogeneous gel, the zone broadening is strongly reduced. The preparation of an appropriate homogeneous gel is, however, not a trivial problem, which probably is the reason why this approach has not been utilized successfully earlier. The difficulty is to synthesize a charged gel with large pores, which is a prerequisite for the high electroendosmotic flow that is required in electrochromatography. The gels synthesized in Hjerte´n’s laboratory approach the ideal chromatographic beds: Eddy diffusion (the first term in the van Deemter equation) is zero; the broadening caused by longitudinal diffusion (the second term) is less than the broadening originating from free diffusion; the resistance to mass transfer (the third term) is less than in a packed bed and is being reduced still more. Hjerte´n and co-workers were the first to succeed in designing continuous beds (also called monoliths) with high resolution and low back pressure [23,24]. Applied research. The utilization of the knowledge and the experiences gained in methodological electrophoretic and chromatographic studies, also in areas other than electrophoresis and chromatography, for instance for the development of: (a) wound dressings; (b) agents against diarrhea; (c) catheters to suppress bacterial adhesion; (d) tooth pastes and gargling water to prevent bacteria from adsorbing to the teeth. References 1.

¨ . Levin, Protein chromatography on calcium phosphate columns, Arch. A. Tiselius, S. Hjerte´n and O Biochem. Biophys., 65 (1956) 132–155.

236 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12.

13. 14.

15.

16. 17. 18. 19. 20.

21. 22. 23.

24.

Chapter 5 S. Hjerte´n, Calcium phosphate chromatography of normal human serum and of electrophoretically isolated serum proteins, Biochim. Biophys. Acta, 31 (1959) 216–235. ¨ . Levin, Protein chromatography on calcium phosphate columns, Current A. Tiselius, S. Hjerte´n and O Contents, Citation Classics, 28 (46) (1985) 21. S. Hjerte´n and R. Mosbach, ‘Molecular-Sieve’ Chromatography of proteins on columns of cross-linked polyacrylamide, Anal. Biochem., 3 (1962) 109–118. S. Hjerte´n, The history of the development of electrophoresis in Uppsala, Electrophoresis, 9 (1988) 3–15. A. Tiselius, Einige neue Trennungmethoden und ihre Anwendung auf biochemische und organischchemische Probleme, Experientia, 17 (733) (1961) 1–11. S. Hjerte´n, Chromatographic separation according to size of macromolecules and cell particles on columns of agarose suspensions, Arch. Biochem. Biophys., 99 (1962) 466–475. S. Hjerte´n, B. Wu and J.-L. Liao, A high-performance liquid chromatographic matrix based on agarose cross-linked with divinyl sulphone, J. Chromatogr., 396 (1987) 101–113. S. Hjerte´n, J. Mohammad, K.O. Eriksson and J.-L. Liao, General methods to render macroporous stationary phases nonporous and deformable, exemplified with agarose and silica beads and their use in high-performance ion-exchange and hydrophobic-interaction chromatography of proteins, Chromatographia, 31 (1991) 85–94. S. Hjerte´n, Agarose as an anticonvection agent in zone electrophoresis, Biochim. Biophys. Acta, 53 (1961) 514–517. S. Brishammar, S. Hjerte´n and B. v. Hofsten, Immunological precipitates in agarose gels, Biochim. Biophys. Acta, 53 (1961) 518–521. S. Hjerte´n, Hydrophobic-interaction chromatography of proteins, nucleic acids, viruses, and cells on non-charged amphiphilic gels, in D. Glick (Ed.), Methods of Biochemical Analysis, Vol. 27, John Wiley and Sons, New York, NY, 1981, pp. 89–108. S. Hjerte´n, Free zone electrophoresis, Chromatogr. Rev., 9 (1967) 122–219. J.-L. Liao, N. Chen, C. Ericson, and S. Hjerte´n, One-step preparation of continuous beds derivatized with alkyl and sulfonate groups for capillary electrochromatography, Anal. Chem., 68 (1996) 3468– 3472. S. Hjerte´n, Zone broadening in electrophoresis with special reference to high-performance electrophoresis in capillaries: an interplay between theory and practice, Electrophoresis, 11 (1990) 665– 690. S. Hjerte´n, M. Sparrman and J.-L. Liao, Purification of membrane proteins in SDS and subsequent renaturation, Biochim. Biophys. Acta, 939 (1988) 476–484. J.-L. Liao, Y. Wang and S. Hjerte´n, A novel support with artificially created recognition for the selective removal of proteins and for affinity chromatography, Chromatographia, 42 (1996) 259–262. S. Hjerte´n, J.-L. Liao, K. Nakazato, Y. Wang, G. Zamaratskaia and H.-X. Zhang, Gels mimicking antibodies in their selective recognition of proteins, Chromatographia, 44 (1997) 227–234. J. Sedzik, R. Zhang and S. Hjerte´n, Ups and downs of protein crystallization: Studies of protein crystals by high-performance capillary electrophoresis, Biochim. Biophys. Acta, 1426 (1999) 401–408. C. Ericson and S. Hjerte´n, Pump based on thermal expansion of a liquid for delivery of a pulse-free flow particularly for capillary chromatography and other microvolume applications, Anal. Chem., 70 (1998) 366–372. ´ . Ve´gva´ri, T. Srichaiyo, H.-X. Zhang, C. Ericson and D. Eaker, An approach to ideal S. Hjerte´n, A separation media for (electro) chromatography, J. Cap. Elec., 5 (1998) 13–26. C. Ericson, J. Holm, T. Ericson and S. Hjerte´n, Electroendosmosis – and pressure – driven chromatography in microchips, using continuous beds, Anal. Chem., 72 (2000) 81–87. S. Hjerte´n, Standard and capillary chromatography, including electrochromatography, on continuous polymer beds (monoliths), based on water-soluble monomers, a review, Ind. Eng. Chem. Res., 38 (1999) 1205–1214. S. Hjerte´n, J.-L. Liao and R. Zhang, High-performance liquid chromatography on continuous polymer beds, J. Chromatogr., 473 (1989) 273–275.

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D.21. Csaba Horva´th Csaba Horva´th, was born in 1930 in Szolnok, Hungary. He graduated with a M.Sc. in chemical engineering from the University of Technical Sciences in Budapest. He moved to Germany in 1956 and was employed as a chemical engineer at Hoechst AG in Frankfurt (M) for the next four years. In 1960, he returned to academia to pursue a Ph.D. in physical chemistry at the J.W. Goethe University in Frankfurt (M). Under the guidance of the late Istva´n Hala´sz, he developed porous layer open tubular columns and porous layer column packings for gas chromatography in his doctoral research. After being awarded his Ph.D. in 1963, he immigrated to the United States to engage in further scientific work and became a Research Fellow in the Physics Research Laboratory of the Massachusetts General Hospital. A year later Horva´th moved to New Haven to be a Research Associate and in 1970 Associate Professor of Physical Sciences at the School of Medicine, Yale University. In 1979, Horva´th was appointed to the post of Professor of Engineering and Applied Science and upon re-establishment of the chemical engineering department in 1981, he became Professor of Chemical Engineering. He chaired the department in 1987–1993, was acting chair in 1994–1995, and in 1998 was granted the endowed chair with the title of Roberto C. Goizueta Professor of Chemical Engineering. Csaba Horva´th was one of the pioneers of biochemical engineering in the area of enzyme technology and biochemical separations. In the laboratory of the late S.R. Lipsky at the Yale School of Medicine, he built the first high pressure liquid chromatograph, introduced new column technology, and was the first to demonstrate the feasibility and potential of what was originally called ‘high-pressure liquid chromatography’, now termed ‘high-performance liquid chromatography’ HPLC, for fast separation of biological substances. In the seventies and eighties he made major contributions to the theoretical understanding and practical applications of reversedphase chromatography and displacement chromatography. His present interest is in the development of capillary electrochromatography, which is based on chromatography combined with an electric field and separations requiring high numbers of theoretical plates. Horva´th has authored and co-authored close to 300 scientific papers and has numerous patents. He has written several books [see Appendix 4-Chem Web Preprint server (http:==www.chemweb.com=preprint)] and organized several chromatography conferences [see Chapter 3 and Appendix 4-Chem Web Preprint server (http:==www.chemweb.com=preprint)]. His teaching at Yale includes courses in chemical engineering about topics such as separation science, biotechnology, bioseparations and biomedical engineering. He is an external member of the Hungarian National Academy of Sciences and member of the Connecticut Academy of Science and Engineering. Csaba Horva´th also has been Fellow of the American Institute of Chemical Engineers since 1994 and is a Founding Fellow of the American Institute for Medical and Biomedical Engineering.

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Awards and honors Cs. Horva´th has received a number of prestigious national and international awards and honors. Some of these are as follows: The Stephen Dal Nogare Award of the Delaware Valley Chromatography Forum (1978), the Anniversary M.S. Tswett Medal of the USSR Academy of Sciences (1979), the M.S. Tswett Chromatography Award of the International Symposia on Advances in Chromatography (1980), The Humboldt Award for US Scientists of the German A.v. Humboldt Foundation (1982), the National Award in Chromatography of the American Chemical Society (1983), the A.J.P. Martin Gold Medal of the Chromatographic Society (1994), First Listing in Who is Who in the World (1995), the Hala´sz Medal of the Hungarian Separation Science Society (1997), the M.J.E. Golay Award of the International Symposium on Capillary Chromatography and Electrophoresis (1999), the Medal of the Connecticut Separation Science Council (2000), and the Michael Widmer Award of the New Swiss Chemical Society (2000). See Chapter 5B, a, b, d, e, h, l, r, t

21.I. MY FOCUS ON CHROMATOGRAPHY OVER 40 YEARS Csaba Horva´th Department of Chemical Engineering, Yale University, Mason Laboratory, 9 Hillhouse Avenue, P.O. Box 208286, New Haven, CT 06520-8286, USA

Nothing begets good science like the development of a good instrument. Sir Humphrey Davy

21.I.1. My acquaintanceship with chromatography The first time I encountered the word chromatography was in 1953 in Budapest where I served on the faculty of the Institute of Organic-Chemical Technology of my alma mater. An older colleague declared that he would analyze certain amino acids in treated wool samples by paper chromatography. We all listened to this proclamation with great awe, since none of us knew what paper chromatography was and almost everybody had difficulty with the pronunciation of the word chromatography. I did not again encounter this word, which has become my destiny, for a long time thereafter. Only when the political maelstrom in Hungary brought me to Frankfurt am Main, Germany, in 1956, where I got a job as a chemical engineer, did it find my ear again. It was gas chromatography, a newfangled analytical technique that required a sophisticated instrument. We now know that the gas chromatograph has set the paradigm for other analytical instruments and thus has brought in a new era of chemical analysis. At that time its future revolutionary role was still shrouded to us. Some of my recollections of the early years are described in the book, “75 Years of Chromatography: A Historical Dialogue” (1979). In this paper I have tried to focus on later developments.

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21.I.2. Growing up I was fortunate to receive, in my hometown gymnasium, an excellent education (in both the humanities and in the sciences), despite the disruptive years during the war. In following my studies of chemical engineering at the Technical University in Budapest have given me a broad, yet solid knowledge not only of engineering, but also of the physical sciences at large. Nevertheless, I did not have much use for it because of the oppressive political situation. This was one of the reasons I left my home country after the end of the revolution in 1956. I was lucky to find employment at Farbwerke Ho¨chst, the giant German chemical company, and a demanding job in Frankfurt am Main Ho¨chst at its central plant. I served in the Zentralversuchsraum of the Dyestuff Division where I learned the scale-up of processes leading to new products. After two years in the pilot plant, I was transferred to a small group working on the applied surface chemistry of color pigments. There I had the good fortune to solve a major technical problem and come up with an economical process to convert large crystals of an important reddish-blue pigment to a very fine powder without expensive milling. The success prompted me to look around to find an opportunity to continue my studies for a Ph.D., something I could not do in Hungary for political reasons. This brought me to gas chromatography when Istva´n Hala´sz, who had just become Professor in the Institute for Physical Chemistry at the University of Frankfurt (M), convinced me that research in gas chromatography can be very rewarding and offered me a place in his research group. Hoechst generously paid three-quarters of my salary for two years facilitating my doctoral thesis. In 1963, I submitted my dissertation entitled “Columns with Thin Porous Layers in Gas Chromatography”. It introduced PLOT (porous-layer open-tubular) columns, which were the subject matters of a patent licensed to Perkin Elmer and prepared by using the apparatus depicted in Fig. 1. It also described the use in gas chromatography of columns packed with glass beads coated with an adsorbent layer [1]. These can be considered the predecessors of the pellicular sorbents that found employment in HPLC at the dawn of modern liquid chromatography.

21.I.3. New life in America After graduation in 1963, I married Valeria Scioscioli of Rome, Italy, and we immigrated in the old fashioned way to the USA where I became a Research Fellow in the Massachusetts General Hospital of Harvard Medical School. It was an exciting experience to get into this high-powered research atmosphere that changed my horizon. Even if I had no inkling of the consequences of my new experience, the stimulating scientific surroundings that I could watch only with awe, made clear to me that the achievements in life sciences will create excellent opportunities also for a chemical engineer with an open mind and a broad knowledge-base to make significant contributions. First of all, life scientists needed new tools for the purification and analysis of complex biological substances. Could such a technique be modeled after gas chromatography and used for non-volatile substances?

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Fig. 1. Apparatus for coating the inner wall of PLOT capillary columns for gas chromatography.

This question came up when I started my research on the products that arose from cholesterol upon irradiation by gamma rays and electron beams. The problem was essentially an analytical one: to separate, to identify, and to quantify the reaction products. My only tool was thin-layer chromatography, but for someone like me who was experienced with gas chromatography, it was not a reliable technique. Of course, gas chromatography was not suitable because of the lability of the oxidation products, many of them containing peroxide groups. This prompted me to propose the construction of a liquid chromatograph, but it was out of the scope of my research and my poorly written proposal did not receive serious consideration. On the other hand, I received encouragement and support from Leslie Ettre, a major figure in the development of gas chromatography, who was chief applications chemist at Perkin Elmer in the late sixties.

21.I.4. Waiting for moon rocks begets HPLC Another decisive moment in my career was a phone call from Sandy Lipsky, Professor at the School of Medicine of Yale University, who was about to assemble a research group with the support of the NIH and NASA to further develop GC–MS for biomolecules. As a pioneer of gas chromatography for biological substances, Sandy was aware of the potential of instrumental liquid chromatography that would not be dedicated to a single analysis as the marvelous amino acid analyzer of Stein and Moore was, but would have the protean features of gas chromatography. The goal of his research laboratory supported by a NASA grant was to be ready for the analysis of moon rock samples the astronauts would bring back several years later,

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Fig. 2. Ad hoc built high-performance pressure liquid chromatography for narrow base columns anno 1964.

for traces of ‘prelife, life or postlife’ on this heavenly body. Sandy’s support-and the long waiting time to receive the lunar samples allowed me to build a liquid chromatograph for biological substances. The, by then, well-developed theory of gas chromatography provided guidance, yet many aspects required experimental verification. Furthermore, the use of high pressure required certain innovative approaches to instrument and column design. It was an exciting period in my life even if high column inlet pressure was needed to propel the liquid mobile phase across the column to carry out high-speed liquid chromatography, and several experts criticized this. Narrow bore columns (i.d. ½1 mm) packed with pellicular ion exchangers were used for the separation of nucleotides. This system was more stable and efficient than those that were employed previously in our laboratory in reversed-phase chromatography with carbon black or with a liquid ion-exchanger as the stationary phase [2–4]. In Figs. 2 and 3, very early homemade high pressure liquid chromatographs are depicted. My case exemplifies a situation in which the funding of a well defined and rather limited project [5] leads to the development of something much broader, and we may say

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Fig. 3. High-performance liquid chromatograph anno 1965. From 75 Years of Chromatography — A Historical Dialogue. L. S. Ettre and A. Zlatkis (Eds.), Elsevier, Amsterdam (1979) pp. 151–158.

Fig. 4. Components of performance.

that waiting for the lunar samples in 1964–1969 has given rise to the birth of a technique which has become known by its acronym HPLC. It stands for high-performance liquid chromatography, where performance means the aggregate of the efficiency parameters shown in Fig. 4.

21.I.5. Reversed-phase chromatography wears the crown The liquid chromatograph eventually had all the features of a gas chromatograph, but was used with liquid mobile phase; this set the stage for high-performance

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chromatography of biological compounds. Yet, it has taken more than a decade to demonstrate that proteins can be rapidly separated by HPLC. At the beginning, I thought that based on experience with biochromatography at that time, ion-exchange chromatography with novel stationary phases would become the dominant technique. Even early works on reversed-phase chromatography, before HPLC, did not presignify the revolutionary impact that would come about upon unfolding the full potential of reversed-phase chromatography. Driven mainly by the need of the pharmaceutical industry for high performance analytical tools, HPLC in the seventies underwent a meteoric growth by using the newfangled reversed-phase chromatography with octadecylated siliceous stationary phases. The simplicity, general applicability, and reliability of this technique were responsible for the triumphant growth of HPLC. Fig. 5 shows some chromatograms obtained by using reversed-phase chromatography with a commercial liquid chromatograph. In searching for theoretical underpinning, we adopted the solvophobic theory of O. Sinano˘glou, Professor of Chemistry at Yale University, and thus provided a framework for treatment of this branch of chromatography [6–8]. We also published two widely cited papers on the adaptation of the theory to liquid chromatography [9,10].

21.I.6. Dawn of biotechnology By then I was appointed Associate Professor in the Department of Engineering and Applied Science with the mandate to teach and conduct research in the area of biochemical engineering. While serving in medical schools for eight years before this appointment, I learned much about life sciences and how to communicate with biochemists and physicians. Indeed, the time had arrived to combine chemical engineering and life sciences. Indeed, a major thrust toward biotechnology commenced with enzyme engineering in the seventies. It had become eminently clear that we were standing at the gate of something big, and I decided to focus my research on the development of separation processes that would be needed in different branches of biotechnology. Suddenly chromatography gained respect as the most suitable separation process in the laboratories and in the plants of the rapidly growing biotechnology industry. Our enzyme engineering work led to a comprehensive treatment of the interplay between enzyme-catalyzed reaction with transport phenomena. Our studies on opentubular enzyme reactors required instrumentation not much different from that employed in liquid chromatography [11]. Later on many enzymatic appliances were developed for Technicon’s clinical analysis systems. Since the small ‘enzyme coils’ made it unnecessary to use perishable and expensive enzyme solution in blood testing by the machines of Technicon, they were successfully commercialized. On the other hand, the expanding role of reversed-phase chromatography called for the preparation and characterization of novel stationary phases, and this endeavor was carried out in an industrial setting by Northgate Laboratories in Hamden, CT. On the scientific end, we explored the use of several linear free energy relationships in reversed-phase chromatography, and after a hiatus, this work was continued in the late nineties. In the early 1980s, my laboratory was engaged in research on displacement chro-

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Fig. 5. Chromatograms obtained by reversed-phase chromatography of urine extract containing added aromatic acids. From I. Molna´r, Cs. Horva´th and P. Jatlow, Chromatographia 11 (1978) 260–264.

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Fig. 6. Csaba Horva´th in the laboratory (mid-eighties).

matography based on the promise of this chromatographic modality in process chromatography. This was an excellent way to learn the idiosyncrasies of non-linear chromatography [12]. But most of the hopes for industrial applications have not yet materialized. Towards the end of the 1980s, our interest was focused on rapid HPLC, which would then advance process monitoring and multidimensional HPLC by using technology developed in our laboratory in the late 1960s, namely: columns packed with pellicular sorbents (2 µm particles) and operated at elevated temperatures, e.g., 80ºC [13,14]. The results were very promising; protein mixtures could be separated in less than 10 s. Yet, the world was not ready for fast chromatography and the chromatographic establishment ignored this opportunity. Times have changed though; upon the arrival of combinatorial chemistry and proteomics, high throughput analytical techniques are widely sought to reach the goals of these undertakings. Finally, the growing demand for rapid analysis offers an opportunity to resume our research in this field with an eye on miniaturization, and on the needs engendered by the high speed analytical machinery which is expected to reach a dominating role in the future.

21.I.7. The expanding scope of electrochromatography The early 1990s have brought about major changes in our research orientation. Advances in instrumentation by miniaturization and by harnessing the electric field to enhance the separating power led to the evolvement of capillary zone electrophoresis, CZE, and capillary electrochromatography, CEC, upon the availability of fused silica capillaries. Capillary columns are a must with these separation methods in order to keep

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Fig. 7. Csaba Horva´th (middle) with Wayne Melander (left) and Imre Molna´r (right) at Pittcon 1994 in Chicago.

Fig. 8. Csaba Horva´th with his research group at Yale 1995.

Joule heating under control. After getting familiar with CZE [15], and applying the technique to carbohydrate analysis, we developed several methods to change favorably the inner surface of the capillaries and a process to form in situ a fluid-impervious tube inside the fused silica capillary. Then the inner wall of the ‘tube in the tube’ was coated with a hydrogel-like layer to suppress protein adsorption or functionalized otherwise. My experience in coating the inner walls of capillary tubes during my doctoral research came very handy in carrying out this work. CEC is a bona fide chromatographic

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analytical method, a hybrid between µHPLC and CZE, which has been the third kind, being closely investigated in my laboratory over the last thirty years. It employs high electric fields to generate electro-osmotic flow of the mobile phase that has been shown to engender less band spreading than laminar flow and thus leads to higher column efficiency [16]. In view of the increasing employment of the mass spectrometer in liquid chromatography as a detector, the idea of an instrument, which encompasses micro-HPLC, CZE, and CEC — each technique employing fused silica capillary columns of very similar dimensions and mobile phases — is quite intriguing. Yet a substantial amount of further equipment with MS detection can be marketed successfully. Our research interest has been refocused again to explore the theoretical underpinning and most promising applications of CEC, so that it becomes a respected analytical technique of wide employment.

Acknowledgments I am indebted to my coworkers and students, whose contributions were essential to the success of our research. Over the years, my research has been generously supported by the National Institutes of Health with a grant GM020993, and by the National Foundation of Cancer Research.

References 1.

I. Hala´sz and Cs. Horva´th, Micro beads coated with a porous thin layer as column packing in gas chromatography. Some properties of graphitized carbon black as the stationary phase, Anal. Chem., 36 (1964) 1178–1186. 2. Cs. Horva´th and S.R. Lipsky, Use of ion-exchange chromatography for the separation of organic compounds, Nature, 322 (1966) 748–749. 3. Cs. Horva´th, B.A. Preiss and S.R. Lipsky, Fast liquid chromatography: An investigation of operating parameters and the separation of nucleotides on a pellicular ion exchangers, Anal. Chem., 39 (1967) 109–116. 4. Cs. Horva´th and S.R. Lipsky, Column design in high pressure liquid chromatography, J. Chromatogr. Sci., 7 (1969) 778–779. 5. S.R. Lipsky, R.J. Cushley, Cs. Horva´th and W.J. McMurray, Analysis of lunar material for organic compounds, Science, 167 (1970) 778–779. 6. Cs. Horva´th, W. Melander and I. Molna´r, Solvophobic interactions in liquid chromatography with non-polar stationary phases, J. Chromatogr., 215 (1976) 129–156. 7. Cs. Horva´th, W. Melander and I. Molna´r, Liquid chromatography of ionogenic substances with nonpolar stationary phases, Anal. Chem., 49 (1977) 142–154. 8. W. Melander and Cs. Horva´th, Salt effects on hydrophobic interactions in precipitation and chromatography of proteins: an interpretation of the lyotropic series, Arch. Biochem. Biophys., 183 (1977) 200–215. 9. Cs. Horva´th and H.J. Lin, Movement and band spreading of unsorbed solutes in liquid chromatography, J. Chromatogr., 126 (1976) 401–420. 10. Cs. Horva´th and H.J. Lin, Band spreading in liquid chromatography, general plate height equation, and a method for the evaluation of the individual plate height contributions, J. Chromatogr., 49 (1978) 43–70.

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11. Cs. Horva´th, A. Sardi and B.A. Solomon, Enzyme reactor tubes: Effect of diffusion and slug flow, Physiol. Chem. Phys., 4 (1973) 125–130. 12. J. Frenz and Cs. Horva´th, High-performance displacement chromatography, in Cs. Horva´th (Ed.), HPLC: Advances and Perspectives, Vol. 5, Academic Press, San Diego, CA, 1988, pp. 212–234. 13. K. Kalghatgi and Cs. Horva´th, Micropellicular sorbents for rapid RPC of proteins and peptides, in Cs. Horva´th and J.G. Nikelly (Eds.), Analytical Biotechnology: Capillary Electrophoresis and Chromatography, American Chemical Society Symp. Ser. 434, ACS, Washington, DC, pp. 162–180. 14. H. Chen and Cs. Horva´th, Rapid separation of proteins by reversed phase HPLC at elevated temperatures, Analytical Methods and Instrumentation I, 4 (1993) 213–222. 15. F. Ka´lma´n, S. Ma, R.O. Fox and Cs. Horva´th, Capillary electrophoresis of S. nuclease mutants, J. Chromatogr. A, 705 (1995) 135–154. 16. G. Choudhary and Cs. Horva´th, Dynamics of capillary electrochromatography: Experimental study on the electroosmotic flow and conductance in open and packed capillaries, J. Chromatogr. A, 78 (1997) 161–183.

D.22. Josef Franz Karl Huber Josef Franz Karl Huber passed away on August 15th, 2000. Many scientists in the world feel his death as a great loss. It is appropriate to devote some words to the significance that his life and work has for the development of analytical chemistry involving separation methods. Josef Huber was born in Salzburg, Germany, January 1, 1925. He was 14 years old when the World War II broke out. He was drafted for the third Reich army, wounded several times and ended up as a prisoner of war. At the age of 25, he started his chemistry study, which he finished with a doctorate under supervision by Erica Cremer in Innsbruek, 1960. This ties his intellectual background to the once flourishing mid-European culture. In 1960, he married Josepha Lu¨ning, born in Emden. Older participants of international meetings will remember her vividly as an extremely congenial lady with an intense interest in history and culture. Huber went through the distress of losing her in 1997, after a life-long happy marriage. They had one son, Wolfgang, who is now a medical oncologist in Vienna. Huber’s career continued in The Netherlands. He was first for four years in Eindhoven Technical University, where a new and strong group in instrumental analysis was founded by A.I.M. Keulemans, and where he had contact with pioneers in separation methods, such as Marcel Golay, and A.J.P. Martin. During that time Huber expanded the basic theoretical considerations [1] from which he could embark on the development of fast column liquid chromatography using small particles, the technique we now know under the name HPLC. Since then, this method was one of his main interests, and he is considered by many as one of the founding fathers of HPLC [2]. In 1964, he moved to Amsterdam University and founded his own group on separation methods. It was there that one of us (HP) marveled at his unbridled energy, his deep theoretical insight and his understanding of matters of organization and society. During his time in Amsterdam (until 1974), many of his most influential papers

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appeared. Anything connected with the possibilities and problems of HPLC would be the subject of research: column design, plate height equations, detectors, data handling, analysis of body fluids, and environmental problems, etc [3–5]. The group expanded rapidly and became an internationally recognized ‘center of excellence’; the place was crowded by visitors and visiting scientists, who wanted to learn or practice HPLC, in particular the method to pack HPLC columns with particle sizes below 10 µm, at that time a pioneering achievement. This manual technique was mastered by his most important co-worker at that time, Johan Kraak, and was later superseded by ‘slurry packing’ for the same type of porous microparticulates. In 1974, he moved to the University of Vienna. The ‘direct democracy’ movement emerging in the sixties in The Netherlands worried him. One of his favorite maxims was that the temperature of an experiment would henceforth be decided by the one-man one vote system. Apart from that, he could obtain a more influential position in Vienna, in command of many more general analytical activities. Here, with co-workers G. Reich, E. Kenndler, and A. Rizzi, his research indeed encompassed a much broader field. Column switching, already studied in Amsterdam as a solution to the ‘general elution problem’, was now harnessed for increased selectivity, by passing the sample over successive columns with differing retention characteristics. In the international forum he became a prominent proponent of such techniques. The approach was later followed by many [2]. This work naturally led to the development of theories regarding multidimensional (‘hyphenated’) analytical systems. The time in Vienna is marked by an important other activity: At the time of the iron curtain, he succeeded in establishing fertile scientific connections between ‘East’ and ‘West’. Even before the time in Vienna, he had been active in this way, and acted as a member of the committee of the ‘Science Exchange Agreement’, an organization funded by Clark Hamilton to support the scientific contacts and the exchange of East and West European scientists as visiting scientists. Numerous people have benefited from this program. One result has been the series of ‘Danube Conferences’, where intense scientific contact across the iron curtain was made possible, see Chapter 3G. The well-known series of large meetings, known since 1984 as ‘HPLC XXXX’, with XXXX standing for the year, should be mentioned here. Willy Simon in Interlaken, Switzerland organized the first meeting of this series in 1973. It was unique in being entirely devoted to liquid phase separations. The initiative for it had been taken by Huber, J.J. (Jack) Kirkland, and John H. Knox. Huber always felt strongly connected with this meeting series; it was ‘his’ meeting. The continuing success of these symposia, usually attracting a thousand or more scientists, demonstrates the vision that these people had in the early seventies. Many international awards and assignments certify the significance of Huber’s work for analytical chemistry, laid down in over 100 scientific papers. He was an editor of more than ten international journals and book series. He acted as chairman of numerous scientific organizations. Awards or honorary memberships were offered to him by G.A.M.S., the French Analytical Chemistry Society, the Russian Academy of Sciences (Tswett medal, 1978) the Delaware Valley Chromatography Forum, USA (Dal Nogare Award, 1981), the Chromatographic Society, UK (A.J.P. Martin Award, 1988), the Technical University of Bratislava, Slowakia (Honorary Medal, 1985), and the Cross

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of Honor for Science and Art from the Austrian Republic (1988). He became honorary doctor at the University of Uppsala, Sweden, the Marie Curie-Sklodowskiej University in Lublin, Poland, and the University for Chemical Technology in Veszprem, Hungary. See Chapter 5B, a, e, h, k, p

Acknowledgments The authors received valuable information from G.A. Guiochon and J.J. Kirkland. Prepared by Ernst Kenndler and Hans Poppe

References 1. 2. 3. 4. 5. 6.

J.H. Quaadras, M.Sc. Thesis, University of Technology, Eindhoven, January 1964. Multidimensionality, Hyphenation and Coupled-Column Techniques, special issue of J. Chromatogr. A, Volume 500 (1995); preface pp. 1–2. J.F.K. Huber and J.A.R.J. Hulsman, Anal. Chim. Acta, 38 (1967) 305; corrections to printing errors ibid, 38 (1967) 581. J.F.K. Huber, Material transport and distribution in chromatographic processes, Ber. Bunsenges. Phys. Chem., 77 (1973) 159–184. J.F.K. Huber, Evaluation of detectors for liquid chromatography in columns, J. Chromatogr. Sci., 7 (1969) 172–176. J.F.K. Huber and H.C. Smit, Information flow and automatic data processing in chromatography, Z. Anal. Chem., 245 (1969) 84–88.

D.23. Daido Ishii and Toyohide Takeuchi Daido Ishii was born November 23, 1926 and, is currently a Professor of Kumamoto Institute of Technology and an Emeritus Professor of Nagoya University. Since he joined Nagoya University as a research associate in 1950, he has been conducting research and education in analytical chemistry. Professor Ishii is the author of over 220 scientific publications in analytical chemistry. He began his career in radioanalytical and nuclear chemistry and became involved in chromatography in the 1970s. His research interests include the development of microcolumns for LC, new detection systems for LC and downsizing of flow analysis systems. Professor Ishii is a pioneer in microcolumn LC. His group developed open-tubular capillary and fused silica micropacked columns for LC. He succeeded in combining microcolumn LC with fast atom bombardment mass spectrometry. Professor Ishii

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proposed unified chromatography, which allows the separation in GC, SFC and LC modes using a single microcolumn by choosing the appropriate operating conditions. Professor Ishii is the Co-editor of “Microcolumn Separations”, published by Elsevier (1985). The book includes eighteen articles, which were presented at the Japan–US Joint Seminar held at Honolulu, Hawaii in June 1982, where thirty-two scientists from Japan and US together with four participants from other countries joined. He is also the Editor of “Introduction to Microscale High-Performance Liquid Chromatography”, published by VCH Publishers (1988). The book includes seven chapters contributed by Japanese leading chromatographers. Professor Ishii is the recipient of national and international awards in analytical chemistry and chromatography: The Japan Society for Analytical Chemistry Award in 1979; the Tswett Chromatography Award in 1987; and the Marcel Golay Award in 1990. Professor Ishii established the HPLC Discussion Group in 1981, and organized thirty-three chromatography seminars and workshops at Nagoya, Japan from 1981 to 1994. He was the chairman of the Seventh International Symposium on Capillary Chromatography held at Gifu, Japan in May 1986. Toyohide Takeuchi was born on May 17, 1954. He studied at Nagoya University, receiving his B.Sc. and M.S. degrees in engineering in 1977 and 1979, respectively, and a Ph.D. in Engineering in 1985. Between 1980 and 1989 he had been a Research Associate at the Department of Applied Chemistry of Nagoya University and then between 1989 and 1992 an Associate Professor at the University’s Research Center for Resource and Energy Conservation. In 1985–1986 he spent one year as a Postdoctoral Fellow at the Ames Laboratory, Iowa State University, Ames, Iowa, USA. Since April 1992, he is affiliated with the Faculty of Engineering of Gifu University, in Gifu, Japan, as an Associate Professor. Dr. Takeuchi received in 1987 the Award for Young Scientist of the Japan Society for Analytical Chemistry. See Chapter 5B, Ishii a, d, e, f, h, k, o, r; Takeuchi d, e, h, r

23.I. DEVELOPMENT OF MICROCOLUMNS FOR LC Daido Ishii 1 and Toyohide Takeuchi 2 1

Department of Industrial Chemistry, Kumamoto Institute of Technology, 22-1, Ikeda 4, Kumamoto 860, Japan 2 Department of Chemistry, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan

In HPLC, 4–6 mm i.d. columns packed with 3–10 µm materials have commonly been employed. Research on miniaturization of the separation column in LC began at nearly the same time as HPLC was introduced. Ishii’s group initiated the work on microcolumn LC in the early 1970s and the first paper written in English appeared in 1977 [1].

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Analytical packed columns in LC can be divided, according to the column diameter, into three categories: conventional columns (4–6 mm i.d.), semi-microcolumns (1–2 mm i.d.), and microcolumns (0.2–0.5 mm i.d.). The volume of the microcolumns is less than one-hundredth of that of conventional columns. There are several advantages arising from miniaturization of the separation columns in LC as follows [1–3]: (a) low consumption of both mobile and stationary phases, which facilitates the use of expensive, hazardous or exotic materials; (b) convenient for direct coupling to mass spectrometers due to low flow rates; (c) increase in mass sensitivity; (d) easy control of column temperature due to low heat capacity; (e) convenient for selecting the operating conditions; (f) easy coupling to secondary chromatographic systems. The increase in the mass sensitivity is especially important when dealing with the analysis of samples of biological origin since such samples often contain very limited quantities of the individual analytes. The low heat capacity of microcolumns allows the easy use of temperature programming in LC like in GC, and this can partly replace solvent gradient elution. The decrease in peak volume facilitates direct coupling with a secondary separation system, e.g., LC–LC. Coupled-column chromatography improves separation efficiencies over single-column chromatography. Various types of column tubing materials involving PTFE, stainless steel, glass, glass-lined stainless steel, and fused silica have been examined for microcolumn LC. Among these tubing materials, fused silica gives higher efficiencies owing to its smooth inert surface and it is convenient to handle owing to its flexibility. Fig. 1 demonstrates the separation of aromatic hydrocarbons on a 10-cm fused-silica column. Around 70,000 theoretical plate numbers per meter were obtained [4].

23.I.1. Open-tubular capillary LC Open-tubular capillary columns were originally proposed by Golay in GC and have widely been utilized as potential separation tools. Good permeability of these columns provides larger theoretical plate numbers in a unit time and unit pressure drop than conventional packed columns in GC. Ishii’s group first studied the feasibility of capillary columns with the dimension of 30–50 µm i.d. for LC [2,3,5]. Band spreading based on the first term of the Golay equation results from the laminar flow, which can be reduced by decreasing the column diameter. In order to appreciate the above advantages the column diameter should be as small as a few micrometers. Typical separations of xylenol isomers and m-cresol on a 33 µm column is demonstrated in Fig. 2 [5]. The stationary phase is β,β0 -oxydipropionitrile physically coated on the soda-lime glass capillary treated with alkaline solution. Although the diameter (33 µm) is much larger than the optimum diameter, it is seen that good separation is demonstrated in the figure. In order to achieve a larger theoretical plate number with open-tubular columns, several problems should be solved: (a) a small volume of sample (nl level) should be injected; (b) detection volume should be as small as 1 nl; (c) techniques for the production of smaller diameter columns should be developed.

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Fig. 1. Separation of polynuclear aromatic hydrocarbons on a fused silica column. Column, 100 ð 0:25 mm i.d., packed with 5 µm ODS; mobile phase, acetonitrile–water (70 : 30); flow rate, 3.3 µl min1 ; sample components: 1 D benzene, 2 D naphthalene, 3 D biphenyl, 4 D fluorene, 5 D phenanthrene, 6 D anthracene, 7 D fluoranthracene, 8 D pyrene, 9 D p-terphenyl, 10 D chrysene, 11 D 9-phenylanthracene, 12 D perylene, 13 D 1,3,5-triphenylbenzene, 14 D benzo[a]pyrene; injection volume, 0.05 µl; wavelength of UV detection, 254 nm. Reproduced from Takeuchi and Ishii [4] with permission from the publisher.

23.I.2. Direct coupling of microcolumn LC with fast-atom bombardment mass spectrometry (FABMS) Direct coupling of LC and MS was expected to provide a versatile analytical method, but many more difficulties have been encountered in the combination of these two mismatched analytical tools than in GC–MS. Nevertheless, many challenging investigations have been devoted to developing new interfaces and reducing the mismatch between the two techniques. FABMS is an excellent method for the analysis of thermally unstable and=or involatile polar compounds with high molecular mass, and it has been playing an

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Fig. 2. Separation of xylenol and m-cresol on an open-tubular column. Column, 8.1 m ð 33 µm i.d., glass capillary coated with β,β0 -oxydipropionitrile (BOP); mobile phase, n-hexane saturated with BOP; flow rate, 0.8 µl min1 ; sample components, 2,6-, 2,4-, 2.3-, 3,5-, 3,4-xylenol and m-cresol eluted in that order; wavelength of UV detection, 280 nm. Reproduced from Ishii and Takeuchi [5] with permission from the publisher.

important role in biopolymer structure elucidation. For the measurement of FAB mass spectra, the analyte, dispersed in a matrix such as glycerol, is placed on a target, which is bombarded by an argon or xenon beam. FAB is a soft ionization technique and generally gives quasi-molecular ions as well as fragment ions, which are useful for identifying the analytes. The interface developed for microcolumn LC–FABMS is composed of fused-silica capillary tubing, the top of which is attached to a thin metal frit [6]. Fig. 3 demonstrates the selective ion monitoring of an artificial mixture of bile acids, where 10 ng amounts of each have been injected. Negative ions are monitored and each m=z value corresponds to the deprotonated ion of each analyte. The mobile phase contains 5% (v=v) glycerol, which is the matrix for FABMS detection. This system is capable of detecting both positive and negative ions.

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Fig. 3. Selective ion monitoring of a reference mixture of bile acids. Column, 100 ð 0:35 mm i.d., packed with 3 µm ODS; mobile phase, acetonitrile–10 mM ammonium bicarbonate–glycerol (18 : 77 : 5 to 39 : 56 : 5 in 55 min); flow rate, 2.1 µl min1 ; sample components: 1 D cholic acid, 2 D glycocholic acid, 3 D taurocholic acid, 4 D glycodeoxycholic acid, 5 D deoxycholic acid, 6 D taurodeoxycholic acid, 7 D glycolithocholic acid, 8 D taurolithocholic acid, 9 D lithocholic acid, 10 ng each injected. Reproduced from Ishii and Takeuchi [6] with permission from the publisher.

23.I.3. Unified chromatography Chromatography is classified into GC, SFC and LC, depending on the physical properties of the mobile phase. The mobile phase in GC is a gas such as helium or nitrogen, that does not have any interaction with analytes and carries them through the column; it is called a carrier gas. On the other hand, the mobile phase in LC dissolves analytes and has a good chance to interact with them. Therefore, the mobile phase in LC significantly affects the selectivity of the separation. In SFC a high-density gas is employed as the mobile phase, where the temperature and pressure in the column are higher than the critical values of the mobile phase employed. Since the separation mode can be selected by changing the pressure in the column and the column temperature, it is possible to demonstrate different-mode separations, viz., LC, SFC and GC separations by using a single chromatographic system. This is the idea of unified chromatography [7]. Capillary columns, packed or open-tubular, facilitate the demonstration of these different-mode separations with a single chromatographic system because mass flow rates for these columns make them convenient to connect to various detectors. Changing the column temperature and the pressure in a single system, different-mode separations can be carried out. Each separation mode can be optimized by the careful selection of the type of the separation column and the stationary phase, the column dimensions, the mobile phase, the detector, and the operating conditions.

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Fig. 4. Separation of an artificial mixture of aromatic hydrocarbons and styrene oligomers. Column, 150 ð 0:5 mm i.d., packed with 5 µm silica; mobile phase, diethyl ether; inlet pressure, programmed as shown in the figure) P D 5 kgf cm1 ; temperature, 220ºC; sample components: 1 D benzene, 2 D naphthalene, 3 D anthracene, 4 D pyrene, 5 D polystyrene A-1000 (weight-average molecular mass D 9:5 ð 102 /; wavelength of UV detection, 220 nm. Reproduced from Ishii and Takeuchi [7] with permission from the publisher.

Fig. 4 demonstrates the separation of an artificial mixture of aromatic hydrocarbons and styrene oligomers on a silica gel packed column using the vapor of diethyl ether as the mobile phase [7]. The analytes are isothermally separated at supercritical temperature, and the pressure is programmed so that the aromatic hydrocarbons can be separated in the GC mode, prior to the SFC separation of the styrene oligomers. The pressure drop across the separation column is kept at 0.49 MPa by using a two-pump system. The inlet and outlet pressures are controlled by a microcomputer. Unified chromatography is good for the separation of analytes with a wide range of physical and chemical properties in a single chromatographic run.

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23.I.4. Future prospects of microcolumn LC Many researchers have experimentally demonstrated attractive features of microcolumn LC. Nevertheless, conventional LC has been generally employed because several problems remain to be solved in microcolumn LC. Advances in modern electronics, and computer and micromachining technology will increase the potential of microcolumn separation techniques in the future.

References 1.

2. 3. 4. 5. 6. 7.

D. Ishii, K. Asai, K. Hibi, T. Jonokuchi and M. Nagaya, A study of micro-high performance liquid chromatography. I. Development of technique for miniaturization of high-performance liquid chromatography, J. Chromatogr., 144 (1977) 157–168. M. Novotny and D. Ishii (Eds.), Microcolumn Separations, J. Chromatography Library, Vol. 30, Elsevier, Amsterdam, 1985. D. Ishii (Ed.), Introduction to Microscale High-Performance Liquid Chromatography, VCH Publishers, New York, 1988. T. Takeuchi and D. Ishii, High-performance micro packed flexible columns in liquid chromatography, J. Chromatogr., 213 (1981) 25–32. D. Ishii and T. Takeuchi, Open-tubular capillary LC, J. Chromatogr. Sci., 18 (1980) 462–472. D. Ishii and T. Takeuchi, Miniaturization in column-liquid chromatography, Trends Anal. Chem., 9 (1990) 152–157. D. Ishii and T. Takeuchi, Unified fluid chromatography, J. Chromatogr. Sci., 27 (1989) 71–74.

D.24. Reed M. Izatt and Jerald S. Bradshaw Reed M. Izatt, Charles E. Maw Professor of Chemistry, Emeritus, was born in Logan, UT, on October 10, 1926. He received his B.Sc. degree at Utah State University and his Ph.D. degree in 1954 with W.V. Fernelius in coordination chemistry at Pennsylvania State University. After two years of postdoctoral work at Carnegie-Mellon University, he joined the Brigham Young University Chemistry Department in 1956. He was recipient of an NIH Career Development Award (1967–1972), the Utah Award of the American Chemical Society in 1971, the Huffmann Award of the Calorimetry Conference in 1983, the Willard Gardner Award of the Utah Academy of Sciences, Arts, and Letters in 1985, the State of Utah Governor’s Medal in Science in 1990, and jointly with Bradshaw, the 1996 American Chemical Society Award in Separations Science and Technology. He is the lead author for many reviews and books on thermodynamic quantities of ligand–metal ion interactions. Jerald S. Bradshaw, Reed M. Izatt Professor of Chemistry at Brigham Young University, was born in Cedar City, UT, on November 28, 1932. He received a B.A. degree at the University of Utah. After four years as an officer in the U.S. Navy, he earned a Ph.D. with Donald J. Cram at UCLA in 1963. He was a postdoctoral fellow with George S. Hammond at the California Institute of Technology. After three years as a research chemist at Chevron Research in Richmond, CA, he joined the faculty

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Reed M. Izatt (left) and Jerald S. Bradshaw (right)

at Brigham Young University in 1966. He was a US National Academy of Sciences Exchange Professor from 1972 to 1973 working with Miha Tisler at the University of Ljubljana, Slovenia. He also was a visiting Professor with J.F. Stoddart at the University of Sheffield, England, in 1978, and a National Science Foundation cooperative Research Fellow with L.F. Lindoy at James Cook University, Townsville, Australia in 1988. He received the 1989 Utah Award from the Salt Lake and Central Utah Sections of the American Chemical Society, the 1991 Utah Governor’s Medal in Science, and, jointly with Izatt, the 1996 American Chemical Society Award in Separations Science and Technology. See Chapter 5B, c, n, s

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24.I. SELECTIVE ION SEPARATIONS USING SOLID-PHASE EXTRACTION PROCEDURES Reed M. Izatt and Jerald S. Bradshaw Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA

The report in 1967 by Pedersen of his discovery of crown ethers and their unique metal ion complexing properties made a profound impression on scientists world-wide [1]. One of us (RMI) and his colleague, J.J. Christensen, were among the first scientists to recognize the significance of Pedersen’s work. The research of Izatt and Christensen was centered on the thermodynamic studies of proton-dissociation processes and metal ion complexation with inorganic anions and neutral organic ligands. During a visit to Pedersen’s laboratory in early 1968, samples of the new crown ethers were obtained. Applying titration calorimetric techniques, Izatt and Christensen determined formation constants and enthalpy and entropy changes for the interaction of various metal ions with the crown ethers. Their work showed an excellent correlation between the formation constants for the interactions of dicyclohexano-18-crown-6 with alkali and alkaline earth cations and the ionic radii of the metal ions [2]. For example, of the alkali metal ions, the ionic radius of KC most nearly matched the radius of the host and KC formed the most stable complex. In the early 1970s, Izatt and Bradshaw began their long-term collaboration on the synthesis and metal ion complexing properties of the crown ethers. Bradshaw and his co-workers prepared new crown ethers where sulfur atoms, pyridine and other nitrogen-containing aromatic subcyclic units and nitrogen atoms replaced one or more oxygen atoms of the crown ethers. Izatt and his co-workers studied the complexation properties of these new macrocyclic ligands with metal ions and neutral molecules. Their work on the thermodynamic properties of these macrocyclic ligands as well as work from other researchers is covered in a series of reviews [3]. In 1986, Izatt and Bradshaw and their coworkers attached the macrocyclic ligands to silica gel covalently by forming new Si–O–Si bonds [4]. The large surface area of silica gel allowed practical amounts of the macrocyclic ligands to be bound to it. The resulting supported material had excellent flow characteristics for solutions containing ions to be separated. Dissociation constants for the immobilized ligand–metal ion complexes were nearly identical to those of the unbound macrocycle analogues. These results showed that the large body of published data for the association of various metal species with the macrocyclic ligands in solution [3] could be used in the design of silica gel-macrocycle systems targeted for specific metal ion separations from aqueous solutions. Izatt and Bradshaw realized the importance of this discovery and patents were prepared covering the work. In 1988, they founded IBC Advanced Technologies, Inc. (IBC, American Fork, UT) for commercial development of silica gel-bound macrocyclic ligands (termed SuperLig7 ) for metal ion separation=recovery systems. IBC has developed a number of SuperLig7 materials for the removal, purification and=or recovery of selected metal ions of commercial value. The IBC SuperLig7 technology has some major advantages over other separations technologies. First, no new species or impurities are added to the system during the metal ion removal process. Second,

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anions are removed along with cations. Third, there is high removal efficiency because of the large binding strengths and high selectivites that the SuperLig7 materials have for the metal ions of interest. Fourth, kinetics are rapid, allowing the processing of large solution volumes of the metal ions of interest in a short period of time. Fifth, the high selectivity for the cation of interest makes possible its recovery in a single stage in a high purity and in a concentrated solution. Sixth, an appreciable reduction in volume is accomplished. Finally, the process is simple, has small space requirements, and uses common inexpensive reagents. A few examples of industrial scale separations will illustrate the use of this new separations process.

24.I.1. Separation of nuclear waste from storage solutions [5,6a] SuperLig7 materials have been perfected to remove radioactive CsC and Sr2C from radioactive waste solutions. The SuperLig7 materials are imbedded into Empore membranes developed by 3M Co. This arrangement allows for a rapid flow of waste solution through the immobilized SuperLig7 . Typical results for the removal of Sr2C using a disk mode are shown in Table 1. Note that Sr2C was removed at the ppm level in the presence of molar quantities of NaC and KC as are present in the nuclear waste solutions stored at the Hanford storage site in the state of Washington. CsC is removed in like manner. 3M and IBC are currently marketing a filter disk to remove analytical quantities of radioactive Sr2C and Ra2C for subsequent measurement [6b]. These filter disks reduce the number of steps involved in sample analysis for Sr2C from over fifty to six. Approximately 20 min are required for the analysis. A similar reduction in the number of steps required for analysis is found in the case of Ra2C .

24.I.2. Separation and purification of mine drainage solutions [6a] The Berkeley Pit in Butte, Montana is a large body of mine drainage water from underground and open pit mining operations over the past century. When mining operations ceased in 1970, the pit started to fill with water. The pit contains an estimated TABLE 1 RAPID REMOVAL OF LOW-LEVEL STRONTIUM FROM HANFORD MIMIC MATRIX a USING 0.2 g OF SUPERLIG7 IN AN EMPORE DISK [5,6] Sample description

Sr2C concentration (mg l1 or ppm)

Original solution 10 l of feed after column treatment b 0-2 ml of 0.03 mol l1 Na4 EDTA eluant b 2-4 ml of 0.03 mol l1 Na4 EDTA eluant b

100 <1 5 ð 105 200

a

Matrix with molar concentrations: NaOH, 3.4; NaNO3 , 0.258; NaF, 0.089; NaNO2 , 0.43; Na2 CO3 , 0.23; Na2 HPO4 Ð7H2 O, 0.025; Na2 SO4 , 0.15; Al(NO3 )3 Ð9 H2 O, 0.43; KNO3 , 0.12; RbNO3 , 0.00005. b Flow rate: 40 ml min1 . EDTA, ethylenediaminetetraacetic acid.

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TABLE 2 SEPARATION AND PURIFICATION OF BERKELEY PIT METAL CONSTITUENTS [5,6] Metal

Feed concentration (mg l1 )

Treated effluent concentration a (mg l1 )

Recovery (%)

Eluent purity (%)

Cu2C Fe3C Al3C Sn(II, IV) Mn(II) Cd(II) As(III, V)

180 994 270 554 194 2 0.3

<0.02 <0.1 <1 <0.05 <1 <0.02 <0.1

>99 >99 >99 b >99 >99 NA c NA c

96-98 99.5 97-98 99-99.5 75-80 b NA c NA c

a

All below detection by the analytical methods used. Some Al3C slipped past the Al3C system but was recovered with the Mn(II). Total Al3C recovery was >99%. c Not analyzed. b

1011 liters of mine waste. In pilot-plant scale tests, the SuperLig7 process was able to perform selective separation, recovery and purification of five selected metal ions as well as removal of environmentally harmful ions. The results are shown in Table 2. Concentrated solutions of the five desired metal ions can be sold to metal refineries to help pay for the clean up process. Actual clean up of the Berkeley Pit will be done at a future time. 24.I.3. Removal of unwanted Bi3+ impurity from a copper concentrate solution [5,6] Bismuth is found as a trace impurity in many copper ore deposits. It concentrates during the refinery process and, when certain levels are reached, the quality of the copper product is not acceptable. Removal of Bi3C from copper solutions by a conventional process is difficult. IBC’s SuperLig7 process to remove bismuth is effective in maintaining acceptable Bi3C levels on a continuous basis as shown in Table 3. It is evident that Bi3C is effectively removed even in very high Cu2C concentrations in this commercial process.

24.I.4. Separation and purification of precious metals [6,7] Precious metals must be near 100% pure for use in catalysis and other purposes. Attainment of this degree of purity is very challenging for the precious metal industry. Using selective SuperLig7 materials, each of the precious metals can be selectively and rapidly purified and recovered. The separation of Pd(II) from other precious metals, for example, is shown in Table 4. The data show that Pd(II) can be separated from a large number of competing metals including the chemically similar Pt(IV) and Rh(III). The SuperLig7 separation process has a bright future for industrial separations of

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TABLE 3 REMOVAL OF BISMUTH IMPURITY FROM A COPPER SOLUTION IN A COPPER REFINERY TANKHOUSE [5,6] Concentration (mg l1 or ppm)

Stream

Feed a Barren feed after passing 0.1 M H2 SO4 Eluate a b

Bi3C

Cu2C

0:28 ð 103 b b 5:65 ð 103

2:96 ð 104 2:91 ð 104 1:48 ð 104 b

Solutions are in a H2 SO4 matrix. Below analytical detection limits.

TABLE 4 SEPARATION OF Pd(II) USING SUPERLIG7 2 FROM A DILUTE FEED CONTAINING Pd(II), Pt(IV), Rh(III), AND BASE METALS a [6,7] Sample description

Volume (column volumes)

Concentration (mg l1 ) b Pd(II)

Original feed Feed and wash Elution c,d Elution c tail

26 30 2 2

610 <1 7,200 710

Pt(IV) 2840 2470 2 1

Rh(III) 345 295 <1 <1

a

Eluant: 2 M NH3 , 1 M HCl. ICP analyzed. [Cl ] in feed D 6 M. c The following elements were also analyzed to be <1 mg l1 : NaC , KC , Ca2C , Mg2C , Fe3C , Cu2C , Zn2C , Ni2C , Co2C , Mn(II), Ir(IV), Ru(III, IV), Cd(II), Pb2C , As(III, V), Se(VI), Bi3C , Sb(III, V), and Sn(II). d Pd(NH ) Cl salt produced by adding HCl to the concentrate yielded a 99.99% pure Pd(II) salt with Pt(IV) 3 2 2 and Si(IV) as the main impurities. b

unwanted trace metal ions or for the purification of valuable metals. The successful use of this technology to design and construct practical systems for use on an industrial scale demonstrates the power of supramolecular chemistry in meeting current industrial and environmental separations needs.

Acknowledgment This work was supported by Office of Naval Research, Department of Energy, National Science Foundation, and the State of Utah Centers of Excellence Program.

References 1.

C.J. Pedersen, Cyclic polyethers and their complexes with metal salts, J. Am. Chem. Soc., 89 (1967) 7017–7036.

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2a.

R.M. Izatt, J.H. Rytting, D.P. Nelson, B.L. Haymore and J.J. Christensen, Binding of alkali metal ions by cyclic polyethers: significance in ion transport processes, Science, 164 (1969) 443–444. 2b. R.M. Izatt, D.P. Nelson, J.H. Rytting, B.L. Haymore, and J.J. Christensen, A Calorimetric Study of the Interaction in Aqueous Solution of Several Uni- and Bivalent Metal Ions with the Cyclic Polyether Dicyclohexyl-18-crown-6 at 10, 25, and 40ºC, J. Am. Chem. Soc., 93 (1971) 1619–1623. 3. R.M. Izatt, K. Pawlak, J.S. Bradshaw and R.L. Bruening, Thermodynamic and kinetic data for macrocycle interaction with cations, anions and neutral molecules, Chem. Rev., 95 (1995) 2529–2586 and references cited therein. 4. J.S. Bradshaw, R.L. Bruening, K.E. Krakowiak, B.J. Tarbet, M.L. Bruening, R.M. Izatt and J.J. Christensen, Preparation of silica gel-bound crown ethers and their cation binding properties, Chem. Soc. Chem. Commun., (1988) 812–814. 5. R.M. Izatt, J.S. Bradshaw, R.L. Bruening, B.J. Tarbet and M.L. Bruening; in: D.N. Reinhoudt (Ed.), Comprehensive Supermolecular Chemistry, Vol. 10, Elsevier, Tarrytown, NY, 1993, pp. 1–16. 6a. R.M. Izatt, Review of selective ion separations at BYU using liquid membrane and solid phase extraction procedures, J. Incl. Phenom., 29 (1997) 197–220. 6b. G.L. Goken, R.L. Bruening, K.E. Krakowiak and R.M. Izatt, Metal ion separations using SuperLig or AnaLig materials encased in Empore cartridges and disks, in A.H. Bond, M.L. Dietz and R.D. Rogers (Eds.), Metal-Ion separation and preconcentration, ACS Symposium Series 716, American Chemical Society, Washington, DC, 1999, pp. 251–259. 7. J.S. Bradshaw and R.M. Izatt, Crown ethers: The search for selective ion ligating agents, Acc. Chem. Res., 30 (1997) 338–345.

D.25. Jaroslav Jana´k Jaroslav Jana´k was born on May 27, 1924 in Uzˇhorod, then Czechoslovakia. He obtained a M.Sc. degree (1947) and a Doctor of Science in chemistry (1964) at the Technical University, Prague. He reached venia docendi in analytical chemistry in Masaryk University, Brno (1965), and the professorship at the same institution in 1992. He started his professional career in the Chemical Works at Most, 1947, having responsibility in the central laboratories for gas and water analysis and development of control methods for new opened pilot and production plants. The idea of chromatographic analysis of gases developed there was further advanced later in The Institute for Petroleum Research, Brno (1951–1956). During this time he added the study of geochemistry to be prepared for application of this new analytical approach in prospection for and analysis of crude oil, gas and natural water sources. Due to the revolutionary nature of gas chromatographic methodology, he joined the Czechoslovak Academy of Sciences, which gave him the opportunity to establish the Laboratory for Gas Analysis in Brno (1956–1964). The goal was to develop research in gas chromatography on a broader basis and in other fields of applications. This laboratory was changed to the Institute of Instrumental Analytical Chemistry (supporting the Czechoslovak production of scientific instruments), and later (1974) in the Institute of Analytical Chemistry (developing a broader spectrum of analytical separation methods such as HPLC, ITP, SFC–SFE, CZE and FFF with emphasis to biochemical and environmental applications). This institute is now a part of the Academy of Sciences of the

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Czech Republic. He served as their head from 1964 to 1970, 1973 to 1980, and 1990 to 1993. During these periods he succeeded in assembling a group of scientists, who realized with him, and later alone a lot of research, works, postgraduate and seminar courses for many hundreds of students, postgraduates and specialists from home and foreign countries as well as keeping close contacts with pioneering laboratories in Europe and other continents (see e.g. J. Chromatography, 119 (1974) 1–612). His scientific productivity constitutes 300 original papers and six book contributions. He served on the Editorial Boards of the Journal of Chromatography (The Netherlands), and Journal of Chromatographic Science (USA) from their establishment and as editor of several symposia on chromatography published in the first journal. His recent activity is as a member of the advisory board of The Encyclopedia of Analytical Science, London, 1997. He is the recipient of the M.S. Tswett Award of the International Symposium on Advances in Chromatography (1975, Munich, Germany), the Anniversary Tswett Medal of the Soviet Academy of Sciences (1978, Tallin, Estonia) and several university medals (1966 Technical University, Gdaˇnsk, Poland; 1984 J. Heyrovsky´-Medal, Prague; 1984 Technical University, Pardubice; 1990 Komenius University, Bratislava; 1991 University of Ferrara, Italy; 1993 Masaryk University, Brno). In 1997, he was honored with a Doctor honoris causa degree at the Technical University, Brno. He is married to Jarmila, born Veˇcerkova´, and they have two sons (1951 and 1954) and one daughter (1966). See Chapter 5B, a, b, d, h, s

25.I. PERSONAL RECOLLECTIONS, ACHIEVEMENTS AND OPINIONS Jaroslav Jana´k Academy of Sciences of the Czech Republic, Institute of Analytical Chemistry, Veveˇri 97, CZ-611 42 Brno, Czech Republic

25.I.1. Introduction I graduated in the very bleak war year of 1942. Czech universities had been closed, laboratories ransacked and teachers displaced. We had to search them out at various technical schools, and it was through them that we gained access to university textbooks. After the reopening of the universities, we had to complete several experimental exercises in industrial laboratories. My diploma work on fuel chemistry was completed in the control laboratories of the Chemical Works in Most. There, the production range, the well-stocked library collection (with extensive German, English and American literature — unfortunately only up to 1941=1942), and good laboratory facilities were what enticed me to return to this factory after obtaining my M.Sc. at the Technical University in Prague. From my studies, I brought with me a keen sense of receptivity for microanalysis and an understanding of chromatographic techniques, such as classical liquid and paper chromatography [1].

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25.I.2. The origin of the gas chromatograph At the Chemical Works, I took on the responsibility for the gas and water laboratory. The methodology of gas analysis was based on volume measurement analysis (Orsat) and on simplified low temperature fractionation (Stock). The shift from the war-time production of gasoline by means of hydrogeneration of brown coal to the refining of waste products (the extraction of bivalent phenols, and bases from the waste water from brown coal carbonization, the production of synthetic methanol and formaldehyde, etc.) brought analytical requirements which the classical methods were able to fulfill only with great difficulty and imprecision. My experience with the separation of monovalent phenols by liquid chromatography on alumina, and the efficient separation of bivalent phenols on paper led me to the idea of also applying the chromatographic principle on a group of gaseous substances, particularly gaseous hydrocarbons, where the shortcomings of gas analysis seemed to be unresolvable. The first experiments (1949), with carbon dioxide as the carrier gas, and the use of a nitrometer filled with potassium hydroxide absorbing CO2 as the detector, and columns containing active charcoal and=or silica gel, all proved to be feasible. At that time (1951), the Institute for Petroleum Research, which had just been established in Brno, offered me the chance to set up analytical laboratories for the prospecting and analysis of crude oil, natural gases and mineral water. It was there that the definitive gas chromatograph was built, gaining excellent results in practical analysis [2]. This method provided some interesting advantages: for each gas, retention volumes were characteristic and they increased in homologous series logarithmically; measuring provided absolute values (real volumes) with what at that time was great precision (0.05%); analysis was quick (in minutes); individual components of gaseous mixtures were visible in the detector and could be prepared; and it was possible for equipment to be set up simply and inexpensively. From a construction point of view, this equipment contained the basic elements of present-day instrumentation (although certainly more sophisticated today): a sampling loop and temperature controlled column heating, which worked in both the gas–solid and gas–liquid mode, in parallel or in series. I succeeded in the separation and analysis of hydrocarbon gases, several permanent but all the rare gases, and their mixtures. In quick sequence, several analytical methods were in the process of being developed: for gas prospecting from ground and mineral waters and soils, for the analysis of gases emitted by living organisms, for the analysis of several gaseous anesthetics, and for freons, etc. Progress was being made in trace analysis, and in automation, etc. This methodological approach made it possible to redefine the origin of crude oil and coal gases, as well as to clarify ideas about the formation of mineral water composition in sedimentary rock. The first publications aroused a lot of interest and they were experimentally verified by world-renowned petroleum companies (D.H. Desty and S.F. Birch of British Petroleum, UK; van der Grats of Shell Laboratories, The Netherlands). At that time, this method surpassed the mass spectrometric analysis of hydrocarbons in both data precision and in cost-effectiveness. This resulted in the method being accepted by the Institute of Petroleum as a tentative method (IP-160-59), and in several producers, putting equipment which was based on this principle (Fig. 1) out on the market (Baird

Fig. 1. (a) One of the first home-made gas chromatograph (1952). (b) British version based on BP design (1956).

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and Tatlock, Griffin and George, UK; Hereus, Germany; Labortechnik, the former GDR; Podbielniak, USA; and Kavalier, Czechoslovakia). During the symposium ‘Gas Chromatography 1960’ held in Edinburgh, Denis Desty stated that the gas chromatograph being presented was really the first example of a gas chromatograph to be industrially implemented in the United Kingdom. Later (1984), he had ordered two historical models to be built based on the original BP designs. One of them is housed in the Technical Museum in London and the second, with a BP dedication plaque, is at the Institute of Analytical Chemistry of the Academy of Sciences of the Czech Republic in Brno. Several significant chemical companies (Badische Anilin und Soda Fabrik (B.A.S.F.), Farbwerke Hoechst, Lurgi, Germany) made use of equipment which was based on this principle even long after the introduction of the modern gas chromatographs. This was because of its reliability, precision and low costs in the determination of hydrogen in gaseous mixtures. Even today, this equipment is still being used in basic curricula at many universities for its high didactic value. I have repeatedly been asked to write a chapter for textbooks about gas analysis by chromatography [3]. Taking into consideration the fact that these events took place in the early fifties, we can still conclude that the above-mentioned mode contributed significantly to the influencing of both the expert and industrial spheres in the conviction that gas chromatography is a practical and useful method with high inner potential. In the branch of gas analysis it represents a real transformation of classical gas analysis. In the first half of the fifties, the significance of gas chromatography expanded greatly beyond the petroleum research and industry (mainly thanks to the concept of partition chromatography by A.J.P. Martin). Therefore, in 1956, the Czechoslovak Academy of Sciences accepted my proposal to establish a laboratory for the further development of this and later of a whole series of analytical separation methods. For this purpose, a team of scientists was assembled, a majority of them were benefiting from the experience with gas chromatography (M. Rusek, J. Nova´k, M. Dressler, K. Tesaˇr´ık, S. Wiˇcar and Vl. Rezl, GC; M. Krejˇc´ı and K. Sˇlais, HPLC; P. Boˇcek, M. Deml, P. Gebauer and L. Kˇriva´nkova´, ITP and later CZE; J. Janˇca and J. Chmelı´k, FFF; M. Roth, SFC; and J. Vejrosta, SFE). The first result of this new situation was the experiment on the thermal degradation of substances under gas chromatographic conditions [4]. The essentials of this approach were as follows: using a microscale (µg), a flow of overloading the amount of molecules of the carrier gas (ml=s) (e.g., hydrogen or a dopped gas), that thermal destruction of any compound happens under conditions near infinite dilution. This fact was able to prove that the primary products of pyrolysis are immediately transferred from the bulk of the substance and the reaction of radicals can be stopped or diminished by the chemical nature of the carrier gas (hydrogenation, derivatization). The pyrograms obtained were fairly consistent, providing characteristic patterns for substances under observation (Fig. 2a). The technique was widely recognized, further improved and now used in the analysis of synthetic and natural polymers, plastics, the analysis of complex natural products, taxonomic studies, etc. Another contribution was a new variant of multidimensional chromatography by means of the application of gas chromatography as a sampling technique to thin-layer chromatography. The objective was to realize the most effective separation in terms

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Fig. 2. (a) Pyrogram of amino acids (see [4]). (b) Two dimensional chromatogram of a mixture of C14 –C22 acids (see J. Chromatogr., 21 (1966) 207).

of volatility (separation according to the vapor pressure of the homologs) and of the polarity (separation according to the functionality of molecules) [5]. If the thin-layer is moved logarithmically under the outlet of a gas chromatograph, the resulting flat

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chromatogram is very easy to read, as it is demonstrated by the GC–TLC chromatogram of a fatty acid mixture (Fig. 2b). As is often the case with a new technique (e.g., high-performance liquid chromatography in the early seventies), many applications began to work ab inicio. This was why we published a book which comprehensively but critically selects the most useful facts and references from the voluminous literature about classical liquid chromatography [6]. Increasing air, water and soil pollution problems directed my interest to chromatographic trace analysis, and therefore to preconcentration techniques. A principal difficulty of air pollution studies is the omnipresence of water vapor in much greater quantities than those of trace pollutants. The use of frontal-chromatographic techniques [7] on non-polar stationary phases effectively reduced the effect of water vapor, and the degree of preconcentration of a trace is as much greater as its vapor pressure is lower. Traces in the chromatogram tend to be in nearly equimolar peaks. This reference reminds me of a visit to Houston, TX where I was a guest of A. Zlatkis, who got permission for me to visit the NASA Space Center and particularly the Apollo laboratory. I was informed that this methodology had been successfully applied to indoor studies of the astronaut’s cabin. This methodology was further improved by the use of a closed circuit and=or by the use of the differences in ad- and absorption rates. These methodologies are now being applied. One of my later significant projects was the development of isotachophoresis with emphasis on qualitative and quantitative analysis. In the seventies, I sent a postdoctoral student of mine, Petr Boˇcek, to Eindhoven (The Netherlands) where A.J.P. Martin was supervising doctoral student Franc Everaets in the evaluation of displacement electrophoresis, as it was called at that time. Boˇcek [8] developed this approach considerably and expanded his activities to include capillary zone electrophoresis.

25.I.3. Opinions One thing that can be said in conclusion is that a good theory verified through experiment can change the level of knowledge, as well as broaden the range of possibilities of society. Chromatography is really one such phenomenon of the 20th century, having taken on an essential role in the methodology of several natural sciences and in the technologies of many industrial branches. The works by botanist Mikhail S. Tswett (1903, dynamic sorption on an adsorbent), and biochemist Archer J.P. Martin (1952, dynamic sorption in a liquid) are the milestones paving the way to further developments. Mathematician Jan J. van Deemter (rate theory), physicist Marcel J.F. Golay (information theory), and physical chemist Calvin Giddings (kinetic theory) provided the main input in the understanding of the parameters of chromatographic separation. According to a statement written by Erika Cremer, physical chemist, in our guest-book, a great many others also contributed, to greater or lesser extent, by adding a brick to the building of chromatography. Furthermore, as Aleksandr A. Zhukhowitskii also wrote in this book, “this scientific tool which separates substances has the ability to unite people”. The chromatographic process is a common process occurring in nature where the

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not fully miscible phase passes along any other phase(s) making diffusion possible. Therefore it is possible to trace such processes or effects in the migration of crude oil, during the flow of ground and mineral waters through rock and=or sediment, in the blood circulation system, on cell membranes, on the surface of lung web and other organs or organisms, when in contact with the environment, during the sedimentation in rivers, lakes or seas, and even during the formation of rocks, if there the time has a geological dimension. From this we can expect that the advances made in the last century in this branch of science and technologies will continue in presenting new potential for discoveries into the present century.

References 1. 2.

3.

4. 5. 6.

7.

8.

J. Jana´k, In: L.S. Ettre and A. Zlatkis (Eds.), 75 Years of chromatography — a historical dialogue. J. Chromatogr. Library, Vol. 17, Elsevier, Amsterdam, The Netherlands, 1979, pp. 175–185. J. Jana´k, Apparatus for quantitative and qualitative analysis of hydrocarbon and other gases — Gas chromatograph, CZ Patent No. 83991, 1952. (Chromatographic semimicroanalysis of gases). Chem. Listy, 47 (1953) 464; English summary — Collection Czechoslovak Chem. Commun., 18 (1993) 798. J. Jana´k, Chromatography of non-hydrocarbon gases; in: E. Heftmann (Ed.), Chromatography. Reinhold, New York 1961, pp. 642–662; 2nd edn. 1967, pp. 761–692; 3rd edn. 1975, pp. 882–914; 4th edn., Elsevier, Amsterdam, 1983, pp. 491–517. J. Jana´k, Nature, 185 (1960) 684–686; in R.P.W. Scott (Ed.), Gas Chromatography 1960, Butterworth, London, 1960, pp. 238–244. J. Jana´k, Nature, 165 (1960) 611, J. Gas Chromatogr., 1 (10) (1963) 20–23; J. Chromatogr., 15 (1964) 15–26. Z. Deyl, K. Macek and J. Jana´k (Eds.), Liquid Column Chromatography. A Survey of Modern Techniques and Applications. Elsevier, Amsterdam, 1975, pp. 1175. Russian translation in 3 volumes. Moscow, Mir 1978. J. Nova´k, V. Vasˇa´k and J. Jana´k, Anal. Chem., 37 (1965) 660–666, J. Nova´k, J. Jana´k and J. Golia´sˇ; in: H.S. Hertz and S.N. Chesler (Eds.), Trace organic analysis: A new frontier in analytical chemistry, U.S. Bureau of Standards, Spec. Publ. No. 519, pp. 739–746, Washington, DC, 1979; and J. Jana´k: Pure Appl. Chem., 61 (1989) 2011–2014. P. Boˇcek, M. Deml and J. Jana´k, J. Chromatogr., 91 (1974) 829–831; P. Boˇcek, M. Demin and J. Jana´k, J. Chromatogr., 156 (1978) 323–326, P. Gebauer, P. Boˇcek, M. Deml and J. Jana´k, J. Chromatogr., 199 (1980) 81–94.

D.26. Egil Jellum Egil Jellum, was born on March 30, 1936 in Drammen, Norway. He received a B.Sc. from Heriott-Watt University, Edinburgh in 1960, and a Ph.D. from the University of Oslo in 1966. He joined the Institute of Clinical Biochemistry, Rikshospitalet, University of Oslo in 1963, and has been there ever since, except for a six month sabbatical at Stanford Medical Center, California in 1969. He was first a Research Fellow of the Norwegian Cancer Society before becoming Associate Professor in 1974. He has been an Adjunct Professor of the University of

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Waterloo, Canada. Jellum is currently Professor at the Institute of Clinical Biochemistry, Rikshospitalet, and also employed by the Norwegian State Hospital as a consultant. A major area of his research has been the development of advanced chromatographic, mass-spectrometric and electrophoretic methods to study body fluids and cells, and the application of these multi-component analytical techniques for clinical diagnoses and studies. The Institute of Clinical Biochemistry in Oslo has become a center for research on metabolic disorders during the last 30 years, and several new diseases have been discovered here by means of chromatographic and mass spectrometric methods. Jellum and his associates use separation methods such as: GC=MS, HPLC, high resolution two-dimensional protein electrophoresis and capillary electrophoresis, also in other biomedical studies, particularly related to cancer, asthma and allergy, microbiology and environmental pollution. He is Chairman of the JANUS-project, which is aimed at the early detection of cancer, using a large serum bank containing prediagnostic blood specimens collected from 293,000 individuals over 25 years. He has authored and co-authored 280 articles and 135 abstracts (invited) and is the recipient of several awards including the M.S. Tswett Medal (USA, 1983), the A.J.P. Martin Award (UK, 1990), the Medinnova Prize (Norway, 1991), the Dimick Award (USA, 1993), and the Latin American Capillary Electrophoresis Award for his work on CE in biomedicine (Buenos Aires, 1997). Jellum has presented a number of invited lectures at meetings and conferences in many parts of the world. He is or has been a member of the Editorial Board of several Journals, including J. Chromatography, Biomedical Applications; J. High Resolution Chromatography and Chromatographic Communications; Spectroscopy; Biochemical Biophysical Methods; J. Pharmaceutical Biomedical Analysis; BioChromatography; Trends in Analytical Chemistry; BioTechniques; Comprehensive Analytical Chemistry (book series), Fresenius’ Journal of Analytical Chemistry, J. Capillary Electrophoresis; and Chromatographia (Honorary Editorial Board Member from 1999). See Chapter 5B, a, b, c, d, g, h, p, r 26.I. CHROMATOGRAPHY, MASS SPECTROMETRY AND ELECTROPHORESIS FOR DIAGNOSIS OF HUMAN DISEASE, PARTICULARLY METABOLIC DISORDERS Egil Jellum Institute of Clinical Biochemistry, Rikshospitalet, 0027 Oslo, Norway

26.I.1. Background My story dates back to 1959=60 when the author was a young, senior student at Heriott-Watt University, Edinburgh. During a special course there, he was engaged in constructing a simple gas chromatograph. After completion of his B.Sc. in the summer of 1960, the author moved back to Norway where he was temporarily employed at the

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Clinical Chemistry Laboratory, Lier Psychiatric Hospital, situated in the neighborhood of Oslo. The Head of this laboratory, O. Skaug listened to the enthusiastic description of gas chromatography, and together he and Jellum built a gas chromatograph for use in the hospital laboratory. The instrument was completed during the autumn of 1960, and everything was home made, even the D.C. amplifier. The flame ionization detector was made from ‘Wassermann’ needles (used for blood sampling in those days) and fitted on top of a stainless steel U-column packed with Kieselguhr coated with Apiezon-L vacuum-grease. The heating system was boiling ethylene glycol (196ºC) or sometimes boiling quinoline (234ºC), which maintained an isothermal temperature suitable for GC analysis of fatty acid methyl esters. Everyone who knows the smell of quinoline can imagine the odor present in our GC laboratory in those days! One of the very first applications of this gas chromatograph was the analysis of fatty acids in serum and spinal fluid from various psychiatric patients in the hospital. The intention was to look for differences in the fatty acid pattern of the normals and the patients. Although we were unable to find any changes, which could be linked to schizophrenia and other psychiatric disorders, the homemade GC proved to work reasonably well. Furthermore, it was the first time we actually carried out ‘metabolic profiling’, although this terminology was not invented until a few years later. Norway has compulsory military service, and the author was drafted for 18 months. During the last 15 months of this period, he worked in the Institute of Aviation Medicine. During this period a new and improved gas chromatograph was constructed, and used for various purposes, including the attempts to analyze dimethyl hydrazine (a rocket fuel) in blood, and attempts to determine bilirubin and its degradation products in serum. A method for the determination of free fatty acids in serum was established. After serving in the military research laboratory, Jellum joined L. Eldjarn’s research group at the newly established Institute of Clinical Biochemistry, University Hospital (Rikshospitalet), Oslo. It is at this place the research leading to the various chromatography awards has been carried out. The first task was to develop a method to isolate and separate thiol-containing compounds. The idea was to attach an ‘arm’ on an immobilized support (Sephadex) which in a specific way could bind SH-containing proteins, and separate them according to the number of SH-groups present on the molecule. This resulted in a paper published in Acta Chem. Scand. in 1963 [1], and is possibly the first example of affinity chromatography, written many years before this term was invented. This new material was used in several papers in the early sixties and included in Jellum’s Ph.D. thesis completed in 1965=66.

26.I.2. GC–MS, HPLC, 2D-PAGE and CE for diagnosis and studies of human disease, particularly metabolic disorders 26.I.2.1. Gas chromatography–mass spectrometry (GC–MS) Having worked with gas chromatography since 1960, and having experienced its weakness (identification of peaks by retention time only), a major breakthrough came

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in 1965 when GC–MS (gas chromatography–mass spectrometry) became commercially available (LKB, Sweden). At this time, our Institute experienced a growing interest in the field of inborn errors of metabolism (metabolic disorders), triggered by studies on patients with Refsum’s disease (phytanic acid storage disease). The studies on this disease were primarily conducted by Eldjarn, Stokke and Try, and resulted in discovery of the enzyme defect responsible for the disease. In 1967, a patient with severe metabolic acidosis was studied in our Institute. Large amounts of an organic acid appeared on the gas chromatogram, but at that time we had not yet purchased our own mass spectrometer and identification of the unknown peak was difficult. The potential of GC–MS was, however, fully realized when the unknown compound was easily identified using a mass spectrometer in Dr. Ryhage’s laboratory in Stockholm. The peak was methylmalonic acid, and the new metabolic disease was called methylmalonic aciduria, as described by Stokke et al. in 1967. In 1968=69, Jellum spent six months as a visiting scientist at Stanford Medical Centre, California, working in B. Halpern’s group. Here, the prototype of Finnigan’s mass spectrometer was used, and GC–MS techniques for analysing biologically important thiols and disulphides, and for analysing phenylalanine in serum were established. After returning to Oslo in 1969, a new GC–MS instrument was installed at the Institute of Clinical Biochemistry. From then on this technology was systematically used to analyze urine and blood samples from patients suspect of suffering from some metabolic disease. This work was done in close collaboration with O. Stokke, and the head of the institute at that time, L. Eldjarn. Soon several new diseases were discovered, the first one being pyroglutamic aciduria in 1970 [2], and the second was 3– methylcrotonyl–CoA carboxylase deficiency, also described the same year. Furthermore, GC–MS proved to be suitable to diagnose a number of previously described diseases. In 1970, we were able to diagnose in an efficient manner up to 40 different metabolic disorders. Consequently, the clinicians consulted our laboratory at increasing rate when facing patients with unclear diagnosis. Samples from many hospitals in Norway and occasionally also from abroad were shipped to us for analysis. A systematic approach was taken, subjecting urine samples to various extraction and derivatization procedures and ending up with eight different gas chromatograms per patient sample. Identification of unusual peaks was done by GC–MS, producing mass spectra on light-sensitive paper. Manual counting of the masses had to be done, and the mass spectral information was matched against a library of known spectra in an off-line fashion. This systematic screening for metabolic disorders using GC–MS was first described in 1971 [3], and was presented at several international meetings. When this work began, many of the GC–peaks on the chromatograms were unknown. In the following years, however, several new metabolites were identified, and artifacts and pitfalls were recognized. For instance, benzoic acid in the urine is due to bacterial contamination; furoylglycine and furandicarboxylic acid are normal constituents of urine; adipic and suberic acid are secondary metabolites produced during ketosis, etc. New information on recognized disorders were found, e.g., the occurrence of 3-hydroxy-n-valeric acid in patients with propionic and methylmalonic acidemia; new findings in a case with 2-methylacetoacetic aciduria; novel data on formiminoglutamic aciduria, and the identification of the glycine conjugate of pyrrolecarboxylic acid

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in the disease hyperprolinemia. Sometimes results were obtained in collaboration with researchers abroad, as in the latter two examples. In the mid-seventies, packed GC-columns were replaced by glass capillary columns resulting in increased resolution and improved diagnostic ability. A system was developed for computer-assisted search for anomalous compounds (CASAC), this made it possible to identify abnormal compounds in a new way. Today, one has automatic identification of all peaks appearing on a gas chromatogram. The state of the art in 1977 of using GC–MS for diagnostic applications was reviewed in an article entitled “Profiling of human body fluids in healthy and diseased states using GC–MS, with special reference to organic acids” [4]. 26.I.2.2. 2D-PAGE and HPLC In 1975, a new method for separating proteins was published — high resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). This technique became commercially available in 1979 after considerable improvement by Anderson and Anderson of the original O’Farrel method. We began to use this ISO-DALT system the same year, with the intention to use GC–MS for analysis of low molecular weight compounds, and 2D-PAGE for proteins. In a series of papers these complementary multicomponent analytical techniques, sometimes combined with pattern recognition data analysis, were used to study a variety of metabolic and other diseases, including rheumatoid arthritis, skin disorders, colon cancer, brain tumors and leprosy. In the mid 1970s, HPLC was also included in the analytical system for diagnosis of metabolic disease, and became an important tool for handling non-volatile diagnostic metabolites, such as orotic acid and adenylosuccinate. In the 1980s, the combined analytical system, GC–MS, HPLC and 2D-PAGE, was extensively used to diagnose diseases and to monitor the effect of therapy. The number of metabolic diseases possible to diagnose with our system had increased to about one half of the 300 metabolic diseases known then; however, failing to detect diseases not leading to accumulation of metabolites in blood and urine, i.e., the storage disorders. We were involved in liver transplantation studies on patients with the metabolic disorders, tyrosinemia and hyperoxaluria, and also discovered in 1986 a new disease, N-acetylaspartic aciduria. Some of this work is overviewed in the article “Profiling cells and body fluids — chromatography and 2D-electrophoresis as complementary techniques” [5]. 26.I.2.3. Capillary electrophoresis (CE) In the mid-1980s, researchers became increasingly aware of CE. Although not being a new technique (first described by Hjerten in 1967), it was, however, not until the 1990s that the technique really took off and is today recognized as a most powerful separation method. We purchased a CE instrument in 1989, and used it for the rapid diagnoses of the diseases homocystinuria, cystinuria, glutathione synthetase deficiency and adenylosuccinase deficiency, as well as to analyze submilligram amounts of tissue biopsies from patients with cardiomyopathy. The results were presented at the Pittcon 1990 meeting and published as a full paper in 1991 [6]. This is possibly the first paper

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Fig. 1. The diagnosis of a disorder called glutaric aciduria due to the defective enzyme glutaryl–CoA dehydrogenase. Organic acid profiles of urine from a patient with glutaric aciduria, type 1 (top) and from a control person (bottom) are shown. The urine samples were acidified, the organic acids were extracted with diethyl ether, and methylated with diazomethane before separation. An HP 5890=5970 GC–MS instrument fitted with a 30-m-long fused silica capillary column coated with SP 1000 was used. Note the large amounts of glutarate present in the patient sample due to the defective enzyme glutaryl–CoA dehydrogenase.

on diagnostic applications of CE. During the last years it has become increasingly clear that CE has an important role in the clinical laboratory, being rapid, simple, sensitive and requires minute sample volumes. Analysis of urine is particularly simple as the sample can be injected onto the capillary without any sample pretreatment. Identification of ‘diagnostic metabolites’ has until recently been done by comparison of migration times and diode array spectra with the same set of data from authentic compounds. Several diseases can readily be diagnosed in this manner [7]. In 1998, CE combined with a single quadrupole mass spectrometer proved useful, and even more powerful was the combination CE–MS=MS allowing rapid and absolute identification of many diagnostic metabolites (presented in Palm Springs at HPCE 99, full paper in press). 26.I.2.4. Chromatography and electrophoresis in cancer studies using a large serum bank In 1973, the Janus Serum Bank was founded, sponsored by the Norwegian Cancer Society. From this year and up to now, blood samples have regularly been collected from 300,000 initially healthy persons. Their sera are stored at 25ºC. As the years have passed, many (1998: 20,000) of the persons donating blood to the bank, have developed cancer. Sera collected from 1 to 25 years prior to cancer diagnosis are thus available. The rationale is to search in these premorbid sera for chemical, biochemical, immunological or other changes that might indicate cancer development at early stages, or be indicative of increased risk of cancer. A number of studies, often in collaboration with international

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research groups, have been carried out using this serum bank. Immunological methods are most frequently used in search for virus and tumor associated proteins and antigens, but chromatography and electrophoresis have also played an important role. GC–MS and 2D-PAGE was used to analyze sequential serum samples from the same persons in order to evaluate possible deterioration of the samples. Fortunately, most components proved to have an acceptable stability at 25ºC. In a study on the role of polyunsaturated fatty acids and risk of breast cancer, fatty acid profiles obtained by GC analysis were analyzed statistically and compared to matched controls. An inverse relation between linoleic acid (C18:2, n-6) and breast cancer was found, but this association was restricted to women who were 55 years and younger. The same analytical technique (GC) was used to study the role of long chain serum fatty acids and risk of thyroid cancer, and prostate cancer. Capillary electrophoresis has recently been used to analyze proteins in sequential sera from persons who developed myelomatosis. We could detect the pathological protein up to 17 years prior to clinical manifestation of the cancer [7]. In a collaboration with NIOSH and CDC in USA, serum organochlorine levels have been determined in Janus sera from women who developed breast cancer. HPLC and GC–MS were used to measure more than 30 different organochlorines, including dioxins. Preliminary data from this investigation were presented in the DOD breast cancer conference in Washington, November 1998. 26.I.2.5. At present, and for the future The Institute of Clinical Biochemistry=Department of Clinical Chemistry, Rikshospitalet, Oslo has become a national center in Norway for diagnosis and studies of metabolic diseases. The analytical system comprises prescreening tests (in urine) for protein, blood, creatinine, reducing substances, cystine, homocystine, acetoacetate and other ketones, guanidoacetate, sulphite, nitrite, mucopolysaccharides and uric acid. Paper chromatography is used to separate carbohydrates (in cases with a positive test for reducing substances) and for analysis of amino acids. Samples giving pathological amino acid patterns are re-analyzed using a quantitative amino acid analyzer. TLC is used for further analysis of samples giving a positive test for mucopolysaccharides. HPLC with a diode array detector is used to determine the profile of purines and pyrimidines; over 30 compounds are separated. GC–MS with automatic library search is used to obtain the profile of urinary organic acids; over 150 can be separated and identified. A tandem MS instrument will be placed into use the summer of 1999. CE and CE–MS=MS are currently being used as research tools, but will be implemented in the routine system at a later stage. Quantitative GC–MS methods using stable-isotope labeled internal standards are used to measure very long chain fatty acids and methylmalonic acid in serum. Quantitative methods for carnitine, lactate, pyruvate and ammonia are routinely carried out. When a patient has been given a tentative diagnosis based on the metabolite analysis described above, the residual activity of the deficient enzyme is measured. We have established about 20 different enzyme assays in our laboratory, but often cultured fibroblast cells are sent to laboratories abroad for determination of special enzymes.

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The diagnosis of a metabolic disease can also be confirmed at the DNA level. PCR amplification followed by digestion with restriction enzymes are routinely used to detect some common mutations in mitochondrial DNA, as well as many different mutations in the genomic DNA. Following the diagnosis of a metabolic disorder, the patient is often treated with diet (e.g. protein restriction), cofactor supplementation, drugs, etc. The chromatography and mass spectrometry methods are used to follow the effect of therapy. Thus, quantitative methods are developed and used to monitor the pathological metabolites, e.g., the excretion of glutaric acid in glutaric aciduria type I. In 1998, about 1700 complete patient evaluations were carried out according to the scheme outlined above. Though such complete investigations are difficult, time-consuming, and have been accomplished thanks to our highly skilled personnel, they are beneficial for the patient and affected family.

References 1. 2. 3.

4.

5. 6. 7.

L. Eldjarn and E. Jellum, Organomercurial-polysaccharide, a chromatographic material for the separation and isolation of SH-proteins, Acta Chem. Scand., 17 (1963) 2610–2621. E. Jellum, T. Kluge, H.C. Borresen, O. Stokke and L. Eldjarn, Pyroglutamic aciduria — a new inborn error of metabolism, Scand. J. Clin. Lab. Invest., 26 (1970) 327–335. E. Jellum, O. Stokke and L. Eldjarn, Screening for metabolic disorders using gas–liquid chromatography, mass spectrometry and computer technique, Scand. J. Clin. Lab. Invest., 27 (1971) 273– 285. E. Jellum, Profiling of human body fluids in healthy and diseased states using gas chromatography and mass spectrometry, with special reference to organic acids (review), J. Chromatogr., 143 (1977) 426–472. E. Jellum and A.K. Thorsrud, Profiling cells and body fluids — Chromatography and 2D-electrophoresis as complementary techniques, J. Chromatogr., 488 (1989) 105–127. E. Jellum, A.K. Thorsrud and E. Time, Capillary electrophoresis for diagnosis and studies of human disease, particularly metabolic disorders, J. Chromatogr., 559 (1991) 455–465. E. Jellum, H. Dollekamp, A., Brunsvig and R. Gislefoss, Diagnostic applications of chromatography and capillary electrophoresis, J. Chromatogr. B, 689 (1997) 155–164.

D.27. Walter G. Jennings Walter G. Jennings was born on March 2, 1922 in Sioux City, IA. His education was at Hoover High School, Glendale, CA 1936–1940; California State University, San Diego 1946– 1948; University of California, Davis, 1948–50 BS; 1950–52 M.S.; 1952–54 Ph.D. The positions he has held: Union Pacific Railroad, Survey Crew 1940–1942; US Army, 1942–1946, Pvt. to 1st Sergeant: University of California, Davis, Instructor to Professor of Food Science and Chemist in the Experiment Station, 1953–1989; and J&W Scientific, Inc., Folsom, CA; Founder to Consultant,

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Walter Jennings, Founder of J&W Scientific, Inc., holding a fused silica capillary column.

1974–date. He founded the following companies: J&W Scientific, Inc., Folsom, CA, 1974; Airtoxics, Ltd. (an Environmental Analytical Company), Folsom, CA, co-founder, 1989. Professor Jennings has some 300 publications and abstracts to his credit; his books include: (1) “Gas Chromatography with Glass Capillary Columns”, Academic Press, 1978; (2) “Qualitative Analysis of Flavor and Fragrance Chemicals by Glass Capillary Gas Chromatography”, (with T. Shibamoto), Academic Press 1980; (3) “Gas Chromatography with Glass Capillary Columns”, second edition, Academic Press, 1980 (also in Russian); (4) “Recent Advances in Capillary Gas Chromatography”, in three volumes (with Co-Eds. Bertsch and R.E. Kaiser), Huethig, 1981; (5) “Comparisons of Fused Silica and Other Glass Columns in Gas Chromatography”, Huethig, 1981; (6) “Applications of Glass Capillary Gas Chromatography” (Ed.), Marcel Decker, 1981; (7) “Sample Preparation for Gas Chromatographic Analysis” (with A. Rapp), Huethig, 1983; (8) “Analytical Gas Chromatography”, Academic Press, 1987 (translated, sans authorization, into Chinese); (9) “Capillary Chromatography. The Applications” (Proceedings ACS Symposium), (with Co-Ed. J. Nikelly), Huethig, 1991; and (10) “Analytical Gas Chromatography”, second edition, (with Co-authors E. Mittlefehldt and P. Stremple), Academic Press, 1997. Dr. Jennings has been singularly honored with a number of Prizes and Awards as follows: Humboldt Prize, ‘Senior American Scientist’, of the Alexander von Humboldt Foundation, 1973; Founders Award in Gas Chromatography, Beckman Foundation; M.J.E. Golay Award, International Symposium on Capillary Chromatography, 1996; Keene P. Dimick Award, Analytical Chemists of Pittsburgh, 1997; A.J.P. Martin Gold

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Dr. Jennings receiving the Keene P. Dimick Award in 1997 from Michael Dimick, son of Keene Dimick.

Medal, Chromatographic Society, 1997. Others include the Award of Merit of the Chicago Chromatography Discussion Group, the National Chromatography Award of the Northeast Regional Chromatography Discussion Group, the L.S. Palmer Award of the Minnesota Chromatography Forum and others from the French Association of Analytical Chemists, the University of Bologna, the Taiwanese Food Chemists Society, and the Society of Flavor Chemists. He has had sabbaticals in Austria, Germany, Switzerland, and served as an ‘Adviser in Gas Chromatography’ to the governments of Poland and Bulgaria for the ‘International Atomic Energy Agency’. He is a past Chairman of the American Chemical Society’s Subdivisions of Flavor Chemistry, and of Chromatography and Separation Science. Professor Jennings regards education as his major contribution. In the early 1970s, he began teaching courses in capillary chromatography for Hewlett Packard. This nation-wide series through the US and Canada endured into the 1980s. Coupled with results he published and reported at scientific meetings, this exposure resulted in innumerable in-house courses for a large number of practitioners from chromatography discussion groups to major corporations. Until 1996, Professor Jennings continued to teach 20–40 one-day courses each year at points all over the world. At a conservative estimate, he has taught gas chromatography to over 30,000 attendees. To this day, he continues these activities albeit at a slower pace. See Chapter 5B, a, d, e

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27.I. CONVERSION OF THE INDUSTRIAL ANALYST TO CAPILLARY GC Walter G. Jennings J&W Scientific Inc., 91 Blue Ravine Rd, Folsom, CA 95630, USA

Notwithstanding the contributions detailed below, Professor Jennings would argue that his most important contribution lies in the field of education. In spite of the fact that Desty’s elegantly simple machine for drawing long lengths of glass capillary tubing spawned a number of ‘centers of excellence’ in glass capillary columns, industry, where gas chromatography was most widely applicable, long continued to favor packed column separations. Utilization of glass capillary columns occurred largely in government and academic laboratories where the cost of labor rarely entered into the equation. While industrial scientists recognized their advantages, they were concerned with their fragility, and the need for sensitive and agile fingers. Industry simply could not afford the perils imposed by unpredictable down times. Exceptions to these generalizations occurred in the petro-chemical and flavor industries, and in a very few major companies that permitted their research departments to explore this area. Nevertheless, this period was characterized by great strides in open tubular gas chromatography, primarily through the efforts of the centers of excellence referred to above. With few exceptions, these were largely confined to the UK and western Europe, with a few scattered throughout the USA. The impetus for change came with the introduction of the much more robust fused silica column by Dandeneau and Zerenner. While industry expressed great interest in this development, the reactions of scientists with established reputations, in the production and use of glass capillary columns was — not surprisingly — less enthusiastic. Almost anyone could afford to make glass capillary columns, while the fused silica column, being both labor and materials intensive, was much more expensive to produce. Many of the centers of excellence that had contributed so much to this developing science suddenly found themselves priced out of the market. More than one investigator lamented that after building his career on glass capillary columns, he was, suddenly and through no fault of his own, redundant. Presentations on fused silica columns were not infrequently received with hostility that was not always veiled. In one major European meeting, the chairman of a session concerned with column developments issued an edict that fused silica columns would be barred from discussion; this was lifted only later in the session in response to objections from industrial attendees in the audience. More than any other one individual or company, Professor Jennings was responsible for converting industrial analysts from packed to open tubular columns. With guidance from Dr. Keene Dimick, then a flavor chemist at the USDA Laboratories in Albany, California, Jennings built the University of California’s first gas chromatograph in 1955 in the Department of Food Science and Technology on the Davis campus. In these early years, it was primarily two groups — petroleum chemists and flavor chemists — that pioneered many of the applications and developments in gas chromatography. In efforts to isolate GC fractions for further off-line characterization, Dr. Jennings developed the hot-wall–cold-wall technique that permitted trapping fractions that otherwise eluted as aerosols [1], and pioneered methods for off-line ozonolysis

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and hydrogenation to establish positions and types of unsaturation in isolated fractions [2]. In 1965, he identified the ‘flavor-impact’ compounds of the Bartlett (or Williams) pear as esters of trans-2-cis-4-decadienoic acid [3–5]. Through the 1960s, he continued the isolation and identification, primarily by infrared spectra generated on thin films of the isolated fraction, a number of constituents in a variety of food products, e.g., [6,7], including saffrole and myristicin [8] in oil of black pepper. In the early 1970s, Professor Jennings pioneered the use of porous polymers as trapping substrates to concentrate air or gas-borne volatiles for subsequent desorption and analysis [9]. This was later expanded to the ‘purge and trap’ techniques that are in wide use today. High among his contributions that led to wide industrial acceptance of capillary chromatography are the first all-glass inlet splitter for capillary columns which he invented in 1971 [10], and patented in 1972 [11]. Incorporating a double flow-inversion mixing device (‘the Jennings’ cup’), the design was later adopted by Hewlett Packard. In 1974, Professor Jennings derived the graphite ferrule, which greatly simplified column installation. To this day, the graphite ferrule has remained the primary mode of interfacing columns to injectors and detectors. During this period, he was also engaged in some seemingly unrelated research in the field of detergency. These studies established that the molecules comprising homogenous thin films on hard surfaces existed only at one of two discrete energy levels, a high energy level that was easier to remove, and a multi-molecular low energy level that resisted displacement [12,13]. Subsequent work established the influence of physical variables that encouraged transition of the high energy form to the low energy state, and vice versa. This information proved useful in later efforts to produce more stable films of stationary phase on glass capillary columns, and led to the design of a column coating machine that produced higher quality columns of much greater stability [14–16]. The machine incorporated a high temperature tubular heater as the entrance to an oven, into which the column was threaded as if it were a massive screw, a modification of a technique first described by Ilkova and Mystryukov. The invention of this device led to the 1974 founding of J&W Scientific, by Professor Jennings and Robert Wohleb, one of his completing doctoral students. The fledgling company grew to become the world’s largest supplier of open tubular columns for gas chromatography. It was then discovered that when this machine was used to coat columns with SE-52 (5% phenyl–1% vinyl polydimethylsiloxane), the columns exhibited even greater stability and much longer lifetimes, and that these improvements correlated with the temperature of the heated inlet tube. Theorizing that crosslinking of the vinyl groups was probably responsible for the increased stability, vinylsilanes were employed in glass deactivation, and also added to the dilute solution with which the column was filled prior to its introduction, via the high temperature inlet tube, into the drying oven. Glass capillary bonded-phase ‘Durabond’ columns were introduced by J&W at the 1978 Expochem Symposium organized by Professor Zlatkis, and held in Houston. It was in this same year that Professor Jennings patented a high pressure housing that permitted using liquid CO2 as the extracting solvent for Soxhlet extraction. The CO2 was then evaporated from the extracted material at sub-zero temperatures, permitting the injection of solvent free extracts [17]. The first coupling of glass capillary columns into a recycling unit was demonstrated

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by Dr. Jennings in 1979 [18,19]. Injections were recycled through two short columns in a valve-controlled reversible sequence, and attained, at the optimum velocity, over two million theoretical plates. Running at the optimum practical gas velocity, the device generated over 4000 theoretical plates per second. Work on methods to re-focus the circulating bands later led to an inexpensive capacitor-discharge device for extremely fast heating of electrically conductive cold traps [20]. Jennings was also the first to develop computer-generated van Deemter curves that permitted assessment of variables (e.g., df , the thickness of the stationary phase film [21]; DS , solute diffusivity in the stationary phase) on column efficiencies. His group was also the first to apply the concepts of window diagramming to high resolution systems [22], a process later used to design the first stationary phase specifically tailored to maximize all separation factors of a given group of analyte [23]. His laboratory served as a chromatographic Mecca, attracting graduate students, post doctoral scholars, and visiting scientists from over twenty-five different countries. Their foci included the elucidation of biosynthetic pathways for volatiles isolated from natural products, e.g., [24–26], techniques of sample preparation, e.g., [27,28], design and modification of instruments and accessories, studies on fundamental chromatographic inter-relationships, and developments in column deactivation and manufacture. Many of the scientists who worked with Professor Jennings are now well known academicians in their own right; some occupy eminent positions in instrument companies, and others direct research in areas as diverse as flavor, forensic, pharmaceutical, petrochemical, and environmental analysis. During the last portion of his 37 years at UC, Professor Jennings was exploring the use of short small diameter (50–100 µm) columns in gas chromatography. In 1989, he fully retired from the University, and is now an Emeritus Professor of Food Science and Technology, and Consultant to J&W Scientific, Inc. His widespread extra-curricular instructional activities that began about 1972 with Hewlett Packard and continue to this day have given Dr. Jennings an extremely broad exposure to practicing analysts in industry and academia. Convinced that the chromatographic expertise of the average practicing analyst has continuously declined since the late 1970s, Professor Emeritus Jennings continues today to focus his professional activities on education. References 1. 2. 3. 4. 5. 6. 7. 8. 9.

R. Teranishi, J. Corse, J. Day and W. Jennings, Volman collector for gas chromatography, J. Chromatogr., 9 (1962) 244–245. M. Sevenants and W. Jennings, Volatile compounds of peach II, J. Food Sci., 31 (1966) 81–86. W. Jennings and R. Creveling, Volatile esters of Bartlett pear II, J. Food Sci., 28 (1962) 91–94. W. Jennings and M. Sevenants, Volatile esters of Bartlett pear III, J. Food Sci., 29 (1964) 158–163. W. Jennings, R. Creveling and D. Heinz, Volatile esters of Bartlett pear IV, J. Food Sci., 29 (1964) 730–734. W. Jennings and R. Wrolstad, Volatile constituents of black pepper, J. Food Sci., 26 (1961) 499–509. W. Jennings, The changing field of flavor chemistry, Food Technol., 26 (1972) 25–34. G. Russell and W. Jennings, Constituents of black pepper: oxygenated compounds, J. Agric. Food Chem., 17 (1969) 107–1112. W. Jennings, paper 99, 160th National Meeting, American Chemical Society, Chicago, IL, Sept. 13–18 (1970).

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10. W. Jennings, Glass inlet splitter for gas chromatography, J. Chromatogr. Sci., 13 (1975) 185–187. 11. W. Jennings, Nonreactive Inlet Splitter for Gas Chromatography and Method, US Patent 4,035,168, July 12, 1977. 12. M. Bourne and W. Jennings, Kinetic studies of detergency. I. Analysis of cleaning curves, J. Am. Oil Chem. Soc., 40 (1963) 517–523. 13. M. Bourne and W. Jennings, Kinetic studies of detergency II. Effect of age, temperature, and cleaning time on rates of soil removal, J. Am. Chem. Soc., 40 (1963) 523–530. 14. W. Jennings and R. Wohleb, Routine production of the glass capillary column, Chem. Mikrobiol. Technol. Lebensm., 3 (1974) 33–35. 15. W. Jennings, R. Wohleb and M. Lewis, Isolation of volatile compounds for GLC analysis, MBAA Technol. Quart. 11 (1974) 104–109. 16. W. Jennings, R. Wohleb and K. Yabumoto, Manufacture and use of the glass open tubular column, J. Chromatogr. Sci., 12 (1974) 344–348. 17. W. Jennings, Vapor-Phase Sampling, J. High Resolut. Gas Chromatogr., 2 (1979) 221–224. 18. W. Jennings, J. Settlage and R. Miller, Multiple short pass glass capillary gas chromatography, J. High Resolut. Chromatogr., 2 (1979) 441–443. 19. W. Jennings, J. Settlage and R. Miller, New approach to chromatographic optimization in glass capillary gas chromatography, J. Chromatogr., 186 (1979) 189–196. 20. J. Settlage and W. Jennings, Inexpensive method for rapid heating of cold traps, J. High Resolut. Chromatogr., 3 (1980) 133–134. 21. D. Ingraham, C. Shoemaker and W. Jennings, Computer comparison of variables in capillary gas chromatography, J. High Resolut. Chromatogr., 5 (1982) 227–236. 22. D. Ingraham, C. Shoemaker and W. Jennings, Optimization of liquid phase mixtures, J. Chromatogr., 239 (1982) 39–50. 23. M. Mehran, W. Cooper, R. Lautamo, R. Freemen and W. Jennings, A new stationary phase for the gas chromatographic separation of volatile priority pollutants and chlorinated pesticides, J. High Resolut. Chromatogr., 8 (1985) 715–717. 24. D. Heinz, R. Creveling and W. Jennings, Direct determination of aroma compounds as an index of pear maturity, J. Food Sci., 30 (1965) 641–643. 25. W. Jennings and R. Tressl, Production of volatile compounds in the ripening banana, Chem. Mikrobiol. Technol. Lebensm., 3 (1974) 52–55. 26. W. Jennings and G. Takeoka, Gas chromatography of isoprenoids, in J. Law and H. Rilling (Eds.), Methods in Enzymology. III. Steroids and Isoprenoids, Part B, Academic Press, New York, 1985. 27. W. Jennings and H. Nursten, Gas chromatographic analysis of dilute aqueous systems, Anal. Chem., 39 (1967) 521–523. 28. W. Jennings and M. Filsoof, Comparison of sample preparation techniques for gas chromatographic analysis, J. Agric. Food Chem., 25 (1977) 440–445.

D.28. James W. Jorgenson James W. Jorgenson was born on September 9, 1952 in Kenosha, WI. His formal education was at Northern Illinois University, 1970 to 1974, receiving a B.Sc. in Chemistry in 1974, then a Ph.D. in Chemistry at Indiana University, 1979. J.W. Jorgenson has had a broad teaching experience as a Teaching Assistant in chemistry at Northern Illinois University (1973–1974) and at Indiana University (1975–1976). At the University of North Carolina he has held the following positions in chemistry: Assistant Professor (1979–1984), Associate Professor

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(1985–1987) and Professor (1987–1994). He was appointed F.P Venable Professor of Chemistry, University of North Carolina (1994–1999), and presently is William Rand Kenan Jr. Distinguished Professor of Chemistry, UNC. He assumed the position as Chair of the Department in July 2000. Jorgenson has received numerous honors and awards as listed following: Lubrizol Graduate Fellowship, Indiana University (1977), DuPont Young Faculty Development Award, (1982), U.N.C. Junior Faculty Development Award (1982), Alfred P. Sloan Foundation Research Fellowship (1984), Bowman and Gordon Gray Professorship in Undergraduate Teaching (1987), Tanner Award for Excellence in Undergraduate Teaching (1989), UNC Arts and Sciences Foundation Professorship, UNC-CH (1991), Award in Chemical Instrumentation, American Chemical Society, Analytical Division (1992), A.J.P. Martin Medal of the Chromatographic Society (1992), Fellow, American Association for the Advancement of Science (1992), American Chemical Society National Award in Chromatography (1993), and the Van Slyke Award, New York Metropolitan Section of the American Association for Clinical Chemistry (1993), ISCO Award for Significant Contributions to Instrumentation for Chemical Separations (1994), Marcel J.E. Golay Medal of the International Organization for the Promotion of Microcolumn Separations (1994), National Chromatography Award of the Northeast Regional Chromatography Discussion Group (1994), Electrophoresis Award of the Frederick Conference on Capillary Electrophoresis (1994), Distinguished Speaker Award, North Carolina Section of the American Chemical Society (1994); Eastern Analytical Symposium Award in Separation Science (1995), Torben Bergman Medal of the Swedish Chemical Society, Analytical Section (1996); Anachem Award, Association of Analytical Chemists of Detroit (1996); Distinguished Alumnus Award of the Indiana University Graduate School (1996), Dal Nogare Award, Chromatography Forum of the Delaware Valley (1998); James B. Himes Merit Award, Chicago Chromatography Discussion Group (1998), California Separation Science Society Award for Major Advances in Separations (1999). J.W. Jorgenson has served as Associate Editor of Analytical Chemistry and a member of the Editorial Boards of: the Journal of Chromatography, Analytica Chimica Acta and Journal of Pharmaceutical and Biomedical Analysis. Presently, he is on the Editorial Boards of the Journal of Microcolumn Separations and the Journal of Capillary Electrophoresis. He has been an invited honorary lecturer at a number of Universities. His research interests are centered on complex analysis of mixtures of natural products using capillary electrophoresis, microcolumn chromatography, two-dimensional separations, ultra-high pressure LC, ultra-high voltage capillary electrophoresis, and combined liquid chromatography-mass spectrometry. See Chapter 5B, a, d, e, f, h, k, l, p, r, s

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28.I. SCIENTIFIC BIOGRAPHY OF JAMES W. JORGENSON James W. Jorgenson Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599–3290, USA

28.I.1. My early formative years I owe a great deal of my early inclination toward science to my parents, who always saw to it that I had stimulating experiences, and did not discourage me from trying crazy things and making messes (as long as I cleaned them up). I also am in great debt to my fifth grade teacher Mr. Palmgren. More than any other teacher, he taught me about the excitement of ideas, about thinking ‘outside the box’. And he taught me that despite living in a huge and complex world, it can all be understood and make sense. He taught me to truly believe that the human mind can take on any question. While growing up, I also learned quite a bit about science on my own, particularly due to my hobbies: these included raising tropical fish, an interest in electronics and radio, and, like many future chemists, an interest in explosives. I am happy to say that, with the exception of explosives, I have maintained a few of these interests right up to the present time. When I entered college at Northern Illinois University, I intended to major in biology, with a plan to have a career in marine biology or ichthyology. As a biology major, I needed to take a considerable number of chemistry courses in addition to biology. In the biology courses (botany and microbiology), I found I was taking lecture classes with hundreds of students. I found the courses to be interesting, but I had essentially no interaction with the faculty. Meanwhile in chemistry, I was put in small advanced sections (about 20 students) of general chemistry and organic chemistry. In these chemistry courses I had a great deal of contact with the faculty teaching the courses. I developed the impression that pursuing a chemistry degree might provide me a better education, and also provide better job possibilities when I graduated. So at the end of my sophomore year I switched to a chemistry major. At this time I thought I would focus on organic chemistry, and natural products chemistry seemed especially interesting to me. The traditional sophomore-level ‘wet’ analytical chemistry I had studied did not interest me in the least. After spending my junior year doing research with an organic chemist, I decided that natural products chemistry was not so interesting after all. I became very interested in biochemistry after taking an introductory course in my junior year. In my senior year, I took more biochemistry courses, and did a research project in biophysical chemistry. I found myself being most fascinated with the instrumentation and electronics aspects of my research. In my final semester of my senior year, I took an instrumental analysis course, and I could not believe how great it was. I finally found a course that absolutely had everything I liked about science. It involved instrumentation, making measurements using electronics, optics, magnets, high vacuum, and all kinds of other really interesting technology. And it was clear that you could use this to study the chemistry of biological systems. Analytical chemistry seemed like a way to be involved with almost all the different interests I had developed over the years. In the fall of my senior year I applied to biochemistry graduate programs at a variety of

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schools, but in the spring, as I was visiting these schools, I realized I was actually more interested in analytical chemistry. Fortunately, one of the schools I was visiting, Indiana University, had both chemistry and biochemistry in the same department. While visiting Indiana, the last faculty member I met (just by a chance meeting in a hallway, not by plan or schedule) was Professor Milos Novotny. He went out of his way and took considerable time to show me what his research group could do with capillary gas chromatography. The chromatograms absolutely took my breath away. I had no idea that such complex mixtures could be resolved into so many components. It was clear to me that capillary GC provided a way to do quantitative analysis on extremely complex mixtures, including those of biological origin. I decided at that point that I wanted to go to graduate school at Indiana and work with Professor Novotny. While working for Professor Novotny, my main thesis project concerned the isolation and identification of mammalian pheromones, chemical substances which are used by animals (within a species) to communicate with each other. The species we focused on were the mouse, fox and rhesus monkey. This work involved a considerable amount of capillary gas chromatography, as well as some preparative liquid chromatography, and even a brief exposure to paper electrophoresis. In fact, this exposure to real-world electrophoresis was an important experience for me. I was struck by the disparity between the practice of techniques such as capillary GC or HPLC versus electrophoresis. It appeared as if no one had taken electrophoresis seriously enough to develop it into an instrumental method of analysis. I was well aware of the work going on in isotachophoresis, but this technique seemed to me (down to this day) as a very odd way of doing electrophoresis, certainly one which no person trained in chromatography would be very comfortable with. While at Indiana, I managed to do a fair amount of research not related to my thesis work on pheromones. This included work on developing new detection methods for LC, studies on column preparation for capillary GC, and even some attempts to do electric-field-flow fractionation in porous glass capillary tubes. Professor Novotny was constantly coming through the laboratories to see what new things his students were up to. To his great credit, he did not discourage me from pursuing odd research ideas that had nothing to do with pheromones. Instead, he encouraged this kind of independent work, even though it might delay progress on the pheromone project. I learned a great many important lessons from Professor Novotny in my time at Indiana University. If I had to pick one single thing that has been most important to me, it was to always center one’s work on a real scientific problem, and to always test new techniques on real samples. A successful analysis requires a combination of both sensitivity and selectivity. It has taken me a long time to realize that, in most cases, the harder thing to achieve is sufficient chemical selectivity. If you only test a new analytical method or device on traces of analyte dissolved in distilled water, you will only be testing the sensitivity of the system, and avoiding the selectivity problem. Surprising as this may sound, I would say that this was common practice in the analytical chemistry of the 1970s. Many fascinating devices were described, but seldom were they challenged with a real sample. In Professor Novotny’s group we prided ourselves on being able to discover and measure substances in blood, urine, and other solutions quite distant from distilled water!

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I went to the University of North Carolina at Chapel Hill in July of 1979, straight from graduate school, without the benefit of any postdoctoral training. I believe the only reason I survived without extra research experience was the richness of the research experience I had as a graduate student, complete with multiple projects, and plenty of interactions with other students in the laboratory. In some sense, I had a postdoctoral experience while in graduate school. The research I proposed (spring of 1978) to do at North Carolina concerned the following topics: ž Separation via reversible covalent interactions between analytes and stationary phase ž Surface analysis of gas chromatographic support materials ž Polymer and particle separations ž Electro-osmotically driven (pumped) liquid chromatography ž Detection in liquid chromatography

28.I.2. Separation and analysis of complex mixtures It is interesting to note that no mention was made of studying zone electrophoresis in capillaries. This idea was developed in the fall of 1979 when I was already at North Carolina. In beginning to develop a theory of band broadening for electro-osmotically driven liquid chromatography, it occurred to me that band broadening in zone electrophoresis in a capillary (or what I later called ‘capillary zone electrophoresis’ or CZE) was a simpler case to consider than electro-osmotically driven chromatography. In the process of considering band broadening in CZE, I became aware of how simple, powerful and attractive such a system might be. It offered a method of separation where only longitudinal diffusion might operate, a system where the faster you go, the better the separations are (a bit like ‘having your cake and eating it too!’). The prospects of what might be possible by doing CZE were so attractive that experiments on electro-osmotically driven chromatography were delayed in favor of trying to do CZE. Krynn DeArman (who later married and changed her name to Krynn DeArman Lukacs) was one of my first three students and was assigned to work on this research problem. The critical piece of equipment required to enable us to really demonstrate the potential of CZE was a sensitive detection system. We did preliminary experiments using a commercial UV absorption detector modified for on-capillary detection, but soon built an on-capillary fluorescence detector. This fluorescence detector served very satisfactorily and allowed us to make the majority of our measurements in our early papers on CZE. The capillaries at this time were not made of fused silica, as fused silica capillaries of suitably small inner diameter were not yet commercially available. Instead, we used borosilicate glass capillaries drawn in our own laboratory using a commercial glass capillary drawing machine designed for drawing glass capillaries for gas chromatography columns. Provided that we restricted our work to UV absorption and fluorescence in the near-UV portion of the spectrum (wavelengths longer than about 350 nm), these capillaries were acceptable. But they had a large outer diameter by today’s fused silica standards, and were essentially not flexible at all. At the same time that we initiated research on CZE, we also began work on open-tubular columns for capillary liquid chromatography. Compared to CZE, this

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research developed more slowly because the preparation of good columns with adequate amounts of stationary phase was difficult (CZE completely avoids this difficulty!). This work began with a student by the name of Edward Guthrie, and continued through many subsequent students, including Robert St. Claire, and eventually evolved into our current efforts using ultra-high pressures to force mobile phase through long capillary columns packed with one micron diameter particles. This project on ultra-high pressure liquid chromatography (UHPLC) was begun in 1994 by a student named John MacNair, and continues with a current student named Kamlesh Patel. We have recently carried out LC separations at pressures as high as 7500 atmospheres or bars (110,000 pounds per square inch), and we believe this level of pressure is probably near the upper limit of useful pressures in LC for a long time to come. We believe that commercial equipment will soon be developed which exceeds the 400 bar limit of most current LC technology, but we expect these moves to higher pressure will be incremental, first to 1000 bar, then 2000 bar, etc. In analogy with the use of ultra-high pressures to improve the separation power in LC, my student Katariina Hutterer began in 1996 the investigation of the use of ultra-high voltages to improve separations in CZE. Here we demonstrated the use of potentials as high as 160,000 volts in CE, and we eventually would like to develop table-top CE systems which work as high as one million volts. All of this work in capillary separations has had its problems, especially in developing suitable detectors sensitive enough to work with the tiny quantities of materials separated on these capillaries. Eventually we wanted to take advantage of the special capability of these small capillaries to handle extremely tiny volumes of samples, particularly in cases where only small volumes of sample were available. The most exciting example of such a sample that we could imagine was the contents of a single cell. In 1985, Robert Kennedy began my group’s research effort on the use of capillary LC to separate the contents of single neurons. This work has continued in my group through the work of students such as Bruce Cooper and Showchien Hsieh. Scott Wills has taken this work to the point of measuring the quantity of both epinephrine and norepinephrine contained in single vesicles. Vesicles are the 100 nanometer sized sub-cellular compartments which store these transmitter substances within single cells! While visiting Cal Giddings at the University of Utah in May of 1987, I presented a seminar on my group’s work in CZE and capillary LC. Afterward, Cal complimented me on our work, but suggested that if we really wanted to separate complex mixtures, we should look into two-dimensional separations. Although people had been doing two-dimensional separations for decades, most of this work was either two dimensional separations done by thin-layer chromatography or slab-gel electrophoresis. Traditional LC or GC columns had been coupled in series, but only in a ‘heart-cutting’ mode, where a small section of the effluent from a first column is diverted to a second column for separation. On the flight home from Utah, I took Cal’s suggestion to heart, but decided that we would investigate what we ended up calling ‘comprehensive’ two-dimensional separations using coupled LC columns. The word comprehensive was chosen to signify that separation of all components in the sample would be subjected to full two-dimensional separation, thus making it distinct from heart cutting, where only a small portion of the sample gets separation by two dimensions. The key to this process is to use an extremely fast separation as the second dimension, so that individual bands

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eluting from the first dimension are each sequentially separated by a very fast run in the second dimension. Michelle Bushey began this work in my group, developing both LC–LC systems and LC–CE systems. The work in LC–CE was continued by John Larmann, Alvin Moore, Anthony Lemmo, Tom Hooker, and Dorothea Jeffery, while LC–LC work was continued by Gregory Opiteck, eventually leading to his work on LC–LC–MS. The unifying theme of my laboratory’s work over the years has been the analysis of complex mixtures. This was a natural result of working in Professor Novotny’s laboratory, since the focus of most of the work there was also complex mixture analysis. I have become intrigued (even obsessed) with this topic over the years. I believe that it is an endless problem, one that will never be completely solved. Nature appears to be unlimited in its ability to provide us with chemical mixtures of extreme complexity, and I am happy to keep tackling these challenges. My group is driven by the goal of providing as much analytical information as possible on complex mixtures. I have never been particularly interested in working out an analytical method to measure a particular substance, say a single peptide hormone, in a complex mixture such as blood. But before I finish my career, I hope we succeed in developing technology enabling us to simultaneously quantify hundreds of peptide hormones from a single drop of blood. I personally believe that the approach of combined liquid chromatography–mass spectrometry will provide such capability, but we are prepared to follow whatever route will lead us to this goal, and most importantly, to have a lot of fun getting there.

28.I.3. Separations of the new millennium I foresee two very novel lines of development for analytical-scale separations that will be particularly important in the next decade. The first is the use of the ‘chip’ format for separations. Such devices will be best suited for relatively simple separations that need to be done in tremendous numbers. The massive parallelism (100s of parallel separation channels) of the chip format will allow rapid sample turn-around and low cost per analysis. At the same time, the chip will permit integration of sample pretreatment, derivitization, and post-column chemistry to be carried out in an automated manner. A second important trend I will call ‘designed nanostructures’. Highly selective separation structures will be designed into nanomaterials. This will be done from the ‘bottom-up’, via chemical synthesis of self-assembling structures, designed to have cavities of precise size and structure. The spatial arrangements of functional groups within the cavity will be designed to specifically host a certain molecule or family of molecules. This trend will not only be important for analytical scale separations, but will be an important trend in preparative scale separations. The other approach will be top-down, where nanomachines will be used to ‘sculpt’ nanomaterials into precise shapes and sizes useful for separations. Other important trends involve revised manifestations of existing separation technologies. This includes the use of multiple conventional columns in parallel, where higher sample throughput is needed, but where the chip format does not provide sufficient separation power or capacity. The separation power of individual columns will

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continue to be improved, through the use of still smaller particles and higher pressures in LC, and higher voltages in CE. These columns will be used for the separation of extremely complex mixtures, and will be routinely coupled to mass spectrometers. Improvements in the sensitivity of capillary LC–MS will be achieved by developing sample vaporization=ionization techniques that occur within the vacuum of the mass spectrometer, allowing complete utilization of the sample. Detection limits for these capillary LC–MS systems will approach the level of single molecules.

References 1.

J.W. Jorgenson and K.D. Lukacs, Zone electrophoresis in open-tubular glass capillaries, Anal. Chem., 53 (1981) 1298–1302. 2. J.W. Jorgenson and K.D. Lukacs, Capillary zone electrophoresis, Science, 222 (1983) 266–272. 3. L.A. Knecht, E.J. Guthrie and J.W. Jorgenson, On-column electrochemical detector with a single graphite fiber electrode for open-tubular liquid chromatography, Anal. Chem., 56 (1984) 479–482. 4. R.T. Kennedy, M.D. Oates, B.R. Cooper, B. Nickerson and J.W. Jorgenson, Microcolumn separations and the analysis of single cells, Science, 246 (1989) 57. 5. M.M. Bushey and J.W. Jorgenson, Automated instrument for comprehensive two-dimensional highperformance liquid chromatography-capillary zone electrophoresis, Anal. Chem., 62 (1990) 978–984. 6. J.E. MacNair, K.C. Lewis and J.W. Jorgenson, Ultra-high pressure reversed-phase liquid chromatography in packed capillary columns, Anal. Chem., 69 (1997) 983–989. 7. G.J. Opiteck, J.W. Jorgenson and R.J. Anderegg, Two dimensional SEC=RPLC coupled to mass spectrometry for the analysis of peptides, Anal. Chem., 69 (1997) 2283–2291. 8. S. Hsieh and J.W. Jorgenson, Determination of enzyme activity in single bovine adrenal medullary cells by separation of isotopically-labeled catecholamines, Anal. Chem., 69 (1997) 3907–3914. 9. J. MacNair, K. Paten and J.W. Jorgenson, Ultra-high pressure reversed-phase liquid chromatography: Isocratic and gradient elution using columns packed with 1.0 µm particles, Anal. Chem., 71 (1999) 700–708. 10. K.M. Hutterer and J.W. Jorgenson, Ultra-high voltage capillary-zone electrophoresis, Anal. Chem., 71 (1999) 1293–1297.

D.29. Rudolf E. Kaiser Rudolf E. Kaiser was born on February 12, 1930 in TeplitzScho¨enau. He studied at both the Technical University of Dresden and the University of Leipzig in the German Democratic Republic and received his doctorate in 1954. In 1952 he joined the Institute of Technical Chemistry of the German Academy of Sciences where he soon became Head of the Department of Separation Sciences. In 1960 he left the GDR for the FRG, where he joined BASF as an Analytical Chemist. Here he stayed until 1972 when he founded his own Institute for Chromatography (IfC) (in Bad Du¨erkheim), which celebrated its 25th jubilee last year and in which thousands of chromatographers from all over the world (from more than 50 countries) have been trained in all aspects of chromatog-

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raphy. R.E. Kaiser has organized and taught many courses in other countries throughout the world. The research activities of R.E. Kaiser span the entire field of chromatography, especially capillary gas and planar chromatography, as well as statistical evaluation of analytical results, and computer applications. R.E. Kaiser has authored more than 200 publications and numerous books, he also acted as Editor and Co-editor of many more. His first book “Gas Chromatographie” was published in 1959 in East Germany. His four-volume series “Chromatographie in der Gasphase” published in 1961 represents the first book on quantitative gas chromatography. It went through three editions in West Germany and was translated into English. One of the volumes of this series represents the first book in the world on capillary gas chromatography. R.E. Kaiser founded the well-known international journals ‘Chromatographia’ (1968), ‘Journal of High Resolution Chromatography and Chromatography Communications’ (1978), ‘Computer Applications in the Laboratory’ (1983). He also played a key role in the founding of the ‘Journal of Planar Chromatography’ (1988). He was for many years Editor-in-Chief or an Editorial Board member of these journals. R.E. Kaiser started the following series of Symposia and was for many years their chairman: International Symposium on Capillary Chromatography (since 1975: Hindelang, now in Riva del Garda); International Symposium on Planar Chromatography (since 1980: Bad Duerkheim, now in Interlaken); International Symposium on Chromatography and Spectroscopy in Environmental Analysis (since 1994: St. Petersburg). R.E. Kaiser’s achievements in chromatography have been recognized by a number of awards: Tswett Medal of the USSR Academy of Science — as a first foreign recipient (Moscow, USSR, 1978); Gold Medal of the Chinese Academy of Science and Chinese Chemical Society (Beijing, PRC, 1988); A.J.P. Martin Award (Brighton, UK, 1989); Marcel Golay Award (Riva del Garda, Italy, 1989); Tswett Medal of the Chromatographic Society of Russia (Du¨sseldorf, 1995); the President of the Federal Republic of Germany, R. Herzog, has decorated R.E. Kaiser in 1996 with the 1st Class German Distinguished Medal for his contribution to environmental analysis and for his international activities in this field. Dr. R.E. Kaiser is one of the pioneers in chromatography, who became active in this field in 1953. He has been an active, enthusiastic, inspiring, creative and original promoter of the development and applicability of chromatography in analytical chemistry for almost 50 years. He has a natural talent for finding fast and simple solutions for many problems encountered in the analysis of complex mixtures. R.E. Kaiser is an extraordinary person whose contributions to chromatography — also his books, his symposia and his journals — are well known and recognized. Chromatography is Rudolf Kaiser’s real hobby. See Chapter 5B, a, b, d, s

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29.I. MY OCCUPATION AND MY HOBBY — CHROMATOGRAPHY Rudolf E. Kaiser * Institut fu¨r Chromatographie, Dr.-Hugo Bischoff-Strasse 1, Bad Du¨erkheim, D-67085 Germany

In the spring of 1953, in Leipzig (the German Democratic Republic, DDR), my first teacher Dr. Otto Mittelstaedt (the physicist who for the first time correctly measured the speed of light) told me that my main job — doing kinetics and organic group analysis of the catalytic oxidation products of hydrocarbons — would be enlarged. “There is a most interesting doctoral thesis work going on at the Boehlen Chemical Complex”, he told me, “E. Koegler is separating hydrocarbons with the help of a glass tube packed with cotton wool, which is impregnated with some high-boiling oil and by flushing the tube with hydrogen. It is something called ‘gas chromatography’. Visit him, build a similar machine here and carry out separation work with solid n-alkanes up to C34 which are the basis of the oxidation process because we have difficulties in separating them by distillation”. This was an order. I did what I was told. In a few weeks we had a ‘machine’ built; my group was also increased to a team of six. One of us injected the gasoline sample with the help of a microliter syringe through a rubber cap into the glass column. The other increased the electric current to the heater stepwise, the third had to look continuously through a very sensitive optical detector, an interferometer, turning the knob to keep the balance of the light-beam and shadow, the fourth recorded the data every 10 s and finally, the fifth gave the time interval commands. After carrying out three ‘chromatographic exercises’ in a day, we needed three days to do all the drawings, calculation and relaxing. There was no recorder available to us in 1953 which could do the job. One year later, we organized a small conference on chromatography in Leipzig. Meanwhile, we had learned from Zhukhovitskii and Turkel’taub (Moscow) how to use adsorption columns, thermochromatography and temperature programming and from Professor Erika Cremer (Innsbruck, Austria) how to use the catharometer, a thermal conductivity detector. We built our own catharometers, including the wire to be used in it. In 1955, we had a homemade instrument containing a large aluminum block with five catharometers in parallel and five GC columns which could be used either parallel or in series, and could be mechanically switched. The instrument also had flow regulators and everything was in an excellent air-thermostatted oven. It was really great. After assembly, it could not be removed from the room: it was now too large in size. However, it worked. Unfortunately, our problem was that carbon dioxide and ethylene gave the same retention times and thus, identification turned out to be too risky. The first London Symposium in 1956 represented a real shock to us; we learned from the lectures that we were doing chromatography in the wrong way: instead of adsorption columns we should use liquid phases. We should also work isothermally and use thermostats with boiling vapors. Meanwhile we carried out systematic studies to improve our thermal conductivity detector. The detector was now shockproof, had practically no baseline drift, had an *

Correspondence address: Postfach 11 41, Bad Du¨erkheim, D-67085 Germany

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excellent sensitivity and could be operated equally well with 1 or 100 ml=min hydrogen flow. With it, we could solve our problem, to separate high-boiling paraffins from C18 to C32 using a 3-m-long packed column. We also made a movie about the manufacturing of the detector and showed it in a number of places in Eastern Europe at various international meetings. Then came the 1958 Amsterdam Symposium where I had my second shock: Professor Keulemans told me that my detector was obsolete — we should use an ionization detector! During those years, we also organized our first national conference on gas chromatography, the first training courses for chemists from the industry and research institutes; we had the first doctoral theses on gas chromatography and we experienced unbelievable cooperation. It was a great time to make friends. Our first series of GC instruments had now been used in a number of laboratories and they produced more-and-more results. One of my colleagues separated amino acids, the other phenols, the third amines, and the fourth carried out pyrolysis–gas chromatography. (We had no helium and thus had to use hydrogen as the carrier gas). At the beginning, we had to change the minds of a number of skeptical persons, but we succeeded. I will never forget the day — it was prior to 1956 — when we had been invited to demonstrate the possibilities of gas chromatography at the BUNA Werke, in Schkopau, one of the largest manufacturers of synthetic rubber in Europe. We started the demonstration by injecting their cleanest butadiene into a polar packed (glass) column, which was mounted in the open on a table so that everybody saw what we were doing. In the first few minutes the column exploded because we had too-high a hydrogen pressure. Fortunately, we had a second system in reserve and, 10 min later we continued the experiment. When the chromatogram was ready, it was clear that the ‘purest’ butadiene was indeed not pure, containing a number of impurities. In November 1958, D.H. Desty accepted the invitation of the Academy of Sciences at our Institute, in Leipzig, to present a paper on new developments in gas chromatography. I served as his interpreter and also as his bodyguard; I remember in great detail the atmosphere of total silence in an overcrowded lecture hall when he showed his over 100,000 theoretical plate gasoline run using a 100-m metal capillary and when he discussed the potentialities of glass capillary columns. At that instant, we were more-or-less converted to continuing our work with capillary columns. We made our own flame ionization detectors, amplifiers and electronic integrators and visited Jaroslav Janak in Brno, in neighboring Czechoslovakia, to learn how to make our own (copper) capillaries. We soon changed to aluminum capillaries. However, our basic problem remained: we had no fast potentiometer recorders. On the 15th of October, 1959, my guests — E. Cremer (Innsbruck, Austria), E. Smolkova (Prague, Czechoslovakia), A.V. Kiselev (Moscow), J.F.K. Huber (Innsbruck), J. Janak (Brno), A.I.M. Keulemans (Eindhoven, The Netherlands), O. Grubner (Prague) and A.A. Zhukhovitskii (Moscow) tried to establish, together with my wife Annemarie, a working group on gas chromatography. I still have my guest book with the entries of all these friends. However, we soon learned officially, that for certain (political) reasons, we should not continue any organization along these lines. In March 1960, my coworkers H. Holzhaeuser, M. Kuhl, M. Hofmann, H.G. Struppe and H.P. Angele plus eight female technicians succeeded in building a temperature-pro-

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grammed gas chromatograph with a 5-m aluminum capillary column (alkaline-etched and coated) connected to a flame ionization detector with a home-made high-speed amplifier and an electronic integrator. With this equipment, Angele and I flew to Moscow, to a research institute, where finally, we had a high-speed recorder available to us. In the first run, we could separate phenols within a few seconds on this (porous-layer) metal capillary column. About that time, M. Mohnke, my coworker since 1952 (in the previous years we had jointly developed urea-inclusion liquid column chromatography for the separation of paraffin waxes by temperature-programmed liquid elution), etched glass capillary columns with aqueous HCl and NH4 OH solutions, and utilized this column for the separation of hydrogen isotopes. We summarized our results in a paper, but this was delayed and then published by changing the name of one of the authors. I, meanwhile, changed my affiliation and left East Germany. In 1960, I joined the Badische Anilin and Soda Fabrik, in Ludwigshafen am Rhein, German Federal Republic. Within a few weeks my new coworker, H. Fiedler, succeeded after a little instruction, in preparing high-resolution capillary columns and was able to carry out high-speed separations of isomeric aromatic compounds, utilizing homemade instruments. Despite the results, I have had colleagues who could not believe the excellent, reliable quantitative and routine analytical data we obtained in such systems and they continued for 10 more years in their disbelief in the power of this new analytical technique. As far as I know, there are still some (although now fewer and fewer) colleagues around who do not believe in high-resolution chromatography; this is, however, more the present than history. At the end of 1960, at a scientific meeting at Dechema, in Frankfurt=Main, my former director from Leipzig, Professor Leibnitz questioned the validity of our own former results. At that time I had sufficient additional high-quality data — obtained under industrial analytical conditions — available to show that high-precision quantitative capillary gas chromatography was indeed the technique of the future. By 1964, we had the first automated instrument ready for sub ppb level analysis of trace impurities in air and water using a special technique basically invented by Zhukhovitskii and Turkel’taub in Moscow. Our results divided the people into three groups: those, who did not believe in the data, those who did believe in them and those who while believing in them, did not like them. At that time, I met D.H. Deans of ICI for the first time and soon combined his column switching technique with capillary columns and sub-ambient conditions. W. Stoll, my coworker, carried out the first routine analysis at the 1010 % level of trace impurities in water and in outdoor air. Backflush, foreflush, on-column enrichment, column transfer, multidetector systems, the use of the first thin-layer glass capillaries impregnated with natural microparticles, superclean carrier gases, as well as concentration and trapping with help from mobile heating fields became our daily tools and technique. I left BASF in 1972, to start my own Institute for Chromatography, in Bad Du¨erkheim. Within a year, my coworker, Rudi Rieder, together with a number of skilled technicians, succeeded in making a dream into a reality: putting the separation system into a ‘black box’ a separation cassette. No longer troubled with installation, separate sample collection and concentration, gas tubing or pneumatic switching, we

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prepared separation cassettes which do the job and which now also work in miniaturized liquid column chromatography under high pressure. During the past few years, we somewhat modified the techniques of classical thinlayer chromatography and, together with D. Jaenchen of CAMAG Muttenz, Switzerland, we succeeded in revitalizing the instrumentation of this technique. It was J. Blome’s (Au=Iller) idea to use the circular development from the center as had been originally done in thin-layer chromatography; Ute B. Hezel, Carl Zeiss, Oberkochen and J. Ripphahn (E. Merck, Darmstadt) developed the method to evaluate such plates quantitatively while H. Halpaap (E. Merck, Darmstadt) can be credited with the new high-efficiency ready-made plate materials. As a result of this collective work, we could now enter a new field: anti-circular multi-component parallel analysis, 48 samples in 4 min. It took only a short time to further improve the technique and to reintroduce the power of a third phase: having the gas phase impregnating the liquid phase dynamically and the solid phase in a real two-dimensional simultaneous separation. We believe that this is the future technique for routine mass analysis of non-volatile compounds. We are now working on its full automation. Optimization in chromatography is of great importance, but sometimes people think that they do not have enough time for it. In my opinion, the present-day computerized techniques will allow us to optimize even the polarity without changing the column in gas chromatography. Everything else will be done by computers, with the help of a very simple multi-component theory, the abt-concept. Chromatography represents a fascinating field of work. We have lovers, believers, and non-believers, active side-by-side. And we also have the great family of real friends, many of them now with white hair, accompanied by the next generation who will take over from us. After so many years, the scientific meetings are still attracting a large number of scientists and the interest is continuously growing. Chromatography is indeed a wonderful technique. However, the next analytical technique which separates and identifies in a few seconds over 10 orders of magnitude in concentration and the number of compounds will come. Whether it will be a sort of chromatography or not, I do not know. I hope it will still be chromatography. Independently of this, I wish all the future pioneers to have the same exciting and happy time in its development as we had in our occupation, love and hobby —chromatography!

29.I.1. Update: 2000 This is what I wrote in 1979 [7] for “75 Years of Chromatography”. Since that time I have had the very good fortune to continue to be involved in many satisfying advances in the field. Moreover, some symposium series and journals that were just a twinkle in my eye 20 years ago are well-established. For my contribution to Chromatography 2000; however, I shall discuss a few new ideas for which we wildly underestimated the development time. I enjoy laughter. Laughing is fun. But I do not recall anyone laughing at us for our over-optimistic projections. This happens to most scientists at some point in their careers.

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29.I.2. First example 29.I.2.1. Background Many years ago, we came to the conclusion that modern planar liquid chromatography (PLC) is superior to most, if not all, chromatographic techniques in terms of speed and performance. PLC can be enhanced by coupling it with HPLC; the initial fractionation conducted by HPLC and the final analysis by PLC. It is not difficult to imagine the magnitude of the quantity of data that can be generated. It was obvious that a completely automated system that provided analytical reports rather than chromatographic data was needed. Such a development appeared to require little more than the assistance of a team of skilled programmers, working in collaboration with the chromatographers. But it was not that simple. As the demands upon our PLC media increased, problems that were previously considered to be minor now exerted a major influence. Not the least of these were baseline aberrations caused by irregularities in the plate structure. But, of course, all that we needed was appropriate software to correct this problem. 29.I.2.2. Forecast Once the software was developed to eliminate systematic errors, we would have an automated system that would be of universal appeal. It might even find acceptance in the United States, which has generally been slow to adopt PLC. Faster computers were being developed, so that complex algorithms including those to be developed for correcting baseline aberrations could be run rapidly enough to be useful. So what went wrong? 29.I.2.3. Reality While a few laboratories are working on this development, none of them is there yet. We underestimated the increase in performance of computers. A one-fold increase in performance might seem significant when you compare the performance of a car with a passenger jet, but it is trivial when dealing with the full automation of the HPLC=PLC combination. Naturally, I am reluctant to make any further projections on the time-frame for full realization of this goal.

29.I.3. Second example 29.I.3.1. Background As we predicted in “75 Years of Chromatography” the potential of serially coupled capillaries is almost limitless [1]. The overall performance of a system can be greatly altered by the temperature, pressure, and flow-rates of the individual capillaries. My wife, Dr. Olga Kaiser, has made significant contributions to this field [2]. But, again, the potential for data production can be overwhelming. Especially when one attempts

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to totally automate the system — coordination of the physical parameters for each capillary with the data that are produced. 29.I.3.2. Forecast The conversion of data again required computing power that was not available 20 years ago. However, we were confident that the data processing tools would soon become available. 29.I.3.3. Reality Is there any instrument yet available for performing multi-chromatography?

29.I.4. Third example 29.I.4.1. Background Gas chromatographers in the 1950s were happy with their packed columns for quantitative analysis. The peaks were broad enough so that their areas could be measured with confidence. When capillary gas chromatography was introduced, two new problems arose. First, of course, some of those broad single peaks were resolved into multiplets. Then there was widespread skepticism about the ability to measure the peak area of a tall narrow peak with any degree of confidence. After the first book on quantitative gas chromatography [3] and the first book on capillary gas chromatography [4] had been written, my former head at the Institute for Organic Industrial Chemistry in Leipzig stated publicly, “Kaiser’s claims about quantitative capillary chromatography are impossible; quantitative results will never be obtained from narrow peaks.” 29.I.4.2. Forecast If we wish to measure individual compounds, we must use high-resolution columns that produce sharp peaks. For those who are working on reducing the analysis time, the peaks are even sharper. As the chromatographic technology evolves, we need faster computers and better software. 29.I.4.3. Reality The goal has been achieved for many applications. Most gas chromatographs can now be purchased with data systems and software that is adequate for quantitation. But we have to ask ourselves what ‘adequate’ means. For many regulatory agencies, ‘adequate’ refers to the current state-of-the-art of analytical systems. Using this definition, all data would be considered to be ‘adequate.’ We are currently striving to upgrade the status of ‘adequate.’ At IfC we are now routinely conducting natural gas analyses with a systematic error as low as 0.01%. Relative standard deviations of š0.003% are achieved

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Fig. 1. Semilog bargraph of chromatogram of a complex hydrocarbon sample. y-axis: mole-%, x-axis: carbon number of the sample components. Total retention time of last peak: 1648 s.

for the major constituents. The standard uncertainty (STU) for these data remains below š0.02% [5]. To achieve this degree of performance, we had to write our own software. We combined the knowledge and skills of true analysts, good chromatographers, mathematical statisticians, well-trained programmers, and up-to-date communication experts. It is advances such as these which boost our confidence in the future value of gas chromatography. We have already moved on to the next step (Fig. 1), which is transmitting chromatographic data in ASCII format by e-mail around the globe [6]. There is still more to be done. Analysis of natural gas is a relatively simple problem because the sample contains so few compounds. Again, greater computing power is needed to extend the applicability to more complex mixtures.

29.I.5. Lessons learned What should we have done with these three projects 20 years ago? We could have decided to wait until adequate computing power was available. But we would still be waiting. This is not research. There is no progress. The true scientist moves ahead, using whatever tools are available at the time. An incremental approach to the problem evolves. If we were still waiting to develop the ultimate system, we would not be conducting our current natural gas analyses. We are constantly encountering problems and learning lessons as we work on early versions of automated systems. It is better to learn these lessons long before the final automated system is developed. But, of course, there is never a final system. Progress never stops.

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Fig. 2. (From right to left): R.E. Kaiser, Erika Cremer, and H.-G. Struppe.

29.I.6. Satisfaction It is not just enough to improve upon the state-of-the-art in the laboratory. Advances must be disseminated to all of those — scientists and nonscientists alike — who can use the results that are produced. It is gratifying that publishers have worked with us on the production of successful new journals and books. To Chromatographia (1968), I could add the Journal of High Resolution Chromatography and Chromatography Communication (HRC and CC) in 1978, the English language journal Computer Application in the Laboratory (CAL) and the German version Computer Anwendung im Labor (CAL) and the latest up to that time missing Journal of Planar Chromatography — Modern TLC (JPC). All of them are now in the best of hands, edited by top chromatographers and colleagues. Unfortunately HRC and CC lost its most informative part ‘CC’, thus the little things or hints making many chromatographers’ lives easier can now only be communicated through the internet. But this may work well in the near future, if we follow the ideas of Slovenian chromatographers (Mirko Prosek and others — see www.itpc.ki.si). It is rewarding to facilitate the congregation of scientists at specialized symposia year after year. The first series I started were the capillary chromatography symposia (Hindelang, 1975 1981), soon described as similar in prestige as the famous Gordon Conferences (USA). Now the Hindelang follow up symposium in the hands of Pat

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Sandra and colleagues will be successfully continued with the 23rd one in Riva del Garda Italy (2001). The First International Symposium on Instrumentalized TLC (HPTLC) started in Bad Duerkheim (1980) will be continued as the 11th in 2001 in Interlaken (CH). My most satisfying achievement of the 1990s has been the closest cooperation with Russian chromatographers: as a result — establishment of the International Foundation for Environmental Assistance to Russia. In addition we are bringing Russian analysts to IfC for intensive training. We have been able to convene conferences in St. Petersburg that are attended by experts from around the world. The First International Symposium on Chromatography and Spectroscopy in Environmental Analysis and Toxicology started in St. Petersburg, Russia in 1994. The fourth conference in this series will be held in the summer of 2001, again in St. Petersburg. Co-working intensively with my many chromatographer friends in the largest country of the world together with my Russian wife Olga is of utmost pleasure. I intend to continue that area of activity, to which A.A. Zhukhovitskii together with me founded a key statement in 1959 in Moscow, “Chromatography separates substances but unites people”. It is this wonderful global chromatographer family which, in a close cooperation, will continue to solve vital problems for the world.

References 1. 2. 3. 4. 5. 6. 7.

R.E. Kaiser and R. Rieder, Multi-chromatography, Labor Praxis, (1985) part 1–9, 1130, 1318, 1465. O. Kaiser and R.E. Kaiser, Chromatographia, 36 (1993) 47. R.E. Kaiser, Chromatographie in der Gasphase, Quantitative Auswertung, Bibliographisches Institut AG-Mannheim, 1964. R.E. Kaiser, Chromatographie in der Gasphase, Kapillar-Chromatographie, Bibliographisches Institut AG-Mannheim, 1961. R.E. Kaiser, Standard Uncertainty STU, paper presented at InCom Duesseldorf-Germany 1999, see www.incom-symposium.de. O. Kaiser and R.E. Kaiser, paper presented at the 19th Symposium on Capillary Chromatography, Riva del Garda, 1998. R.E. Kaiser, in: L.S. Ettre and A. Zlatkis (Eds.), 75 Years of Chromatography — A Historical Dialogue, Elsevier, Amsterdam, 1979, pp. 187–192.

D.30. Barry L. Karger Barry L. Karger was born in Boston, MA, April 2, 1939. He received his B.Sc. in Chemistry from MIT in 1960 and his Ph.D. in Analytical Chemistry from Cornell University in 1963. His Ph.D. thesis was in the field of gas chromatography. In 1963, he was appointed an Assistant Professor of Chemistry at Northeastern University, Boston, MA, attaining Full Professorship in 1972. In 1973, he founded the Institute of Chemical Analysis, Applications and Forensic Science. In 1983, the name was changed to the Barnett Institute of Chemical Analysis and

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Materials Science with a generous endowment gift from Lou Barnett and his family. The Institute, a leading bioanalytical research center, recently celebrated its 25th Anniversary. B.L. Karger has been the Director of the Institute since its inception. In 1985, through a generous gift of James and Faith Waters, he was appointed the first holder of the James L. Waters Chair in Analytical Chemistry. B.L. Karger has been an active researcher with over 250 publications in the field of separation science, with particular emphasis in liquid chromatography and capillary electrophoresis. He also is the holder of 22 patents in these fields, a number of which have been commercialized. With Lloyd Snyder and Csaba Horva´th, he is the co-author of “An Introduction to Separation Science” (1973), the leading graduate textbook in the field for a number of years. He has trained more than 150 students at the Ph.D. and postdoctoral levels. Recent research interests have focused on bioanalysis, including high performance DNA separation and analysis for DNA sequencing in the Human Genome Project, new separations — mass spectrometric methods for proteome analysis, binding assays for drug lead identification and separations using microfabricated devices (chips). B.L. Karger has received numerous honors and awards including: the Stephen Dal Nogare Award (1975), the ACS National Chromatography Award (1982), the ACS National Award in Analytical Chemistry (1990), the A.J.P. Martin Award of the Chromatographic Society (1991), the ACS National Award in Separations Science (1998), the Frederick Conference Award for Capillary Electrophoresis (1997), and the Eastern Analytical Symposium Award in Separation Science (1998). See Chapter 5B, a, b, d, h, l, p, r

30.I. REMINISCENSES OF 40 YEARS IN SEPARATION SCIENCE Barry L. Karger Barnett Institute, Northeastern University, 360 Huntington Ave, Boston, MA 02115-5000, USA

In 1959, as an undergraduate senior at MIT, I had to select a thesis advisor for a year of research study. My choice was between an analytical chemist, L.B. ‘Buck’ Rogers, and a biochemist. I selected Buck Rogers because of his enthusiasm for his science. While the biochemist went on to win the Nobel Prize, I never regretted my decision. My undergraduate thesis dealt with separations based on differential gas–liquid adsorption using foams (foam fractionation). I published my first paper in 1961 in this field, and my career in separation science was launched. Buck Rogers was a friend and mentor to me until his passing in 1992. It is my honor to dedicate this article to his memory for all his help over many years to encourage and uplift me. I went to Graduate School at Cornell to study with Don Cooke in the new and emerging field of gas chromatography. Part of my thesis dealt with building a flame ionization detector, at about this time James Lovelock invented and introduced the argon ionization detector. However, the main thrust of my work dealt with the issue

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of separation optimization. We recognized that time of separation must be considered in any optimization scheme and introduced the concept of time normalization to determine optimum conditions for a fixed separation time in chromatography [1]. Georges Guiochon was stimulated by our work and followed up with a detailed analysis of optimization in gas chromatography. Our discussions together in the late 1960s led to a friendship that has lasted to the present time. In the early years of my career at Northeastern University, I worked in the field of gas chromatography, as this was the ‘hot’ topic at the time (mid to late 1960s). We worked in the field of chiral separations and became friendly with Emanuel Gil-Av from the Weizmann Institute in Israel. In addition, we did some work in the study by gas chromatography of adsorption of nonpolar compounds at the gas–liquid interface of water. At this time I met Csaba Horva´th, who was a postdoctoral fellow at Harvard Medical School. Csaba encouraged me to continue studies on the interaction of nonpolar groups with water (i.e., hydrophobic interactions), since these were so important for biological systems. In addition, he encouraged me to move into the emerging field of high performance liquid chromatography, which we did in the late 1960s. Much of the next 25 years were spent focused on HPLC. My friendship with Csaba continues to the present. Because of our interest in hydrophobic interactions, we became quite intrigued with the approach of reversed phase chromatography (RPC). This no doubt, was encouraged from my interaction with the late Istva´n Hala´sz, who in 1970 spent 3 months as a Visiting Professor at the Barnett Institute. It was here that Heinz Engelhardt, my first postdoctoral fellow, and Istva´n Hala´sz met. After Dr. Engelhardt’s fellowship, Istva´n invited Heinz to Saarbru¨cken where he now is a Professor at the University in Chemistry. I have remained a close friend with Heinz and his family since those days. In 1977, we highlighted the importance of reversed phase chromatography to separations in the biological sciences in a review article for Analytical Chemistry [2], co-authored with my colleague in the Barnett Institute, Roger Giese. We focused on the use of chemical equilibria manipulating separations. This arose in part from our interaction with Goren Schill from Uppsala, who had done a great deal of work in ion pair extraction. So, we began studying ion pair chromatography [3]. This led to the logical idea of using chiral reagents as ion pairing systems, particularly in metal complexes. In 1978, we presented the first results on direct chiral separation in HPLC at the International Chromatography Symposium in Baden-Baden, Germany [4]. In the late 1970s and 1980s, HPLC research moved from the small molecule domain to peptides and proteins. We, along with Fred Regnier, Csaba Horva´th, and others, became quite interested in achieving high performance separation for proteins, both for analysis and purification. Our laboratory did a great deal of work on RPC and hydrophobic interaction chromatography (HIC). Interestingly, HIC arose from a negative aspect in size-exclusion chromatography. It was known that the use of high concentrations of chaotropic salts such as ammonium sulfate led to retention of proteins on size-exclusion packings. This was deemed to be a problem since the proteins would no longer separate based solely on size. However, we recognized that this could be put to positive use, and, employing hydrophilic bonded phases, we were able to show that a gradient of decreasing concentration of ammonium sulfate [5] led to good separation

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Fig. 1. RPLC behavior of RNase A as a function of detection wavelengths at 287 nm (A) and 254 nm (B). (From Lu et al. [7].)

with retention of protein activity. Of course, HIC is widely used today, particularly in preparative scale separations. Our interest in maintaining proteins in an active state after separation led us into the discovery that proteins can undergo time-dependent conformational changes while in contact with hydrophobic surfaces. Thus, in reversed phase chromatography, it was possible to obtain several peaks for both active and inactive forms, the extent of each peak being a function of the contact time with the surface. We showed that one could measure the unfolding rate of proteins on surfaces by this approach [6]. Additionally, this multiple peak behavior was important to understand or the protein would be assumed to be impure. We were able to show the reversibility of solution conformational changes using reversed phase chromatography. Fig. 1 shows ribonuclease A in which the two forms of the protein are discriminated based on the ratio of UV absorbance at two wavelengths. One could then study solution phase conformational changes using this approach [7].

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(From left to right): Faith Waters, Sefa Huber (deceased), Jack Kirkland, Barry Karger, Trudy Karger, Jim Waters, Josef Huber.

It was at this time in the mid-1980s that we began our interaction with Bill Hancock, who was at Genentech. I appreciated greatly the interaction in my many visits to Genentech, as it helped to calibrate me in doing meaningful studies useful to the biotechnology industry. Our work with growth hormone was important in this regard [8]. Bill and I have remained close friends since that time. In 1984, I attended the International Chromatography Symposium in Nu¨rnberg at which a discussion session was held comparing electrophoresis with chromatography. Fred Regnier had the unfortunate task of defending electrophoresis in a room filled with chromatographers. The attacks on electrophoresis were most surprising to me and, given the defensive nature of these comments, I decided that it would be interesting to look into this field. Of course, slab gel electrophoresis was in the domain of the biochemists; however, with the introduction of capillary electrophoresis (CE) by Jim Jorgenson in the early 1980s, it was clear that, from an analytical point of view, CE was a new approach. After some early studies, we wrote a broad review article in 1989 on this topic based on our experience in chromatography [9]. We recognized from the beginning that the true advantage of CE would be as a substitute for slab gel electrophoresis, not for HPLC, which by this time had become fully ingrained in the analyst’s repertoire of techniques. Hence, we began looking in 1987 at the use of gel-filled capillaries for separation of proteins and DNA, two major fields of slab gel electrophoresis. Our paper in 1988 reported the separation of DNA molecules using crosslinked polyacrylamide gel filled capillaries [10]. This work demonstrated the high-resolution capabilities of CE and set the stage for where we are today. Capillary array electrophoresis is currently being used to sequence the human genome.

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Crosslinked gels turned out to be too unstable to work with on a routine basis. We then introduced in 1990 noncrosslinked linear polyacrylamide as a CE matrix for the separation of DNA [11]. At the time, we polymerized the monomer in the column; however, we and others, especially Stellen Hjerten of Uppsala, realized that with appropriate pressure and manipulation of the polymer, the matrix could be replaced after each run. We recognized that this would be very important for DNA sequencing where automation was necessary and, in 1993, introduced the first separations of DNA sequencing using replaceable polymer matrices [12]. At that time we were able to separate 300 bases in roughly 0.5 h with high accuracy. Today, with further improvements, we now sequence over 1000 bases with high accuracy in less than 1 h, see Fig. 2 [13]. CE led us into studies in mass spectrometry, some work related to what we had done in the early 1980s in LC=MS. CE=MS is an important tool and is becoming more widely used with the passing of time. As an example, we have used CE=MS for analysis of binding of 300 or more combinatorial library compounds in a single run using affinity capillary electrophoresis coupled to electrospray MS [14]. The advantage of the capillary system is that all binding is done in solution, which reduces ambiguities that may exist when the binding is done at surfaces. Additionally, we have been interested in high throughput mass spectrometry, and one of our activities now is the study of interfacing microchips to mass spectrometry for high throughput analysis [15]. We believe that microfluidics in mass spectrometry will be important hybrid techniques in the coming years. With this rather incomplete survey of my past 40 years in the field of separation science, a logical question to conclude this paper is: What have I learned? The first important thing that I have learned is that one needs to continually change one’s focus. Problems emerge and are actively pursued. After a period of time they are solved, and it is time to move in new directions. The difficulty, of course, is what to bet on; that is, what fields to move into? Here, my only suggestion is to go with your gut feelings. Sometimes you will guess wrong and bet on the wrong horse, but many times, based on your experience and knowledge of the field, you will bet right. Another interesting thing that I have learned is that it is best to devote your efforts to presenting your results and not to get into arguments with others. We have a saying in our laboratory — “Let the data speak for itself.” Finally, continually ask the question “Why do this?” You need to understand the needs of the field and what you really can potentially bring to the table. When I began in 1960, the biological sciences were far removed from the type of chemistry that I was studying. Indeed, in the field of gas chromatography, we were dealing with volatile substances, and most biological substances are hardly volatile. Today, however, there is a strong integration of the biological and chemical sciences. If I were a student beginning my career today, I would certainly make sure that I would have a strong background in the biological sciences. Then, using my knowledge in chemistry, I would probably end up all right. Finally, my advice to students is not to be afraid of change. Change is inevitable and we can either be reactive or proactive. For my part, it is much better to be proactive and be part of the change rather than simply to react after the facts. I should end by saying that separations will always be with us. Even if we can analyze very

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Fig. 2. Electrophoretic separation of DNA sequencing fragments generated on ssM13mp18 with BigDyelabeled universal (21) primer and AmpliTaq FS (From Salas-Solano et al. [13].)

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(From left to right): Ray Scott, Agnes Hala´sz, Istva´n Hala´sz (deceased), Trudy Karger, Barry Karger, Geoffrey Davies, Georges Guiochon, Lois Beaver.

complicated mixtures directly using tools hitherto unknown, there will always be a need for obtaining purified substances. Separation science plays a critical role in purification. The fundamentals of separation — selectivity and efficiency — will always be needed and should be a part of a student’s education in the chemical sciences.

30.I.1. A look at separation: science in the 21st century As we move into the new millenium, it is clear that separations will become faster, more automated, operated in a microfluidic environment and handle more complex problems. Some trends are already discernable. Capillary electrophoretic separations on a microfabricated device (lab-on-a-chip), multidimensional chromatography=electrophoresis coupled to high resolution mass spectrometry=nuclear magnetic resonance, and separation and analysis at the attomole level. It is likely that nanotechnology will revolutionize some forms of separation. By nanotechnology, I mean designing structures on a molecular level. Such structures are just beginning to emerge; for example, the use of posts, appropriately spaced, to mimic obstacles in gels for DNA separation. Separation speeds not yet imaginable may be achieved. Besides these instrumental advances, creative new approaches using chemistry and biology can be envisioned. For example, combinatorial chemistry offers us the possibility of synthesizing thousands of compounds rapidly and automatically. Just as these libraries are screened for drug leads, they can be screened for chemical selectivity. This already exists using phage display to find compounds for purification of proteins by affinity chromatography. This field will only grow in the years ahead. Additionally, as we understand life processes that inevitably involve molecular recognition (e.g.,

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protein–protein interactions), our ability to design selective phases or surfaces will be enhanced. These and other separation advances will have a major impact on all fields of science, especially the life sciences. To provide in depth predictions, even for 5 years from now, would be foolhardy. Nevertheless, scientists one to two generations in the future may view our separation era as primitive. References 1a. 1b. 2. 3. 4.

5.

6.

7.

8.

9. 10.

11.

12.

13.

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15.

B.L. Karger and W.D. Cooke, Effect of column length on resolution under normalized time conditions, Anal. Chem., 36 (1964) 985–991. B.L. Karger and W.D. Cooke, Effect of particle size and average velocity on resolution under normalized time conditions, Anal. Chem., 36 (1964) 991–995. B.L. Karger and R.W. Giese, Reversed phase liquid chromatography and its application to biochemistry, Anal. Chem., 50 (1978) 1048A. B.A. Persson and B.L. Karger, High performance ion pair partition chromatography: The separation of biogenic amines and their metabolites, J. Chromatogr. Sci., 12 (1974) 521–528. J.N. LePage, W. Lindner, G. Davies, D.E. Seitz and B.L. Karger, Resolution of the optical isomers of dansyl amino acids by reversed phase liquid chromatography with optically active metal chelate additives, Anal. Chem., 51 (1979) 433–435. N.T. Miller, B. Feibush and B.L. Karger, Wide-pore silica-based ether-bonded phases for separation of proteins by high performance hydrophobic interaction and size-exclusion chromatography, J. Chromatogr., 316 (1985) 519–536. P. Oroszlan, R. Blanco, X.-M. Lu, D.M. Yarmush and B.L. Karger, Intrinsic fluorescence studies of the kinetic mechanism of unfolding of α-lactalbumin on weakly hydrophobic chromatographic surfaces, J. Chromatogr., 500 (1990) 481–502. X.-M. Lu, K. Benedek and B.L. Karger, Conformational effects in the HPLC of proteins: further studies of the reversed phase chromatographic behavior of ribonuclease A, J. Chromatogr., 359 (1986) 19–29. P. Oroszlan, S. Wicar, G. Teshima, S.-L. Wu, W.S. Hancock and B.L. Karger, Conformational effects in the reversed phase chromatographic behavior of recombinant human growth hormone (rhGH) and N-methionyl recombinant human growth hormone (Met-hGH), Anal. Chem., 64 (1992) 1623–1631. B.L. Karger, A.S. Cohen and A. Guttman, High performance capillary electrophoresis in the biological sciences, J. Chromatogr. Biomed. Appl., 492 (1989) 585–614. A.S. Cohen, D.R. Najarian, A. Paulus, A. Guttman, J.A. Smith and B.L. Karger, Rapid separation and purification of oligonucleotides by high performance capillary gel electrophoresis, Proc. Natl. Acad. Sci., USA, 85 (1989) 9660–9663. D.N. Heiger, A.S. Cohen and B.L. Karger, The separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields, J. Chromatogr., 516 (1990) 33–48. M.C. Ruiz-Martinez, J. Berka, A. Belenkii, F. Foret, A.W. Miller and B.L. Karger, DNA sequencing by capillary electrophoresis with replaceable linear polyacrylamide and laser-induced fluorescence detection, Anal. Chem., 65 (1993) 2851–2858. O. Salas-Solano, E. Carrilho, L. Kotler, A.W. Miller, W. Goetzinger, Z. Sosic and B.L. Karger, Routine DNA sequencing of 1000 bases in less than one hour by capillary electrophoresis with replaceable linear polyacrylamide solutions, Anal. Chem., 70 (1998) 3996–4003. Y.-H. Chu, Y.M. Dunayevskiy, D.P. Kirby, P. Vouros and B.L. Karger, Affinity capillary electrophoresis-mass spectrometry for screening combinatorial libraries, J. Am. Chem. Soc., 118 (1996) 7827– 7835. Q. Xue, Y.M. Dunayevskiy, F. Foret and B.L. Karger, Integrated multichannel microchip electrospray ionization mass spectrometry: Analysis of peptides from on-chip tryptic digestion of imelittin, Rapid Commun. Mass Spectrom., 11 (1997) 1253–1256.

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D.31. Jerry W. King Jerry W. King was born on February 19, 1942, in Indianapolis, IN. He graduated from Butler University in 1965, with a B.Sc. in Chemistry. J.W. King continued with graduate studies at Butler University and the University of Utah, where he worked with J. Calvin Giddings on supercritical-fluid chromatography. In 1973, J.W. King received his Ph.D. from Northeastern University in Boston, MA under the direction of Barry Karger. He then conducted postdoctoral research in physical chemistry under Dan Martire at Georgetown University in Washington, DC. J.W. King has worked with several industrial companies and R&D organizations prior to his appointment as a research scientist at USDA (United States Department of Agriculture). These have included Arthur D. Little, Inc. (Cambridge, MA), Union Carbide Corporation (Bound Brook, NJ), and CPC International (Summit-Argo, IL). While at CPC he was in charge of HPLC methods development for biotechnology, installation of a process monitoring HPLC in the pilot plant, and industrial products analysis. Since 1988, he has been the Lead Scientist of the Critical Fluid Technology Group at the National Center for Agricultural Utilization Research (NCAUR) in Peoria, IL. His research interests there have included the development of critical fluid technology for food and agrimaterial processing, as well as for the analysis of toxicants, nutrients, and fats=oils. He has authored over 125 publications (including two patents) in SFE (supercritical-fluid extraction), SFC (supercritical-fluid chromatography), and related separation techniques, and has lectured extensively on these subjects over the past 14 years at national and international symposia, including the ACS Shortcourse on SFE=SFF=SFC with Larry Taylor. J.W. King has organized many symposia on SFE and SFC, including the well-known International Symposia on SFC and SFE. He serves on the Editorial Board of the Journal of Supercritical Fluids, Italian Journal of Food Science, Seminars in Food Science and is a member of the American Chemical Society (ACS), American Oil Chemists Society (AOCS), Institute of Food Technology (ITF), Association of Official Analytical Chemists (AOAC), and regional=international supercritical fluid technology groups. In 1993, J.W. King was named Scientist of the Year at NCAUR, and in 1994 was elected a corresponding member of the Academia dei Georgofili in Florence, Italy. J.W. King has been awarded the Chicago Chromatography Discussion Group’s Merit Award for significant contributions to chromatography and was elected to Who’s Who in America. He was awarded the Harvey Wiley Award of the AOAC in 1997 for his research in analytical SFE. In 1998, received the Merit Award from the Midwest SFC Group=Tri-State Discussion Group for consistent contributions in the supercritical fluid technology field, and the Award of Excellence at the 8th International Symposium on SFC=SFE for pioneering achievement, leadership, and enthusiasm in the development of supercritical fluid technology and the education of others. In 2000, he received the Keene P. Dimick Award for his contributions to the field of gas and supercritical-fluid chromatography. See Chapter 5B, a, d, h, o, s

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31.I. THE USE OF SUPERCRITICAL FLUIDS IN ANALYTICAL CHEMISTRY Jerry W. King National Center for Agricultural Ultilization Research, 1815 North University St., Peoria, IL 61604-3999, USA

My first initiation into the realm of supercritical fluid technology occurred in 1967 when I was considering at which university I should continue my graduate studies. Reading a publication by J. Calvin Giddings sparked my interest in the University of Utah and I subsequently visited Cal that summer to find out more about this interesting topic. Late in the summer of 1968, I migrated to Salt Lake City and began research in this area, which would later become the focal point of my research career. Supercritical-fluid chromatography (SFC) in those days went under the titles as ‘dense phase gas chromatography’. Giddings’ research group at Utah was laying the basic groundwork, not only for the theoretical advantages that were afforded by operating gas chromatographic columns at high velocities (and hence high pressure), but the unique solvent-like characteristics these gaseous (fluid) mobile phases took on under tremendous pressure. I became involved under the watchful eye of Giddings’ research collaborator, Marcus Myers, in conducting solute migration studies at pressures up to 2000 atmospheres using carbon dioxide as the mobile phase. In those days, no columns existed that were packed to withstand such enormous pressures; likewise all of the experimental equipment had to be designed and fabricated from scratch. With the help of my colleagues, we overcame the barriers to working under such adverse conditions, but this was not without considerable pain and suffering in a figurative sense. The result was my first coauthored publication [1] entitled, “Dense gas chromatography at pressures to 2000 atmospheres”. In this publication, we had made differential migration experiments for simple solutes dissolved in what would later become known as supercritical carbon dioxide (SC-CO2 ), and related the shift in chromatographic retention factors with pressure, to the changing ‘solubility parameter’ of the carrier gas. At 2000 atmospheres, carbon dioxide was beginning to show the solvency characteristics of moderately polar organic liquid solvents. Attempts at migrating or solubilizing macromolecular solutes, such as long-chained alkanes, various synthetic polymers, and proteins were partially successful. In today’s light, these experiments would complement developments in a parallel field, supercritical-fluid extraction (SFE). However, the use of the Hildebrand’s ‘solubility parameter concept’ explained the qualitative trends we saw in our data and rather fascinated me, as it did many chromatographers. I would make use of this concept almost 20 years later, as I will comment on shortly. After a brief stint in industry, I resumed my graduate studies under two excellent mentors, Barry Karger at Northeastern University, and Dan Martire at Georgetown University. Research under these two mentors in physico-chemical surface and solution thermodynamic measurements by gas chromatography cemented by a physical chemistry approach to the separation sciences which had begun under Giddings. Unlike most of Karger’s students who were involved in HPLC and capillary electrophoresis, my

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research involved making sorption isotherm and precise retention volume measurements by GC using water, a rather unusual stationary phase [2]. As it turned out, I would revisit water over 25 years later in a slightly altered form. From 1976 to 1986, I worked at several industrial positions as a separations specialist doing proprietary research that involved considerable use of GC, HPLC, and the development of process methodology. One memorable aspect during this time in my career was my involvement at Arthur D. Little, Inc. in Cambridge, MA in a venture development team that later became the nucleus of Critical Fluid Systems, Inc. The company purchased a crude SFE system that I had developed as an Assistant Professor in academia for their initial extraction studies. The design template for this apparatus would be repeated many times in slightly altered formats by many researchers and companies, resulting eventually in a hybrid system known as the Spe-ed unit [3] offered by Applied Separations, Inc. of Allentown, PA. In 1986, I joined the Agricultural Research Service Northern Regional Research Center in Peoria, IL, where my interest and passion in supercritical fluid technology was rekindled. USDA had been exploring the use of supercritical fluids, such as SC-CO2 , as an alternative solvent media, to replace hexane as an extraction solvent. Research by John Friedrich at the Peoria laboratory had shown that at high pressures and temperatures, SC-CO2 could readily solubilize significant quantities of vegetable oil from seeds, and lead to quick and efficient extractions in an environmentally benign matter [4]. I took up the charge of working in this area invoking the solubility parameter concept as I had done under Giddings to explain the salient features of oilseed solubility in supercritical fluid media [5]. However in 1988, I was approached by a sister agency, the Food Safety and Inspection Service (FSIS) regarding the possibility of using SC-CO2 extraction to replace organic solvents for the extraction of lipid phases from meat products for pesticide residue analysis. This proved to be an ideal opportunity for transferring our processing methodology down to an analytical scale and to reduce the analyst’s dependence on organic solvents for sample preparation. This transfer of critical fluid technology from process application to analytical use, and vice versa, has been a seminal theme in research conducted under my direction over the past 11 years. Commensurate with the above, development was the first offering of capillary SFC systems by the Lee Scientific Company of Salt Lake City, UT. I convinced USDA to purchase one of these chromatographs on the basis that we just might be able to detect pesticide residues via flame ionization detection (FID), and that SFC could help define some of the physico-chemical parameters vital to achieving optimum results in SFE. It also turned out that SFC is a quick and efficient tool for the separation of many different types of lipid species; hence the technique has been used for the last decade to support our process SFE program as an analysis tool [6]. I became intrigued as to how the separation mechanism of SFC worked and eventually wrote a paper entitled, “Fundamentals and applications of supercritical-fluid extraction in chromatographic science” [7], and which has been widely cited ever since. This paper invoked the solubility parameter theory coupled with Flory-Huggins solution theory to offer a plausible explanation of the retention trends observed in capillary SFC. By the early nineties it was apparent that simple analytical SFE was not a panacea for all of the sample preparation woes facing the analyst. It occurred to me about

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this time that there might be some advantage in coupling SFE with a crude form of sorbent chromatography to allow in-situ cleanup of the resultant SFE extract. I reasoned by analogy that if the pesticide residue analyst could use non-polar eluents with tempered surface activity sorbents, that a form of supercritical fluid ‘normal’ phase chromatography could provide the same function. I believe that we were probably the first research group to demonstrate this principle [8], but many other researchers eventually took up the method. Today, both on-line and off-line use of sorbent beds in analytical SFE are routinely used for environmental, food, and drug analysis. A fortunate and productive collaboration with Marvin Hopper of the FDA’s Total Diet and Pesticide Research Center in Lenexa, KS, led to some other developments in analytical SFE that are based on chromatographic principles. Hopper had been using for some time a diatomaceous earth sorbent called Hydromatrix for liquid phase cleanup of extracts prior to pesticide residue analysis. This material was not unlike the early packed column GC support materials. Utilizing this as a sample matrix dispersant as well as extraction cell filler facilitated very efficient SFE of a variety of different types of food matrices. Eventually a USA patent [9] was issued to the author and Mr. Hopper for this development, and the material is widely used in many laboratories. The experience with Hydromatrix and like sorbents only wetted my appetite to see how other sorbents faired in the presence of dense SC-CO2 . Since SC-CO2 was not the best solvent for polar molecules, I reasoned that there might be some advantage in looking at the separation process in a different light; perhaps one could remove unwanted coextractives obtained in the total extract from polar target analytes. Because I was aware of the research studies of polymer researchers using gas chromatography to determine solute–polymer interactions in molten polymers (i.e., ‘inverse’ GC), I coined the term ‘inverse’ SFE [10] to characterize the above process. Due to circumstances beyond our control, we did not fully exploit this principle, but others filled the void and it has proven particularly facile for the analysis of drugs and active principles in pharmaceutical formulations. Inverse GC, or using gas chromatography for physico-chemical measurements, has played a key role in my career since graduate school. I have authored or coauthored papers that have investigated solute probe interactions with volatile stationary phases, such as water and carbon tetrabromide, molten sulfur, and polystyrene above its glass transition temperature, and recently, vegetable or seed oils as stationary phases. The last study, along with research characterizing sorbent resins by injecting sorbate probes, has been completed to support concurrent studies in analytical and process SFE. One noteworthy citation [11], often ignored by others studying high pressure adsorbate– adsorbent interactions, correlated sorbate breakthrough volume trends with molecular interactions at the gas–solid interface. Further studies in our laboratory using gas– solid chromatography to study the effect of carbon dioxide versus other carrier fluids, on solute breakthrough volumes and adsorption coefficients, have shown that CO2 substantially reduces retention volumes on many common analytical sorbents relative to helium and more ‘ideal’ fluids. Thus, CO2 even close to ambient conditions, behaves in a non-ideal manner through increased gas phase interactions (i.e., second mixed virial coefficients) with solutes and its ability to displace sorbates from sorbent surfaces. The above technique can be used to mimic the behavior of high pressure gas-adsor-

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Fig. 1. SFE–SFC separation of the on-line CO2 extract from a single dried-fruit beetle (live specimen).

bent systems such as those that occur in SFE schemes. Utilizing a modified inverse GC approach, we measured breakthrough phenomena for selected odoriferous probe molecules that mimicked components we wanted to remove from a recycle SC-CO2 in our pilot plant operations at NCAUR (USDA’s Northern Regional Research Center became the National Center for Agricultural Utilization Research in the early 1990s through an Act of Congress). The derived data permitted an estimate to be made on the effective service lifetime of sorbents, such as activated carbon, for purifying the SC-CO2 , recycled back into the extraction vessel. Returning to our use of chromatography with supercritical fluids, we have practiced both open tubular and packed column SFC techniques. Although most of our studies have employed capillary SFC, we have also found niches for the packed column mode, and find arguments by proponents for one technique over the other, rather immature. I believe that one of the great benefits to using analytical SFC that often goes untouted, is its elimination of sample cleanup techniques that normally must be performed prior to GC. The versatility of pressure or density programmed SFC allows one to optimize a separation for the determination of target analytes, and to then ‘program out’ other species that interfere with the analysis. Capillary SFC also allows the establishment of signature profiles that can be used by analysts in product deformulation schemes. For certain specific micro-analysis problems, SFC can be combined with on-line SFE using small extraction cells. This principle is nicely demonstrated by the extraction of a live insect in a SFE cell with transfer of extract containing an active pheromone principle and cuticle lipids to a capillary SFC column (Fig. 1) [13]. A similar approach to the one described above was also developed for the on-line methylation of oils extracted from seeds in the extraction cell, prior to packed microbore SFC analysis. This early attempt was the beginning of a long association with the formation of fatty acid methyl ester (FAME) derivatives, not only for analytical purposes, but for process reaction chemistry in supercritical fluids. Several years ago we were asked to look into the feasibility of determining speciated fat levels in food products via FAME–GC analysis as mandated by the Nutritional Labeling and Education Act (NLEA). We developed a rather novel approach to this analytical problem

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Fig. 2. Development and utilization of lipase reaction in SC-CO2 at NCAUR.

by employing an enzyme (lipase) as an integral part of the extraction step, thereby facilitating the formation of the derivative after extraction with SC-CO2 , all in one step. The quantitative results were most gratifying and eventually the whole process was converted into an entirely automated scheme permitting SFE–SFR (supercritical fluid reaction) GC to be accomplished unattended overnight. This saga is nicely recounted in my Harvey Wiley Award address manuscript, and is amply described in Fig. 2. Many of the principles learned from analytical supercritical-fluid chromatography have been incorporated into some of our processing schemes for fractionating or isolating high value chemicals from natural product matrices. For example, we have used a normal phase silica gel preparative chromatography step in enriching tocopherol extracts from a front end SFE step. Significant enrichments can be achieved to prepare solute-enriched extracts for the nutraceutical marketplace. Similarly, the same theme has recently been employed to isolate phospholipid-rich extracts, using an ‘all-green’ eluent consisting of pressurized SC-CO2 , ethanol, and water. Preparative and production scale SFC will play an increasing role in processing schemes employing critical fluids; the most obvious example being the new fish oil ethyl ester SFE-SFC plant in Tarragona, Spain. No researcher is an island onto themselves and I have been aided over the years by my association with many excellent colleagues. One deserves special mention, Janet Snyder, who has shared her expertise on lipid analysis with me for the past decade. We have coauthored many analytical SFC publications together; but one not directly involving SFC, but rather SFE-GC, deserves special mention. We were asked on an emergency basis to explore the feasibility of using analytical SFE to determine the presence of contaminants from smoke in various items of produce that had been exposed to fire in a storage cave. The problem was daunting, since the compounds (aromatics) that would indicate such exposure were at the parts-per-billion level in meat and cheese products. Mrs. Snyder, with some advice from me, developed an excellent SFE-GC method that clearly showed, relative to background chromatograms, that the presence of such aromatic contaminants as naphthalene and ethylbenzene, were indicative of the food matrix being exposed to smoke and fire conditions. As with many researchers in the field of chromatography, the use of tandem techniques has always been intriguing. Unfortunately analytical methods developed using hyphenated techniques are not always warmly received by the practicing analyst because of their complexity. For this reason, we have in recent years pursued off-line analytical SFE methodology as the methodology of choice, and the one that is most

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likely to be adopted for routine use. However some of our earlier work and the efforts of others, has shown the SFE–SFC–GC, SFE–SFC–MS, and similar tandem techniques can fulfill a specific role. Our work has shown the SFC, both low and high resolution options, can be used to advantage for sample and extract cleanup, particularly in the field of trace toxicant analysis. Unpublished studies in our laboratory on using cross-linked polymer media (traditionally used for size exclusion chromatography), have revealed a better understanding of the fundamental basis on how these packing materials behave in the presence of supercritical fluids, in affecting solute separation. Interestingly, sight glass measurements have shown that highly cross-linked styrene=divinylbenzene resins do not swell in the presence of quite high pressure SC-CO2 , and that resultant separations are more the result of adsorption interaction of the solutes with the column packing than any molecular sieving effect. Finally, an example of our research that indicates the value of a physico-chemical approach to chromatography or extraction, are studies on the effect of using binary supercritical fluids on solute solubility. Initial studies in this area were undertaken to answer the vexing question, does the presence of helium in SC-CO2 reduce solute solubility in the compressed medium? The use of helium with SC-CO2 had also been implicated in the instability of retention times in SFC and we were anxious to resolve the question. With the help of an excellent postdoctoral researcher, Zhouyao Zhang from Canada, we precisely measured the solubilities of several solutes in neat SC-CO2 , and SC-CO2 containing a defined amount of helium in the cylinder. These solubility measurements were also accompanied by GC analysis of the binary gas mixture by traditional thermal conductivity detection and high pressure densitometer measurements of the density of the binary fluid mixture, both as a function of the time of use of the gas cylinder source. Our combined measurements definitely showed that helium in SC-CO2 depressed solute solubilities. After pondering this result for sometime, I decided that, if this were a general phenomena (and it is!), it might be put to good use in achieving more selective analyte extraction in SFE. Hence I instructed Zhang to try various mixtures of nitrogen with SC-CO2 at different extraction pressures and temperatures to see if he could obtain a lipid-free extract from a food matrix (poultry peritoneal fat containing pesticide residues). Indeed, the suppression of the lipid solubility that we had seen in our initial solubility studies held in this case and we obtained an extract that could be used directly for GC-electron capture detection analysis of pesticide residues. Over the last year we have built several devices for generating these binary fluid phases and are using them not only in our analytical SFE research, but to generate binary fluid mixtures of utility in conducting reaction chemistry (i.e., hydrogenation) in supercritical fluid media.

31.I.1. In summary I believe that the astute scientist should always be aware of the technology transfer possibilities in the research that she=he is developing. This is true whether one is considering SFC to SFE or vice versa, or conveying useful results from analytical to process application. The techniques and results developed in one form of chromatography may

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have implications in similar separation techniques, be they small (analytical) or large (engineering) in magnitude. These rules have guided my approach to research over the years and provided gratifying extensions to my career in chromatography and separation science.

References 1. 2.

3. 4. 5.

6.

7. 8. 9. 10. 11.

12. 13.

J.C. Giddings, M.N. Myers and J.W. King, Dense gas chromatography at pressures to 2000 atmospheres, J. Chromatogr. Sci., 7 (1969) 276–283. J.W. King, A.K. Chatterjee and B.L. Karger, Adsorption isotherms and equations of state of insoluble vapors at the water–gas interface as studied by gas chromatography, J. Phys. Chem., 76 (1972) 2769–2777. J.W. King, Analytical-process supercritical-fluid extraction: A synergistic combination of solving analytical and laboratory scale problems, Trends Anal. Chem., 14 (1995) 474–481. G.R. List, J.P. Friedrich and J.W. King, Supercritical CO2 extraction and processing of oilseeds, Oil Mill Gazetteer, 95 (6) (1989) 28–34. J.W. King, Critical fluids for oil extraction, in: Technology and Solvents for Extracting Oilseeds and Nonpetroleum Oils. P.J. Wan and P.J. Wakelyn (Eds.), AOCS Press, Champaign, IL, 1997, pp. 283–310. J.W. King and J.M. Snyder, Supercritical fluid chromatography — A shortcut in lipid analysis, in: R. MacDonald, and M. Mossoba, (Eds.), New Techniques and Applications In Lipid Analysis. AOCS Press, Champaign, IL, 1997, pp. 139–162. J W. King, Fundamentals and applications of supercritical fluid extraction in chromatographic science, J. Chromatogr. Sci., 27 (1989) 355–364. J.E. France, J.W. King and J.M. Snyder, Supercritical fluid-based cleanup technique for the separation of organochlorine pesticides from fats, J. Agric. Food Chem., 39 (10) (1991) 1013–1016. M.L. Hopper and J.W. King, Supercritical fluid extraction enhancer, U.S. Patent 5,151,188, issued on September 29, 1992. J.W. King, Integration of sample cleanup methods into analytical supercritical fluid extraction, Am. Lab., 30 (8) (1998) 46–58. J.W. King, Supercritical-fluid adsorption at the gas-solid interface, in: T.G. Squires and M.E. Paulaitis (Eds.), Supercritical Fluids — Chemical and Engineering Principles and Applications, ACS Symposium Series #329, American Chemical Society, Washington, D.C. 1987, pp. 150–171. J.W. King, Analytical supercritical fluid techniques and methodology: Conceptualization and reduction to practice, J. Assoc. Off. Anal. Chem. Int., 81 (1998) 9–17. J.W. King, Applications of capillary supercritical-fluid chromatography-supercritical fluid extraction to natural products, J. Chromatogr., 28 (1990) 9–14.

D.32. J.J. Kirkland J.J. Kirkland was born in Winter Garden, FL on May 24, 1925. After receiving A.B. and M.S. degrees in chemistry from Emory University in 1948 and 1949, respectively, Joseph J(ack) Kirkland worked for Hercules from 1950 to 1951. He left to earn a Ph.D. in Analytical Chemistry at the University of Virginia in 1953. He was employed by E.I. DuPont de Nemours Co. at the Experimental Station, Wilmington, Delaware, until 1992, when he retired as a DuPont Fellow. He then was co-founder of Rockland Technologies, Inc., where he was Vice-President, Research and Development. In 1997,

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this organization merged into the Hewlett-Packard Co., where he currently is Manager, Research and Development, Newport Site. Jack is on the Editorial Advisory Board of the Journal of Chromatography and the Journal of Chromatographic Science. He edited the book, “Modern Practice of Liquid Chromatography” (1971), co-authored “Introduction to Modern Liquid Chromatography” (1974), second edition, (1979), “Modern Size-Exclusion Liquid Chromatography” (1979), and “Practical HPLC Method Development” (1988), second edition (1997). He was co-professor of the American Chemical Society (ACS) Short Course, Practical HPLC Method Development, and coauthor of two taped ACS Audio Short Courses on liquid chromatography. Kirkland has authored over 125 major publications, mainly in separation sciences, and holds twenty-six US patents. Recent research interests involve high-resolution separations, HPLC method development, silica supports and silane bonding reactions. He received the 1972 American Chemical Society Award in Chromatography, the 1973 Delaware Section ACS Publication Award, the 1974 Dal Nogare Memorial Award in Chromatography from the Chromatography Forum of the Delaware Valley, the 1979 Anachem Award, the 1982 Torbern Bergman Medal in Analytical Chemistry from the Swedish Chemical Society, the 1988 Delaware Section ACS Award, the 1993 Eastern Analytical Symposium Award in Separation Science, DuPont’s Lavoisier Award in 1997, the A.J.P. Martin Chromatography Award Medal in 1997 and the 1999 Merit Award of the Chicago Chromatography Discussion Group. In 1974, he was awarded the honorary D.Sc. degree by Emory University. Kirkland was an Adjunct Professor of Chemistry at the University of Delaware. Kirkland significantly contributed to the development of both gas and liquid chromatography. His many awards testify to his distinguished work in all phases of chromatography: for his development of equipment, techniques, and special column packing for analytical liquid chromatography. His efforts have also been directed to education on modern liquid chromatography through his widely successful books and teachings of a short course on HPLC for the American Chemical Society to thousands of students and chromatographers. See Chapter 5B, a, d, e, g, h, k

32.I. SEARCH FOR NEW MEASUREMENT TECHNOLOGIES Joseph Jack Kirkland Hewlett Packard Co., Little Falls Analytical Division, Newport Site, 538 First State Boulevard, Newport, DE 19804, USA

This summary is in response to the Editors’ request to provide an overview of my technical career, primarily as a supplement to an earlier account given in Ref. [1].

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Perhaps best known for efforts in the separation sciences, I have always preferred to be considered as a generalist in analytical chemistry because of training and a range of experiences. As examples, studies for a Master’s degree at Emory University involved organic polarography; two years of research at the Hercules Powder Company used ultraviolet and infrared spectroscopy; dissertation studies for a Ph.D. degree at the University of Virginia employed the trace analysis of noble metals with organic complexing reagents. This early broad training served to keep me open to new technology opportunities for analytical measurements. I began my DuPont research career in 1953, primarily working in molecular spectroscopy. Studies with insoluble organic pesticides led to the development of the first quantitative techniques in infrared spectroscopy with pressed potassium bromide disk techniques [2]. However, interactions with DuPont’s Steve Dal Nogare (see Chapter 2) in 1954 convinced me that separation methods were the important future in analytical chemistry. In three hours, using a new technique called gas chromatography (GC), Steve solved a problem for me that I had unsuccessfully worked on for several weeks with other approaches. Intensive work on several GC projects in DuPont’s Biochemical Department followed in rapid succession. Preparative GC was developed for isolating highly purified materials to be used as standards and for field tests [3]. Volatile derivatives of nonvolatile agricultural chemicals allowed GC analyses that were previously not adequately handled by other methods [4]. The first reported method using GC for the trace analysis of pesticides was devised [5]. Fluorocarbon-based supports were developed for analyzing samples that would attack and degrade the then-used silica-based column packing supports [6]. Porous layer open tubular (PLOT) capillary columns were designed and tested [7]. However, with all its great separation power, GC was not the total answer for solving many problems associated with the agrochemical business. Many compounds of interest were not only nonvolatile, but also did not lend themselves to the formation of acceptable volatile derivatives that allowed GC analysis. The breakthrough for solving many difficult agrochemical problems came in 1964. During a fortunate trip to Europe, I visited the Eindhoven University laboratories in The Netherlands. There I found. J.F.K. Huber just initiating studies that ultimately led to the technique now called, high-performance liquid chromatography (HPLC). Returning to the United States, I quickly convinced my research manager that we should undertake a major project to develop this promising new technique for analyzing agrochemical, pharmaceuticals and related compounds. If this new HPLC technology were ever to be the answer to our difficult problems two sorely needed practical developments were apparent: a sensitive and reliable detector, and better column packing materials. Fortunate interactions with scientists in DuPont’s Engineering Department made me aware of a high-quality in-house UV photometer developed to monitor liquid process streams for trace components. Modification of this instrument with suitable optics and a low-volume cell immediately solved the requirement of a stable, sensitive UV detector suitable for HPLC separations [8]. Solving the need for better column-packing materials was more difficult. Then, separations were done with diatomaceous earth supports of the type used in GC, but with particle sizes in the 30–50 µm range. Fortunately, expertise within DuPont again

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Fig. 1. Prototype DuPont Model 820 liquid chromatograph, circa 1969. Right, J.J. Kirkland; left, R.E. Leitch.

came to the rescue. In the adjacent laboratory was a close friend and a world-renown expert in silica chemistry, Ralph K. Iler. Ralph had already consulted with me in developing PLOT columns for GC. The basic technology involved in this work was then adapted to create superficially porous particles, that consisted of a thin, porous shell coated on a solid silica core of about 25 µm. These particles showed excellent mass transport characteristics, resulting in the first packing materials that produced fast, high-quality HPLC separations in keeping with the theory proposed years before by Cal Giddings and others. Based on this and other new technology, DuPont’s Instrument Products Division in the late 1960s began to market HPLC instrumentation and columns based on the superficially porous particles called Zipax controlled porosity chromatographic support [9]. (Fig. 1 is a circa 1969 photograph of the prototype for the DuPont Model 820 liquid chromatograph subsequently marketed world wide by DuPont’s Instruments Products Division. Note the tall oven (with the 820 label) required to house the one meter Zipax columns commonly used then). Using the Zipax model, ion-exchange packings also were devised so that this powerful technique could be used for needed fast, high-resolution separations. Using these new column-packing materials to develop needed analytical data, DuPont filed the first petition based on HPLC for approval of a new fungicide to the FDA (now an EPA responsibility) [10]. HPLC separations in the late 1960s (other than ion exchange) generally were done by liquid–liquid chromatography. Here, the column support contained mechanically held liquid stationary phases for a partitioning process needed for separations. This technique was effective but awkward to use routinely, since the stationary liquid was difficult to maintain unchanged in the presence of a moving mobile phase. Realizing

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that a chemically bonded stationary phase would offer a significant practical advantage, I undertook work with Paul C. Yates that ultimately resulted in Zipax particles coated with a covalently bonded, three-dimensional porous silicone polymer [11]. This new column-packing material allowed stable separations that were easily repeated routinely by operators. These new packings eliminated precolumns and presaturating of the mobile phase with the stationary phase, techniques required when using mechanically held liquid stationary phases. Columns of these materials quickly were used routinely within DuPont laboratories, and versions of these materials were subsequently marketed by DuPont’s Instrument Products Division under the Permaphase trademark. During this period, I also was investigating preparative liquid chromatography to solve some of our difficult problems. Here I had a particularly satisfying relationship as a mentor to Joseph J. DeStefano, who later used this topic for a dissertation to obtain a doctorate at the University of Delaware. My close relationship and friendship with Joe have continued, first in DuPont’s Biochemicals (Agricultural Chemicals) Department, the Instrument Products Division, the Central Research and Development Department, in our own company, Rockland Technologies Inc., and now with Hewlett-Packard. Theoretical studies by others in the early 1960s had predicted that much faster, higher resolution separations would result if particle sizes much smaller than the traditional 25 µm materials that were used then. Practice with much smaller particles proved difficult. However, J.F.K. Huber again led the way and showed that rapid separations could be obtained with columns of 10 µm diatomaceous-earth particles. Unfortunately, the techniques for preparing such columns were highly technique-dependent and this approach did not result in a general following. Nevertheless, the success of this approach encouraged attempts to use small particles. Again with the assistance of Ralph Iler, I developed a method for preparing narrow particle size, porous silica microspheres of <10 µm. However, for practical use, an efficient method for preparing packed columns of these materials was needed. Fortunately, a modification of the slurry packing method used previously in preparing columns of Permaphase packings [11] proved satisfactory. This development then led to the first successful columns with high-performance porous silica microspheres, and such columns were quickly used within DuPont in 1970 to produce separations such as that shown in Fig. 2. A paper on these new porous silica microspheres was given as my ACS Chromatography Award address at Boston in April 1972 [12], and DuPont subsequently made columns of these materials commercially available as Zorbax porous silica microsphere chromatographic packing. Initial studies with these new particles used liquid–liquid partition chromatography, but liquid–solid (adsorption) technology quickly followed [13]. Work on the porous silica microspheres continued after my transfer to DuPont’s Central Research and Development Department in 1973. With Paul E. Antle, these materials were used extensively for high-resolution preparative separations in a variety of important projects [14]. Covalently bonded monofunctional silane stationary phases were developed and similar materials ultimately were marketed by DuPont’s Instrument Products Division [15]. Interest in high-speed polymer separations then led to a series of fruitful collaborations with Wallace W. Yau, who was primarily concerned then with the fundamentals of polymer characterization. After I developed wide-pore silica microspheres suitable for

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Fig. 2. Separation of hydroxylated aromatics by liquid–liquid chromatography. Column, 250 ð 3:2 mm, 6 ˚ ; stationary liquid, β,β0 -oxydipropionitrile, 30% by weight; mobile µm porous silica microspheres, 350 A phase, hexane saturated with stationary phase; pressure, 600 psi; flow rate, 1.00 ml=min; temperature, 27ºC; sample, 4 µl of mixture in hexane. Reprinted with permission from J. Chromatogr. Sci. 10 (1972) 593.

high-speed size-exclusion polymer separations [16], Wallace Yau, Charles R. Ginnard and I showed for the first time that only two properly designed pore sizes are required to produce columns with wide-range linear molecular weight calibrations [17]. This technology now is widely used to produce linear molecular weight calibration columns employed for most polymer characterizations by size-exclusion chromatography. Studies with Yau and others produced insights on optimized parameters, limitations, and other experimental aspects of size-exclusion chromatography. Out of these experiences later came a book co-authored with Yau and Donald D. Bly dedicated to modern size-exclusion chromatography [18]. The need for characterizing macromolecules led to new interest in a separation method pioneered by J. Calvin Giddings: field flow fractionation (FFF). With Yau and other talented DuPont people, we developed a high force field sedimentation FFF

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Fig. 3. High force-field sedimentation field flow fractionation at DuPont, 1984. Right, J.J. Kirkland; left, W.W. Yau.

instrument, which showed effective characterization of microparticulates such as polymer latices, and certain macrobiomolecules. DuPont’s Instrument Products Division ultimately commercialized an instrument based on this work. Several basic studies of sedimentation FFF followed, defining the opportunities and limitations of this approach (e.g., [20]). An SFFF instrument with the force-field capability of 100,000 gravities subsequently was designed and constructed [21]. This high-field SFFF separating force (never since approached, to my knowledge) permitted the separation of samples that were previously not feasible with this method. Fig. 3 shows this instrument with diligent workers about to mount a different rotor into the system. Using this new SFFF instrument, the separation of a variety of biomacromolecules (DNAs, plasmids, proteins, etc.) was investigated in cooperation with a visiting young scientist, Luke E. Schallinger [22]. During the middle 1980s, continued interest in silica-based columns for HPLC led to important basic studies with another visiting young scientist, Ju¨rgen Ko¨hler. Out of this work came a much improved understanding of the surface chemistry of chromatographic silica supports [23]. This insight ultimately resulted in the development and patenting of a new form of silica column support, Zorbax Rx-SIL. This is an ultra-pure, less acidic,

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chromatographically ‘friendly’ material for separating a wide range of highly polar and ionizable materials, including peptides, proteins, etc. [24]. Starting in 1988, DuPont’s Instrument Products Division again made new products commercially available based on this new (‘Type B’) silica support. During this time, cooperative research with another DuPont colleague, Joseph L. Glajch, resulted in approaches that were significant in the emergence of systematic HPLC method development technology. Using Lloyd R. Snyder’s selectivity triangle method for describing solvent selectivity, we devised systematic reversed-phase (and adsorption) chromatographic approaches for predicting separation resolution for a sample throughout the range of solvents selected for the separation [25]. This technology was used by DuPont in a new ‘Sentinel’ HPLC instrument specifically designed for automatic HPLC method development. Glajch and I also collaborated in another discovery that resulted in important chromatographic and commercial implications: sterically protected, covalently bonded silane stationary phases. Studies showed that column packings made with these materials were impressively more stable against stationary phase hydrolysis and loss at low pH than conventional monofunctional silane stationary phases [26]. Materials with such stable phases were patented and commercialized by DuPont as StableBond chromatographic columns, which are widely used today. During the late 1980s and early 1990s my interest continued in the potential of FFF for doing a variety of difficult macromolecular separations and characterizations. Besides SFFF studies, two different thermal FFF (TFFF) instruments were constructed. These devices were used to study the fundamental properties of polymers and to characterize materials of DuPont interest [27]. One TFFF instrument was designed to permit very high temperature operation (greater than 250ºC), and to allow stable and reproducible temperature programming during the separation. This feature permitted the measurement of wide molecular weight distributions for polymers that are only soluble at high temperatures, such as polyolefins. Unfortunately, details of this work were never published. The last project before my retirement from DuPont in 1992 was in flow FFF (FFFF). This work was undertaken to support a large DuPont effort in the life sciences. We were successful in building an FFFF instrument that for the first time permitted a programmed force field to be generated during the separation. Such capability allowed good resolution of samples with a wide range of particle sizes and molecular weights in a single run [28]. Proteins, plasmids, DNAs, silica sols and polymer latices were all nicely characterized with this approach. After retirement from DuPont in 1992, I became a principal and R&D director for Rockland Technologies Inc., an HPLC column business divested from DuPont. During the succeeding years, my time was largely spent in developing and expanding the Zorbax product line, particularly the sterically protected columns, and a new series of products based on densely reacted, double-endcapped packings. During this time, a series of fundamental studies on silica-based columns also took place, sometimes in cooperation with H.A. Claessens of Eindhoven University, The Netherlands. Particular interest was focused on the parameters for optimum silica-based column stability and performance at intermediate and high pH (e.g., see [29]). Concurrently, I also was attempting to devise

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superior approaches for developing rugged HPLC methods for ionizable compounds [30]. New bidentate stationary phases have been of recent interest as materials for high pH reversed-phase separations with silica-based column packings [31]. Hewlett-Packard purchased Rockland Technologies Inc. in early 1997, ending my career as an entrepreneur. However, research in separations for Hewlett Packard has continued, with new HPLC column packings and other media on their way for use by the separations scientists. Some of the most challenging technology confronted me during the later months of my career as an analytical chemist. Much of the scientific work carried out during my career has been with the collaboration of highly skilled coworkers. The longest and most fruitful relationship has been with Lloyd R. Snyder. Over the years, Lloyd and I have collaborated on a wide range of research studies. However, most of our interaction has been involved in teaching for the Continuing Education Department of the American Chemical Society and in the writing of HPLC books. Lloyd and I first met in 1970 at the first symposium on HPLC, held in Wilmington, DE. We both were on the program, the material from which was supposed to be collected into a book. Much to my surprise, during this meeting the proposed Editor informed me that he was leaving the area for another job, so that he could not prepare the book. This left me with the job of collecting manuscripts, editing and completing responsibilities with the publisher. One of my tasks was to convince Lloyd Snyder to prepare two chapters for this book, for which he finally agreed. The authors of the various chapters for this book eventually completed their assignments, but I found that the mathematical symbols and terminology used by these contributors were often different. This led me to decide unilaterally on common symbols and terminology throughout the book, published in 1971 as the first treatise on HPLC, “Modern Practice of Liquid Chromatography”. This book ultimately was translated into six languages for international distribution. Our interaction in this widely successful book led Lloyd Snyder to contact me to see if I was interested in co-professoring a short course on HPLC for the American Chemical Society. The result was the first course on “Modern Liquid Chromatography”, given in Chicago in June 1971. Relationship with Lloyd and the ACS continued with two other updated course series on HPLC until 1997. During this period, more than 5000 attendees attended the Snyder-Kirkland ‘road-show’, and Lloyd and I had many dinners together, mostly discussing separations. Success of this short course series also led to the issuing of two taped audio courses for ACS, “Modern Liquid Chromatography” and “Solving Problems in Modern Liquid Chromatography”. With our experiences of the short courses, Lloyd Snyder and I in 1974, co-authored a source book entitled, “Introduction to Modern Liquid Chromatography”. This was followed in 1979 with a much-expanded second edition. Now more than 30,000 copies of these two editions have been distributed, with the second edition still in print. Interest in method development in HPLC led us not only to an ACS short course, but also a 1988 book, “Practical HPLC Method Development”, co-authored with Joseph Glajch, who also participated in the teaching of this short course. The success of this book, coupled with additional insights based on developments in this field, led to a largely expanded second edition in 1997. Now, distributions of the two editions of this book have totaled more than 15,000 copies.

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After forty-five years in separations, I still find that this technology still challenges and interests me greatly. Each day I realize how little I know and how much there still is to learn. I look forward to challenges that may come my way, and to the interesting people and experiences that will result.

32.I.1. Separation potpourri During the past fifteen years or so, strong efforts have been made by researchers to understand the fundamental properties of column packing materials for HPLC, so that separation materials could be optimized for a wide range of needs. Because of its overall compromise of desirable qualities, porous silica remains as the preferred support material for HPLC columns. Users now mostly choose columns with the newer less acidic, highly purified (‘Type B’) porous silica microspheres that have been specially prepared with low-activity surfaces. Columns of these silica particles elute basic compounds and other highly interactive structures, such as peptides and proteins efficiently and with good peak shapes. Covalently bonded silane stationary phases on these silicas are now prepared with excellent reproducibility and column efficiency; monofunctional coatings are generally preferred for these advantages. Most reversed-phase HPLC separations of ionizable compounds are done at low pH, where ionizable solutes and residual silanol groups on the silica support are fully protonated. Under these conditions, most compounds exhibit excellent peak shapes and high separation efficiency. Excellent retention time reproducibility also can be expected, since the least change in solute interaction with the stationary phase can occur with small changes in operating parameters, such as percent organic phase, pH and temperature. Because the siloxane bond that binds these groups to silica can be hydrolyzed at low pH, additional column stability is obtained by using bonded silanes with bulky side chains. These bulky groups sterically protect the silane against degradation by hydrolysis even at temperatures as high as 90ºC. Some polymerized and bidentate-attached silanes also show good stability at low pH. At times, separations of ionizable compounds are attempted at intermediate pH to take advantage of the additional band spacing possibilities afforded by pH effects. Here, such separations should be carefully buffered for good separation reproducibility. Ionizable solutes and surface silanol groups can be partially ionized in the intermediate pH range, resulting in conditions that often produce lower column efficiencies and poorer peak shapes. Small changes in operating conditions (pH, percent organic modifier, temperature) also can seriously affect separation resolution and reproducibility, so these parameters must be carefully controlled for good results. Since the solubility of the silica support in aqueous mobile phases begins to be appreciable above about pH eight, column degradation with continued use is caused by slow silica support dissolution with ultimate collapse of the packed bed. To reduce silica support dissolution and subsequent column failure, densely reacted and highly endcapped alkyl stationary phases have been developed. The resulting low surface energy coatings apparently present an effective high surface tension barrier that retards the rate of silica support dissolution in aqueous mobile phases, significantly increasing column lifetime and

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Fig. 4. Column, 150 ð 4:6 cm bidentate–C-18; mobile phase, 55% methanol–45% 0.05 M pyrrolidine buffer, pH 11.5; flow rate, 1.5 ml=min; temperature, 40ºC; UV detector, 215 nm; sample, β-blocker drug mix with toluene. Reprinted with permission of the American Chemical Society.

generally improving column performance. Column lifetimes are favored when longer alkyl ligands are used for the stationary phase. Studies have shown that column lifetime at intermediate and high pH also is greatly increased by using organic buffers, avoiding phosphate and carbonate buffers where possible. The rate of silica support dissolution also is reduced by using lower temperatures, so that separations no higher than 40ºC are recommended. Although a traditional recommendation that silica-based column should not be used above pH 8, recent studies have shown that these materials can be used routinely up to at least pH 11.5, providing certain operating conditions are used. Here, densely bonded and highly endcapped long-chain alkyl stationary phases are required for good column stability. Polymerized and bidentate stationary phases appear to further increase stability to degradation. Organic buffers should be used and phosphate and carbonate buffers avoided. When separated at least one pH unit (preferable 1.5 pH units) above the pKa , basic solutes exist as free amines and unreacted silanol groups are fully ionized. Now, unwanted ion exchange interaction cannot occur, and the result is excellent column efficiency and peak shapes. Under these high pH conditions, small variations in pH, percent organic modifier and temperature do not seriously affect retention and peak resolution, much for the same reasons that low pH separations are often preferred. Fig. 4 shows the separation of β-blocker drug mixture (pKa 9.5–9.5) with a bidentate–C18 column and a pH 11.5 buffered mobile phase at 40ºC. Note the excellent peaks, shapes, and column efficiency that can be obtained with high pH conditions. These and other results now suggest that reproducible analyses can be routinely done at high pH with proper silica-based packing and operating conditions. Experience has shown that no single HPLC column can perform optimally at all pH ranges and in all mobile phases. Accordingly, it would appear that a different kind or type or column should be selected for each pH range for optimum performance and stability. This may be the reason that so many types of commercial HPLC columns

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Fig. 5. Column, 150 ð 2:1 mm Zorbax Eclipse dsDNA; mobile phases: A, 0.1 M triethylammonium acetate, 0.1 mM EDTA, pH 7.0; B:A, mobile phase with 25% acetonitrile; gradient, 45–75% B in 90 min; flow rate, 0.2 ml=min; temperature, 50ºC; sample, 200 ng restriction digest standards.

are popular with users. What remains is the development of a convenient rationale to help the user in rapidly making a judgement which column type to select for optimum performance. Reversed-phase HPLC has recently been adapted to allow the separation of very large biomolecules, such as dsDNAs with resolution usually only found in slab gel separations. The advantage of the HPLC approach is that simple quantitative analyses can be conducted in automated, unattended runs with less working time. Direct fraction recovery and easy sample preparation and detection also eliminate tedious gel recovery procedures. The capabilities of the HPLC approach are illustrated in Fig. 5 for the separation of restriction digest fragments ranging from 51 to 1353 base pairs (almost 900,000 Da) with unit resolution or better. The molecular size limit of reversed-phase separations with porous particles is usually limited by the size of available pores in the column packing particles. Based on realistic extrapolation, one would expect a few megadaltons as the ultimate practical limit for high-resolution separations by this method. When high-resolution separations are required for very large macromolecules or particulates, it is likely that one of the FFF methods originally devised by J. Calvin Giddings will be the answer. This less well known family of separation methods has been under study for more than thirty years, but is just now beginning to gain the deserved interest and application. The principle of FFF is too detailed for a description here, but pointing out that separations are conducted in a very thin open channel is sufficient. A force field (sedimentation, flow, electrical, thermal, etc.) directed perpendicular to flow in this channel creates a differential migration pattern that can produce of high resolution separations for macromolecules that almost rival those of the chromatographic methods. Thermal FFF is commonly used to characterize organic-soluble polymers, while sedimentation, flow and electrical FFF are more suited for separating biomacromolecules (e.g., proteins, DNA) and particulates (e.g., polymer latices, liposomes, blood cells) in aqueous systems. Because of the wide range of capability for FFF, it is likely that applications with this family of methods will continue to expand, especially with the recent strong interest in genetics and related sciences.

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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15. 16. 17.

18. 19. 20. 21. 22. 23.

24.

L.S. Ettre and A. Zlatkis (Eds.), Seventy-five Years of Chromatography, Journal of Chromatography Library, Vol. 17, Elsevier, New York, 1979, p. 209. J.J. Kirkland, Quantitative application of potassium disk technique in infrared spectroscopy, Anal. Chem., 27 (1955) 1537. J.J. Kirkland, An apparatus for laboratory preparative-scale vapor phase chromatography, in: A. Zlatkis (Ed.), Gas Chromatography 1958, Academic Press, New York, NY, Chapter XXI. J.J. Kirkland, Analysis of sulfonic acids and salts by gas chromatography of volatile derivatives, Anal. Chem., 32 (1960) 1388. J.J. Kirkland, Trace analysis by programmed temperature gas chromatography, Anal. Chem., 34 (1962) 428. J.J. Kirkland, Fluorine-containing polymers as solid supports in gas chromatography, Anal. Chem., 35 (1963) 2003. J.J. Kirkland, Some modified gas chromatographic adsorbants and supports, in A. Goldup (Ed.), Gas Chromatography 1964, The Institute of Petroleum, London, 1965, p. 285. J.J. Kirkland, A high-performance ultraviolet photometric detector for use with efficient liquid chromatographic columns, Anal. Chem., 40 (1968) 391. J.J. Kirkland, Controlled surface porosity supports for high speed gas and liquid chromatography, Anal. Chem., 41 (1969) 218. J.J. Kirkland, R.F. Holt and H.L. Pease, Method for high-speed liquid chromatographic analysis of benomyl residues in soils and plant tissues by high-speed cation-exchange liquid chromatography, J. Agric. Food Chem., 21 (1973) 171. J.J. Kirkland, High speed liquid-partition chromatography with chemically bonded organic stationary phases, J. Chromatogr. Sci., 9 (1971) 206. J.J. Kirkland, High-performance liquid chromatography with porous silica microspheres, J. Chromatogr. Sci., 10 (1972) 593. J.J. Kirkland, Porous silica microsphere column packings for high-speed liquid-solid chromatography, J. Chromatogr., 83 (1973) 149. J.J. Kirkland and P.E. Antle, High-performance size-exclusion chromatography of small molecules with columns of porous silica, microspheres, J. Chromatogr. Sci., 15 (1977) 15. J.J. Kirkland, Microparticles with bonded hydrocarbon phases for high-performance liquid chromatography, Chromatographia, 8 (1975) 661. J.J. Kirkland, Porous silica microspheres for high-performance size-exclusion chromatography, J. Chromatogr., 125 (1976) 231. W.W. Yau, C.R. Ginnard and J.J. Kirkland, Broad-range linear calibration in high-performance size-exclusion chromatography using column packings with bimodal pores, J. Chromatogr., 149 (1979) 465. W.W. Yau, J.J. Kirkland and D.D. Bly, Modern Size-Exclusion Liquid Chromatography, J. Wiley and Sons, New York, NY, 1979. J.J. Kirkland, W.W. Yau, W.A. Doerner and J.W. Grant, Sedimentation field flow fractionation of macromolecules and colloids, Anal. Chem., 52 (1980) 1944. J.J. Kirkland, S.W. Rementer and W.W. Yau, Time-delayed exponential field-programmed sedimentation field flow fractionation for particle-size–distribution analyses, Anal. Chem., 53 (1981) 1730. J.J. Kirkland, C.H. Dilks, Jr. and W.W. Yau, Sedimentation field flow fractionation at high force fields, J. Chromatogr., 255 (1983) 255. L.E. Schallinger, W.W. Yau and J.J. Kirkland, Sedimentation field flow fractionation of DNA’s, Science, 225 (1984) 434. J. Ko¨hler, D.B. Chase, R.D. Farlee, A.J. Vega and J.J. Kirkland, Comprehensive characterization of some silica-based stationary phases for high-performance liquid chromatography, J. Chromatogr., 352 (1986) 275. J. Ko¨hler and J.J. Kirkland, Porous silica microspheres having a silanol enriched surface, US Patent, 4,874,518, October 17, 1989.

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25. J.L. Glajch, J.C. Gluckman, J.G. Charikofsky, J.M. Minor and J.J. Kirkland, Simultaneous selectivity optimization of mobile and stationary phases in reversed-phase liquid chromatography for isocratic separations of phenylthiohydantoin amino acid derivatives, J. Chromatogr., 318 (1985) 23. 26. J.J. Kirkland, J.L. Glajch and R.D. Farlee, Synthesis and characterization of highly stable bonded phases for high-performance liquid chromatography column packings, Anal. Chem., 61 (1989) 2. 27. J.J. Kirkland, S.W. Rementer and W.W. Yau, Polymer Characterization by Thermal Field Flow Fractionation with a Continuous Viscosity Detector, J. Appl. Polymer Sci., 38 (1989) 1383. 28. J.J. Kirkland, C.H. Dilks, Jr., S.W. Rementer and W.W. Yau, Asymmetric-channel flow field flow fractionation with exponential force-field programming, J. Chromatogr., 593 (1992) 339. 29. J.J. Kirkland, J.W. Henderson, M.A. van Straten and H.A. Claessens, Stability of silica-based, endcapped columns with pH 7 and 11 mobile phases for reversed-phase high-performance liquid chromatography, J. Chromatogr. A, 762 (1997) 97. 30. J.J. Kirkland, Practical method development strategy for reversed-phase HPLC of ionizable compounds, LC–GC, 14 (1996) 486. 31. J.J. Kirkland, J.B. Adams, Jr., M.A. van Straten and H.A. Claessens, Bidentate silane stationary phases for reversed-phase high-performance liquid chromatography, Anal. Chem., 70 (1998) 4344.

D.33. Ernst Klesper Ernst Klesper was born in 1927 in Cologne, Germany. After receiving the Vordiplom at the University of Kiel, Germany, in 1949, and the Diplom at the University of Hamburg, Germany, in 1951, Ernst Klesper obtained the Dr. rer. nat. degree from the latter university in 1954, submitting a thesis in inorganic chemistry. Working from 1955 in the United States on a three-year contract he was responsible for the chemistry in establishing a polyurethane foam division for Wm. T. Burnett, a smaller company in Baltimore, Maryland, until 1958. E. Klesper then joined the group of Alsoph H. Corwin, Johns Hopkins University, Baltimore, as a research associate. The research centered on the identification of porphyrins found in crude oil with the aim to elucidate the processes of the formation of oil. It was during this time that he was able to do the first experiments in supercritical-fluid chromatography. In 1961, Klesper taught physical chemistry at the University of Maryland in Baltimore as an assistant professor. During his activities at the universities, he continued as a consultant to his former company. After returning to Germany in 1962, he was at first employed as a research chemist for organic chemistry by the company Henkel and Cie in Du¨sseldorf, working among others, on the synthesis and polymerization of oxetanes and partaking in the development of rubber–metal bonding. In continuation of his university career, he joined the newly founded chair of macromolecular chemistry of H.J. Cantow at the University of Freiburg in 1965 as a scientific assistant. Up to 1979, his research in Freiburg involved the investigation of reactions on polymers by chemical, statistical, kinetic, and spectroscopic means, pursuing also his research in supercritical-fluid chromatography. He obtained his habilitation and left Freiburg as a Privatdozent and apl. Professor to follow a call to assume the newly founded chair of macromolecular chemistry at the RWTH Aachen, University of Technology. This position he held until his retirement in 1993. Klesper

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has authored and co-authored about 140 publications and a number of patents. He is a member of professional organizations and academies in Germany, USA, and Brazil and received an honorary doctorate from the Universidade Federal do Rio de Janeiro. See Chapter 5B, a, d, h, o

33.I. PROGRESS DURING THE FIRST 20 YEARS AND SOME LATER DEVELOPMENTS Ernst Klesper RWTH Aachen, University of Technology, Katterbachstr. 73, D-51467 Bergisch Gladbach, Germany

In 1958, Klesper joined the group of Alsoph H. Corwin at Johns Hopkins University in Baltimore as a research associate. The research of this group was concerned with the separation, identification and synthesis of porphyrins, particularly of porphyrins which are found in crude oil. The aim was to obtain information on the conditions of the formation of crude oil. Toward this end the chemical reactions of the precursors of the porphyrins and of the porphyrins found in oil were investigated, particularly reactions the porphyrins may have undergone during the formation of oil. If the reaction conditions are known, information is obtained about the conditions of the formation of crude oil. It soon became obvious, however, that the separation of mixtures of porphyrins found in oil could not always be satisfactorily achieved by the adsorption and partition chromatographic methods on gravity columns which were in use at this time. High-pressure liquid chromatography (HPLC) on small size particles, later renamed high-performance liquid chromatography, was at this time not yet available. Gas chromatography (GC), then already quite effective, would have been applicable had it not been for the insufficient vapor pressure of the porphyrins. At temperatures where the vapor pressure becomes higher the porphyrins decompose. Corresponding experiments were done by us in the gas chromatography laboratory of Mt. Sinai Hospital, Baltimore, headed by D.A. Turner. This lack of vapor pressure and the degradation behavior the porphyrins share with other large molecules and with oligomers of higher degree of oligomerization, polymers representing the extreme. As a consequence, Klesper was led to look for possibilities to modify the gaseous mobile phase to impart a higher degree of solubility for the porphyrins. The assumption was that the solubilizing power of a gas for an analyte should not only be a function of the chemical structure of the gas molecules but also of the denseness of the gas, the latter being greatly variable by pressure. A low boiling solvent in its gaseous state at higher pressure and temperature, preferably above the critical temperature to avoid condensation, was thought to be a promising possibility. A search of the literature about the solubilizing property of dense gases yielded a considerable number of references, both in basic as well as in applied physical chemistry. The earliest reference found was already remarkable with respect to the information contained therein. Hannay and Hogarth [1] reported in 1879 and 1880 the dissolution of organic compounds and 1960 Ehrlich and Graham reported on the

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solubility even of polymers in dense gases [2]. The solubility of different materials in dense gases had also been observed and utilized early in industry. Among other instances, engineers found precipitated silica and salts on steam turbines in places where the superheated steam expanded, and the decaffeination of coffee by dense carbon dioxide gas has been an industrial process for many years. Having obtained from the literature evidence about the solubilizing ability of dense gases, particularly of gases which in their liquid state could be classified as solvents in a wider sense, Klesper consulted Corwin about his opinion on the possibility of using dense gases, inorganic salts, and of a derivative of chlorophyll in dense, gaseous carbon dioxide, ammonia, sulfur dioxide, ethanol, and ether. The solubility was far in excess of the thermodynamic increase in vapor pressure of the substrate, as caused by the pressure exerted by the dense gas. Later in as mobile phases in chromatography. Seeing the avenues the concept opened, Corwin encouraged him in the plan to build a simple apparatus for a feasibility study. Interestingly, and unknown to us, the idea of employing dense gases as mobile phases in chromatography was already set forward by J.E. Lovelock in 1958 by way of a one page notarized, unpublished suggestion. The apparatus which we built is shown in Fig. 1. It was of simple design but sufficient to confirm the feasibility of employing dense gases as mobile phases in chromatography. In several experiments with dichlorodifluoromethane and monochlorodifluoromethane as mobile phases, porphyrins could be separated on packed columns at pressures up to 2000 psi and supercritical temperatures up to 170ºC. The results of this short investigation were published soon afterwards, because in the opinion of Corwin, Turner, and the present author, the finding was of importance to the science of chromatography in general [3], particularly in view of the expected higher diffusion and lower viscosities when compared to liquids. In order to build a new, more capable, and also versatile apparatus for high pressure gas chromatography, as this type of chromatography was called by us at the time, Klesper obtained together with Turner a research grant from the Department of Health, Education and Welfare. At the same time Klesper had been engaged in teaching physical chemistry at the University of Maryland in Baltimore as an Assistant Professor. Soon afterwards he returned to Germany and he therefore asked Corwin to take over the project in the United States. Because our grant could not be transferred, Corwin obtained a grant for himself and started building a new high-pressure gas chromatography apparatus in collaboration with the company of Perkin-Elmer. The paper on his and his coworkers efforts [4] appeared in 1968. In the publications which followed, Karayannis and Corwin reported on the separation of porphyrins, metal porphyrins and metal acetylacetonates by the new chromatograph [5–7]. After Klesper had established himself at the Institute of Macromolecular Chemistry at the University of Freiburg by taking up research in polymers, he could also resume work in Supercritical-fluid Chromatography (SFC) in 1975. A rather elaborate chromatograph was built using commercially available individual parts. It was changed and improved several times because of technical difficulties and because we could not be certain at this time about all the capabilities which would be needed in the future for this type of chromatography. The apparatus could maximally reach 3000 bar, an automated pressure cascade controlled the pressure in different parts of the apparatus, an extra oven raised

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Fig. 1. First supercritical-fluid chromatograph, schematic: 1, pressure vessel as a reservoir for the mobile phase; 2,3,8,9, shut-off and regulating valves; 4, coil of copper tubing for temperature equilibration of the mobile phase; 5, pressure tube containing the separation column, the tube being equipped with pressure resistant windows allowing to observe the movement of the porphyrin bands; 6, relief valve; 7, separation column, glass tube packed with Chromosorb coated with polyethylene glycol; 10, pressure gauge; 11, stirrer for air circulation; 12, lining of heating foil for heating the air inside the insulated box; 13, insulated box; 14, transformer for the heating foil to regulate the temperature; arrows indicating the location of thermocouples. Reprinted by permission [3].

the mobile phase to the supercritical state before entering the column oven. The column oven was large enough to accommodate columns of greater length and larger diameter. The effluent stream could be taken up in an auxiliary solvent to collect the fractions of the effluent in the liquid state, even if the mobile phase had evaporated after expansion to ambient pressure at the outlet, and other features [8–10]. A photograph of the apparatus is seen in Fig. 2 and the corresponding flow scheme in Fig. 3. The chromatograph occupied approximately one third of the laboratory space. The reasons for this large size was the lack of commercial modules specifically designed for the purpose and the wish for versatility. Later, with the advent of a broad range of commercial HPLC apparatus available, we demonstrated the applicability of a commercial HPLC unit after modifying it for SFC [11]. Our first work with the chromatograph of Fig. 2 was the separation of a number

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Fig. 2. Photograph of SFC apparatus built in Freiburg in 1975–1976.

of oligostyrenes for an initial investigation of the effects of temperature and pressure, and the corresponding programs e.g., a pressure ramp [10–12]. Thereby, semipreparative separations were found feasible by SFC [13]. The mobile phase was n-pentane containing 5 or 10% methanol, the methanol being useful for the partial deactivation of the unbonded porous silica used as a stationary phase for the packed columns. The oligostyrenes could be separated into all its individual degrees of oligomerization, that is maximally into 49 individual oligomers which amounts to molecular masses of up to 5000 daltons. A chromatogram for a semipreparative separation of an oligostyrene using a pressure program is seen in Fig. 4. The individual oligomers could be identified by mass spectrometry having the expected molecular weights. Rechromatographing mixtures of the individual oligomers obtained by the semipreparative separation, also shown in Fig. 4, confirmed the purity. Later we could show the separation of polymer samples [14], specifically polystyrene samples of different weight average molecular weights, Mw , of up to 600,000 as seen in Fig. 5. These separations were, however, not separations in individual molecular species as had been the case for the oligostyrenes – since this would be a most demanding task at such high molecular weights where the differences in solubility between degrees of polymerization n and n C 1 become extremely small – but separations between the samples themselves, for instance, a separation of the polymer samples of 254,000 Mw (peak 7) and 600,000 Mw (peak 9) in Fig. 5(a). The two chromatograms of Fig. 5 were not obtained by pressure programming as before in Fig. 4, but by introducing composition programming to SFC. In this case a

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Fig. 3. Flow scheme of SFC apparatus shown in Fig. 2.

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Fig. 4. Semipreparative SFC chromatogram of an oligostyrene of a weight-average molecular weight of Mw D 2200 (lower trace) using a pressure program as indicated by the pressure line. The numbers at the peaks represent the degree of oligomerization. Individual oligomers of different degrees of oligomerization were collected and analyzed by MS for molecular weight. Then different individual oligomers were remixed, i.e. 3,5,7 and 1,2,4,6,8, and the two mixtures again chromatographed using the same conditions as before for checking the purity. Reprinted by permission [13].

binary mixture of n-pentane=1,4-dioxane was used as the mobile phase. The dioxane being the better supercritical solvent, its content in the mobile phase was increased from 40 to 50% during the chromatogram. In Fig. 5b the composition program of an industrial polystyrene produced in bulk shows a broad molecular weight distribution as is seen by the large peak width. The molecular weight distribution is non-Gaussian as is already obvious from the splitting of the peak. Later work of our group was also directed towards composition programming [15–16]. It could be shown, for instance, that a temperature program run simultaneously to a combined composition-pressure program improved the separation (Fig. 6). Besides the group of Corwin and the group of Klesper, other research groups contributed strongly to the development of SFC instrumentation during the first some 20 years. Jentoft and Gouw described a chromatograph [17] for SFC in 1972 and also introduced pressure programming to SFC, applying a ramp of increasing pressure [18–19]. They also wrote two useful early review articles on SFC [20–21]. A stepwise increase of pressure had been used i.a. by the group of Corwin before [7]. Later, simultaneous pressure and temperature programs were found useful by us for improving the resolution [9].

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Fig. 5. (a) SFC chromatogram of polystyrene samples of different weight-average molecular weights Mw : 1 D 3600, 2 D 10,000, 4 D 53,700, 5 D 93,000, 7 D 254,000, 9 D 600,000 (b) SFC chromatogram of a commercial polystyrene sample produced in bulk possessing a broad molecular weight distribution. Reprinted by permission [14].

Positive pressure programs, i.e., a continuous increase of pressure, obviously increases the solubility of the analyte in the mobile phase, leading to elution in a shorter time. The counterpart in gas chromatography is the positive temperature program which however, leads to faster elution by an increase in vapor pressure instead by an increase in solubility. Remarkably, both positive and negative temperature programs have been employed for SFC. A positive temperature program at constant pressure leads to a lower density of the mobile phase decreasing its solubility power and at the same time increasing the interdiffusion coefficient. This may be desirable for a fine tuning in the separation of a difficult to separate analyte mixture. A negative temperature program at constant pressure, on the other hand, leads to increasing density and therefore to faster elution, unless the decrease in vapor pressure of the analyte is the overriding factor. This latter type of program has been used, for instance, in cases when a chromatograph possessed no facilities for pressure programming or when it was desired to leave the SFC region for entering the HPLC region. Composition programs have a similar effect as positive pressure programs. With increasing content of the more strongly solvating component in the mobile phase the speed of elution is increased.

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Fig. 6. (a) SFC chromatogram of an oligostyrene of a molecular weight of 800 (PS 800) separated by a composition program employing CO2 =dioxane, the dioxane content increasing from 5 to 50%. Temperature constant at 145ºC. (b) as before but with an additional temperature program, the temperature rising from 145 to 230ºC. Pressure rising for (a) and (b) from 250 to 295 bar.

Another research group which became interested very early in SFC was the group of Giddings and coworkers. They started with high pressure gas chromatography in a restricted sense of the word. Employing permanent gases (He, A, N2 ) and high inlet pressures at the column, the column outlet was kept at ambient pressure, [22–23]. One may be of the opinion that this pioneering work belongs more to the realm of Gas Chromatography (GC) than to SFC because both the use of permanent gases and of ambient pressure at the column outlet are not typical for SFC. Mobile phases for SFC usually belong to the category of solvents in a wider sense, having low boiling points like CO2 , n-pentane or dimethylether, or even water. Moreover, the pressure at the column outlet is kept greatly above ambient pressure in order to retain the solubility power of the supercritical mobile phase in the last part of the column, otherwise the elution of analytes which possess little or no vapor pressure would be slowed or not take place at all.

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It may be reasoned that the term supercritical-fluid chromatography (SFC) itself is misleading to some extent. The so called permanent gases are also supercritical-fluids in the strict sense of the word, because they are both above the critical temperature, Tc , and they are fluids, regardless of the pressure. A term like supercritical-solvent chromatography (SSC) might actually be more descriptive. While there is no sharp border line between supercritical-fluids with and without solvent power one may arbitrarily choose a specific Tc as a border line e.g. 0ºC or lower. As a possible alternative for defining a border line, one may specify: c.total/ D c.vp/ C c.so/ (1) where c(total) D total concentration of an analyte in the mobile phase, c(vp) D partial concentration of the same analyte in the mobile phase due to vapor pressure and c(so) D partial concentration of the same analyte in the mobile phase due to dissolution. Assuming the phase ratio (approximately) and the concentration of the analyte in the stationary phase c(st) to be the same for all three terms, one may divide Eq. 1 by c(st) and obtains a relation between capacity ratios k 0 : 1=k 0 .total/ D 1=k 0 .vp/ C 1=k 0 .so/ (2) Specifying arbitrarily k 0 (vp) to be greater than k 0 (so) for defining SFC, and k 0 (vp) to be smaller than k 0 (so) for GC, it is possible to determine which mode, SFC or GC, prevails for a given single analyte. The term k 0 (total) is known by the SFC experiment, k 0 (vp) may be approximated by lowering the pressure of the mobile phase to normal GC conditions, both terms then yielding k 0 (so) according to Eq. 2. As is obvious; however, the term supercritical-fluid chromatography (SFC) has become commonly accepted and it is now rather late to change to another name. Moreover, a change may not be necessary, given a common consent about the specific features of SFC. In work following the investigation on GC with permanent gases at high column inlet pressure, Giddings group used supercritical solvents, i.e., NH3 and CO2 , to study the dissolution of high molecular weight compounds in supercritical solvents and to separate larger molecules, including polar molecules and biomolecules on columns [24–25]. Also, a step by step pressure program was employed. The solvent power of supercritical solvents was rated by the Hildebrand solubility parameter and the information collected then available about SFC. Another group, Sie and Rijnders at the Shell company in The Netherlands, belonged also to the early investigators in SFC, making considerable contributions in the years from 1966 to 1969. Using CO2 as the mobile phase, a quantitative experimental and theoretical study on the effect of pressure on partition coefficients and retention ratios [26] was followed by work on the influence of pressure, flow rate and other factors on plate height [27]. Moreover, using pentane as the mobile phase the effects of partition and adsorption were treated in detail [28–30]. The efforts of this group were directed to a large extent toward quantitative measurements of chromatographic data at a time already when SFC was just starting. Later quantitative work was carried out by Rogers and coworkers [31– 32] by determining, for instance, Kovats and McReynolds indices on Rohrschneider solutes using different batches of a commercial polysiloxane stationary phase. This was followed by a study on the effects of column length, particle size, flow rate and pressure programming rate [33–34].

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A considerable amount of quantitative work on general physicochemical aspects is due to the group of Schneider in Germany. SFC separations using CO2 as the mobile phase were presented in connection with phase diagrams, transport properties, and plate height [35]. With respect to transport properties the emphasis was on retention ratios [36] and binary diffusion coefficients [37–38]. Similar aims were followed some time later by Lauer et al. [39], and Chester et al. [40]. Chester et al. applied van’t Hoff plots to split the behavior of analytes in SFC, into GC and LC behavior, i.e., into transport by vapor pressure and by solubility. This is a useful subdivision since many analyte mixtures which may be chromatographed by SFC contain analytes which exhibit at least some vapor pressure at the temperatures used. Novotny and coworkers applied themselves to van Deemter plots and to the measurement of binary diffusion coefficients using the method of Giddings and Seager for uncoated capillary columns. The lower alkanes were found to be favorable as mobile phases because of the relatively high interdiffusion coefficients [41–42]. Remarkably, the diffusion coefficients of polycyclic aromatic hydrocarbons in the alkanes were quite high, this is possibly connected to an intermolecular interaction of the analytes with the alkane mobile phases which is of fast kinetics in contrast to mobile phases which tend to stronger intermolecular interactions with analytes, particularly with polar analytes. Such mobile phases would be at the extreme H2 O and NH3 . In another Novotny paper the conclusion was reached that the independent control of the mobile phase velocity, i.e., independence of the velocity from pressure during pressure programming, is desirable for capillary columns [43]. For capillary columns the desired independence has been a greater instrumentation problem than for packed columns, because the pressure at the outlet of a capillary is more difficult to control than for a packed column where the amount of effluent, usually in the liquid state, is larger. Our apparatus of Figs. 2 and 3 for packed columns possessed already the hardware to keep the pressure at the column outlet constant or programmable and independent of the amount of effluent. Another chromatograph having the capability for programming the pressure had been reported by Jentoft and Gouw [17], as already mentioned. It is to be concluded that in addition to pressure control at the column outlet, the linear velocity of the mobile phase in capillary and packed columns should be freely programmable including the possibility to keep the linear velocity constant. This applies also to temperature or composition programming. Expressed differently, the velocity should be determined by the programmable feed rate of the pumps which deliver the mobile phases, but not by pressure or temperature or composition programs. The pressure at the column outlet and the average velocity should be controllable by the chromatographer as desired, independent from any other program. This will allow to control velocity, pressure, composition, and temperature as independent parameters whenever this is desirable for a separation. In an early paper on packed columns Novotny et al. dealt with a negative (inverse) temperature program and with capacity ratio and plate height. They pointed out that the addition of moderators to the mobile phase, moderators being usually stronger solvents than the main component, may be used to advantage for increasing the mobility of analytes in SFC [44]. Gere et al. also investigated packed columns and emphasized the advantages of small particle size for the packing [45]. The high performance of

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small particle packings in HPLC indicated beneficial results to be obtained in SFC also. While the development of SFC had started and then also continued with packed columns, the high performance of capillary columns in GC suggested their application to SFC, considering i.a. the gaseous nature of the mobile phases for both GC and SFC. Therefore, a still relatively high interdiffusion coefficient in SFC mobile phases can be expected when compared to the high interdiffusion coefficient of the low pressure gases employed by GC and more so when compared to the low interdiffusion coefficients of the liquid mobile phases for HPLC. The use of capillary columns for SFC separations was introduced by Novotny and coworkers [43], and later by Lee and colleagues, the latter group also comparing density programming to pressure programming for capillary columns [46]. Density programming for packed columns had been investigated by Giddings and coworkers [47]. Particularly at temperatures near Tc where the pressure–density isotherm is of a strongly sigmoid shape, it is advantageous to use density programs, because density is more linearly related to solubility than is pressure. In actual practice, however, one will still run a pressure program but a program which has been calculated by pressure– temperature–density data to yield the desired density–time curve. This calculation procedure will be followed whenever pressure is more easily monitored or controlled than density. While it is possible for a given mobile phase to relate density to solvent power, for comparing the solvent power of different mobile phases we suggested the free volume of the supercritical phase. This eliminates the dependence on molecular weight which in contrast to chemical structure is incidental to the solvent power [48]. Lee and coworkers [49] and the group of Wahrhaftig [50–51] contributed also by introducing a generally applicable detector for SFC, connecting the SFC apparatus to a mass spectrometer. This is of importance because MS is sensitive, widely applicable to different analytes and mobile phases, and delivers mass data which are useful for the identification of analytes. The first 20 years of SFC were in large part devoted to exploratory work. The years following saw a much increased activity in SFC as documented by a greatly increased number of publications including books. It would be impossible to detail this work and even begin to do justice to this large amount of research within limited space. During this time the use of SFC spread to university and industry laboratories finding a niche in special applications. Because this type of chromatography fills a gap between low pressure non-solvent gases of GC and the liquids in HPLC, it supplements the arsenal of separation methods. SFC makes it even possible to circle the critical point without phase separation, starting the chromatogram, for instance, in the realm of GC, crossing the SFC region, and arriving finally in the HPLC domain [52]. Standard methods like GC, HPLC, TLC, or electrophoretic methods by virtue of their widespread use and their acknowledged performance will remain, however, the most widely used tools for separations. SFC is more demanding with respect to hardware, handling and method development. It is, however, more versatile, mainly because of a higher number of parameters influencing the separation which leads to a higher number of programming modes for optimizing resolution and analysis time, a feature not found to this extent in GC or HPLC. Given suitable hardware and software, all parameters of the mobile phase are subject to programming procedures, resulting in a large effect on separations.

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Fig. 7. Three-dimensional plot of average resolution R Łm plotted versus column end pressure p(e) and column temperature T . For exhibiting R Łm more clearly, different areas of R Łm are shown as shaded bands. The data have been collected by a series of isobaric–isothermal chromatograms. Reprinted by permission [53].

The past has shown that pressure (density), temperature, composition and linear velocity of the mobile phase have a considerable or even striking effect on SFC separations. It should be stressed, however, that good results can be achieved without great effort by a suitably devised isothermal–isocratic pressure program or by an isothermal–isobaric composition program, or even with a temperature program, all with a constant feed rate of the mobile phase. Conversely, the optimum for the quality of an analysis is likely to be found by combining different programming possibilities either simultaneously or consecutively. As already mentioned, a pressure (density) program may be overlaid by a temperature program to improve the separation and the same holds true for a composition program. It is to be considered that simultaneous multiple programs are interdependent. For instance, a given pressure (density) program or a specific composition program, even if chosen to give the best attainable separation by itself, may require a specific temperature program for optimal results and vice versa. A case in point is given by Figs. 7 and 8 and the discussion which follows, citing results obtained by our group after the 20 year period. The data of Figs. 7 and 8 have been obtained with CO2 as the mobile phase, delivered as a liquid at 1 ml=min., unbonded silica gel as the stationary phase, and a mixture of naphthalene, anthracene, pyrene, and chrysene as the analytes [53–54]. Fig. 7 represents a perspective three dimensional plot of the average resolution R Łm between the four analytes versus the column end pressure p(e), and the column temperature T . The maxima of the resolution are found at the ‘crest’ of the three dimensional contour surface. Because the R Łm are also given in form of shaded areas on the contour surface, it is immediately seen that the resolution decreases to lower values with increasing pressure. This is to be expected because an increasing pressure increases the density and decreases the diffusion. With respect to the optimum temperature for a pressure program a constant temperature between 100 and 150ºC would be a reasonable choice, a simultaneous temperature program does not result in a significant improvement in resolution. Considering now the retention ratio k 0 , which is related to the time requirement of a chromatographic

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Fig. 8. Three-dimensional plot of retention ratio k 0 plotted versus column end pressure p(e) and temperature T . The R Łm are shown as shaded bands as in Fig. 7. Reprinted by permission [53].

run, the k 0 is shown in Fig. 8 in a three dimensional plot similar to Fig. 7. The k 0 (C), the retention ratio of the last eluting analyte chrysene, has replaced the R Łm on the z-axis. However, the R Łm remain to be seen in Fig. 8 by way of shading the contour surface. Although the contour surface of Fig. 8 looks different than in Fig. 7, it is not much different in principle. There is a maximum seen for k 0 (C) which decreases strongly with increasing pressure, again forming a crest, even if the elevation of the crest is low and flat at higher pressures. The crest of this maximum stays in about the same temperature range, here 100–125ºC. Because the time requirement for an analysis should be as short as is compatible with the required resolution R Łm , a k 0 -value of a hundred or more is very high. A possibility for reducing the time of analysis would be to start the pressure program at a high enough pressure to reduce k 0 sufficiently, the temperature being either higher or lower than that of the crest, and use during the pressure program a negative or a positive temperature program, respectively. These simultaneous temperature programs keep the resolution at a higher level than the isothermal pressure programs by themselves. Which one of the programming possibilities is selected depends on the desired resolution of the individual peak pairs, R Łm being only an average resolution. For the conclusions drawn from Figs. 7 and 8 it is of importance to realize that the mobile phase has been delivered by the pump at a constant feed rate of 1 ml=min as a liquid. While this is a frequently used feed rate for 4.6 mm diameter packed columns, a constant feed rate does not represent per se an optimized linear velocity of the mobile phase in the column. For such an optimization additional data are desirable which show the dependence of capacity ratio and resolution on the linear velocity besides their dependence on pressure and on temperature [55–57]. Also, when composition programs are used and an optimization is desired going beyond that which follows from general principles, additional data are required [58–60]. A third consideration is the vapor pressure of the analytes. The analytes of Figs. 7 and 8 possess significant vapor pressures which cannot be neglected for SFC. Analytes without vapor pressures exhibited a steady rise in k 0 and no maxima when going to higher temperatures

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at constant pressures [61], a finding which necessitates for such analytes differently designed programs. Despite the larger number of variables for SFC when compared to GC or HPLC, the use of SFC requires only a reasonable experimental effort in method development, provided one does not aspire to a full optimization. The role of SFC in the future development of chromatography in the 21st century has several aspects. For one, SFC closes the gap between HPLC and GC by employing a mobile phase which possesses properties both of liquids and gases. As a consequence, SFC allows to separate many nonvolatile compounds which are not accessible by GC, this separation being more efficient than by HPLC. Moreover, the phase behavior in the supercritical region allows to consecutively combine GC, SFC, and HPLC in a single uninterrupted chromatogram.This leads to the separation of mixtures of widely different vapor pressures and solubilities in one chromatographic analysis. In addition, SFC increases the number of chromatographic parameters which influence a separation. Not only temperature and linear velocity, as in GC, influence the separation, but also the composition of the mobile phase is of major impact and can be changed, as is the case in HPLC. Particularly, the pressure i.e. the density determines the quality of the separation in SFC. Programs of these parameters are interdependent and simultaneous programs open possibilities not accessible to this extent for GC or HPLC. They constitute a challenge to the chromatographer. Fortunately, much of the progress in GC and HPLC, e.g., the development of stationary phases, columns, detectors, and hardware in general, is of great value for SFC also. As to hardware, SFC might lend impetus to the development of generally applicable chromatographs allowing to combine GC, HPLC, and SFC. By their very nature they will not be in competition to the instruments designed specifically for GC, HPLC or even SFC, but will be of use for certain types of analyte mixtures.

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

J.B. Hannay and J. Hogarth, On the solubility of solids in gases, Proc. Roy. Soc. (London), 29 (1879) 324–326; Chem. News, 41 (1880) 103. P. Ehrlich and E.B. Graham, Solubility of polymers in compressed gases, J. Polymer Sci., 45 (1960) 246–247. E. Klesper, A.H. Corwin and D.A. Turner, High-pressure gas chromatography above critical temperatures, J. Org. Chem., 27 (1962) 700–701. N.M. Karayannis, A.H. Corwin, E.W. Baker, E. Klesper and J.A. Walter, Apparatus and materials for hyperpressure-gas chromatography of nonvolatile compounds, Anal. Chem., 40 (1968) 173–1738. N.M. Karayannis and A.H. Corwin, Volatilization and separations of metal acetylacetonates at 115ºC by hyperpressure-gas chromatography, J. Chromatogr. Sci., 8 (1970) 251–256. N.M.Karayannis and A.H. Corwin, Hyperpressure-gas chromatography. IV. gas chromatography of etioporphyrin II metal chelates, J. Chromatogr., 47 (1970) 247–256. N.M. Karayannis and A.H. Corwin, Hyperpressure-gas chromatography. III. Gas chromatography of porphyrins and metalloporphyrins, Anal. Biochem., 26 (1968) 34–50. E. Klesper, Chromatography with supercritical-fluids, Angew. Chem., Intern. Ed. Engl., 17 (1978) 738–746. E. Klesper and W. Hartmann, Apparatus and separations in supercritical-fluid chromatography, Eur. Pol. J., 14 (1978) 77–88.

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10. E. Klesper and W. Hartmann, Supercritical-fluid chromatography of styrene oligomers, J. Polym. Sci., Poly. Lett. Ed., 15 (1977) 9–16. 11. F.P. Schmitz and E. Klesper, Separation of styrene oligomers by supercritical-fluid chromatography (SFC) using a modified HPLC-Instrument, Polym. Bulletin, 5 (1981) 603–608. 12. E. Klesper and W. Hartmann, Parameters in supercritical-fluid chromatography of styrene oligomers, J. Polym. Sci., Polym. Lett. Ed., 15 (1977) 707–712. 13. W. Hartmann and E. Klesper, Preparative supercritical-fluid chromatography of styrene oligomers, J. Polym. Sci., Polym. Lett. Ed., 15 (1977) 713–719. 14. F.P. Schmitz and E. Klesper, Polystyrene separation by supercritical-fluid chromatography, Polym. Commun., 24 (1983) 142–144. 15. F.P. Schmitz and E. Klesper, Supercritical-fluid chromatography of oligostyrenes by eluent gradients, Makromol. Chem., Rapid Commun., 2 (1981) 735–739. 16. F.P. Schmitz and E. Klesper, Gradient elution in supercritical-fluid chromatography, J. Chromatogr., 267 (1983) 267–275. 17. R.E. Jentoft and T.H. Gouw, Apparatus for supercritical-fluid chromatography with carbon dioxide as the mobile phase, Anal. Chem., 44 (1972) 681-686. 18. R.E. Jentoft and T.H. Gouw, Supercritical-fluid chromatography of a monodispersed polystyrene, J. Polymer Sci., Part B, 7 (1969) 811–813. 19. R.E. Jentoft and T.H. Gouw, Pressure-programmed supercritical chromatography of wide molecular weight range mixtures, J. Chromatogr. Sci., 8 (1970) 138–142. 20. T.H. Gouw and R.E. Jentoft, Supercritical-fluid chromatography, J. Chromatogr., 68 (1972) 303–323. 21. T.H. Gouw and R.E. Jentoft, Practical aspects in supercritical-fluid chromatography, Adv. Chromatogr., 13 (1975) 1–39. 22. M.N. Myers and J.C. Giddings, High inlet pressure micro column system for use in gas chromatography, Anal. Chem., 38 (1966) 294–297. 23. M.N. Myers and J.C. Giddings, Ultra-high-pressure gas chromatography in microcolumns to 2000 atmospheres, Separation Sci., 1 (1966) 761–776. 24. L. McLaren, M.N. Myers and J.C. Giddings, Dense-gas chromatography of nonvolatile substances of high molecular weight, Science, 159 (1968) 197–199. 25. J.C. Giddings, M.N. Myers, L. McLaren and R.A. Keller, High pressure gas chromatography of nonvolatile species, Science, 162 (1968) 67–73. 26. S.T. Sie, W. van Beersum and G.W.A. Rijnders, High-Pressure Gas Chromatography and Chromatography with Supercritical-fluids. I. The effect of pressure on partition coefficients in gas–liquid chromatography with carbon dioxide as a carrier gas, Separation Sci., 1 (1966) 459–490. 27. S.T. Sie and G.W.A. Rijnders, High-pressure gas chromatography with supercritical-fluids. II. Permeability and efficiency of packed columns with high pressure gases as mobile fluids under conditions of incipient turbulence, Separation Sci., 2 (1967) 699–727. 28. S.T. Sie and G.W.A. Rijnders, High-pressure gas chromatography and chromatography with supercritical fluids. III. Fluid-liquid chromatography, Separation Sci., 2 (1967) 729–53. 29. S.T. Sie and G.W.A. Rijnders, High-pressure gas chromatography and chromatography with supercritical-fluids. IV. Fluid-solid chromatography, Separation Sci., 2 (1967) 755–777. 30. S.T. Sie and G.W.A. Rijnders, Chromatography with supercritical-fluids, Anal. Chim. Acta, 38 (1967) 31–44. 31. F.J. van Lenten, J.E. Conaway and L.B. Rogers, Gas chromatographic retention characteristics of different polysiloxane oligomers, Separation Sci., 12 (1977) 1–28. 32. J.A. Nieman and L.B. Rogers, Supercritical-fluid chromatography applied to the characterization of siloxane-based gas chromatographic stationary phase, Separation Sci., 10 (1975) 517–545. 33. J.A. Graham and L.B. Rogers, Effects of column length, particle size, flow rate, and pressure programming rate on resolution in pressure-programmed supercritical-fluid chromatography, J. Chromatogr. Sci., 18 (1980) 75–85. 34. B.P. Semonian and L.B. Rogers, Unusual gas chromatographic behaviors of naphthalene, pyrene, and phenanthrene using pressurized n-pentane as a carrier gas, J. Chromatogr. Sci., 16 (1978) 49–60. 35. D. Bartmann and G.M. Schneider, Experimental results and physico-chemical aspects of supercritical-

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fluid chromatography with carbon dioxide as mobile phase, J. Chromatogr., 83 (1973) 135–145. 36. U. van Wasen and G.M. Schneider, Pressure and density dependence of capacity ratios in supercritical-fluid chromatography (SFC) with carbon dioxide as mobile phase, Chromatographia, 8 (1975) 274–276. 37. I. Swaid and G.M. Schneider, Determination of binary diffusion coefficients of benzene and some alkylbenzenes in supercritical CO2 between 308 and 328 K in the pressure range 80–160 bar with supercritical-fluid chromatography (SFC), Ber. Bunsenges., 83 (1979) 969–974. 38. R. Feist and G.M. Schneider, Determination of binary diffusion coefficients of benzene, phenol, naphthalene and caffeine in supercritical CO2 , Separation Sci. Technology, 17 (1982) 261–270. 39. H.L. Lauer, D. McManigill and R.D. Board, Mobile phase solute mass transfer in supercritical-fluid chromatography, Anal. Chem., 55 (1983) 1370–1375. 40. T.L. Chester and D.P. Innis, Retention in capillary-supercritical fluid chromatography, J. High Resolut. Chromatogr., Chromatogr. Communications, 8 (1985) 561–566. 41. S.R. Springston and M. Novotny, Mobile phase solute mass transfer in supercritical-fluid chromatography, Anal. Chem., 56 (1984) 1762–1766. 42. M. Novotny and S.R. Springston, Fundamentals of column performance in supercritical-fluid chromatography, J. Chromatogr., 279 (1983) 417–422. 43. S.R. Springston and M. Novotny, Kinetic optimization of capillary-supercritical fluid chromatography using carbon dioxide as the mobile phase, Chromatographia, 14 (1981) 679–684. 44. M. Novotny, W. Bertsch and A. Zlatkis, Temperature and pressure effects in supercritical-fluid chromatography, J. Chromatogr., 61 (1971) 17–28. 45. D.R. Gere, R. Board and D. McManigill, Supercritical-fluid chromatography with small particle diameter packed columns, Anal. Chem., 54 (1982) 736–740. 46. J.C. Fjeldsted, W.P. Jackson, P.A. Peaden, and M.L. Lee, Density programming in capillary supercritical-fluid chromatography, J. Chromatogr. Sci., 21 (1983) 222–225. 47. L.M. Bowman Jr., M.N. Myers and J.C. Giddings, Supercritical-fluid (dense gas) chromatography= extraction with linear density programming, Separation Sci. Technology, 17 (1982) 271–287. 48. E. Klesper, D. Leyendecker and F.P. Schmitz, Comparison of carbon dioxide and pentane as eluents in supercritical-fluid chromatography, J. Chromatogr., 366 (1986) 235–242. 49. R.D. Smith, W.D. Felix, J.C. Fjeldsted and M.L. Lee, Capillary-column supercritical fluid chromatography=mass spectrometry, Anal. Chem., 54 (1982) 1883–1885. 50. L.G. Randall and A.L. Wahrhaftig, Dense-gas chromatography=mass spectrometer interface, Anal. Chem., 50 (1978) 1703–1705. 51. L.G. Randall and A.L. Wahrhaftig, Direct coupling of a dense-supercritical gas to a mass spectrometer using a supersonic molecular beam interface, Rev. Sci. Instrum., 52 (1981) 1283–1295. 52. B. Gemmel, F.P. Schmitz and E. Klesper, Consecutive gradients in chromatography. Circling the critical point under isocratic conditions, J. High Resolution Chromatogr., Chromatogr. Commun., 11 (1988) 901–903. 53. A. Hu¨tz, F.P. Schmitz, D. Leyendecker and E. Klesper, Effects of temperature, pressure, and density on the chromatographic behavior of supercritical carbon dioxide, J. Supercritical-fluids, 3 (1990) 1–7. 54. A. Hu¨tz, D. Leyendecker, F.P. Schmitz and E. Klesper, Isocratic networks in supercritical-fluid chromatography. IIIa . Dependence of capacity factor, selectivity, and resolution on temperature, pressure, density, and free volume of pentane as shown in multidimensional plots, J. Chromatogr., 505 (1990) 99–108. 55. S. Ku¨ppers, M. Grosse Ophoff and E. Klesper, Influence of linear velocity and multigradient programming in supercritical chromatography, J. Chromatogr., 629 (1993) 345–359. 56. A. Hu¨tz and E. Klesper, Efficiency in supercritical-fluid chromatography as a function of linear velocity, pressure=density, temperature, and diffusion coefficient employing n-pentane as the eluent, J. Chromatogr., 607 (1992) 79–89. 57. U. Ko¨hler and E. Klesper, The influence of linear velocity, column length and pressure drop in SFC. II. Plate numbers, effective plate numbers, and resolution, J. Chromatogr. Sci., 32 (1994) 525. 58. D. Leyendecker, F.P. Schmitz, D. Leyendecker and E. Klesper, Three-dimensional network plots in supercritical-fluid chromatography with n-pentane-1,4-dioxane, J. Chromatogr., 393 (1987) 155–167.

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59. S. Ku¨ppers, D. Leyendecker, F.P. Schmitz and E. Klesper, Three-dimensional network plots using colours for a fourth variable with binary mixtures of pentane and 1,4-dioxane under sub- and supercritical conditions, J. Chromatogr., 505 (1990) 109–117. 60. S. Ku¨ppers, D. Leyendecker, F.P. Schmitz and E. Klesper, Three-dimensional network plots using colours for a fourth variable for binary mixtures of CO2 or ethane and 1,4-dioxane under sub- and supercritical conditions, J. Supercritical-fluids, 3 (1990) 121–126. 61. D. Leyendecker, D. Leyendecker, F.P. Schmitz and E. Klesper, Elution behavior of styrene oligomer fractions in supercritical-fluid chromatography, Chromatographia, 23 (1987) 38–42.

D.34. John H. Knox John Henderson Knox was born on October 21, 1927 in Edinburgh, United Kingdom. He received a B.Sc. (1st Class Honours, in Chemistry) from the University of Edinburgh in 1949, the Ph.D. from Cambridge University in 1953 and a D.Sc. from Edinburgh in 1963. He is a Fellow in the following Societies: Royal Society of London (1984), Royal Society of Edinburgh (1971), Royal Society of Chemistry and a member of the Chromatographic Society. Some of the major events in his life are: an appointment as Lecturer in Chemistry, University of Edinburgh; a NSF fellowship as a Senior Visiting Research Scientist Fellow at University of Utah, where he did research in collaboration with J. Calvin Giddings on band spreading in liquid chromatography. This was followed with an appointment as Director of Studies in Chemistry, University of Edinburgh, then a promotion to Personal Chair in physical chemistry at the University of Edinburgh. John has been the recipient of a number of prestigious awards: M.S. Tswett Anniversary Medal (Tallin, USSR), Dal Nogare Award of the Chromatographic Forum of the Delaware Valley, M.S. Tswett Chromatography Medal awarded by the Committee of the Symposium Series on Advances in Chromatography, the Mackdougall Brisbane Prize of the Royal Society of Edinburgh, the Martin Award of the Chromatographic Society, the American Chemical Society National Award in Chromatography sponsored by Supelco Inc., and the 2000 ‘Golay Medal’ at the 23rd International Symposium on Capillary Chromatography. He has been in high demand to share his broad expertise with the corporate sector as a consultant: in high-speed liquid chromatography to Du Pont Inc. in Wilmington, Delaware, USA, to Rank Hilger in high-performance liquid chromatography, to Kratos Scientific Instruments, Ltd. in relation to equipment for high-performance liquid chromatography, to Shandon Southern Instruments, now Hypersil on column packing materials for HPLC, to Glaxo in electrochromatography, and to Hewlett Packard on electrochromatography. In 1984, he was awarded the title of Emeritus Professor of Chemistry at Edinburgh University. His research activities have been directed to unravelling the kinetics of combustion and chlorination reactions using gas chromatography for product analysis, theoretical aspects of band dispersion in liquid chromatography, fundamental mechanisms in

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chromatography, preparative HPLC and size exclusion chromatography. Lately his efforts are centered on electrochromatography. See Chapter 5B, a, d, g, h, k, i

34.I. 40 YEARS EXPLORING CHROMATOGRAPHY John H. Knox Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh Eh9 3JJ, UK

34.I.1. Early days I first learned of chromatography during my undergraduate degree course at Edinburgh University from which I graduated with an honors B.Sc. in Chemistry in 1949. Professor Sir Edmund Hirst and his carbohydrate team were using both paper and column chromatography to separate methylated sugar derivatives. From the results they could infer the structure of the carbohydrates whose methylation and subsequent hydrolysis gave these methylated sugars. Paper chromatography was carried out in tall glass jars with the paper hanging downwards from a reservoir containing the eluent. Column chromatography, for preparative purposes, was carried out in columns several cm in diameter using gravity feed. This was a slow, inefficient and cumbersome procedure. I then went on to Pembroke College Cambridge, where I worked for my Ph.D. (1949–53) on the mechanism of low temperature (300–400ºC) combustion of propane under the formidable Professor R.G.W. Norrish. One of my main tasks was to determine the quantities of different aldehydes (propionaldehyde, acetaldehyde and formaldehyde) in the reaction products. Norrish regarded these as the key intermediates through which hydrocarbons were sequentially degraded. My training in Edinburgh suggested to me that chromatography was the way to do it. I oxidized the aldehydes to the silver salts of their acids using a short column of silver hydroxide, freed the acids and separated them on a column of homemade silica gel using titration for detection. This was reported in my first scientific paper (1951). My other big problem at the time was to analyze the gaseous products of combustion, especially the various hydrocarbon products. The best I could do at the time was to isolate the hydrocarbon gases, and get an empirical formula. At the same time Howard Purnell, also doing a Ph.D. under Norrish on the mercury photosensitized decomposition of hydrocarbons, had the same problem. Howard and I were good friends and remained so until he sadly died some years back. Over the years, we had long discussions on scientific and other topics. Our mutual interest in gas chromatography came as a result of various job interviews during my final year at Cambridge. During one such, at ICI Heavy Chemicals Division (Billingham), I met the team working on gas–liquid chromatography, Bradford, Harvey and Chalkley. I was keen to see the gas chromatograph, but unfortunately it had been dismantled for some reason and the only thing to be seen was the glass heating jacket for the columns, about 1 meter long and

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about 3 cm internal diameter. But at least this gave us an idea of the dimensions of the columns for GC. Later I visited the Alkali Division at Northwich. The research manager, Dr. Ray Richards, mentioned that one of their young scientists N.H. Ray had developed a method of determining trace quantities of acetylene in ethylene using thermal conductivity. This did not make sense to me, but, on discussion with Howard, we concluded that Ray must be using GC with a thermal conductivity detector. We decided that this was just what we needed for our hydrocarbon analysis, and built our first gas chromatograph. Our TC gauges were tungsten electric lamp-bulb filaments, hard soldered in narrow glass tubes between platinum electrodes. These tubes were mounted in a thermos flask to maintain constant temperature. We successfully separated the lower alkanes including the two butanes. A fellow research student was sceptical. He made up a mixture of butanes and asked us to analyze it. When we obtained the correct result, he accused us of secretly looking at his lab book. So we had to do it again under conditions of better security! Subsequently, Howard and I refined our methods and were able to separate many further hydrocarbon isomers. We also invented temperature programming but our paper, in 1953, proposing it, was rejected on the grounds that it was “too speculative”!

34.I.2. Mainly GC in Edinburgh When I came back to Edinburgh, I was determined to apply GC to analyze the products of hydrocarbon combustion, both the hydrocarbon and the oxygenated products; I knew this would be difficult so I decided to apply GC initially to a simpler problem, namely the relative chlorination rates of methane and ethane and of the two positions in propane. The beauty of GC in this regard was that I needed to go only to very small degrees of chlorination of the starting hydrocarbon; whereas, previous methods involved extensive chlorination followed by fractional distillation to separate the products. I obtained (1955) very accurate Arrhenius parameter differences for the pairs of reactions. This was the first use of GC as a quantitative tool. This line of work was continued by a sequence of research students who studied competitive chlorinations of hydrocarbons and of various chlorinated alkanes and alkenes. On the combustion front the great advantage of GC could again be exploited. For the first time, it was possible to analyze the very early stages of reactions. The main discovery I made was that the initial products from low temperature (300–400ºC) combustion were the alkenes having the same carbon number as the parent hydrocarbon, not the aldehydes which Norrish so fervently believed in, for example: C3 H8 C O2 D C3 H6 C H2 O2 The alkene went on to react to form carbonyl compounds: CH3 CH : CH2 C O2 D CH3 CHO C CH2 O Norrish’s aldehydes were secondary not the primary products, but he was right in supposing that they were important intermediates and largely responsible for chain branching once the reaction was under way.

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Although working mainly on gas kinetics, I began to make contributions in gas chromatography about this time. The famous paper by van Deemter, Klinkenberg and Zuiderweg, had laid the foundation of our understanding of the main band broadening processes in chromatography. Their equation for plate height, H , as a function of linear flow velocity, u, was popularized by A.I.M. Keulemans in the form: H D A C B=u C Cu Following the Edinburgh Symposium in 1960, which I helped to organize, Al Zlatkis invited me to attend the first Houston Symposium on Gas Chromatography in 1963, where Lilian McLaren and I presented a paper comparing band spreading in packed and open tubular columns. We used a piece of equipment built specially for us by the Electronics Division of Bruce Peebles in Edinburgh, which we called “Sir Hector”. This enabled us to measure the widths of very narrow peaks electronically rather than with a conventional recorder. At the Houston meeting I met Calvin Giddings, and we struck up an immediate rapport. During a taxi ride we thought of a novel way of determining the B-term in the van Deemter equation, or rather the obstructive factor. This involved eluting an unsorbed band, part-way down a GC column, allowing it to stand there for a few minutes with the flow stopped, and then eluting it. The additional broadening during the arrest time gave us the apparent diffusion coefficient, which could be compared with the unencumbered value to get . The method worked beautifully and is still, I believe, the best way to determine the obstructive factor. The results were delivered at Houston in 1964 [1]. Hugh Scott (1983) used the same method to determine effective diffusion coefficients in LC. Another interest about the same time was in the speed of chromatography. M. Saleem and I (1969), concluded that to make LC as fast as GC, it would be necessary to use particles of about 2 µm in diameter and pressures of around 200 bar [2]. Our conclusions in 1969 were ‘rediscovered’ many times over the subsequent years, but still represent the current state of the art in HPLC. Later Mary Gilbert and I (1979), analyzed the conditions necessary to make open-tubular LC compete with packed column LC. We concluded that to do this, open tubes would have to be around 1 µm in bore or less. At the time this seemed impracticable but the subsequent work of Tijssen and later Poppe has proved that spectacularly fast separations can be achieved in very narrow tubes, even if detection is a problem.

34.I.3. Liquid chromatography – band dispersion studies At Houston in 1963, Cal Giddings invited me to spend a sabbatical period with him in Salt Lake City. We eventually made it as a family in January 1964. We were attracted to Salt Lake City not only because Cal worked there, but because he also assured us that the best skiing in the US was to be found just a few miles away at Alta! He was right and we had a fabulous eight months enjoying the outdoor activities, which Utah provided, and which Cal and his group of eccentrics so much enjoyed. Shortly before this, Cal had proposed his coupling theory for eddy diffusion and mobile zone mass transfer. He had also introduced the concepts of the reduced plate height and reduced

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velocity. These enabled the theories of GC and LC to be unified, so that experiments in one field could be interpreted for the other. It also drew together the different modes of LC, showing that they should all obey the same fundamental laws. We decided that I should look at the validity of the coupling theory by doing experiments on band broadening using a liquid eluent and glass beads as column packing. This combination would allow us to work at very high-reduced velocities where the effects of axial diffusion would be negligible. The results showed that the reduced plate height, h, rose gradually as the reduced velocity (¹) increased from 1 to 10,000, at which point the curve flattened off with the onset of turbulence. Roughly speaking, h rose with a power of ¹ close to 1=3. While this broadly confirmed the coupling theory, the change over from mass transfer-dominated dispersion at low velocities to the eddy diffusion-dominated dispersion at high velocities was much more gradual than the original simple theory predicted. My original results were amplified and confirmed by Jon Parcher in Edinburgh (1969). To date there has been no theoretical explanation of this dependence. Jon also showed that trans-column spreading of a sample injected centrally into a glass bead column was very slow. This work led to the concept of the ‘infinite diameter’ column. Paul Raven (1976) measured the rate of radial dispersion directly. He showed that the plate height for radial dispersion was much less than the axial plate height and resulted from two processes: firstly normal molecular diffusion hindered by the packing, as for axial dispersion, and secondly a velocity independent contribution from stream splitting by the particles of packing. h radial D 2 =¹ C 0:1 My early work in GC plus my experiments in Utah showed how the A- and B-terms could be measured and confirmed broadly the explanations given by van Deemter and Giddings. When we came to look at optimizing column packings in the 1970s and 1980s, the use of reduced parameters was at the core of our analyses. Following the original work of Lilian McLaren (1962) we routinely plotted log h against log ¹. As a result of extensive work with a wide range of packing materials we proposed what has come to be known as the Knox equation. This provided a dimensionless way of comparing the performance of HPLC columns. h D B=¹ C A¹ n C C¹ In practice, when the Knox equation is fitted to experimental data, one normally gets a reasonable fit with B ³ 2, A ³ 1, n D 1=3, and C ³ 0:1. The value B ³ 2 is in accord with theoretical expressions for B, although the full expression for B should take into account axial diffusion in both the mobile and stationary phases. The values A ³ 1 and n D 1=3 are in accord with the results originally obtained in the glass beads experiments. By contrast, the theoretical expression for C derived by Giddings and others, shows that C should not be greater than about 0.01. The empirical value C ³ 0:1 is an order of magnitude too high. Hugh Scott [3] looked at this anomaly in 1983. Because the A- and contributions C-term to h are both velocity-dependent, it is necessary to work at very high reduced velocities in order to separate them. Hugh achieved this by using 50 µm laboratory-made particles of ODS silica gel. One of his

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Fig. 1. Plate height-velocity curve at very high reduced velocity, showing that A D 2:5 and C D 0:0125. Packing 50 µm ODS bonded silica gel; solute, phenol, k 00 D 1:86; eluent methanol=water (60=40 v=v). Source: Fig. 3 from Ref. [3].

(h; ¹) plots is shown in Fig. 1. Fitting the Knox equation gave A D 2:5, but the value of C was now only 0.0125 in excellent agreement with theory. This means that most of the dispersion which is actually seen in LC under practical operating conditions, arises from A-term dispersion, not, as was previously thought, from C-term dispersion. This surprising conclusion is discussed in more detail in two papers I wrote in 1998.

34.I.4. Column packings and the Wolfson Liquid Chromatography Unit When I returned from Utah in the late summer of 1964, I became much more interested in LC, which seemed to me to be ripe for development. It was not long before Josef Huber (1967), showed how very small particles could be packed to make highly efficient columns. Ray Scott was also active at this time in promoting the ideas of pressurized LC using small particles. But the major breakthrough came from Jack Kirkland who not only devised a way of making very high quality pellicular particles which Du Pont marketed under the name Zipax, but also an HPLC system incorporating a pressure intensifier pump and UV detector. Kirkland showed separations on Zipax, which astonished the world of chromatography, although nowadays they look quite modest. Subsequently he disclosed his 5-µm porous spherical silica gel particles at the Montreux Symposium in 1972, and the modern phase of HPLC had begun. In 1970, I was appointed as a consultant to Du Pont in the HPLC field, and John Done (1972) joined me as Du Pont Research Fellow. John’s main achievement was to show that 30–100 µm batches of Zipax conformed perfectly with the reduced parameter

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approach giving coincident (h; ¹) curves. Gordon Kennedy (1972), applied the same method to examining the fully porous materials Porasil and Corning Porous Glass and showed that, as expected, they give somewhat higher (h; ¹) curves. Just about this time (late 1971), I learned about some spherical particles of alumina available from AERE Harwell. These were made by the sol–gel process and were extremely uniform in size. George Laird (1974), showed just how good they were by determining their (h; ¹) curves which displayed a minimum h-value of below 2 for both retained and unretained solutes. These materials eventually became the basis of the Spherisorb range first commercialized by Phase Separations, and now marketed by Waters Ltd. In 1972, Richard A. Wall, Jadwiga Jurand and I were fortunate to be awarded a grant from the Wolfson Foundation to set up a unit for the development and commercialization of HPLC in collaboration with a UK instrument company. This was part of the Wolfson Foundation’s scheme to ‘Link Scientific Research with Industry’. The basis of our proposal was that all the running in HPLC was then being made by USA companies. We felt that it was high time UK companies got in on the act. My experience with Du Pont suggested that the best way to do this would be for a company to develop an HPLC instrument along with suitable columns and packing materials. At the same time it would be necessary to develop applications of the new technique. Dr. Jurand was primarily involved in this area and developed many separations in the clinical and pharmaceutical field. Some highlights of her work were the analysis of morphine alkaloids (1973), tetracyclines (1975 and 1979), tricyclic antidepressants (1975) catecholamines (1976), paracetamol and its metabolites (1978), nucleotides (1981), LSD and related drugs (1981). Our first appointment to the staff of the Wolfson Unit was Andrew Pryde, a superb organo-metallic chemist, whose job was to develop a group of microparticulate packing materials. Andrew’s method involved emulsifying acidified silica sol in a hydrocarbon, followed by drying and separating the hardened particles. Their size range was 3– 10 µms. Subsequently he reacted these with silanes to give methyl- ODS-, amino-, cyano- and ion-exchange-silica gels. After one false start we forged links with Shandon Southern Instruments (now Hypersil Ltd., a Division of Thermoquest) in 1974. This collaboration resulted in an HPLC instrument of advanced design for its time with a thermostatted enclosure housing a heat exchanger, injection valve, column and detector cell. The pump was a small capacity dual pressure intensifier unit. There were the usual teething troubles but the instrument worked well. Regrettably the company cancelled this part of the project for financial reasons, but fortunately the range of packings developed by Andrew Pryde [4] was continued and became the basis of the Hypersil range, now one of the popular brands of HPLC packing material. The new HPLC particles were assessed (1975) in the standard way by determining (h; ¹) curves. Minimum h-values of around 3 showed that the new 5-µm packings could be efficiently packed. In 1979, I had one of those bright ideas that are supposed to come to scientists in the bath. It was obvious by this time that silica based packings had their disadvantages, primarily instability at extreme pH. There was a need for a more chemically robust support. Several groups were looking at carbon. The best form of carbon was graphite, but it was difficult to make and intractable, especially if based on carbon black, a dirty and fragile material. My idea was to exploit the excellent structural properties of silica,

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which could be made with a wide variety of pore sizes and porosities. An appropriate silica gel would be impregnated with a polymer, which could then be pyrolysed to form carbon. The silica would be dissolved out by alkali, and the resulting carbon graphitized. I expected that the original structure of the silica gel would be preserved, while the micropores in the carbon skeleton would be removed by graphitization to give a porous but strong graphite. When my chromatographic colleagues unanimously said that this could never work, I knew that I was on to a good idea. Mary Gilbert, who succeeded Andrew Pryde, had the job of putting my ideas to the test, and devising appropriate chemistry. She did this brilliantly [5], but we nevertheless had a lot of teething problems, which took a good 10 years to solve. They were mostly associated with the graphitization process and surface contamination. Bulvinder Kaur made a major breakthrough in 1984 when she produced the first really good chromatograms on porous graphitic carbon (PGC). The peaks for the first time showed little or no tailing while h was in the region of 3. Shandon took on the product in 1989 and, after a formidable amount of further development work, Hypersil Ltd. successfully marketed PGC under the trade name Hypercarb. Paul Ross of Hypersil (1999) has recently studied some of the extraordinary properties of Hypercarb. Contrary to our expectations that graphite would behave as a kind of super-ODS phase, it shows remarkable retentive properties for polar and ionized solutes. These are attributed to the ready polarization of graphite which, with its huge pi-electron systems, can readily accommodate extra electronic charge or equally readily donate it.

34.I.5. Mechanisms in chromatography The word ‘mechanism’ to a physical chemist normally means the kinetic mechanism of a particular overall reaction, but to a chromatographer it means the thermodynamic basis for a particular type of retention. I have been interested in both types of ‘mechanism’ in chromatography. One of our first studies of a thermodynamic mechanism was that of ion-pair chromatography using detergent species such as sodium lauryl sulphate and cetrimide. George Laird (1976), showed that the adsorption of such substances by ODS silica gel was essentially irreversible and that the resulting material then acted as an ion exchanger. We called this ‘soap chromatography’. Later, Richard Hartwick (1991), examined the thermodynamic mechanism of ion-pair chromatography in more detail using alkyl sulphonates. At high concentrations and=or high Mw , they behaved as ion-exchangers, while at low concentrations and=or Mw , the mechanism was best described as ion-pair adsorption (the ion pairs comprising a sulphonate anion and an analyte cation). Very recently a similar situation has been demonstrated with porous graphite coated with polyethylene imine, which can be used as an ion exchanger for inorganic anions, as shown by Qian Hong Wan (1996). Coating does not, of course need to be by an ionic compound. Qian (1995), also showed that porous graphite coated with naphthalene sulphonyl phenylalanine could separate amino acid enantiomers by copper complexation, while Jadwiga Jurand in 1982, demonstrated enantiomeric separation with a chiral zwitterion adsorbed on to ODS Hypersil.

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In regard to kinetic mechanisms, slow mass transfer or slow equilibration are reflected in the C-term contribution to h. Masami Shibukawa (1991) discovered a beautiful example of slow chemical equilibration in his study of the HPLC of ethylenediamine tetraacetate–chromium complexes. He found that peaks of certain of these complexes were extremely wide but nevertheless symmetrical. Giddings had previously derived equations for the effect on h of slow kinetics and Masami was able to demonstrate that these applied precisely to the wide peaks he observed. The rate constants and activation energies for the processes in question agreed well with literature values measured independently.

34.I.6. Preparative HPLC We had only one excursion into preparative HPLC where column overloading is the name of the game. Hazel Pyper (1986) examined the practice and theory of overload chromatography. We came up with a simple way of determining the optimal conditions for prep. HPLC on the basis that overload peaks, which obeyed the Langmuir isotherm, were essentially triangular. A consequence of this is that the peak width increases with the square root of the distance migrated along the column, in exactly the same way as it does for normal kinetic band broadening (i.e., van Deemter type broadening). Accordingly it was possible to apply standard plate height theory to the process, and come up with simple guidelines for the optimum particle size and column length to be used for a given resolution in preparative LC. Cox and Snyder have used this idea in their extensive work on optimization of preparative LC.

34.I.7. Size-exclusion chromatography (SEC) Calvin Giddings pointed out that all forms of chromatography should obey the same basic kinetic theory, and that SEC was no exception. Nevertheless peaks in SEC are generally wide, and little seemed to be known about band dispersion for polymers. In 1977, Forbes McLennan and I published a short paper correcting an error in a publication dealing with how the polydispersity of a narrow range polymer (such as polystyrene standard) contributed to the peak width. This led on to an experimental study of kinetic band spreading and polydispersity in SEC [6] using silica gel as packing material and narrow Mw range standards. We showed for the first time, how these two contributions to band spreading could be separated. The (h; ¹) curves showed C-values of about 0.1 independent of the Mw of the polymer. We concluded that the higher Mw polymers had more difficulty (relative to their unobstructed diffusion rates) in moving around the pores of the packing material than the lower Mw polymers. However at 0.1, these C-values are very high, and it now seems to me that we were most probably observing broadening due to mobile zone effects not slow mass transfer rate within the particles. To prove this, it would be necessary to adopt the method of Scott with large particles and very high-reduced velocities. It would be an interesting project. Later Hugh Scott (1984) and Harry Ritchie (1987), studied how the SEC calibration

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curve (the plot of log Mw against elution volume) was related to the pore structure of a SEC packing material, this being found by mercury porosimetry. Assuming that a polystyrene behaved as a random sphere, and that the pores in the silica gel were cylindrical, the SEC calibration curve of log Mw against elution time could readily be calculated, and excellent agreement was obtained between the calculated and experimental curves. The task of calculating the pore size distribution from the SEC curve was more difficult, but again fairly good agreement was achieved. The work fully confirmed that the SEC exclusion coefficient K D .V Vo /=.Vm Vo / was entirely predictable on a steric basis.

34.I.8. Dead volumes and solvent peaks Roman Kaliszan spent a sabbatical year in my laboratory and studied a topic which had long intrigued me, namely how best to determine the dead volume of an HPLC column. He used isotopically labelled eluents to trace the retention or exclusion of different components of mixed solvents, as well as measuring the retention of solvent disturbance peaks. These behaved according to straightforward adsorption theory, and we came up with the simple measure of the dead volume of any column on the basis of isotopic labelling (1985) namely: Vm D ya Va C yb Vb C Ð Ð Ð where Vm is the dead volume, ya ; yb etc. are volume fractions of the solvent components, and Va; Vb etc. are the retention volumes of the isotopically labelled solvent components. This seemed to us an elegant solution to the problem, and showed that neither solvent disturbance peaks, nor excluded ion peaks, nor peaks of so called unsorbed solutes would give the correct answer.

34.I.9. Miniaturization of HPLC and electrochromatography Keith Freebairn (1984), carried out a study of miniaturization of HPLC using a specially constructed detection and injection systems to minimize dead volumes. This showed just how poor current commercial systems were, and gave us experience in handling very narrow bore columns. The project was continued by Iain Grant who developed new methods for making drawn packed capillaries, as well as slurry methods for packing quartz tubes with conventional column packing materials. He also carried out our first experiments on capillary electro-chromatography (CEC) using a power supply capable of delivering 100 kV. We found that above 50 kV arcing occurred so we restricted further experiments to a 30 kV maximum. Iain (1991) found [7], that there was no reduction in the linear electroosmotic velocity when the particle diameter was reduced to 1.5 µms, contrary to what had been found by some other workers. This opened the way to using very small particles for packing CEC columns. Iain found, not surprisingly, that electrically induced flow provided higher plate efficiencies than pressure driven flow using the same packed columns. The reason for this is that

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Fig. 2. Capillary electrochromatogram of aromatic hydrocarbons. Column, 500 mm long, 40 µm bore drawn packed capillary containing 5 µm Hypersil derivatized with dimethyloctadecylsilylchloride; solutes, aromatic hydrocarbons from naphthalene to methyl anthracenes. Source: Fig. 11 from Ref. [7].

electroosmotic flow is unaffected by channel diameter, so that electrically driven flow is considerably more uniform than hydraulically driven flow. Fig. 2 shows the beautiful separations, which can be achieved by CEC with peaks that are exceptionally narrow and symmetrical. In CEC as in capillary electrophoresis (CE), there is a certain amount of ohmic heating. If this is too severe bubbles form in the column and electrical breakdown occurs. I worked out an approximate theory for this self-heating in 1988. This was confirmed experimentally for electrophoresis by Kathleen McCormack (1994) who also refined the theory, and very recently by Bob Boughtflower for CEC. The main reason for the self-heating is the failure of convection to remove the heat fast enough. It is therefore important that commercial equipment for CE and CEC incorporates vigorous air cooling of the capillary. CEC is becoming increasingly reliable, and offers speeds and efficiencies that can be ten times those of pressure driven LC. The technique will surely develop into a major method in the future where it will rival capillary GC in its overall performance. The future for purely electrically driven chromatography lies in the use of particles which are in the 1 µm diameter range where the effects of A-, and C-term dispersion are minimized. The use of non-porous particles in electrophoresis should also result in improved performance and reliability. Finally one envisages that all the miniaturized liquid separation methods will be carried out on silicon chips.

34.I.10. Postscript Being a part of the international chromatographic community for 40 years has been a privilege. Many of the main participants have become my personal friends whose support and interest I have greatly valued over the years. Throughout this long period, my research students and post-doctoral colleagues have made the running and produced some excellent work, of which I am very proud. Without them little could have been achieved. But overall it has been fun, and immensely enjoyable.

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References 1. 2. 3. 4. 5. 6. 7.

J.H. Knox and L. McLaren, A new gas chromatographic method for measuring gaseous diffusion coefficients and obstructive factors, Anal. Chem., 36 (1964) 1477–1482. J.H. Knox and M. Saleem, Kinetic conditions for optimum speed and resolution in column chromatography, J. Chromatogr. Sci., 7 (1969) 614–622. J.H. Knox and H.P. Scott, B and C terms in the van Deemter equation for liquid chromatography, J. Chromatogr., 282 (1983) 297–313. J.H. Knox and A. Pryde, Performance and selected applications of a new range of chemically bonded packing materials in HPLC, J. Chromatogr., 112 (1975) 171–188. M.T. Gilbert, H.J. Knox and B. Kaur, Porous glassy carbon, a new column packing material for gas chromatography and high-performance liquid chromatography, Chromatographia, 16 (1982) 138–146. J.H. Knox and F. McLennan, Band dispersion in high-performance exclusion chromatography, J. Chromatogr., 185 (1979) 289–304. J.H. Knox and I.H. Grant, Electrochromatography in packed tubes using 1.5 to 50 µm silica gels and ODS, Chromatographia, 32 (1991) 317–328.

D.35. Milton L. Lee Milton L. Lee was born on July 20, 1946 in Salt Lake City, Utah. He received a B.A. degree in chemistry from the University of Utah in 1971 and a Ph.D. in analytical chemistry from Indiana University in 1975. M.L. Lee spent one year (1975–1976) at the Massachusetts Institute of Technology as a postdoctoral research associate before taking a faculty position in the chemistry department at Brigham Young University, where he is presently the H. Tracy Hall professor of analytical chemistry. M.L. Lee is an author or co-author of over 400 scientific publications, and is a co-author of two books, “Analytical Chemistry of Polycyclic Aromatic Compounds”, Academic Press, 1981, and “Open Tubular Column Gas Chromatography”, John Wiley, 1984. He is also a Co-editor of a comprehensive text entitled “Analytical Supercritical Fluid Chromatography and Extraction”, 1990. He is the founder and an Editor of the Journal of Microcolumn Separations and is on the Editorial Advisory Boards of Chromatographia, Polycyclic Aromatic Compounds, Field Analytical Chemistry and Technology and Fresenius’ Journal of Analytical Chemistry. Since 1980, he has given over 500 presentations on various aspects of his research, of which approximately one-third were invited lectures at major conferences and symposia. He is a member of the Scientific Committee for the International Symposia on Capillary Chromatography. In addition, he has organized a number of symposia sessions on specialized topics in various national and international meetings. Following is a list of scientific awards he received for his achievements in research and professional activities: 1982 1984 1985

Karl G. Maeser Research and Creative Arts Award, Brigham Young University. M.S. Tswett Chromatography Medal. H. Tracy Hall Professor of Chemistry, Brigham Young University.

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Lee Scientific Founder’s Award. American Chemical Society, Utah Award. Keene P. Dimick Chromatography Award. American Chemical Society Award in Chromatography. 5th Annual Research Achievement Award, Brigham Young University. Tswett Award of the Russian Chromatographic Society. Analytical Supercritical Fluid Technologies Award. Merit Award of the Chicago Chromatography Discussion Group. Northeast Regional Chromatography Discussion Group, National Chromatography Award. A.J.P. Martin Gold Medal. COLACRO Medal. M.J.E. Golay Award. ACS Award in Chemical Instrumentation. Doctor of Philosophy honoris causa, Uppsala University. Stephen Dal Nogare Award of Delaware Valley Chromatography Forum. Distinguished Alumni Award Indiana University Graduate School. Eastern Analytical Symposium Award for Achievements in Separation Science.

M.L. Lee is also an entrepreneur, and has been involved in transferring technology from his university research laboratory to the private sector. In 1982, he founded Alpine West Laboratories, which is a Utah Corporation specializing in contract research in the area of high resolution chromatography and chromatography=mass spectrometry. In 1984, he founded Lee Scientific to manufacture and market supercritical-fluid chromatographic instrumentation. In 1991, he founded another analytical instrument company, Sensar Corporation, to manufacture and market unique time-of-flight mass spectrometric instrumentation. In addition, he acquired ownership of the Journal of Microcolumn Separations in 1991, and became the publisher as well as editor. These activities have earned him the Mountain West Venture Group Award for Most Promising Company Developed from University Based Technology in 1987, the Utah Governor’s Medal for Science and Technology in 1987, and the award for Outstanding Achievement in Technology Transfer at Brigham Young University in 1994. He is listed as an inventor on ten patents. He is best known for his research in microcolumn separations and time-of-flight mass spectrometry. Specific areas of research include: (a) development of instrumentation, column technology, and mobile phases for capillary supercritical-fluid chromatography, (b) development of instrumentation for time-of-flight mass spectrometry, (c) development of element-selective detection systems, (d) synthesis and characterization of selective polysiloxane stationary phases for capillary chromatography, (e) surface chemistry of fused silica as it applies to the preparation of efficient, well-deactivated, and thermally stable capillary columns, (f) column technology for capillary electrophoresis, (g) separation and identification of polycyclic aromatic compounds in coal-derived products, and (h) high resolution chromatography–mass spectrometry. See Chapter 5B, a, d, h, j, l, o, p

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35.I. QUARTER OF A CENTURY OF RESEARCH IN CAPILLARY COLUMN SEPARATIONS AT BRIGHAM YOUNG UNIVERSITY Milton L. Lee Department of Chemistry and Biochemistry, Brigham Young University, P.O. Box 25700, Provo, UT 84602-5700, USA

My academic career began in 1976 when I was offered a position as an assistant professor of chemistry at Brigham Young University. Since that time, I have supervised the research of 37 students that have earned masters or doctoral degrees and co-authored 430 papers that describe this work. The major achievements in my laboratories can be divided into four categories: ž capillary column technology, ž high resolution chromatographic analysis of polycyclic aromatic compounds, ž capillary column supercritical-fluid chromatography, and ž time-of-flight mass spectrometry. I will attempt to summarize the most important accomplishments in each of these areas.

35.I.1. Capillary column technology In 1980, at an early stage in my academic career, I published with the help of an excellent graduate student, the most extensive and complete review to date on the preparation of glass capillary columns for gas chromatography. This effort provided the foundation for research on capillary column technology in my laboratory that has spanned over 23 years. We have made contributions in the areas of column surface deactivation, wettability, stationary phase film stability, free-radical cross-linking, and synthesis of selective polysiloxane stationary phases. In the late 1970s, we were studying the deactivation of glass capillary columns using Auger electron spectroscopy with the help of David Hercules at the University of Pittsburgh. Hydrochloric acid leaching was currently being used to remove Lewis acid components in Pyrex glass tubing before silylation and coating with the stationary phase. Auger electron spectroscopy was used to obtain depth profiles of the glass inside the capillary to determine how deep the ions were leached from the surface, and how soon they migrated back to the surface during heat treatment. Just at the time that we had completed our study and were preparing to publish our findings, the landmark work of Dandeneau and Zerenner on fused silica capillary columns was reported in Hindelang, Germany, and published soon thereafter. We immediately turned our attention to deactivating and coating fused silica tubing. The wettability of the fused silica surface was of primary concern at the time. With the help of Keith D. Bartle at the University of Leeds, we measured the contact angles of different treated surfaces using the capillary rise method to determine the most effective methods of deactivating the surface (blocking acidic silanol groups), while at the same time producing a surface that was wettable by the stationary phase of

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concern. High temperature reactions with chlorosilanes, alkoxysilanes, cyclic siloxanes, and polysiloxanes produced well-deactivated surfaces; however, the high temperatures required for the reactions caused some oxidation of the polyimide coating on the fused silica tubing. Our major contribution in this area was with the first use of silicon hydride-containing polysiloxanes for deactivation. The silicon hydride groups were very reactive with surface silanol groups; the by-product of the reaction was hydrogen gas instead of acidic or basic compounds. The deactivation layer was a densely bonded and cross-linked polymeric film, and the reactions were conducted at temperatures below 250ºC. The polyhydrosiloxane deactivation reagents contained substituent groups (methyl, phenyl, and cyanopropyl) that provided the wettability needed for the stationary phases of interest. The column surface wettability was a major factor that affected the stationary phase film stability in the coated capillaries; however, other factors also contributed. Even when a uniform film was initially coated on the capillary, droplets often formed on the surface after using the column. We as well as others recognized the importance of stationary phase viscosity on column stability, and began to synthesize and work with ‘gum’ polysiloxanes. The temperature–viscosity properties of these polymeric phases were critical. Gum phases at room temperature could become very low viscosity liquids at even 100ºC. The polysiloxanes were found to be superior in their temperature– viscosity properties because the helical structure of the polysiloxane backbone minimizes the reduction in viscosity with temperature. This also explained why the C87 branched hydrocarbon synthesized by Kovats to be a standard stationary phase for gas chromatography was not successful. While it was a waxy solid at room temperature, it had the viscosity of water near 100ºC. It was not until 1987 that the subject of film stability was completely understood with the help of a physical chemist, Robert S. Hansen, at Iowa State University. We explained how, regardless of wettability, any film on the inner surface of a cylindrical tube is inherently unstable and tries to rearrange in order to decrease the interfacial energy of the liquid=gas interface, i.e., to reduce the area of the inner surface of the film and eventually produce droplets. This Raleigh instability can decrease the efficiency of a column during the processes of deactivation with polymeric reagents, coating with polymeric stationary phases, and cross-linking. It explains the difficulties encountered in preparing columns with small internal diameters and with thick films. It explains the need to prepare and immobilize the stationary phase as rapidly as possible. Free-radical cross-linking for immobilizing the stationary phase film on the wall of the capillary column was discovered in the early 1980s by several research groups. We were interested in this area because of our work in supercritical-fluid chromatography where the stationary phase had to resist the solvating power of the mobile phase. Our first paper on this subject appeared several months after similar work was reported by two other groups. Our most significant contribution in this area was the first report of the use of azo compounds as free-radical initiators. These compounds produced efficient cross-linking of polysiloxane stationary phases without producing reactive or polar by-products. Much of the work on capillary column technology during the past 20 years has involved the development of stationary phases that have the right chemical structures and properties to produce highly efficient and stable columns, as well as desired selectivity.

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This includes the incorporation of functional groups for selectivity, for cross-linking or immobilization, functional groups or polymer backbone modification for increased thermal stability, and adjustment of polymer length for optimizing viscosity and solubility in coating solvents. In a collaborative effort with a synthetic organic chemist, Jerald S. Bradshaw, we synthesized, tested, and reported in the literature over 150 new polysiloxane stationary phases. These new polymeric phases contain a wide variety of functional groups including phenyl, cyanopropyl, biphenyl, n-octyl, methoxyphenyl, pentafluorophenyl, nitrophenyl, cyanophenyl, crown ether, oligoethylene oxide, (methylsulfonyl) phenyl, cyanobiphenyl, liquid crystalline biphenylcarboxylate ester, chiral cyclohexylene benzamide, and cyclodextrin. Fig. 1 shows representative structures of the functional groups of the different phases that were synthesized. In recent years, we have directed our attention to producing immobilized polymer coatings on spherical particles for packed capillary chromatographic separations. These polymer-encapsulated particles have shown superb inertness and selectivity, but with some reduced capacity compared to conventional LC packing materials. Furthermore, we have developed a number of polymer coatings for CE that are tailored to provide specific electroosmotic flow or no flow at all, and that produce inert surfaces for sensitive analytes. We even investigated the use of polymeric hollow fibers extruded from polypropylene and polymethylmethacrylate for CE. We recently achieved very high efficiencies in CEC with continuous-bed columns made by bonding large-pore particles together using sol–gel technology.

35.I.2. High resolution chromatographic analysis of polycyclic aromatic compounds My doctoral dissertation was concerned with the analysis of polycyclic aromatic hydrocarbons in air particulate matter, tobacco smoke, and marijuana smoke using capillary column gas chromatography. Complex mixtures of these compounds were difficult to analyze because of the numerous isomers that were present. My chromatograms demonstrated the best resolution obtained for such compounds at that time. I continued work in this area as a post-doctoral research associate at Massachusetts Institute of Technology, studying compounds produced from turbulent diffusion flames, and demonstrated the differentiation of polycyclic aromatic hydrocarbon isomers using mixed charge-exchange=chemical ionization mass spectrometry. My first major research grant at Brigham Young University was a grant from the Department of Energy to identify the sulfur heterocycles in coal-derived liquids. At that time, there were only three commercially available sulfur heterocyclic compounds, benzothiophene, dibenzothiophene, and a benzonaphthothiophene to use as standard compounds for comparison with the sulfur heterocyclic fractions that we isolated from coal liquids. I teamed with a synthetic organic chemist, Raymond N. Castle, and over the next twelve years, synthesized over 220 new sulfur heterocyclic compounds that were predicted to be possible in coal liquids. Separations were performed using capillary column gas chromatography. This work still stands as the most comprehensive analytical study of sulfur heterocycles in coal-derived materials.

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Fig. 1. Representative structures of the synthesized polysiloxane stationary phase functional groups.

Particularly noteworthy was the development and use of a smectic liquid crystal polysiloxane stationary phase that afforded shape selectivity for the polycyclic aromatic compound isomers in gas and supercritical-fluid chromatography. The polymer was unique in that it remained smectic from room temperature to 300ºC, and could be

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used in temperature programmed gas chromatography. An intense summer at the Pittsburgh Energy Technology Center with Curt M. White led to the development of a retention index system, similar to the Kovats Retention Index, for polycyclic aromatic hydrocarbons that was based on naphthalene (200), phenanthrene (300), chrysene (400), and picene (500) as standard reference compounds. The retention indices of over 300 polycyclic aromatic compounds were measured and tabulated for use by other laboratories.

35.I.3. Capillary column supercritical-fluid chromatography The development of capillary supercritical-fluid chromatography occupied much of our time in the 1980s. This work began in collaboration with Milos Novotny during a summer sabbatical at Indiana University. During the following decade, we developed the fundamental theory, instrumentation, and column technology for capillary supercritical-fluid chromatography, and demonstrated its potential for the analysis of a wide range of sample types. Open tubular columns could be maintained under supercritical conditions along the total column length with only a small pressure drop because of the openness of the column. Some of the problems that were solved included the construction of pumps and seals that could withstand high pressures, stable capillary columns that could withstand the solvating power of the supercritical mobile phase, efficient restrictors to maintain supercritical pressures in the column, and computer control of pressure=density programming. Major effort was spent in interfacing the supercritical-fluid chromatograph to several different mass spectrometers. Our research in supercritical-fluid chromatography produced 16 Ph.D. dissertations and over 100 scientific publications. The technology was commercialized and sent to laboratories around the world for a variety of applications. It was found that supercritical-fluid chromatography was particularly applicable for separations that were typically accomplished using normal phase liquid chromatography, such as separations of low molecular weight polymers, polymer additives, petroleum fractions, agrochemicals, and chiral compounds. A by-product of our work in supercritical-fluid chromatography was the development of a supercritical fluid slurry packing system for packing capillary columns with small particle packings. We demonstrated that 250 µm i.d. capillaries as long as 10 meters could be packed with 12 µm diameter particles. We have used this packing method for preparing packed capillary columns for current research in supercritical-fluid chromatography, ultra-high pressure liquid chromatography, and capillary electrochromatography. Particles as small as 1 m diameter have been efficiently packed in fused silica tubing.

35.I.4. Time-of-flight mass spectrometry In the late 1980s, we began work to develop a time-of-flight mass spectrometer to use as a detector for microcolumn liquid-phase separations. Time-of-flight mass

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spectrometry was the mass spectrometer of choice because of its inherent sensitivity and speed. We decided to use an atmospheric pressure ion source and to use orthogonal acceleration geometry because of the high duty cycle of this configuration. The result was the first reported atmospheric pressure ionization time-of-flight mass spectrometer system. The instrument could produce 5000 spectra per second with excellent sensitivity, and could be used as a detector for high-speed separations for which the eluting peaks were very narrow. This new mass spectrometer system was soon commercialized, and in 1996, we received an R&D 100 award for its invention. Work continued in our laboratory to interface the time-of-flight mass spectrometer to the various capillary separation techniques using an electrospray ion source. We developed both sheath flow and microelectrospray configurations, and demonstrated their use with capillary electrophoresis, isotachophoresis, capillary liquid chromatography, and capillary electrochromatography. We are currently modifying the mass spectrometer for application to high through put screening.

References: key papers selected 1. 2. 3. 4. 5. 6. 7.

J.C. Fjeldsted and M.L. Lee, Capillary supercritical fluid chromatography, Anal. Chem., 56 (1984) 619A–624A. B.A. Jones, K.E. Markides, J.S. Bradshaw and M.L. Lee, Contemporary capillary column technology for chromatography, Chromatogr. Forum, 1 (1986) 38–44. M.L. Lee and K.E. Markides, Chromatography with supercritical fluids, Science, 235 (1987) 1342– 1347. C.H. Sin, E.D. Lee and M.L. Lee, Atmospheric pressure ionization time-of-flight mass spectrometry with a supersonic ion beam, Anal. Chem., 63 (1991) 2897–2900. A. Malik, W. Li and M.L. Lee, Preparation of long packed capillary columns using carbon dioxide slurries, J. Microcol. Sep., 5 (1993) 361–369. M. Lazar, B. Xin and M.L. Lee, Design of a time-of-flight mass spectrometer as a detector for capillary electrophoresis, Anal. Chem., 69 (1997) 3205–3211. B. Xin and M.L. Lee, Design and evaluation of a new capillary electrochromatography system, Electrophoresis, 20 (1999) 67–73.

D.36. Hendrik Lingeman Hendrik Lingeman was born in 1954, educated at the State University of Utrecht for his B.A. (1975), his M.A. (1981) and his Ph.D. (1986). His thesis title was ‘Selective Pre Chromatographic Derivatization Methods for Carboxylic Acids (HPLC with fluorescence detection and GC with nitrogen selective detection)’. Other research included LC analysis of basic compounds on non-modified silica and alumina in aqueous solvent mixtures). From 1985 to 1988, he was a post-doctorate fellow at the Division of Analytical Chemistry, Center for Bio-Pharmaceutical Sciences, State University of Leiden. His next role as an

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assistant professor (1988–1991) was in the Department of Analytical Chemistry, Faculty of Chemistry, Vrije (Free) Universiteit, Amsterdam, The Netherlands. He has been an associate professor at the same university since 1991, teaching (courses in bioanalytical strategies, chromatography, separation sciences, electrochemical analysis and environmental analysis=chemistry), and advising eight Ph.D. students, serving on Ph.D. examination committees and on several faculty committees. He has been involved in two international joint research projects as coordinator: GC for the analysis of environmental samples (1994–1998, his university and one in India), and automated determination of micropollutants in industrial and communal waste water (1998–2001, his University plus partners in Spain, Greece, Austria, United Kingdom and Lelystad, The Netherlands). He has been active in six international societies, such as the Board of Trustees of the International Association for Pharmaceutical and Biomedical Analysis (USA), as Acting Secretary of the European Foundation of Pharmaceutical and Biomedical Analysis. He has served as a member of the organizing committees for 10 international conferences on chromatography or pharmaceutical areas, including being co-chairman of the Organizing and Scientific Committees of the First International Symposium on Micropollutants (Amsterdam, May 1998), and First International Symposium on Separations in the Biosciences. He serves on the Editorial Board of three chromatography journals and one pharmaceutical journal. H. Lingeman is the co-author of ca. 110 original scientific papers, ca. 35 review papers, ca. 20 book chapters; co-editor of 5 books (including dissertation), and has presented many oral presentations and posters at international meetings, seminars and symposia. He received the Jubilee Medal in 1990 from the Chromatographic Society, UK. See Chapter 5B, a, d, f, h, k, o, r, s

36.I. AUTOMATION OF ALL STEPS IN CHROMATOGRAPHY Hendrik Lingeman Department of Analytical Chemistry, Faculty of Chemistry, Vrije Universiteit, Amsterdam, The Netherlands

After his educational years, Dr. Lingeman joined the faculty of Vrije (Free) University as an assistant professor in 1988, and then continued as an associate professor in 1991. He, his students and colleagues have focused their research on the development of ž On-line systems combining GC detectors with LC and SFC; ž Post-column reaction=detection systems based on post-column ion-pair formation and fluorescence detection, including photochemical reactions, for acidic compounds in biological and environmental samples; ž Automated on-line pre-concentration systems with LC and photodiode-array detection for pesticides in tap, surface and waste water; ž Automated on-line sample preparation techniques (e.g., dialysis, immunoaffinity precolumns, column switching, automated SPE, membrane-extraction disks) for LC; ž Diode laser-induced fluorescence detection systems for LC and sensor devices;

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ž On-line monitoring systems for fermentation processes based on ultrafiltration and LC; ž Injection and detection systems in CZE and bioseparations of collagen proteins and protein fragments; ž Chemiluminescence systems for the determination of drugs in biological fluids; ž Automated sample preparation procedures for organic compounds in combination with CZE; ž Membrane-based extraction procedures for the in-field sampling of organic pollutants in industrial waste water; ž Multidimensional-separation systems for peptides and protein in biological samples. His research interests have centered on the development of automated procedures for the determination of all kinds of analytes in biomedical, pharmaceutical, and environmental samples using sophisticated chromatographic and electrophoretic separation techniques in combination with on-line sample preparation, derivatization, and reaction-detection systems. The emphasis is on the development of new on-line sample preparation techniques (e.g., dialysis, immunoaffinity, solid-phase isolation, column-switching, supercritical-fluid extraction), automated pre-column derivatization techniques, combined separation techniques (e.g., chromatography and electrophoresis), detection techniques (e.g., laser-induced fluorescence), and post-column derivatization techniques.

References for original papers (11 cited of 110 published) 1.

2.

3.

4.

5.

6.

7.

8.

C.M.B. van den Beld, H. Lingeman, G.J. van Ringen, U.R. Tjaden and J. van der Greef, Laser-induced fluorescence detection in liquid chromatography after preliminary derivatization of carboxylic acid and primary amino groups, Anal. Chim. Acta., 205 (1988) 15–27. H. Irth, G.J. de Jong, H. Lingeman and U.A.Th. Brinkman, Liquid chromatographic determination of azidothymidine (AZT) in human plasma using on-line dialysis and preconcentration on a silver(I)-loaded stationary phase, Anal. Chim. Acta., 236 (1990) 165–172. A. Farjam, A.E. Brugman, A. Soldaat, P. Timmerman, H. Lingeman, G.J. de Jong, R.W. Frei and U.A.Th. Brinkman, Immuno- precolumn for selective sample pretreatment in column-liquid chromatography: Immunoselective desorption, Chromatographia, 31 (1991) 469–477. N.C. van de Merbel, J.M. Teule, H. Lingeman and U.A.Th. Brinkman, Dialysis as an on line sample-pretreatment technique: Influence of experimental variables using benzodiazepines as model compounds, J. Pharm. Biomed. Anal., 10 (1992) 225–233. J. Slobodnik, E.R. Brouwer, R.B. Geerdink, W.H. Mulder, H. Lingeman and U.A.Th. Brinkman, Fully automated on-line liquid chromatographic separation system for polar pollutants in various types of water, Anal. Chim. Acta., 268 (1992) 55–65. A.J.G. Mank, E.J. Molenaar, H. Lingeman, C. Gooijer, U.A.Th. Brinkman and N.H. Velthorst, Visible diode-lasers induced fluorescence detection in liquid chromatography after pre-column derivatization of thiols, Anal. Chem., 65 (1993) 2197–2203. N.C. van de Merbel, P. Zuur, M. Frijlink, J.J.M. Holthuis, H. Lingeman and U.A.Th. Brinkman, Automated monitoring of amino acids during biotechnological processes using on-line ultrafiltration and column-liquid chromatography: Application to fermentation medium improvement, Anal. Chim. Acta., 303 (1995) 175–185. J.R. Veraart, S.J. Kok, J.M. te Koppele, C. Gooijer, H. Lingeman, U.A.Th. Brinkman and N.H. Velthorst, The separation of two collagen crosslinkers with capillary electrophoresis and laser-induced fluorescence diode-array detection, Biomed. Chromatogr., 12 (1998) 226–231.

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References for review papers (4 cited of ca. 35 published) 1. 2. 3. 5.

H. Lingeman, W.J.M. Underberg, A. Takadate and A. Hulshoff, Fluorescence detection in high-performance liquid chromatography, J. Liq. Chromatogr., 8 (1985) 789–874. H. Lingeman, R.D. McDowall and U.A.Th. Brinkman, Guidelines for bioanalysis using column liquid chromatography, Trends Anal. Chem., 10 (1991) 48. N.C. van de Merbel, H. Lingeman and U.A.Th. Brinkman, Sampling and analytical strategies in on-line bioprocess monitoring and control, J. Chromatogr. A, 725 (1996) 13–27. H. Lingeman and S.J.F. Hoekstra, Particle-loaded membranes for sample concentration and=or clean-up in bioanalysis, J. Chromatogr. B, 689 (1997) 221–237.

References for book chapters (ca. 20) or books (5) (partial list) 1.

2.

3.

4.

C.M.B. van den Beld and H. Lingeman, Laser-based detection in liquid chromatograph with emphasis on laser-induced fluorescence detection; in W.R.G. Baeyens, D. De Keukeleire and K. Korkidis (Eds.), Luminescence Techniques in Chemical and Biomedical Analysis, M. Dekker, Inc., New York, NY, USA, 1990. H. Lingeman, C. Gooijer, N.H. Velthorst and U.A.Th. Brinkman, Chromatography–fluorescence= luminescence spectroscopy; in A.M.C. Davies and C.S. Creaser (Eds.), Analytical Applications of Spectroscopy II, Royal Society of Chemistry, Cambridge, UK, 1991. C. de Ruiter and H. Lingeman, Analytical applications of phase-transfer catalysis, in Y. Sasson and R. Neumann (Eds.), Handbook of Phase-Transfer Catalysis, Blackie Academic and Professional, London, UK, 1997, pp. 405–423. H. Lingeman and W.J.M. Underberg (Eds.), Detection-oriented derivatization techniques; in Liquid Chromatography, M. Dekker, Inc., New York, NY, USA, 1990, 389 pp.

D.37. Per G.M. Flodin Per G.M. Flodin was born in 1924 and received a fil.kand. degree in chemistry at the University of Uppsala in 1950 and a fil.lic. degree in biochemistry in 1953. He was employed by AB Pharmacia 1954 to 1962 and earned a Ph.D. in biochemistry at the University of Uppsala in 1962. He was Research Director at Perstorp AB in 1962 to 1972 and Associate Professor in polymer technology at the Royal Institute of Technology in Stockholm 1972 to 1977. Between 1977 and 1991 he was Professor and Head of Department of Polymer Technology at Chalmers University of Technology in Gothenburg. He was a cofounder of Artimplant AB (publ.) and is presently member of its board and advisor in polymer research. Flodin is a member of the Swedish Academy of Engineering Sciences, the American and Swedish Chemical Societies and has been active in IUPAC, the ad hoc group on polymer science of the European Science Foundation, the Swedish Board for Technical Development, the Swedish Board for Higher Education. He has been consultant of a number of Swedish companies and member of several boards and advisory boards. He has authored more than 100 scientific papers and is inventor of more than 40

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patents and pending patent applications. His present research interests are synthesis, characterization, and technology of biodegradable polymers for use in orthopedic surgery. Flodin received in 1963 the Arrhenius Gold Medal of the Swedish Chemical Society, in 1968 the Gold Medal of the Swedish Academy of Engineering Sciences; in 1979, the M.S. Tswett award; and in 1986 the Distinguished Medal of the University of Helsinki. See Chapter 5B, a, c, g, l, r

37.I. SEPHADEX AND GEL FILTRATION Per G.M. Flodin Chalmers University of Technology, Gothenburg, Sweden

Ever since the existence of macromolecules was acknowledged, scientists have wanted to separate molecular species according to their molecular weights. The first successful approach was by ultracentrifugation as elaborated by Svedberg and coworkers. However, no complete separation could be achieved and it was not until the appearance of size exclusion chromatography (SEC) that such results could be obtained. In SEC the separation takes place according to differences in hydrodynamic volume which property is closely related to the molecular weight. SEC was originally called gel filtration – a term still used by many scientists. The early development of gel filtration took place in the laboratories of AB Pharmacia and at the Institute of Biochemistry both in Uppsala, Sweden. Since the author was deeply involved in all phases of the early development and still possesses much of the early documentation in the form of laboratory notebooks, reports etc., the following account of the history is as true as possible more than 40 years after it happened. To fully describe the circumstances under which gel filtration became a reality I shall start with the work on electrophoresis in packed columns.

37.I.1. The Institute of Biochemistry (1950–1953) Attracted to biochemistry by the fame of Arne Tiselius after he had received the Nobel Prize in 1948 I began my graduate studies in 1950 under his supervision. He had made his fundamental work on boundary electrophoresis and electrophoresis was still one of his main interests. There was work going on to make electrophoresis a preparative and not just an analytical method, as was the boundary method. Attempts had been made to use columns packed with glass beads to avoid disturbances due to convections but they were only occasionally successful. Another approach was to perform electrophoresis in paper sandwiched between glass plates. It had been investigated for a year by an American post doc when I was assigned to the project in the fall of 1950. I tried a great number of treatments of paper and of the glass plates and tested them with separations of blood serum proteins, but the results were meager.

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However, one observation was made which proved to be valuable later and it was that filter paper on treatment with methanolic HCl disintegrated into a powder. After more than a years of fruitless experimentation I began to look for another approach and it was then that Jerker Porath and I started a fruitful cooperation on zone electrophoresis. Half a year after me, in the fall of 1950, Jerker Porath who was a licentiate in organic chemistry, came to the Institute to work for a Ph.D. in biochemistry. His research field was peptide hormones from the pituitary gland, and the goal was to isolate, characterize, and ultimately, synthesize them. In the first step of isolation, he wanted to use all methods available at the Institute, and understandably a preparative electrophoresis procedure was of great interest for him. Thus in our frequent discussions the idea was born to use starch instead of glass beads in column electrophoresis. In connection with the move in 1952 of the Institute into a new modern laboratory building we (Jerker and I) secured some old equipment and started experimenting with starch as column packing material. Since the procedure involved the introduction of a narrow zone into a column, displacing it with buffer to a position suitable for electrophoresis, running the electrophoretic separation, and finally displacing the zones formed out of the column into a fraction collector, the utmost care was necessary while packing the column to avoid zone distortion. For the future development of column electrophoresis and later gel filtration the early work on column packing and handling was very important. To study the result of the packing procedure we used a zone of hemoglobin to be able to visually observe the behavior of a zone while it was transported down the column by elution with buffer. It was when using hemoglobin together with a yellow DNP-amino acid that we observed that they migrated at different rates. After a number of experiments made to exclude adsorption as the cause we concluded that it was the difference in molecular size which determined the difference in the rate of migration. In our publication on starch electrophoresis we did not dare to say this but reported it as “anomalous behavior” [1]. The observation was an important piece of information for the future since it provided the ground for the prepared mind considered necessary for a serendipitous discovery like that of gel filtration. The starch method made it possible to use electrophoresis to separate proteins and peptides preparatively. The next step in the development of column electrophoresis was to make a packing material in which the ‘anomalous behavior’ of starch was avoided. For this purpose I used my earlier observation that treatment of paper with methanolic HCl yielded a powder. By using purer alpha-cellulose like cotton linters it was possible to lower the adsorption to the cellulose and use it only as the anticonvection medium it was meant to be [2]. Excellent separations of serum proteins and many other proteins and peptides were obtained with the cellulose powder and both the apparatus and the paper powder were commercially available for many years. In the fall of 1953 I got my licentiate degree with a thesis named ‘Zone Electrophoresis in Packed Columns’. Shortly after I accepted an offer from AB Pharmacia to work in their research laboratory.

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37.I.2. AB Pharmacia (1954–1962) In 1954 Pharmacia was a small pharmaceutical company which just had moved from Stockholm to Uppsala to be close to the university. It was mainly home market oriented but had at that time two products on the international market. One of them was Macrodex, a blood plasma extender first used in the Korean war. Its main ingredient was derived from the polysaccharide dextran. Now that the war was over the management felt that new products based on dextran were needed and it became my task to look for such opportunities. Dextran is a very high molecular weight bacterial polysaccharide. To be used as plasma extender it had to be hydrolyzed and fractionated to a well defined molecular weight range just above the kidney threshold (around 30,000 dalton). The high molecular weight fractions could be reprocessed while the low molecular weight ones had to be discarded. From a process economical point of view the waste was a considerable cost factor the minimization of which was important. My supervisor, Bjo¨rn Ingelman (co-inventor of Macrodex) had already in the late 1940s tried to increase the molecular weight of the waste by reaction with epichlorohydrin but always got insoluble three dimensional networks, gels. I continued his work seeking to find methods to obtain soluble products. To make a long story short, it was only with mono-or oligosaccharides that it was possible to get soluble polymerization products, all higher oligomers or polymers yielded gels.

37.I.3. Sephadex In the summer of 1956 Jerker Pora´th was working on his Ph.D. thesis which was to be defended in public in 1957. He was using zone electrophoresis in cellulose columns as one of his methods to separate the peptide hormones referred to above. When we met he mentioned that he was not content with the adsorption properties of the cellulose powder he used but looked for alternatives. I suggested that he try a new type of insoluble material that I had synthesized. To be able to custom make material for electrophoresis I had to get permission from my employer, AB Pharmacia, to devote part of my time for the purpose. Commercially it was not interesting for a pharmaceutical company but as a gesture to the Institute of Biochemistry I was allowed to use a small part of my time in this pursuit. However, I was not allowed to mention how the material was made and strict secrecy and exclusive rights to the products were conditions to which we had to comply. During the summer of 1956 I made the first batch of packing material for an electrophoresis column and Jerker made the first experiments with it. Afterwards he told me that the adsorption properties for basic peptides had improved but the material had the same disadvantage as starch in that small molecules migrated at lower rates than the large ones when just eluting them through the column. The question was whether the material was useful at all for the intended purpose. The observation did not immediately lead to the concept of gel filtration. In fact it took several months of mental and practical work before it materialized. For me it was the realization that gels of this kind could be used for dialysis type separations that

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gave the impetus to further work. Thus during the fall of 1956 and early 1957 I used hemoglobin and sodium chloride as model substances to evaluate the use for dialysis type separations. In an internal report dated May 1957 I presented the results of Jerker Porath’s and my experiments and surveyed the future uses of dextran gels. It is amazing to see today how many of the future applications that were visualized at that early stage. The report was well received by the Pharmacia management and led to authorization of a project to which some laboratory resources were allocated. We (the people at Pharmacia) were given one year to show results which would indicate commercial possibilities. A hectic period followed comprising preparation of patent applications, finding limits for applications, suitable crosslinking degrees, particle sizes and many other things. Already in the fall of 1957 the two basic patent applications were filed. The first one was the Sephadex patent with Bjo¨rn Ingelman and me as inventors and the second, the Gel Filtration patent with me and Jerker Porath as inventors. From the summer of 1957 on, more and more people became involved in the project and in the end of 1958 ten people were engaged. Since the costs of the project increased rapidly, in 1958 a steering committee of three was appointed with the object to put the first product on the market in 1959. It was a tough task but understandable since the project took resources from research on pharmaceuticals which was the main business of the company. The advocates of pharmaceuticals were not too happy about the project but the top management saw the possibilities, probably influenced by Tiselius who had been consulted since the project was authorized. Due to the limited time and money at our disposal we (the steering committee) decided to concentrate on products for dialysis-like separations and to limit the introduction to two markets, the USA and Sweden. Thus Sephadex G-25 supplemented by G-50 became the products to concentrate the resources on until the market introduction. Among the tasks to be done before the introduction were to establish specifications for the products, arrange for production, make booklets and brochures, prepare advertising, and not the least create names for the product and the method. We chose Sephadex (Separation Pharmacia Dextran) for the material and Gel Filtration (as suggested by Tiselius) for the method. It was decided that the introduction in the USA was to take place at the Gordon Conference on Proteins in New Hampshire in July 1959. Tiselius arranged an invitation for me to the conference. We also tried to get a short paper in Nature published just before the Gordon Conference. All these measures worked perfectly, I gave my lecture at the conference, the paper in Nature appeared as expected [3], and when I returned to New York two weeks after the conference all the Sephadex we had had in stock there had been sold. In fact throughout 1959 and into 1960 we had difficulties to satisfy the demand for the products. Now that Sephadex had proven to be a success the development of new products based on the concept could start. A number of such had been made in the laboratory and could now be brought to the market. I believe that the most important one was an invention I made in 1959, the making of gels as spherical beads. It was made by new kind of inverse suspension polymerization where the dextran–NaOH solution was suspended in an organic solvent containing a stabilizing polymer and the epichlorohydrin added to the solvent. Thus every droplet acted as a reactor, the reaction ran smoothly at 40ºC,

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and the heat of reaction was easily taken care of. Further benefits in the production were very high product yields and very narrow particle size distributions. Since the spherical form also gave lower resistance to flow in packed columns more loosely crosslinked gels (Sephadex G-200) could be used allowing separation of species with larger sizes. Furthermore products were made based on reactions of the hydroxyl groups of the various Sephadexes. By etherification both anion and cation exchanging derivatives were developed (DEAE-, CM-, SE-Sephadex) as well as propoxylated gels for use with other solvents than water (the Sephadex LH series).

37.I.4. Gel filtration The development of the gel filtration method took place both in Jerker Porath’s laboratory at the Institute of Biochemistry and at Pharmacia. Due to the strict secrecy requirements of the Company, we unfortunately had to restrict the information given to Jerker Porath before 1959. Thus two parallel developments took place. Jerker’s main interest was in peptide hormones and small proteins while we at Pharmacia emphasized dialysis type separations, polysaccharide fractionation and later (with G-200 and ion exchangers) separation of blood serum proteins. As the contact person I had the possibility to coordinate the efforts of the two groups. As mentioned above the dialysis type separations were the ones for which we expected a market large enough to motivate commercialization for laboratory use. There was also a possibility of production scale use which was judged as most promising. Thus much of my early work was devoted to what we called group separation. The technique of packing columns I had learned the hard way during the electrophoresis period now proved useful again. The first factors to be evaluated were the degree of crosslinking and the particle size versus the efficiency and rate of separation. Since in the beginning I was both the synthesizer and the user feedback was no problem. Soon I had assistants in both roles and after numerous experiments what became called Sephadex G-25 (25 standing for water regain of 2.5 g=g dry gel) was found to be a good compromise for group separation gels [4]. When the gel type had been chosen it remained to determine how large sample volumes and how high solute concentrations could be used. In contrast of most other chromatographic method the concentration per se proved of little importance as long as the viscosity did not exceed a certain limit. Thus very high concentrations of globular proteins could be separated from salts in volumes up to about 30–40% of the bed volume. High flow rates could be used and the column was regenerated when the experiment was finished. This paved the way for automatization and scaling-up of the process. In Fig. 1 the first automatic gel filtration apparatus is shown. With it the sucrose–epichlorohydrin copolymer Ficoll was separated from salt in 398 cycles of two hours each. The principle has since been used on much larger scale for separations in many production scale processes [5]. For the main product of Pharmacia, Macrodex, it was of great interest to get a fast method to characterize the molecular weight distribution of the dextran fractions used. Earlier characterization was made by tedious fractional precipitation which could take

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Fig. 1. Schematic drawing of an automatic gel filtration apparatus. The column A with sample distributor B is fed with sample from pump C1 and eluant from pump C2. The pumps and solenoid valves D2, D3 for collection of fractions are regulated by a timer E. From [5].

several weeks to complete for a single sample. The first report published on molecular weight characterization by SEC was presented at the IUPAC meeting on polymers in Wiesbaden in 1959. It was found that plotting the elution volumes vs. the logarithm of molecular weight yielded straight lines [6]. For the company the important thing was the time to obtain a complete molecular weight distribution was reduced from several weeks to less than a day. At the Institute of Biochemistry I had separated blood plasma proteins by electrophoresis in columns. Then the different rates of electrophoretic migration was the separation factor. With the advent of Sephadex G-200 size became an additional dimension. The difference in separation behaviour of blood plasma proteins in three Sephadex types is illustrated in Fig. 2. The position of a number of proteins in the G-200 diagrams were recorded using various detection methods (chemical, immunological etc.) on the fractions [7]. By combination of gel filtration with electrophoresis or ion exchange chromatography still more advanced separations could be obtained [8]. Almost pure components like transferrin and 7S γ-globulin could be isolated. 37.I.5. Some characteristic features of SEC In contrast to many other chromatographic methods the mechanism of SEC is well understood. It is the hydrodynamic volume of a solute which determines its elution volume provided non-specific adsorptive forces are absent. Reduction of the latter has been and still is an important task in the development of media for SEC. The hydrodynamic volume is closely related to the molecular weight and thus to one of

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Fig. 2. Elution pattern of bovine plasma proteins from three Sephadex types. Top: G-75; center G-100; bottom: G-200. In the bottom diagram the first peak contained lipoproteins and macroglobulins, the second one all the 7S antibodies, and the third one albumin, transferrin, and α1 -glycoprotein. From [5].

the most fundamental concepts in chemistry. It makes it conceptually easy to design experiments and to interpret experimental results. In a complex mixture of solutes, like in many biological fluids, a SEC experiment sorts the solutes in groups of similar molecular weights which facilitates further separation by other methods. Another characteristic feature of SEC is that the column used is regenerated in the experiment and a new experiment can be started when or even before the slowest solute in the preceding one has emerged. No change of solvent is needed. Unlike other chromatographic methods SEC is insensitive to solute concentration per se provided the viscosity of the samples is low. Thus globular proteins can be handled at fairly high concentrations without undue zone broadening. This paves the way for preparative use of SEC in many biological systems like for example separation of blood serum or the change of buffer for protein solutions. The regeneration without solvent change and the possibility to use high concentrations of solutes make automation of SEC for preparative purposes easy. It has made industrial scale processes possible and in fact a large number of such are in use today.

37.I.6. Final remarks Many of the results described above were summarized in my Ph.D. thesis which was defended in public in May 1962 [4]. Shortly afterwards I left Pharmacia for a leading position at another company and thus my years with Sephadex and gel filtration ended (a review is given in Ref. [9]). At that time the project generated sufficient money to sustain its own development. It was made a division of Pharmacia and later a subsidiary

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called Pharmacia Fine Chemicals. A number of acquisitions and mergers later it was Pharmacia Biotech and now it is Amersham Pharmacia Biotech AB with more than 3000 employees and a turnover of $500 million. Many great improvements have been made and the SEC method has become a standard method in many branches of science including biochemistry, molecular biology, and polymer science. In particular I should like to mention the use of agarose and macroporous gels which have greatly expanded the range of the method. I believe size exclusion chromatography will be used for a long time in the future. Finally, I would like to emphasize that although the original inventions was were made by a few people the major part of the developments were from teamwork involving many persons all of whom were essential for the success of the project.

References 1. 2. 3. 4. 5. 6. 7. 8.

9.

P. Flodin and J. Porath, Zone electrophoresis in starch columns, Biochim. Biophys. Acta, 13 (1954) 175. P. Flodin and D.W. Kupke, Zone electrophoresis in cellulose columns, Biochem. Biophys. Acta, 21 (1956) 368. J. Porath and P. Flodin, Gel filtration: a method for desalting and group separation, Nature, 183 (1959) 1657. P. Flodin, Dextran Gels and Their Applications to Gel Filtration, PhD thesis, Uppsala (1962). P. Flodin, Methodological aspects of gel filtration with special reference to desalting operations, J. Chromatgr., 5 (1961) 103. K. Granath and P.Flodin, Fractionation of dextran by the gel filtration method, Makromol. Chem., 48 (1961) 160. P. Flodin and J. Killander, Fractionation of human serum proteins by gel filtration, Biochim. Biophys. Acta, 63 (1962) 403. B. Gelotte, P. Flodin and J. Killander, Fractionation of human plasma proteins by gel filtration and zone electrophoresis or ion exchange chromatography, Arch. Biochem. Biophys., Supplement, 1 (1962) 319. P. Flodin, The sephadex story, Polymer Eng. Sci., 38 (1998) 1220.

D.38. James S. Fritz James S. Fritz was born in Decatur, Illinois on July 10, 1924, and received a B.Sc. degree from James Millikin University. He obtained an M.Sc. degree in 1946 and a Ph.D. in 1948 from the University of Illinois under G. Frederick Smith. He was Assistant Professor at Wayne State University (1948– 1951), and then moved to Iowa State University where he has been Professor of Chemistry since 1960. James Fritz is known for his research in several areas of analytical chemistry and has more than 289 published papers. He is also author or co-author of several books including the textbook “Quantitative Analytical Chemistry” (5 editions) and “Ion Chromatography”

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(2nd edition, 1987). J. Fritz has received several major awards: the A.C.S. National Award in Chromatography in 1976, the A.C.S. Award in Analytical Chemistry in 1985, the L.S. Palmer Award of the Minnesota Chromatography Forum in 1987, the First International Chromatography Forum Award in 1988, and the Dal Nogare Award of the Delaware Valley Chromatography Forum in 1991. He was named Distinguished Professor at Iowa State University in 1990. See Chapter 5B, c, h, l, g, s

38.I. ADVANCES ON SEVERAL FRONTS James S. Fritz Department of Chemistry and Ames Laboratory, US Department of Energy, Iowa State University, Ames, IA, USA

38.I.1. Introduction Not all successful research fits neatly into any single category. I have selected four topics to include in this brief paper. The first, solid-phase extraction, is a concept of sample preconcentration that has become recognized as a necessary adjunct to several kinds of chromatography. Ion chromatography has also stood the test of time. The last two topics, HPLC modifiers and combined ion chromatography–capillary electrophoresis, describe research done in the later 1990s that may have an impact on the chromatography to come.

38.I.2. Solid-phase extraction Around 1970, a diverse group of chemists at Iowa State University asked themselves: How can we identify and quantify organic pollutants in drinking water when their concentration is well below the detection levels of the best chromatographic methods (then) available, by gas chromatography? Concentration of the pollutants is the obvious answer. Repeated partial freezing and discarding the pure ice particles was briefly considered. Extraction with an immiscible organic liquid gave only partial extraction of test compounds and a questionable degree of extraction of pollutants of unknown identity. The approach taken was to use small particles of a porous polystyrene resin (Rohm and Haas XAD-2) as a solid extractant. This resin had a very high surface area (¾300 m2 =g) and thus approximated the condition in liquid–liquid extraction where the temporary emulsion has a high interfacial area between water and liquid solvent for efficient mass transfer. Thus the solid polymer particles could be packed into a column and the water sample passed through. In essence, the packed column generated

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a number of theoretical plates and thereby gave a much more complete extraction of trace organics than liquid–liquid extraction, which was only a single-stage equilibrium. Following extraction of the organic pollutants by the resin in the packed column, the extracted materials could be completely desorbed by elution with a relatively small volume of diethyl ether. After careful evaporation of most of the ether, a portion was injected into a gas chromatograph for separation of the individual organic compounds. In the early 1970s, identification of completely unknown GC peaks presented a real problem. Fortunately two co-investigators (Harry Svec and Gregor Junk) had one of the first GC–MS setups in existence and were able to identify the major pollutants in a contaminated well that then supplied part of the drinking water for Ames, Iowa. Following an initial publication in 1972, a detailed study was made covering all aspects of using a resin extraction column to concentrate trace organic compounds from water samples. More than 100 test compounds covering a wide variety of chemical structures were shown to be essentially quantitatively recovered. A comprehensive paper published in 1974 [1] was later identified as one of the 20 most cited papers ever published in the Journal of Chromatography. It was some years later that the name, solid-phase extraction (SPE), was coined to describe this type of extraction. Now at the beginning of the 21st century, SPE has undergone a surge of popularity and has become recognized as an essential adjunct to chromatographic analysis.

38.I.3. Ion chromatography Ion-exchange chromatography has been in existence for much of the 20th century but its development languished for lack of a suitable automatic detector. All this changed in 1975 when a commercial instrument was introduced that used a conductivity detector for an improved form of ion-exchange chromatography that was dubbed ‘Ion Chromatography’. The reason this system worked was that an ion-exchange ‘suppressor’ column was placed between the separation column and the conductivity detector. The suppressor column converted the basic eluent ion primarily to a molecular form and thereby reduced the background conductance. Sample ions remained in their ionic form and could be easily detected. But was the use of a second ‘suppressor’ column (which initially was somewhat awkward to use and maintain) the only way that a conductivity detector could be used in ion-exchange chromatography? in 1979, we produced a series of anion exchangers with capacity, it became possible to use a much lower ionic concentration in the eluent for anion-exchange chromatography. Later in 1979 Gjerde, Fritz and Schmuckler [2] devised a form of chromatography that used a conductivity detector but did not require the use of a second suppressor column. The anion-exchange resin used had a capacity of only 0.007–0.07 meq=g and required only a very dilute solution (ca. 104 M) of an organic salt in the mobile phase [See Figs. 6 and 7, Ref. 2]. The organic anion (benzoate or phthalate) had a much lower equivalent conductance than the typical inorganic anions to be separated. Thus, the detection was based on an increase in conductance when a sample anion passed through the detector. While the detection sensitivity was not as good as the suppressed system, it was quite adequate for most separations. The

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non-suppressed system also allowed greater flexibility in the eluent ions that could be used. The practical aspects of this simple, rugged method were recognized, as illustrated by this excerpt from an early paper [2]: Practical application: determination of sulfate in water The quantitative determination of parts per million concentrations of sulfate in water is a widely studied problem; often a turbidimetric or spectrophotometric procedure is used for the analysis. Low concentrations of sulfate can be separated rapidly by anion chromatography, as shown in Fig. 9 [2]. Using an appropriate calibration graph, an accurate quantitative determination of the sulfate is possible. The efficacy of the chromatographic method was demonstrated by separating a series of samples containing chloride, nitrate and sulfate on a 50-cm XAD-1 column with 6:5 ' 104 M potassium phthalate (pH 6.2) as the eluent. The chloride and nitrate concentrations were held constant at 5.12 and 12.2 ppm, respectively, while the sulfate concentration was varied from 2.75 to 13.75 ppm. A plot of sulfate peak height versus concentration proved to be perfectly linear, so that an accurate quantitative determination of sulfate was possible. The chromatograms obtained indicate the possible use of the new chromatographic system for routine water analysis for the simultaneous determination of three anions. In the intervening years many improvements have been made in ion chromatography, which in its various forms is now considered as an essential chromatographic method in a contemporary analytical laboratory. Modern ion chromatography does use resins of quite low exchange capacity and includes non-suppressed as well as suppressed detection techniques.

38.I.4. Mobile-phase modifiers in HPLC: when bigger is better Separations in HPLC are based on differences in partitioning of the various analytes between the mobile and stationary phases. It is common practice to use a C18 silica stationary phase and vary the composition of an aqueous–organic mobile phase so that the retention times of the sample analytes will be within a desired range for a good separation. For the most part, methanol, acetonitrile, or another common solvent is used as the organic component in the mobile phase. However, incorporation of an organic modifier of higher molecular weight in the mobile phase can be a real advantage. Separation of formic, acetic, propionic butyric and valeric acids illustrates this point [3]. With a hydrophobic stationary phase (polystyrene or C18 silica), pure water may be used as the mobile phase to separate formic and acetic acids, but propionic and butyric acids elute late with extremely poor peak shape. A good separation of all five acids required a mobile phase containing 60% methanol–40% water. A similar separation on the same column was obtained with 40% ethanol and with 20% n-propanol (80% water). An aqueous mobile phase containing only 5% n-butanol gave an excellent separation of all five acids in <5 min. For the separations with propanol–water and

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especially with butanol–water, the amount of organic modifier in the mobile phase is too small to explain the observed effects on the basis of increased solvation in the liquid phase. Adsorption of a monomolecular layer of butanol on the stationary phase surface was shown to explain the observed results. The modified surface greatly reduces the partitioning between the predominately aqueous eluent and the stationary phase. Conductivity detection of the sample acids gave much better sensitivity with the butanol–water eluent than with those containing a much higher proportion of an organic solvent. Another case where ‘bigger is better’ is illustrated by the HPLC separation of 18 organic analytes covering a broad range in polarity and size, from benzene to a PAH compounds containing six or more fused benzene rings [4]. Isocratic elution with acetonitrile–water could not resolve all of the sample compounds in any reasonable amount of time. But addition of only a 50 mM concentration of a suitable surfactant (Brij 30, for example) to a 60% acetonitrile mobile phase showed a drastic reduction in the retention times of the larger polycyclic aromatic hydrocarbons (PAH) compounds while still allowing good resolution of the earlier peaks. An effect much like that obtained with gradient elution was obtained with this unconventional isocratic eluent! Studies with several surfactants in 60% acetonitrile showed no appreciable adsorption of the surfactant surface, and micelle formation seemed unlikely due to the high acetonitrile concentration. It was speculated that the analytes formed association complexes of vary stabilities with the surfactant in the liquid phase. Later work showed that mixtures of two or even three different surfactants could provide even more dramatic reductions in retention times and changes in selectivity.

38.I.5. Ion chromatography–capillary electrophoresis (IC–CE) In capillary electrophoresis (CE) separation of analyte ions is based on differences in their electrophoretic mobilities. CE is a powerful separation method with actual theoretical plate numbers often >100,000. Nevertheless, the electrophoretic mobilities of many ions are too similar to be resolved by CE alone. So why not combine CE with ion chromatography (IC) where the elution order of sample ions is often different? For example the migration order of halide ions in CE is I , Br > Cl > I while the order in IC is F > Cl > Br > I . In thinking of ways to combine these two techniques, the use of solid ion exchange resins has the disadvantage of contributing significantly to peak broadening. A little known technique that used a soluble polymer with quaternary ammonium groups combines ion-exchange and electrophoretic separation functions in a single phase seemed to be a much better approach. Operationally, a soluble polymer (represented as PC Cl ) is simply added to the background electrolyte in a standard setup for capillary electrophoresis. The following equilibrium occurs between the polymer and a sample anion (A ): PC Cl C A $ PC A C Cl

K D

PC A ð Cl A ð PC Cl

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Fig. 1. Separation of 17 inorganic and organic anions. Peaks: 1 D bromide; 2 D nitrate; 3 D chromate; 4 D iodide; 5 D molybdate; 6 D phthalate; 7 D 1,2,3-tricarboxylate; 8 D 1,2-benzenedisulfonate; 9 D terephthalate; 10 D isophthalate; 11 D benzoate; 12 D p-toluenesulfonate; 13 D 1,3,5-tricarboxylate; 14 D 2-naphthalenesulfonate; 15 D 1-naphthalenesulfonate; 16 D 3,5-dihydroxybenzoate; 17 D 2,4-dihydroxybenzoate; x D unidentified impurity. From Ref. [5].

Separation of anions is based on differences in the ratio of the free anions (A , fast moving) and ‘complexed’ anions (PC A , slow moving) as well as differences in the electrophoretic mobilities of the free anions. We found that a polymer concentration of 0.05–0.6% together with a high salt concentration (120–150 mM) in the electrolyte provides the excellent conditions for separation of anions. Bromide-iodide, 1- and 2-naphthalene sulfonic acids, and other ion pairs that cannot be separated by CE alone were baseline resolved [5]. A mixture of 17 inorganic and organic ions could be separated in <6 min, generating an average of nearly 300,000 plates=m. [Fig. 1, Ref. 5]. IC–CE is elegant in its simplicity as well as its effectiveness. The ion-exchange contribution may be readily strengthened or weakened by varying the type and concentration of the salt used as well as the concentration of polymer.

References 1.

G.A. Junk, J.J. Richard, M.D. Grieser, D. Witiak, J.L. Witiak, M.D. Arguello, R. Vick, H.J. Svec, J.S. Fritz and G.V. Calder, The use of macroreticular resins in the analysis of water for trace organic contaminants, J. Chromatogr., 99 (1974) 745–762.

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D.T. Gjerde, J.S. Fritz and G. Schmuckler, Anion chromatography with low-conductivity eluents, J. Chromatogr., 186 (1979) 509–519. J. Morris and J.S. Fritz, Eluent modifiers for the liquid chromatographic separation of carboxylic acids using conductivity detection, Anal. Chem., 994, 66 (1994) 2390–2396. X. Li and J.S. Fritz, Novel additives for the separation of organic compounds by high-performance liquid chromatography, J. Chromatogr. A„ 728 (1996) 235–247. J. Li, W. Ding and J.S. Fritz, Separation of anions by ion chromatography–capillary electrophoresis, Abstract No. 1178, Pittcon 99, Orlando, FL.

D.39. Charles H. Lochmu¨ller Charles H. Lochmu¨ller was born May 4, 1940 in New York City. He received his B.Sc., in Chemistry, Manhattan College (1962); M.Sc., Ph.D., Fordham University (1964,1967) (with M. Cefola). Professional Experience: USAEC Postdoctoral Fellow, Purdue University (1967–1969) (L.B. Rogers); Assistant Professor (1969–1974), Associate Professor (1974–1978), Professor (1978–present), Duke University, Department of Chemistry; Professor of Biochemical Engineering, Duke University, School of Engineering (1985–1998.); Director of Graduate Studies, Center for Biochemical Engineering (1990); Director of the Center for Biochemical Engineering (1991–1993); Chairman, Department of Chemistry (1982–1987); Elected Member, Committee of Revision – United States Pharmacopeial Convention (1985–1990,1990–1995, 1995–2000); Chairman, Division of Analytical Chemistry, American Chemical Society (1983–1984); Member, Committee to Review NBS=NIST Programs: Center for Analytical Chemistry–National Research Council (1987–1990). Editorships and Editorial Boards: ž Critical Reviews in Analytical Chemistry – Editor-in-Chief (1994–present); ž Isolation and Purification – Editor (1991–1994), Associate Editor (1994–present); ž Journal of Chemical Information and Computer Sciences; Journal of Chromatographic Sciences; Journal of Chemometrics; Chemically Modified Surfaces; ž Proceedings of the Estonian Academy of Science–Chemistry –Advisory Board. Lectureships: Dow Lecturer – Bucknell University – 1985. Scientific Lecturer – Academy of Sciences, Peoples Republic of China – 1985, 1987. SACP Lecturer – Penn State University, 1987. Scientific Exchange Lecturer – Academy of Science, USSR – 1987; 1988; 1989, 1990. ž Special Summer Lecturer – University of Wyoming – 1988. ž Summer Lecturer 5th Nordic Summer School in Polymer Chemistry – University of Helsinki, Finland – 1994. ž ž ž ž

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Honors: American Microchemical Society Student Award – 1964. Pioneer in Laboratory Robotics Award – 1985. American Chemical Society Award in Chromatography – 1987. North Carolina Distinguished Chemist, American Institute of Chemists – 1988. Life Member with Honors, Estonian Chemical Society –1996. EKS Societal Medal No. 12 of the Estonian Chemical Society 1997, and membership in ž Phi Lambda Upsilon. ž ž ž ž ž ž

Affiliations: American Chemical Society; Fellow, Royal Society of Chemistry (Faraday Division); Fellow, American Institute of Chemists; International Chemometrics Society – Treasurer (1995–96). Research interests: Chemical separations; spectroscopy of surfaces, modified surfaces and surface-bound species; chemically-modified surfaces; chemometric methods and robotics; large-scale separations. See Chapter 5B, a, e, h, q

39.I. DISCOVERIES, CONTRIBUTIONS, EVENTS AND RECOLLECTIONS Charles H. Lochmu¨ller Duke University, Department of Chemistry, Durham, NC 27708, USA

My graduate training was under the tutelage of Dr. Michael Cefola, who earned his degree with Benedetti-Pichler in the area of microchemistry. He was the person selected to set up the microchemistry laboratories at Chicago Stagg Field [Manhattan Project] and was a participant in the first isolation of plutonium and its physical and chemical characterization on a microgram scale in that facility. My work for him was on the effect of mixed solvents on the ketoenol tautomerism of betadiketonates used for uranium extraction. I did the first use of NMR for that kind of study. Cefola encouraged me to accept L.B. Rogers’ offer of a post-doc at Purdue University to study protein stabilization at high temperatures using high system pressures. I accepted, and while I did many things with ‘Buck’ Rogers, I never got to do the protein work. At Purdue, I designed and oversaw the construction of the equipment for the very first truly high-pressure liquid chromatograph where system pressures were kept in the 5000 atm range. We showed the first example of the pressure dependence of adsorption equilibria in liquid chromatography. We also showed the first successful example of the use of an electrical field to orient the liquid crystalline phase for GC and its effect on selectivity. The next 30 years, and my career at Duke University, can be divided arbitrarily into three emphasis areas: (a) The resolution of enantiomers, (b) The nature of

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covalently-bound, bonded phases for liquid chromatography, and (c) The use of factor analysis and target factor methods to study complex chemical systems and to predict retention behavior in reversed-phase chromatography. Along the way there were other discoveries and milestones such as: the first use of photo-thermal spectrometry to study acid–base behavior of silica bound molecules; pioneering work in the use of proton beams for proton-induced X-ray emission (PIXE) in environmental and protein chemistry, and the first use of laboratory robots driven by SIMPLEX optimization algorithms and by Expert systems. We also published the first use of the analysis of peak variance for the design of high efficiency detectors for open tubular or capillary chromatography. I became interested in the resolution of enantiomers in Dr. Rogers’ group at Purdue. We did work with a carbonyl-bis-amino acid esters, which was incorrectly called a ‘ureide’ by its first discoverer. One evening while studying the mechanism of association of such esters with chiral amides, Rex Souter noticed that our samples gave narrow NMR lines 20 degrees below the isotropic melting point [1]. We pursued this and discovered that these esters exhibited liquid crystalline behavior and were smectic not cholosteric in nature. We also showed that when these stationary phases were in the smectic state, the largest Þ values ever seen for GLC separation of enantiomers were observed. The pursuit of this discovery and the developments from it consumed the better part of a decade of graduate student effort. Independent of the liquid-crystal work, I became interested in the physical and chemical nature of ‘bonded phases’ used for high-performance liquid chromatography. For a variety of reasons, we chose to pursue charge transfer acceptor and donor phases in the first work done. They were spectroscopically interesting and there was a chance we could combine spectroscopy and retention measurements in the development of a new model for such phases. Along the way, I reasoned that it should be possible to resolve enantiomers and that it would be a goal to resolve enantiomers of hydrocarbons with no substituent groups. A candidate class of hydrocarbon was the helicenes. These molecules are helical coils formed by aromatic rings bonded in a continuation of the structure of phenanthrene. At about 6-rings there is overlap of the rings to form what could be called, by analogy, a lock-washer conformation. The right and left helical forms are enantiomers. We succeeded in separating the hexa- and hepta-helicenes and related compounds using a chiral charge transfer acceptor bonded phase of our own creation [2]. The most commonly used phases for HPLC are hydrocarbon phases consisting of alkyl silane substituents bound to silica gel. The fundamental questions we asked beginning in the late 1970s include: (a) Are these hydrocarbon phases, or is the effect that of creating a graphite adsorbent? (b) Do molecules intercalate into the bonded phase? (c) Are the phases dynamic in terms of organization and do they respond to changes in mobile phase contribution? While the chase involved many sophisticated spectroscopic experiments, the first discovery was the result of simple shake flask experiments with pure paraffins. From those results, we proposed a collapsed model in which the chains were associated to reduce the surface area exposed to the hostile mobile phase components such as water. Ultimately we used time-dependent excited state spectroscopy to prove the model [3]. This led to many more experiments on the

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effect of prior thermal history of the silica on the texture of the phase formed on the reversibility of thermal dehydration in terms of phase texture after silanization. There are experiments on mixed phases both polar and non-polar, and evidence was published for the enhancement of mass-transport kinetics. If the long chains were spaced by insertion of trimethylsilyl groups on the surface, dramatic increases in net plate number were seen. The binding of large molecules to the bonded phase in RPLC is an area of some importance. One, it is important to understand e.g., protein hydrophobic bonded phase-interaction because more people are interested in recovering native protein, than are excited about analytical methods, which deliberately denature the protein because only quantitation is of interest. Two, proteins immobilized on the surfaces were used as catalysts for reactions, offering the easy removal of the catalyst by filtration or the use of packed beds as flow reactors. Is the reduced activity often seen for bound enzymes a result of denaturation, or do proteins form multi-layers of sorbed molecules where only the top layer is exposed and active? The first observations of a dynamic change in conformation of a protein sorbed on a hydrocarbon phase were reported in the Journal of the American Chemical Society, by Lochmu¨ller and Saavedra [4]. This work followed a series of equilibrium studies where the native fluorescence of apomyoglobin was seen to undergo significant shifts depending on the length of the bound chain, and the nature of the bound hydrocarbon [4]. Later studies showed that not all enzymes present on the packed bed were accessible to small substrate molecules, and that this was true both for esterase and oxides proteins. Finally, we were led to use the factor analytical methods to attempt to understand the complex nature of solute and mobile phase determined retention in RPLC. The goal was to see if abstract methods free of historical chromatography models could give either confirmation of accepted views or provide new ones. The first work of its kind is to be found in the work of Breiner, Koel and Lochmu¨ller [5]. The importance of this first paper was the discovery that retention was indeed factor analyzable. This opened the opportunity for classifying behavior based on mobile phase independently of the solute effect and vice versa. Perhaps even more importantly, it opened the opportunity to perhaps build basis sets of retention and to use these to predict the likely behavior of a new, previously unstudied solute. The impact on method development by practical users of RPLC could be dramatic. Full detail of the factor analysis work would fill a book. Since [5] was published, we have demonstrated: (1) It is possible to predict the behavior of a new solute in retention terms in 50 solvents using retention measurements made in 3 key solvents (solvents which best span solvent space); (2) It is possible to predict across column types (C18 to C18, C18 to C8, etc.) with precisions of 2% or better in observed vs. predicted retention values; (3) Addition of a sorbed buffer component, such as acetic acid, leads to the appearance of another abstract factor in the analysis, and suggests that a mixed bonded phase is formed, or that the standard state of the sorbed state changes with buffer concentration and mobile phase composition; and (4) It is possible to construct libraries of factor loadings for a given column type, and to use 3 measurements as a minimum to predict retention over a wide range of mobile phase composition to a precision of about one percent.

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References 1. 2. 3. 4. 5.

6.

C.H. Lochmu¨ller, J. Harris and R. Souter, Chromatographic resolution of enantiomers, 1H and 13C NMR studies of ureide–amide systems, J. Chromatogr., 71 (1972) 405–413. C.H. Lochmu¨ller and R.R. Ryall, Direct resolution of enantiomers by high-performance liquid chromatography on a bonded chiral stationary phase, J. Chromatogr., 150 (1978) 511. C.H. Lochmu¨ller, A.S. Colborn, M.L. Hunnicutt and J.M. Harris, Organization and distribution of molecules chemically bound to silica, Anal. Chem., 55 (1983) 1344. C.H. Lochmu¨ller and S.S. Saavedra, Intrinsic fluorescence characteristics of apomyoglobin adsorbed to microparticulate silica, Langmuir„ 3 (1987) 433. C.H. Lochmu¨ller and S.S. Saavedra, Intrinsic florescence characteristics of apomyoglobin adsorbed to microparticulate silica, Langmuir, 3, 433, (1987); Interconversion of conformation of apomyoglobin adsorbed on hydrophobic silica gel, J. Amer. Chem. Soc., 109 (1987) 1244–1245. C.H. Lochmu¨ller, S. Breiner, C. Reese and M. Koel, Characterization and prediction of retention behavior in reversed-phase chromatography using target factor modeling, Anal. Chem., 61 (1989) 367–375.

D.40. James E. Lovelock James Ephraim Lovelock was born on July 26, 1919 in Letchworth Garden City in the United Kingdom. He graduated as a chemist from Manchester University (1941) and received a Ph.D. degree in medicine from the London School of Hygiene and Tropical Medicine in 1948. He received the D.Sc. degree in biophysics from London University (1959). After graduating from Manchester, he started employment with the Medical Research Council at the National Institute for Medical Research in London, but five years between 1946 and 1951 were spent at the Common Cold Research Unit at Harvard Hospital in Salisbury, Wiltshire. In 1954, he was awarded the Rockefeller Traveling Fellowship in Medicine and chose to spend it at Harvard University Medical School in Boston. He visited Yale University for a similar period (1958). He resigned from the National Institute in London in 1961 to take up full time employment as Professor of Chemistry at Baylor University College of Medicine in Houston, Texas, where he remained until 1964. During his stay in Texas, he collaborated with colleagues at the Jet Propulsion Laboratory, Pasadena, California on Lunar and Planetary Research. Since 1964, he has conducted an independent practice in science, although continuing honorary academic associations as a Visiting Professor, first at the University of Houston and then at the University of Reading in the U.K. He has been associated with the Marine Biological Association at Plymouth (1982), first as a council member, and from 1986 to 1990 as its president. James Lovelock is the author of approximately 200 scientific papers, distributed almost equally among topics in medicine, biology, instrument science and geophysiology. He has filed more than 50 patents, mostly for detectors for use in chemical analysis. One of these, the electron capture detector, was important in the development of environmental awareness. It revealed for the first time the ubiquitous distribution of pesticide

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residues and other halogen–bearing chemicals. This information enabled Rachel Carson to write her book, “Silent Spring” (1962), often said to have initiated the awareness of environmental disturbance. Later it facilitated the discovery of the presence of PCBs in the natural environment. More recently the electron capture detector was responsible for the discovery of the global distribution of nitrous oxide and of the chlorofluorocarbons, both of which are important in the stratospheric chemistry of ozone. NASA adopted some of his inventions in their program of planetary exploration; he was awarded by NASA three certificates of recognition for these. He is the originator of the Gaia Hypothesis (now Gaia Theory) and has written three books on the subject: “Gaia: A New Look At Life on Earth” [1]; “The Ages of Gaia” [2]; and “Gaia: the Practical Science of Planetary Medicine” [3]. He was elected a Fellow of the Royal Society in 1974, and in 1975 received the Tswett Medal for Chromatography. Earlier he received a CIBA Foundation Prize for Research in Aging. In 1980, he received the American Chemical Society’s National Chromatography Award, and in 1986, the Silver Medal and Prize of the Plymouth Marine Laboratory. In 1988, he was a recipient of the Norbert Gerbier Prize of the World Meteorological Organization, and in 1990 was awarded the first Amsterdam Prize for the Environment by the Royal Netherlands Academy of Arts and Sciences. In 1996, he received both the Nonino Prize and the Volvo Environment Prize, and in 1997 Japan’s Blue Planet Prize. He has received honorary Doctorates in Science from the University of East Anglia (1982), Exeter University (1988), Plymouth Polytechnic (now Plymouth University) (1988), Stockholm University (1991), University of Edinburgh (1993), University of Kent and the University of East London (1996), and the University of Colorado (1997). Her Majesty the Queen made him a C.B.E. in 1990. James Lovelock’s first interest is the Life Sciences, originally as medical research, but more recently in geophysiology, the systems science of the Earth. His second interest, that of instrument design and development, has often interacted with the first to their mutual benefit. He is at present an Honorary Visiting Fellow of Green College, Oxford University. See Chapter 5B, a, d, f, k, r, s

40.I. SOME HISTORICAL COMMENTS James E. Lovelock Coombe Mill, St. Giles on the Heath, Launceston, Cornwall PL15 9RY, UK

When devising a series of ionization detectors for gas chromatography in the mid 1950s, I had no notion that one of them, the electron capture detector, would significantly affect the development of environmental thinking. It was invented in 1957, and is still among the most sensitive detectors of chemical analytical methods in existence; moreover it is specifically sensitive to those chemicals that are a threat to the environment. Its use led to the discovery of the ubiquitous distribution of pesticide

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residues in the natural environment, and to Rachel Carson’s book, “The Silent Spring” (1962) [4], which can be said to have started the environmental movement. It was later used to discover and measure the abundance of PCBs, chlorofluorocarbons and nitrous oxide in the atmosphere. Most recently, the detector has made possible a system of atmospheric and oceanic tracer technology. Perfluorocarbons, which are otherwise inert and harmless, are easily detected tracers by electron capture. This system has enabled meteorologists to follow the movement of air masses across continents and is now finding use in ocean research. In 1961, having heard of these new detectors, NASA invited me to join the team at Jet Propulsion Laboratory who were developing lunar and planetary landers. Initially the invitation concerned the development of methods for analyzing lunar soil, but soon I became involved with NASA’s quest to discover whether there was life on Mars. In a letter to Nature in 1965, I proposed some physical tests for the presence of planetary life. One of these was a top down view of the whole planet instead of a local search at the site of landing. The test was simply to analyze the chemical composition of the planet’s atmosphere. If the planet were lifeless, then it would be expected to have an atmosphere determined by physics and chemistry alone and be close to the chemical equilibrium state. But if the planet bore life, organisms at the surface would be obliged to use the atmosphere as a source of raw materials and as a depository for wastes. Such a use of the atmosphere would change its chemical composition. It would depart from equilibrium in a way that would show the presence of life. Dian Hitchcock joined me then, and together we examined atmospheric evidence from the infrared astronomy of Mars, in 1967 [5]. We compared this evidence with that available about the sources and sinks of the gases in the atmosphere of the one planet we knew bore life, Earth. We found an astonishing difference between the two atmospheres. Mars was close to chemical equilibrium and dominated by carbon dioxide, but the Earth was in a state of deep chemical disequilibrium. In our atmosphere carbon dioxide is a mere trace gas. The coexistence of abundant oxygen with methane and other reactive gases, are conditions that would be impossible on a lifeless planet. Even the abundant nitrogen and water are difficult to explain by geochemistry. No such anomalies are present in the atmospheres of Mars or Venus, and their existence in the Earth’s atmosphere signals the presence of living organisms at the surface. Sadly, we concluded, Mars was probably lifeless. Thinking about the profound difference between the Earth’s atmosphere and those of the other planets led me to my other principal research during the past twenty years, a hypothesis that the Earth is a self regulating system able to keep its climate and chemical composition comfortable for the organisms that inhabit it. In the course of expeditions to gather evidence for tests of the Gaia hypothesis, I made several interesting discoveries. One, made in 1971, was that the chlorofluorocarbons were distributed throughout the atmosphere and at an average abundance of 50 parts per trillion, suggesting the absence of any sink for these gases. This was the evidence that allowed Molina and Rowland [6] to develop their theory of ozone depletion. On this expedition I also found the ubiquitous distribution in the ocean of methyl iodide, dimethyl sulphide, carbon disulphide and carbon tetrachloride. The presence of methyl iodide and dimethyl sulphide was sought as confirmation of a prediction from Gaia that there should be a large enough emission of these gases from the oceans to balance

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the natural sulphur and iodine budgets. Preliminary confirmation came from these first measurements in 1971 to 72 [7]. Other environmental contributions were the discovery in 1975 of methyl chloride as a natural atmospheric gas [8]. An estimate (1977) of the hydroxyl abundance of the atmosphere from measurements of the abundance of methyl chloroform, a man-made chemical whose principal sink is reaction with hydroxyl [9]. The first atmospheric halocarbon monitoring station was established at Adrigole in Ireland in the 1970s. It later became one of the five globally distributed stations that established the atmospheric lifetimes of the chlorofluorocarbons.

40.I.1. Significant scientific contributions Medical research. In 1952, I developed a quantitative theory of the damage suffered by living cells when frozen and thawed. My experiments had shown that damage was due to the concentration of salt and other solutes when ice separated as a pure substance. I was also able to explain the protective action of glycerol and neutral solutes and predicted successfully that dimethyl sulphoxide would be an excellent protective agent. I participated in the team that successfully froze and thawed whole animals, hamsters. My other researches included an investigation of the pathways for the spread of respiratory infection, especially the common cold, and the design of means for its prevention. Inventions. Among my inventions are detectors and other devices for use in gas chromatography. The argon detector was the first practical sensitive detector. It realized the potential of gas chromatography. The electron capture detector was invented in 1957 [10], and is still among the most sensitive of chemical analytical methods in existence. Its use led to the discovery of the ubiquitous distribution of pesticide residues in the natural environment. The same detector was later used to discover and measure the abundance of chlorofluorocarbons and of nitrous oxide in the atmosphere. Another invention was the palladium transmodulator, a device whose use was crucial for the gas chromatograph–mass spectrometer experiment aboard the Viking space craft that landed on Mars. Most recently, I developed a tracer method for mass transport measurements in the atmosphere and oceans. It uses perfluorocarbons as tracers and detects them by electron capture. It has enabled meteorologists to follow the movement of air masses across continents and is now finding use in ocean research. Geophysiology. Twenty years ago, I postulated that the Earth is a self-regulating system able to keep the climate and chemical composition comfortable for organisms. This, the Gaia Hypothesis, is now the Gaia Theory, with a mathematical basis, and is still up for trial. A common criticism is of teleology. This accusation is unjust; neither purpose nor foresight were ever claimed. Whether right or wrong, it is a testable theory and capable of making ‘risky’ predictions. One of these was that there should be a large enough emission of dimethyl sulphide from the oceans to balance the natural sulphur budget. Preliminary confirmation came from my own measurements in 1972 [7]; complete confirmation was made independently by M.O. Andreae [11]. Later, when considering the prediction from Gaia of climate regulation, Charleson, Lovelock,

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Andreae and Warren proposed that cloud density was modulated by the abundance of atmospheric dimethyl sulphide, and that this in turn changed the Earth’s albedo and mean surface temperature. This proposal was published as a Nature paper in 1987 [12] and is still under test. Gaia Theory also offered an interpretation of the long-term regulation of carbon dioxide and climate through biologically assisted rock weathering. Schwartzman and Volk confirmed this proposal in 1989 [13]. Lovelock (1991) presented a review, with 31 references, which covers the geophysiology of the oceans, including geophysiology and the Gaia hypothesis, CO2 and climate, and algae in the oceans and clouds [14].

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

J.E. Lovelock, Gaia: A New Look at Life on Earth, Oxford University Press, Oxford, UK, 1979, 157 pp. J.E. Lovelock, The Ages of Gaia: A Biography of Our Living Earth, W.W. Norton, New York, NY, 1988, 252 pp. J.E. Lovelock, Healing Gaia: Practical Medicine for the Planet, Harmony Press, New York, NY, 1991, 192 pp. Rachel Carson, Silent Spring, Houghton Mifflin, Boston, MA, 1962, 368 pp. Dian R. Hitchcock and James E. Lovelock, Life detection by atmospheric analysis, Icarus, 7 (1967) 149–159. M.J. Molina and F.S. Rowland, Stratospheric sink for chlorofluoromethanes: chlorine-atom-catalysed destruction of ozone, Nature, 249 (1976) 810–816. J.E. Lovelock, R.J. Maggs and R.A. Rasmussen, Atmospheric dimethyl sulfide and the natural sulfur cycle, Nature, 237 (1972) 452–453. J.E. Lovelock, Natural halocarbons in the air and in the sea, Nature, 256 (1975) 193–194. J.E. Lovelock, Methyl chloroform in the troposphere as an indicator of hydroxyl radical abundance, Nature, 32 (1977) 267. J.E. Lovelock, A sensitive detector for gas chromatography, J. Chromatogr., 1 (1958), 35–46. M.O. Andreae, Dimethylsulfide in the water column and the sediment porewaters of the Peru upwelling area, Limnol. Oceanogr., 30 (1985) 1208–1218. R.J. Charlson, J.E. Lovelock, M.O. Andreae and S.G. Warren, Oceanic phytoplankton, atmospheric sulfur, cloud albedo and climate, Nature, 326 (1987) 655–661. D.W. Schartzman and T. Volk, Biotic enhancement of weathering and the habitability of Earth, Nature, 340 (1989) 457–460. J.E. Lovelock, Geophysiology of the oceans, Phys. Chem. Earth Sci. Res. Report, 9 (1991) 419–431.

D.41. Karel Macek Karel Macek was born on October 31, 1928 in Prague, Czechoslovakia. He studied at the Charles University, Prague, where he received the degree Dr. Rerum Naturalium in 1951. In 1983 he received the degree D.Sc. from the Technical University of Prague. His professional career started in 1950 at the Research Institute for Pharmacy and Biochemistry, Prague, in the biochemical laboratories. In 1957, he took postdoctoral study in the University of Go¨ttingen. In 1968, he became Associate Professor of Analytical Chemistry at the University of Pardubice. In 1966–1967, he was guest

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Professor at the Institute of Pharmacognosy of the University of Munich, and in 1968 at Chelsea College of Science and Technology, London. In 1968–1969 he spent a sabbatical year at the Consiglio Nazionale delle Recerche in Rome. In the seventies he was Head of the Clinical Biochemistry Laboratories at the 3rd Internal Clinic of Faculty of Medicine, Charles University in Prague. From 1977 he was appointed to the Institute of Physiology of the Czechoslovak Academy of Sciences where he remained until 1992. Since 1962, he had been the Editor of the Bibliography Section of the Journal of Chromatography. In 1968, he was offered the post of Associate Editor, and from 1989 up to 1993 he was one of the Editors of the Journal of Chromatography. In 1977, he established an independent section, Journal of Chromatography, Biomedical Applications, which he has been leading as the Editor for the last twenty years. In 1960, K. Macek founded the Chromatography Section of the Czechoslovak Chemical Society and was Chairman of this section up to 1991. Since 1996, he is vice president of the Society for Connective Tissue Research and Application, Prague. From 1961 until 1993, he organized and was Chairman of 19 international symposia dealing first with general problems of planar chromatography, and starting 1966, about biomedical applications of chromatography. Dr. Macek is the author and co-author of over 160 scientific papers and 29 books or chapters in monographs dealing with all types of chromatographic techniques and their applications in biochemistry, medicine and pharmacy. The most popular was the extensive monograph Paper Chromatography which he edited together with Ivo Hais in 1954. He is a recipient of the M.S. Tswett Medal of the Academy of Sciences of the U.S.S.R., the J. Hanusˇ Medal of the Czechoslovak Chemical Society, the M.S.Tswett Chromatography Award of the International Symposium on Advances in Chromatography, the Leonard Michaelis Medal of the Gesellschaft fu¨r klinische Chemie der D.D.R., the Jean-Servais-Stas Medal of the German Society for Toxicology and Forensic Chemistry, and the J.E. Purkynı` Medal of the Czechoslovak Academy of Sciences. See Chapter 5B, a, b, d, e, i, s

41.I. RESEARCH AND EDITORIAL ACTIVITY IN CHROMATOGRAPHY Karel Macek Lukesˇova 16, CZ-14200 Prague 4, Czech Republic

My interest in chromatography began at the end of the forties. This was the pioneering time where nobody could expect what directions chromatography would expand into and how important this scientific discipline would become. I was introduced to chromatography in 1948 by Professor Josef Kosˇtı´ˇr, who was the supervisor of my

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Fig. 1. K. Macek working with sample collecter in his laboratory (about 1949).

thesis at Charles University in Prague. In this thesis (1949–1951) I exploited column chromatography for the study of a new pigment produced by Penicillium lilacinum Thom which I named lilacin. In 1949, I met Dr. Ivo Hais who had just come back from his long sabbatical at the University College in London. In his cellar laboratory saturated with butanol and phenol vapors, I became acquainted with paper chromatography. His enthusiasm for this technique was so infectious that I immediately became involved. Two years later we met again in the newly established Research Institute for Pharmacy and Biochemistry in Prague, which in the fifties became the leading center of paper chromatography for the entire Central Europe. In the early fifties the main interest was directed to paper chromatography. At the end of the forties paper chromatography was used generally for the separation and identification of structurally related compounds. The identification was based on the mobility in a chromatographic system and on results of detection reactions. I worked out procedures for the separation, identification, and purity estimation of many synthetic

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as well as natural drugs of pharmaceutical interest. But I was convinced that the chromatographic data could supply us with much more information dealing with the structure of compounds under analysis. My first attention was paid to the optimization of chromatographic conditions. I studied the characteristics of more than 50 kinds of chromatographic papers and developed a new type of glass fiber paper. I have devoted a great interest to this chromatographic system and to the conditions for good reproducibility. In this respect the most suitable system proved to be a system with stationary polar organic solvents. The next more theoretical step made was a study of the mechanism of separation and the relationship between structure of compounds and their chromatographic behavior. Based on A.J.P. Martin’s relationship dealing with the additivity of functional groups and, in addition, on the thermodynamic presumption made by Reichl and Bush which led to the equation for the calculation of RF values from group constants of some simple compounds, I proposed another equation which could be used for more complicated structures. This equation included not only the functional group constants, but also the so-called position factors, which represented the contribution of the position of these groups in the molecule [1]. I have calculated many group constants, further on constants for basic structure and position factors for various types of heterocyclic compounds, like veratrum alkaloids [2], ergot alkaloids [3], barbituric acid derivatives, purines, indoles and many others. The use of our equation made it possible to predict RM and hence RF values for the above compounds in various chromatographic systems. Later, I attempted to exploit the chromatographic data for the confirmation of several functional groups, for a structural feature of the molecule or for a steric arrangement. Choosing various experimental conditions, it was possible to emphasize or to suppress interactions of some groups with one component of the chromatographic system. Such changes led to the determination of not only a functional group, but also in some cases the number and position of such a group. This procedure was utilized for the structural study of some veratrum alkaloids [2] and for the determination of the configuration of some reserpoids [4]. All of the procedures mentioned dealt with the analysis in a closed category of structurally related compounds. But quite a different problem was faced in the analysis of biological materials where tens and hundreds of compounds of various chemical structure should be analyzed side by side. This was the main problem in the analysis of, e.g., alkaloids in plant material, or some noxious compounds in toxicology and forensic medicine. In 1956, I worked out a scheme for the systematic gradient analysis of alkaloids by paper chromatography [5]; this system was modified in 1965 by introducing the so-called fraction development. Using this procedure the compounds analyzed were assorted into specific groups and followed by identification with an additional system of paper or thin-layer chromatography, by using selective or specific detection reactions, and further, by applying a combination of some other physicochemical procedures, like measuring UV-spectra in situ [6]. In 1957, I met Egon Stahl in Mainz, who acquainted me with his new procedure that was published one year later, i.e., thin-layer chromatography. I was impressed by the possibilities offered by this new method and soon afterward we turned in many cases to this procedure. From the early sixties we turned our attention to gas chromatography

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Fig. 2. Karel Macek and Michael Lederer during the international symposium on planar chromatography in Liblice (1961). Here started their cooperation with the Journal of Chromatography which lasted almost 30 years.

and we worked out many procedures for gas chromatographic analysis of synthetic drugs following their synthesis and stability, then in the seventies to high-performance liquid chromatography. In 1969, I changed my position from pharmaceutical research to clinical biochemistry. Here I was interested in biomedical applications of all types of chromatographic techniques especially in the monitoring of drug levels, in the analysis of biogenic amines, polyamines, steroid hormones, amino acids and proteins, in the analysis of collagens and use of chromatography for diagnostic purposes. Another part of my scientific career has been connected with publishing. At the beginning of the fifties I worked with Dr. Ivo Hais to write a monograph about paper chromatography which was published in 1954: this was a most complete monograph on this topic. This book was translated into German, English, Russian, Hungarian and Romanian [7]. Books summarizing the bibliography of paper and thin-layer chromatography (1943–1956, 1957–1960, 1961–1965, 1966–1969, 1970–1973) [8] followed. Thereafter the bibliography was published in a special section of the Journal of Chromatography, it was enlarged later with GC, HPLC and electrophoresis; I have edited this section from 1961 until 1991. From several other books dealing with chromatography, let me mention at least “Pharmaceutical Applications of Thin-Layer and Paper Chromatography”, published in 1972 [9] and “Liquid Column Chromatography”, edited together with Drs. Zdenek Deyl and Jaroslav Jana´k in 1975 [10]. In 1968, after the Soviet Armies occupied Czechoslovakia, I moved to Rome and

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Fig. 3. Symposium on planar chromatography in Liblice (1967): one of the pioneers of paper chromatography, Ivo Hais, with the discoverer of thin-layer chromatography, Egon Stahl (right).

joined the University and worked with Michael Lederer. He offered me the post of Associate Editor of the Journal of Chromatography, an activity that I have been involved in for the following 20 years. After the retirement of Professor Lederer in 1989, I became one of the Journal’s editors for which I was involved until 1993. Because of an increasing number of contributions from the area of biosciences we then established in 1977 an independent section, Journal of Chromatography, Biomedical Applications, the Editor of which I have been until 1995. Another activity that took considerable time was organizing international symposia. Since 1965, I organized 19 international symposia: the first meetings (1961 until 1972) were devoted to the general topic of planar chromatography, and since 1966 until 1993, the symposia were devoted to biomedical applications of chromatography. These symposia took place in Czechoslovakia, Italy, Germany, and the Soviet Union and the proceedings of a majority of them were published in the Journal of Chromatography. In 1992, I retired from the Institute of Physiology of the Czechoslovak Academy of Sciences, and in 1995 I stepped down as the Editor of Journal of Chromatography, Biomedical Applications. In conclusion, I am very happy that for fifty years I could be a witness of the history of chromatography and I would be pleased if my scientific, editorial and organizational work contributed to the development of this certainly one of the most important analytical methods of the 20th century.

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Fig. 4. International meeting about Biomedical Applications of Chromatography 1988 in Riva del Garda in honor of 60th birthday of K. Macek. The photo presents mostly the members of the Journal of Chromatography Editorial Board: (1) M. Lederer; (2) I.M. Hais; (3) P. Padieu; (4) K. Macek (behind him, J. Macek, his son); (5) I.M. Chaiken; (6) A.A. Boulton; (7) J. Sjo¨vall; (8) E. Jellum; (9) P. Kissinger; (10) M. Novotny´; (11) J.K. Haken; (12) R.A. DeZeeuw; (13) A. Frigerio; and (14) J. Wagner.

References 1. 2. 3. 4. 5. 6. 7.

8.

K. Macek and Z.J. Vejdı`lek, Application of paper chromatography to structural problems in the Veratrum alkaloid group, Nature, 176 (1955) 1173–1174. K. Macek, S. Vanı`e`ek and Z.J. Vejdı`lek, Veratrum-Alkaloide. VI. Beitrag der Papierchromatographie zur Lo¨sung von Strukturfragen, Coll. Czech. Chem. Commun., 22 (1957) 253–261. K. Macek, Mo¨glichkeiten der Verwendung der Papierchromatographie in der Alkaloidchemie, Abhandl. Deut. Akad. Wiss., Berlin, Nr. 7 (1957) 51–56. K. Macek, Papierchromatographie-Beitrag zur Bestimmung der C3 -Konfiguration bei Reserpoiden, Experientia, 17 (1961) 162–163. K. Macek, J. Hacaperkova´ and B. Kaka´e`, Systematische Analyse von Alkaloiden mittels Papierchromatographie, Pharmazie, 11 (1956) 533–538. K. Macek, J. Vee`erkova´ and J. Stanislavova´, Ein neues Analysengang von Arzneimitteln mittels Papierchromatographie, Pharmazie, 20 (1965) 605–616. ˇ I.M. Hais and K. Macek (Eds.), Paper Chromatography, Nakl. CSAV, Prague 1954; Fischer Verlag, Jena 1958; Academic Press, New York 1963; Izd. Inostr. Liter., Moscow 1962; Akad. Kiado´, Budapest 1961; Edit. Tehn., Bucarest 1960, pp. 955. K. Macek, et al., Bibliography of Paper and Thin-Layer Chromatography, 1943–1956, 1957–1960, 1961–1965, 1966–1969, 1970–1973, Publ. House of Czech. Acad. Sci. and Elsevier, Amsterdam 1960, 1962, 1968, 1972 and 1976.

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9.

K. Macek, Pharmaceutical Applications of Thin-Layer and Paper Chromatography, Elsevier, Amsterdam 1972, pp. 743. 10. Z. Deyl, K. Macek and J. Jana´k (Eds.), Liquid Column Chromatography, Elsevier, Amsterdam 1975, pp. 1175%Chromatography from Utah to Uppsala

D.42. Karin E. Markides Uppsala University, Institute of Chemistry, Department of Analytical Chemistry, Box 531, S-75121 Uppsala, Sweden

Karin Erika Markides was born on November 24, 1951 in Sweden. She received her Ph.D. in Analytical Chemistry at the University of Stockholm, Sweden in 1984 and directly joined the research group of Professor Milton Lee at Brigham Young University, Utah, USA for a postdoctoral research period. During her Ph.D. work, K.E. Markides developed novel cyancontaining stationary phases for capillary-gas chromatography, including surface deactivation, synthesis of novel siloxane stationary phases and column technology showing high temperature stability and resolution. At Brigham Young University, he was actively involved in a number of diverse research projects in separation science. Deactivation methods for fused silica capillaries were refined and basic research was performed to understand the detailed surface chemistry. Stationary phases with flexible siloxane backbone and substitution of novel and useful side-groups, for use in open tubular GC and SFC, were developed together with Drs. Lee and Bradshaw and of which several were protected with patents. Among the most useful high-resolution stationary phases developed were the octylsiloxane (true boiling point elution), biphenylsiloxane (universal), dicyclohexyl-amide and siloxane copolymer (for chiral separation) and the smectic liquid crystal siloxane (size and shape selective). Detection systems for capillary-column separations were refined and one most useful detection system developed was the multi-element selective radio frequency plasma detector that showed robustness and was not quenched by solvent or sample matrices. The main effort in research at Brigham Young University was, however, the development of supercritical-fluid separation and extraction techniques and methods. K.E. Markides stayed on as Assistant Professor at Brigham Young University and also became Full Professor before she, in 1989, received the appointment from Uppsala University in Sweden to Chair the Department of Analytical Chemistry. With ten years as Chair in analytical chemistry at Uppsala University, the research projects represents a well functioning creative research environment for basic and applied research in miniaturized sample pretreatment and separation on-line with atmospheric-pressure ionization, mass spectrometric detection and chemometric data handling. Research on electrospray and chemical ionization mass spectrometry at atmospheric pressure from liquid samples are of major interest in the group with the aspects of novel materials, microfabricated and column formats, as well as chemical,

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electrochemical and analyte mechanistic and transmission aspects. The aim is to control the ionization for higher ordered structures, adducts, complexes and interactions including biomolecules, by understanding the basic effects in the ionization process itself. This is also a prerequisite for the interest to study dynamic events in real time for higher ordered biomolecules and complexes. The research group develops knowledge and tools to make this possible. At present, the research group of analytical chemistry at Uppsala University consists of one Professor, seven senior Lecturers, two Research Assistant Professors, one Postdoc and sixteen graduate students. Today, K.E. Markides is an internationally leading scientist in the areas of column technology, separation sciences, mass spectrometry and extraction in miniaturized systems. She has a solid scientific publication record that is well cited and comprises 175 publications in international leading journals on a range of subjects as: siloxanes as stationary phases; capillary-column gas chromatography and SFC; deactivation of fused silica columns; synthesis and properties of siloxane stationary phases; SFC using NH3 as mobile phase; SFC of hydrocarbon groups; two dimensional separation of peptides; on line SFE-SFC for analysis of vitamins; RP-ion pair chromatography coupled to electrospray MS and a variety of other subjects. She has also presented close to 200 lectures and seminars at conferences and symposia. Her research has been acknowledged with prestigious awards such as the Norblad-Ekstrand Medal, Sweden (1990), Jubilee Medal, Great Britain (1992) and the Senior Individual Grant, Sweden (1997). In parallel to her scientific career, K.E. Markides has also been active and successful in the area of technology and knowledge transfer between university and industry. In the mid-1980s she was involved in establishment of Centers of Excellence and development of effective links between research at the university and startup companies (Lee Scientific and Sensar) from research patents. In the late 90s she started to develop novel recipes for an improved technology transfer policy also at universities in Sweden, with the driving goal to enhance the number of high-tech startup companies in Sweden and enhanced room for basic research at the universities. Scientific organizations – It is noted that K.E. Markides has over the years been strongly involved in scientific organizations of different kinds to influence and take responsibility for the development of separation science and to spread the diverse vision of analytical chemistry to different parts of society. The main activities in this respect comprise: ž Member of the American Chemical Society, 1987. ž Scientific Referee for Journals in Separation Science and Mass Spectrometry, 1990. ž Member of the Board for Analytical Chemistry in the Swedish Chemical Society, 1990. ž Member of IVA, Royal Swedish Academy of Engineering Science, 1992. ž Member of Chemistry Board of Natural Science Foundation, 1996. ž Dean of Chemistry at Uppsala University, 1996. ž Member of the Swedish Ministry of Education Advisory Board 1997–98. ž Member of the Board of ‘College Dalarna’, 1998. ž Member of the Board of Smelink Foundation for Technology Transfer IT-tools, 1998. ž Chairman of the Board of the Swedish Mass Spectrometry Society, 1998.

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ž Chairman of the Board of the Center for Mass Spectrometry, Uppsala University, 1999. ž Member of KVA, the Royal Swedish Academy of Science, 1999. ž Member of the Board of SSF, the Swedish Foundation for Strategic Research, 2000. See Chapter 5B, a, d, e, f, h, l, o, q

References: selected publications 1.

2.

3.

4.

5.

6. 7. 8.

9. 10. 11. 12. 13. 14. 15.

16. 17. 18.

K. Markides, L. Blomberg, J. Buijten, S. Hoffmann and T. Wa¨nnman, Cyanosilicones as stationary phases in gas chromatography, III. Synthesis, characterization and evaluation, J. Chromatogr., 302 (1984) 319–340. J.C. Kuei, B.J. Tarbet, W.P. Jackson, J.S. Bradshaw, K.E. Markides and M.L. Lee, A new n-octylmethyl polysiloxane stationary phase for capillary-column gas and supercritical-fluid chromatography, Chromatographia, 20 (1985) 25–30. K.E. Markides, M. Nishioka, B.J. Tarbet, J.S. Bradshaw and M.L. Lee, Smectic biphenylcarboxylate ester liquid-crystalline polysiloxane stationary phase for capillary-gas chromatography, Anal. Chem., 57 (1985) 1296. K.E. Markides, B.J. Tarbet, C.L. Woolley, C.M. Schregenberger, J.S. Bradshaw, K.D. Bartle and M.L. Lee, Deactivation of fused-silica capillary columns with phenylhydrosiloxanes, HRC and CC, 8 (1985) 379. K.E. Markides, M.L. Lee and D.W. Later, Capillary supercritical-fluid chromatography: Practical aspects; in F.J.Yang (Ed.), Microbore Column Chromatography: A Unified approach to Chromatography, M. Dekker, New York, NY, 1988.K.E. Markides, S.M. Fields and M.L. Lee, Capillary supercritical fluid chromatography of labile acidic compounds, J. Chromatogr. Sci.„ 24 (1986) 254–257. M.L. Lee and K.E. Markides, Chromatography with supercritical fluids, Science, 235 (1987) 1342. M.L. Lee and K.E. Markides, Supercritical-fluid chromatography, Nature, 327 (1987) 441–442. J.S. Bradshaw, S.K. Aggarwal, C.A. Rouse, B.J. Tarbet, K.E. Markides and M.L. Lee, Polysiloxanes containing thermally stable chiral amide side-chains for capillary gas and supercritical-fluid chromatography, J. Chromatogr., 405 (1987) 169–177. M.L. Lee and K.E. Markides (Eds.), Analytical Supercritical-fluid Chromatography and Extraction, Brigham Young University Press, Provo, Utah, 1990. L.Q. Xie, K.E. Markides and M.L. Lee, Bioanalytical applications of multidimensional open-tubular column supercritical-fluid chromatography, Chromatographia, 35 (1993) 363. D.E. Raynie, K.E. Markides and M.L. Lee, Boiling range distribution of petroleum and coal-derived heavy ends by supercritical-fluid chromatography, J. Microcol. Sep., 3 (1991) 423. L.M. Svensson and K.E. Markides, Fiber optic-based UV-absorption detector cell using high-temperature liquid chromatography, J. Microcol. Sep., 6 (1994) 409. M. Stefansson, P. Sjo¨berg and K.E. Markides, Regulation of multimer formation in electrospray-mass spectrometry, Anal. Chem., 68 (1996) 1792. U. Petersson and K.E. Markides, Stability and purity of low-polarity adsorbent for coupled SFE-SFCFID, J. Chromatogr., 734 (1996) 311. G.B. Jacobsson, R. Moulder, L. Lu, M. Bergstro¨m, K.E. Markides and B. La˚ngstro¨m, Supercriticalfluid extraction of 11 C-labeled metabolites in tissue using supercritical ammonia, Anal. Chem., 69 (1997) 275. Z. Juvancz, L. Jicsinszky and K.E. Markides, Phosphated cyclodextrins as new acidic additives for capillary electrophoresis, J. Microcol. Sep., 9 (1997) 581. S.R. Wallenborg, K.E. Markides and L. Nyholm, Development of an amperometric detector for packed capillary-column supercritical-fluid chromatography, Anal. Chem., 69 (1997) 439. L.M. Nyholm and K.E. Markides, Column preparation for reversed-phase high-temperature open-tubular column LC, J. Chromatogr. A., 813 (1998) 11.

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19. P.J.R. Sjo¨berg and K.E. Markides, New supercritical-fluid chromatography interface probe for electrospray and atmospheric-pressure chemical-ionization mass spectrometry, J. Chromatogr., 785 (1997) 101. 20. A. Sandberg, K.E. Markides and E. Heldin, Lasalocid as a chiral selector, adsorbed onto porous graphitic carbon, in capillary-liquid chromatography, J. Chromatogr. A., 828 (1998) 149–156. 21. D.R. Barnidge, S. Nilsson, H. Rapp, K. Hjort and K.E. Markides, Metallized sheathless electrospray emitters for use in capillary-electrospray orthogonal time-of-flight mass spectrometry, Rapid Communications in Mass Spectrometry„ 13 (1999) 1. 22. D.R. Barnidge, S. Nilsson and K.E. Markides, A design for low flow sheathless electrospray emitters, Anal. Chem., 71 (1999) 4115. 23. J.N. Alexander, J.B. Poll and K.E. Markides, Evaluation of automated-isocratic and gradient-nano-liquid chromatography and capillary electrochromatography, Anal. Chem., 71 (1999) 2398.

D.43. Michel Martin Michel Martin was born on July 6, 1947 in Saint-Sulpicesur-Rille (Orne), France. He received the Inge´nieur diploma from E´cole Nationale Supe´rieure de Chimie de Paris in 1969 and his Doctorat d’Etat (Ph.D.) e`s Sciences Physiques from the Universite´ de Paris VI in 1975. M. Martin was a Research Assistant and an Instructor in organic chemistry at the E´cole Polytechnique, Paris, then Research Attache´ at the National Center for Scientific Research (CNRS), a Research Charge´ at CNRS, and presently a Research Director at CNRS from 1986 to present. He conducted studies at the Laboratoire de Chimie Analytique Physique of the E´cole Polytechnique, Paris (1969–1976); at the Department of Chemistry, University of Utah, Salt Lake City, USA (1976–78); then for ten years again at the E´cole Polytechnique, and presently since 1988 at the Laboratoire de Physique et Me´canique des Milieux He´te´roge`nes, E´cole Supe´rieure de Physique et de Chimie Industrielles, Paris. In 1992, M. Martin received the Mikhail S. Tswett Medal of the Russian Tswett Chromatography Association, and in 1997 the Silver Jubilee Medal of the Chromatographic Society for his contribution to the field of separation science. He has been a member of various committees of professional societies: 1983–1993

Board member of GAMS (Groupement pour l’Avancement des Me´thodes Spectroscopiques et Physico-Chimiques d’Analyse); adjunct-secretary (1983–1985) then secretary (1985–1991) 1981–1987 Founder and chairman of the Particle Size Analysis Division of GAMS 1984–1995 Founding member and board member of the Aerosol Division of GAMS 1987–1995 Chairman of the Chromatography Division of GAMS 1997–present Founding member and chairman of AFSEP (Association Franc¸aise des Sciences Se´paratives).

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M. Martin’s research is directed to HPLC and field-flow fractionation. In HPLC, he investigated the function of pressure, the effects of the pressure dependence of liquid density and viscosity and their influences on instrument design and plate height; and the dispersion in non-classical chromatographic systems. His research on field-flow fractionation (FFF) is directed to thermal FFF and high speed separations of macromolecules, polymers, colloids, and particles, and various physico-chemical studies addressing the theory of FFF. See Chapter 5B, a, h, r

43.I. CONTRIBUTION TO THE FIELD OF SEPARATION SCIENCE Michel Martin E´cole Supe´rieure de Physique et de Chimie Industrielles, Laboratoire de Physique et Me´canique des Milieux He´te´roge`nes (UMR CNRS 7636), 10, rue Vauquelin. 75231 Paris Cedex 05, France

43.I.1. High-performance liquid chromatography I started my doctorate research studies in 1969 in the Laboratoire de Chimie Analytique Physique at the Ecole Polytechnique in Paris. I was asked by my thesis supervisor, Professor Georges Guiochon, who was directing the laboratory, to perform investigations on HPLC. This was a new technique, known at that time as ‘high-pressure liquid chromatography’, and my thesis was the first devoted to this technique in France. The lab was just recently equipped with a homemade LC instrument. The column packing was obtained by dry-sieving relatively large amounts of alumina or silica and the columns were home-packed by pouring the dry particles in the columns under vibration. The objective was then to separate petroleum steam cracking fractions into families of components of varying aromaticity. In order to improve the resolution, essentially by increasing the plate number, I spent more time in sieving a larger amount of alumina to get a finer range of particle sizes, 80–100 µm, and even 50–63 µm. The performance of the columns was characterized by plotting plate height curves (plate height vs. eluent velocity). In order to scan a relatively wide velocity range for these plots, the column inlet pressure had to be relatively high. The maximum pressure at which one could make a syringe injection was then uncertain. It was depending on the finger strength (volleyball players had then an advantage) and on the ratio of the outer to inner surface areas of the syringe piston. Thanks to the HP 305 Hamilton syringe, which had two side guides to avoid bending the piston when piercing the septum, reproducible syringe injections up to 200 bar could be made. Later on, injection valves became available, but syringe injection offered the possibility to introduce the sample just on the top of the column packing with minimal contribution to band broadening. I then got perfect straight-line plate height curves with intercepts and slopes respectively proportional to the particle size and to its square, as predicted by theory. Later on,

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30 µm superficially porous particles became available, offering improved efficiencies. Then, one day, my thesis director, Professor Guiochon, came to me with a sample of 5 µm silica particles, large enough to slurry pack a 6 cm long, 4 mm i.d. column. The shapes of the efficiency curves changed significantly from straight lines to convex curves, then to curves with a minimum plate height. Within a few years, the plate height values I got had dropped by a factor of 1000. I believe we were the first to publish a plate height value as low as 10 µm, corresponding to a reduced plate height of about 2 [1]. 43.I.1.1. The pertinence of pressure in HPLC This finding contributed to re-orientate the topic of my thesis work toward the kinetic optimization of separations in liquid chromatography and to investigate the role of pressure on column performances. Theoretical calculations, based on typical values of the coefficients on the Knox plate height equation showed that the minimum pressure which is necessary to obtain a separation is generally much lower than currently used. In an experimental verification of this finding, I obtained exactly the same separation, in terms of analysis time and resolution, of a test mixture on a 2 m long column packed with 25–31.5 µm Spherosil particles with a pressure drop of 305 bar, as that on a 6 cm long column packed with 7 µm Spherosil particles with a pressure drop of 1.75 bar [1]. It thus appeared that the key factor that has led to the renewal of liquid chromatography in the late sixties was much more the availability of very fine particles than the use of high pressures. Accordingly in the seventies, the meaning of the acronym HPLC has progressively changed from ‘high-pressure liquid chromatography’ to ‘high-performance liquid chromatography’. 43.I.1.2. Compressibility of liquids and influence on instrument design For most chromatographers, liquids are considered as incompressible. In a theoretical investigation we found out effectively that the effect of the compressibility of liquids on retention time is most often not noticeable (less than 0.1%). However, it appeared that the effect on the operation of high pressure pumps in HPLC is far from being negligible. In the seventies, several LC manufacturers had equipped their chromatographs with syringe pumps with a capacity of several hundreds milliliters, as it was supposed that a constant displacement rate of the piston would provide a smooth and constant flow rate. We found that the duration of the transient period to reach a steady-state pressure at the inlet of a column can typically reach 15–60 minutes. During this transient period, the retention times greatly differ from their steady state values. Thus analysis must be avoided during this period. The situation is worse in gradient elution operation with two syringe pumps since then, a pressure steady state is never reached. Then, because of the effect of liquid compressibility on the effective rate of solvent flow from each syringe, the composition profile of the mixture entering the column may greatly differ from the expected one. In extreme cases, a 0–100% step gradient can be obtained while a linear gradient is searched for [2]. At the end of the seventies, syringe pumps were withdrawn from commercial instruments. They were re-introduced some years later for use with

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microbore columns, but then equipped with devices for correcting the compressibility effects by minimizing pressure fluctuations in the reservoirs. The compressibility effect is also present with reciprocating pumps and more or less sophisticated devices are used by manufacturers to minimize the flow rate fluctuations and deviations arising from this effect. 43.I.1.3. Pressure dependence of liquid viscosity and plate height in liquid chromatography Another pressure-dependent physico-chemical liquid parameter involved in LC is viscosity. While the compressibility of common liquids is about 104 =bar, the rate of change of viscosity with pressure is about 10 times larger, so that with a pressure drop of 200 bars, the retention time of a compound differs by about 10% from that it would have if the carrier viscosity was constant. This deviation somewhat affects theoretical predictions on the basis of a constant viscosity, but not the proper column operation. However, the pressure dependence of viscosity has an indirect effect on a topic of debate in the seventies and eighties: what is the exact form of the plate height expression in LC? Several plate height equations have been proposed, noticeably by van Deemter et al., Giddings, Huber, and Horva´th and Lin. They differ by the exponent of the velocity in the term called eddy diffusion or anastomosis or hydrodynamic dispersion (the A term in the semi-empirical Knox equation). Experimental HPLC plate height determinations, in which the plate height is measured as a function of flow rate from the chromatogram, were not able to point out the most correct of the various expressions because of the limited precision of the data. In addition, we have shown that these types of plate height determinations are, because of the pressure dependence of viscosity, intrinsically biased and, in practice, unable to help answering the above question [3]. Indeed, the variation of the viscosity leads to a variation of the solute diffusion coefficient, and thus of the chromatographic reduced velocity (the Pe´clet number of chemical engineers). Accordingly, because of the pressure dependence of viscosity and of the pressure drop along the column, a chromatographic experiment at a given flow rate is associated not to a single value of the reduced velocity but to a range of reduced velocities, so that the experimental plate height curve is an apparent curve which does not reflect the true plate height vs. reduced velocity curve. The above problem was solved later by experiments done with a narrow pressure drop, thus with a narrow reduced velocity drop along a column, even at the largest velocities, using particles of a much larger size than those of HPLC columns. It was shown that the Giddings expression fits the best the experimental data [4]. The same conclusion was later derived from experiments performed on HPLC columns by pulsed-field gradient NMR [5]. It is interesting to note that, in this technique, the plate height and the diffusion coefficient are experimentally determined locally, at a nearly constant pressure, so that, like in our experiments, they are not subject to the bias of the averaging process encountered with classical HPLC plate height determinations.

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43.I.1.4. Dispersion in non-classical chromatographic systems Interested in the dispersion aspects of the chromatographic process, I have performed several theoretical and=or experimental studies of band broadening in unusual situations, like in chromatography with turbulent flow or, still, in capillary LC with electroosmotic flow (the open tubular version of what is today known as capillary electrochromatography) [6]. Our experiments on GC in an open channel with a rectangular cross-section of large aspect ratio have led Marcel Golay in 1981 to reformulate his 1958 theory for chromatography in open systems of parallel plate geometry by taking into account the significant effect of the presence of the small edges. This was in excellent agreement with our data.

43.I.2. Field-flow fractionation In fact, these latter experiments were performed in a field-flow fractionation (FFF) channel, the walls of which had been coated with a very thin layer of liquid stationary phase. I got involved in FFF during a post-doctoral stay at the University of Utah with Professor J. Calvin Giddings who had invented FFF ten years earlier. This was a fascinating period because, first, I had the chance to work with one of the most eminent chromatographers, second, I had the opportunity to learn on a new separation method, and third, I was brought in a new area of applications for me (macromolecules, colloids, particles). 43.I.2.1. Thermal FFF and high-speed separations I was asked to perform investigations on thermal FFF, the FFF sub-technique which uses a thermal gradient as the cross-field. Initial analyses were quite long and lasted often several hours or even days, which had given the impression that FFF separations were inherently slow. Still, Cal Giddings had shown that very high rates of generation of theoretical plates could be achieved in properly optimized FFF systems. The line of attack for fast and high resolution separations in FFF is similar to that of HPLC: one has to operate under conditions minimizing the characteristic distance over which solute molecules have to diffuse in order to relax flow inequalities within the separation medium. In HPLC, this has been achieved by using very fine packing particles. In FFF, this requires channels of narrow thickness and relatively strong fields (in order to constrain solute zones in narrow layers near the accumulation wall). An implicit additional requirement is that the surface of this wall is smooth enough for not disturbing the solute layers. With the advice and help of Dr. Marcus N. Myers, I spent a rather long time hand-polishing the plates with decreasing grit size down to sub-µm diamond paste. I could then use a 51 µm thick spacer for making the channel, which was about five times thinner than in previous channels. With this channel, I was able to perform the baseline separation of a three-component polymer mixture in less than 5 minutes, instead of 20 hours some years earlier. The separation was even performed in less than one minute by increasing the flow rate, but at the expense of some resolution

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loss [7]. This demonstrated the high-speed capability of FFF, which, in fact, is not restricted to the thermal subtechnique. Later on, fast macromolecular or particulate separations were also achieved in sedimentation FFF and in flow FFF. 43.I.2.2. Physico-chemical investigations in FFF Returning to France after my post-doctoral stay in Utah, I pursued research on field-flow fractionation, and established the first group involved in FFF in Europe, with various analytical interests ranging from synthetic polymers and copolymers, to environmental particles, heavy fuel products, emulsions, or, still, living blood cells. Besides, I have always been interested by the fundamental aspects of this method and by the understanding of the physico-chemical mechanisms underlying separation and resolution by FFF. One of the simplest FFF method, thermal FFF, is based on thermodiffusion (called thermophoresis in the case of particulate materials or the Soret effect for binary mixtures), a process in which the transport of material is induced by a temperature gradient. While it is understood in the gas phase on the basis of the kinetic theory of gases, this process is hardly known and poorly understood in liquids. Even its sign (the direction of migration) cannot be predicted. This sign cannot be determined from classical thermal FFF experiments because of the non-monotony of the carrier velocity profile in the channel. We have shown that, when the channel is oriented vertically, one can take profit of the thermogravitational effect to determine the wall (hot or cold) at which the sample accumulates by thermodiffusion. A fundamental question about the thermodiffusion of polymers concerns the eventual molar mass dependence of the Soret coefficient. It was shown in earlier thermal FFF studies that this dependence, if it exists, should be weak. But, since any thermal FFF experiment is associated with a range of temperatures and, consequently, a range of values of the Soret coefficient, the answer to the above question requires a refined FFF model taking into account the dependence of the various relevant physico-chemical parameters (viscosity, thermal conductivity, Soret coefficient) with temperature. We have recently developed a retention model, which allows these dependencies to be taken into account and the Soret coefficient at a specific temperature to be extracted from retention time [8]. Its eventual molar mass dependence is currently under investigation. More generally, the classical retention model in FFF is based on some limiting assumptions, which have to be relaxed for accurate sample characterizations and=or physico-chemical determinations [9]. For some of them, this is done by including in the retention model well established scientific results. In other cases, a full description of the sample behavior is not possible because of the lack of understanding of some features of the migration process. For instance, we have shown in 1982 that the specific retention behavior of micron-sized particles can be explained by the interplay of the field force and of a hydrodynamic lift force of inertial origin. But the exact nature of this lift force acting on relatively large particles (in the super-micron range or upper sub-micron range) migrating in the vicinity of a wall, and its quantitative value, is not entirely known and is currently a topic of intense research work in fluid mechanics. Another area of interest in FFF concerns the behavior of sample solutions or

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suspensions at finite concentrations. Even quite dilute samples can become highly concentrated near the accumulation wall when strong field forces are applied. The influence of sample concentration on retention results from the balance of two opposite effects: first, concentrated zones, due to their higher viscosity, tend to move more slowly than dilute ones, and, second, the mean distance of the particles from the accumulation wall increases with increasing concentration, which tends to move particles, in average, toward the faster flow streamlines away from the wall. Using the theoretical approach developed by Einstein and Batchelor for describing the diffusion process, we have established the equilibrium concentration profile in the case of sedimentation of hard colloidal spheres [10]. Then, we predicted that the second of the above two effects is dominating so that retention of hard spheres in sedimentation FFF should decrease with increasing concentration. This is well what is generally observed for particulate matter in any FFF subtechnique, although electrostatic and van der Waals interactions should also influence the particle behavior at finite concentrations.

Acknowledgment In my scientific carrier, I had the great chance to work with two exceptional scientists, Professor Georges Guiochon, my thesis supervisor, in France, and the late Professor J. Calvin Giddings during my postdoctoral stay in the United States. At their contact, I have learnt chromatography and separation science. I recognize all what I owe to them, and, especially, the awards I received. I would like also to acknowledge the contributions to my activity of all my students, my laboratory colleagues, especially Dr. Mauricio Hoyos, who has been closely associated with my research activity for the past ten years, and all scientific colleagues, from France or abroad, with whom fruitful and enjoyable collaborations have been established.

References 1.

2. 3. 4. 5. 6. 7.

M. Martin, C. Eon and G. Guiochon, Study of the pertinency of pressure in liquid chromatography. I. Theoretical analysis, J. Chromatogr., 99 (1974) 357–376. II. Problems in equipment design, J. Chromatogr., 108 (1975) 229–241. M. Martin and G. Guiochon, Theoretical study of the gradient elution profiles obtained with syringetype pumps in liquid chromatography, J. Chromatogr., 151 (1978) 267–289. M. Martin and G. Guiochon, Pressure dependence of the diffusion coefficient and effect on plate height in liquid chromatography, Anal. Chem., 55 (1983) 2302–2309. P. Magnico and M. Martin, Dispersion in the interstitial space of packed columns, J. Chromatogr., 517 (1990) 31–49. U. Tallarek, K. Albert, E. Bayer and G. Guiochon, Measurement of transverse and axial apparent dispersion coefficients in packed beds, AIChE, 41 (1996) 3041–3054. M. Martin and G. Guiochon, Axial dispersion in open-tubular capillary liquid chromatography with electroosmotic flow, Anal. Chem., 56 (1984) 614–620. J.C. Giddings, M. Martin and M.N. Myers, High-speed polymer separations by thermal field-flow fractionation, J. Chromatogr., 158 (1978) 419–43.5.

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8.

M. Martin, C. Van Batten and M. Hoyos, Retention in field-flow fractionation with a moderate nonuniformity in the field force, Anal. Chem., 69 (1997) 1339–1346. 9. M. Martin, Theory of field flow fractionation, in P.R. Brown, E. Grushka (Eds.), Advances in Chromatography, Vol. 39, Marcel Dekker, New York, NY, 1998, pp. 1–138. 10. M. Martin, M. Hoyos and D. Lhuillier, Sedimentation equilibrium of suspensions of colloidal particles at finite concentrations, Colloid Polym. Sci., 272 (1994) 1582–1589.

D.44. Daniel E. Martire Daniel E. Martire was born in New York City on June 3, 1937. He received his elementary school education at St. Gregory School in New York City, New York where he enjoyed playing in Central Park and developed a life-long passion for museums and cinema. He graduated from Teaneck Jr. and Sr. High Schools in New Jersey. He received his Bachelor of Engineering (1959) and Ph.D. in Chemistry (1963) degrees from Stevens Institute of Technology. During 1963–1964, he was a NSF postdoctoral fellow at Cambridge University, U.K. He has been on the faculty of the Department of Chemistry at Georgetown University since 1964 (Assistant Professor, 1964– 1969; Associate Professor, 1969–1974; Professor, 1974–present; Acting Chair, 1987– 1988; Chair, 1996–present). In 1972, he was a Visiting Professor at the Colle`ge de France and Universite´ de Paris Sud. In 1976, he was a Senior Visiting Fellow at University College of Swansea. In 1980, he was a Visiting Professor at E´cole Polytechnique Fe´de´rale de Lausanne. D.E. Martire is the author or co-author of over 130 publications in the areas of chromatography, liquid crystals, thermodynamics and statistical mechanics. His main area of research since 1960 has been the fundamental aspects of analytical chromatography (GC, LC and SFC), culminating in a unified theory of chromatography in 1987. His most fulfilling accomplishment has been mentoring the research of some 30 Ph.D. recipients. D.E. Martire is one of the founders and a past president of the Washington Chromatography Discussion Group. He received the 1992 Eastern Analytical Symposium Award in Chromatography ‘in recognition of his outstanding contribution to the field of chromatography’ and the 1997 Stephen Dal Nogare Award ‘in recognition of significant contributions to the field of separation science’. He was cited twice (1987 and 1992) for ‘special creativity’ by the National Science Foundation. He also received Georgetown University Faculty Senate Service Awards in 1992 and 1997. See Chapter 5B, a, b, d, h, o

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44.I. THERMODYNAMICS AND THEORY OF CHROMATOGRAPHY: DEVELOPMENT OF NEW METHODS AND A UNIFIED APPROACH Daniel E. Martire Department of Chemistry, Georgetown University, Washington, DC 20057, USA

My introduction to research in chromatography came in the summer of 1960 at the end of my first year of graduate studies in physical chemistry. It was not uncommon at the time for graduate students to take summer positions in federal or industrial laboratories. I had the very good fortune, as it turned out, of landing a position in the Analytical Division of what was then the Esso Research and Engineering Company (now Exxon Research). On my first day there I was handed A.I.M. Keulemans’ ‘new’ book (published in 1959), “Gas Chromatography”, with the instruction that I should choose a relevant GLC research problem on which to work that summer. After a few weeks of reading and library work, it became clear that a major problem in the field was the a priori selection of an appropriate stationary liquid phase (SLP) to effect the desired separation. Accordingly, my first summer at Esso (I returned in 1961 and 1962) was spent developing an extended solubility-parameter approach to apply to the selection of SLPs for GLC. It was moderately successful and resulted in my first publication in chromatography [1]. Very excited about the untapped possibilities in the field, I returned to my graduate studies at Stevens Institute of Technology where my advisor, L.Z. Pollara, wisely allowed me to pursue this newly found interest which, as it turned out, developed into a life-long commitment to the science of chromatographic separations. The fundamental approach initiated then is one that has continued throughout my professional career in the areas of GC, LC and SFC, culminating in the unified theory of chromatography that will be described later. With respect to GLC, work was begun on the development and application of GLC as a reliable method to study the thermodynamics of non-electrolytic solutions. Through systematic studies on well-chosen solute plus solvent (SLP) systems, thermodynamic data (transfer and excess enthalpies, entropies and free energies of solution) were obtained and analyzed to test and extend (through statistical mechanics) theories of solution, eventually leading to a more quantitative molecular-level description of the effects of molecular interactions, and of SLP molecular weight and composition (for mixed phases) on GLC retention. Empirical methods of predicting thermodynamic and retention parameters were also explored, resulting in the first (and successful) application of factor analysis to chromatographic data [2]. In later years this general approach was expanded to include the development and utilization of a GLC method for investigating the thermodynamics of hydrogen-bonding and charge-transfer molecular association. A statistical mechanical theory was derived to enable quantitative interpretation of the GLC results and their reconciliation with results from NMR and UV=VIS spectroscopy. During my postdoctoral year at Cambridge University, where I collaborated with Howard Purnell and Bob Pecsok (who was there on sabbatical leave from UCLA), I

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became interested in liquid crystals. I began research on these intriguing orientationally ordered materials upon my appointment to the faculty of Georgetown University in 1964. In addition to extensive statistical–mechanical and static thermodynamic studies of bulk- and surface-phase transitions of liquid crystals and mixtures thereof, we developed, validated and applied GLC as an effective method of studying solution thermodynamics in cholesteric, nematic and smectic liquid crystals. Novel separations, primarily for di-substituted benzenes, were carried out on nematic liquid-crystalline stationary phases and a theory of selectivity based on solute molecular structure and shape was proposed and tested. Also, inverse GC was applied to give rapid methods for assessing solute-induced anisotropic-to-isotropic phase transitions and for correlating solute-induced shifts in the wavelength of maximum reflectance from cholesteric films with thermodynamic solution parameters, studies with relevance to liquid crystal display and sensing devices, respectively. In recent years research on orientationally ordered systems was extended to shapeselective separations using capillary column SFC with smectic polymeric stationary phases. Retention parameters were determined and subjected to theoretical analysis using a novel molecular theory. Our results and the GC and SFC results of others were correlated and interpreted through the derived, simple equation for the capacity factor, k, of rigid isomeric solutes having roughly the same molecular volume and surface area: ln k D c1 Am C c2

(1)

(where Am is the minimum projection area of the solute, and c1 and c2 are constants reflecting the solute series and column [3]). Our work in the area of LC began in the mid-1960s and resulted in two early papers with Dave Locke on the theory of solute retention and selectivity in LLC [4] which are still relevant today. Following a hiatus, research in the early 1980s in collaboration with my associate of 25 years, Dr. Richard Boehm, produced detailed molecular theories of retention and selectivity in HPLC with mixed mobile phases, for liquid adsorption chromatography (including heterogeneous surfaces), and for reversed-phase LC (RPLC) with bonded phases (introducing the concept of dynamic or ‘breathing’ bonded phases responding to the solvent environment) [5]. This work clarified the effect of solvent sorption on=in the stationary phase on solute retention and marked the beginning of a unified approach. More recently, thermodynamic and theoretical investigations of common hydroorganic mobile phases and a range of stationary phases have demonstrated that, in addition to capacity factor or retention volume data (free energy quantities), a full understanding of the retention process in RPLC requires knowledge of the solvent sorption isotherms (an LC method for such was developed and successfully applied), and both 1H and 1S of solute transfer (which exhibit unexpected behavior) [6]. Other noteworthy LC work has involved excursions into the theory of homopolymer and oligomer retention and separation by gradient elution LC, and the theory of separation of enantiomeric solutes, both done in collaboration with Richard Boehm and Dan Armstrong, a former Georgetown colleague. The unified theory of chromatography came about when I brought my thermodynamic and statistical–mechanical kits over to SFC in the mid-1980s. I began to notice

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similarities in the experimental and theoretical results between the dependence of ln k on temperature and mobile-phase composition in LC, and the dependence of ln k on temperature and mobile-phase density in SFC. I was groping for an explanation. Then the light came on. Without getting too technical and risk losing the reader in a morass of theory, it became clear that by putting the previously derived LC equations into reduced form and then exploiting the isomorphism in the critical-point behavior of binary liquid systems (LC) and compressible pure-fluid systems (SFC), retention equations for SFC could be readily obtained. What this revealed in practical terms was that the physical space or volume fraction occupied by the molecules in a supercritical fluid in SFC played the same role as the volume fraction of the stronger eluting solvent in LC, while the fraction of void or unoccupied volume in SFC paralleled the volume fraction of the weaker solvent in LC. Taking a purist’s approach, we then returned to first principles and derived, using statistical mechanics and a compressible=expansible lattice model, general equations applicable to any mobile-phase fluid (gas, liquid or supercritical fluid), to adsorbent and=or absorbent stationary phases, and to both capillary and packed column systems. The theoretical treatment necessarily included the effects of mobile-phase fluid sorbed on=in the stationary phase. The details of the derivation, the final results and some applications can be found in two review chapters [7,8]. The retention equation obtained, the basis of the unified approach, can be written in the following general form: ln K a.bCc/ D ln K a.c/ C F.T; b.m/ / C Ð

(2)

(where K is the distribution coefficient, a is the solute, b refers to the ‘good’ solvent (LC) or actual molecules (SFC), c refers to the ‘poor’ solvent (LC) or void (SFC), and

b.m/ is the mobile-phase volume fraction of (b). (Note that b.m/ is proportional to the mobile-phase density, ², in SFC.) K a.c/ , which depends on the nature of the stationary phase (adsorbent and=or absorbent) and contains molecular size and interaction-energy parameters, is the distribution coefficient when b.m/ D 0 ( c.m/ D 1). It corresponds to LC with component c as a neat mobile phase or to ideal GC in the explicit SFC retention equation. It is independent of b.m/ . F.T; b.m/ / is solely a mobile-phase term. It also contains molecular size and interaction-energy parameters, thus opening the retention equation to molecular-level interpretation. It describes the linear dependence of ln K a.bCc/ on reciprocal absolute temperature and the quadratic dependence on b.m/ (² for SFC). The ‘coupling’ term, Ð, which, alas, cannot be ignored, arises from sorption of the mobile-phase fluid on=in the stationary phase. It produces a weaker dependence on T and b.m/ than does the F.T; b.m/ / term. In general, plots of ln K (or ln k) vs. b(m) reflect the variation of both terms with

b.m/ , but, provided the sorption isotherm of b is not in the rapidly rising (low b.m/ / region, the rate of change of Ð with b.m/ is negligible compared to that of F.T; b.m/ / with b.m/ . (Fortunately, LC and SFC separations are seldom performed in this low b.m/ region.) Note as a caution, however, that in both LC and SFC the intercept of a plot of

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Fig. 1. Plots of the natural logarithm of the net retention volume (Vn ) of n-hexylbenzene vs. the volume fraction of water in methanol=water mixtures in liquid chromatography at 25ºC, for the monomeric and self-assembled monolayer (SAM) stationary phases shown [9].

Fig. 2. Plots of the natural logarithm of the capacity factor (k) of n-octylbenzene vs. the reduced density of CO2 in supercritical-fluid chromatography at a reduced temperature of TR D 1:161, for the following bonded phases: (a) C18 , 10 µm (), (b) C18 , 5 µm (), (c) C2 , 10 µm (Ž), (d) C2 , 5 µm (ž) [10].

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ln K vs. b.m/ , determined by extrapolation to b.m/ D 0, is approximately equal to the sum, ln K a.c/ C Ðs , where Ðs corresponds to a stationary phase virtually saturated with component b (slowly changing region of the sorption isotherm). Under such near-saturation conditions, an interesting and useful implication of Eq. 2 is that, independent of the stationary phase used, plots of ln k vs. b.m/ for a given type of mobile phase at fixed T should display nearly the same dependence on b.m/ , governed by the F.T; b.m/ / term. This prediction (one of many) has been successfully tested in recent LC and SFC studies with different mobile phases [9,10], as illustrated in Figs. 1 and 2. Although not shown, capillary-column SFC results run parallel to the packed-column results in Fig. 2, as predicted. Lastly, the spirit of the unified approach found its way to our generalized treatments of (a) spatial and temporal column parameters and (b) plate-height theory of compressible fluids, both applicable to GC, LC and SFC. Judging from the books and articles that have appeared since our initial publication in 1987, a unified approach to the practice and theory of chromatography is an idea whose time has come. In closing, I have been fortunate in my scientific lifetime to have had wonderful mentors, dedicated and resourceful graduate students, excellent collaborators, and such friends and colleagues in the field as Pete Carr, Georges Guiochon, Ervin Kovats and Ray Scott, who have thankfully challenged me in the best sense.

References 1.

D.E. Martire, Application of the theory of solutions to the choice of solvent for gas–liquid chromatography, Anal. Chem., 33 (1961) 1143–1147. 2. P.T. Funke, E.R. Malinowski, D.E. Martire and L.Z. Pollara, Application of factor analysis to the prediction of activity coefficients of non-electrolytes, Separation Sci., 1 (1966) 661–676. 3. C. Yan and D.E. Martire, Determination and theoretical analysis of supercritical-fluid chromatographic retention of PAHs in a polymeric smectic phase, J. Phys. Chem., 96 (1992) 3505–3512. 4. D.C. Locke and D.E. Martire, Theory of solute retention in liquid–liquid chromatography, Anal. Chem., 39 (1967) 921–925; D.E. Martire and D.C. Locke, Selectivity in liquid–liquid chromatography, Anal. Chem., 43 (1971) 68–73. 5. D E. Martire and R.E. Boehm, A unified theory of retention and selectivity in liquid chromatography, J. Phys. Chem., 84 (1980) 3620–3630 (Part I), 87 (1983) 1045–1062 (Part II). 6. A. Alvarez-Zepeda, B.N. Barman and D.E. Martire, A thermodynamic study of the marked differences between ACN=H2 O and MeOH=H2 O mobile-phase systems in RPLC, Anal. Chem., 64 (1992) 1978– 1984. 7. D.E. Martire, in F. Dondi and G. Guiochon (Eds.), Theoretical Advancement in Chromatography and Related Separation Techniques, Kluwer, Dordecht (Holland), 1993, pp. 261–274. 8. D.E. Martire, R.L. Riester and X. Zhang, in F. Dondi and G. Guiochon (Eds.), Theoretical Advancement in Chromatography and Related Separation Techniques, Kluwer, Dordecht (Holland), 1993, pp. 275–288. 9. J.E.N. Gutierrez, A thermodynamic comparison of monomeric C18 and self-assembled monomeric (SAM) C18 and C2 chemically bonded phases in reversed-phase liquid chromatography, Ph.D. Dissertation, Georgetown University, 1998, p. 118. 10. M. Osonubi, A systematic thermodynamic investigation of retention and selectivity in packed column supercritical-fluid chromatography, Ph.D. Dissertation, Gerogetown University, 1998, p. 135.

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D.45. Robert B. Merrifield Robert B. Merrifield was born in Fort Worth, Texas, on July 15, 1921. He grew up in California and received his bachelor’s degree in chemistry at the University of California – Los Angeles (UCLA) in 1943. He earned his Ph.D. in biochemistry under Max Dunn at UCLA (1949). Soon thereafter, he began a long fruitful career at the Rockefeller Institute for Medical Research, which became the present Rockefeller University; he became a full Professor in 1966 and retired in 1993. Beginning in the 1950s, Merrifield developed the sequence of reactions – ‘solid phase synthesis’ – to produce the very specific sequence of amino acids in proteins. This stepwise synthesis has the growing peptide chain attached to an insoluble polystyrene bead. An amino acid with a blocked amino group is attached to the polymer through its carboxyl group. After removing the amino blocking group, a second amino acid also blocked on the amino group and R group if necessary, is condensed with the first amino acid on the polymer. These sequential steps may be repeated over and over again to synthesize the desired peptide, and followed by selective removal of the blocking groups from the synthesized peptide [1]. These steps were next automated and, now with added microcomputer controls of all the reaction steps. After synthesizing a variety of smaller peptides (hormones [2,4]), Merrifield performed the first total synthesis of an enzyme, specifically ribonuclease, a protein with 124 amino acids [3]. The citation for his 1984 Nobel Award reads – ‘for his development of methodology for chemical synthesis on a solid matrix’ [5–7]. This basic research serves as the foundation for his later investigations and now used by many other scientists [8]. One evaluation stated, “The solid phase technique has enabled investigators to elucidate the effect of structure on biological function of many proteins, including enzymes, hormones, and antibodies : : : helped in the development of monoclonal antibodies : : : [and may help to develop] synthetic vaccines against such viral diseases as influenza, rabies and hepatitis.” R.B. Merrifield’s own description of the inseparable relation of chromatography and peptide synthesis follows. By Robert L. Wixom See Chapter 5B, b, c, h 45.I. THE ROLE OF CHROMATOGRAPHY IN SOLID PHASE PEPTIDE SYNTHESIS Bruce Merrifield Rockefeller University, 1230 York Avenue, New York, NY, 10021–6399, USA

Nobel Prize in Chemistry, 1984 I first used alumina and silica gel chromatography in 1944 for separation of plant pigments, then, beginning about 1954, I have continually used paper and thin layer

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chromatography as well as ion-exchange chromatography for separation and purification of natural and synthetic peptides and small proteins. Currently, reversed phase HPLC is my method of choice for purification of synthetic peptides. The technique of Solid Phase Peptide Synthesis was conceived and developed between 1959 and 1963; further development and refinement has continued up to the present time. The idea was not based on chromatography, but I believe my previous experience with chomatographic methods contributed to the concepts behind the new synthetic approach. Chromatography was, of course, of great value in the purification and evaluation of the synthetic products. HPLC, along with NMR and mass spectrometry, continues to be indispensable to my work.

References 1. 2. 3. 4. 5. 6. 7. 8.

R.B. Merrifield, Solid phase peptide synthesis I. The synthesis of a tetrapeptide, J. Am. Chem. Soc., 85 (1963) 2149–2154. R.B. Merrifield, Solid phase peptide synthesis III. An improved synthesis of bradykinin, Biochemistry, 3 (1964) 1385–1390. B. Gutte and R.B. Merrifield, The synthesis of ribonuclease A, J. Biol. Chem., 246 (1971) 1922–1941. S. Mojsov and R.B. Merrifield, An improved synthesis of crystalline mammalian glucagon, European J. Biochem., 145 (1984) 601–605. Nobel Lectures – Chemistry 6 (1992) 146–175. Nobel Laureates in Chemistry (1901–1992) 667–673. R.B. Merrifield, The concept and development of solid phase peptide synthesis, in J.I. Seeman (Ed.), Profiles, Pathways and Dreams, American Chemical Society, Washington, D.C., 1993. R.B. Merrifield, Life During a Golden Age of Peptide Chemistry: The Concept and Development of Solid-Phase Peptide Synthesis, American Chemical Society, Washington, DC, USA, 1993, 200 pp.

D.46. Hiroshi Miyazaki Hiroshi Miyazaki was born on June 1, 1929, in Yokohama, Japan. He studied at Tokyo College of Pharmacy, graduating in 1950. In 1976, he received a Ph.D. from Tohoku University. In 1950, H. Miyazaki joined Nippon Kayaku Co., Ltd., and had been associated with this company, and in 1982, he became the Vice Director of the research laboratories of the pharmaceutical division of the company; in 1983 he was appointed as General Manager of research and development for the pharmaceutical group, and in 1984 as the Director of the corporate planning office. In 1988, he joined Kotobuki Pharmaceutical Co., Ltd., as a Director of the research laboratories. From 1974 to 1978, he was also a lecturer at the Pharmaceutical Institute of Tohoku University, and in 1978 to 1979 at the Science Institute of Tokyo Metropolitan University. From 1988 to 1992 he was appointed a Professor at Showa University, School of Medicine, and since 1997 he has been Professor at Niigata College of Pharmacy, his present position. H. Miyazaki is a member of the Pharmaceutical Society of Japan, the Japanese

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Society of Gastroenterology and the Japanese Society of Radioisotopes; he was a member of the Executive Boards of the Japanese Society for the Study of Xenobiotics and the Japanese Society of Mass Spectroscopy; he was an editor of the Japanese journals Pharmacia and Xenobiotic Metabolism and Disposition. H. Miyazaki is the author or co-author of over 150 scientific and technical papers dealing with gas–liquid chromatography, mass spectrometry, isotachophoresis, drug metabolism, pharmacology, and organic synthesis. He is also the co-author of two books: “Practical Mass Spectrometry in the Medical and Pharmaceutical Science” (with A. Tatematsu, M. Suzuki and Y. Maruyama), and “Isotachophoresis” (with K. Katoh) which were published in English and Japanese by Kodansha Scientific Co., in 1979, respectively. H. Miyazaki received the M.S. Tswett Chromatography Medal in 1986 and the Kitagawa Award from the Japanese Society for the Study of Xenobiotics in 1999. See Chapter 5B, a, d, e, l, p, r, s

46.I. MICROANALYSES OF BIOLOGICALLY IMPORTANT SUBSTANCES AND DRUGS IN BIOLOGICAL SPECIMENS BY GAS CHROMATOGRAPHY, MASS SPECTROMETRY, AND ISOTACHOPHORESIS Hiroshi Miyazaki 19–12 Chitose-shin, Takatsu-ku, Kawasaki, Kanagawa 213–0021, Japan

In 1968, I started my activities on the microanalysis of endogenous compounds and drugs in biological specimens by gas chromatography–mass spectrometry. At that time endocrinological and chemical studies on the insect molting hormone encountered the elementary question of whether the prothoracic glands are really the site of ecdysone biosynthesis. In order to clarify the above question, an excellent in vitro system was developed by Chino et al., using isolated prothoracic glands of Bombyx silkworm and a medium containing hemolymph. Then, the microanalysis of the molting hormone in the resulting culture medium was successfully carried out in our laboratory by gas chromatography (GC) – mass fragmentography (MF) after conversion to the trimethylsilyl (TMS) ether derivatives [1]. This GC–MF research revealed that the hormone in the medium was identical with an ecdysone [2]. Trimethylsilylation has been extensively used as a derivative suitable for GC=MS analysis in electron ionization (EI) mode of a polar and nonvolatile compounds. However, the TMS ether derivative has an inevitable disadvantage in GC=EI=MS; in that characteristic ions in the high mass region may be of extremely low intensity, when many hydroxyl groups are present in the parent molecule because of the successive eliminations of trimethylsilanol. To overcome this disadvantage of the TMS ether derivatives, a series of silylating agents shown in Fig. 1 were synthesized in our laboratory since 1975. The resulting silylether derivatives exhibited excellent GC and EI=MS properties.

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Fig. 1. Newly synthesized silylating agents.

(1) in most cases, the fragment ion of [M-29]C or [M-43]C , due to the loss of an ethyl or n-propyl group from the DMES and DMnPS ethers, was more prominent than that of [M-15]C in the corresponding TMS ethers. (2) The number of hydroxyl groups in a hydroxylated steroid could be estimated easily by the use of new retention indices which are defined by differences in the methylene unit values of the dimethyl ethylsilyl (DMES) or dimethyl propylsilyl (DMnPS) and trimethylsilyl (TMS) ether derivatives. ∆[Um]E D MUE MUT ∆[Um]P D MUP MUT where MU, MUE and MUP are the methylene unit values of TMS, DMES and DMnPS ethers of the hydroxylated steroids, respectively. In particular, the round number of 1[Um]E is in good agreement with the number of hydroxyl groups as shown in Table 1 [3]. (3) It has been thought to be almost impossible to distinguish by gas chromatography a difference between phenolic and alcoholic hydroxyl groups. However, a phenolic TMS ether group in estradiol was selectively exchanged for a DMES group ether on a GC TABLE 1 INCREMENT OF 1[Um ] WITH INCREASING NUMBER OF HYDROXYL GROUPS AND ITS MEAN VALUE AND COEFFICIENT OF VARIATION 1[Um ]

Number of hydroxyl groups

Number of samples

Mean value

Coefficient of variation (%)

1 2

18 15

1.22 2.38

6.56 6.30

1[Um ]E

3 4 1

6 5 18

3.53 4.35 1.93

3.40 4.14 5.70

1[Um ]P

2 3 4

15 6 5

3.72 5.23 6.23

7.80 5.16 1.93

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Fig. 2. Selected ion recordings of the Dips ether derivatives of PGD2 and PGF2 , methyloxime-methyl esters in the extract from human brain (arachinoid).

column by use of sandwich injection with DMES-imidazole. This selective exchange reaction was considered to be caused by a difference in the lability between the ethereal TMS linkages to phenolic and alcoholic hydroxyl groups, respectively [4]. (4) A number of methods for prostaglandins (PGs) and thromboxane B2 (TXB2 ) have been developed to investigate their occurrence and distribution in biological fluids or tissues. Of these methods, GC=EI selected ion monitoring (SIM) has been extensively used as the most sensitive and specific method for microanalysis of PGs and TXB2 after their conversion into the TMS ether derivatives of the methyl esters (ME) or methoxime–methyl esters (MO–ME). To enhance their GC separation and production of the characteristic fragment ions of high relative abundance in high mass region, the corresponding DMiPS ether derivatives were synthesized and the resulting derivatives provided not only complete separation but also the production of [M-43]C , with prominent intensity. In early 1980, I started a collaboration with Dr. Hayaishi’s group (Kyoto University, Japan) for the quantiation of trace amounts of PGs, especially PGD2 in human brain by GC=EI=SIM using the ME–DMiPS and ME–MO–DMiPS ether derivatives. At that time, the presence of PGD2 , in human brain was debatable because there was no direct evidence by GC=SIM. It was very difficult to quantify very low concentration of PGs in human brain by GC=low resolution (LR) SIM due to the large amounts of interfering substances giving ions with the same nominal masses that coexisted in the eluate after elaborate purification. The problem was satisfactorily solved by the use GC=EI=high resolution (HR) SIM. The GC=EI=HR-SIM made it possible to improve greatly the GC separation and the sensitivities of the corresponding DHiPS ether derivatives. Thus, the presence of PGD2 , E2 and F2 Þ in human brain was identified and quantified by using specific radioimmunoassys and the GC=EI /HR-SIM as shown in Fig. 2 [5]. (5) Although the DMiPS ether derivatives of hydroxysteroids such as pregnanediols have the characteristics ion of [M-43] with prominent abundance in high mass region, the DMiPS ethers of those containing the sterically hindered 17α-hydroxyl group such

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as 5β-pregnane-3α, 17α, 20α-triol were not silylated completely due to the bulkiness of the isopropyl group in the DMiPS moiety. On the other hand, reagents for selective derivatization of 1,2- and 1,3-diols, diacetoxydimethylsil (DADMS) and alkyl or aryl boronic acids have been reported. However, the resulting products have disadvantages in that the former is hydrolytically unstable and the latter requires further derivatization for other hydroxyl groups that are unable to yield the cyclic boronate. Thus, N,O-bis-(diethylsilyl)-trifluoroacetamide (DEHS-BSTFA) was synthesized. The resulting DEHS- and=or diethylsiliconide (DES)-DEHS ether derivatives obtained by one-step operation gave good GC and MS properties in GC=EI=MS. In particular, the DES-DEHS ether derivative of 5β-pregnane-3a, 17α, 20α-triol gave the molecular ion at m=z 506 as a principal ion in GC=EI=MS [6]. Thus, the above silylating agents have been used widely for the GC=MS analyses of steroids, bile acid, prostaglandins, drugs and their metabolites in biological specimens. Since early 1970, the studies of drug metabolism utilizing GC=MS have been carried out in our laboratory. The possible pathways of drug metabolism are now considered to be almost completely known, and most new chemical compounds are found to be metabolized through one or another of the recognized pathways. However, we discovered a new and quite unexpected metabolic pathway during our research into the metabolism of 5-(40 -chloro-n-butyl) picolinic acid (CBPA). The chemical structures of the unexpected metabolites of CBPA could be estimated by mass spectral analysis utilizing characteristic reaction products after derivatization without the aid of authentic metabolites. These unexpected metabolites in rat urine were elongated by a C2 unit in the carboxyl group at the 2-position on the pyridine ring. This novel metabolic pathway of CBPA corresponded well with the biosynthetic pathway of fatty acids. This pathway was confirmed in man, rabbit, guinea pig and mouse by the aid of the stable isotope technique using an equimole mixture of [12 C] and [13 C] CBPA [7]. In 1978, in order to investigate the pharmacokinetics of the individual enantiomeric pair of D-and L-chlorpheniramin (D-and L-CPA) in human, D-CPA-d6 and DL-CPA-d13 were synthesized in our laboratory. Then a 1 : 1 mixture of D-CPA-d6 and L-CPA-d0 which is called ‘pseudoracemate’ was administered in a dose of 1 mg each to healthy volunteers after no biological isotope effect on the pharmacokinetics between D-CPAd6 and D-CPA-d0 was confirmed. The serum concentration of L -CPA-d0 and D-CPA-d6 were quantified by GC=positive ion chemical ionization MF using trimethylamine as a reagent gas and DL-CPA-d13 as an internal standard. The serum level of the D-isomer was higher than that of the L-isomer, and the biological half-lives of the D-and L-isomers were 24 h, and 15 h, respectively. This first pharmacokinetic study of enantiomers in humans may generally enable one to discriminate the enantiomers which can not be separated by GC without the influence of individual differences [8]. In 1976, a capillary tube isotachophoretic study of peptides was carried out in our laboratory. In 1975, Deml et al., proposed the ‘inverse relative mobility’ as a new qualitative index for isotachophoresis. This index has excellent reproducibility under identical operating conditions, but is considerably influenced by the current, inner diameter of the capillary tube and re-preparation of the electrolyte. This inverse index is only as reliable as the relative retention time in GC; that is, it

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At the 1988 International Symposium on Advances in Chromatography in Minneapolis, USA. Left-to-right sitting: W.J.A. VandenHeuvel, M.G. Horning, the late E.C. Horning; standing: C.J.W. Brooks, H. Miyazaki, J. Sjo¨vall.

differs with the apparatus and laboratory used. Then, we proposed a new index that is relative to the leading and terminating ions, in the same manner as the methylene unit (MU) value in GC, which is the retention time relative to two standard substances. We termed this new index the ‘PU value’. PU D .PGS PGL /=.PGT PGL / Where PGS is the potential gradient of the sample, PGL the potential gradient of the leading ion, and PGT the potential gradient of the terminating ion. The validity of the new index was verified by comparing the inverse index under various conditions [9]. Capillary-tube isotachophoresis has been used as a rapid and specific method for the simultaneous qualitative and quantitative analysis of a mixture of peptides and amino acids; we applied this method for measuring urokinase activity using N-α-acetyl-L-lysine methyl ester as a synthetic substrate. A comparison between the urokinase activities of the same sample determined by the present method and Walton’s modified plate method gave excellent agreement [10]. The above research was made possible by the help of a number of my co-workers who are Drs. M. Ishibashi, Y. Hashimoto, H. Abuki, H. Takayama, K. Katoh, M. Koyama, M. Itoh, K. Yamashita, G. Idzu, N. Asakawa, M. Inoue, Y. Minatogawa, C. Mori, K. Watanabe, and J. Irie in the research laboratory, Nippon Kayaku, Co., Ltd. I would like to express my sincere thanks to Drs. E.C. Horning (dec.), M.G. Horning, N. Ikekawa, T. Nambara and W. Tanaka for their encouragement and their valuable suggestions throughout our studies.

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References 1.

H. Miyazaki, M. Ishibashi, C. Mori and N. Ikekawa, Gas phase microanalysis of zoo edysones, Anal. Chem., 45 (1973) 1164–1168. 2. H. Chino, S. Sakurai, T. Ohtaki, N. Ikekawa, H. Miyazaki, M. Ishibashi and H. Abuki, Biosynthesis of α-ecdysone by prothoracic glands in vitro, Science, 183 (1974) 529–530. 3. H. Miyazaki, M. Ishibashi, M. Itoh and T. Nambara, Use of new silylating agents for identification of hydroxylated steroids by gas chromatography and GC=MS, Biomed. Mass Spectrom., 4 (1977) 23–35. 4. H. Miyazaki, M. Ishibashi, M. Itoh and K. Yamashita, Use of silylating agents for indentification of hydroxylated steroids by gas chromatography and gas chromatography-mass spectrometry. Discrimination between phenolic and alcoholic hydroxyl groups, J. Chromatogr., 133 (1977) 311–318. 5. M. Ishibashi, K. Yamashita, K. Watanabe and H. Miyazaki; pp. 423–441, in S.J. Gaskell (Ed.), Mass Spectrometry in Biomedical Research, John Wiley and Sons, Chichester 1986. 6. H. Miyazaki, M. Ishibashi, M. Itoh, and K. Yamashita, Diethylsilyl ether and diethylsiliconide derivatives in gas chromatography mass spectrometry of hydroxylated steroids, Biomed. Mass Spectrom., 11 (1984) 377–382. 7. H. Miyazaki, H. Takayama, Y. Minatogawa and K. Miyano, A novel metabolic pathway in the metabolism of 5-(40 -chloro-n-butyl) picolinic acid, Biomed. Mass Spectrom., 3 (1976) 140–145. 8. H. Miyazaki and H. Abuki, Mass fragmentographic determination of D- and L-chlorpheniramine with aid of the stable isotope technique, Chem. Pharm. Bull., 24 (1976) 2572–2574. 9. H. Miyazaki and K. Katoh, Isotachophoretic analysis of peptides, J. Chromatogr., 119 (1976) 369–386. 10. K. Katoh and H. Miyazaki, Assay method for urokinase activity by capillary-tube isotachophoresis using a synthetic substrate, J. Chromatogr., 188 (1980) 383–390.

D.47. E. David Morgan E. David Morgan was born on March 4, 1930, and raised in Newfoundland. David Morgan began his university education there, with a Diploma in Arts and Science, 1948 from the (then) Memorial University College, and completed his B.Sc. at Dalhousie University, Halifax, Nova Scotia in 1950, winning the Governor-General of Canada medal and the Rhodes scholarship for Newfoundland for 1950. At Oxford, he studied chemistry for the Honors B.A., 1952 and continued there, obtaining the M.A. and D.Phil degrees for work on the lipids of Mycobacterium tuberculosis in 1956. He then joined the group of J.W. (later Sir John) Cornforth (Nobel Prize for Chemistry, 1975) at the National Institute for Medical Research, London, working on the chemotherapy of tuberculosis and leprosy. His first encounter with gas chromatography was in 1957 while working there. Tony James and James Lovelock were colleagues in the same building, and there was collaboration between the Cornforth and James groups. He made polymers for them to try out as stationary phases, and later made for them poly(ethylene glycol) esters of various dicarboxylic acids, probably the very first polyester phases. In 1959, he joined his former Professor from Oxford, Sir Robert Robinson (Nobel Prize for Chemistry, 1947), who was now a director of Shell Chemical Company and had a personal laboratory at Egham, Surrey. There he acquired a temperature-programmed

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E. David Morgan at the time of the award of the Jubilee Medal, with former student Athula Attygalle and Post-Doctoral Fellow Anne-Genevie`ve Bagne`res-Urbany.

gas chromatograph, and began to realize the possibilities that it offered. In 1966, he moved to the academic world as lecturer in the Department of Chemistry at Keele University. He immediately made his first GC–MS link-up, with a Pye 104 GC and a Hitachi-Perkin Elmer RMU-6 mass spectrometer, and has had a GC–MS continuously in operation now for 33 years. He was successively Senior Lecturer, 1972; Reader, 1979; and from 1990 Professor of Entomological Chemistry until he retired as Professor Emeritus in 1995. He was Visiting Professor at Memorial University of Newfoundland, 1976; invited speaker at the 150th Anniversary Symposium of the Royal Entomological Society, 1983; he received the Jubilee Medal of the Chromatographic Society for 1989 (see photograph), a Leverhulme Fellowship, 1988–1990; the Neem Mission Award, 1996; and was the president of a Jacques Monod Conference on Chemical Communication, 1997. Speaker at numerous international conferences. He has served on the Editorial Boards of the Journal of Chromatography, Journal of Chemical Ecology, and Chromatography and Analysis. He has held various posts in the Royal Society for Chemistry, including Chairman of the North Staffordshire Section and Chairman of the Micro and Chemical Methods Group. He is a Fellow of the Royal Society of Chemistry; a Chartered Chemist; Fellow of the Royal Entomological Society; Life member of the International Society for Chemical Ecology; Member of the British Section; International Union for the Study of Social Insects; Fellow of the Chromatographic Society, and a member of the Pye 104 Club. He has co-authored over 250 papers, reviews, contributions, patents, editorship and

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authorship of books, excluding book reviews, posters, ephemeral publications, letters, etc. His special interest has been in the chemistry of the pheromones and secretions of insects, especially those of ants, bees, and wasps; the chemistry of the moulting hormones (ecdysteroids) of insects; the chemistry of plant substances affecting feeding in insects, and the application of chemical methods to archaeology. He has also done work on the analytical chemistry of atmospheric pollution. His teaching interests have concentrated on analytical methods and organic spectroscopy, mass spectrometry, biosynthesis, pheromones, natural products, environmental pollution, and the origins of life on earth. See Chapter 5B, d, f, k, s

47.I. ISOLATION, STRUCTURE AND QUANTIFICATION OF INSECT SUBSTANCES E. David Morgan Keele University, Department of Chemistry, Staffordshire ST55BG UK

My first encounter with gas chromatography was in 1957 while working at the National Institute for Medical Research in London. Drs. Tony James and James Lovelock were working in the same building, and I remember visiting their laboratory to see demonstrations of their prototype detectors. In May of that year I made some polymers for them to try as stationary phases, and later made for them poly(ethylene glycol) diesters, probably the very first polyester phases. A few years later I received my very own temperature programmed gas chromatograph, and took to the technique with enthusiasm. On taking up my academic post at Keele, I linked up a GC-MS in my first year, made from a new Pye 104 GC and a Hitachi-Perkin Elmer RMU-6 mass spectrometer – a beautiful machine, all stainless steel and glass. What if one did have to count the peaks on the light sensitive recorder paper, and measure all the heights of the peaks with a ruler, to draw the spectrum! We have had a GC–MS continuously in operation ever since for 33 years. My contributions to chromatography have been mostly in applying it to the very small samples available when analyzing insect material. Therefore, the group has produced many ideas and special techniques for very small samples, particularly the Keele solid sampling methods (see Fig. 1), the Keele micro-reactor with a volume of 100 µl (see Fig. 2), and various microchemical reactions that can be carried out on nanogram quantities of material, all for use with gas chromatography. Research has taken me for longer periods to Nigeria, with the Scrub Savannah Project, to Brazil on several visits for lectures and research, supported by the British Council and the Brazilian government, and Korea for the National Instrumentation Center for Environmental Management. Currently, I am a partner in the European Community Training and Mobility of Researcher Network ‘Social Evolution’, in which

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Fig. 1. Total ion current from the gas chromatography of a single gland from the abdomen of an ant of the species Cataglyphis savignyi, obtained with the Keele solid sampling method [8]. Note that at least 38 substances can be identified and quantified, and that no solvent has been used.

our group provides the chemical expertise for studies on social insects (bees, wasps and ants). The highlights of my research have been the isolation of the powerful natural insecticide, azadirachtin in 1967, and subsequent work on determination of its structure, and analytical methods for its determination; the identification of the moulting hormone of the desert locust (1975), the first identification of this hormone in a hemi-metabolous insect; followed by development of sensitive analytical methods for measuring moulting hormone levels (1971–1976). Our group made the first measurement of the moulting hormone levels throughout the complete life cycle of an insect (1976–1979); and achieved the identification of the moulting hormone of the common barnacle (1977). From 1970 onwards we developed special nanochemical techniques and apparatus (e.g., the Keele solid-sampler of GC, and the Keele micro-reactor for nanochemical reactions) for the study of insect pheromones using single insect glands. We made the first microscale identification of an ant trail pheromone (1981). About three quarters of all of the known trail pheromones of ants have been identified in our laboratory. I have always regarded my role as a teacher to be as important as that of a research. I am therefore as proud of the subsequent achievements of my former students as I am of the work we did together. To mention a few is not to dismiss the great

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Fig. 2. The Keele micro-reactor showing how it is used for extraction or reaction. (A) water or reaction medium (a) (50 µl) is placed with the extraction solvent, hexane (b) (10 µl) in the bottom chamber. (B) The liquids are withdrawn from and flushed back into the bottom chamber with a syringe (c) (100 µl) to effect extraction between the phases. (C) The liquid layers are transferred to the top chamber by filling the bottom chamber with air or more water, and allowed to separate. (D) The level of liquids is adjusted by removing air or water from the bottom chamber, so that the upper, organic layer is left in the narrow middle chamber (d). (E) The upper layer is ready for removal with a syringe. (See Anal. Chem., 58 (1986) 3054.)

contributions of the others. Colin F. Poole has gone on to be a prolific contributor to chromatography in both books and papers. Ian D. Wilson has pioneered many hyphenated methods, particularly HPLC–NMR and HPLC–NMR–MS; he was the first to combine TLC and MS. Both these researchers have themselves won chromatography awards. Lester J. Wadhams has become a world expert in linking a gas chromatograph to an electro-antennograph, and especially in studying the response of a single sensillum on the antenna of an insect to pure compounds delivered to it from the GC of plant and insect extracts. Athula B. Attygalle has done remarkable work in GC–MS on identification of insect substances at Erlangen and Cornell University.

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References 1. 2. 3.

4. 5. 6.

7. 8.

J.H. Butterworth and E.D. Morgan, Investigation of the locust feeding inhibition of the seeds of the Neem tree (Azadirachia indica), J. Insect Physiol., 17 (1971) 969–977. E.D. Morgan and L.J. Wadhams, Gas chromatography of volatile compounds in small samples of biological materials, J. Chromatogr. Sci., 10 (1972) 528–529. E.D. Morgan and C.F. Poole, The extraction and determination of ecdysones in arthropods, Chapter 2, in J.E. Trehern, M.J. Berridge and V.B. Wigglesworth (Eds.), Advances in Insect Physiology, Academic Press, London, UK, 1976, pp. 17–62. R.P. Evershed, E.D. Morgan and M.C. Cammaerts, Identification of the trail pheromone of the ant Myrmica rubra (1), and related species, Naturwissenschaften, 67 (1981) 374–376. A.B. Attygalle and E.D. Morgan, A versatile micro-reactor and extractor, Anal. Chem., 58 (1986) 3054–3058. A.B. Attygalle and E.D. Morgan, Pheromones in nanogram quantities: Structure determination by combined microchemical and gas chromatographic methods, Angew. Chemie, Internat. Ed. Engl., 27 (1988) 460–478. E.D. Morgan, Preparation of small scale samples from insects for chromatography, Anal. Chim. Acta., 236 (1990) 227–235. M.F. All, J.P.J. Billen, B.D. Jackson and E.D. Morgan, Secretion of the Dufour glands of two African desert ants, Camponotus aegyptiacus and Cataglyphis savignyi (Hymenoptera: Formicidae), Biochem. Systemat. Ecol., 16 (1988) 647–654.

D.48. Milos V. Novotny Milos Novotny was born on April 19, 1942 in Brno, Czechoslovakia. He received his scientific training at the University of Brno: undergraduate in Chemistry=Physics, doctorate in Biochemistry (1965). During the following four years, M.V. Novotny held research positions in Czechoslovakia (Academy of Sciences), Sweden (Royal Karolinska Institute) and the United States (University of Houston), all in various areas of analytical chemistry. Since becoming a faculty member at Indiana University (1971), Novotny has developed a strong research program with emphasis on analytical separation science and bioanalytical chemistry. He became a full Professor in 1978 and the James H. Rudy Professor of Chemistry in 1988. In 1999, he was named a Distinguished Professor. During his professional career of over 30 years, he has played a pivotal role in conceptualization and application of analytical separation methods at very small sample scale. Beginning with capillary gas chromatography in the mid-1960s, Novotny made substantial contributions to the area through preparation of high-efficiency separation capillaries, and an understanding of their surface chemistry and conceptual applications of the method to biochemical and environmental problems. The 1975, Viking Lander contained a miniature GC column he designed for the analysis of the soil on Mars. M.V. Novotny pioneered the methods of microcolumn liquid chromatography, its coupling with electrospray mass spectrometry and microscale biochemical investigations. Together with Milton Lee, Novotny published in 1981 a pioneering study on capillary supercritical-fluid chromatography. Also, he has been among the key scientists developing the field of capillary electrophoresis (CE). Particularly noteworthy are carbohydrate separations, ultrasensitive peptide mapping, and pulsed-field CE of large biomolecules.

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While Novotny’s role in developing modern separation tools for the benefit of other scientists has been substantial, he is known to go beyond the development of methodologies and instrumentation. For example, he has made visible contributions to the biochemistry of olfaction, glycobiochemistry, and biochemistry of the oxidative stress. He has been responsible for the first definitive pheromones ever identified in a mammal (house mouse). At the interface of analytical and physical chemistry, he has contributed to a better understanding of the processes associated with separations. Novotny is the author of over 350 journal articles, reviews, books and patents. Novotny’s international reputation in analytical chemistry and separation science is reflected in more than 27 awards, medals and other distinctions. These include three national American Chemical Society awards (Chromatography, 1986; Chemical Instrumentation, 1988; Separation Science and Technology, 1992), an Eastern Analytical Symposium Award (1988) and the 1992 Anachem Award. He was named the 1994 Scientist of the Year by R&D Magazine. He is the recipient of the 2000 Pittsburgh Analytical Chemistry Award. Overseas, M.V. Novotny was recognized by two honorary doctorates (Uppsala University, 1991; Masaryk University, Czechoslovakia, 1992), and the M.J.E. Golay Medal (1991). He was also honored by the Czech Academy (J.E. Purkynje Medal), the Russian Academy (M.S. Tswett Memorial Medal), the Royal Society of Chemistry of Great Britain (Theophilus Redwood Lectureship), and the A.J.P. Martin Gold Medal, from the Chromatographic Society. In 1999, he was elected as a foreign member of the Royal Society of Sciences (Sweden). At Indiana University, M.V. Novotny has been the mentor to numerous graduate students and postdoctorals who have later assumed leadership in separation science.

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In 1997, he was awarded the Distinguished Teaching and Mentoring Award of the University Graduate School. In 1999, he received the College of Arts and Sciences Distinguished Faculty Award. See Chapter 5B, a, b, d, f, h, l, o, p, r, s

48.I. CHROMATOGRAPHY, A JOURNEY FROM CENTRAL EUROPE TO AMERICA Milos V. Novotny Department of Chemistry, Indiana University, 800 E. Kirkwood Ave., Bloomington, IN 47405–7102, USA

Over the course of my life and scientific career, I have seen many changes and had to make many adjustments. Most of these, in retrospect, I do not regret. During my formative teenage years, I had two main goals: to figure out how the things of nature work, and to see the world, including even some of its most distant parts. Somewhat naively, I assumed that the first goal would be easy to reach, little knowing how hard and tedious the pursuit of science can be – a task requiring, as they say, “some inspiration, and lots of perspiration.” But, today, having experienced the joys of scientific discovery, I would not change my job for any other. My second desire has also persisted to this very day. Although growing up in postwar Czechoslovakia taught me quickly the hard reality of national borders, my perspective broadened after I finished my studies and moved elsewhere. Perhaps, my first significant lesson was that education means freedom – both spiritual liberty and the ability to move around freely. In the end, my fondness for travelling interferes little with my scientific interests; in fact, science still brings me to some very distant corners of our planet. Adding to my ‘good, old-fashioned’ European basic education, my parents provided me with an invaluable background and a wealth of ideas. My father, a self-taught botanist whose dreams of advanced studies in plant sciences, were interrupted by the Germans closing of the Czech universities during the war, taught me many valuable early lessons in life sciences. His spacious library, which included a few chemistry books from his deceased uncle, a chemical engineer, sparked my early interest in chemistry and biology. My mother taught me to speak my conscience whenever necessary. In communist-dominated Czechoslovakia, this often meant walking a fine line between the ‘comfortable’ and the outright dangerous. While I survived there without ever being labeled as a rebel or an anti-establishment person, I draw from this early experience to this day. I still believe a degree of ‘stubbornness’ (in my case, presumably, a combination of the ideas instilled in me through my parents and the genes of tenacious, survival-driven Czech ancestors) is often necessary in fostering the less conventional ideas in science. Through my ‘gymnasium’ (high school) years, I excelled in science and also developed affection for other fields, such as history, geography, and languages. But I was hardly a bookworm and I even played a musical instrument then. However, when I entered the University of Brno, I started to view my university studies as a more serious business and developed more concerted efforts to study what I considered to

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be important. Initially, I had believed that my career objectives would best be met through becoming a medical scientist. After being denied admission to a medical school for political reasons, I chose chemistry (which was to coincide soon with my first encounters with chromatography) at the University of Brno. As a prelude to my planned graduate studies in biochemistry, I chose an undergraduate project in that area; this was around 1960. Most certainly, chromatography was already a key methodology in biochemical studies, albeit in very different ways than we practice it today. I can easily recall a basement room at the Institute of Biochemistry, in which were kept fish tanks (chromatographic chambers) filled with smelly solvents for the benefits of paper chromatography. My undergraduate project was to speed up the very slow separations of certain plant alkaloids through the use of the then new technique, thin-layer chromatography (TLC). During the late 1950s, Professor Egon Stahl in Saarbru¨cken, West Germany, had demonstrated extensively the advantages of TLC over paper chromatography. Yet, TLC was still simple and inexpensive. After reading the key articles by Stahl, I rolled up my sleeves and started from scratch: preparing silica gel from water glass, grinding the gels, sieving adsorbents, testing their activities, pouring the adsorbent slurries on glass plates, impregnating the layers, running the standard and real mixtures in different solvent systems, etc. I do not know to this date whether it was chromatography per se, or the possibility of doing something ‘new’ and, at any rate, so much more interesting than the standard laboratory experiments, which caused me to devote a disproportionate part of my time to undergraduate research. Admittedly, my physics classes suffered a bit from this activity, but I was even willing to forfeit my weekend tennis games and other extracurricular activities to my research. Since no one else practiced TLC at the university at that time, in a year or so I became something of a local TLC guru. Working between my undergraduate classes, I recall trying to solve separation problems for others: nucleotides and nucleosides on the modified cellulose layers, and a less successful project on separating azodye-labeled proteins. I happened to use chromatography several times during my graduate research; I even perhaps tried to force ‘chromatographic solutions’ on several problems where others would try these techniques only as the last resort. By then, I dealt with small molecules as well as proteins and felt quite comfortable with diverse separation methodologies. In retrospect, these years initiated my analytical orientation – in my view, this blended well with the primary objectives of biochemical research, as evidenced, for example, by a long succession of world-class research activities in biochemical separations at Uppsala University in Sweden. I still owe considerable gratitude to my research advisor, Dr. Ladislav Skursky, who allowed me to do research in my own way. While my thesis centered around the biosynthesis of alkaloids, I considered my acquired knowledge of chromatography to be my strongest selling point. To the dismay of my biochemistry teachers, I accepted a postgraduate fellowship in chromatography at the Institute of Analytical Chemistry (Czechoslovak Academy of Sciences). This was my genuine entrance into the field of chromatography, an important decision that I do not regret. I had several reasons for the decision. The then available jobs in biochemistry were seldom research-oriented and clearly subservient to clinical diagnosis and agriculture. With my increasing aspirations to do ‘big science’ one day, that did not seem the way to go. My desire to learn more of the fundamentals of

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separation science was also a lucky coincidence. And, furthermore, I did not have to leave my hometown; I merely relocated to a lab across the street. During the 1960s, the Czechoslovak Academy of Sciences was widely considered to be a step ahead of the Czech universities and other research institutions – much less ideologically rigid and more free-spirited, better equipped with scientific instruments, and, importantly, maintaining connections with the outside world. During the previous decade, the Institute’s director, Dr. Jaroslav Jana´k (a pioneer of gas–solid chromatography), had managed to gather an impressive team of relatively young and enthusiastic scientists. The atmosphere was generally conducive to learning among the group of people who ‘played hard and worked hard.’ This turned out to be the most intensive learning period of my life. Chromatography was now a far bigger area than what I had known during my university studies. I rapidly became familiar with the new jargon and the theoretical aspects of the field, and enlarged the number of my ‘scientific heroes,’ namely J. Calvin Giddings, John Knox, Istva´n Hala´sz, Victor Pretorius, and others. I was a bit frustrated by the lack of available journals, but this was amply compensated for by much collegial advice and help from my senior co-workers. Among these, I owe a special gratitude (in memoriam) to Dr. Josef Nova´k, my unofficial mentor in the Institute. I learned a lot from him. Dr. Jana´k had correctly predicted that high-pressure-driven separations were one of the waves of the future. We worked on some aspects of high-pressure gas chromatography (GC), only to be beaten in 1967 by the elegant studies by Sie and Rijnders in The Netherlands, in what became known as the supercritical-fluid chromatography (SFC). While our modest work in the area was not to be my last word on SFC, my immediate attention became directed to capillary GC. In collaboration with Dr. Karel Tesa˘rik, we were lucky to solve a long-standing problem of poor surface wettability in glass capillary columns. Although the details of our work were published during 1968, the review of activities in this emerging area of importance (strengthened further by additional work during my stay in Sweden) was published at a later date [1]. Later in Sweden, I also learned for the first time about the parallel efforts by a Swiss chemist, Dr. Kurt Grob. My farewell to Czechoslovakia in 1968 preceded by several months all the important events of that very eventful year, including the invasion of my country by the Russian troops. Through my work at the Royal Karolinska Institute in Sweden and my collaboration with a Swedish instrument company, LKB Produkter, I was introduced to the field of mass spectrometry. After all, I went to Sweden to learn the techniques of GC=MS, which Dr. Jana´k had encouraged me to do for the benefits of a future research in Brno. The political events after the August of 1968 changed our plans altogether. My research experience in Sweden was hardly a one-way street: while I was learning GC=MS and became aware of certain directions of biomedical research at Karolinska Institute, my co-workers were appreciative of my chromatography experience. Surprisingly, capillary GC was then little known in Sweden and my previous work on glass capillary columns received considerable attention. The use of glass capillary columns in steroid analysis was constantly on my mind, but I managed to use them instead in separation and identification of the complex mixture of tobacco smoke. Besides doing GC=MS studies, I found two enthusiastic co-workers at the University of Stockholm: Dr. Keith Bartle and an undergraduate, Lars Blomberg, who both helped me to advance some of my

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earlier ideas in technology of glass capillary columns (also mentioned in Ref. [1]). We managed to publish together a couple of nice papers, but perhaps more importantly, both individuals have continued to be active in separation science. I have had the distinct pleasure of meeting them repeatedly at various symposia since that time. After receiving his doctorate at Stockholm, Blomberg became a Swedish academician, while Keith Bartle assumed a faculty position at the University of Leeds. Keith and I got together again after I got my faculty position at Indiana University, and we have co-authored additional papers and a book. Keith’s residence in Bloomington overlapped with that of my first graduate student, Milton Lee, and they formed a long-lasting friendship. In turn, Blomberg’s former student, Dr. Karin Markides, later became Milton Lee’s associate. As the late Professor Zhukhovitskii once remarked, “chromatography is an excellent means of separating compounds and uniting people!” Perhaps my most notable scientific contribution dating from the period I spent in Sweden was the coupling of capillary columns to a mass spectrometer via a modified jet separator [2]. While it was not the first time that such connection was made (Gohlke certainly did it first), it was the first convincing report that it is feasible to use capillary columns in GC=MS without a decrease in separation efficiency – in fact, even faster due to a shift of the optimum velocity at a reduced outlet pressure. I eagerly sent the report to Analytical Chemistry, but it was promptly returned without a review because it was deemed by the Editor as uninteresting to a wider readership. I made many long-term friends in Sweden and my fondness for that country only continues to grow with each return visit. While at the Karolinska Institute, I met for the first time some well-known chromatographers visiting from abroad: Charles Brooks, Gerhard Schomburg, the Hornings, and Chuck Sweeley. In fact, it was Chuck Sweeley who recommended me to Al Zlatkis for another postdoctoral stint at the University of Houston. To spend some time in America had been my life dream; I accepted at once Al Zlatkis’s job offer. On August 12, 1969, I viewed with mixed emotions the New York City skyline: Is America the place I wish to live? A few days later, I reported at the University of Houston, starting another chapter in my scientific career. Association with Al Zlatkis was enjoyable; I appreciated the combination of his casual and friendly manners and his contagious enthusiasm for chromatography and all it can do. He gave me considerable freedom to continue my work in capillary GC, but we did many other things together as well. This included my brief return to SFC while helping to supervise graduate research of Wolfgang Bertsch, now a chemistry professor at the University of Alabama. A real treat to me in Houston were the frequent visitors with big names in chromatography: Archer Martin, Istva´n Hala´sz, Ernst Bayer, Howard Purnell, Emanuel Gil-Av, and Leslie Ettre, just to mention a few. And, yes, Jim Lovelock, some of whose GC ionization detector projects I inherited. With no documentation available, I could not make heads and tails of most of his instrumental setups. Al Zlatkis also forgot what they had once been for, so we moved on with other things. Within less than a year of my arrival in Houston, I was standing in front of a large audience in Miami Beach, delivering my first international symposium lecture. Shortly after my arrival, Al Zlatkis had politely but firmly imposed on me a deadline, insisting that “we must have something good” for the next symposium on Advances in

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Chromatography. So I rose to the occasion and finally ‘made’ capillary GC work with complex steroid mixtures. A lesson to graduate students? To some degree, deadlines help you progress and much can be done if you work hard and plan your research activities carefully. I owe to Al Zlatkis considerable gratitude for many other things as well. In Houston, I learned to teach and write grant proposals. Through his excellent chromatography symposia series, I became gradually connected to the ‘world of chromatography’ and the people who were a part of it. Dr. Zlatkis also encouraged me to seek a university position, something that was not my original intention. He advised me on the interview trips and became very pleased when I accepted the offer from Indiana University. My early research at Indiana University was mostly on capillary GC and HPLC. I was hired primarily as an analytical faculty member, but I was told that my biochemical orientation had earned me some points in the interview process as well. Earning tenure at a major university is widely viewed as a major struggle, but I personally never felt that way. It is clear that you need to start a serious research program right away, get the necessary equipment, and attract good students immediately – your ‘time-clock’ has already been set. And the research area has to be your own. I was lucky in several respects. Serendipitously, capillary GC was finally getting its due attention at the time, my postdoctoral work was widely known in the chromatography community, and, importantly, Al Zlatkis never made major claims to certain research directions that I had imported to his laboratory. To “keep up the good work” was all I had to do. In entering HPLC, I chose to work on the chemically bonded stationary phases; this work promptly earned me a lecture invitation to the first HPLC international conference in Interlaken, Switzerland. The bonded-phase column technology using specialty silane reagents was just a step from my earlier efforts to produce tailor-made surfaces in capillary GC. We emphasized HPLC selective phases, which, I realize in retrospect, was not the best way to go. As Csaba Horva´th, John Knox, Go¨ran Schill and others demonstrated years later, the principle of the reversed-phase HPLC, combined with ion pairing and other mobile-phase effects, has far more to offer. Good graduate students and postdoctoral associates are the bloodline of any successful academic research. Needless to say, having Milton Lee as my first graduate student set my laboratory right on target. As soon as I received my first major grant, I invited Keith Bartle to come to Bloomington as a visiting scientist. To this day, the succession of numerous other talented graduate students and postdoctorals continues unabated. The students of the 1970s, including such excellent individuals as Jim Jorgenson, Milton Lee, Mike McConnell, Sharon Smith, and Jerry Rhodes, received most of their primary ‘intellectual nourishment’ from capillary GC and HPLC. In my early years at Indiana University, I was able to work in the laboratory alongside my students. One of my most memorable projects started when my faculty colleague John Hayes brought in a group of NASA people, who were interested in a chromatographic column to be sent to the planet Mars as a part of the miniaturized GC=MS. I accepted the analytical challenges of the Viking 1975 chromatographic column and started a truly international project [3]; we were aided by ideas from Italy (Fabrizio Bruner) and England (Peter Simmonds, a former Lovelock=Zlatkis student), and we were going to export GC to Mars!

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As my analytical orientation continued to develop, I started to pay more attention to certain instrumental aspects of chromatography–detectors and ancillary techniques. This orientation became particularly pertinent due to my interest in miniaturization (microcolumn LC and SFC). Not only did columns with drastically reduced dimensions demand the best of the then available detection technologies, but here was the chance to develop unconventional detection techniques as well. My understanding of the impact of modern chromatographic instrumentation was greatly supplemented through my consulting contracts with instrument companies: first with Varian, and later with Perkin-Elmer, a mutually rewarding experience that lasted a decade. At Perkin-Elmer, I fondly remember my many enlightening discussions with John Atwood, Leslie Ettre, the late Marcel Golay, John Purcell, and Walter Slavin. In accordance with this developing research emphasis, my analytical chemistry students from the late 1970s on were more instrumentally oriented than before. My preoccupation with small separation columns started with the work of Dr. Takao Tsuda, a postdoctoral associate. I had long been stimulated in my thoughts by the Giddings’ papers from the early 1960s, which pointed at the efficiency discrepancies between GC and LC. Could some small column designs with unique geometries help us overcome the slow diffusion in liquids and the heat-of-friction problems encountered in the conventional HPLC? We also predicted several other advantages of microcolumns as well: (a) unique detection opportunities, perhaps best exemplified in LC=MS; (b) the possibility of using ‘exotic’ solvents at microliter-per-minute flows; and (c) negligible solvent disposal problems. Takao Tsuda (now a full professor at Nagoya Institute of Technology in Japan) is one of the best experimentalists that I have ever known. In my laboratory, he was the right man at the right time (1976) to initiate the technically demanding projects in capillary LC. His dedication and innovative spirit soon paid off. We first produced glass, packed capillaries while using our favorite glass-drawing machine, and miniaturized a UV detector and sample introduction techniques. At a typical flow-rate of one microliter per minute, these first microcolumns were producing astounding efficiencies close to 100,000 theoretical plates, albeit at the analysis times of several hours. While this was encouraging, it took us a decade or so to do significantly better. I reported Dr. Tsuda’s experiments for the first time at the 1977 Pittsburgh Conference, and a few months later to a purely chromatographic audience at the 1977 Advances in Chromatography meeting in Amsterdam. To my great surprise, most leaders in HPLC appeared incensed: why bother when HPLC works so well? Our first paper on microcolumn LC appeared in February 1978 [4]. For some, microcolumn LC was the ugly duckling of the field for at least several years. Supporters were few (most notably, Cal Giddings and John Knox), but eventually additional laboratories started to pursue miniaturization on their own. Interestingly, more appreciative audiences were found in the field of capillary GC, which meanwhile experienced a boost due to the development of fused silica columns. The small-diameter, slurry-packed capillary columns reported in 1988 by Dr. Karlsson and I [5], were a significant improvement in terms of efficiency. Interestingly, the ‘final word’ in terms of column efficiency may well come from the work in ultrahigh-pressure microcolumn LC initiated recently by the Jorgenson group. The early years of microcolumn LC overlapped significantly with the development

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of additional microcolumn techniques: capillary SFC and capillary electrophoresis. My second return to SFC was not accidental. During my early work in Czechoslovakia, I had already thought that generating large pressures together with minimum volumes would be technologically easy, with the diffusion-controlled sorption=desorption kinetics facilitated by small-bore open tubular columns. But how would one avoid an instantaneous stripping of the very small amount of a stationary phase by a supercritical fluid? During my early time at Indiana University, I even considered starting a project in SFC, but decided not to be ‘confrontational’ in my pre-tenure years; the success of HPLC largely overshadowed the early gains of SFC and no one in the chromatography community seemed to care. The appropriate time came during the summer of 1979 when Milton Lee came back to Bloomington to spend a period of time with us. At that time, chemically bonded polysiloxane phases were being developed for the benefit of capillary GC and we had just learned, through our involvement with microcolumn LC, how to deal with the low-flow systems and miniaturized detection. Through a collaboration between the Lee group at Brigham Young University and us, the first report on capillary SFC was prepared for publication [6]. At the time, a renewed interest in SFC was also emerging from other laboratories. Milton Lee later became involved with the first commercialization efforts in the area. The ‘SFC era’ in our laboratory had significant educational attribute. It offered the opportunity for the development of unconventional detection techniques (flame and plasma-based detectors and a combination to FTIR spectroscopy) and brought us, full-speed, to more instrumentation. Due to the complex behavior of supercritical fluids, we became also interested in the physicochemical aspects of SFC and published several papers in the area. This brought me as close as I probably will ever be to physical chemistry. I remember several excellent students and associates working on SFC throughout the 1980s: Paul David, Debbie Luffer, Susan Olesik, Michal Roth, and Steve Springston. SFC is clearly a niche technique that does not cover the molecular mass range as HPLC does, and thus, with my increasing interests in biomolecules, we eventually lost interest in SFC. The present activities in my separation science career are focused almost exclusively on electromigration techniques. Capillary electrophoresis (CE), in my mind and now most certainly in the minds of numerous symposia organizers, has become increasingly interlinked with the other microcolumn techniques. Yet, not so long ago, the fields of chromatography and electrophoresis were marching to their own distinctly different tunes! The now classic Jorgenson-Lukacs paper from 1981 has done much to change that situation and has brought us all a major step closer to the point of ‘unification’ in separation science, a situation long viewed as desirable by the late Cal Giddings. I still recall quite vividly viewing one of the first high-performance electropherograms, shown to me by my former student at the 1980 Gordon Conference on Analytical Chemistry. I knew at once that Jim Jorgenson was onto something big! in retrospect, I feel quite lucky that a research finding of a former student later enriched my own research program to such a degree. While the so-called high-performance CE had its additional instrumentalized prelude in isotachophoresis and Stellan Hjerte´n’s rotating tube experiment, Jorgenson’s work unleashed a number of additional investigations in which not only separation scientists, but also electrochemists and spectroscopists started to participate. The microcolumn

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sample manipulation and detection techniques were now ready for implementation and further development in CE. This was quite evident during the 1982 US–Japanese Joint Seminar on Microcolumn Separations in Hawaii that Professor Daido Ishii and I organized [7]. In an atmosphere conducive to exploring new, exciting avenues, we discussed emerging trends (microcolumn LC and SFC, as well as CE), laser-based detection techniques, LC–MS, and microelectrodes. As one of the meeting’s highlights, the late Willy Simon discussed the potential of microelectrochemical detection and demonstrated some impressive results of his student, Andreas Manz. Dr. Manz later became a pioneer of the CE on a microchip. The Honolulu seminar brought the then practitioners and potential users of microcolumn techniques together and started new friendships and collaborations. Daido Ishii, a pioneer in microcolumn LC, has provided me with several capable postdoctoral associates over the years. Microcolumn LC and CE, with their unique instrumental aspects and biochemical applications, were high on our bioanalytical agenda for the remaining years of 1980s and throughout the 1990s. We made contributions to column design, fundamentals of biopolymer electromigration, pulsed-field CE, affinity CE, laser-induced fluorescence detection, and capillary electrochromatography. There were several outstanding students and postdoctorals in the area, most notably Kelly Cobb, Vladislav Dolnik, Jinping Liu, Anders Palm, Jan Sudor, Helena Soini, and Morgan Stefansson. We have been engaged in the separations of peptides, proteins, nucleotides, large DNA, small molecules of pharmaceutical interest, and, perhaps most notably, carbohydrates. The current success of microseparation techniques has much to do with their unique capability to address the important problems of modern biology and biomedical research. This is primarily why we can predict many important developments in the years to come. Over the years, only a few of my separation science friends have been aware of my two additional ‘scientific hobbies,’ mammalian pheromones and glycobiology. While both areas have their deep roots in my biochemical background, our significant progress in these fields would not have been possible without their close connection to ‘microcolumn technologies,’ particularly capillary GC=MS and CE. I hope that this will continue to provide a significant rationale for the activity that I have enjoyed most, the future training of additional scientists. I feel fortunate in having participated in several interesting stages of the development of modern separation science. Since 1960, when I did my first chromatographic experiment, much has happened in the field. I also feel fortunate in knowing the leaders who have shaped it. While the methods became gradually more and more instrumentalized, we have spanned a considerable range of molecular masses of solutes: from small molecules to the ‘giants’, such as chromosomal DNA and various molecular aggregates. As a passive participant of the 1968 conference in Copenhagen, I heard Jim Lovelock using the astonishing term femto in relation to minimum detectable quantities. Thanks to laser-induced fluorescence and microelectrodes, going through atto, we are down to the impressive zepto. In terms of ‘dilute solutions’ to analyze, p.p.m. and p.p.b. ranges do not appear to be any longer a significant issue, as the combined efforts of preconcentration, advanced column technologies and ultrasensitive detection seem to be setting us to the p.p.t. range. Enormous strides have been made in the component resolution, virtually in all forms of chromatography and electrophoresis. Importantly,

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some techniques have often been made an integral part of special-purpose analyzers (e.g., sequencing machines or ‘sniffers’) so that the people operating them no longer think of them in terms of chromatography or electrophoresis. Is this a compliment or an insult to separation science? What will be the next frontier in separation science at the beginning of the new millennium? Scientists seem no better than meteorologists in forecasting long-term trends. The self-propelling nature of our field seems to favor future developments, and there is much unfinished business in separating very large molecules. The late Cal Giddings showed us, through his dedicated, almost heroic efforts in developing field-flow fractionation methodologies, what a difficult and complicated task it is. But, when there is a need, we must develop the means. Over the years, we have become quite capable of manipulating the separatory channels pneumatically and, more recently, electrically. And this has been happening at increasingly smaller scale (small capillaries and microchips). These capabilities, when combined with the continuing advances in sensing devices, provide numerous incentives toward solving some of the most interesting and challenging tasks of modern biology and materials science. I just wish to be active for some time into the future to enjoy these directions.

References 1. 2. 3. 4. 5. 6. 7.

M. Novotny and A. Zlatkis, Glass capillary columns and their significance in biochemical research, Chromatogr. Rev., 14 (1971) 1–44. M. Novotny, Coupling of open tubular columns with a mass spectrometer through the jet-type molecule separator, Chromatographia, 2 (1969) 350–353. M. Novotny, J.M. Hayes, F. Bruner, and P.G. Simmonds, Gas-chromatographic column for the Viking 1975 molecular analysis experiment, Science, 189 (1975) 215–216. T. Tsuda and M. Novotny, Packed microcapillary columns in high-performance liquid chromatography, Anal. Chem., 50 (1978) 271–275. K.-E. Karlsson and M. Novotny, Separation efficiency of slurry-packed LC microcolumns with very small inner diameters, Anal. Chem., 60 (1988) 1662–1665. M. Novotny, S.R. Springston, P.A. Peaden, J.C. Fjelsted and M.L. Lee, Capillary supercritical-fluid chromatography, Anal. Chem., 53 (1981) 407A–411A. M. Novotny, The U.S.–Japan joint seminar on microcolumn separation methods and their ancillary techniques, Anal. Chem., 55 (1983) 1308A–1310A.

D.49. Janusz Pawliszyn Janusz Pawliszyn was born in Gdansk, Poland on May 16, 1954. He attended secondary school in Gdynia, Poland, received his B.Sc. in Chemical Engineering from the Technical University of Gdansk in 1977 and M.Sc. in 1978. His doctoral degree was obtained in 1982 from Southern Illinois University, Carbondale, Illinois. He has moved through the ranks of academia starting at Utah State University from Assistant Professor in 1984 to full Professor at the University of Waterloo, Ontario, Canada in 1997. The primary focus of J. Pawliszyn’s research program is the design of highly automated and integrated instrumentation for the isolation of organic materials from

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complex matrices and the subsequent separation, identification and determination of these species. J. Pawliszyn is exploring application of the mathematical modeling and data processing techniques to enhance performance of chromatographic separations and detection. The major area of his interest involves the development and application of imaging detection techniques for microcolumn chromatography and capillary electrophoresis. He is an author of over 150 scientific publications and recently published a book on “Solid Phase Microextraction”. He is a member of the Editorial Board of Analytical Chemistry, Journal of Microcolumn Separations, Analyst, Canadian Journal of Chemistry, Field Analytical Chemistry and Technology. He received the 1995 McBryde Medal, the 1996 Tswett Medal, the 1996 Hyphenated Techniques in Chromatography Award, the 1996 Caledon Award and the Jubilee Medal 1998 from the Chromatographic Society, U.K. He presently holds the Supelco–Varian– NSERC Industrial Research Chair in New Analytical Methods and Technologies. Currently his research is focusing on elimination of organic solvents from the sample preparation step to facilitate on-site monitoring and analysis. Several alternative techniques to solvent extraction are under investigation including use of supercritical fluids, membranes and coated fibers. See Chapter 5B, p, k, n

49.I. SOLID PHASE MICROEXTRACTION Janusz Pawliszyn Department of Chemistry, University of Waterloo, Waterloo, ON N2L 3G1, Canada

Solid phase microextraction (SPME) was developed to address the need for fast, solvent-free and field compatible sample preparation technologies facilitating convenient introduction of the extraction components to a chromatographic instrument [1,2]. The basic principle of this approach is to use a small amount of the extracting phase, usually less than 1 µl. The sample volume can be very large, when the investigated system, for example air in a room or lake water, is sampled directly. The extracting phase can be either a high molecular weight polymeric liquid, similar in nature to stationary phases in chromatography, or it can be a solid sorbent, typically of a high porosity to increase the surface area available for adsorption. To date the most practical geometric configuration of SPME utilizes a small fused silica fiber, usually coated with a polymeric phase. The fiber is mounted for protection in a syringe-like device. The analytes are absorbed or adsorbed by the fiber phase (depending on the nature of the coating) until an equilibrium is reached in the system. The amount of an analyte extracted by the coating at equilibrium is determined by the magnitude of the partition coefficient (distribution ratio) of the analyte between the sample matrix and the coating material.

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In SPME, analytes typically are not extracted quantitatively from the matrix. However, equilibrium methods are more selective because they take full advantage of the differences in extracting-phase=matrix distribution constants to separate target analytes from interferences. Exhaustive extraction can be achieved in SPME when the distribution constants are large enough and sample volume does not exceed a few ml. This can be accomplished for most compounds by the application of an internally cooled fiber or on-fiber derivatization [3,4]. In exhaustive extraction, selectivity is sacrificed to obtain quantitative transfer of target analytes into the extracting phase. One advantage of this approach is that, in principle, it does not require calibration, since all the analytes of interest are transferred to the extracting phase. On the other hand, the equilibrium approach usually requires calibration when dealing with complex matrices. This is accomplished by using surrogates or standard addition to quantify the analytes, and compensate for matrix-to-matrix variations and their effect on distribution constants. Since equilibrium rather than exhaustive extraction occurs in the microextraction methods, SPME is ideal for field monitoring. It is unnecessary to measure the volume of the extracted sample and therefore the SPME device can be exposed directly to the investigated system for quantitation of target analytes. In addition, the extracted analytes are introduced to the analytical instrument by simply placing the fiber in the desorption unit (Figs. 1B,C). This convenient, solvent free process facilitate sharp injection bands and rapid separations [5,6]. These features of SPME result in integration of the first steps in the analytical process: sampling, sample preparation, and introduction of the extracted mixture to the analytical instrument. The equilibrium nature of the technique also facilitates speciation in natural systems since the presence of a minute fiber, which removes small amounts of target analytes, is not likely to disturb the system. Because of the small size, coated fibers can be used to extract analytes from very small samples. For example, SPME has been used to probe for substances emitted by a single flower bulb during its lifespan. Fig. 1A illustrates the commercial SPME device, manufactured by Supelco, Inc. (Bellefonte, PA). The fiber, glued into a piece of stainless steel tubing, is mounted in a special holder. The holder is equipped with an adjustable depth gauge, which makes it possible to control repeatably how far the needle of the device is allowed to penetrate the sample container (if any) or the injector. This is important, as the fiber can be easily broken when it hits an obstacle. The movement of the plunger is limited by a small screw moving in the z-shaped slot of the device. For protection during storage or septum piercing, the fiber is withdrawn into the needle of the device, with the screw in the uppermost position. During extraction or desorption, the fiber is exposed by depressing the plunger, which can be locked in the lowered (middle) position by turning it clockwise (the position depicted in Fig. 1A). The plunger is moved to its lower most position only for replacement of the fiber assembly. Each type of fiber has a hub of a different color. The hub-viewing enables a quick check of the type of fiber mounted in the device. If the sample is placed in a vial, the septum of the vial is first pierced with the needle (with the fiber in the retracted position), and the plunger is lowered, which exposes the fiber to the sample. The analytes are allowed to partition into the coating for a predetermined time, and the fiber is then retracted back into the needle. The device is

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Fig 1. (A) Design of the commercial SPME device. (B) SPME=HPLC interface: (a) stainless steel (SS) 1=1600 tee, (b) 1=1600 SS tubing, (c) 1=1600 polyetheretherketone (PEEK) tubing (0.0200 i.d.), (d) two-piece finger-tight PEEK union, (e) PEEK tubing (0.00500 (i.d.) with a one-piece PEEK union. (C) SPME=GC interface.

next transferred to the analytical instrument of choice. When gas chromatography (GC) is used for analyte separation and quantitation, the fiber is inserted into a hot injector, where thermal desorption of the trapped analytes takes place (Fig. 1C). The process can be automated by using an appropriately modified syringe autosampler. For HPLC applications, a simple interface mounted in a place of the injection loop can be used to re-extract analytes into the desorption solvent (Fig. 1B). The SPME device is capable for both spot and time averaged sampling. As described

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Fig. 2. Modes of SPME operation: direct extraction (a), headspace extraction (b) and membrane-protected SPME (c).

above, for spot sampling, the fiber is exposed to a sample matrix until the partitioning equilibrium is reached between sample matrix and the coating material. In the time average approach, on the other hand, the fiber remains in the needle during the exposure of the SPME device to the sample. The coating works as a trap for analytes that diffuse into the needle, resulting in integral of concentration over time measurement. SPME sampling can be performed in three basic modes: direct extraction, headspace extraction, and extraction with membrane protection. Fig. 2 illustrates the differences between these modes. In direct extraction mode (Fig. 2a), the coated fiber is inserted into the sample and the analytes are transported directly from the sample matrix to the extracting phase. To facilitate rapid extraction, some level of agitation is required to transport the analytes from the bulk of the sample to the vicinity of the fiber. For gaseous samples, natural flow of air (e.g., convection) is frequently sufficient to facilitate rapid equilibration for volatile analytes. However, for aqueous matrices, more efficient agitation techniques, such as fast sample flow, rapid fiber or vial movement, stirring or sonication are required to reduce the effect of ‘depletion zone’ produced close to the fiber as a result of slow diffusional analyte transport through the stationary layer of liquid surrounding the fiber. In the headspace mode (Fig. 2b), the analytes are extracted from the gas phase equilibrated with the sample. The primary reason for this modification is to protect the fiber from adverse effects caused by non-volatile, high molecular weight substances present in the sample matrix (e.g., humic acids or proteins). The headspace mode also allows matrix modifications, including pH adjustment, without affecting the fiber. In a system consisting of a liquid sample and its headspace, the amount of an analyte extracted by the fiber coating does not depend on the location of the fiber, in the liquid phase or in the gas phase, therefore the sensitivity of headspace sampling is the same as the sensitivity of direct sampling as long as the volumes of the two phases are the same in both sampling modes. Even when no headspace is used in direct extraction, a significant sensitivity difference between direct and headspace sampling can occur only for very volatile analytes. However, the choice of sampling mode has a very

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Fig. 3. Professor Pawliszyn in the laboratory at the University of Waterloo.

significant impact on the extraction kinetics. When the fiber is in the headspace, the analytes are removed from the headspace first, followed by indirect extraction from the matrix. Therefore, volatile analytes are extracted faster than semivolatiles. Temperature has a significant effect on the kinetics of the process, since it determines the vapor pressure of the analytes. In general, the equilibration times for volatile compounds are shorter for headspace SPME extraction than for direct extraction under similar agitation conditions, because of the following three reasons: a substantial portion of the analytes is present in the headspace prior to the beginning of the extraction process, there is typically a large interface between the sample matrix and headspace, and the diffusion coefficients in the gas phase are typically higher by four orders of magnitude than in liquids. The concentration of semivolatile compounds in the gaseous phase at room temperature is small, headspace extraction rates for those compounds are substantially lower. They can be improved by using very efficient agitation or by increasing the extraction temperature. In the third mode (SPME extraction with membrane protection, Fig. 2c), the fiber is separated from the sample with a selective membrane, which lets the analytes through while blocking the interferences. The main purpose for the use of the membrane barrier is to protect the fiber against adverse effects caused by high-molecular weight compounds when very dirty samples are analysed. While extraction from headspace serves the same purpose, membrane protection enables the analysis of less volatile compounds. The extraction process is substantially slower than direct extraction because the analytes need to diffuse through the membrane before they can reach the coating. Use of thin membranes and increase in extraction temperature result in shorter extraction times.

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Acknowledgment Development work on Solid Phase Microextraction in my laboratory was supported by Natural Sciences and Engineering Council of Canada, Varian and Supelco.

References 1. 2. 3. 4. 5. 6. 7.

J. Pawliszyn, Solid Phase Microextraction. Theory and Practice, Wiley-VCH, New York, NY, 1997, 247 pp. J. Pawliszyn (Ed.), Application or Solid Phase Microextraction, RSC Chromatography Monographs, RSC, Cambridge, UK, 1999, 655 pp. C. Arthur and J. Pawliszyn, Solid phase microextraction with thermal desorption using fused silica optical fibers, Anal. Chem., 62 (1990) 2145. Z. Zhang and J. Pawliszyn, Quantitative extraction using an internally cooled solid phase microextraction device, Anal. Chem., 67 (1995) 34. P. Martos and J. Pawliszyn, Sampling and determination of formaldehyde using solid phase microextraction with on-fibre derivatization, Anal. Chem., 70 (1998) 2311. T. Gorecki and J. Pawliszyn, Sample introduction approaches for solid phase microextraction, Anal. Chem., 67 (1995) 3265. R. Eisert and J. Pawliszyn, New trends in solid phase microextraction, Crit. Rev. Anal. Chem., 27 (1997) 103.

D.50. William H. Pirkle and Christopher J. Welch William Howard Pirkle was born on May 2, 1934 in Shreveport, LA. His initial educational training was at Modesto Junior College in 1952 to 1953 and 1955 to 1956. He received his B.Sc. degree from the University of California, Berkeley, in 1959, a doctorate from the University of Rochester, in 1963; followed by an appointment as a Postdoctoral Research Associate under E.J. Corey at Harvard University, 1963 to 1964. His professional activities were as Laboratory Assistant at the University of California, Berkeley, 1956 to 1959; Assistant Professor at the University of Illinois (1964 to 1969), Associate (1969 to 1980) and Professor at the University of Illinois from 1980 to present. He was a visiting Professor at the University of Wisconsin, Madison in 1971. William Pirkle received pre and postdoctoral fellowships from the National Science Foundation (1961 to 1964), and the Alfred P. Sloan Fellowship in 1970 to 1973. He has been honored with numerous awards such as the A.J.P. Martin Medal by the Chromatography Society (1990), the Merit Award by the Chicago Chromatography Discussion Group (1991), the American Chemical Society National Award in Chromatography (1994), the Chirality Medal by the Swedish Academy of Pharmaceutical Sciences (1994), the Eastern Analytical Symposium Award for Achievements in Separation Science (1998), the Robert Boyle Medal for Analytical Chemistry, from the Royal Society

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of Chemistry (1998), the ISCO Award (1998), and the Dal Nogare Award awarded by the Chromatography Forum of Delaware Valley (March 2000). He is a member of the following Editorial Advisory Boards: The Journal of Liquid Chromatography, Chirality, Supramolecular Chemistry, and Enantiomer. He is in high demand as a speaker. Since 1980 he has made at least 15 invited presentations each year on chiral separations at universities, in the corporate sector, and at international symposia. He has received nine patents on chiral liquid membranes and chiral selectors for the separation of enantiomers. Dr. Pirkle has published 233 papers in referred journals. Christopher Welch was born on May 27, 1960 in Chicago and raised in the west central Illinois village of Smithfield. He is the second son of noted Illinois muralist and painter, Harold Kee Welch. He began his longstanding collaboration with Professor William Pirkle in 1981 while still an undergraduate at the University of Illinois. Following a few years of industrial research experience, Welch returned to the University of Illinois taking a Ph.D. with Dr. Pirkle in 1992. His research investigations involve the use of laboratory automation and robotics in process research, with special emphasis on chirality and stereochemistry. He is also involved in the development of automated robotic analyzers for use in planetary exploration. His notable research accomplishments include the invention of a number of useful reagents for improved immunodiagnostic assays (while at Abbott Laboratories) and the invention and commercialization of several useful chromatographic stationary phases (while at University of Illinois and Regis Technologies). He is the author of more than 50 scientific publications and 20 patents, Dr. Welch is also the American Editor in Chief of Enantiomer: A Journal of Stereochemistry. See Chapter 5B, a, q, r

50.I. CHIRAL HPLC AND ENANTIOSEPARATION OF PHARMACEUTICALS William H. Pirkle 1 and Christopher J. Welch 2 1

School of Chemical Sciences, University of Illinois, Urbana, IL 61801, USA 2 Merck and Co., Inc., Rahway, NJ 07065, USA

50.I.1. Introduction Tswett’s discovery of adsorption chromatography around the turn of the 20th century must surely rank as one of the most important contributions to chemistry for the subsequent years [1]. Hardly a laboratory exists today in which some form of chromatography is not employed. Early users of column-liquid chromatography

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employed readily available stationary phases, such as calcium carbonate, sugar, starch, paper, wool, silk, alumina and silica. In recent years, a number of materials have been developed as more selective adsorbents, and nowhere has the pace of discovery been greater than in the development of enantioselective adsorbents for the chromatographic separation of enantiomers.

50.I.2. Background Willsta¨tter proposed the idea that the two enantiomers of a racemic dye might be differentially adsorbed by a biopolymer, such as wool or silk in 1904 [2]. Shortly thereafter there followed several reports dealing with this phenomenon [3–7]; however it was not until 1938 that the first chromatographic separations of enantiomers were reported by two different groups, one reporting a partial separation of the enantiomers of a camphor derivative using a lactose stationary phase [8], and the other reporting a partial separation of the enantiomers of an organometallic chromium complex using a stationary phase consisting of optically active quartz powder [9]. Subsequently, Prelog and Wieland separated the enantiomers of Troeger’s base using starch as a stationary phase [10], and Senoh and co-worker’s reported the chromatographic separation of amino acid enantiomers using paper chromatography [11]. Pauling’s idea that polymerization in the presence of a ‘template’ molecule could lead to a stationary phase possessing some selectivity for the template molecule was demonstrated for enantiomers in 1952 [12,13], and the first ‘brush type’ bonded phase consisting of a chiral selector immobilized on a silica support was reported by Klemn and Reed in 1960 [14]. The first separation of amino acid enantiomers using ligand exchange chromatography was reported by Davankov and Rogozhin in 1971 [15], the same year that the first attempt at the development of a chiral stationary phase designed specifically for the enantioseparation of a particular analyte molecule (DOPA) was reported [16]. In 1974 Cram and co-workers reported the preparation of a chiral stationary phase covalently attached to a silica support which showed high enantioselectivity for amino acid enantiomers [17]. As an outgrowth of studies on NMR chiral solvating agents [18], Pirkle and co-workers reported the first in a long line of chiral stationary phases in 1979 [19]. The first commercial chiral stationary phase (CSP) was introduced in 1980, followed rapidly by the general acceptance of the technique and by the introduction of a number of commercial chiral stationary phases [20]. The explosion of research interest in chirotechnology (the science of making and measuring enantiopure materials), which began in the early 1980s and continues unabated to this day, can in some measure be attributed to the availability of tools for the rapid and reliable quantitation of enantiopurity. Where it once took days or weeks to obtain oftentimes questionable results, accurate and reliable measurements can now be obtained in a matter of minutes. In the decade leading up to the new millennium, the technique of chromatographic enantioseparation has become the method of choice for analytical determinations of enantiopurity. The availability of a variety of CSPs and the existence of automated chromatography equipment have made analytical determinations of enantiopurity almost routine. This method is very widely used, particularly in the

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Fig. 1. Example of how chiral HPLC is used in pharmaceutical analysis. Note the high degree of enantiopurity seen in the over-the-counter medication, Aleve.

pharmaceutical sector, where most new chiral drugs are now manufactured and sold in enantiopure form.

50.I.3. Pirkle-type CSPs Among the many types of chiral stationary phases (CSPs) which have been developed, the Pirkle-type, or brush-type CSPs, have proven to be among the most useful for many liquid chromatographic enantiomer separations. These CSPs consist of an enantioenriched, small molecule selector immobilized on an inert chromatographic support, typically silica gel. Separation is achieved when the two enantiomers of the analyte are differentially adsorbed by the CSP. A combination of simultaneous, geometrically constrained, intermolecular interactions utilizing forces, such as hydrogen bonding, π–π attraction, ionic interactions, and steric repulsion can result in diastereomeric adsorbates with differing free energies, the prerequisite for enantioseparation. The design and development of CSPs in the Pirkle laboratories stems from a research program aimed at developing a better understanding of molecular interactions. Pirkle has been a pioneer in demonstrating that chromatography can be an exquisitely sensitive tool for the study of molecular recognition. A rational model constructed from a constantly evolving understanding of molecular interactions guides his research. These models are constantly tested and refined, ultimately yielding tools of great utility and predictive power. Several reviews trace the historical development of CSPs in the Pirkle laboratories [21,22]. Here we will present only a brief overview, highlighting some of the more significant developments that show greatest promise for the future.

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Fig. 2. Enantiomer separation on CSPs is made possible by formation of transient diastereomeric adsorbates with differing free energies. In this illustration, the analyte enantiomers are depicted as right and left hands, and the CSP is depicted as immobilized right-handed gloves.

50.I.4. Advantages of Pirkle-type CSPs In general, Pirkle-type CSPs possess a number of advantages relative to other CSP types. Since the selector is a small molecule which is often completely synthetic, a structure which contains no labile or reactive components can usually be developed. In addition, the mode of attachment of the selector to the chromatographic support can be chosen for durability. Brush-type CSPs are typically covalently attached to the chromatographic support. Thus, most brush-type CSPs are chemically robust, and are generally quite long lived. Longevity is useful for an analytical CSP, but truly essential for a preparative CSP, where continuous operation for several years may be required. The chemical robustness of brush-type CSPs results in the ability of these CSPs to be used in a wide variety of mobile phases, which provides greater flexibility in method development, especially when poorly soluble analytes are being investigated. An additional advantage of brush-type CSPs stems from the fact that the selectors are typically small molecules with molecular weight less than 1000. Consequently, the selectors can be very densely arrayed on the chromatographic surface, resulting in a CSP which is highly resistant to sample overload and which has a very high preparative capacity. Finally, most synthetic CSPs are available in either enantiomeric form. Consequently, either elution order (C before , or before C) can be chosen. Elution of the minor enantiomer before the major is generally preferred in analysis, while elution of desired component before the undesired can greatly increase productivity in preparative HPLC.

50.I.5. Preparative enantioseparation by HPLC Recently, the use of chromatography for the preparative separation of pharmaceutical enantiomers has become increasingly popular. While often expensive, chiral HPLC of-

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Fig. 3. Gram scale preparative enantioseparation are routinely used in the pharmaceutical industry to provide enantiopure material for biological testing. Chromatogram courtesy of Les Dolak, Pharmacia and Upjohn.

fers the tremendous advantage of speed. Consequently, many pharmaceutical companies now use preparative chiral HPLC as an integral part of the drug development process. Very early in the drug discovery process, small amounts of a candidate drug can be separated for initial screening, animal testing or metabolism and toxicology studies). Once a candidate has been selected for larger scale development, a search for less expensive manufacturing routes can be initiated, but preparative HPLC can continue to supply material for advanced development, early clinical studies, or even for full scale manufacturing. For preparative separations on the multi-kg level, a highly enantioselective CSP is of great importance. All other things being equal, a highly enantioselective CSP will afford the least costly separation. There has been considerable recent progress in the design of highly enantioselective CSPs, with several reports of enantioselectivities in excess of 100. With this level of enantioselectivity relatively low-tech, inexpensive, separation processes such as batch adsorption can be utilized. However, there are a number of difficulties in developing new highly enantioselective CSPs for given separations problems.

50.I.6. Discovery of new CSPs The discovery of new CSPs in the Pirkle group has relied heavily upon the principle of reciprocity (Fig. 5). Simply stated, this principle suggests that if a ‘gloves’ CSP is capable of separating enantiomeric hands, then a ‘hands’ CSP should be able to separate enantiomeric gloves. Of course there are sometimes exceptions; for example, the manner in which the selector is tethered to the support can influence enantioselectivity. Nevertheless, this principle has proven to be quite useful in the discovery and development of CSPs. In practice, evaluation of the original anthryl carbinol CSP showed that a number of 3,5-dinitrobenzamide (DNB) derivatives of amino acids were well resolved. Subsequent

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Fig. 4. An example showing an easy preparative chiral HPLC separation. Factors which are important for convenient preparative enantioseparation include high enantioselectivity, good solubility of the analyte in the mobile phase, elution of the desired component before the undesired, and the ability of the product enantiopurity to be upgraded via crystallization.

Fig. 5. Illustration of the principle of reciprocity.

preparation and evaluation of DNB amino acid-based CSPs showed that in addition to separating the enantiomers of aryl carbinols, the enantiomers of analytes from a number of other compound classes were also separable. This gave rise to yet another generation of CSPs, each of which showed interesting separation properties and some of which inspired subsequent generations of new CSPs. This almost biblical history weaves a tangled web, which is chronicled elsewhere [21,22], but it can be summarized more or less as follows: in the beginning there was Pirkle, who begat the anthryl carbinol chiral shift reagent, which was immobilized to form the anthryl carbinol CSP, which begat the DNB amino acid CSPs, which were fruitful and did multiply. The DNB amino acid CSPs begat the hydantoin CSPs, N-arylamidoalkane CSPs, the phthalide CSPs and the N-aryl amino acid CSPs. And the N-aryl amino acid CSPs were fruitful and begat both the α-Burke and the β-GEM, which begat the immobilized naproxen CSPs, which begat the Whelko, the polyWhelko and the co-polyWhelko. And the Whelko was fruitful and did multiply, begetting the Turbo CSPs, the Snapper, the Pirkle 1-J and the Murray : : : etc.

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Fig. 6. Summary of ‘genealogy’ of some Pirkle CSPs.

The pace of discovery and development of new chromatographic phases depends upon the time required for synthesis and evaluation of new candidate materials. Historically, CSP development has been a labor intensive process in which a candidate CSP is designed, synthesized on the multigram scale, immobilized onto a support, packed into a column and evaluated chromatographically. Such an approach can take weeks, months, or even years, and is clearly too slow to develop rapid solutions for actual separation problems in the fast paced pharmaceutical industry. Several years ago we described the specific application of the principle of reciprocity to the design of a CSP for a particular analyte molecule [23,24]. In this approach, termed the immobilized guest method, a single enantiomer of the compound of interest is immobilized on a chromatographic support, which is then packed into a chromatography column. Small amounts of candidate selectors are then screened for enantioselective binding using conventional chromatography. Submilligram amounts of candidate selectors can readily be analyzed, and the material need not be absolutely pure. The most promising candidates are selected and synthesized in enantiopure form and used to prepare new CSPs which should be useful for separating the enantiomers of the compound of interest.

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In terms of development time, the immobilized guest method represents a great improvement on the traditional method used to develop new CSPs. Disadvantages of the method include the fact that gram scale amounts of the enantiopure drug are required for preparation of the immobilized guest CSP and the very serious drawback that screening results cannot be directly translated to chromatographic performance. For instance, screening of extensive racemate libraries can lead to the discovery of an ‘optimal’ selector, which when immobilized on a solid phase and packed into a column may show dismal performance. There are at least two ways in which this can happen, the first being that the ‘optimal’ selector is actually the best for enantioselective binding to the tethered guest, and may display little enantioselectivity for the free guest. The second situation occurs when selector immobilization destroys some key interactions required for molecular recognition. Recently, several groups have become interested in the use of combinatorial chemistry approaches for the rapid preparation and evaluation of new CSPs. Still and coworkers have developed an interesting method in which candidate selectors from combinatorial libraries are evaluated using analogs of the target enantiomers to which dye molecules have been conjugated [25]. In another approach, phage display technology, or phage planning has been used to aid in the discovery of new CSPs [26]. Apart from other drawbacks, both of these methods suffer from the same fundamental problem we see in the immobilized guest method: Because a tethered version of the analyte is used in the screening process, the ‘optimal’ selector which is discovered may actually work best for the tethered, rather than the free analyte enantiomers. The existence of this ‘tether effect’ means that screening results obtained using these methods cannot be confidently extrapolated to chromatographic performance.

50.I.7. A new approach to CSP development We recently reported a strategy for synthesis and screening for libraries of CSPs, which offers a tool for the rapid assessment of which CSP will work best for preparatively resolving the enantiomers of a given compound. The technology has two parts: (1) parallel synthesis of libraries of milligram quantities of diverse stationary phases on porous silica particles [27], and (2) screening the resulting libraries for chromatographic performance [28]. This method for synthesis and screening for CSP libraries has a number of advantages when compared to alternative methods used for developing new chromatographic stationary phases. For example, CSP library synthesis on milligram scale results in tremendous savings in materials and reagents. In addition, the parallel nature of the library synthesis means that tens or hundreds of new CSPs can be prepared in the time required for making one full sized CSP. Furthermore, the CSP library screening technique is simple, rapid, inexpensive, and can be automated. Another advantage is that after evaluation, the CSP libraries can be washed and reused. Finally, and most importantly, the compound mixture of interest can be directly used in the screening assay without the need for purification, derivatization, immobilization, or formation of conjugates. Since both the CSP and the analyte being evaluated are exactly the same as what will be used in the full scale chromatographic separation, a

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Fig. 7. Five amino acids were used to prepare a library of 50 dipeptide DNB CSPs.

successful screening result can be confidently translated into successful chromatography at the large scale without worrying about things such as tether effects or selector immobilization. We initially worked at a multigram scale, packing the CSPs into HPLC columns for conventional evaluation. While the cost and time required to make each of these materials on a 5-g scale is less than that of conventional CSP development, we required an even more rapid way of sampling the structural diversity of our candidate CSPs. Consequently, we developed a method in which each candidate CSPs is prepared on 50 mg scale and then evaluated using an ex-column screening technique [27,28]. In order to demonstrate proof of principle, we chose first to prepare a library of 50 dipeptide DNB CSPs using all possible combinations of the 5 amino acids; valine (V), glutamine (Q), phenylalanine (F), phenylglycine (PG) and proline (P). This set includes sterically bulky, strong hydrogen bonding and aromatic amino acids. Evaluation of the library was first tested using a model racemate, which is known to be well resolved on DNB amino acid CSPs [29]. The results of the screen are presented in Fig. 8. The vertical axis represents enantioselectivity, with the tallest bars indicating the best separations. From this graph we can deduce that a sterically bulky group in the aa 2 position is very important for enantioselectivity, and that a hydrogen bonding group in the aa 1 position may be of secondary importance. A subsequent ‘focused’ library of 87 different peptide DNB CSPs was then prepared using amino acids with the hydrogen bonding sidechains in the aa 1 position and amino acids with bulky sidechains in the aa 2 position. Screening showed that many of the members of this focused library performed better than the best members of the first library. One of the best candidates CSPs (DNB Leu–Glu) was selected for evaluation at a larger scale using HPLC (Fig. 9). Full scale HPLC evaluation of the selected dipeptide DNB CSP shows an enantioselectivity of greater than twenty for the separation of the enantiomers of the test racemate. An enantioselectivity of greater than 18 was still obtained when a mobile phase of pure ethyl acetate was used. Preparative evaluation showed that the analytical column .4:6 ð 250 mm) was capable of baseline resolution of 100 mg of the test racemate in a single injection (Fig. 10). Further increase in load could almost certainly be obtained by optimization of certain parameters such as temperature or flow rate. Nevertheless, even based on this unoptimized separation, a single kilogram of this CSP could be used to separate the enantiomers of nearly a ton of racemate in a year, a clear illustration of the power of the approach.

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Fig. 8. Results of screening of a library of 50 dipeptide DNB CSPs for the separation of the enantiomers of a test racemate.

Fig. 9. HPLC evaluation of one of the selected dipetide DNB CSPs.

50.I.8. Summary and conclusion During the course of the 20th century, we have seen the science of chromatography advance from a laboratory curiosity to an invaluable tool of modern technology. In

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Fig. 10. (A) Preparative separation using analytical column: 100 mg injection. Conditions: mobile phase D ethyl acetate; flow rate D 2.0 ml=min; detection D UV 380 nm. (B) Analysis of fractions from 100 mg injection on analytical column. Conditions: column D (S) DNB Leu CSP (4.6 mm ð 26 cm); mobile phase D MeOH; flow rate 1.5 ml=min; detection D UV 254 nm.

the last few years chromatographic enantioseparation has become an indispensable analytical tool in the pharmaceutical industry, and has received increasing attention as a technique for the preparation of bulk scale enantiopure material. For the coming years, we anticipate that as new, inexpensive, and highly enantioselective CSPs are discovered and developed, the use of large scale preparative chromatographic enantioseparation will continue to grow.

References 1. 2. 3. 4. 5.

L.S. Ettre and C. Horva´th, Anal. Chem., 47 (1975) 423A. R. Willsta¨tter, Ueber einen Versuch zur Theorie des Farbens, Chem. Ber., 37 (1904) 3758. C.W. Porter and C.T. Hirst, Asymmetric drugs, J. Am. Chem. Soc., 41 (1919) 1264. C.W. Porter and H.K. Ihrig, Asymmetric drugs, J. Am. Chem. Soc., 45 (1923) 1990. A.W. Ingersoll and R. Adams, Optically active dyes, I, J. Am. Chem. Soc., 44 (1922) 2930.

452 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22. 23. 24. 25.

26. 27. 28. 29.

Chapter 5 W.R. Brode and R. Adams, Optically active dyes II. Adsorption, absorption spectra and rotation, J. Am. Chem. Soc., 46 (1924) 2032. W.R. Brode and R.Adams, Optically active dyes III. Physical properties, dyeing reactions and mechanism of dyeing, J. Am. Chem. Soc., 48 (1926) 2193. G.M. Henderson and H.G. Rule, Resolving a racemic compound, Nature, 141 (1938) 917. G. Karagune and G. Coumoulos, A new method of resolving a racemic compound, Nature, 142 (1938) 162–163. V. Prelog and P. Wieland, The resolution of Tro¨ger’s base into its optical antipodes, a note on the stereochemistry of trivalent nitrogen, Helv. Chim. Acta„ 27 (1944) 1127–1134. M. Kotake, N. Nakamura, T. Sakan and S. Senoh, Resolution into optical isomers of some amino acids by paper chromatography, J. Am. Chem. Soc., 73 (1951) 2973–2974. F.H. Dickey, Preparation of specific adsorbents, Proc. Natl. Acad. Sci. USA, 35 (1949) 227–229. R. Curti and U. Columbo, Chromatography of stereoisomers with ‘tailor-made’ compounds, J. Am. Chem. Soc., 74 (1952) 3961. L.H. Klemm and D. Reed, Optical resolution by molecular complexation chromatography, J. Chromatogr., 3 (1960) 364–368. S.V. Roghzhin and V.A. Davankov, Ligand chromatography on asymmetric complex-forming sorbents as a new method for resolution of racemates, J. Chem. Soc., 10 (1971) 490. R.J. Baczuk, G.K. Landram, R.J. Dubois and H.C. Dehm, Liquid chromatographic resolution of racemic β-3,4-dihydroxyphenylalanine, J. Chromatogr., 60 (1971) 351–361. G. Dotsevi, Y. Sogah and D.J. Cram, Chromatographic optical resolution through chiral complexation of amino ester salts by a host covalently bound to silica gel, J. Am. Chem. Soc., 97 (1975) 1259–1261. W.H. Pirkle, S.D. Beare and R.L. Muntz, Assignment of absolute configuration of sulfoxides by NMR. Solvation model, Tet. Lett., 26 (1974) 2295–2298. W.H. Pirkle and D.W. House, Chiral high-performance liquid chromatographic stationary phases. 1. Separation of the enantiomers of sulfoxides, amines, amino acids, alcohols, hydroxy acids, lactones, and mercaptans, J. Org. Chem., 44 (1979) 1957–1960. A.S. Allenmark, Chromatographic Enantioseparation: Methods and Applications, Ellis Horwood, New York, NY, 1988, 224 pp. C.J. Welch, Evolution of chiral stationary phase design in the Pirkle laboratories, J. Chromatogr., 666 (1994) 3–26. C.J. Welch, pp. 171–197, in Advances in Chromatography, Crawling out of the chiral pool: the evolution of Pirkle-type chiral stationary phases, Vol. 35, 1995, M. Dekker Inc., New York, NY. W.H. Pirkle, C.J. Welch and B. Lamm, Design synthesis and evaluation of an improved enantionselective naproxen selector, J. Org. Chem., 57 (1992) 3854–3860. W.H. Pirkle, C.J. Welch, J.A. Burke and B. Lamm, Target-directed design of chiral stationary phases, Anal. Proc., 29 (1992) 225–226. M.D. Weingarten, K. Sekanina and W.C. Still, Enantioselective resolving resins from a combinatorial library, kinetic resolution of cyclic amino acid derivatives, J. Amer. Chem. Soc., 120 (1998) 9112– 9113. J. Maclennan, Engineered microprotein ligands for large-scale purification, Spec. Chem., 16 (1996) 267–269. C.J. Welch, G.A. Bhat and M.N. Protopopova, Silica based solid phase synthesis of chiral staionary phases, Enantiomer, 3 (1998) 463–469. C.J. Welch, M.N. Protopopova and G.A. Bhat, Microscale synthesis and screening of chiral stationary phases, Enantiomer, 3 (1998) 471–476. C.J. Welch, M.N. Protopopova and G.A. Bhat, Selection of an optimized adsorbent for preparative chromatographic enantioseparation by microscale screening of a second-generation chiral stationary phase library, J. Comb. Chem., 1 (1999) 364–367.

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D.51. Colin F. Poole Colin Poole was born on April 7, 1950 in Huyton, Merseyside, United Kingdom. He was educated in the United Kingdom receiving a B.Sc. in chemistry from the University of Leeds (1971), followed by graduate studies at the University of Bristol, M.Sc. in analytical chemistry (1972), and Ph.D. under E.D. Morgan at the University of Keele (1975) on the analysis of insect molting hormones. This was followed by postdoctoral appointments with D.G. Wibberley at the University of Aston in the UK, M. Verzele at the University of Ghent in Belgium and A. Zlatkis at the University of Houston in the USA. Since 1980 he has been at the Department of Chemistry, Wayne State University, Detroit, Michigan, USA, except for 1995–1996, spent as the Governors’ Lecturer and Professor of Analytical Chemistry at Imperial College of Science, Technology and Medicine, London, in the UK. He is an Editor of the Journal of Chromatography A and serves on the Editorial Advisory Boards of the Journal of Planar Chromatography, Journal of High Resolution Chromatography, Chromatographia, and LC=GC Magazine. With his wife Salwa he co-authored the book “Chromatography Today” (1991). Earlier he co-authored the book “Contemporary Practice of Chromatography” (1984) and edited “Electron Capture. Theory and Practice in Chromatography” (1981). He has presented hundreds of short courses for professional societies, instrument manufacturers and Government agencies on chromatographic techniques and sample preparation technology in the USA and Europe. Since 1989, he has been a Science Advisor to US Food and Drug Administration, initially with the Pesticide and Industrial Chemicals Research Center, and more recently with the Division of Field Science. C.F. Poole has authored about 275 publications, mainly in the separation sciences and related technology. Recent research interests include computer-aided methods development, characterization of chromatographic materials, new sample preparation procedures, and the application of data analysis techniques to chromatographic results [1]. C.F. Poole received the 1980 Camille and Henry Dreyfus Award for newly appointed young faculty in chemistry, the 1985 M.S. Tswett Chromatography Medal of the International Symposium on Advances in Chromatography, the 1991 Jubilee Medal of the Chromatographic Society, and was elected a Fellow of the Chromatographic Society in 1992. In 1997 the University of Leeds awarded him a D.Sc. C.F. Poole has made significant contributions to the practice and theory of chromatography through his research papers, short courses, and widely used textbooks. He is recognized for his early studies on derivatization techniques for trace analysis using electron-capture detection, contributions to modern thin-layer chromatography and packed column supercritical-fluid chromatography, the development of techniques for characterizing stationary phase properties, and advances in sample preparation technology. He remains active in many fields of separation science and is a frequent invited speaker at international symposia. See Chapter 5B a, d, e, l, o, p, s

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51.I. REFLECTIONS OF A POLYCHROMATOGRAPHER Colin F. Poole Department of Chemistry, Wayne State University, 5101 Cass Ave., Detroit, MI 48202, USA

From an early age, chemistry was the subject I enjoyed most at school. I graduated from university in the United Kingdom with a typical background in chemistry for that time; roughly characterized as broad enough to include everything but analytical chemistry. When it was time to start graduate studies, I wanted to try analytical chemistry. I am not sure why, except that I liked the idea of applying chemical knowledge to the solution of practical problems. I went to the University of Bristol to study for a taught M.Sc. in analytical chemistry under the tutelage of G. Nickless. This was really my first contact with analytical chemistry, and from that time onwards, I knew I had found what I wanted to do. Opportunities to proceed to a Ph.D. degree in analytical chemistry were rare in the United Kingdom at that time. I was lucky to be accepted into the group of Professor E.D. Morgan at the University of Keele to work on the isolation of insect molting hormones from the desert locust. It was here that I commenced research in gas chromatography and gained valuable experience in natural product isolation techniques. During my studies on the desert locust, I became interested in the electron-capture detector, new derivatizing reagents for trace analysis, and mass spectrometry; all these techniques became the backbone of my attempts to develop an independent research career in later years. The identity of the molting hormones were known when my studies commenced, but microchemical methods for the determination of these complex steroids (ecdysones) were poorly developed. The sensitive assay I developed involving formation of trimethylsilyl ether derivatives and gas chromatography with electron-capture detection remained in use for several years, although better methods exist today. I don’t miss the daily activities associated with the maintenance of an active insect colony, but I regret that circumstances have never directed my research work back to this fascinating field. After completion of my graduate studies, I succumbed to the nomadic life of a postdoctoral research fellow. Real jobs in industry were hard to find at that time. A brief stay at the University of Aston was followed by a period in Professor M. Verzele’s research group at the University of Ghent, where I became proficient in the making of glass capillary columns, at that time more of an art form than an exact science. The wizards of Ghent had mastered the spells needed to perform the impossible trick of taking a glass tube and turning it into a capillary column with a stable stationary phase film capable of exquisite separations of complex mixtures. These columns I used for the separation of amino acids and other compounds extracted from beer. My early training in microchemistry was invaluable in ensuring that so little beer was required for analysis that sufficient remained to maintain a happy research group in the manner I suspect my current research group would be happy to get accustomed to. In Ghent I met Professor A. Zlatkis who invited me to join his renowned research group at the University of Houston. This was the start of a very productive three-year association in which I met many of the great names of the day in chromatography and became involved in a diverse range of projects. Gas chromatography remained Al’s passion, but

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at that time high performance thin-layer chromatography had been introduced recently in Europe, and shortly afterwards we acquired equipment in Houston for this technique. I became involved in the TLC projects as well as directing my own little research sub-group on the synthesis and evaluation of new derivatizing reagents for use with the electron-capture detector. At this time the bread and butter project within the group was the identification of volatile marker compounds in biological fluids suitable for the early diagnosis of metabolic disorders. During my stay at the University of Houston, I was invited to spend six months in Sweden at the analytical research laboratories of Astra Hassle by Professor J. Vessman working on several projects involving trace analysis. Eventually, I was considered trained and the United States Immigration and Naturalization Service gave me the ultimatum of finding a real job or being deported. So I ended up at Wayne State University where I have worked all my professional life except for two years spent as a Governors’ lecturer and Professor of analytical chemistry at Imperial College of Science, Technology and Medicine in the United Kingdom. My early training had not fitted me up to specialize in anything in particular. I still have difficulty when asked by students or colleagues for my specialty. I don’t think I have one and have never tried to acquire one. I was once introduced to an audience in Sydney, Australia, as a polychromatographer. I remember thinking that that was a big word for an Australian, but if poly is taken to mean many and not heavy, then I am happy with that label. There is probably more glory in being identified with a single technique, but there is more fun in using them all. For that reason I have tried to maintain a separation science research group focused on a wide range of problems that require the use of different chromatographic techniques for their solution. For the reader I will try and highlight some of my activities in a (semi) orderly fashion that perhaps suggests more long term planning than I could own up to. One thing I have learned over the years is that you never finally solve any problem. For no sooner do you feel the breeze from the summit of the mountain, then the mists lift and you realize that the solution was only the beginning of another problem. In science no one can be sure which is the highest mountain and our destiny is to keep climbing until we get to the top of a big one and then fall off. The central theme of my research in the late 1970s was the development of derivatizing reagents containing electrophores for various trace analysis problems using gas chromatography with electron-capture detection. The electron-capture detector is one of the most sensitive detectors for gas chromatography with selectivity to compounds containing electronegative groups. My idea was to expand the scope of the detector to compounds with a low response by devising reagents to selectively tag them with an electrophoric group. Two general routes were successful. A number of pentafluorophenylalkylsilyl reagents were synthesized for the trace analysis of compounds containing functional groups with replaceable hydrogen atoms. These reagents became commercially available under the trade name ‘flophemesyl’ and are still in use today. Boronic acids with alkyl or aromatic groups were well established for the selective formation of cyclic derivatives with bifunctional compounds, but none of these reagents were suitable for use with the electron-capture detector. A number of new boronic acids with electrophoric groups were synthesized, several of which became commercially available. New reaction strategies for their use including extractive

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derivatization and the transboronation reaction were developed and applications to pharmaceutical compounds in biological fluids followed. The weakness of these reagents remained their poor hydrolytic stability and they are little used today. An interesting problem in developing this chemistry was to devise methods for purity assessment of these reactive compounds. At times this required more ingenuity than their synthesis. From work that commenced in the early 1980s with the aim of developing standard reference phases for gas chromatography, a number of projects emerged. When this work commenced, nearly all polar stationary phases with reasonable thermal stability were polymeric materials subject to dubious batch to batch reproducibility. Uncertainty of structure and composition are undesirable properties in a reference stationary phase, and in addition, confused attempts to quantitatively determine the contribution of polar interactions to retention. Initially a number of interesting compounds were synthesized by introducing cyano groups onto a poly (phenyl) ether backbone containing a defined number of rings, but real success was obtained with the development of liquid organic salts. While browsing through one of those journals, you wonder why the library subscribed to them; while waiting for a break in the rain to facilitate the return to my office, I came across an article describing the (claimed) unusual properties of ethylammonium nitrate. This compound enjoyed a cult following for awhile based on the hypothesis that it possessed solvent properties resembling water. Its chromatographic properties were poor, but not discouraged, I embarked on a decade of research on liquid organic salt chemistry. We demonstrated that certain low-melting point alkylammonium and alkylphosphonium salts, with suitable anions, formed stable liquids possessing low vapor pressure at temperatures up to 150ºC above their melting point. This provided a sufficient working temperature range to explore their properties as novel ionic solvents. Over the years a number of techniques were developed to study their physical and solvent properties, including solvatochromic parameters, thermodynamic constants for solvation, spectroscopic properties (including the development of FAB mass spectrometry for the identification of impurities), etc., [4]. Room temperature liquid organic salts were tried as mobile phases in liquid chromatography and for extraction. More importantly we were able to demonstrate that liquid organic salts possessed unique solvent properties that cannot be duplicated by common non-ionic solvents in any of their applications. However, they were developed too late in the evolution of stationary phase chemistry to have any real impact on the practice of gas chromatography. One rainy day in a library somewhere, I suspect someone not engaged in separation science will rediscover them, wondering why the library subscribes to all those chromatography journals. Their further technological evolution will depend on matching their chemistry to suitable applications. Of more interest to separation scientists was a project that came about as a direct result of our studies of liquid organic salts. Simply put, standard methods of characterizing stationary phases classified liquid organic salts as moderately polar. Either these hallowed methods were incorrect or these salts did not live up to expectations. It turned out to be the former, but this was no easy, or uncontroversial research problem. We rediscovered the importance of interfacial adsorption as a retention mechanism in gas–liquid chromatography and proceeded to use gas–liquid distribution constants to correct the various solvation scales in general use at that time. This was insufficient

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and kicking and screaming I found myself immersed in chemical thermodynamics. Two factors were slowly identified as critical problems in established methods. Use of the retention index scale was inappropriate because retention index differences were governed more by the solute properties of the n-alkane index standards than the solutes used to evaluate specific intermolecular interactions. The use of solution free energies of individual select solutes failed because of the size-dependence of their solution properties and because individual solutes with singularity in their taste for intermolecular interactions are virtually non-existent. At last a free energy-based model that took size differences into account and used chemometrics to isolate the contribution of individual intermolecular interactions to retention was developed. Thankfully, this model logically ranked phases according to their capacity for defined intermolecular interactions. I was quite pleased with this and ready to quit this field while the going was good only to come into contact with Dr. Michael H. Abraham at University College London. Abraham had proposed an alternative and more general model for predicting solubility-dependent properties in chemistry. Initially we worked together to show that the two models predicted the same results for gas chromatography, which they did, and since then we have co-operated on many projects to characterize bonded phases for liquid chromatography, micelles for micellar electrokinetic chromatography, and sorbents for solid-phase extraction. This was also the first step for me into a new field of quantitative structure-property relationships. Some of my research efforts have moved into devising models for the estimation of biopartitioning, aquatic toxicity, and soil–water sorption, etc., as well as the design of chromatographic systems that emulate biological systems. Supercritical-fluid chromatography re-emerged as a separation method in the early 1980s based largely on open tubular column technology. Along with others I saw advantages for the packed column format in terms of sample capacity and fast separations if column deactivation could be improved. Significant developments in SFC technology were made by my group in recognizing the connection between packing density and column performance, in column activity test methods, and in the introduction of a solventless injector that overcame additional band broadening due to the strong solvent effect. The solventless injector was later developed into a general interface for at-column derivatization and as an accumulation interface for on-line supercritical-fluid extraction and chromatography [7]. Separation methods for organometallic compounds were developed and formamide introduced as a polar modifier for carbon dioxide compatible with flame ionization detection. Studies in supercritical-fluid extraction resulted in the development of solvent-assisted supercritical-fluid extraction, which was applied to the isolation of flavor compounds from plant materials. This approach is complementary to accelerated solvent extraction and recognizes the primacy of temperature in overcoming analyte–matrix interactions that is a major cause of low analyte recovery. A natural progression of this work was the use of pressurized hot water for the extraction of contaminants from foods and the development of a theoretical model to explain the influence of temperature on the solvation properties of water. Over the years I have tried to lend some respectability to thin-layer chromatography by either developing applications or contributing to its evolutionary advances in performance and methodology. In the late 1980s, interest started to wane in modern TLC as

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high-pressure liquid chromatography became the dominant form of liquid chromatography. There is good reason to utilize TLC as a complementary technique to HPLC and this has maintained my interest in this area. Early instrumentation for scanning densitometry left a lot to be desired. Comprehensive studies in my group of the parameters that affected the recording of in situ chromatograms in the reflectance, transmission and fluorescence modes were helpful in establishing TLC as a quantitative technique [5]. Polycyclic aromatic compounds are common environmental contaminants, and were of great interest in the mid-1980s due to their potential adverse health effects. Methods for their general analysis were not well developed and it was thought that TLC could play an important role as a high sample throughput screening technique for their detection in environmental samples. Sample preparation procedures, separation conditions and emission response ratios for qualitative identification were developed. A wide range of environmental samples were successfully analyzed but the lower resolving power of TLC compared to column methods and the need for more sample preparation steps than was desirable made this approach less useful than we had hoped for. This project taught us a lot about developing TLC methods and the need to improve separation capacity using forced flow and multiple development strategies that formed a significant part of future projects. Using forced flow development and computer models for data analysis, we were able to fully characterize the kinetic properties of modern TLC plates. These measurements revealed that the binder used to stabilize the layer resides predominantly in the pore volume, where it serves no useful purpose, and is responsible for an order of magnitude increase in the plate height contribution from resistance to mass transfer. Poor mass transfer characteristics of modern TLC layers limit both the performance and separation speed for forced flow TLC. Mass transfer properties are not critical for capillary flow controlled conditions because zone broadening is dominated by molecular diffusion. Capillary forces are incapable of providing adequate mobile phase velocities for optimum separation performance. The instrumentation and methodology developed to characterize precoated layers proved ideal (with minor modifications) for studies of sampling properties of particle-loaded membranes and particle-embedded glass fiber disks, introduced recently for solid-phase extraction. Unidimensional multiple development strategies were explored to enhance the separation potential of TLC by exploiting the zone refocusing mechanism that accompanies sequential development and allows zones to be migrated for greater distances, while maintaining a small average zone size. Under favorable conditions an increase in zone capacity of about six fold is possible. The mechanical aspects of the zone refocusing mechanism have been well-characterized and practical rules stated, but a comprehensive theory still eludes us due to the complex relationship between the many parameters involved. Multiple development with decreasing solvent strength gradients is useful for the separation of mixtures of wide polarity. This approach proved valuable in developing approaches for classifying the botanical origin and flavor potential of spices. That brings me to current research projects. These can be capped by three headings: computer-aided method development; sample preparation technology; and data analysis [2,3,6]. Good ideas and sparks of inspiration don’t have a habit of providing advance warning. They just seem to emerge from chaos and require persistence over a long time

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to establish. Thus I am not sure what I will be doing after the millennium celebrations, only that it will involve some aspect of separation science. References 1. 2. 3.

4.

5. 6. 7.

C.F. Poole and S.K. Poole, Chromatography Today, Elsevier, Amsterdam, 1991. C.F. Poole and S.K. Poole, Interphase model for retention and selectivity in micellar electrokinetic chromatography, J. Chromatogr. A 792 (1997) 89–104. C.F. Poole, S.K. Poole, D.S. Seibert and C.M. Chapman, Determination of kinetic and retention properties of cartridge and disk devices for solid-phase extraction, J. Chromatogr. B, 689 (1997) 245–260. C.F. Poole, T.O. Kollie and S.K. Poole, Recent advances in solvation models for stationary phase characterization and the prediction of retention in gas chromatography, Chromatographia, 34 (1992) 281–302. C.F. Poole and S.K. Poole, Instrumental thin-layer chromatography, Anal. Chem., 66 (1994) 27A–37A. C.F. Poole and I.D. Wilson, Planar electrophoresis and electrochromatography: time to revisit these techniques? J. Planar Chromatogr., 10 (1997) 332–335. J.W. Oudsema and C.F. Poole, Some practical experiences in using a solventless injection system for packed column supercritical-fluid chromatography, J. High Resolut. Chromatogr., 15 (1992) 65–70.

D.52. Jerker Porath Jerker Porath was born in Sala, Sweden on October 23, 1921. He received the following degrees from Uppsala University, B.Sc. in 1946, Ph.D. in 1950, and Fil. Dr. in 1957. J. Porath was appointed as an Assistant Professor in 1957 at Uppsala University, Associate Professor in 1964, and Professor in 1968 to the present. J. Porath has had a distinguished career and received numerous fellowships and guest professorships – some are listed as following: a fellow of Swedish Natural Science Research Council 1960 to 1964; Visiting Fellow, Institut fu¨r Krebsforschung, Heidelberg, 1950; Visiting Fellow, University of California, Berkeley, 1951 to 1952; Visiting Professorships at Universities of Tokyo, Copenhagen, Prague, Moscow, and Bucharest; Visiting Professor, University of California, San Francisco, 1962 and 1967; Jacobsonian Professor in Biochemistry, 1968 to 1988; Visiting Professor, University of California, 1974; Visiting Professor, Canadian Medical Research Council, Montreal, 1975; Visiting Professor, Universite´ Pierre et Marie Curie, Paris, 1981; Fogarty Scholar at NIH, Bethesda, 1985; and Research Professor in Biochemistry, The University of Arizona, 1989 to present. Some of his honors and awards include: The Gold Medal of the Royal Swedish Academy of Engineering, MD h.c., University of Uppsala; Knighthood of the Swedish Order of the Northstar, the Honorary Plaquette of the French Society of Biochemistry, and the Bjorken’s medal, all in 1972; the Biochemical Analysis Prize and Sir Frederich Gowland Hopkins Medal, the Tswett Medal, and recipient as a Fogarty Scholar, all in 1978; in 1987, the Grand Old Medal, Swedish Academy of Engineering, and the

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Eric Fernstrom Grand Prize. In 1994, he was awarded a Ph.D. h.c. at the University of Compie`gne, France, and the Pehr Edman Award. J. Porath is the discoverer of Sephadex and extensively researched the dextran gels and cross-linked agarose. His research led to the development of two new branches of affinity chromatography, electron-donor–acceptor chromatography (EDAC) and immobilized metal affinity chromatography (IMAC). His work now centers on cascade-mode multiaffinity chromatography (CASMAC) for difficult separations of proteins and protein pharmaceuticals. J. Porath is a member of numerous learned societies, i.e., Royal Swedish Society of Science, International Society of Toxicology, American Association for the Advancement of Sciences (AAAS), The New York Academy of Sciences, The Royal Swedish Academy of Engineering, Foreign Member of the Russian Academy of Science, The Royal Swedish Academy of Science, Honorable Member, Japanese Biochemical Society, Germany Society for Clinical Chemistry, and Honorable Member of ‘Group Francais de Bio-Chromatographie.’ His publications number more than 260 and he has received approximately 25 patents. See Chapter 5B, a, e, g, i

52.I. HALF A CENTURY OF STRUGGLING WITH MOLECULAR SIEVING AND AFFINITY CHROMATOGRAPHY Jerker Porath Uppsala University, Center for Surface Biotechnology, Box 577, S-751 23 Uppsala, Sweden

Try to imagine the stage of chromatography in the year 1950. History at that time comprised the following most important events: Tswett’s invention, rediscovery of his work, introduction of partition- and displacement chromatography. The theory was not far advanced. The adsorbents, to some extent useful for organic substances, were inadequate for proteins and peptides. 52.I.1. Preliminaries About 1950, I entered the scene for reasons I would like to tell you. I started out as a young organic chemist, with mathematics, physics and some geology in the background. Soon I realized that for a career in organic chemistry, I had to master the art of product purification and my shortcomings may be illuminated by the following story: During and just after World War II, there was a shortage of basic chemicals. We had to synthesize them from scratch. On one occasion I had to prepare glutaric acid. Synthesis was no problem but purification was. I obtained a brown syrup containing some floating dark-colored crystals. The disgusting sample resisted all my attempts to improve its quality. Finally, Arne Fredga, my teacher, took pity on me. He suggested that my product should be divided into two equal parts and we should compete in purification and compare our accomplishments. The next day Fredga entered my lab with white, beautiful crystals to be exhibited side by side with my yellow

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mass (mess!) of crystals melting over a broad temperature interval and of unesthetic appearance. Eventually, however, I discovered part of the answer to his extraordinary cleverness in achieving successful crystallizations. He, himself, ruled over two very large interconnected laboratory halls in which he had exposed on open display hundreds or perhaps thousands of various crystallized substances. The air was filled with dust apparently consisting of minute crystals that served as nuclei for the crystallization processes. After some years as one of his favorite students, I had the privilege to be placed in a corner of his ‘Holiest of Holies’ and behold, my competence in separation science took a significant step forward. Fredga was an artist — a wood carver — and he was also a ‘separation artist’, although of an old fashioned kind. For an apprentice like me to reach his level of skill appeared impossible — or would take a very long time. I therefore looked around for other opportunities to become productive. In the same complex of buildings — the Kemikum — resided my teachers in physical chemistry and biochemistry; Theodor (‘The’) Svedberg and Arne Tiselius, both Nobel Laureates and specialists in separation science. Chromatographic activities were in full swing in Tiselius’ small team. Future Nobel laureates came and went. I joined him to gain inspiration in chromatography. Tiselius had entered the field from two directions: through his work on zeolites in the early 30s and through his interest in serum proteins. He realized that electrophoresis did not have the power to resolve the complex mixtures of gamma globulins, now called immunoglobulins. Instead of ‘chromatography’, he preferred the term ‘adsorption analysis’, the reason being the fact that ‘chromatography’ was a nonsense word when used to describe separation of colorless substances. Active charcoal was the adsorbent most used by Tiselius’ team. It was a difficult material to work with. New chromatographic inventions had to be made; namely, gradient elution and carrier displacement. Even so, progress for practical applications was modest, but eventually the new techniques paid off when more selective adsorbents became available. At the same time progress was made elsewhere. For example, by chromatography on starch and sulfonated cross-linked polystyrene, Stanford Moore and William Stein succeeded to completely separate all of the amino acids present in protein hydrolysates.

52.I.2. Discovery of Sephadex [1–3] We all understood that to discover or invent adsorbents which could separate the components in a peptide or protein mixture into discrete zones on columns by elution (linear chromatography) would be an ‘open sesame’. Such adsorbents were badly needed. Per Flodin and I had packed our electrophoresis columns with starch grains to suppress convection in the buffer media. To me the enlightenment came one day about four years after I had joined Tiselius. When testing the efficiency of bed-packing, I discovered that some dyes (DNP-amino acids for example) migrated as discrete zones, and of course that was a certain sign of isotherm linearity. My curiosity further increased after an accident when Noris Siliprandi, an Italian research guest, absent-mindedly forgot to turn on the current over night. The next morning his assisting wife eluted the substances from the column and found that a strange unexpected kind of separation had

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occurred. I had certain suspicions about what had happened, made systematic studies, and discovered that test substances often migrated in order of their molecular size within a volume less than that of the column. Some of them moved with stronger retention because they were linearly or non-linearly adsorbed. Unfortunately, the eluates were contaminated by extracts from the starch. I deemed the results not to be recommended as a suitable fractionation procedure. I did not publish. At that time premature publication was considered risky. Bo¨rje Lindqvist at Mjo¨lkcentralen — a Swedish center for milk and milk products — learned from Per Flodin about my study results. He and his director, Torsten Storga˚rds, were less cautious and published a paper in Nature describing fractionation of peptides and amino acids from maturing cheese by starch chromatography. In a short note to Biochemical Journal these studies were criticized by Lathe and Ruthven. The latter authors later wrote a full article and became ‘the discoverers of size-exclusion chromatography.’ I considered myself to be the discoverer, later to find that I also had some predecessors. In any case, I believe that Per Flodin and I were the only ones who really understood the implications of the discovery. For years, nobody paid any attention to the articles on what now is called size exclusion. From 1954 on we had prepared our minds, to cite Pasteur, for the discovery of a chromatographic medium which should behave as an ideal molecular sieve. I will now digress to what happened before my time at the Institute of Biochemistry in Uppsala; in the 1940s, Tiselius gave one of his students, Bjo¨rn Ingelman, the task to undertake a study of a slimy product that had become a great nuisance in a sugar factory in southern Sweden. Sugar beet extracts were contaminated by some unknown substance that made crystallization of sugar very difficult. Ingelman identified the mucous substance as dextran. As a blood plasma expander, the dextran later became the most important product of Pharmacia, at that time a small company, located in Stockholm. Pharmacia moved to Uppsala to come closer to our Institute. Ingelman made a comprehensive study on dextran for his doctoral thesis. He cross-linked dextran and got a lump of gel which, as a curiosity, was placed on a remote shelf of the factory lab, where it remained for several years. Eventually this gel came into my possession, thanks to Flodin. The introduction of cross-linked dextran has been described elsewhere [1,2]. I will only make some brief comments. Already in my first experiments with cross-linked dextran, I observed both molecular sieving and linear adsorption, just as I had done on starch about two years earlier. The cross-linked dextran was stable and no leakage was observed. It appeared that we had found the chromatographic medium we had been looking for. We also tried other hydrogels, among them cross-linked polyvinyl alcohol, but decided to continue to work with the dextran gels. Our publication was delayed to give Pharmacia time to develop the necessary knowledge in technology and to build a factory. The new separation method using dextran gels appealed to me for the following reasons: ž Solutes migrated as compact zones analogous to those obtained under the influence of linear adsorption. ž We had a much simpler alternative to the ultracentrifuge (the complicated machine designed by The Svedberg) for estimation of molecular size — and it could be used for smaller molecules.

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ž It could be applied on an analytical or preparative scale and had a potential for industrial applications. From the beginning, Flodin and I had a clear perception of the molecular mechanism behind molecular sieving, but we never formulated a fully fledged theory. I refer to a recent review on gel filtration theory by Laurent [3]. My interest in size-exclusion chromatography (SEC) soon faded, but only temporarily. I tried to combine precipitation and molecular sieving in a salt concentration gradient on Sephadex columns. The method was published under the name of ‘zone precipitation’, later to be called ‘reverse gradient salting-out chromatography’ [2]. About 35 years later, in 1998, the method was independently rediscovered under an even fancier name: ‘on column precipitation– redissolution chromatography’.

52.I.3. From ion exchange — to adsorption — to affinity chromatography [4–6] My narrative has to be kaleidoscopical in order to make it historically reasonably coherent. I will therefore now turn to contributions made in ion-exchange chromatography. They were started at about the same time as the first studies on molecular sieving. In the mid 1950s, Herbert Sober and Elbert Peterson, at the National Institutes of Health; Bethesda, MD, USA, introduced cellulosic ion exchangers as protein adsorbents. This achievement is a milestone in the history of protein chromatography. Herbert and Elbert became my close friends and their work inspired me to follow in their footsteps. I added a new variant of cellulose derivative: sulfonate ion exchanger. On one occasion the synthesis went astray. Too high a degree of sulfonate substitution was obtained leading to an excellent wall paper paste, but this is another story. I also introduced a quarternary ammonium derivative of cellulose. These studies were included in my doctoral thesis. Pharmacia commercialized the same kinds of adsorbents, but they were based on dextran and later on agarose. (I was close to obtaining royalties on wall paper paste, but not on the adsorbents)! Sober’s and Peterson’s work, together with our discoveries on Sephadex paved the way to modern biochromatography. Is this a subjective overestimation? Aromatic adsorption on Sephadex attracted my particular attention and its discovery initiated my work in various fields of affinity chromatography. According to my provisional working hypothesis, aromatic adsorption must depend on some kind of aromatic hydrogen bonding coupled with (pi)-electron interactions. These ideas were discussed in several articles before aromatic hydrogen bonding was observed in internal regions of protein molecules by X-ray analysis. The adsorption was particularly strong in the presence of alkali sulfates and phosphates. At a Nobel Symposium in Stockholm in June of 1967 [4], I reported some experimental work that was going on in my research team at that time. I mentioned briefly a one-step purification of avidin from egg white by chromatography on biotinSephadex. More than a 10,000-fold purification was obtained with excellent recovery. The cyanogen method for coupling was also mentioned before the articles in Nature appeared. Concluding my Symposium lecture, I expressed my view on our research projects on bioselective adsorption as follows: “Stereochemical complementarity and

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molecular flexibility are responsible for the coordination forces that confer specificity upon the adsorption phenomena we are studying. We expect that the studies of the primitive models used in adsorption chromatography will reveal important effects that are obscured in the more complicated living systems.” The statement still holds true. A prerequisite for well-functioning affinity chromatography is not only a suitable gel matrix, but also the availability of synthetic methods for reliable ligand attachment. We therefore introduced the following coupling reagents: isothiocyanate, cyanogen bromide, epichlorohydrin, butanediol-bisglycidylether, p-benzoquinone, divinylsulfone and the Ugi reagents. I will tell you about some of the events that lead to the discovery of the CNBr-method [6]. My postdoc Rolf Axe´n had just returned from Bernhard Witkop’s laboratory in Bethesda where he had learned to cleave peptides with cyanogen bromide. He suggested that we should convert amino-Sephadex to a cyanamide derivative by cyanogen bromide treatment, thus activating the gel for coupling. Sverker Ernback, one of my students, would assist us. Ernback succeeded beyond our expectations. He obtained a yield far above 100%! I had to suspect either Ernback or the concept. Since the high recoveries were reproducible, I concluded that the matrix itself reacted with cyanogen bromide and became activated for coupling. The method was patented, and for a very modest royalty the patent was transferred to Pharmacia. As a university teacher I was the owner of my inventions. We divided the forthcoming royalty into three equal parts, which to some extent changed our future lives. Axe´n eventually could buy a farm in the countryside outside Uppsala where he built a private laboratory. He left the university and Pharmacia to enjoy independence from the bureaucrats. He spent his post academic life as a ‘gentleman scientist’ a` la Robert Boyle. Ernback also disappeared from the academic scene. None of my other students ever received such a profit for so little work as they did. Armed with the CNBr method my students and collaborators, Tore Kristiansen, Lars Sundberg and Ka˚re Aspberg started under my guidance to develop bioaffinity chromatography. The future looked bright. Unknown to us, however, the catastrophe for our program lingered around the corner. A few months after our two reports on the CNBr method had appeared in Nature, Pedro Cuatrecasas, Meir Wilcheck and Christian Anfinsen published their work on ‘affinity chromatography!’ [5]. All of the world’s protein chemists became excited. Finally, the time for dull fractionation work could be shortened! This turned out not to be quite true, but my students with more than a years experience in the field became very discouraged. I tried to calm them. After all, the Anfinsen team had proved that our research was of good quality. Our contributions were summed up in some review articles. In 1972=1973, I resumed my work on salting-out chromatography. We first prepared uncharged agarose with nonionic hydrophobic ligands; these adsorbents were later further improved by cross-linked alkyl-sulfide agarose gels.

52.I.4. Cross-linked agarose [7] Agar was introduced for chromatography by Poulsen and agarose by Hjerte´n. With this matrix SEC could be extended to large molecules. However, agar and agarose

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derivatives have both advantages and shortcomings. We solved most of the problems by cross-linking the gels. Bed compaction at moderate pressures was practically eliminated. This was an important step forward in our attempts to improve hydrogels for chromatography. A series of systematic studies by Jan-Christer Janson, Torgny Laas and myself [7] showed that the rigidity of agarose products depends strongly on the length of the cross-linker used to stabilize the gel. The patent right to exploit this discovery was sold to Pharmacia in the hope to increase our chance of receiving financial support from The Swedish Board of Technical Development. Royalties caused mixed blessings. Financial help from elsewhere dried up and we had to pay back the major portion of the royalties to the Board. However, Pharmacia prospered and their products found widespread use under such trade names as Sepharose and Superose, etc. Cross-linking of dilute agarose with a high concentration of divinylsulfone (DVS) extends the molecular sieving range upwards, but no DVS-agarose products ever appeared on the market, few further studies were carried out and no optimization trials were made. The knowledge of DVS-agarose went into oblivion. This unfortunate situation is a consequence of the industrial take-over of almost all research on adsorption media for chromatography. Using DVS-cross-linked 0.5–1.0% agarose, SEC can be extended to particles of molecular mass exceeding 106 daltons, to small viruses such as Tobacco Mosaic Virus. Maybe we discouraged followers by pointing out chemical instability, but DVS-agarose synthesized by us was in fact shown to be completely stable in the pH-range 3–8. Furthermore, it can be safely used without carbohydrate leakage between pH 3 and 12. Excess vinyl groups remaining after the cross-linking reaction can be deactivated by hydrolysis. When instead, we removed the vinyl groups by mercaptoethanol treatment, we discovered a new kind of protein adsorbent, the ‘T-gel’, to which I will return shortly [8]. The improved adsorbent matrix came in handy and in time for our projects aimed at the development of two new branches of affinity chromatography: electron donor–acceptor chromatography (EDAC) and immobilized metal (ion) affinity chromatography (IMAC), respectively.

52.I.5. EDAC [8–10] EDAC is based on weak polar interactions and in attraction between (pi)-electron rich structures in the analytes and gel immobilized ligands. These attractions can be reinforced or weakened depending on the composition of the solvent medium. Selectivity and capacity thus depend on the kind and concentration of salt added to the solvent. Karin Dahlgren-Caldwell and I discovered at an early stage that the electron acceptor efficiency of aromatic ligands is amplified by thioether sulfur adjacent to the aromatic nucleus. Dinitrophenylthioether Sephadex is thus a strong adsorbent, whereas the corresponding DNP-O-ether is not. Like hydrophobic-interaction-based affinity EDA-adsorption is promoted by strongly water-structuring salts like sodium and potassium sulfates and phosphates. To demonstrate the efficiency of biorecognition-dependent chromatography, a biotin– avidin system was used. I believe that bioaffinity in this case depends on electron-donor–

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acceptor interaction similar to that behind aromatic adsorption in a T-gel [8]. The argument is as follows: in the case of formation of a biotin–avidin complex, the tetrahydrothiophene-sulfur in biotin is transferred into a region of low polarity caused by a cluster of tryptophan residues. In the case of tryptophan being adsorbed to a T-gel (or an aromatic thioether gel), it is forced by a strongly water-structuring salt into the vicinity of the polymer matrix with its S-ether ligand. This environment has a lower polarity than the bulk water. Thus, in both cases tryptophan and the thioether-S are coming close together in an environment of low polarity which permit short-range attractive forces to cause the adsorption. The common denominator is the electron donor–acceptor contact in a region of low polarity and this is in essence the condition for EDA and EDAC. Hydrophobic interaction (HI) and EDA represent different ‘dimensions’ in separation technology which is clearly demonstrated by experiments with composite columns consisting of tandem coupled beds of hydrophobic and EDA-adsorbent. Aromatic substances can be selectively extracted from a complex mixture. A clean cut separation of serum proteins into albumin and immunoglobulin fractions can be achieved on such tandem columns. We have synthesized and studied quite a few aromatic and heteroaromatic thioagaroses [9]. Sven Oscarsson and I introduced 3-(2-pyridylmethylenethio)-2-hydroxypropylagarose, which is now on the market. Aromaticity is not necessary for donor or acceptor properties, as shown by the ‘T-gel’ with the ligand CH2 –CH2 –SO2 –CH2 –CH2 –S–CH2 –CH2 OH. I would like to mention a successful application of EDAC for one-step purification of monoclonal antibodies from mouse ascites fluid by one of my research teams under the seniority of Makonnen Belew. Although EDAC is superbly well suited to concentration and group isolation of immunoglobulins, it has a wider potential field of application. In a study by Robert Scopes and myself, we showed that under the experimental conditions used, 43% of the proteins in an extract from Zymonas mobilis were adsorbed to a T-gel, and 77% to epichlorohydrin coupled α-mercaptothiazole-agarose. Many other non-aromatic EDA-gels have been synthesized. They are all characterized by ligands of very high (pi)-electron density. For further information, I refer to a recent article by Patrick Berna et al. [10]; EDAC, which still is in its infancy, seems extremely promising. The weak point with EDAC in aqueous and hydroorganic solvents is that this kind of chromatography has not yet been ‘rediscovered.’

52.I.6. IMAC [11,12] IMAC, immobilized metal affinity chromatography, is another area of separation methology with many origins. My contributions started a quarter of a century ago. In 1974, I was appointed guest professor by the Canadian Medical Research Council and was located at the Hormone Laboratory of the Clinical Research Institute in Montreal. After many years of absence from the laboratory bench, I lacked experimental skills and decided therefore to choose an easy project. I came up with the working hypothesis that perhaps protein precipitation and adsorption might have a similar, if not identical, affinity background. If so, a precipitant may, in immobilized form, serve as an affinity

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ligand. Alkaloids and certain heavy metal ions are protein precipitants. Why not select metal ions? That appeared lazy enough and I made the choice. Serum was fractionated on gel-immobilized zinc and copper ions. The capacity was high, elution was easy and quantitative, and the adsorbent could be regenerated easily and it appears indefinitely. After my return to Uppsala I found that people at Pharmacia had lost their confidence in me and rejected collaboration. I was told that Pharmacia already had sufficient competence to develop new chromatographic media and IMAC was not likely to compete with ion-exchange chromatography. Besides, the market was already saturated. Disappointed, I left IMAC for a few years only to take it up seriously again in the early 1980s. Many of my collaborators and students have made valuable contributions. We first concentrated our studies on the transition elements Co2C , Ni2C , Cu2C and Zn2C and found histidine residues in proteins and peptides to play a dominant role. Thanks to Eugene Sulkowski’s contributions, IMAC has now in many peoples mind become synonymous with protein chromatography on borderline metal ions, copper and nickel in particular. IMAC, outside the quartet of metal ions mentioned is largely unknown despite some published orientation studies. According to Ralph Pearson’s generally accepted nomenclature soft and hard metal ions have easily and not easily polarized outer electron clouds. Hard metal ions are, for example, Ca2C , Mg2C , Al3C ; whereas, Hg2C , AgC , Pt2C are soft, and Cu2C , Ni2C are borderline metal ions [13]. Immobilized soft ions have presented problems. Pd2C , Au3C and AgC are presently studied by M. Vijayalakshmi and myself. The affinity seems to be too broad and the selectively not good enough for protein purification. Their applications to protein immobilization appears promising. Chromatography using immobilized hard metal ions, also a virgin field, is more promising than the soft ions and can be applied selectively to biomolecules of great importance, such as phosphoproteins and calcium-binding proteins. It is remarkable that IMAC — so effective in the one step isolation of many histidine-tagged recombinant proteins — has not yet been used much for isolation of calcium binding proteins. There are a few exceptions. A number of calcium-binding serum proteins were isolated by Tuula Mantovaara in my team. Grigory Chaga, Bo Ersson and I later found that the required experimental conditions, at least in some cases, are strange and unexpected. Calmodulin present in the extract from bovine testes was selected as our model. Electrophoretically pure calmodulin was isolated by chromatography on Fe(III) IDA agarose followed by Eu(III)-TED agarose (TED D triscarboxymethylethylenediamine); Fig. 1a,b. It is interesting to note that CaCl2 and NaCl at high concentration in the buffer are necessary for high capacity adsorption. Selective separation of calmodulin from other calcium-binding proteins is accomplished by removal of calcium and inclusion of malonate in the phosphate buffer eluent. A hypothetical role for the excess calcium was suggested. Immobilized Fe3C and Al3C have been used to fractionate phosphoproteins, phospho-peptides and nucleotides. Glycogen phosphorylase and lactic dehydrogenase were both isolated in a single chromatographic step from crude chicken breast extracts. Fractionation could be scaled up directly from the milligram to the gram scale according to the same elution scheme.

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Fig. 1. IMAC as applied to immobilized hard metal ions: isolation of calmodulin. Sample: Three hundred milligram of crude calmodulin dissolved in 1.9 ml of 20 mM PIPES, I M NaCl, pH 6.1 (Buffer A). Column: 6:5 ð 1 cm i.d. containing Fe(III) chelating Sepharose FF equilibrated in Buffer A. Chromatography: The sample was applied to the column and washed with Buffer A, followed by Buffer A adjusted to pH 6.9, which eluted calmodulin in peak II. Sample: 50 ml of peak II from (a) was saturated to 2.0 M NaCl, 0.2 M CaCl2 . Column: 6:3 ð 0:5 cm i.d. containing Eu(III) TED Novarose equilibrated with Buffer B 0.1 M Tris-HCl, 2 M NaCl, 0.2 M CaCl2 , pH 7.5. Chromatography: The sample from peak II (a) was applied to the column and washed with Buffer B, followed by Buffer B to which Na2 SO4 was added to 0.6 M. Concanavalin was obtained electrophoretically pure in quantitative yield in peak III by adding 40 mM malonate to the buffer. The adsorbents were regenerated by elution with 0.1 M EDTA, pH 7.0, followed by recharging with respective metal ions. More details are given in J. Chromatogr. A, 732 (1996) 261–269.

52.I.7. Strategies and future prospects [14] The diversified supply of different adsorbents makes it exceedingly difficult to decide what kind of strategy should be chosen to solve a particular protein fractionation problem. We may distinguish between three kinds of chromatographic strategies to deal with such problems: (1) To maximize the separation in a single bed. (2) To separate the substances according to their affinities for a number of selected adsorbents: differential affinity chromatography. (3) Combination of 1 and 2. Almost all chromatographic applications have been made in single beds, while only a few have been performed by differential chromatography and, as far as I know, none by the combined use of strategy 1 and 2. Cascade-mode multiaffinity chromatography, CASMAC, of human serum exemplifies a particular mode of differential chromatography [12,13,14]. There are many problems waiting in the future that require improved chromatographic techniques. Further development of strategies according to principles (1) and (2) can be anticipated. Dis-

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Fig. 2. One of the first experiments by J. Porath on size exclusion chromatography made on cross-linked dextran (Sephadex) in my laboratory in the 1950s.

placement and spacer-displacement chromatography offer extremely powerful ultimate purification methods for protein pharmaceuticals of natural origin. Synthetic proteins and peptides for medical use will also require extraordinarily effective chromatographic methods. It should be possible to use all the adsorbents I have described in micro- and submicroscale chromatography. My interest is now going in the opposite direction, towards macro- and megascale, with other demands on the matrix. A new kind of matrix based on cross-linked polyethyleneimine and agarose has recently been synthesized by Roberto Guzman, Bo Ersson and myself. Granular beds of this material which also acts as an IMA-adsorbent permit very high linear flow-rates (>30 ml=hour) and very high capacities (>150 grams of Cu(II) per liter gel swelled). Metal ions are entering and leaving the gel at very high diffusion rates. Eventually, adsorbents based on this new matrix may find use for concentration of low grade ore extracts and mine tailing leachates, as well as for environmental clean-up processes such as removal of aromatic contaminants (with EDA-adsorbents) and toxic metal ions (with IMA-adsorbents) from industrial and natural waters. Only the first steps in such a pretentious program have been taken and practical applications have to wait far into the 21st century.

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References 1. 2. 3. 4.

5. 6.

7.

8. 9. 10. 11. 12. 13. 14.

J. Porath and P. Flodin, Gel filtration: A method for desalting and group separation, Nature, 183 (1959) 1657. J. Porath, The twelfth Hopkins memorial lecture. Molecular-sieving and non-ionic adsorption on polysaccharide gels, Biochem. Soc. Trans., 7 (1979) 1197. T.C. Laurent, History of a theory. Chromatography classic, J. Chromatogr., 633 (1993) 1. J. Porath, Recent advances in separation methods in gamma globulin, in Proc. of the Third Nobel Symposium, Stockholm 1967, J. Killander, Almqvist and Wiksell (Ed.), Stockholm and Interscience Publishers, New York, NY, 1967, p.287. P. Cuatrecasas, M. Wilchek and C.B. Anfinsen, Selective enzyme purification by affinity chromatography, Proc. Natl. Acad. Sci. USA, 61 (1968) 636. J. Porath and R. Axe´n, Immobilization of enzymes to agar, agarose and Sephadex supports, in Methods in Enzymology XLIV: Immobilized Enzymes, K. Mosbach (Ed.), Academic Press, New York, NY, 1976, p. 19. J. Porath, T. La˚a˚s and J.C. Janson, Agar derivatives for chromatography, electrophoresis and gel-bound enzymes. II. Rigid agarose gels cross-linked with divinyl sulphone (DVS), J. Chromatogr., 103 (1975) 49. J. Porath, F. Maisano and M. Belew, Thiophilic adsorption a new method for protein fractionation, FEBS Lett., 185 (1985) 306. J. Porath and S. Oscarsson, A new kind of thiophilic electron-donor–acceptor adsorbent, Makromol. Chem. Macromol. Symp., 17 (1988) 359. P. Berna and J. Porath, Salt-independent adsorption of human serum proteins on cyanocarbon gels, J. Chromatogr. B., 693 (1997) 277. J. Porath, Protein Expr. Purif., 3 (1992) 263. L. Ka˚gedal, Immobilized metal ion affinity chromatography, in J.-C. Janson and L. Ryde´n (Eds.), in Protein Purification, 2nd Ed., Wiley-Liss New York, NY, 1988, p. 311. R.G. Pearson, Hard and soft acids, HSAB, part I. Fundamental principles, J. Chem. Ed., 45 (1968) 581. J. Porath and P. Hansen, Cascade-mode multiaffinity chromatography. Fractionation of human serum proteins, J. Chromatogr., 550 (1991) 751.

D.53. Michel Prost (invited paper) Michel Prost was born September 23, 1948 in Autun, France. He studied biochemistry, physiology and molecular biology, first at the University of Strasbourg, then at the University of Dijon where he received his M.Sc. degree in Physiology — Molecular and Chemical Biology, in 1969. He joined the Medical Biochemistry Laboratory of the Medical School of Dijon where he started research in biochemistry under the direction of Professor Prudent Padieu. His research was centered on the development of well-adapted and sensitive gas chromatographic methods coupled to mass spectrometry and mass fragmentography for the study of corticosterone synthesis and metabolism in newborn rat adrenal cells in cultures. He received his degree of ‘Doctor in Biochemistry’ in 1973.

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In 1975, M. Prost was the principal founder of the S.P.I.R.A.L. Co. (Socie´te´ de produits pour l’Industrie, la Recherche et les Analyses de Laboratoires), a high technology company for the study, manufacture and marketing of materials and products for industry, research and chemical analysis laboratories. At the same time, he was the founder of a training center for the biological investigations in humans and mammals, the C.E.F.I.B.R.A. Co. (‘Centre de Formation et d’Investigations Biologiques’). In 1984, he was also the co-founder of C.E.D.R.A. (Centre Europeen de Recherche et d’Analyses). As President of the above three companies, M. Prost has focused and developed for the past sixteen years the activities of these companies and analytical centers in three ways: ž Manufacture of materials and reagents for the analytical biochemistry laboratory, especially for gas chromatography and mass spectrometry in the fields of water and air pollution control, agronomy, nutrition and clinical chemistry. ž Analytical studies and pharmacological studies of various medicines for their expert valuation and marketing approval; ž Research, setup, development and manufacture of new materials and apparatus in analytical chemistry, cell biology and biotechnology. M. Prost developed a ‘Biological Test to Measure the Human Resistance Capability to Free Radical Attack.’ Today, this biological test, a SPIRAL patent pending, has widely proved its availability for the simple sensitive measurements of the total anti-oxidative defense status of individuals. M. Prost is author and co-author of many scientific publications and has been awarded ten international patents in analytical biochemistry, cellular biology and biotechnology. He is also co-author=editor of two treatise books entitled “Les Nouvelles Dimensions de la Chromatographie en Phase Gazeuse”, published by Labo-France, Editor (1982), and “Me´thodes Chromatographiques Couple´es a` la Spectro-me´trie de Masse”, published by Masson S.A. (1986). M. Prost is an expert analyst at the Common Bureau of Reference of the European Economic Community for the chemical quality monitoring of irradiated foods. He is also an ‘expert analyst’ attached to the French courts and to the insurance companies for the control and=or determination of chemical environmental pollutions, fire origins, wood treatments, adulterations in enology, etc. He has many scientific connections and collaborative works with the French Research Organizations, i.e., C.N.R.S. (Centre National de la Recherche´ Scientifique), I.N.S.E.R.M. (Institut National de la Sante´ et de la Recherche´ Me´dicale), I.N.R.A. (Institut National de la Recherche´ Agronomique), and with many French and foreign academia. Dr. Prost served as general chair for the NASA International Conference and Workshop for the Future of Humans in Space, Dijon, France (1993). See Chapter 5B, a, d, e, h, k, p, r, s

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53.I. CAPILLARY–GAS CHROMATOGRAPHY COUPLED-MASS SPECTROMETRIC ANALYSIS OF STEROIDS, BIOGENIC AMINES AND AMINO ACIDS IN BIOLOGICAL MATERIALS Michel Prost Pre´sident des Socie´te´s ‘S.P.I.R.A.L.’, et du Centre de Recherche´s et d’Analyses ‘C.E.D.R.A.’, 21560 — Couternon, France

On the French side, SPIRAL was the first company involved in the production of capillary columns with GC producers and GC–MS manufacturers (US, Dutch, Finnish, French, German). Knowledge came from the experience that Michel Prost acquired as a pioneer with B. Maume and P. Padieu for the first coupling of GC–MS with a capillary column with or without solid injector [1–2]. His first papers were written between 1970 and 1975 using capillary column–gas chromatography–mass spectrometry with a multiple ion detector for analysis of steroids, catecholamines and amino acids in biological material at the nanogram and picogram level [3,4]; these studies opened a new area for the selective,

Fig. 1. Mass fragmentogram of total steroid extract from 4-day-old male rats. Corticosterone (B) and 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) are detected by the m=e 517 ion. Each reference hormone (200 ng) as their MO-perdeuterated-TMS derivatives are injected with the sample. They are detected by the m=e 535 fragment ion and used as internal standards. Three THB isomers are detected by the fragment ion at m=e 564; 3α, 11β, 21-trihydroxy-5β-pregnane-3, 20-dione (THB III). 3β, 11β, 21-trihydroxy-5β-pregnane-3, 20-dione (THB IV), and 3β, 11β, 21-trihydroxy-5α-pregnane-3, 20-dione (THB II). The 18-hydroxycorticosterone (18-OH B) is recorded by the m=e 605 fragment ion.

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Fig. 2. Mass fragmentogramme of a quantitative analysis of 18 OH DOC and corticosterone extracted from 2 rats adrenals (male, 15 days old.) Detection of extracted hormones by m=e 517 ion (unbroken line). Detection of 200 ng of each deuterated reference hormone corresponding to (Et. Int. 1 and Et. Int 2.) playing the role of internal standard by ion at m=e 535 (broken line). The multiple ion detection (Dim) is obtained by the peak matching comparator (LKB 9060) [14]. The energy of the electron beam is at 28 eV. The detection sensitivity is 3 times more for m=e 517 (ð600) compared to ion m=e 535 (ð200).

sensitive and specific methodology which is now in world wide daily practice in all research and control laboratories. Fig. 1, presented as a reference of qualitative mass fragmentography analysis, has been published in different papers or books [4–7]. This mass fragmentogram of a total steroid extract represents a typical quantitative analysis of the hormones in adrenal and liver cell cultures using perdeurated derivatives or isotopic compounds as the internal standards. This fragmentogram shows all the possibilities presented by the coupling of the separation power of GC (for isomers) with the sensitivity and the detection of specific fragment ions. In this figure, we can detect two different steroids under the same peak plus the deuterated internal standard added before extraction to the biological material. The other specific ions show the detection of different corticosteroids present at a very low level in the biological material. The fragmentogram (Fig. 2), presents a quantitative analysis of 2 hormones (position isomer 18 OH-DOC and corticosterone) with their respective deuterated internal standard. We can appreciate the quality of the fragmentogram obtained from 2 adrenals extracted from a newborn rat [8].

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Our 1972 conclusion [1] was gas chromatography–mass spectrometry and mass fragmentography allow automated analysis of biological materials, a high level of resolution, and selectivity and sensitivity. At this time, the preparation of the analytical sample remains a non-automated step. Sophisticated gas phase methods are highly suitable for quantitative work in advanced biology, while gas chromatography is more and more used in biological routine analysis. In this field of modern physico-chemical analysis, fundamental research is very quickly followed by application to such practical problems as from the environment, clinical analysis, food chemistry and nutrition. The next steps of capillary gas-phase analysis will be application to solving biological problems using the new methodologies and instrumentation of hyphenated techniques, as combined GLC–MS-computers, chemical ionization detection, coupled glass capillary column–MS and peak matching-multiple ion detection on GLC effluents.

References 1.

2.

3.

4.

5. 6.

7.

8.

9.

B.F. Maume, P. Bournot, J. Durand, J.C. Lhuguenot, G. Maume, M. Prost and P. Padieu, New developments in steroid and catecholamine analysis in biological media by gas chromatography– mass spectrometry using a multiple ion detector, L’expansion Scientifique Franc¸aise, Organisation des Laboratoires Biologie Prospective lle Colloque de Pont a` Mousson, 1972 pp. 637–654. B.F. Maume and J.A. Luyten, Evaluation of gas chromatographic mass spectrometric and mass fragmentographic performance in steroid analysis with glass capillary columns, J. Chromatogr. Sci., 11 (1973) 607–610. B.F. Maume, P.Bournot, J.C. Lhuguenot, C. Baron, F. Barbier, G. Maume, M. Prost and P. Padieu, Mass fragmentographic analysis of steroids, cate´cholamines and amino acids in biological materials, Anal. Chem., 45 (1973) 1073–1082. M. Prost and B.F. Maume, Hormonal steroids in biological materials. A study by mass fragmentography, in A. Frigerio and N. Castagnoli, Jr. (Eds.), Mass Spectrometry in Biochemistry in Medicine, Raven Press, New York, 1974, pp. 139–150. J. de Graeve, F. Berthou and M. Prost (Eds.), Me´thodes Chromatographiques Couple´es a` la Spectrome´trie de Masse, Masson, Paris, 1986, p. 61. P. Padieu and B.F. Maume, Evaluation by mass fragmentography of metabolic pathways of endogenous and exogenous compounds in eukaryote cell cultures, in Quantitative Mass Spectrometry in Life Sciences, Elsevier, Amsterdam, The Netherlands (1977). P. Bournot, M. Prost and B.F. Maume, Separation and characterization of the reduced metabolites of 18-hydroxy-deoxycorticosterone hormone by gas–liquid chromatography–mass spectrometry; occurrence of stereoisomeric forms in rat adrenals and liver, J. Chromatogr., 112 (1975) 617–630. M. Prost and B.F. Maume, Hormones steroides de la surre´nale de rat. Analyse des 18-hydroxycorticoste´roides par chromatographie gaz liquide couple´e a` la spectrome´trie de masse et par fragmentographie de masse, J. Steroid Biochem., 5 (1974) 133–144. D. Blache and M. Prost, Free radical attack: Biological Test for Human Resistance Capability, in C. Ponnamperuma and C.W. Gehrke (Eds.), A Lunar-Based Chemical Analysis Laboratory, A. Deepak, Hampton, VA, 1992, pp. 82–98.

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D.54. Jacques Rijks Development and instrumentation in capillary gas chromatography– chromatographic education in the Third World, Foundation EDC International, DeKoppele 391, 5632 LN Eindhoven, The Netherlands

Jacques Rijks, the recipient of the 1993 Stephen Dal Nogare Award, presented by the Chromatography Forum of the Delaware Valley, was honored for his contributions to fast-GC at the femtogram level, and for his efforts to expand the application of chromatography to third world countries. Born in Brunssum, The Netherlands in 1934, he graduated from St. Joseph College in Sittard in 1951 and began his career in the production control laboratory of the Fertilizer Plant Group of the Dutch Coal Mines in Geleen. In 1952 (the early days of chromatography), he became involved in method development, instrument design, automation and research in gas chromatography at that company. He started graduate studies at the Faculty of Chemical Engineering at the Eindhoven University of Technology in 1962, while working full time. In 1965, he joined the group of Professor Dr. Ir. A.I.M. Keulemans and was employed as the leader of the Instrumental Analytical Service Group until he graduated in 1970. While there, he started his Ph.D. studies on ‘Identification of Hydrocarbons by Capillary Gas Chromatography’. He received his Ph.D. degree in Technical Sciences in 1973, and became head of the GC group in the Laboratory of Instrumental Analysis of the Faculty of Chemical Engineering of the Eindhoven University of Technology in 1975. There he continued his work, initially on development of preparation methods

Dr. Rijks in his office at Eindhoven, Netherlands (1986).

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Dr. Rijks at a training course on capillary GC in Ho Chi Minh City, Vietnam (1998).

for glass capillary columns, and later in fused silica capillaries and sample introduction systems for various application areas. Since 1980, Dr. Rijks has focused on the development and instrument design of high-speed capillary-gas chromatography (15 peaks per second and femtogram detection limits) in combination with small-bore capillary columns (<50 µm). The design, development and evaluation of modular, massflow controlled, multidimensional systems that allow the introduction of large sample volumes, speed-programmed sample introduction, and cold-temperature programmed sample introduction, were other major subjects of interest. Dr. Rijks has published over 130 co-authored papers during the past 15 years in international journals. He was frequently sought as plenary lecturer, seminar speaker and consultant of industries, instrument companies etc., all over the world. He has been an active Editorial Board member of chromatography journals and organizing committees of international symposia. He was one of the pioneers in funding of the research in Dutch universities by national and international contract research with industries, institutes and instrument companies. After his early retirement from the Eindhoven Technical University in 1991, he took the initiative to set up Centers of Excellence for Education and Development of Chromatography (EDC centers) in Third World industrial development countries. The first of these EDC centers, located in Hanoi Vietnam, was established in November 1993. The second one in Ho Chi Minh City was started in 1997. More than 250 Vietnamese analytical chemists and laboratory managers from various Vietnamese industries, institutes and universities have been trained in these centers in the past years. Various Vietnamese laboratories have been efficiently assisted at source with method

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development, introduction of new methods, selection, service, maintenance and repair of equipment in the domain of chromatography. Entering this Third World, which can be characterized by an extremely poor economy with substandard working and living conditions, has reinforced his dedication to education and development in the field of chromatography. See Chapter 5B, d, h, k, l

References 1.

J. Curvers, J. Rijks, K.A. Cramers, K. Knauss and P. Larson, Temperature programmed retention indices: Calculation from isothermal data. Part I: Theory, J. High Resolut. Chromatogr. Chromatogr. Commun., 8 (1985) 607–611. 2. J. Curvers, J. Rijks, K.A. Cramers, K. Knauss and P. Larson, Temperature programmed retention indices: calculation from isothermal data. Part II: Results with non polar columns, J. High Res. Chromatogr. Chromatogr. Commun., 8 (1985) 611–618. 3. J. Staniewski and J.A. Rijks, Solvent evaporation rate in temperature-programmed injection of large sample volumes in capillary gas chromatography, J. Chromatogr., 623 (1992) 105–113. 4. C.P.M. Schutjes, E.A. Vermeer, J.A. Rijks and K.A. Cramers, Increased speed of analysis in isothermal and temperature-programmed capillary gas chromatography by reduction of the column inner diameter, J. Chromatogr., 253 (1982) 1–16. 5. C.P.M. Schutjes, P.A. Leclercq, J.A. Rijks, K.A. Cramers, C. Vidal Madjar and G. Guiochon, Model describing the role of the pressure gradient on efficiency and speed of analysis in capillary gas chromatography, J. Chromatogr., 289 (1984) 163–170. 6. C.P.M. Schutjes, Ph.D. Thesis, High Speed, High Resolution Capillary Gas Chromatography, Eindhoven University of Technology, 1983. 7. K.A. Cramers and P.A. Leclercq, Considerations on speed of separation, detection and identification limits in capillary GC and MS, CRC Crit. Rev. Anal. Chem., 20 (1988) 117–147. 8. A.J.J. van Es, J.A. Rijks and K.A. Cramers, Turbulent flow in capillary gas chromatography, J. Chromatogr., 477 (1989) 39–47. 9. A.J.J. van Es, J.A. Rijks, K.A. Cramers and M.J.E. Golay, Turbulent flow in capillary gas chromatography, J. Chromatogr., 517 (1990) 143–159. 10. A.J.J. van Es, H.G.J. Janssen, K.A. Cramers and J.A. Rijks, Sample Enrichment in high speed narrow bore capillary gas chromatography. J. High Resolut. Chromatogr. Chromatogr. Commun., 11 (1988) 852–857. 11. P.A. Leclercq and K.A. Cramers, High Speed GC–MS, Mass Spectrom. Rev., 19 (1998) 37–49. 12. K.A. Cramers, H.G. Janssen, M.M. van Deursen and P.A. Leclercq, High-speed gas chromatography: an overview of various concepts, J. Chromatogr. A, 856 (1999) 315–329.

D.55. Pat J. Sandra Pat J. Sandra was born in 1946 in Kortrijk, Belgium. He studied at the University of Ghent and received his Ph.D. in Sciences in 1975, under M. Verzele. Since then, he has been on the Faculty of Sciences at the same University where he is presently Professor in Separation Sciences at the Department of Organic Chemistry. In 1986, he founded the Research Institute for Chromatography in Kortrijk, Belgium, a center of excellence for research and education in chromatography, capillary electrophoresis and

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mass spectroscopy. He is currently also Visiting Professor at the Eindhoven University of Technology, The Netherlands. In 1998, he accepted a part-time position at the University of Stellenbosch, South Africa to establish a center of excellence in separation sciences. A major area of his research has been the development of microcolumn separation methods to study natural products and pollution. Innovative contributions were made in capillary GC with the introduction of silanol terminated stationary phases, the immobilization of the stationary phases, and the development of high temperature GC. His present research activities include automated sample preparation methods, hyphenated systems and electromigration methods. Pat Sandra is a frequent traveler presenting lectures throughout the world and is appreciated for his high standard of teaching. He is the author of more than 300 scientific papers in the international literature, contributed as author or co-author to books on “High Resolution GC”, (Ed. 3, 1989), “Sample Introduction in Capillary GC” (1985), “Essential Oil Analysis, Introduction to Micellar Electrokinetic Chromatography” (1992) and “Water Analysis”, and is Editor of several books and journals, including the Journal of Microcolumn Separations. He is the Chairman of the International Symposium on Capillary Chromatography, the Chairman of the Foundation ‘International Organization for the Promotion of Microcolumn Separations’ and member of the permanent committee for the promotion of chromatography in Latin America. He was also the coordinator of an educational program, sponsored by the European Community, in High Resolution Chromatography for Central European Countries. He was the recipient of the 1989 Tswett Award (Russian Chromatography Society), the 1994 Dal Nogare Award, the 1994 Martin Gold Medal, the 1995 Golay Award and the 1996 COLACRO Medal. See Chapter 5B, a, d, e, h, l, p, q, s

55.I. THIRTY YEARS IN SEPARATION SCIENCES (1969–1999) Pat J. Sandra University of Ghent, Vakgroep Organische Chemie, Krijgslaan 281 (S.4) 9000 Ghent, Belgium

My introduction to the fascinating field of separation sciences started in 1969 when Maurice Verzele, Director of the Department of Organic Chemistry at the University of Ghent, Belgium invited me to join his research team and to prepare a Ph.D. thesis on ‘The Contribution of Hops to the Flavour of Beer’. At that time there was a controversial discussion going on in how far the essential oil of hops was contributing to the flavor of our ‘national product’ beer. From the beginning, it was clear that to unravel the complexity of a beer extract, high resolution techniques were needed which explains why my earliest work was in capillary GC.

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Bouche and Verzele developed in the late sixties the static coating procedure for glass capillary columns and when Bouche left the department, Verzele decided that, as part of my Ph.D. work, I had to continue the work in capillary column technology. Priorities were the development of polar columns and the increase of the maximum allowable operating temperature (MAOT) for apolar columns. My research immediately focused on capillary column making and I considered the essential oils of hops and beer as perfect model mixtures to evaluate the performance of the prepared columns. Nevertheless, my Ph.D. was defended in 1975 [1] and I had my hands free to develop column technology further, although I never lost my interest in natural product research [2]. In the next ten years, several developments were made and our research contributed to the state-of-the-art in column technology. To mention a few contributions: surface treatment by leaching, dendrite and whisker formation, deactivation by persilylation and with amino-alcohols, immobilization of the stationary phases, introduction of new stationary phases, e.g., superoxes, crosslinkable biscyanosilicone phases, silanol-terminated silicone phases, etc. During our work, we immediately recognized the importance of flexible fused silica columns introduced by Dandeneau and Zerenner, and several developments were realized on this material. A nice anecdote, in this respect, concerns a letter I received from the late Kurt Grob, at that time an authority in capillary column making. The letter was sent to my private address from the University of Stellenbosch, South Africa, where Kurt was teaching a course in column making in the laboratory of Ben Burger (what a coincidence, see further). The reason why the letter was sent to my private address was twofold. First of all my former boss, Verzele, and Grob were not at all good friends after a hard discussion at the First International Symposium on Glass Capillary Columns organized by Rudolf Kaiser, in Hindelang, Germany, May 1975, and secondly, I was ready to quit from the university (see further). A paragraph of the letter is given in Fig. 1. Kurt was not at all in favor of fused silica columns for several reasons. Firstly, by his leaching, silylation or BaCO3 deposition procedures developed for borosilicate glass,

Fig. 1. Letter sent by Kurt Grob.

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he could create surfaces wettable for a wide variety of stationary phases which often could be immobilized. Secondly, he considered fused silica columns too expensive and last but not least, and because he was very handy, he could not understand why the installation of a flexible fused silica column was easier than that of a rigid glass column. As a remark to the paragraph of the letter, Sandy Lipsky indeed published the first papers on high temperature capillary GC, but the columns used were coated with silanol terminated silicone phases that I secretively sent him. Sandy invited me to be co-author of his publications, but my situation at the University did not allow me to do so. I cannot conclude this ‘column making period (1969–1986)’ without mentioning some other anecdotes and events. Some of them had an important impact on my scientific career. I remember in the mid-seventies a visit of Dr. Stuart Cram, at that time R&D manager at Varian, to our laboratory. Stuart was visiting different laboratories in Europe (Grob, Schomburg, Cramers, Verzele, etc.) to prepare Varian for the post Perkin-Elmer period in capillary GC (what an obsession at that time?). For our routine analyses of natural products and essential oils, we were mainly working with 0.5 mm i.d. glass columns, 50–100 m in length and this for obvious reasons, e.g., large sample capacity, direct injection, coupling with MS via the all glass jet separator, odor evaluation and fraction collection [3], etc. Stuart was disappointed with his visit, because the future was, as confirmed by our European colleagues, the use of 0.2–0.3 mm i.d. columns. Fifteen years later, Hewlett-Packard introduced megabore 0.53 mm i.d. fused silica columns and Stuart, now an HP employee, presented several lectures on their features — i.e., ease of use, direct injection, coupling via the jet separator to MS, etc. Looking back to this period (1969–1986), I had the fortune of meeting and working with many outstanding scientists. Walt Jennings was the first to invite me to the US, the late Al Zlatkis and Leslie Ettre offered me the possibilities to present our work at the symposium series ‘Advances in Chromatography’ and the ‘Tarrytown’ meetings, Sandy Lipsky was opening several doors for the neophyte, Denis Desty was collaborating in our study on capillary columns with unconventional cross-sectional geometry, Harold McNair invited me to be an instructor for the American Chemical Society. The laboratory of Gerhard Schomburg (the most outstanding laboratory in capillary GC in Europe at that time), had an open door for me and I had several in depth discussions with Gerhard which were eye-openers for me; a fruitful collaboration was started with Karel Cramers and Jacques Rijks, and last but not least there was Rudolf Kaiser. I met Rudolf Kaiser the first time at the second International Symposium on Glass Capillary Gas Chromatography in Hindelang, May 1977, and I immediately admired his knowledge, enthusiasm and entrepreneurial skills. Rudolf invited me to become a member of the scientific committee for the 1979 and 1981 meetings and, two years later to join the Editorial Board of Journal High Resolution Chromatography and Chromatographic Communications. After the closing address of the 1981 meeting, Rudolf invited me to have a walk in the beautiful mountains of the Alps and asked me, to my great surprise, to take over the chairmanship of the meeting. After some hesitation, I decided to accept and together with Jacques Rijks and Sorin Trestianu, I started the ‘Riva meetings’. This symposium series has been very successful and maybe this is my most important contribution to capillary chromatography — bringing scientists together

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Fig. 2. Some thoughts of Marcel Golay jotted down on the back of a program of the 6th International Symposium on Capillary Chromatography, in Riva del Garda, Italy, May 15, 1985.

to exchange ideas in a beautiful and relaxing surrounding. The 23rd meeting will take place in Riva in 2000. Meetings were also organized in the US (Monterey, Baltimore, Wintergreen, Park City) and in Japan (Gifu, Kobe) with respectively, Milton Lee and Kiyokatsu Jinno, as chairpersons. As a spin-off of the Riva-meetings, COLACRO was founded to promote chromatography in Latin-America with Fernando Lanc¸as as chairman and organizer of a biennial meeting. The organization of the Riva symposium series has not only tremendously influenced my scientific career, but also strengthened ties of friendship with scientists like Milos Novotny, Milton Lee, Jim Jorgenson, Keith Bartle, Shigeru Terabe, Marcel Golay, Arnaldo Liberti, to mention only a few. As an illustration of the scientific atmosphere at the Riva meetings, Fig. 2 shows some thoughts of Marcel on turbulent flow in capillary GC jotted down on the back of a program of the 1985 Riva meeting during a very late informal discussion with Karel Cramers and myself at the bar of hotel du Lac et du Parc.

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Fig. 3. Presentation of the Marcel Golay Award to Ray Dandeneau and Ern Zerenner, the inventors of fused silica columns, at the 10th International Symposium on Capillary Chromatography, Riva de Garda, May 22, 1989.

In 1989, the scientific committee of the meeting together with Leslie Ettre and John Hinshaw from Perkin-Elmer decided to establish the Marcel Golay Award for outstanding contributions in capillary chromatography. Marcel was very pleased with the award and accepted our invitation to present himself, for the first time, awards to Rudolf Kaiser, as initiator of this symposium series and to Ray Dandeneau and Ern Zerenner for the invention of fused silica capillary columns on the occasion of the 10th Riva meeting held May 22–25, 1989. With deep regret we learned of his sudden death in the night of April 28. As chairman of the meeting, I had to take over Marcel’s task and presented the awards in an emotional session (Fig. 3). Coming back to my academic career, starting from the beginning of 1985, I was pushing very hard at the University to broaden our research field in separation sciences and to start investigations in supercritical-fluid chromatography and capillary electrophoresis. Unfortunately, the director of the department was not willing to support me with the necessary funds to purchase a syringe pump, a nanoliter valve and a high power supply and I decided to quit the university on February 1, 1986. Under the influence of entrepreneur Rudolf Kaiser, I started a Research Institute for Chromatography and very soon my activities continued in the garage of our house. I survived through sponsorships, in the beginning by Carlo Erba, and later by Hewlett-Packard. Research in column technology was slowing down, because, by the introduction of fused silica columns, column-manufacturing companies intensified their research (Chrompack, Hewlett-Packard, J&W, Quadrex, Alltech, SGE, Supelco, etc.) and it was senseless to compete with them. Our contributions since 1986 are restricted to

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Fig. 4. FAME analysis of milk fat on a 25 m ð 0.25 mm and a 10 m ð 0.1 mm i.d. column.

the introduction of some high temperature phases, biscyanopropyl silicone phases, chiral stationary phases and apolar and polar capillary columns with i.d’s equal or smaller than 100 µm for fast GC [4]. In 1986, we did some experiments with resistant heated capillaries. Fused silica tubing was enveloped in a stainless steel jacket, insulated like an electrical wire and the metal column ends were connected to a power supply. Retention time reproducibility was not excellent, but nevertheless the principle was patent pending for one year. Because of lack of funds, I could not continue the patent application. A missed opportunity — flash GC. Our recent data in fast capillary GC, however, illustrate, that at present GC instrumentation with fast separations can be realized in a reproducible way maintaining the resolution like on conventional columns. The method translation software developed by Leon Blumberg and Matthew Klee is extremely useful in this respect. Fig. 4 compares the analysis of the fatty acid methyl esters of milk fat on a 25 m ð 0.25 mm i.d. (top) and on a 10 m ð 0.1 mm i.d. (bottom) column both coated with polyethylene glycol applying the method translation software. The gain in analysis time is a factor of 8!

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In order to fully exploit the potential of capillary GC, developments were needed in sample inlets, sample preparation, selectivity tuning and multidimensional systems, enhanced hyphenation with spectroscopic detectors, etc. Only two important areas of our activities involving hyphenated systems will be discussed, namely polydimethylsiloxane (PDMS) sorption and coupling capillary GC to ICPMS. Carlo Bicchi, Professor at the University of Turin, Italy visited the Institute to discuss an interesting and challenging problem, namely capturing volatiles emitted by living plants. Our joint research efforts resulted in the development of open tubes coated with a 20-µm film of PDMS. Retaining the volatiles by sorption rather than by adsorption offered a number of advantages, the most appreciated at that time being the prediction of breakthrough volumes and the inertness. In one of our publications [5] on this subject, we concluded that: “The knowledge of the chemical composition of the atmosphere surrounding an odorous plant is of great importance in many fields : : : The emitted compounds constitute a system with which the plant maintains its ‘public relations’ with the surrounding environment. In fact the life and mutual interactions between vegetable and animal species (i.e., allelopathic phenomena) in a certain ecosystem are often controlled by chemical signs (messages), normally governed by the emission of volatiles from the plant itself. In this contribution, the sampling device, used to capture the volatiles directly from the living plant is described and the application of partition chromatography as a trapping technique is reported.” Much later, I received an important grant from the University to develop this further, in collaboration with the Department of Biotechnology. The study of volatiles emitted by living plants under biotic or abiotic stress (chemical analysis) can lead to the identification of the genes which are responsible for their production, and of the genes which react on the stimulus (genetic analysis) which eventually can lead to genetic engineering. A picture of the system we recently developed to monitor continuously the headspace surrounding living plants put under stress and allowing static and dynamic sampling is given in Fig. 5. In collaboration with the Eindhoven University of Technology, the capacity of open tubular traps coated with PDMS was strongly enhanced by synthesizing PDMS particles of 0.1–0.5 mm size. The sorption principle was further broadened by applying, besides the breakthrough mode, the equilibrium mode. Studies on the mechanism (sorption or adsorption) finally led to the development of stir bar sorptive extraction (SBSE), which in principle is the same like solid phase microextraction (SPME), but with a sensitivity increase by a factor of 100–1000. Another subject of intensive research was related to the determination of organometallics in environmental samples. This project started in 1982 in collaboration with the Analytical Department of our University (remember I am an organic chemist). The project was recently finalized [6] with the development of the hyphenated system SPME (or SBSE)–capillary GC–ICPMS. Quantities down to the ppt level of organo-mercury, -tin, and -lead can be determined in environmental and marine samples. Most of the developments we made in capillary GC in the period 1988–1999 were results of collaboration between the Research Institute for Chromatography and the Separation Sciences Group at the University of Ghent. On February 1988, I indeed accepted the invitation of the new director of the Department in Organic Chemistry

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Fig. 5. Automated system for the analysis of volatiles emitted by living plants.

to restart my activities at the University of Ghent and to lead the group of Separation Sciences. Because of my duties at the Institute, I could only accept a half time position and my ex-Ph.D. student, Frank David took over most of my R&D tasks at the Institute. Under the influence of Frank and my co-workers, the Institute was growing. Fig. 6 shows a picture of its present premises. Besides method development, the Institute presently organizes courses in all fields of separation sciences. The most successful courses are Advances in Capillary GC, Capillary GC-MS, and Environmental Analysis. Back at the University, I decided to focus our research on capillary electrophoresis (CE), supercritical-fluid chromatography (SFC), and hyphenated systems. An important activity of the group, however, was and still is ‘service’ for the real organic chemists. This is a challenging task because the Department of Organic Chemistry has more than 100 co-workers, of which 50–60 are preparing their Ph.D. A large number of developments are therefore made directly to meeting the analytical needs of the department. When I took over the Separation Sciences Group, one of the co-workers was Jo Vindevogel. Jo was a brilliant student, but did not finish his Ph.D. dissertation because his research had been too diverse. I proposed to him to restart completely his Ph.D. work with micellar electrokinetic chromatography (MEKC) of natural products as the subject. MEKC was more important than CE for our department because relatively small and often neutral solutes were synthesized. The first analytical problem we tackled was the analysis of the hop and beer bitter principles. Part of the thesis of Vindevogel

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Fig. 6. The Research Institute for Chromatography, Kannedypark 20, B-8500 Kortrijk, Belgium.

has been published as the first book on CE: “Introduction to Micellar Electrokinetic Chromatography” [7]. Under the direct guidance of Vindevogel, the group in CE was very successful, but unfortunately, Jo left my group in 1995, because I could not offer him a permanent academic position. Our research in SFC focused on the separation of enantiomers and lipids. Separation of enantiomers was of high priority in our department to follow enantioselective synthesis. Over the years a strategy was developed which, until now, we could separate more than 95% of the racemates. Volatile solutes are analysed by capillary GC on 2,3-di-O-acetyl-6-O-TBDMS β- or γ-cyclodextrins. The solubility of involatile solutes is decisive to select either SFC or CE. Enantiomers soluble in organic solvents are analysed by packed column SFC on polysaccharide or macrocyclic antibiotic chiral stationary phases [8], while water soluble sample solutes are analysed by CE with a tetracarboxylic acid crown ether, highly sulfated α-, β- and γ-cyclodextrins, or vancomycin as chiral selectors [10]. As for the last scientific contribution, I would like to mention our work in capillary electrochromatography (CEC). Fig. 7 shows the CEC separation of the triglycerides in corn oil [9]. To the best of our knowledge, this is the most complete separation obtained until now for this kind of sample and most probably the first real application published illustrating the enormous potential of CEC. In 1991, I accepted an invitation by Dr. Carel Cramers to be Visiting Professor at the Eindhoven University of Technology, The Netherlands and until today, I am teaching the course, Environmental Analysis. Environmental analysis has always crossed our research [10]. I felt very honored with this invitation, because other previous temporary

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Fig. 7. CEC chromatogram of corn oil.

associates with Keulemans and Cramers, included Archer Martin, Marcel Golay, Harold McNair and Joseph Huber. Finally in January 1998, I was appointed Professor in Analytical Chemistry at the University of Stellenbosch, South Africa with the aim to establish, following the footsteps of Kurt Grob, a center of excellence in separation sciences. In my years in separation sciences, my work always has been exciting. Separation sciences has allowed me and my wife to travel around the world and to develop friends that I never would have met. I am looking forward to the next (thirty) years. References 1. 2. 3.

4. 5. 6.

P. Sandra and M. Verzele, Contribution of hop-derived compounds to beer aroma, Proc. 15th European Brewery Convention, Elsevier, Amsterdam, 1975, pp. 107–122. P. Sandra and C. Bicchi (Eds.), Capillary GC in Essential Oil Analysis, Huethig Verlag, Heidelberg, 1987. P. Sandra, T. Saeed, G. Redant, M. Godefroot, M. Verstappe and M. Verzele, Odor evaluation, fraction collection and preparative scale separations with glass capillary columns, J. High Resolut. Chromatogr. Chromatogr. Commun., 3 (1980) 107–119. P. Sandra, Fast capillary gas chromatography, LC–GC, 5 (1987) 236–244. C. Bicchi, A. D’Amato, F. David and P. Sandra, Direct capture of volatiles emitted by living plants, Flavours Fragrance J., 2 (1987) 49–58. L. Moens, T. de Smaele, R. Dams, P. Van Den Broek and P. Sandra, Sensitive, simultaneous determination of organomercury-, lead and tin compounds with headspace solid phase microextraction capillary gas chromatography coupled with inductively coupled plasma mass spectrometry, Anal. Chem., 69 (1997) 1604–1611.

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7.

J. Vindevogel and P. Sandra, Introduction to Micellar Electrokinetic Chromatography, Huethig Verlag, Heidelberg, 1992. 8. A. Medvedovici, P. Sandra, L. Toribio and F. David, Chiral packed column subcritical-fluid chromatography on polysaccharide and macrocyclic antibiotic chiral stationary phases, J. Chromatogr. A, 785 (1997) 159–171. 9. P. Sandra, A. Dermaux, V. Ferraz, M. Dittmann and G. Rozing, Analysis of triglycerides by capillary electrochromatography, J. Microcolumn Sep., 9 (1997) 409–419. 10. R. Soniassy, P. Sandra and C. Schlett, Water analysis, Hewlett-Packard, No. 5962-6216, 1994.

55.II. SOLVING THE BELGIAN DIOXIN CRISIS BY ANALYZING PCBs IN FATTY MATRICES P. Sandra 1,2 , F. David 2 and T. Sandra 2 1

Laboratory of Organic Chemistry, University of Gent, Krijgslaan 281 S4, B-9000 Gent, Belgium, 2 Research Institute for Chromatography, Kennedypark 20, B-8500 Kortrijk, Belgium

55.II.1. Introduction In Belgium, 1999 will be remembered as the year of the dioxin crisis. Early in the year (January–February), chicken farmers observed premature death (up to 25%) and nervous disorders among chicks, combined with a high ratio of eggs failing to hatch. Initially, different hypotheses were forwarded, including the possibility of mortal doses of an antibiotic (salinomycin) used as growth promoter in animal feed. One month after the start of these problems, a producer of animal feed took the initiative to send a sample of suspected animal feed and of chicken fat to a laboratory specialized in the analysis of polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). These compounds are often referred to as ‘dioxins’, but in fact consist of two classes of chlorinated aromatic compounds. It has been shown in the past that chickens are very sensitive to the toxicity of chlorinated aromatic compounds. The response time for the dioxin analysis, however, was 4 weeks. At the end of April, the Belgian authorities were informed that very high concentrations of dioxins were detected in animal feed and chicken fat, but hardly any measurements were taken at that time. Another set of samples was taken and sent to the same laboratory for confirmation, which took another 4 weeks. Only at the end of May, 4 months after the problems at the farms started, the public was informed about a food contamination and strong measurements were taken, including the destruction of several lots of eggs, chicken meat and related food products. At that stage, the source of the contamination was still unknown. The analyses, however, showed a 1000-fold higher concentration of PCDDs and PCDFs in the animal feed fat and in the chicken fat (1–2 ng=g fat) versus normal background values (1–5 pg=g fat). It was also observed that the PCDFs were present in much higher concentrations than the PCDDs. PCDDs and PCDFs are not technical chemicals but are by-products in the synthesis of several chlorinated compounds like pentachlorophenol (PCP, an insecticide, mainly

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used in wood preservation), polychlorinated biphenyls (PCBs) or the chlorophenoxy acid herbicides 2,4-D and 2,4,5-T. Dioxins are also formed during badly performed combustion of chlorinated material and can therefore also be present in emissions of waste incinerators. At the Research Institute for Chromatography (RIC), we did not believe that dioxins as such could contaminate food products at ppb level without the presence of other chlorine-containing contaminants in much higher levels. Moreover, the high PCDF=PCDD ratio supported a hypothesis of a PCB contamination. Polychlorinated biphenyls are, in contrast to PCDDs and PCDFs, technical products that have been used intensively in electric capacitors, transformers, vacuum pumps and even as adhesive, fire retardant, in inks, etc. Although their production has been banned for many years, they are still widely present in old electric installations. Since the disposal costs of PCB containing materials is very high, mixing PCB oils with mineral or edible oils becomes an attractive and very profitable alternative. For many years we have noticed on several occasions that fats used in the production of animal feed were of low quality, not only containing polymerized lipids but also contaminants such as mineral oil. The link between dioxins, PCBs, contaminated oils, fat, animal feed and finally chicken and eggs was very logical to us. Consequently we analyzed some samples for PCBs instead of for dioxins. Within 1 day we could prove that high ppm levels of PCBs were present in animal feed, chicken fat and eggs. The link was made and it was very clear that, in this case, used PCB oil was the source that later indeed could be traced and confirmed. Polychlorinated biphenyls consist of a group of 209 possible congeners ranging from mono- up to decachloro-biphenyls. Although their acute toxicity is lower than that of dioxins, several studies have shown that PCBs are linked to several negative health effects including endocrine disrupting activity, reproductive function disruption, developmental deficits in newborns and decreased intelligence in school-aged children who had in uteri exposure. Moreover, the toxicity of dioxins and organochloro pesticides increases in the presence of PCBs. PCBs accumulate in fat and build up in the food chain. Levels from 50 to 500 ppb (ng=g fat) have also been detected in human fat samples [1] and in mother’s milk. Because PCBs are present at higher levels than dioxins, PCB analysis is much faster and cheaper (by a factor 10) than dioxin analysis. The analysis of PCBs was finally accepted by Belgian and European authorities. The norm for PCBs in fatty food (more than 2% fat content) was set at 200 ppb (200 ng=g fat) for the sum of seven PCB congeners (PCBs 28, 52, 101, 118, 138, 153 and 180). Using the much faster PCB monitoring analysis, some 50,000 analyses were performed in various laboratories. This enabled us to promptly deliver certificates for non-contaminated food and to localize all contaminated farms. An overview is given of the sample preparation and analytical methods that can be used to perform PCB analysis in fatty matrices. A short description of the fast screening method developed at RIC is also presented. The complete method details and validation will be described elsewhere [2]. During the crisis, the GC analysis was further improved by using the retention time locking concept and the time was reduced by using narrow bore capillary columns and optimization via the method translation software [3].

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55.II.2. Methodologies for the analysis of PCBs in fatty matrices The analytical scheme for the analysis of PCBs in fatty matrices consists of three steps: extraction, clean-up and analysis. In the first step, the lipophilic contaminants are extracted from the matrix using an apolar solvent. The extract contains the lipids, PCBs, PCDDs, PCDFs and other apolar solutes such as pesticides, polycyclic aromatic hydrocarbons and mineral oil. For the extraction of fat and apolar solutes from meat, eggs and fatty food matrices in general, different extraction techniques are available. These include Soxhlet extraction (or the automated versions Soxtec or Soxtherm), solvent extraction using ultrasonic agitation, accelerated-solvent extraction (ASE), microwave assisted solvent extraction (MASE) and supercritical-fluid extraction (SFE). All these techniques perform similarly and are efficient in extracting fat and contaminants such as PCBs. In general, classical sample extraction techniques (Soxhlet) can handle larger samples (several tens of grams) than the more recent techniques such as ASE or MASE, but the latter have the advantages of automation, speed and low solvent consumption. Selection of the best technique is therefore based on sample throughput, laboratory history, experience with PCBs in other (environmental) samples, etc. In the second step, the PCBs are fractionated from the (co-extracted) fat matrix. For this fractionation, several techniques can be applied, including treatment with sulfuric acid (fat destruction), gel permeation chromatography (GPC), column chromatography on a polar adsorbent (silica, aluminum oxide), and solid-phase extraction (SPE). A critical parameter in the selection of the sample clean-up method is sample capacity. Column chromatography can handle more fat than GPC or SPE, but the latter techniques can be fully automated. In combination with state-of-the-art PCB analyzers sufficient sensitivity can be reached using miniaturized sample preparation techniques. Finally, the cleaned extract can be analyzed using capillary-gas chromatography with electron capture detection (CGC–ECD) or capillary-gas chromatography with mass selective detection (CGC–MS). CGC–ECD is extremely sensitive and in most cases sufficiently selective for the detection of PCBs extracted from fat. CGC–MS in the selected-ion monitoring mode is somewhat less sensitive, but more specific since the presence of PCBs is confirmed by the detection of several ions per congener in a well-defined relative ratio.

55.II.3. Sample preparation method for PCB analysis developed at RIC At RIC, a fast screening method was developed to detect PCBs at the ppb level in fat matrices. The technique includes ultrasonic extraction, followed by clean-up using matrix solid phase dispersion [4]. The extract is analyzed by CGC–µECD. Positive samples are confirmed by CGC–MS. At the end of the food crisis, high speed CGC was also used. Samples are homogenized using a blender. From fat samples (chicken or pork fat) 1 g sample is weighed in a 20 ml headspace vial. From eggs, 3 g egg yolk is taken. For animal feed samples or other meat products, a sample size corresponding to 200–500

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Fig. 1. CGC–MS ion extracted chromatograms of animal feed extract (upper window) and Aroclor 1260 reference sample (lower window) unpublished.

Fig. 2. Mass spectrum of peak at 16.4 min, identified as hexachlorobiphenyl (unpublished).

mg fat is taken. To the sample, 2 g anhydrous sodium sulfate and 10 ml petroleum ether are added. Tetrachloronaphthalene, octachloronaphthalene or Mirex may be used as internal standard and are added to the sample at this stage. The headspace vial is closed and placed in an ultrasonic bath at 30ºC during 30 min. In this step, the fat

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Fig. 3. Retention time locked CGC–µECD analyses of pork fat (upper window) and mink fat (lower window). Minks were fed with contaminated eggs (unpublished).

and PCBs are transferred from the matrix in the petroleum ether phase. The sodium sulfate adsorbs the water present in the sample. After extraction, the sample is allowed to settle. An aliquot is transferred to a test tube and another aliquot is used to determine gravimetrically the fat content of the extract. To the test tube, 2 g of acidic silica gel is added. This technique is called matrix solid-phase dispersion (MSPD) [4]. In column chromatography and in SPE, the analytes are eluted through a bed and the fat is retained.

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Fig. 4. Fast CGC–µECD analysis of Aroclor 1260 reference sample (upper window) and contaminated egg extract (lower window) unpublished.

In MSPD, the fat matrix is allowed to bind on the adsorbent that is mixed with the sample, while the solutes of interest stay in solution. For the fractionation of fat from PCBs, this method works very efficiently. After settlement of the adsorbent, an aliquot of the clear solution is transferred to an autosampler vial. The final volume is not important, since the concentrations are calculated to the initial 10 ml solvent and the sample or fat weight. The whole sample preparation takes approximately 2 h and several

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TABLE 1 ROUND ROBIN TEST FOR THE NEW SAMPLE PREPARATION METHOD PCB

Certified values

28 52 101 118 153 180

22.5 62 164 142 317 73

Sum

778.5

Laboratory 1

Laboratory 2

Laboratory 3

Laboratory 4

ECD

MS

ECD

MS

ECD

MS

ECD

MS

25 75 152 163 337 73

24 72 181 152 345 79

21 56 175 117 319 75

21 56 175 117 319 75

21 65 152 138 287 76

19 71 143 125 350 83

19 54 150 131 290 70

16 63 172 134 316 60

825

853

763

763

739

791

714

761

samples can be prepared in parallel. One technician can handle more than 50 samples per day. The extracts are analyzed by CGC–ECD and CGC–MS. For CGC–ECD, an HP 6890 GC (Agilent Technologies, Wilmington, DE, USA) equipped with split=splitless inlet and µECD detection is used. Standard separations are performed on a 30 m ð 0.25 mm i.d. ð 0.25 µm HP-5MS column. Injection of 1 µl is done in the splitless mode at 250ºC. The carrier gas is hydrogen at 71 kPa. The oven is programmed from 70ºC (2 min) to 150ºC at 25ºC=min, to 200ºC at 3ºC=min and to 300ºC at 8ºC=min., then held for 4 min. Nitrogen at 40 ml=min is used as detector make-up gas. For CGC–MS, the same conditions apply, except that helium is used as carrier gas at 1 ml=min constant flow. Detection is done in selected-ion monitoring mode using two ions per congener group. The first ‘dioxin’ sample analyzed at RIC was an animal feed taken from a farm where most chickens died after eating that food. The sample was prepared using the sample preparation method described above and the analysis was performed by CGC– MS in scan mode. For the first sample, GC conditions were not yet optimized and the sample was screened on a GC–MS configuration available. The scan mode was selected in order to detect other contaminants as well. The extracted ion chromatogram on ion m=z 360 (hexachlorobiphenyls) is shown in Fig. 1. For comparison, the extracted ion chromatogram for the same ion of an Aroclor 1260 reference PCB mixture is shown in the lower window. The correspondence is very clear. Spectra taken at the peaks clearly confirm the presence of PCBs (Fig. 2). After this positive sample, more than 5000 samples, including animal feed, eggs, chicken fat, pork fat, pork meat, meat products (ham, sausages), etc. were analyzed using optimized conditions. The detection limit of the method is 5 ng=g fat per congener. The performance of the sample preparation method was compared with Soxhlet, ASE, MASE and SFE and the results with all techniques were very similar [2]. The accuracy and reproducibility of the sample preparation method was determined by the analysis of a certified reference material CRM350 (mackerel oil), from IRMM (Geel, Belgium) in four laboratories to which the method was transferred. The results are summarized in Table 1. Most values, both using GC–ECD and GC–MS are within

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80 and 110% of the certified values. The values outside these ranges are indicated in italics. For all laboratories and both techniques, the sum values were always within these limits. During the crisis, two relatively new concepts were introduced to improve repeatability and speed of analysis, namely retention time locking and fast capillary GC. Fig. 3 shows the extracts of a pork fat (upper window) and of a mink fat (lower window) with retention time locking in CGC–µECD. Full details will be published elsewhere [2]. The use of narrow bore columns [3] could reduce analysis speed. For fast CGC analysis of PCBs, a 10 m ð 0.1 mm i.d. ð 0.1 µm HP-5 column was used in combination with splitless injection and µECD. The carrier gas was hydrogen at 233 kPa. The oven was programmed from 70ºC (0.45 min) to 150ºC at 110ºC=min, to 200ºC at 13.2ºC=min, and finally to 300ºC at 35.2ºC=min. Head pressure and temperature program were calculated using the method translation software program. Fig. 4 (lower window) shows the CGC–µECD chromatogram obtained for the extract of a contaminated egg using fast CGC. Also in this sample, the Aroclor 1260 profile (Fig. 4, upper window) is clearly present. The analysis time is less than 8 min.

55.II.4. Conclusion The contamination of Belgium food products has initially been referred to as a ‘dioxin crisis’. The contamination source, however, were polychlorinated biphenyls. PCBs can be analyzed much faster and cheaper than PCDDs and PCDFs and therefore the analysis of PCBs was the only possible solution to the crisis. A new method, using ultrasonic extraction, followed by matrix solid phase dispersion clean up and CGC–µECD or CGC–MS, was used for fast screening of the samples. Retention time locking and fast capillary GC completed the introduction of new methodologies in the PCB screening.

References 1.

2. 3. 4.

A. Pauwels, F. David, P. Schepens and P. Sandra, Automated gel permeation chromatographic clean-up of human adipose tissue for analysis of organichlorine compounds, Intern. J. Environ. Anal. Chem., 73 (3) (1999) 171–178. P. Sandra, T. Sandra and F. David, J. Chromatogr., in preparation. F. David, D.R. Gere, F. Scanlan and P. Sandra, Instrumentation and applications of fast high-resolution capillary gas chromatography, J. Chromatogr. A, 842 (1999) 309–319. S.A. Barker, Matrix solid-phase dispersion, Current Trends and Developments in Sample Preparation, Supplement to LC–GC, May 1998, p. 37.

D.56. Gerhard Schomburg Gerhard Schomburg was born on August 22, 1929 in Bochum, Germany. After finishing high school, he first graduated as chemical engineer in 1951 at the School of

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Engineering in Essen, Germany and then studied chemistry at the University of Bonn; in 1954 he moved to the Max-Planck Institut fu¨r Kohlenforschung and became a Ph.D. student of the then director of this institute, the inventor of the catalysts for the synthesis of polyethylene and polypropylene and later Nobel Laureate, Karl Ziegler. His doctoral thesis dealt with the infrared spectroscopy of aluminum alkyls and hydrides and the results of this work were a contribution to the understanding of the ‘electron deficient bonds’ in aluminum and boron compounds formed via alkyl groups, halogenes and hydrogene anions. The thesis was finished in 1956, and he obtained his Ph.D. from the Technical University of Aachen, where K. Ziegler was affiliated as an honorary professor. From 1956 to 1996, G. Schomburg was head of the Chromatography Department in the Max-Planck Institut which was founded by him and which later became the Department of Chromatography and Capillary Electrophoresis. This department was further developed and operated in combination with a similar group for analytical chromatography in the neighboring Max-Planck Institut fu¨r Strahlenchemie. Besides the more than 230 publications which were the result of the work performed with professional analytical chemists and about 25 Ph.D. and diploma students over the entire period of his research work, G. Schomburg wrote three textbooks on capillary GC and two book chapters in the field of capillary electrophoresis. He was the co-founder of the series of the International Symposia on Capillary Chromatography first in Hindelang, Bavaria and then in Riva Del Garda, Italy, and acted as chairman and member of scientific organization committees of several other international symposia on chromatography and capillary electrophoresis. In 1982, he became academic lecturer at the Universities of Wuppertal and Bochum and was appointed Professor in 1989. He also received an Honorary Doctorate from the University of Duisburg in 1987. In 1996, he had a guest Professorship with Professor Gu¨nther Bonn, at the Institut fu¨r Analytische Chemie und Radiochemie University of Innsbruck, Austria. Dr. Schomburg was chairman of the Chromatography Group of the German Chemical Society from 1986 to 1989. He was a member of the Editorial Boards of the Journal of Chromatography and Chromatographia for several years and is still Editor of the Journal of High Resolution Chromatography. Dr. Schomburg received the following scientific awards: Anniversary Tswett Medal of the USSR Academy of Sciences, 1978; Tswett Chromatography Medal of the Symposium on Advances in Chromatography, 1983; A.J.P. Martin Award of the Chromatographic Society, 1984; the Stephen Dal Nogare Award of the Chromatography Forum of Delaware Valley, USA, 1986; M.J.E. Golay Award of the Symposium on Capillary Chromatography, 1990; Tswett Medal of the Russian Chromatography Society, 1992; Ruhrpreis fu¨r Kunst and Wissenschaft (Award for Art and Science) given by the city of Mu¨lheim-Ruhr, Germany, 1995; Keene P. Dimick Award, Pittsburgh Conference, 1996; and the Alexander v. Humboldt Prize of the Fond National de la Recherche Scientifique, Belgium, for a stay as visiting senior scientist at the Laboratoire

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Gerhard Schomburg (center) in the laboratory of his group in the Max-Planck Institut fu¨r Kohlenforschung, talking to Nobel-Laureates Karl Ziegler (left, then Director of the Institute) and Adolf Butenandt (right, then President of the Max-Planck Society) about gas chromatograms obtained in October 1963. Professor Karl Ziegler was born November 26, 1899, and died in 1973. Since 1943 he was director of the Kaiser Wilhelm Institut fu¨r Kohlenforschung in Mu¨lheim a.d. Ruhr. The name of this institute was changed after the war in 1955 into the Max-Planck Institut fu¨r Kohlenforschung. Karl Ziegler received the Nobel Prize for the discovery of the metallorganic (‘Ziegler’) catalysts for the industrial production of low pressure polyethylene, polypropylene and other copolymers together with E. Natta from Italy. Gerhard Schomburg was his Ph.D. student from 1954 to 1956. The title of his thesis was ‘Infrared Spectroscopy of Aluminiumalkyls and Hydrides’. Professor Adolf Butenandt was born March 24, 1903, and died January 18, 1995. He was the Director of the Kaiser Wilhelm-Institut fu¨r Biochemie, the later Max-Planck Institut fu¨r Biochemie in Mu¨nchen. He was the President of the Max-Planck Society since 1960 as a successor of Nobel Laureate Otto Hahn. He received the Nobel Prize in 1939 for the isolation and structure identification of sexual hormones.

d’Analyse des Me´dicaments at the University of Lie`ge with Professor J. Crommen in the years of 1996 to 1997. See Chapter 5B, a, b, d, e, f, i

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56.I. SURFACE MODIFICATION IN SEPARATION SYSTEMS OF CHROMATOGRAPHY AND ELECTROPHORESIS: 30 YEARS OF PROGRESS Gerhard Schomburg Max-Planck Institut fu¨r Kohlenforschung und fu¨r Strahlenchemie, Stiftstr. 39, D-45470 Mu¨lheim a.d. Ruhr, Germany

56.I.1. Introduction Chromatographic separations are based on partition equilibria of solutes (separands) between a mobile and a stationary phase during the separation of these solutes by differential migration in the mobile phase. The equilibrium between the mobile and the stationary phase is diffusion controlled and proceeds in a direction vertical to that of the mobile phase flow and of the related solute migration. Therefore in chromatographic systems, separations of high efficiency and=or speed can only be achieved with fast mass transfer. In GC, the diffusion paths of solutes are made short in narrow bore capillaries, in LC by means of small particle packings. Electrophoretic separations are achieved by differential migration of charged species in an electrical field which is maintained in a buffer contained in the separation tool (capillary). In principle, differential migration in the buffer, in which an electrical field exists, and without movement of the buffer, is possible without ‘vertical’ equilibration as in chromatographic systems. In many (capillary) electrophoretic systems used in analytical practice, the buffer may also be moving as a consequence of electro-endoosmotic effects, and intermolecular interaction of the analytes between the moving buffer and the surface of the capillary walls can never be completely suppressed. In recent years hybrid separation methods advantageously based on both chromatographic and electrophoretic mechanisms such as micellar electrokinetic chromatography (MECC), and especially capillary electrochromatography (CEC) have been recognized as separation methods with a mobile liquid phase, which might supply easy to modify special selectivities of the analyte(s) at high efficiencies and=or speed of separations.

56.I.2. Surface modification in GC, SFC, HPLC, CZE and CEC Coatings of support of capillary surfaces in open tubular separation columns (capillary), and respectively, the porous microparticle packed type columns are created to realize a stationary phase which, in chromatography systems, acts with selective intermolecular interaction, for the partition process of the separands. In most cases, contributions of the inorganic silica support surface to the analyte=stationary phase interaction should be suppressed as much as possible or well-defined; however, in CZE and CEC surface coatings have to meet the special and=or additional requirement of high electro-endoosmotic (mobile phase) flow in parallel to their properties for selective chromatographic partition as in CEC.

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Coatings must be stable against high operation temperatures as in GC, or against dissolution in the liquid mobile phase or buffer, and against decomposition by chemically aggressive mobile phase buffers as in LC and CE. They must also cover the support surface completely in order to avoid irreversible and non-linear adsorption of analytes. Especially in CEC these different requirements have already been recognized as difficult to meet. In the history of method development in GC, LC and CE in the past 40 years, the separation technology in chromatography was revolutionized by the introduction of the principle of open tubular (capillary) columns in gas chromatography, and later also by the introduction of columns packed with porous microparticles for liquid chromatography (HPLC). Today, mainly fused silica capillaries are applied in GC, CE and also in HPLC but with microparticle packings in the latter. Both approaches, that of capillary GC and of HPLC, or micro-LC, are characterized by ‘miniaturization’ of either the capillary diameter or the size and porosity of the packing material, which is related to very short diffusion path lengths in the equilibration process. However, in capillary electrophoresis (CE) the approach of miniaturization in narrow-bore fused silica capillaries had to be adopted mainly for low generation and fast dissipation of heat due to the applied strong electrical fields.

56.I.3. Capillary-gas chromatography (CGC) In gas chromatography systems realized in narrow bore capillaries a considerable increase of separation efficiency could be achieved by miniaturization, but at the unavoidable expense of a limited range of sample capacities (from trace to main components) of the common column types used in capillary GC. In the latter, the early period of development of the new separation technologies was related with the necessity of obtaining temperature-stable coatings of variable film thickness in narrow-bore stainless steel, copper, glass and finally, today, exclusively quartz (fused silica) capillaries for analytical GC. Oligomers or polymers with low vapor pressure at elevated separation temperatures, which have to be applied for the separation of less volatile solutes, were firstly deposited on the surfaces of the different types of capillary (or porous particle) surfaces. In the beginning, homogeneous coating layers could be achieved by use of special coating techniques as the dynamic or the static method using solutions of the stationary phase polymers which were to be fixed to the smooth or rough surfaces by mere adhesion or adsorption. Various procedures of basic or acidic etching and=or roughening developed by Grob [1,2] and Alexander and Rutten [3], of the surfaces were tried to improve the fixation, e.g., the homogeneity and stability of the polymeric surface layers during usage over the entire range of separation temperatures. Today, the mentioned roughening procedures have been recognized as not necessary for the generation of homogenous films of stationary phases except for the fixation of the highly polar cyano alkysiloxanes. After many years of experimental work in this field, it was found that preferably non-polar and, with some difficulties, a few polar (cyanoalkyl substituted), also chiral, alkylpolysiloxanes and high molecular weight poly (ethylene or propylene glycols) were suited to form stable stationary phases in fused

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silica capillaries. These were successfully used for separations at elevated temperatures and with the mainly applied wide range temperature programming. The coatings on unpretreated glass or fused silica surfaces were not suited, however, for separations of strongly basic solutes which gave rise to very undesirable non-linear adsorption to the acidic surface underneath the different types of coatings; or to insufficiently covered regions of the capillary surface, especially when low thicknesses of the stationary phase films are applied to achieve fast separations. In capillary columns for GC various deactivation (precoating) procedures, such as high temperature trimethylsilylation or the polysiloxane degradation, ‘PSD’ methods were applied before coating with the stationary phase, especially when non-polar alkylpolysiloxane phases were deposited (Welsch et al. [4]; Grob Jr. et al. [5] and Schomburg and colleagues [6,7]). These pretreatment steps were based on lowering of the concentration of the acidic silanols on the surfaces by derivatization with non-acidic preferably neutral groups, or by shielding through different polar and especially basic polymers adsorbed in a precoating step. It was recognized as important that the generated stationary phase coatings were homogeneous at variable thickness in order to achieve the optimum separation efficiency: films could be stabilized against droplet formation by crosslinking of the polymeric layers and=or chemical bonding to the surface, e.g., by fixation of Si–H groups to the surfaces by silanization which could then serve for the fixation of SiOH or vinyl groups containing alkylsiloxane oligomers. Deposition of thin layers of adsorptive microparticle silicas before the coating has also been considered as effective for improved fixation of the liquid polymer films especially in the case of cyano silicone phases. In the commercial field of capillary gas chromatography, chemical bonding of the stationary phase coatings has been claimed as a significant feature of stable and versatile columns for routine analysis. It was intended to fix the coating layers so well that contaminated columns could be re-conditioned by solvent extraction of contaminating deposits originating from the sample. Another reason was that bonding to the surface would also generally stabilize the coatings. No information can be found which kind of chemical bonding had been effective or even could be proved. Si–O–Si bonding to the oligomer chains seems to be involved in most cases either to the surface, or most probably between the alkylsiloxane chains effected by crosslinking.

56.I.4. High-performance liquid chromatography (HPLC) Problems arose in HPLC when porous microparticles of silica had been modified by the classical silanization of the silanols which were to act as anchor groups for the fixation of a layer of hydrophobic groups or of varying chemical structure, e.g., polarity. With this silanization procedure the anchor silanol groups can only be reacted to about 40% of maximum (C18 alkyl groups), mainly depending on the stereochemistry of the silanization reagents. The residual and different types of free silanols generally give rise to non-linear (Langmuir type) interaction of basic solutes (originating from the pharmaceutical or biochemical field, for example) with these acidic groups, thus causing undesirable tailing of the analyte peaks which make such phases unusable for routine

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analyses. Porous silica particles produced according to different processes, and which were to be modified for reversed-phase HPLC, had to be optimized with regard to the type and the concentration of silanols resulting from the different production processes of making silica particles and subsequent surface treatment. It became clear that modified silica surfaces were never free of the before-mentioned secondary interaction of basic analytes, which had to be suppressed by different measures of ‘deactivation’ of the silica surfaces in capillaries and on porous silica particles, or by using a special composition of the mobile phase. Mobile phases were modified by special additives which deactivate the surface by strong interaction with the ‘active’ surfaces in competition to the ‘weaker’ interaction of the analytes. Modification of the pH of aqueous mobile phases and the resulting change of the dissociation of charged analytes is another approach of suppression of undesirable analyte-support phase interaction. Besides the nonpolar reversed-phase coatings of silica, polar stationary phases, e.g., for chiral or ion-exchange separations, can also be produced by application of silanization but also by a ‘polymer coating’ method. In analogy to capillary GC polymer coating procedures, the generation of homogenous and not too thick layers of well-defined thickness on the surfaces of porous small particles for HPLC were achieved by Schomburg [8] and Engelhardt et al. [9]. The methods described proved to be specially suited for the deposition of multilayers. A sublayer, with different chemical structure than the top layer, can be made to serve herewith the purpose of shielding the inorganic support surface (e.g., the acidic silanols); and should be better suited for the fixation of more stable films or the actual stationary phase. The polymer coating method has the advantage of the possibility of making defined variations of film thickness by using solutions of different concentrations of polymers to be deposited. By silanization, the thickness of the non-polar coating layer can only be varied by choosing silanization reagents with variable length of the substituting alkyl groups. The polymer coating method can be successfully applied for the synthesis of special ion-exchange phases; e.g., a crosslinked poly (butadienemaleic acid) coated silica for the fast separation of all alkali and alkali earth cations [10]. Crosslinked polybutadiene coatings proved to be well-suited to convert porous alumina into non-polar reversed phase materials which are characterized by very high stability at pH up to 12 [11]. Alkylpolysiloxanes can also be deposited on porous silica particles. By crosslinking with dicumylperoxide or other radical initiators the fixation of the layers can be very much improved, see Fig. 1.

56.I.5. Capillary zone electrophoresis (CZE), and micellar electrokinetic chromatography (MECC) Separations in CE systems are possible without involvement of chromatographic equilibria of analytes between the buffer and the capillary walls, or any kind of undesirable analyte=wall interaction as in CZE and MECC; surface modification serves primarily as a method of manipulation of the electro-endoosmotic flow which can be decreased, increased, completely suppressed or even reversed for different reasons of performance optimization with regard to efficiency, resolution or speed [12,13]. The

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Fig. 1. Fast isocratic separation of the four alkali- and ammonium ions and the four alkaline earth cations in a single run. The stationary phase for this ion-exchange HPLC system had been synthesized by ‘polymer coating’ with the copolymer poly(butadiene-maleic anhydride); 30% by weight of this polymer ˚ pore diameter silica (Nucleosil) and immobilized by radical crosslinking. was deposited on 7 µm, 100 A The aqueous mobile phase was 5 Mm citric acid C 0.5 mM pyridine-2,6-dicarboxylic acid.

surface modification can be done dynamically by additives contained in the buffer or also by permanent chemical modification using coating procedures as known from stationary phase technology in CGC and HPLC. Problems arise with the stability of permanent coatings considering the wide range of buffer pH which are used for the typical CE separations of charged analytes. In CZE and MECC, the maximum performance can only be achieved if analyte=wall interactions are suppressed as much as possible because of the slow diffusion in the liquid media and the related band broadening. These effects of analyte=wall interaction impair the high speed or efficiencies of analyses which are based on differential migration in electrophoretic systems and without involvement of the slow vertical equilibration mechanisms. Fig. 2

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Fig. 2. Electrophoretic CGE separation of more than 400 homologues of poly(uridine 50 -phosphate) using a polyacrylamide gel filled FS capillary of 100 µm i.d. The capillary surfaces were specially coated for suppression of the electroosmotic flow. There was no chemical bonding between the surface modification layer and the separation gel. The field strength was 300 V=cm.

shows an electrophoretic CGE separation of more than 400 homologues of poly(uridine 50 -phosphate) using a polyacrylamide gel filled fused-silica capillary of 100 µm i.d. 56.I.6. Dynamic surface modification Dynamic surface modification, preferably by charged (basic) and uncharged (hydrophilic), in many cases oligomeric or polymeric buffer additives which compete with

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the analyte molecules at wall interaction, or which form strongly adsorbed coating layers, can easily be applied in analytical routine work; whereas, permanent modifications are sophisticated to perform in most cases and are better generated in a commercial environment. Charged coating layers lead to either anodic or cathodic electro-endoosmotic flow (EOF); whereas, uncharged mainly hydrophilic coating layers from PEG or PVA, provide a complete suppression of the EOF by shielding. Hydrophilic coating layers have been found best suited for biochemical separations, e.g., of proteins of different size, charge and hydrophobicity [14–17].

56.I.7. Permanent coatings Permanent coatings, for CZE can be achieved, as in HPLC, by silanization and further chemical reactions with functional groups of the silanization layer. In fused-silica capillaries; however, the concentration of anchor silanols per unit area are quite low. Permanent coatings can also be achieved by strong adsorption of preferably polar oligomers or polymers, which, moreover could be crosslinked for a decrease in dissolution in the buffer. Perfect immobilization of coating layers is important when a large series of repetitive separations in analytical routine work is done and high precision is required. Chemical bonding of coating layers via silanization, i.e., via Si–O–Si bonds suffers from the instability of this bond at high pH. Generally the wide range of buffer pH used in CE requires a special highly stable coating preferably of the crosslinked type.

56.I.8. Capillary electrochromatography (CEC) In the extensive research work on the recent and present development of capillary electro-chromatography, which is a hybrid of chromatographic and electrophoretic separation mechanisms, it was found that specially modified surfaces on porous small (silica) particles have to be realized, which are optimal with regard to the desired chromatographic selectivity and also for the generation of a strong electro-endoosmotic flow which is to drive the buffer as mobile phase at high speed through a microparticle packing. Electrically driven LC allows for separations at much higher efficiency than with pressure driven separation systems. It was found that under the following conditions high efficiencies in CEC can be achieved: ž In the use of buffers containing solvents of high dielectric constants, such as acetonitrile, methanol, etc., but with preference for low viscosity solvents. ž With stationary phases that are charged: e.g., silicas with a certain concentration of residual silanols, cation or ion-exchange groups, etc. It is obvious that phase systems which also give rise to high EOF are not very well suited for a linear, tailing-free chromatography of strongly basic pharmaceuticals at high efficiency; for example, phase systems which contain special surface modifiers; and other measures have been used to overcome these difficulties. Reversed phase separations of hydrophobic uncharged compounds in systems with C18 modified silica

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phases, and with acetonitrile or methanol containing buffers (mobile phases) could be performed at much higher efficiencies than in HPLC systems. Coating procedures of different kinds that have been developed for GC and HPLC could perhaps be suited to overcome the characterized difficulties.

References 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13.

14.

15. 16. 17. 18.

K. Grob, Polare Impra¨gnierung von Glaskapillaren fu¨r die Gaschromatographie, Helv. Chim. Acta, 48 (1965) 1362. K. Grob, Glaskapillaren fu¨r die Gaschromatographie. Verbesserte Erzeugung und Pru¨fung stabilier Trennflu¨ssigkeitsfilme, Helv. Chim. Acta, 51 (1965) 718. G. Alexander and G.A.F.M. Rutten, Surface characteristics of treated glasses for the preparation of glass capillary columns in gas-liquid chromatography, J. Chromatogr., 99 (1974) 81. Th. Welsch, W. Engewald and Chr. Klauke, Zur Desaktivierung von Glaskapillaren mittels Silanisierung, Chromatographia, 10 (1977) 22. K. Grob, G. Grob and K. Grob Jr., deactivation of glass capillary columns by silylation, Part I, J. High Res., 23 (1979) 1. G. Schomburg and H. Husmann, Methods and techniques of gas chromatography with glass capillary columns, Chromatographia, 8 (1975) 517. G. Schomburg, H. Husmann and H. Borwitzki, Alkylpolysiloxane glass capillary columns combining high temperature stability of the stationary liquid and deactivation of the surface. Thermal treatment of dealkalinized glass surfaces by the stationary phase itself, Chromatographia, 12 (1979) 651. G. Schomburg, Polymer coating of surfaces in column liquid chromatography and capillary electrophoresis, Trends Anal. Chem., 10 (1991) 163. H. Engelhardt, H. Lo¨w, W. Eberhardt and M. Mauss, Polymer encapsulated stationary phases. Advantages, properties and selectivities, Chromatographia, 27 (1989) 535. P. Kolla, J. Ko¨hler and G. Schomburg, Polymer coated cation exchange stationary phases on the basis of silica, Chromatographia, 23 (1987) 465. G. Schomburg, U. Bien-Vogelsang, A. Deege, H. Figge and J. Ko¨hler, Synthesis of stationary phases for reversed phase LC using silanization and polymer coating, Chromatographia, 19 (1984) 170. G. Schomburg, Coated capillaries in high-performance capillary electrophoresis, in (Ed.), M.G. Khaledi, High Performance Capillary Electrophoresis, John Wiley and Sons, 1998, p. 481. G. Schomberg, Technology of separation capillaries for capillary zone electrophoresis and capillary gel electrophoresis: The chemistry of surface modification and formation of gels, in A. Guzman (Ed.), Capillary Electrophoresis Technology, Marcel Dekker, New York, 1993, p. 311. H. Lindner, W. Heiliger, A. Dirschlmayer, M. Jacquemar and B. Puschendorf, High-performance capillary electrophoresis of core histone and their acetylated modified derivatives, Biochem. J., 238 (1992) 437. G. Schomburg, D. Belder, M. Gilges and St. Motsch, Ionic and non-ionic polymers as wall modifiers in capillary electrophoresis, J. Capillary Electrophor., 1 (1994) 3. M. Gilges, M.H. Kleemiss and G. Schomburg, CZE separation of basic and acidic proteins using poly(vinylalcohol) coatings in FS capillaries, Anal. Chem., 66 (1994) 2038. B.L. Karger and W. Go¨tzinger, Polyvinyl alcohol (PVA) based covalently bonded stable hydrophilic coating for capillary electrophoresis, US Patent 5,840,388 (1997). G. Schomburg, Oligonucleotides, in P. Camillieri (Ed.), Capillary Electrophoresis, Theory and Practice, CRC Press, Boca Raton, FL, 1993, p. 255.

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D.57. Raymond P.W. Scott Raymond P.W. Scott was born in June 1924 in Erith, Kent, England, married to Barbara (ne´e Strange) with two sons, Kerry Raymond and Kevin Francis. His elementary and secondary education was at The Erith Grammar School, Kent, England. Professional: The University of London, B.Sc. 1946, D.Sc. 1960, Associate of the Royal Institute of Chemistry 1946–1958, Fellow of the Royal Institute of Chemistry 1958 to date, Fellow of the Royal Society of Chemistry 1972 to date, Fellow of the American Institute of Chemistry 1983 to date, Member of the American Chemical Society 1969 to date, Chartered Chemist of the Royal Society of Chemistry (C. Chem UK) 1962 to date, Certified Chemist of the American Institute of Chemists (C. Chem. USA) 1983 to date. R.P.W. Scott has spent the majority of his professional life in industry starting in the Armament Research Department, Woolwich Arsenal in 1944 and retiring from the Perkin Elmer Corporation at the age of 63 in 1987. He is now associated with both Georgetown University, Washington, DC and Birkbeck College, University of London as a visiting professor. He practices as a consultant to various companies in the area of separation science. R.P.W. Scott is the author and co-author of over 180 peer reviewed scientific papers, largely involving the theory and practice of gas and liquid chromatography. He received the America Chemical Society Award in Chromatography (1977), the M.S. Tswett Chromatography Award (USA) (1978), the Tswett Anniversary Medal (USSR) (1978), the A.J.P. Martin Award in 1982 and the Royal Society of Chemistry Award in Analytical Instrumentation in (1987). He has taken part in the teaching of many courses in Chromatography in the USA, UK, The Netherlands and Australia. He is a member of the Editorial Boards of the Journal of Chromatographic Science, and the Journal of Liquid Chromatography, Editor of the Separation Science Series of J. Wiley and Sons, and cited in Who’s Who in America, Who’s Who in the North East, and Who’s Who in Technology. R.P.W. Scott has been the Editor of the following books: “Gas Chromatography 1960”, Butterworths, London (1960); “Small Bore Liquid Chromatography Columns”, J. Wiley and Sons, New York (1984); and author of a number of books: “Contemporary Liquid Chromatography”, J. Wiley and Sons, New York (1976); “Liquid Chromatography Detectors”, First Edition, Elsevier, Amsterdam (1977); “Liquid Chromatography Detectors”, Second Edition, Elsevier, Amsterdam (1986); “Liquid Chromatography Column Theory”, J. Wiley and Sons, New York (1992); “Silica Gel and Bonded Phases”, J. Wiley and Sons, New York (1993); “Liquid Chromatography for the Analyst”, M. Dekker Inc., New York (1994); “Techniques and Practice of Chromatography”, M. Dekker Inc., New York (1995); “Chromatographic Detectors”, M. Dekker Inc., New York (1996); “Tandem Techniques”, J. Wiley and Sons, New York (1997); “An Introduction to Analytical Gas Chromatography”, M. Dekker Inc., New York (1998); “Chiral Chromatography”, J. Wiley and Sons, New York (1999). See Chapter 5B, a, d, f, h, k, p, q

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57.I. FORTY-FIVE YEARS OF CHROMATOGRAPHY RESEARCH AND DEVELOPMENT Raymond P.W. Scott 2 Sprucewood Lane, Avon, CT 06001, USA

My first introduction to chromatography was at a lecture given by A.J.P. Martin at the laboratories of the Medical Research Council at Mill Hill, London, in 1954. This was one of the first lectures given by Dr. Martin on his new invention of gas chromatography (or Vapour Phase Chromatography as it was known then) which would, in due course, revolutionize the world of analytical chemistry. The essentials of a chromatograph, a gas supply, a thermostatted packed column and a detector were carefully described and most of the audience returned to their laboratories to construct a gas chromatograph. The detector Archer Martin described, the gas density balance, consisted of a Wheatstone network of capillary tubes drilled out of a solid block of high conductivity copper. I eventually succeeded in constructing a working density balance, but the problem of working in high conductivity copper, and the ease in which drills snapped in the material, provoked me to develop an alternative detection system. The flame thermocouple detector [1], my alternative to the density balance, and a forerunner to the flame ionization detector, was my first original contribution to chromatography. I described it at the initial meeting of the Chromatography Discussion Group at Ardeer, Scotland in 1955. In its original form, it consisted of a thermocouple (supported in a cocoa tin) and situated over a jet through which the carrier gas from the column passed. The carrier gas consisted of hydrogen or a mixture of hydrogen and nitrogen, which was burned at the jet so the thermocouple, was heated to a steady temperature. When solute vapor was eluted from the column, the temperature rose and the thermocouple output increased which was fed to a potentiometric recorder. It had a sensitivity of about 106 to 107 g=ml, not a great sensitivity in modern terms, but was relatively high and very useful in those early days. The simpler detection system with its higher sensitivities supported the development of long, high efficiency packed columns, and in 1957, I was the first to demonstrate the partial separation of m- and p-xylene [2] on a packed column 25 ft long having 12,000 theoretical plates. This was achieved on a dispersive stationary phase with no polar selectivity and relied almost entirely on the high efficiency of the column. The advent of the argon detector developed by Lovelock provided even greater sensitivity and permitted further development of high efficiency packed columns. At the 1958 Amsterdam symposium on gas chromatography, I described a 50 ft packed column having an efficiency of 30,000 theoretical plates, operated at an inlet pressure of 200 psi, that would separate all the isomeric heptanes and thirteen of the isomeric octanes. At the same meeting, I also described the first successful moving bed continuous preparative gas–liquid chromatography system [3] and this concept is now being actively developed for large scale liquid chromatography separations. At Amsterdam, Golay described the theory of the capillary columns and subsequently in 1959, using 1000 ft of Nylon capillary tubing, I achieved the goal of one million theoretical plates. My early work on gas chromatography was carried out at the research laboratories of Benzole Producers

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Fig. 1. A photograph of myself with Bob Maggs and Ernie Ormerod taken ca. 1957–1958 at Benzole Producers Research Laboratories during the height of GC development.

and during this period of my research, my co- workers were Bob Maggs, Ernie Ormerod, Graham Hazeldean, Neville Coup and Colin Cummings. Exploiting the simplicity of Nylon tubing further, we used short lengths (2–3 m) of the plastic tubing to pioneer work on high speed separations. At the 1960 symposium on gas chromatography held in Edinburgh, we demonstrated the separation of a five component mixtures in 5 s, displaying the chromatograms on a cathode ray tube. In the early 1960s, I moved to the research laboratories of Unilever at Sharnbrook in Bedfordshire where my co-workers included, Trevor Wilkins, Ian Fowliss, Colin Cummings and Graham Lawrence. One of our main interests was the development of chromatography hardware and, while continuing work on gas chromatography, we also initiated development work on liquid chromatography techniques and apparatus. The renaissance of liquid chromatography had begun. Using the basic principles put forward by Purnell, we developed the first practical flow programmer and described it in the 1964 symposium on gas chromatography, which was held in Brighton, UK. Unfortunately it was based on pneumatic logic and was rapidly superseded by electro-mechanical devices. At the same meeting, Tony James, John Ravenhill and I described the first LC transport detector, crude in form, but functional and which established the viability of the transport concept for LC detection and other uses. Later Graham Lawrence and I were to develop this concept into a far more effective detection device. In the early 1960s, we were actively investigating tandem systems and Ian Fowliss, Trevor Wilkins, David Welti and I developed the first in-line GC=IR=MS combined instrument that would provide IR and MS, spectra (simultaneously) of each peak eluted from the gas chromatograph. We described this instrument at the 1966 symposium on gas chromatography in Rome [4], and exhibited it in operation, producing IR and MS spectra from eluted peaks during the entire meeting. The chromatograph used with the

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Fig. 2. New generation high speed chromatography in 3.5 s.

spectrometers contained a glass packed column, 50 ft long. The eluent was split, a small portion passed directly to the mass spectrometer whereas the remainder was trapped, and thus concentrated. The sample was automatically regenerated into an IR vapor cell and scanned. In 1969, just before I left the Unilever Research Laboratories to join Hoffman La Roche in the USA, Graham Lawrence and I developed the transport detector further using a stainless steel wire as the transport medium. The LC column eluent passed over the wire leaving a coating from which the solvent was evaporated. The solute layer was

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then oxidized in an enclosed atmosphere of air, and the carbon dioxide formed entrained into a hydrogen stream that was passed over a heated catalyst converting the carbon dioxide to methane. The methane was detected by means of a flame ionization detector. This modification increased the sensitivity by over an order of magnitude. At Hoffman La Roche my co-workers included, Cliff Scott, Charles Reese, Ben Buglio, Paul Kucera, Fe Chang and Marion Munroe. We now concentrated on the development of LC and spent considerable time clarifying the mechanism of retention and in column design. As LC column efficiencies improved, so the concept of on-line LC=MS became more plausible. In 1973, during discussion at the International Meeting on Advances in Chromatography that was held in Toronto, Canada, I suggested that the transport system used in the transport detector could also be used as an effective LC=MS interface. With two exceptions, namely, Archer Martin and T.Z. Chu, this suggestion was greeted with amused disbelief. However, Mr. Chu, at that time CEO of the Finnigan Corporation, offered me the loan of a quadrupole mass spectrometer to use for the development of the interface. During the rest of the year, Cliff Scott, Marion Munroe and I developed the transport system into an effective LC=MS interface and published the results early in 1974 [5]. We used two differential pumping chambers situated on either side of the ion source of the mass spectrometer separated, and isolated, by sapphire jewels through which the wire passed. The column eluent passed over the wire leaving a coating on the surface. A current was passed through the wire that was insufficient to volatalize the solutes, but sufficient to volatalize the solvent at atmospheric pressure. However, when the wire entered the MS source at very low pressure, heat could no longer be convected or conducted away from the wire and its temperature rose to about 350ºC and thus volatilized the solute. In 1977, I was accorded the American Chemical Society-National Chromatography Award for the LC=MS interface and my other contributions to the field of chromatography. In the mid-1970s, we worked on retention mechanisms in LC, including solvent selection, surface characteristics of silica and bonded phases and the role played by solvent concentration in the probability of solute–solvent interactions. In 1978, I was accorded both the International Symposia on Advances in Chromatography — M.S. Tswett Award and the Scientific Council of Chromatography, M.S. Tswett 75th Anniversary Chromatography Medal from the Soviet Academy of Sciences for my contributions to the field of chromatography. In the late 1970s, Paul Kucera and I turned our attention to the production of very high efficiencies from LC columns and developed the first long microbore columns. In 1979, we described a small bore column, 10 m long that provided 750,000 theoretical plates [6]. In 1980, I joined the Perkin Elmer Corporations as their Director of Applied Research and my new co-workers were Elena Katz, Ken Ogan and Gary Schmidt. Naturally, our work was centered on instrument development. However, we were able to develop columns for high speed LC analysis and critically examined many of the dispersion theories and equations identifying the van Deemter equation as the most pertinent to use for LC column design. In addition we re-examined the argon detector and developed a novel form, that would function at high sensitivity without the need of a radioactive source. In 1982, I was accorded the Chromatographic Society’s A.J.P. Martin Gold Medal for my work in the field of gas and liquid chromatography.

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I retired from the Perkin Elmer Corporation in 1986 and since that time have worked as a consultant and written 7 books on various aspects of chromatography (one on “Tandem Techniques” [7]) with the 8th currently in press and the 9th in process. I have had the privilege and pleasure of being associated with the Chemistry Department at Georgetown University, Washington, DC and the Chemistry Department at Birkbeck College, London University as Visiting Professor and I am still carrying out original research although my publications are less frequent. At present I am working on some unique aspects of chiral separations, the argon detector and the development of yet another improved transport detector. In retrospect, I am very thankful that I attended that lecture by Professor A.J.P. Martin in 1954 and was able to appreciate its significance. But, as with every success, serendipity also played an important part, I had the good fortune to be in the right place at the right time. As a result, I have had the most satisfying and rewarding scientific career that I could possibly have wished. Even more important, it has all been great fun!

References 1. 2. 3. 4.

5. 6. 7.

R.P.W. Scott, A new detector for vapor phase partition chromatography, Nature, 176 (1955) 793. R.P.W. Scott and J.G. Chesire, High efficiency columns for the analysis of hydrocarbons by gas–liquid chromatography, Nature, 180 (1957) 702–703. R.P.W. Scott, A new detector for vapor phase partition chromatography, in D.H. Desty (Ed.), Gas Chromatography 1958, Butterworths Scientific Publications, London, 1958, 145–287. R.P.W. Scott, I.A. Fowliss, D. Welti and T. Wilkins, Interrupted-elution chromatography, in A.B. Littlewood (Ed.), Gas Chromatography, 1966, published by the Institute of Petroleum, London, 1967, pp. 318–320. R.P.W. Scott, C.G. Scott, M. Munroe and J. Hess, The poisoned patient, in The Role of the Laboratory, Elsevier, New York, NY, 1974, 395–408. R.P.W. Scott and P. Kucera, Mode of operation and performance characteristics of microbore columns for use in liquid chromatography, J. Chromatogr., 169 (1979) 51–72. R.P.W. Scott, Tandem Techniques, J. Wiley and Sons, Chichester, New York, NY, 1997, 526 pp.

D.58. Robert E. Sievers Robert E. Sievers was born on March 28, 1935 in Anthony, KS. He received his Bachelor of Chemistry from the University of Tulsa, Tulsa, OK, and his M.Sc., and Ph.D. degrees from the University of Illinois, Urbana, IL. His professional experiences include the following: Director, CU Environmental Program (1993–1999) Professor of Chemistry, Department of Chemistry and Biochemistry, University of Colorado (1975– 2001); Director, Cooperative Institute for Research in Environmental Sciences, University of Colorado (1980–1993); Regent, University of Colorado (Elected to represent the 2nd Congressional District) (1990–1999); Interim Dean of the Graduate School and Associate Vice-Chancellor for Research, University of Colorado (1986–

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1987); Co-Chairman, Department of Chemistry, University of Colorado (1978–1980); Senior Scientist (GS-16), Aerospace Research Laboratories, Wright-Patterson A.F.B., Ohio (1969–1975); Visiting Professor, Tu¨bingen University, Germany (1968–1969); Research Chemist and Group Leader, Aerospace Research Laboratories, Wright-Patterson A.F.B., Ohio (1960–1967).

ž ž ž ž ž

Professor Sievers’ awards and honors include: Keene P. Dimick Award in Chromatography, PITTCON, New Orleans (1992); Gold Medal Award, Colorado Section, American Chemical Society (1985); Tswett Chromatography Medal, Barcelona, Spain (1981); Decoration for Exceptional Civilian Service, Department of Air Force (1975); Distinguished Alumni Award, University of Tulsa (1973).

Selected professional and service activities are given: 1991 Chairman, IUPAC-CHEMRAWN Conference on Changes in the Atmosphere 1987–1990 Chair, Committee on Atmospheric Chemistry, NAS=NRC. 1987–1989 Member of National Academy of Sciences’ NRC Committee on Global Change. 1985–1988 Editorial Advisory Board, Chromatographia. 1979–1982 Member of Science Advisory Board, EPA. 1984 Chairman, 22nd International Conference on Coordination Chemistry. 1970–1975 Principal Investigator for NASA Apollo Lunar Analysis Project. 1983 Co-founder, Sievers Instruments, Inc.; acquired by Ionics, Inc., 1996. 1980–1983 Editorial Advisory Board, Analytical Chemistry. 1978–1982 Science Advisor, Rocky Mountain Region of FDA. 1960–1999 Co-authored 184 journal publications and 26 patents. Selected professional publications: 1965 R. Sievers and R. Moshier, “Gas Chromatography of Metal Chelates”, Pergamon Press, New York, NY, 1965. 1978 R. Sievers and J. Sadlowski, Volatile metal chelates, Science, 201 (1978) 217–223. 1992 J. Calvert, J. Birks and R. Sievers (Eds.), “The Chemistry of the Atmosphere: Its Impact on Global Change”, American Chemical Society, Washington, 1992, 163 pp. 1995 R.E. Sievers (Ed.), “Selective Detectors: Environmental, Industrial, and Biomedical Applications”, J. Wiley and Sons, Inc., New York, NY, 1995, 261 pp. 1997 R. Sievers and U. Karst, Methods for Fine Particle Formation, US Patent 5,639,441, June 17, 1997. Professor Sievers’ research interest in chromatography dates to the mid 1950s with a wide spectrum of developments and applications in inorganic chemistry, analytical chemistry, space sciences, environmental sciences, material sciences, pharmaceutical sciences, gas chromatography of metal chelates, separation of isotopes of carbon and sulfur by GC, ozonization of water pollutants, selective complexing sorbents for

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GC, pollutants from oil shale refining, supercritical-fluid chromatography with special chemiluminescent detection, and pulmonary drug delivery of fine aerosols. NSF, EPA, DOE, DOD, NASA, private industry, and others have supported his research. Table 1 presents the historical evolution of chromatographic research by Professor Sievers and colleagues for the period of 1955–2000. The subject areas include chromatography, environmental studies, coatings, novel metal chelates, supercritical-fluid technology and fine particle synthesis. See Chapter 5B, a, d, e, f, k, o, q, r, s

58.I. THE REACH AND STIMULUS OF CHROMATOGRAPHY IN DIVERSE REALMS Robert E. Sievers Department of Chemistry and Biochemistry and CIRES, Campus Box 215, University of Colorado, Boulder, CO 80309, USA

58.I.1. Early experiments In the mid-1950s, my first experiments with gas chromatography with home-made equipment so inspired me that they have shaped my professional career spanning nearly a half century. If anyone had told me at the time that chromatography would stimulate so many explorations in other realms, I would never have believed them. As an Awardee, the Editors have provided me with an opportunity to share the threads of stimulus that chromatography has provided to lead us down the many paths we have followed.

58.I.2. The reach and influence of chromatography Through the years, my co-workers and I have employed chromatography in inorganic chemistry, analytical chemistry, space sciences, environmental sciences, materials sciences, and pharmaceutical sciences. This has led us into subjects as diverse as the separation of optical isomers by gas chromatography [1], gas chromatography of metal chelates [2], metal organic chemical vapor deposition (MOCVD) [3], trace analysis of metals by electron capture gas chromatography [4], separation of isotopes of carbon and sulfur by packed column gas chromatography [5], trace measurement by GC of chromium and beryllium in the first moon rocks brought to Earth by the Apollo astronauts [6], the invention of fluorinated NMR shift reagents [7], studies of the interactions of nucleophiles with coordinatively unsaturated metal chelates [8], the trace analysis of aqueous nitrates and nitrites in saliva, blood, and suspended particulates in ambient air by GC–ECD [9], ozonization of water pollutants [10], vinyl chloride measurement at sub-ppb levels using chemically sensitized electron capture detection [11], selective

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complexing sorbents for GC [12], gold-catalyzed reactions forming NO for selective detection [13], chromatographic measurement of organic compounds in ambient air [14], pollutants from oil shale refining [15], selective retention of oxygen using GC columns containing metal chelate polymers [16], selective chemiluminescence chromatography detectors [17], sources of aerosols in the troposphere [18], supercritical-fluid chromatography with selective chemiluminescent detection [19,20], pulmonary drug delivery of fine aerosols assisted by supercritical fluids [21], synthesis of sub-micron phosphor spheres by supercritical carbon dioxide-assisted aerosolization and pyrolysis [22], and the invention of new methods for aerosolizing aqueous solutions for spray drying pharmaceuticals for dry powder inhalers [23]. To the casual reader some of these subjects may appear unrelated to chromatography, but, if you read further, the creativity and inspiration links will become more apparent.

58.I.3. Environmental studies and needs My first experiments with gas chromatography in the mid-fifties inspired me to attempt to apply chromatography to all kinds of complex and challenging systems. For example, chromatography provides the tools to trace the identities, amounts, and sources of a wide variety of environmental pollutants through complex matrices and systems [24]. The powers of separation and detection are so impressive that even when analytes were non-volatile, many of the chromatography pioneers used their skills and knowledge of chemistry to form derivatives that would be stable and volatile enough to be rapidly separated and sensitively measured. At the same time, the pioneers were drawing on physics to push back the frontiers of selective and sensitive detection.

58.I.4. Gas chromatography and NMR of metal chelates In the early days of the Lovelock electron capture detector, my group sought to convert mixtures of metal ions into volatile fluorinated metal chelates that we separated in what was at that time a trace metal analysis method with unprecedented sensitivity [25,26]. We applied this chromatographic approach to the trace determination of chromium and beryllium in matrices as diverse as lunar rocks [27], blood [28], and aerosol particles in the atmosphere [29]. Once the right chelating agents and chiral sorbents were in hand, it became possible to separate optical enantiomers of metal chelates by gas chromatography [1]. We synthesized new unsymmetrical fluorinated chelating agents [30], that allowed us to perform other studies in inorganic stereochemistry [31]; developing skills in stereochemistry has allowed me to pursue a side career as a marble sculptor (an example of more than 50 works, some of which are permanently installed in public settings, is shown in Fig. 1). We discovered that some of the same fluorinated metal chelates that we synthesized and studied as volatile species that might be suited for metal analysis were even more valuable as nuclear magnetic resonance shift reagents [32–34]. Not only were NMR spectra greatly simplified, but also optical enantiomers could be distinguished and quantitated by NMR. Our papers

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Fig. 1. Professor Robert E. Sievers with ‘Colorado Rose’, which he carved from marble.

on these subjects became among the most frequently cited in the scientific literature during that era, and we were awarded US and foreign patents [35]. Few of the thousands of users of Aldrich’s ‘Sievers Eu-fod Shift Reagent ’ (and related products) know that the reagents that they have found so useful in NMR spectroscopy grew out of chromatography studies.

58.I.5. Thin films and coatings Similarly, our other chromatography studies led to new processes for forming thin films and coatings. In 1967, and later in 1990, my students and I developed new processes [3,36,37] that utilize metal chelates that are volatile (and soluble in supercritical fluids) to form metal and metal oxide films and coatings at heated surfaces. These processes are of interest in materials science because they can rapidly coat

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large surface areas at atmospheric pressure, and they are not limited to line-of-sight deposition. Again this is work that was inspired by our earlier studies of the gas chromatography of metal chelates [38,39].

58.I.6. Selective detectors and sorbents Our work on selective detectors [13,17,19,40–49] and on selective sorbents based on the interactions of nucleophiles with coordinatively unsaturated metal chelates was driven by the need to simplify the analysis of extraordinarily complex environmental samples. As sensitivity of detection improved, the number of compounds that could be detected skyrocketed and outstripped our abilities to separate all of the detected species, even with the great improvements in column resolution afforded by highly efficient capillary columns. So chromatographers studying environmental and biomedical systems had to become more inventive, both in selective detection and in sample pretreatment. Fortunately, these goals in chromatographic analysis provided the stimulus for the creation, development, and marketing of a wide array of selective detectors. In 1984, Drs. Misha Plam and Ric Hutte, and I formed Sievers Instruments, Inc., to develop selective and sensitive instrumentation for chemical analysis. Initially, the selectivity of the detectors was based on chemiluminescence, but as the company evolved, it benefited from advances by many scientists and by analytical needs in fields as diverse as medicine, pharmaceuticals, foods and beverages, water analysis, petrochemicals, environmental sciences, and space sciences. Sievers Instruments has manufactured thousands of selective detectors which are now in use throughout the world; one has been launched and used to continuously monitor water quality by total organic carbon (TOC) analysis for the astronauts on a platform in space. Other Sievers instruments are used to selectively measure trace levels of sulfur or nitrogen by chemiluminescence in petrochemical plants and in health science centers. Tracking how nitric oxide moves and acts in the body as a messenger and plays roles in vasodilation, asthma, angina and impotence was the subject of 1998 Nobel Prizes in physiology and medicine, many researchers in this field have used instruments that grew out of earlier selective chemiluminescence chromatography detectors. By 1996, Sievers Instruments, Inc. had grown to more than 100 employees and was acquired by Ionics, Inc. (New York Stock Exchange symbol ‘ION’). It now develops chromatographic and other instrumentation as a subsidiary under the name, Ionics-Sievers.

58.I.7. Aerosolization and fine powder synthesis Our most recent advances in aerosolization, fine particle synthesis, and spray-drying can be traced to our earlier experiments in the 1980s with supercritical-fluid chromatography [19,20], or as it has been sometimes called, dense-gas chromatography. We have developed [21,23] and patented [50–53] new processes for forming microspheres and microbubbles with micron and sub-micron size diameters. In one process [23] therapeutic drugs are dissolved in water and an emulsion is formed with supercritical carbon

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dioxide. Approximately one mole percent of carbon dioxide can become dissolved in the aqueous solution, and this greatly facilitates the formation of very fine bubbles and droplets when the emulsion is ejected through a nozzle and the droplets return to atmospheric pressure. As the small bubbles swell and burst, and mix with dry air or dry nitrogen, they are dehydrated to form dry powders that are small enough to be used in pulmonary drug delivery to the deep, most distal alveoli. Drying of the microbubbles occurs more rapidly than spray drying of droplets formed by conventional methods. In some instances, hollow spheres are formed from the drying of micro bubbles. The gentle nebulization and drying of protein, peptide, and small molecule drugs has made it more likely that a wide array of drugs can be delivered by inhalation rather than by injection.

58.I.8. Waste clean-up By developing a new method of extracting organic compounds from atmospheric aerosol particles with supercritical carbon dioxide [53], to complement thermal desorption [18], preparatory to gas chromatographic analysis, we extended this application in chromatography to the clean-up of wastes. It was demonstrated that several organic compounds could be removed from waste matrices and that certain metal chelates have unusually high solubilities in liquid and supercritical carbon dioxide [54]. In 1967, we reported that many metals and metal oxides can undergo reactions directly with a fluorinated chelating agent [55], and these chelated products can be removed from wastes by extractions with carbon dioxide.

58.I.9. The influence of chromatography in broadly diverse publications As support for the hypothesis that chromatography has influenced work in many diverse realms, one might examine the 40 different journals in which our group has published its findings from 1962 to the present. There are the expected journals in which chromatographers usually publish their work: 30 papers in Analytical Chemistry and 19 in the Journal of Chromatography, but there were also 13 papers in the Journal of the American Chemical Society and 22 in Inorganic Chemistry. There were also papers in Science, Nature, Journal of Applied Physics, Journal of Geophysics, Earth and Planetary Science Letters, Chemistry of Materials, Polyhedron, Environmental Science and Technology, Environmental Geochemistry and Health, Aerosol Science and Technology, and Journal of Aerosol Medicine. Chromatography has had a broad influence, indeed, on the work of my colleagues and me, and on science in general.

Acknowledgments Research is a costly enterprise, and it is appropriate to thank those who have provided the funding for our studies of chromatography and the many paths it has led us along. We are grateful to NSF, NOAA, EPA, DOE, AFOSR, NIST, DARPA,

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NASA, USGS, Genentech, Boehringer-Ingelheim Pharma, Cires, Alza Pharmaceuticals, Rhone-Poulenc-Rorer, Ford, Monsanto, Hewlett-Packard, Spire, Sievers Instrument Co., and others for financial support.

58.I.10. Kudos Space does not permit me to thank all of my many students and collaborators, but their contributions, hard work, and wonderful stimulation have been a source of great satisfaction to me. We all stand on the shoulders of others who have worked with us and gone before us. Our legacies will be those whom we have encouraged and helped to learn, and the advances that we have made together. My Doktorvater, the late Prof. John C. Bailar, Jr., was a source of great inspiration to me while I was studying at the University of Illinois; and my postdoctoral year with Prof. Dr. Ernst Bayer at Tu¨bingen Universita¨t deepened my love of chromatography.

References 1. 2. 3. 4. 5. 6.

7. 8.

9. 10.

11.

12. 13.

R.E. Sievers, R.W. Moshier and M.L. Morris, Resolution of chromium(III) hexafluoroacetylacetonate by gas chromatography, Inorg. Chem., 1 (1962) 966–967. R.W. Moshier and R.E. Sievers, Gas Chromatography of Metal Chelates, Pergamon Press, Oxford, 1965 (Russian translation, Moscow, 1967). R.E. Sievers, R.L. Van Hemert and L.B. Spendlove, Vapor deposition of metals by hydrogen reduction of metal chelates, J. Electrochem. Soc., 112 (1965) 1123–1126. W.D. Ross and R.E. Sievers, Ultra-trace analysis of beryllium by gas chromatography, in Gas Chromatography, Butterworths Scientific Publications, London, UK, 1966, pp. 272–295. E. Bayer, G. Nicholson and R.E. Sievers, Separation of isotopes of carbon and sulfur by gas chromatography with a packed column, J. Chromatogr. Sci., 8 (1970) 467–470. K.J. Eisentraut, D.J. Griest and R.E. Sievers, Ultratrace analysis for beryllium in terrestrial, meteoritic and Apollo 11 and 12 lunar samples using electron-capture gas chromatography, Anal. Chem., 43 (1971) 2003–2007. R.E. Rondeau and R.E. Sievers, New superior paramagnetic shift reagents for spectral clarification, J. Am. Chem. Soc., 93 (1971) 1522–1524. B. Feibush, C.S. Springer, Jr., M.F. Richardson and R.E. Sievers, Studies of interactions of weak donors with rare earth chelates by gas–liquid chromatography, J. Am. Chem. Soc., 94 (1972) 6717– 6724. J.W. Tesch, W.R. Rehg and R.E. Sievers, Microdetermination of nitrates and nitrites in saliva, blood, water, and suspended particulates in air by gas chromatography, J. Chromatogr., 126 (1976) 743. R.E. Sievers, R.M. Barkley, G.A. Eiceman, R.H. Shapiro, H.F. Walton, K.J. Kolonko and L.R. Field, Environmental trace analysis of organics in water by glass capillary column chromatography and ancillary techniques: products of ozonolysis, J. Chromatogr., 142 (1977) 745–754. P.D. Goldan, F.C. Fehsenfeld, W.C. Kuster, M.P. Phillips and R.E. Sievers, Vinyl chloride detection at sub-ppb levels using a chemically sensitized electron capture detector, Anal. Chem., 52 (1980) 1751–1754. J.E. Picker and R.E. Sievers, Applications of selective complexation by a europium (III) coordination polymer sorbent for the pre-fractionation of volatile compounds, J. Chromatogr., 217 (1981) 275–288. M.J. Bollinger, R.E. Sievers, D.W. Fahey and F.D. Fehsenfeld, Conversion of NO2, HNO3 , and n-propyl nitrate to NO by a gold-catalyzed reduction with CO, Anal. Chem., 55 (1983) 1980–1986.

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14. J.M. Roberts, F.C. Fehsenfeld, D.L. Albritton and R.E. Sievers, Measurement of monoterpene hydrocarbons at Niwot Ridge, Colorado, J. Geophys. Res., 88 C15 (1983) 10667–10678. 15. S.B. Hawthorne and R.E. Sievers, Emission of organic air pollutants from shale oil wastewaters, Environ. Sci. Technol., 18 (1984) 483–490. 16. J.N. Gillis and R.E. Sievers, Selective retention of oxygen using chromatographic columns containing metal chelate polymers, Anal. Chem., 57 (1985) 1572–1577. 17. R.E. Sievers, S.A. Nyarady, R.L. Shearer, J.J. DeAngelis, R.M. Barkley and R.S. Hutte, Selectivity of the redox chemiluminescence detector for complex sample analysis, J. Chromatogr., 349 (1985) 395–403. 18. R.C. Greaves, R.M. Barkley, R.E. Sievers and R.R. Meglen, Covariations in the concentrations of organic compounds associated with springtime atmospheric aerosols, Atmos. Env., 21 (12) (1987) 2549–2561. 19. W.T. Foreman, R.E. Sievers and B.W. Wenclawiak, Supercritical-fluid chromatography with redox chemiluminescence detection, Fresenius Z. Anal. Chem., 330 (1988) 231–234. 20. W.T. Foreman, C.L. Shellum, J.W. Birks and R.E. Sievers, Supercritical-fluid chromatography with sulfur chemiluminescence detection, J. Chromatogr., 465 (1989) 23–33. 21. B.M. Hybertson, J.E. Repine, C.J. Beehler, K.S. Rutledge, A.F. Lagalante and R.E. Sievers, Pulmonary drug delivery of fine aerosol particles from supercritical fluids, J. Aeros. Med.: Depos. Clear. Effects Lung, 6 (1993) 275–285. 22. C. Xu, B.A. Watkins and R.E. Sievers, Submicron sized spherical yttrium oxide based phosphors prepared by supercritical CO2 -assisted aerosolization and pyrolysis, Appl. Phys. Lett., 71 (12) (1997) 1643–1645. 23. R.E. Sievers, U. Karst, P.D. Milewski, S.P. Sellers, B.A. Miles, J.D. Schaefer, C.R. Stoldt and C.Y. Xu, Formation of aqueous small droplet aerosols assisted by supercritical carbon dioxide, Aerosol Sci. Technol., 30 (1999) 3–15. 24. P.R. Veltkamp, K.J. Hansen, R.M. Barkley and R.E. Sievers, Principal component analysis of summertime organic aerosols at Niwot Ridge, Colorado, J. Geophys. Res., 101: D14 (1996) 19,495– 19,504. 25. W.D. Ross, G. Wheeler and R E. Sievers, Quantitative ultra-trace analysis of mixtures of metal chelates by gas chromatography, Anal. Chem., 37 (1965) 598–600. 26. W.D. Ross and R.E. Sievers, Rapid ultratrace determination of beryllium by gas chromatography, Talanta, 15 (1968) 87–94. 27. K.J. Eisentraut, M.S. Black, F.D. Hileman and R.E. Sievers, Beryllium and chromium abundances in Fra Mauro and Hadley-Apennine lunar samples, Geochim. Cosmochim. Acta, 2 (1972) 1327–1333. 28. L.C. Hansen, W.G. Scribner, T.W. Gilbert and R.E. Sievers, Rapid analysis for sub-nanogram amounts of chromium in blood and plasma using electron capture gas chromatography, Anal. Chem., 43 (1971) 349–353. 29. W.D. Ross, J.L. Pyle and R.E. Sievers, Analysis for beryllium in ambient air particulates by gas chromatography, Environ. Sci. Tech., 11 (1977) 469–471. 30. C.S. Springer, D.W. Meek and R.E. Sievers, Rare earth chelates of 1,1,1,2,2,3,3-heptafluoro-7,7dimethyl-4,6-octanedione, Inorg. Chem., 6 (1967) 1105–1110. 31. B. Wenclawiak, R.M. Barkley, E.J. Williams and R.E. Sievers, Gas chromatographic and liquid chromatographic separations of geometrical isomers of tris (2,2,7-trimethyloctane-3,5-dionato)chromium(III) and cobalt(III) chelates and other Cr-β-diketonates, J. Chromatogr., 349 (1985) 469–479. 32. T.J. Wenzel and R.E. Sievers, Nuclear magnetic resonance studies of terpenes with chiral and achiral lanthanide(III)-silver(I) binuclear shift reagents, J. Am. Chem. Soc., 104 (1982) 382–388. 33. T.J. Wenzel, T.C. Bettes, J.E. Sadlowski and R.E. Sievers, New binuclear lanthanide NMR shift reagents effective for aromatic compounds, J. Am. Chem. Soc., 102 (1980) 5903–5904. 34. R.E. Rondeau and R.E. Sievers, New NMR shift reagents, Anal. Chem., 45 (1973) 2145–2147. 35. R.E. Sievers, Rare earth complexes as nuclear magnetic resonance shift reagents, US Patent 3,846,333, Nov. 5, 1974. 36. R.E. Sievers, S.B. Turnipseed, L. Huang and A.F. Lagalante, Volatile barium β-diketonates for use as MOCVD precursors, Coord. Chem. Rev., 128 (1993) 285–291.

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37. B.N. Hansen, B.M. Hybertson, R.M. Barkley and R.E. Sievers, Supercritical-fluid transport–chemical deposition of films, Chem. Mater., 4 (1992) 749–752. 38. K.J. Eisentraut and R.E. Sievers, Volatile rare earth chelates, J. Am. Chem. Soc., 87 (1965) 5254–5256. 39. R.E. Sievers, B.W. Ponder, R.W. Moshier and M.L. Morris, Gas phase chromatography of metal chelates of acetylacetone, trifluoroacetylacetone and hexafluoroacetylacetone, Inorg. Chem., 2 (1963) 693–698. 40. R.E. Sievers (Ed.), Selective Detectors: Environmental, Industrial, and Biomedical Applications, J. Wiley and Sons, Inc., New York, NY, 1995, 261 pp. 41. T.B. Ryerson, R.M. Barkley and R.E. Sievers, Selective chemiluminescence detection of sulfur-containing compounds coupled with nitrogen-phosphorus detection for gas chromatography, J. Chromatogr. A, 670 (1994) 117–126. 42. T.B. Ryerson, A.J. Dunham, R.M. Barkley and R.E. Sievers, Sulfur-selective detector for liquid chromatography based on sulfur monoxide-ozone chemiluminescence, Anal. Chem., 66 (1994) 2841– 2851. 43. A.J. Dunham, R.M. Barkley and R.E. Sievers, Aqueous nitrite ion determination by selective reduction and gas-phase nitric oxide chemiluminescence, Anal. Chem., 67 (1995) 220–224. 44. A.J. Dunham, and R.E. Sievers, in R. Sievers (Ed.), Selective Detectors, John Wiley and Sons, Inc., New York, NY, 1995, pp. 71–95. 45. N. Pourreza, S.A. Montzka, R.M. Barkley and R.E. Sievers, Chemistry occurring in redox chemiluminescence detectors for chromatography, J. Chromatogr., 399 (1987) 165–172. 46. W.T. Foreman, C.L. Shellum, J.W. Birks and R.E. Sievers, Supercritical-fluid chromatography with sulfur chemiluminescence detection, J. Chromatogr., 465 (1989) 23–33. 47. R.S. Hutte, R.E. Sievers and J.W. Birks, Gas chromatography detectors based on chemiluminescence, J. Chromatogr. Sci., 24 (1986) 499–505. 48. S.A. Nyarady and R.E. Sievers, Selective catalytic oxidation of organic compounds by nitrogen dioxide, J. Am. Chem. Soc., 107 (1985) 3726–3727. 49. M.A. Wizner, S. Singhawangcha, R.M. Barkley and R.E. Sievers, Selective electron-capture sensitization of water, phenols, amines and aromatic and heterocyclic compounds, J. Chromatogr., 239 (1982) 145–157. 50. R. Sievers, B. Hybertson and B. Hansen, Methods and apparatus for drug delivery using supercritical solutions, US Patent 5,301,664, April 12, 1994. 51. R. Sievers, B. Hybertson and B. Hansen, Methods and apparatus for drug delivery using supercritical solutions, European Patent EP 0 627 910 B1, Jan 10, 1997. 52. R. Sievers and U. Karst, Methods for fine particle formation, US Patent 5,639,441, June 17, 1997. 53. K.J. Hansen, B.N. Hansen, E. Cravens and R.E. Sievers, Supercritical-fluid extraction–gas chromatographic analysis of organic compounds in atmospheric aerosols, Anal. Chem., 67 (1995) 3541– 3549. 54. A.F. Lagalante, B.N. Hansen, T.J. Bruno and R.E. Sievers, Solubilities of copper(II) β-diketonates in supercritical carbon dioxide, Inorg. Chem., 34 (1996) 5781–5785. 55. R.E. Sievers, J.W. Connolly and W.D. Ross, Metal analysis by gas chromatography of chelates of heptafluorodimethyloctanedione, J. Gas Chromatogr., 5 (1967) 241–247.

D.59. Colin F. Simpson Colin F. Simpson was born on March 1, 1932, on the Overcliffe Drive, Southbourne, Bournemouth and now lives at Eaton Place, Kemp Town, Brighton, UK. Following the award of an external University of London B.Sc. degree in Special Chemistry in 1956, Colin Simpson obtained a position as a research chemist with British Petroleum where he worked until 1964. He moved to Brunel College of Advanced Technology as a research assistant and there he obtained his external University of London M.Sc. Degree.

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He was then appointed to the University of Sussex in June 1966 as a senior experimental officer, and remained there until 1979 when he gained a University of London Ph.D. and joined Chelsea College, University of London as a lecturer in Analytical Chemistry. In 1984, he moved to the Department of Chemistry, Birkbeck College, University of London, as Lecturer, Senior Lecturer (1989) and Head of Department (1990–1994). He retired from Birkbeck College in September 1997 and joined King’s College, London as a research worker and part-time lecturer. Colin Simpson is a member of the Editorial Boards of the Journal of Chromatographic Science, Chromatographia, Separation Science Series (John Wiley and Sons), Royal Society of Chemistry Chromatography monographs series and Chromatography and Analysis. He has been the author, editor and=or contributor to eight books on chromatography and electrophoresis, viz.: “Gas Chromatography” (1970), “Practical High Performance Liquid Chromatography” (1976), “Techniques in Liquid Chromatography” (1982), “HPLC in Food Analysis” (1982), “Techniques in Electrophoresis” (1983), “Characterization of Proteins” (1988), “HPLC in Biotechnology” (1992) and “The Application of Capillary Electrophoresis for Environmental Monitoring” (1996). For the Chromatographic Society, he organized workshops in HPLC, 1972 and 1973, and for the Royal Society of Chemistry, he organized a review Symposium on HPLC, 1974; followed by ten RSC summer schools on HPLC commencing in 1975 then biennially to 1993. Organized a Faraday Symposium on chromatography, equilibria and kinetics in 1980, and several other meetings on chromatographic methodology. He has published about eighty scientific papers, mainly on chromatography and electrophoresis and made (with students) an equivalent number of presentations at scientific meetings. Dr. Simpson was the recipient of the Royal Society of Chemistry Silver Medal for contributions to separation science and detection in 1991, and the Chromatographic Society’s Jubilee Medal in 1997. He was placed fourth in the Desty Memorial Prize for Advances in Separation Science, with a submission entitled: ‘High Speed Separations using Serpentine Capillaries.’ Dr. Simpson has been responsible for the teaching of separation science in analytical chemistry at both the undergraduate and post-graduate (M.Sc.) level. He has been responsible for the guidance of some 250 M.Sc. theses and about 50 undergraduate projects. He has been the advisor to 26 Ph.D. students who have all been awarded their degrees and been the external=internal examiner of about 50 Ph.D. theses drawn from a number of UK universities. In addition to his University teaching duties, he has frequently been invited to speak at meetings and schools in the USA, Germany, France, The Netherlands, Ireland, UK and Thailand. See Chapter 5B, a, d, e, f, h, l, q, r, s

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59.I. RESEARCH AND DEVELOPMENT OF INTEREST IN CHROMATOGRAPHY AND ELECTROPHORESIS Colin F. Simpson Department of Pharmacy, King’s College, London, 150 Stamford Street, Waterloo, London SE1 8WA, UK

My first contact with the chromatographic technique occurred shortly after joining British Petroleum as a research chemist in 1958. I was asked to synthesize a cyclic boron–nitrogen tetramer as a pre-ignition additive for gasolines and was advised to estimate its purity on a Metrovick gas chromatograph, a process which achieved only limited success. This was followed by synthesizing cyclic boron–phosphorus compounds as pre-ignition additives for gasolines (for obvious reasons) and again estimating their purity by GLC. This was more successful. Subsequent to these rather limited initial attempts to use GLC, I met C.L.A. (Happy) Harbourne and together we analyzed by GLC the various typical component streams for gasolines. This was considerably more successful (due to Happy’s expertise) and we accumulated a dossier of the composition of fuel constituents from which it was possible to estimate their approximate proportion in finished gasolines. Following this initial experience with GLC, I must confess to have been slightly disappointed initially. Then I became very interested and enthusiastic about the ability to separate and identify the components of gasolines, which previously had been done by distillation procedures to produce a hydrocarbon type analysis, a rather wearisome procedure. Then I was assigned a project to discover suitable additives for gasolines to prevent carburetor icing during the initial 5 min or so from start-up on cold dark winter mornings. After screening a very large number of different chemicals, it was determined that dipropylene glycol (DPG) had the best carburetor anti-icing performance, although it showed a tendency to be leached out of solution by the water bottoms in storage tanks. I decided to estimate the amount of DPG in gasolines using GLC and commenced work to determine its retention volume (time). It turned out that the sample of DPG analyzed was rather impure, containing at least three components. At that time (ca. 1962) Dennis Desty, Alan Goldup, Terry Swanton and others had developed equipment for performing capillary GC on a semi-routine basis with Carbowax 20 M as the stationary phase; five components were determined. (It is strange that I did not know Dennis at BP, that had to wait until later). Other samples of DPG from different sources all gave similar results, but with different ratios of the components. I rather suspected that what was being separated were the three possible structural isomers of DPG (there were three major peaks) and the two (minor) peaks were impurities. After a considerable amount of work involving high efficiency distillation, preparing the p-nitrobenzoyl derivatives of DPG followed by fractional crystallization, and undertaking structure determination by NMR, it was determined that what was present was only DPG, and that two pairs of diastereoisomers had been separated, the third, unseparated pair consisted of two secondary alcohol groups on the chiral centers, i.e., CH3 ÐCH(OH)ÐCH2 ÐOÐCH2 ÐCH(OH)ÐCH3 . The minor impurities were, in fact, the

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diastereoisomers of the diprimary alcohol, i.e., CH3 ÐCH(CH2 ÐOH)ÐOÐCH(CH2 ÐOH)CH3 . The other two components were the mixed primary=secondary diastereoisomers. I was interested in pursuing this work and the opportunity arose for getting a research fellowship sponsored by Wilkens Aerograph, Walnut Creek, CA, USA to continue the study with Stuart Thorburn at Brunel College of Advanced Technology. I started there in October 1964 and followed a series of postgraduate lectures on chromatography given by Desty, Goldup, Welti and importantly, R.P.W. Scott who enthused me enormously in the technique; indeed this was the start of a long-lasting collaboration and friendship. Also present at Brunel at that time was Terry Gough, who was undertaking research on the mass detector and Dr. Badami who was very keen on performing separations by reversed phase TLC (which was to form a large part of my research later). Following a year of research on attempting to provide an improved separation of the DPG isomers (and other diols containing two asymmetric centers) and the optical enantiomers present, my progress was monitored in early 1966 for Wilkens Aerograph by Dr. Harold McNair, who was of the opinion that it was not possible to separate enantiomers and recommended that my fellowship, be terminated. In June of 1966, E. Gil-Av demonstrated the chiral separation of amino acid derivatives by capillary GLC on a chiral stationary phase, (N-TFA-L-isoleucine lauryl ester). I therefore wrote up my work for a Master’s degree which proposed that enantiomers could be separated by the formation of transient diastereoisomers and the ease of separation depended on the separation between the chiral centers and the size of any groups attached to the chiral center [1]. The work was also presented to the Gas Chromatography Discussion Group Meeting in September 1971. In June 1966, I joined the School of Molecular Sciences at the University of Sussex as a senior experimental officer to run a laboratory devoted to separation science in general and chromatography in particular. I had to set up a laboratory capable of analyzing almost anything, volatile and non-volatile, that the chemical laboratories could produce. The School at that time was concerned principally with natural product chemistry and organo-metallic chemistry so that the range of analyte types was large. After careful consideration I decided to buy the gas chromatographs capable of providing the maximum versatility in the maximum number of units to maximize throughput: these were the Pye 104 series of gas chromatograph. I also purchased a Varian Aerograph 1520 GC for its versatility, but it turned out not to give the high running time required and which was possible to obtain on the much cheaper Pye machines. The cost of one 1520 was sufficient for 3–4 Pye machines! A wide variety of different analytes were investigated, but of course, GLC was not capable of undertaking all the work submitted for analysis. In 1967, I therefore commenced work on HPLC using a moving wire detector. I initially started work on LLC and obtained satisfactory results, but was frustrated by the instability of the column systems through stripping the stationary phase from the column resulting in varying retention times. It occurred to me that it might be possible to prepare stationary phases chemically bonded to a silica support similar to the way silicous supports were deactivated by silanization for GLC applications. Two methods were employed: (1) through the formation of silicon esters, and (2) using the silanization technique. Both methods gave excellent results in normal phase operation, but the silicon ester technique failed for reversed phase work

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through hydrolysis of the Si–O–C bond. A patent application was filed in January 1968 [2] and granted April 1975. A paper was presented to the (then) Gas Chromatography Discussion Group informal meeting held in March of that year. At the same time I constructed a GC–MS system to provide a means of identifying the various components separated by GLC. The molecular separator used was initially a Watson–Biemann porous glass device followed by a variable jet separator based on Rhyage’s jet separator. In addition with Terry Gough, a miniature membrane separator [3] was constructed based on the Llewellyn and Littlejohn double membrane device. All of these systems worked well, but had some drawbacks compared with direct introduction from capillary columns. Two other advances were made during my time at Sussex; one direct, the other provided a basis for subsequent work. During a visit of a Bulgarian worker, Penko Russev, when we were investigating the operation of the flame ionization detector (FID) with oxygen as the supporter of combustion, some unusual results were obtained. It was shown that it was possible to detect inorganic gases using the FID operated in this way. This work was repeated in Terry Gough’s laboratory. Furthermore, it was possible to obtain direct quantitative results on a molar and weight basis. In addition, by operating the detector at high hydrogen flow rates, it was possible to obtain selective responses to the halogens and, to a lesser extent, to nitrogen and phosphorus containing compounds whilst minimizing the response to carbon. However, the flame temperature achieved burnt out a conventional detector quite quickly and hence a new design was constructed with concentric alumina jets and called the FIDOH detector, i.e., FID operated with Oxygen and Hydrogen (with thanks to Ron Hurrell for the name). This work was written up in a series of papers and the new detector patented [4]. It was presented at the St. Louis meeting on Advances in Chromatography and, at this meeting, Ray Scott asked me if I would like to spend some time working in his laboratories at Hoffman-La Roche, an opportunity I accepted. The other advance was concerned with some work being undertaken by I.G. Nixon on the use of a rotating tubular furnace for the preservation of the pore structure of low grade iron ores on reduction with butane. The equipment worked well but was subject to numerous breakdowns. I suggested that instead of using this type of furnace it would be preferable to use a fluidized bed, which had no moving parts and achieved excellent gas–solid contact. This proved to be far superior to the previous method and suggested to me that it would be interesting to investigate whether the fluidized bed method could be used for the production of chemically bonded phases for liquid chromatography. However, this had to wait because I was in the process of leaving Sussex and moving to the University of London. One final item most worthy of note occurred during the mid 1970s; Professor A.J.P. Martin moved to Sussex University to carry out a research project on preparative isoelectric focusing. I had many fascinating discussions with him which were causal in many ways but, most importantly, he stimulated me to do work on electrophoresis, which he believed was a superior separation technique to chromatography! I commenced work on isotachophoresis (ITP), which indeed I am continuing to investigate. Before moving to the University of London at Chelsea College, I spent six months working with Dr. Ray Scott in the USA. We worked on a project concerned with the

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mechanism of separation by reversed-phase chromatography and produced sufficient work on the effect of mobile phase modifiers to produce a paper for a Faraday Symposium in 1980 [5]. It was quite clear that the use of mobile phase additives could change the nature of the surface available for interaction with the solute molecules, and this became the subject of research when I joined the University of London and several Ph.D. theses and papers resulted. I started work at Chelsea College in December 1979 and immediately became immersed in the teaching of analytical chemistry and separation science at both the undergraduate and postgraduate (M.Sc.) level together with starting to build up a research group. Fortunately, the Master’s teaching also included a research topic, which extended over a period of three to six months. This enabled me to undertake feasibility studies on various topics that could, if successful, be written up for a grant application. Money in UK Universities was tight and in the early 1980s Analytical Chemistry was rarely supported by the funding councils. Nevertheless, it was possible to undertake work with the limited equipment available and I had several M.Sc. students to do feasibility studies. The proposed fluidized bed idea was researched and initially it was uncertain that the method would work as silica gel is a colligative (sticky) material and not easy to fluidize. It was shown that the nature of the bottom plate was important in that it was necessary to have a moderate pressure drop across the plate to promote an even flow of fluidizing gas up the fluidization tower. Preliminary work indicated that the scheme was feasible and from the graduating students I found one who was happy to progress the work. Unfortunately, it was impossible to start this investigation immediately because, following a major study of the University of London, it was decided to close the Department of Chemistry at Chelsea. After lengthy negotiations, it was decided that the analytical group transfer to Birkbeck College and occupy laboratories in University College, Department of Chemistry into which the whole of Birkbeck’s chemistry department eventually moved with office space in Gordon House next door. This move eventually led to the demise of the Department of Chemistry at Birkbeck College, which was a shame because excellent work had been carried out in the Department. In 1980, I spent a further period of three months working in Ray Scott’s laboratory at Hoffman-La Roche, this time considering extra-column band broadening. Various configurations of connecting tubes were considered and it was clear that it should be possible to eliminate or at least, reduce band broadening to a minimum. Time prevented this from being achieved, but it was subsequently, following Ray’s move to Perkin-Elmer in 1981 when he and Elena Katz produced serpentine tubing which effectively completely removed extra column band broadening (see later under capillary LC). At Roche, Charlie Reese asked me if he could undertake research with me in London, and I was delighted to accept him as a student and subsequently he became a close friend. Charlie arrived in London in 1983, and commenced work on the FIDOH detector and subsequently properties of the Nitrogen Phosphorus Detector for Perkin-Elmer who were kindly supporting him. The projected move to Birkbeck College came about in August 1984. I was able to set up my laboratories once more and get equipment constructed for the fluidized bed project. Meng Khong moved with me from Chelsea to undertake research on this topic

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and the following year, Sam Akapo joined the group to work alongside Meng. This proved to be a most fruitful collaboration which demonstrated the worth of the fluidized bed technique in being able to provide highly repeatable and reproducible packings with a high surface coverage. The rather nice part of the process was that there was no solvent to be removed and the packing was automatically fined during the process, so that at the end of the reaction the prepared phase could be packed immediately into a column without any further work-up. This process was patented [6]. Through my teaching of separation science (see later), I was asked to run a short course on chromatography at the 1981 Symposium on Advances in Chromatography at Barcelona, and to participate in the meeting subsequently. Jim Jorgenson presented his paper on capillary zone electrophoresis at this meeting which caused a considerable stir, and although a large number of papers had been published on capillary electrophoresis (mainly isotachophoresis), this particular paper caught the imagination of the audience including myself; (because it showed separated peaks — in ITP the output is a series of steps). I determined to commence work on the topic as soon as possible. I had about five years of background in isotachophoresis, but not in the depth I wished to have and certainly not in zone electrophoresis. In 1984, following the move to Birkbeck College, during student admissions the Head of the Department made one studentship available to assist the course get off the ground at its new home. This position was taken by Kevin Altria, who obtained his Master’s degree in 1985, and I had advised him to do a research project on electrophoresis. Subsequently Kevin undertook research in capillary electrophoresis under my direction and he was awarded the Ph.D. degree for his work in 1990. Over the period 1984–1990, my research group expanded to an average of twelve students working on different aspects of separation science. However attention continued on the fluidized bed technique and variations, the mechanism of chiral separations by both chromatography and electrophoretic methods, capillary electrophoresis and the mechanism of reversed phase separation and the use of mobile phase additives. One of the disadvantages with the fluidized bed process is that it is a batch process; even though the quality in terms of repeatability and reproducibility between batches was excellent, the setting up cost for the process was excessive. An attempt was made to solve this problem by using a counter-current reaction process with the rate of reaction accelerated by catalyzing the process with microwave energy. This concept has been investigated using M.Sc. student projects and shown to be feasible but requires further work to investigate all the parameters. The success of using microwave stimulation prompted me to investigate whether microwave energy would catalyze the hydrosilylation reaction in the absence of the platinum catalyst usually required. This could be of importance because the platinum catalyst remains in the product after reaction; it is very difficult to remove it entirely and it could have a deleterious effect on solute molecules. By taking silica gel, brominating with thionyl bromide, and reducing the resultant surface with hydrogen, one obtains a silicon hydride surface which reacts with terminal olefins in the hydrosilylation reaction when stimulated by microwave radiation at 2.45 GHz. The product shows excellent chromatographic properties and potentially could lead to a high density of surface ligands, far greater than that currently achieved using conventional techniques.

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In 1990, I was concerned with the characteristics of the UV detector used to monitor the separations obtained by capillary electrophoresis; I believed that it would be possible, with proper design, to produce a detector with superior properties. Using either one or two reflecting objectives of short focal length, it proved possible to construct a detector which was one to two orders more sensitive than conventional detectors available [7]. Furthermore, by using faster electronics it was also possible to use a far higher peak sampling rate, thus making it possible to precisely monitor the extremely fast peaks obtained in CEC by Norman Smith, which had efficiencies measured in the tens of millions [8]. The success of this project stimulated me to reconsider capillary liquid chromatography which, because of the slow diffusion rates in liquids, necessitate columns about 10 µm in internal diameter. The serpentine capillaries produced by Scott and Katz clearly provided radial mixing, and I believed that by using serpentine tubing the problems associated with detectability, sample loading, and mobile phase flow rate with 10-µm capillaries could be eliminated while maintaining a reasonable flow rate of mobile phase. I had a first class student, Pawel Grochowicz, who had undertaken the work on constructing the UV detector who was keen to undertake this work. We decided to investigate the flow characteristics in the serpentine tubes using computational fluid dynamics which demonstrated that the optimum flow rate to achieve the best mixing was about 0.5–1.5 cm3 min1 . This came as a bit of a surprise to me, because I had been hoping to minimize mobile phase usage thus cutting down the expense of operation. However, calculations showed that, in fact, the mobile phase usage would be minimized because the separations would be performed in a few tens of seconds. A device was constructed to produce the serpentine shape, and it proved possible to wind out the tubing very easily when the correct grade of stainless steel capillary was used. The product was not quite serpentine in character, but evaluation of its performance showed a minimum of band broadening, indicating that the original idea had been correct. There was an additional problem — which was obvious on considering the relatively high flow rates necessary to minimize band broadening — the high linear mobile phase velocities gave rise to high shear forces at the wall of the capillary which would quickly strip off an adsorbed stationary phase coating. Thus the stationary phase had to be bonded to the internal walls of the capillary and on stainless steel, this is difficult to achieve. On browsing the literature in 1989, I had come across work on self-assembled monolayers where mercaptans would self-assemble on to a gold or silver surface. The problem then was to achieve a gold or silver layer on the inner surface of the capillary. Preliminary electrolysis experiments were unsuccessful principally because of the narrow bore of the capillary (250 µm); however, experiments on flat surfaces of stainless steel showed that ‘liquid gold’ or the use of ammonical silver nitrate and a reducing sugar solution (Tollen’s reagent) would do the job, provided the surface was flat and an even layer of metal was formed to which it was possible to self-assemble alkyl mercaptans as a monolayer on the surface. This was transferable to capillaries. Then the main problem was what would be the phase ratio possible, because this would determine the loadability of the sample and hence detectability, and this was a function of the surface area exposed on the inner walls of the capillary. With a self-assembled C16 alkyl chain, calculations showed that it was very low, thus retention would be low.

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However, it was felt that for high speed separations this could be an advantage and it was shown that it was possible to obtain a partial separation of five alkyl benzoates on a two meter, 250 µm i.d. capillary in a few tens of seconds. In order to evaluate the concept of self-assembly when applied to chromatographic stationary phases, and to get a feel for the phase ratios obtained which could be used to determine the length of capillary necessary, a Master’s student investigated the production of stationary phases using 5-µm silica gel and 5-µm glass beads coated with gold or silver followed by self-assembly. The performance of these phases was excellent and with the beads, high speed separations were possible [9]. In the early 1990s, through the suggestion of a colleague, Marianne Odlyha, who was working with and developing thermal analysis techniques, I became interested in the study of the binding media used in works of art. The oxidative degradation and cross-linking which occurs on the drying of the oil film produces highly complex mixtures which make it difficult to determine the nature of the drying oil used by an artist. The problem was exacerbated by the tiny sample it is possible to obtain from a painting (a few micrograms). The problem was approached using a variety of techniques by two students supported by the Tate Gallery (Tom Learner and Caroline Mathews). Tom used GC–MS, infra-red, and thermal analysis in his study, and Caroline used capillary methods of separation, both in LC and CE. Both of these students achieved a reasonable degree of success with their approaches. The final project concerned the problem of sample loading in capillary electrophoresis particularly with regard to trace analysis and matrix effects. Everaerts and Mikkers have demonstrated the efficacy of a dual column ITP system as has Kaniansky. Bocek has demonstrated that much higher sample loads=volumes could be applied to an ITP column which has been used as a method of sample introduction for CZE. I believed that it would be useful to produce a triple column system where the sample would be applied in the ITP mode, which could mean that samples up to about 1 cm3 could be applied and thus the problem of sample size would be eliminated. Because of the Kohlrausch regulating function, even very dilute solutions would adapt their concentrations to that of the leading electrolyte. Unwanted matrix components of the sample could be effectively eliminated by ground switching the applied potential, and the components of interest transferred to the second column which would also operate in the ITP mode. Further clean up of the sample is then possible, and finally the components of interest are transferred to the final column where they would be separated in almost any of the CE modes of analysis. This last step would get over the problem in ITP of contiguous zones which can be difficult to distinguish. The equipment has been built and preliminary results look interesting; it appears that the design of the equipment will enable separation scientists to take raw samples from most sources, perform the sample clean-up on-line in the first and second dimensions; followed by obtaining quantitative results in either the ITP mode (which would be the most convenient) or from conventional peaks in the third dimension (see Figs. 1–3).

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Fig. 1. Isotachophoretic multidimensional separation of a 25 : 1 dilution of urine. Conditions: Leading electrolyte: 20 mM histidine, 10 mM hydrochloric acid, 0.1% vol hydroxyethyl cellulose (to minimize electroendosmotic flow). Terminating electrolyte: 0.5 mM glutamic acid, 1.5 mM TRIS. Column 1: 0.8 mm bore, 140 mm long. Applied current: 250 µA. Column 2: 0.3 mm bore, 200 mm long. Applied current: 60 µA. Column 3: Conductivity detector trace of the output from the first, pre-separation column 2. Conductivity detector trace of the output from the first, pre-separation column.

59.I.1. Teaching of separation science In the late 1960s and early 1970s, the committee of the (then) Gas Chromatography Discussion Group was perturbed by the lack of activity in liquid chromatography in the UK. At a meeting held at the Esso Research laboratories under the chairmanship of Sid Perry, it was decided to rename this ‘new’ form of liquid-column chromatography and the term ‘High-Performance Liquid Chromatography’ was coined. (Often called High Pressure, High Speed and High Price! Liquid Chromatography — I have now gone back to calling it Liquid Chromatography — LC.) At the meeting it was also decided to run some workshops on HPLC at (then) Trent Polytechnic, Nottingham, UK, and I was asked to organize two workshops in September 1972; because they were successful and stimulated considerable interest, two further workshops the following year. It is of interest to note that Siemens Chromatography, Germany were using 10-µm silica particles at that time; it is often not recognized! In October 1973, I was approached by the Education Officer of the Royal Society of Chemistry (RSC) to organize a Review Symposium on the ‘new’ technique of HPLC. I accepted the invitation and invited four lecturers, Drs. Dennis Saunders, Ray Scott, Josef Huber and John Knox to speak. The meeting was held at the University of Sussex in 1974 and was exceptionally well attended; it was a case of standing room only (in spite of fire regulations!). In the light of this success, I was asked by the RSC to organize a Residential School in HPLC, and was given a free hand to organize it

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Fig. 2. Conductivity detector, its differential and UV traces of the separation achieved on the second column. Conditions: see Fig. 1

Fig. 3. Comparison of the UV detector traces from the second (upper) and third (lower) columns. The benefit of using the third column is clear. Conditions: see Fig. 1

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Professor Colin Simpson and postdoctoral Melissa Hanna showing his triple column ITP system, King’s College, London, 1999.

as I wished. Clearly, there are several requirements for such a school. (1) First class lecturers were required; (2) the participants required practical experience; and (3) they also required question and answer sessions with the leading chromatographers present. Finally, to be really good, the venue should ensure that all the above points could be met and (for chromatographers in the UK at least) there should be a good bar! This requirement was amply met at the University of Sussex. The venue, lecturers, support from the manufacturers in supplying equipment provided an excellent school, so good that it was repeated on nine further occasions (although the last two were held at University College, London). The success of the schools is undoubtedly due to the enthusiasm of the lecturers: Drs. Ray Scott, John Knox, Csaba Horva´th, Josef Huber, Hans Poppe Klaus Unger, Johan Kraak, Ron Majors, Geoff Cox, Ann-Marie Ohlson, Elena Katz, Keith Bartle, John Done, Douglas Anderson, Norman Billingham, Frans Everaerts, Brian Wheals, Tony Hamnett and David Taylor on various occasions. In addition, there were several seminar leaders from various industrial and academic sources, who participated in the 120 seminars and practicals held during the week. Johan Kraak has arranged a similar series of schools at the University of Amsterdam, in which I have been fortunate to participate, and lately has organized courses on capillary electrophoresis. Dr. Klaus Unger similarly organized schools at the Johann Gutenberg Universita¨t, Mainz. All of these schools have been immensely successful; because of them (and lately the equipment manufacturers schools), the practice of separation science in Europe has been in the forefront of advances in chromatography and electrophoresis.

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My time working in and teaching Separation Science has been most enjoyable. I have met a large number of people and I hope helped them in some small way to understand the processes taking place. Above all — it has been fun.

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

C.F. Simpson, M.Sc. Thesis, University of London, 1966. C.F. Simpson, Chemically Bonded Phases for LC and GC, British Patent No. 1,310,872, 13th April 1975. A.M. Greenway and C.F. Simpson, Gas chromatography–mass spectrometry combinations, J. Phys. E. Sci. Instrum., 13 (1980) 1131–1147. T.A. Gough and C.F. Simpson, ‘FIDOH’ British Patent No. 2,037,066, 18=2=1983. R.P.W. Scott and C.F. Simpson, Solute interactions on the surface of reversed phases, Proc. Faraday Symp. Chem. Soc., 15 (1980) 69–82. T.M. Khong and C.F. Simpson, A fluidized bed method for the preparation of chemically bonded phases, British Patent No. GB 8618322, 28=7=86. C.D. Flint, P.R. Grochowicz and C.F. Simpson, The design, construction and evaluation of a UV detector for capillary electrophoresis, Anal. Commun. R. Soc. Chem., 31 (1994) 117–121. P.R. Grochowicz, Ph.D. Thesis, Birkbeck College, University of London, 1997. N. Sorafan, M.Sc. Thesis, Birkbeck College, University of London, 1996.

D.60. Jan Bertil Sjo¨vall Jan Bertil Sjo¨vall was born in Lund, November 28, 1928. He received the M.D. degree from Karolinska Institutet in 1960, the Ph.D. degree in 1955 in physiological chemistry at Lund University under Professor Sune Bergstro¨m, Nobel Laureate in Physiology and Medicine. His postdoctoral training was at the National Institutes of Health, Bethesda, MD, in 1960; at Sinai Hospital of Baltimore in 1960 and 1962; and at the Lipid Research Center, Baylor College of Medicine, Houston, TX, in 1963. His academic appointments progressed from Assistant Professor in 1956 at the Department of Physiological Chemistry, University of Lund; to Associate Professor in 1959 at the Government Laboratory for Forensic Chemistry, Stockholm; then Associate Professor to Professor and chairman, Department of Physiological Chemistry at Karolinska Institutet, Stockholm; now Professor Emeritus since 1994. J. Sjo¨vall’s research areas were separation methods and mass spectrometry, and their use to solve biomedical problems. The analysis of bile acids and steroids formed from cholesterol and studies of the biochemistry and clinical chemistry of bile acids has been a central theme throughout the years. New chromatographic methods have been developed, leading to basic information about normal and pathological conditions in bile acid synthesis and metabolism. In recent years new inherited and acquired diseases of bile acid synthesis have been discovered, partly due to the new ionization methods in mass spectrometry. In 1954, he received the Magnus Blix Prize. In 1958 the Alvarenga

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Prize for the use of analogous chromatographic systems which led to the first isolation of crystalline prostaglandins with S. Bergstro¨m. The Arrhenius Gold Medal of the Swedish Chemical Society in 1966, the Berzelius Gold Medal of the Swedish Society of Medicine in 1991, the Eppinger Prize in 1976, and the M.S. Tswett Chromatography Medal in 1987, were awarded for various aspects of this work. The metabolism and clinical chemistry of steroid hormones was studied by liquid– gel, gas chromatography and mass spectrometry; new compounds, metabolic reactions and abnormalities in disease were discovered. The compartmentation and contribution of carbons and hydrogens from ethanol metabolism to the biosynthesis and metabolism of cholesterol, steroids, bile acids and glycerolipids was studied using labeling with stable isotopes. Sample preparation methods for many studies were based on the development and use of lipophilic, neutral, and ion-exchange derivatives of Sephadex. Recent studies include the use of nano-electrospray mass spectrometry, also combined with capillary column high-pressure liquid chromatography for the structural studies of steroids, bile acids, polyisoprenoids, xenobiotics, peptides and novel types of conjugates. See Chapter 5B, c, d, e, g, h, p, r

60.I. NON-POLAR NEUTRAL AND ION-EXCHANGING DERIVATIVES OF SEPHADEX FOR EXTRACTION AND SAMPLE PREPARATION Jan Sjo¨vall * Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden

My first contact with chromatography was in 1950 when I was accepted as a student in Sune Bergstro¨m’s group at the Department of Physiological Chemistry at the University of Lund. Bergstro¨m and von Euler had recently isolated noradrenaline by counter-current distribution and a simpler method was needed for separating it from adrenaline. I was asked to try column partition chromatography using hydrochloric acid as the stationary phase on Kieselguhr and phenol as the mobile phase. Pehr Edman, working on his sequencing method, lent me a simple fraction collector designed by him. The separation was successful and I became fascinated by chromatography. When Bergstro¨m initiated a program on the biochemistry of bile acids, I was given the task of developing chromatographic methods for the separation of bile acids. A.J.P. Martin and G.A. Howard had just described the separation of fatty acids by reversed-phase chromatography using siliconized Kieselguhr as support for the non-polar phase. By increasing the polarity of their solvent system, the more polar bile acids could be separated according to number and positions of hydroxyl groups. This method was used extensively by Bergstro¨m’s and other groups to elucidate the biosynthesis, metabolism and enterohepatic circulation of bile acids. I also developed quantitative paper chromatographic methods for separation of conjugated as well as *

Correspondence address: Sveava¨gen 111, S-113 50 Stockholm, Sweden

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unconjugated bile acids. These were based on the use of acetic acid as stationary phase, introduced via a liquid or vapor phase. Studies with these methods gave the first information about bile acid metabolism in humans, changes occurring in pathological states, bacterial 7-dehydroxylation, and about levels and composition of bile acids in the human intestine during fat absorption. Paper chromatography also revealed the presence of ursodeoxycholic acid in humans. This bile acid, first found in bear’s bile, which was used as a traditional Chinese and Japanese medicine, is now extensively used in the treatment of cholestatic liver diseases. When Bergstro¨m turned his interest to prostaglandin, a biological activity independently discovered by M.W. Goldblatt and U. von Euler, the bile acid chromatographic systems were applied to the fractionation of extracts of sheep prostate glands. Sequential use of the reversed- and straight-phase systems, and monitoring with paper chromatography and UV-spectra in sulfuric acid, showed the biological activity to separate into components having partition coefficients similar to that of cholic acid. In this way crystalline prostaglandins F and E were obtained from fractions of the final chromatographies [1]. Using these methods the occurrence of a number of different prostaglandins in different organs could be established. They were also useful in the structure determinations by Bergstro¨m, Bengt Samuelsson, Ragnar Ryhage and myself (Fig. 1). In 1959, Evan Horning, Charles Sweeley and E.A. Moscatelli described the gas chromatographic separation of steroids. With William VandenHeuvel they showed that bile acid methyl esters could be separated by this method. I had now moved to Karolinska Institutet in Stockholm and was given the opportunity in the fall of 1960 to spend a few weeks, with Evan and Bill at the NIH and to learn more about the new technique in David Turner’s laboratory at the Sinai Hospital of Baltimore. After returning home I set up a laboratory for gas chromatographic analysis of bile acids, which gave us opportunities to study the occurrence, metabolism and kinetics of bile acids with much higher resolution than had been possible with the liquid chromatographic methods. QF-1 was found to be particularly well suited as stationary phase for separation of positional and configurational isomers which are so abundant among steroids and bile acids. In 1962, I returned to Baltimore where we developed the first GLC method for analysis of serum bile acids. Solid-phase extraction was used for the first time as the initial step in the sample preparation, and in Evan Horning’s laboratory at Baylor, we applied this principle to the analysis of steroid sulfates in plasma. In the early 1960s, Ragnar Ryhage at the Mass Spectrometry Laboratory at Karolinska Institutet, was doing his pioneering work on mass spectrometers for the analysis of biomolecules. He had invented the jet separator for sample enrichment, and was designing the GC=MS instrument that became the first commercially available one, the LKB 9000. By using steroids as the most challenging test compounds, we could optimize the interface construction and eliminate problems due to cold spots and pressure gradients. Using prototypes of the instrument, we characterized the complex mixture of bile acids in feces which was important for the understanding of cholesterol metabolism. New bile acids, formed by the action of bacterial enzymes, were identified, which increased our understanding of the formation of potentially toxic bile acid structures.

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Fig. 1. The author and Sune Bergstro¨m in Ragnar Ryhage’s office, December 1973. Picture taken by The Upjohn Company following their release of the first prostaglandin derivative approved for human use.

The work with complex matrices like plasma and feces made it clear that appropriate sample preparation procedures were essential for a successful analysis of complex mixtures of bile acids and steroids in biological materials. Chromatography with conventional two-phase solvent systems were not practical for this purpose because of the need for solvent equilibration. With a graduate student, Ernst Nystro¨m, I began to study how stationary phases could be prepared by chemical modification of cross-linked dextran gels. Methylated Sephadex was prepared that permitted molecular sieving in organic solvents, but it was also clear that partitioning between the mobile phase and the solvent-gel phase took place. Depending on the solvent polarity, reversed-or straight-phase partition separations were obtained. In straight-phase systems, separations of lipids, protected peptides and other lipophilic compounds were amplified by the combined action of partitioning and size exclusion. High column efficiencies could be obtained by recycling chromatography or with capillary columns made with superfine particles (17–23 µm). Sterols differing by a CH2 group in the side chain could

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Fig. 2. Peter Eneroth applying a sample to the top of a 6-m column of methylated Sephadex in an organic solvent. (Photo by E. Nystro¨m in about 1968.)

be separated with a separation factor of 1.017 using recycling chromatography in heptane=chloroform=methanol systems. Reusable capillary columns with an injection port could easily be prepared in 1.5 mm i.d. Teflon tubing and used with a moving-chain flame ionization detector constructed by Eero Haathi in Turku, with whom we had a most stimulating collaboration [2]. Preparative columns extending through two floors (Fig. 2) and sometimes containing 2 kg of gel were also useful. Sample preparation procedures could be based on these liquid–gel systems. One of the most important early applications was in the analysis of steroid sulfates in plasma and urine. With my graduate student Reijo Vihko, I made the observation that sodium and potassium salts (but not the protonated acids or other salts in analogous systems), of steroid sulfates were strongly retained on these gels in non-aqueous mixtures of chloroform=methanol containing sodium or potassium chloride, respectively. This provided the basis for a rapid isolation procedure for these steroid conjugates which could then be analyzed by GC=MS after solvolysis and derivatization. Vihko’s thesis, published as a supplement in Acta Endocrinology in 1964, provided the first information

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Fig. 3. Separation of cholesterol and its C2 –C18 fatty acid esters by reversed-phase chromatography on a 34% hydroxyalkoxy (C11 –C14 ) propyl–Sephadex G-25 superfine column, 3000 ð 1:5 mm, in heptane=acetone=water, 4 : 15 : 1, by vol. The effluent was monitored with a moving chain-flame ionization detector [2]. Values on the x-axis are % total column volume. (From Ellingboe, Nystro¨m and Sjo¨vall, Biochim. Biophys. Acta, 152 (1968) 803–805, with permission). Refer to figure in reference.

about sex- and age-related differences in plasma levels of dehydroepiandrosterone sulfate (now a popular compound in geriatric research) and other steroid sulfates ˚ ke Gustafsson and Ha˚kan in humans. In a series of studies with my students Jan-A Eriksson, we could characterize new steroids in rats and humans, and, using the germ-free rats reared by Bengt Gustafsson, show the importance of steroid metabolism by intestinal microorganisms. With Tomas Cronholm, the pronounced redox effect of ethanol on steroid sulfate metabolism could be demonstrated using deuterated ethanol given to humans. At this time, Pharmacia had started to market the hydroxypropylated derivative Sephadex LH-20, which later became a widely used medium for preparation of steroid samples prior to radioimmunoassays using solvent systems analogous to those described by us [2,3]. It soon became clear that a wider range of substituted Sephadexes were needed for sample preparation purposes. Less polar derivatives were required for establishment of better reversed-phase systems, and ion exchangers of variable polarity were also desirable for group separations of biological extracts, in which metabolites occurred in different forms of conjugation. A postdoctoral fellow, Jim Ellingboe, observed that long-chain olefin oxides were commercially available in bulk quantities from Ashland Chemical Company. These were used to synthesize hydrophobic derivatives of Sephadex LH-20 in a BF3 -catalyzed reaction [4,5]. Fig. 3 shows a reversed-phase separation of cholesterol esters on a capillary column of hydroxyalkoxypropyl–Sephadex G-25 (see Chapter S-10 — Sjovall). In a similar way chloro- and bromohydroxypropyl derivatives could be prepared with epichloro- and epibromohydrins, respectively. These derivatives, whose polarities could be modified by appropriate hydroxyalkylations, served as intermediates in the preparation of ion exchangers and other stationary phases for ligand exchange purposes [5]. A variety of these were synthesized, of which those substituted with diethylamino, triethylamino, and sulfonic acid groups were the most useful ones [6]. The neutral hydrophobic, hydroxyalkyl derivatives could be used for reversed- or straight-phase partition chromatography, the mechanism depending on the solvent used. Extreme reversed- and straight-phase conditions were obtained with aqueous methanol and heptane, respectively [4]. Ethylene chloride or chloroform could be used as modifiers to adjust the polarity to the desired level in either case. When the polarities of the solvent and the gel-solvent phases were about the same, size exclusion was the predominant separation mechanism [2]. Our and other laboratories applied the hydrophobic Sephadex gels to the separation of a wide range of lipid

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soluble compounds, and the separations achieved were in most cases superior to those previously described. Non-polar compounds such as triglycerides and polyisoprenoids were readily separated according to carbon number. Conditions were mild, adsorptive losses were not observed, and it was possible to separate labile compounds, such as trimethylsilyl ethers of 1,2-diacylglycerides in a reversed-phase system prior to GC=MS analysis. This was done by the student Tore Curstedt who studied the participation of hydrogen atoms from ethanol metabolism in the biosynthesis of glycerolipids by giving deuterated ethanol to rats and analyzing deuterium enrichment and localization in individual molecular species by liquid-gel separations and GC=MS. The lipophilic gels were also useful for rapid clean-up of reaction mixtures in the preparation of trimethylsilyl ethers for GC=MS [3,6]. Reversed-phase chromatography on hydrophobic Sephadex gels was an essential step in the more recent discovery of lung surfactant polypeptides by T. Curstedt, J. Johansson and coworkers. These polypeptides are functionally important components of natural lung surfactants which are prepared commercially by reversed-phase chromatography on such gels and successfully used in the treatment of the respiratory distress syndrome in preterm infants. After our development of the hydrophobic gel phases, reversed-phase high-performance chromatography as we know it today, began to develop. Our soft gels did not allow the use of high pressures, and separation times in high efficiency columns were very long. It was therefore evident that, if not synthesized on a solid matrix, these gels would find their main applications in low-pressure open column systems, mostly for the fractionation of crude biological extracts prior to analysis with faster high-resolution methods such as GC=MS. With some exceptions this has also become their major use. With graduate students B. Alme´, P. Thomassen and M. Axelson and the postdoctoral fellow K. Setchell, strategies were developed in the early part of the 1970s for the preparation and group fractionation of biological samples for multicomponent analysis (metabolic profiling) of steroids, bile acids, and metabolites of other lipophilic compounds in urine, plasma, feces, and tissues by GC=MS [6]. Analogous methods were later applied with B. Egestad and K. Nore´n to the analysis of chlorinated aromatic pollutants and unconjugated and conjugated metabolites of other xenobiotics in biological matrices [7,8]. An important point is that the non-polar neutral gels are efficient extractants for compounds of low and medium polarity. Solid-phase extraction of biological fluids with these gels is different from other solid-phase extractions based on adsorption. For example, a fatty acid is extracted only in the protonated form, not as a soap, and the bile acids can be extracted as ion pairs with decyltrimethylammonium as a highly specific counter-ion [6]. Thus an extraction with the lipophilic gels is much more selective than an extraction with C18 or other bonded silica or a polystyrene resin. Because of the tight cross-linking of the matrix, proteins and other large molecules are excluded from the extraction (today referred to as limited access extraction). The extraction of protein-bound compounds depends on the dissociation constant of the complex, which can be controlled by the temperature of the gel bed and sample. We have used this effect in studies of ligands bound to steroid receptors. The gels could also be used in combination with solvent extraction of tissues. The solvent extract is evaporated or water is added in the presence of the hydrophobic gel, resulting in transfer of the

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analytes to the gel which is slurried into a column and eluted with appropriate solvents to give a purified sample [6,7]. Our sample preparation procedures are based on passage of the sample through an appropriate sequence of lipophilic, neutral and ion-exchanging gel beds. The strategy is to retain either the analytes or the interfering material (digital chromatography). Groups of unconjugated and conjugated metabolites differing in charge and acidity are sequentially eluted by stepwise displacement from the ion-exchanging beds. This process can be automated [8]. Following desalting of the fractions by solid phase extraction (which can also be done on line), the conjugates can be analyzed by nano-electrospray mass spectrometry, capillary column HPLC=MS and by GC=MS after deconjugation. Selective gels can be prepared for group isolation of compounds with a common structural feature, e.g., synthetic steroids carrying an ethynyl group (retained on a lipophilic sulfonic acid gel in silver form), ketonic steroids as their oximes (retained on a lipophilic sulfonic acid gel in a nonaqueous solvent), organomercury compounds (retained on sulfhydryl gels) [5,6]. Gels can be prepared with controlled polarity and appropriate ion-exchanging groups for combined ion exchange and partition chromatographic purification of analytes [6,9]. However, in applications involving multicomponent analyses, passage of the sample through a sequence of beds, each with a desired property and separation mechanism, is more flexible than the use of beds with multiple functional groups. Non-polar and ion-exchanging derivatives of Sephadex have served important functions in the preparation of samples for subsequent analysis by high-resolution methods. They have also found important preparative applications. Obviously, over the years the new technologies in stationary phase chemistry, columns and coupled systems have led to sample preparation methods that are much superior, especially when large numbers of samples have to be analyzed, e.g., in clinical chemistry and in the monitoring of drugs, other xenobiotics and their metabolites. However, there are still situations with fewer samples when the Sephadex derivatives can accomplish the same or better results than phases based on silica or polystyrene resins, at a lower cost and with less contamination problems in open columns. This may be the case for initial sample handling in research concerned with analyses of very complex mixtures, e.g., in metabolic studies, and in the characterization of partly unknown mixtures and compounds. Thus, it may still be of interest to evaluate the potential of the gel phases in such applications. Sufficient quantities of a derivative to last for years can be synthesized in two days, and the gels are reusable after washing.

References 1. 2. 3.

S. Bergstro¨m and J. Sjo¨vall, The isolation of prostaglandin F (E) from sheep prostate glands, Acta Chem. Scand., 14 (1960) 1693–1700 (F), 1701–1705 (E). J. Sjo¨vall, E. Nystro¨m and E. Haahti, Liquid chromatography on lipophilic Sephadex: Column and detection techniques, Adv. Chromatogr., 6 (1968) 119–170. J. Sjo¨vall and M. Axelson, Newer approaches to the isolation, identification, and quantitation of steroids in biological materials, Vitam. Horm., 39 (1982) 31–144.

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J. Ellingboe, E. Nystro¨m and J. Sjo¨vall, Liquid chromatography on lipophilic-hydrophobic Sephadex derivatives, J. Lipid Res., 11 (1970) 266–273. J. Ellingboe, B. Alme´ and J. Sjo¨vall, Introduction of specific groups into polysaccharide supports for liquid chromatography, Acta Chem. Scand., 24 (1970) 463–467. J. Sjo¨vall and M. Axelson, Sample work-up by column techniques, J. Pharm. Biomed. Anal., 2 (1984) 265–280. K. Nore´n and J. Sjo¨vall, Liquid–gel partitioning and enrichment in the analysis of organochlorine contaminants, J. Chromatogr., 642 (1993) 243–251. J. Uusija¨rvi, B. Egestad and J. Sjo¨vall, Manual and automated enrichment procedures for biological samples using lipophilic gels, J. Chromatogr., 488 (1989) 87–104. M. Axelson and J. Sjo¨vall, Strong non-polar cation exchangers for the separation of steroids in mixed chromatographic systems, J. Chromatogr., 186 (1979) 725–732.

D.61. Hamish Small Hamish Small was born on October 5, 1929, in Newtowncrommelin Northern Ireland. He was educated in Northern Ireland where he received B.Sc. and M.Sc. degrees from the Queen’s University of Belfast. He worked from 1949 to 1955 for the United Kingdom Atomic Energy Authority at Harwell in England. From 1955 to 1983 Small was employed by the Dow Chemical Company in Midland, MI, USA, where he attained the rank of Research Scientist. Since leaving Dow in 1983, he has remained active in research and invention. His main areas of research activity have been in separation science and technology with a career-long emphasis on ion exchange and its applications. He has published extensively (32 papers) and is the author of a book “Ion Chromatography”. His papers on Ion Chromatography and Indirect Photometric Chromatography were chosen for inclusion in “Milestones in Analytical Chemistry” (American Chemical Society, Washington, DC, 1994). H. Small is credited with 34 USA patents, several of which cover key inventions on ion chromatography. His recent discovery of the principle of ion reflux (Anal. Chem., 70 (1998) 2205), has opened up new ways of performing ion chromatography. H. Small has received the following honors and awards for his pioneering work in ion chromatography and hydrodynamic chromatography ž 1976 Midland Chapter, Sigma Xi Outstanding Research Award for hydrodynamic chromatography. ž 1977 Midland Chapter, Sigma Xi Outstanding Research Award for ion chromatography. ž 1977 Applied Analytical Chemistry Award (given by the Society of Analytical Chemists of Pittsburgh for ion chromatography). ž 1978 Albert F. Sperry Award of Instrument Society of America (for ion chromatography). ž 1981 Investors Award of the Saginaw Valley Patent Lawyers Association for inventive contributions in chromatography, particularly ion chromatography.

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Hamish Small (third from left) and his colleagues, William Bauman (second from left) and Timothy Stevens (right) receive the 1978 Sperry Award from the Instrument Society of America for their pioneering work on Ion Chromatography.

ž ž ž ž ž ž ž ž

1983 Arnold O. Beckman Award of the Instrument Society of America. 1983 Herbert H. Dow Gold Medal 1 . 1984 Stephen Dal Nogare Chromatography Award. 1985 Midland Chapter Sigma Xi Outstanding Research Award for indirect photometric chromatography. 1988 Achievement Award, presented by the First International Ion Chromatography Forum. 1991 The American Chemical Society Award in Chromatography. 1992 Merit Award of the Chicago Chromatography Discussions Group. 1998 Achievement Award of the International Ion Chromatography Symposium.

See Chapter 5B, c, g, m

1

The citation on the Herbert H. Dow Gold Medal reads “The Herbert H. Dow Medal recognizes and honors those rare individuals whose inventiveness and pioneering technology have had an outstanding impact on the growth and well-being of The Dow Chemical Company”.

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61.I. ION CHROMATOGRAPHY AND HYDRODYNAMIC CHROMATOGRAPHY Hamish Small 4176 Oxford Drive, Leland, MI 49654, USA

“With his introduction of ion chromatography, Hamish Small offered a technique to the scientific world that has proven to be an invaluable and broadly applied tool. His earlier work developing the hydrodynamic chromatography method and his research on ionexchange chelating resins and other new techniques in ion exchange have had enormous scientific, environmental, and commercial impact.” (from the ACS Award announcement, C&E News, November 12, 1990)

61.I.1. Ion chromatography At one time, the quantitative determination of common inorganic species such as the halide, sulfate, nitrate, phosphate, and carbonate ions, etc., involved much tedious and time-consuming ‘wet-chemistry’. Since each analytical method was often applicable to just one ionic species or to a small set of ions, many different methods were in use. Furthermore, by today’s standards, much of inorganic quantitative analysis was relatively insensitive. In the late 1950s, a few of us in Dow’s Physical Research Laboratory foresaw the benefits of replacing these many methods with a single chromatographic technique that would be universally applicable to all ionic species. Contrary to some accounts, there were no task forces or teams set up with the mission to extend chromatography to inorganic analysis. Nor was there any clamor at scientific meetings for chromatography to be expanded in this direction. Inorganic chromatography, as we called it then, was just a dream that I shared with colleagues like Bill Bauman, Bob Wheaton and Mel Hatch. All of us were active, in one way or another, in ion-exchange technology. None of us were trained as analytical chemists. At first, the chromatographic separation of the inorganic analytes did not seem to be a major problem; after all, there was an abundance of ion-exchange methods that we could draw on. But detection of the resolved analytes presented serious difficulties. Since many of the ions of interest lacked suitable chromophores or convenient means of generating chromophores, they were deemed to be undetectable by light-absorption methods (later we showed that commonly held idea to be false) [1]. Electrical conductivity monitoring had promise; electrical conductance was a universal property of ions in solution. However, conductivity detection in combination with ion-exchange separation introduced what we perceived to be a serious complication: how to distinguish minute conductance changes due to the analytes from the conductance ‘noise’ in the highly conducting ionic eluents that ion-exchange chromatography demanded. These considerations led to ion chromatography (abbreviated IC). The first breakthrough came in late 1971. In order to ‘see’ the smallest conductance changes contributed by the analytes, we felt that we must subdue the noise in the relatively highly conducting eluents. To do that we introduced a device called the

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‘stripper’, later called the ‘suppressor’. The suppressor was a column of ion-exchange resin that converted the eluent to a low conductivity form, while leaving the conductance of the analytes relatively unaffected — or in some cases enhancing it [2,3]. We were reducing the background noise simply by reducing the background signal. The suppressor was a key concept in the evolution of IC, but it was a component of the chromatographic system whose capacity to function was partly consumed in each run and therefore required periodic interruptions of the chromatography to restore it to its original capacity. This idea of a consumable component was radically new to chromatography and some doubted that it would be accepted. Because I shared that skepticism, I knew that we must make suppressor regeneration as unobtrusive as possible. To this end the new separating phases that we developed were critical. There were two properties that a useful ion-exchange separator needed above all others: first, a low capacity to match the low concentration of eluent that suppressor longevity dictated, and second, stability in the strongly basic (or acidic) eluents that we proposed to use. In 1971, commercially available low-capacity ion exchangers had been developed for the then burgeoning field of HPLC but they were based on silica and had very poor stability in our eluents. So I tapped my background in ion-exchange resins to develop more rugged alternatives. For the first cation separations, carefully controlled surface-sulfonation of crosslinked polystyrene resin gave cation exchangers of requisite low specific capacity. The analogous separator for anions was produced in a very different way. My involvement some fifteen years earlier with the problem of ‘resin clumping’ had taught me that anion-exchange particles will adhere strongly to oppositely charged cation-exchange particles. Drawing on that experience, I prepared the first successful anion exchanger for IC by treating surface-sulfonated polystyrene substrate particles with a colloidal dispersion of anion-exchange particles that adhered to the substrate. Initially these colloidal dispersions were prepared by grinding large anion-exchange particles, but later we developed anion-exchange resins in latex form as a superior means for controlling the size, uniformity, and ion-exchange properties of the colloidal materials. Some questioned the physical stability of these pellicular composites, but I had developed faith in the tenacity of the electrostatic linkage between colloid and substrate; in some serious attempts to separate them I had never been successful. Because of the excellent physical and chemical stability of these ‘surface agglomerated’ products and the advantages that the method offered to manufacturing, this route to resin preparation was eventually adopted and very successfully elaborated by Chris Pohl and his group at Dionex. Finding suitable eluents was another key to IC’s success, particularly for anion analysis. The history of these developments is described in Ref. [3]. In these early days we showed how the ion exchange, eluent-suppression technique, while greatly simplifying inorganic analysis, also enabled the analysis of many organic ions that had previously been inaccessible to chromatography because they lacked suitable chromophores. Aliphatic acids, amines and quaternary ammonium ions were notable examples. In fact nowadays, suppressed conductometric detection, because of its universal ability to detect most ionic species with high sensitivity, is often the chosen method of detection regardless of whether the species are inorganic or organic. In the pioneering work on IC we also demonstrated how ion-exchange membranes

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could provide continuous suppression thus avoiding interruptions for regeneration [4]. Today, the great majority of ion chromatographs employ membrane-based suppression devices. The most direct and measurable commercial impact of ion chromatography was the formation of the Dionex Corporation, begun in 1975 under a licensing agreement to exploit the Dow technology, which was then described in several key patents. Since then, Dionex has become the acknowledged world leader in the commercialization of IC, with annual worldwide sales of about 150 million dollars. While this is the most obvious and measurable impact of IC, its impact on industry and on science is immeasurable. There is virtually no industry in which IC is not used, whether it is to control processes, to assure product quality or monitor waste streams. A good example is the electrical power generating industry where IC monitoring protects massively costly installations from the effects of corrosive ions in boiler feed-waters. In this application, where potentially damaging ions are successfully determined at the parts per trillion level, IC displays its unprecedented sensitivity. The ion content of electro-plating baths, the acids in wine, toxic ions in infant foods, corrosive ions in packaging film, acid rain, blood and urine analysis, soil analysis, trapped ions in polar ice-caps are applications of IC that show its widely diverse penetration into modern chemical analysis.

61.I.2. Hydrodynamic chromatography In the late 1960s, I was asked by my management to attempt to develop a rapid means for the determination of the particle size and size distribution of a particular polymer colloid. The particles were in the neighborhood of one micrometer in diameter. To this end I invented what became known as hydrodynamic chromatography (HDC). The technique is based on my discovery that colloidal particles, when carried through a bed packed with solid packing particles, display different rates of migration, with the larger particles exiting ahead of smaller particles [5,6]. HDC instruments — at my last count some thirty — are in use throughout the Dow Chemical company. Applied mainly in the polymer latex business, HDC provides product definition and quality control and is an important tool in providing process control of latex synthesis. This was impossible with previous particle size techniques. The HDC technology is also widely used in colloid research studies.

Acknowledgments In concluding this brief account of my activities in chromatography, I would like to acknowledge the invaluable assistance and collaboration of such colleagues and co-inventors as Bill Bauman, Ted Miller, George Rock, Dewey Scheddel, Jitka Solc and Tim Stevens, and the entrepreneurial drive of the Dionex Corporation that has converted our cluster of inventions in IC into an important innovation in chemical analysis.

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References 1. 2. 3. 4. 5. 6.

Ł H.

Small and T.E. Miller, Indirect photometric chromatography, Anal. Chem., 54 (1982) 462. Small, T.E. Stevens and W.C. Bauman, Novel ion exchange chromatographic method using conductimetric detection, Anal. Chem., 47 (1975) 1801. H. Small, Ion Chromatography, Plenum Press, New York, 1989. T.S. Stevens, J.S. Davis and H. Small, Hollow fiber ion exchange suppressor for ion chromatography, Anal. Chem., 53 (1981) 1488. H. Small, Hydrodynamic chromatography. A technique for size analysis of colloidal particles, J. Colloid Interface Sci., 48 (1974) 147 H. Small, Discoveries concerning the transport of colloids and new forms of chromatography, Acc. Chem. Res., 25 (6) (1992) 241. Ł H.

Ł

These papers were chosen for inclusion in “Milestones in Analytical Chemistry”; Washington Staff of Analytical Chemistry, American Chemical Society, Washington, DC 1994.

D.62. Roger M. Smith Roger M. Smith was born in December 1943. His present position is Professor of Analytical Chemistry, Department of Chemistry, Loughborough University, Loughborough, Leics, UK. He received a First Class B.Sc. (Honours) in Chemistry, University of Manchester, UK (1964), a M.Sc. in Chemistry from the University of Manchester, UK (1965), a Ph.D. in Organic Chemistry at the Research School of Chemistry, Australian National University, Canberra, Australia (1968). R.M. Smith is a Chartered Chemist and a Fellow of the Royal Society of Chemistry. Professional experiences R.M. Smith’s professional experiences include the following. In 1968, as Postdoctoral Research Assistant, Department of Pharmaceutical Chemistry, University of Wisconsin, Madison, WI, USA; then in 1969 in the Department of Chemistry, University of Virginia, Charlottesville, VA, USA; this was followed by an ICI Research Fellowship in the Department of Molecular Sciences, University of Sussex, Brighton, UK. From 1972, he was a Lecturer, then Senior Lecturer in Chemistry at the University of the South Pacific, Suva, Fiji. From 1978 to the present, he has lectured at Loughborough University, UK and was appointed Professor of Analytical Chemistry in 1995. Awards R.M. Smith has been honored in receiving the Royal Society of Chemistry Award for Analytical Separation Methods sponsored by Roche Products (1990), and the Silver Jubilee Medal of the Chromatographic Society (1998). He is presently the Secretary of the Commission V3 on Separation Methods in Analytical Chemistry of the Analytical

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Presentation of the Silver Jubilee award by Dr. Derek Stevenson, President of the Chromatographic Society to Dr. Roger M. Smith for his contributions to the field of separation science.

Division of the International Union of Pure and Applied Chemistry (IUPAC) (1999). Smith has published four books, 14 chapters, and over 185 research publications in analytical chemistry and chromatography. See Chapter 5B, a, b, e, h, l, o

62.I. RETENTION AND SELECTIVITY IN LIQUID AND SUPERCRITICAL/SUPERHEATED SEPARATIONS Roger M. Smith Department of Chemistry, Loughborough University, Loughborough, Leics LE11 3TU, UK

My early interest in chromatography was as a natural product chemist who used separation methods for the isolation of compounds, usually with a biological activity from plant or microbial sources. Over the last 20 years, my research has concentrated on the investigation of separation techniques, initially high-performance liquid chromatography (HPLC), but more recently supercritical chromatography, superheated-liquid chromatography, and capillary electrophoretic methods. More recently superheated water for HPLC and extraction have become my main research theme.

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62.I.1. Introduction and application of retention indices in liquid chromatography My research on liquid chromatography has centered on three principal themes, the effect of the structure of the analyte on retention, the influence of stationary phase interactions on retention, and the changes in retention caused by the presence of additives to the mobile phase. When we first started to examine the relationship of structure and retention in HPLC, it was clear that a method was needed to separate the retention contributions from the column=eluent, and enable the selective interactions due to the functional groups to be identified. We therefore proposed that a retention index scale should be introduced for liquid chromatography, which would enable relative effects of different functional groups to be measured. The Kovats indices could not be adopted because the n-alkanes were very nonpolar and therefore highly retained in reversed-phase liquid chromatography. The absence of a chromophore would also make detection difficult. In 1982, we proposed the use of an alternative scale based on the moderately polar alkyl aryl ketones, with the origin at acetophenone (I D 800). These homologues were readily detected and were similar in polarity and thus retention to many aromatic analytes. This approach gave reproducible results for the retention of analytes and enabled direct comparisons to be made between different systems. For many analytes the indices were consistent on different ODS phases but differed markedly as the mobile phase altered, reflecting changes in selectivity. This work led to a detailed study of the effect of different functional groups on the retention of analytes in reversed-phase liquid chromatography and to derive a database of increments that could be employed for retention prediction. The driving force behind this study was the concern that in most HPLC separations the initial conditions for new compounds were determined by trial and error. However, frequently the structure of the analyte was known and it should be possible to predict the conditions to give a desired retention based solely on the molecular structure. The proposed method was based on the summation of a retention index value, based on the alkyl aryl ketones, for a parent skeleton with contributions for alkyl chains, for substituents on aliphatic and aromatic carbon atoms, and for interactions between substituents. In each case a quadratic expression relating the retention contribution to the proportion of modifier in the eluent was determined. This work led to CRIPES (Chromatographic Retention Index Prediction Expert System), an expert system, which would propose retention indices for multifunctional compounds in different eluents. These studies were reported in a series of original papers, and an overview and reviews of related studies elsewhere have been compiled in a book, “Retention and Selectivity in HPLC” [1]. This interest also led to an extensive review of the relationship between the functional groups in an analyte and its retention in reversed-phase HPLC [2]. This review examined the use of functional group constants for retention prediction and surveyed a wide range of published papers containing data from different sources. The paper includes a re-analysis of the extensive data collected in our retention index studies. A range of functional group contributions were calculated and correlations were derived between the functional group contributions (− values) and Hansch ³ constants for octanol–water partitioning. Also, the effect of the proportion of organic modifiers in the mobile phase was studied and general expressions derived for the retention increments.

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62.I.2. Effects of mobile phase additives in liquid chromatography One theme of my research has been the effect of additives to the mobile phase, in particular interactions between metal ions and analytes containing groups capable of chelation. Initial studies looked at the use of dithiocarbamates to analyse metal ions by on-column complexation reactions. We then reported a detailed examination of novel systems in which metal ions are incorporated in the mobile phase and could reversibly interact with analytes containing chelating groups. The effect of complexation was to produce a more polar product whose retention in reversed phase was reduced compared to the original compound. This contrasted with conventional ion-pair formation, which normally creates less polar complexes with longer retention times. The selectivity of the separation could be adjusted by using different transition metal ions, and varying their concentration and the pH of the mobile phase. We studied the theoretical background for these secondary chemical equilibria, using 2-aminophenol and the more complex β-diketone system [3]. 62.I.3. Separation of basic drugs on silica In collaboration with the Home Office Forensic Science Service, we carried out a series of studies to examine the long-term reproducibility of methods for the separation of basic drugs. Much of this work examined the separation of basic drugs on a silica column using an eluent containing an ammonia-based high-pH buffer and high proportion of methanol. The mechanism of separation is primarily ion-exchange chromatography using the acidity of the silanol groups on the silica. In a series of studies we examined the effect of varying the operating conditions to identify critical parameters, and the conclusions based on a principal components analysis have been tested in national and international collaborative studies. Subsequently, we employed an alternative high pH buffer, which is prepared using solid organic components defined by weight. The retention results were more reproducible, and because the overall ionic strength was lower, the analytes were retained longer and a better discrimination was obtained. Although the method is highly robust, long-term changes in the selectivity of the silica were observed [4]. The observations implied that changes were occurring in the chemistry of the silica surface on contact with the mobile phase.

62.I.4. Packed column supercritical-fluid chromatography We took an early interest in supercritical-fluid chromatography (SFC) on packed columns, and this led us to run a Workshop=Short Course in 1986, which led to the first monograph on the use of SFC [5]. This book included a chapter on our initial work which concentrated on the use of different HPLC column materials in packed columns. We compared the retention of analytes and homologues, and reported detailed studies of the separation of alkyl aryl ketones, model test-, and pharmaceutical compounds on a number of different bonded-stationary phases. Later studies examined the influence

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of mobile phase additives which altered the surface of the silica. By examining a homologous series of analytes, we were also able to demonstrate that retention on polar columns in SFC was a combination of normal-phase like polarity effects and analyte volatility. We also carried out studies of the chiral resolution of isomeric analytes to determine which structural features led to high resolution.

62.I.5. Supercritical-fluid extraction of natural products A related series of studies examined the application of supercritical-fluid extraction (SFE) to the extraction of natural products [6], initially from herbal and plant material medicines and subsequently from fungi as part of a drug discovery programme. In addition we examined the kinetics of extraction from biocompatible polymers.

62.I.6. Retention prediction in capillary electrophoresis and electrochromatography Although much has been written on separations by capillary electrophoresis, there is very little predictive ability of other than a broad size-mobility relationship. However, this is unable to explain the resolution of homologues and structural isomers with the same overall volumes. We have examined this problem in a series of studies and have proposed that the analytes are oriented by the applied electrical field and so cannot tumble freely. Structural differences can then be predicted to show different retention, as they will possess different effective volumes. This theory can explain most of the

Fig. 1. Separation of the dimethylpyridines at pH 6.5. Buffer, 50 mM lithium phosphate, 25ºC. Compounds: 1, 3,4-DMP; 2, 3,5-DMP; 3, 2,3-DMP; 4, 2,5-DMP; 5 2,4-DMP; 6 2,6-DMP.

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differences between the isomeric dimethylpyridines or isomeric monoalkyl pyridines [7] (see Fig. 1). Current work is examining mixed mode elution of ionised analytes in electrochromatography.

62.I.7. Superheated-water chromatography Our recent research has drawn on a number of these earlier experiences to examine the potential for the novel use of superheated water as an environmentally clean eluent for reversed-phase liquid chromatography [8]. The approach has a number of interesting properties. The mobile phase, unlike carbon dioxide in SFC, is inherently polar, but as water is heated its polarity decreases with increased temperature. We have demonstrated that the method can be used for the separation of phenols, esters, and pharmaceuticals, vitamins and other groups. Additional scope in selectivity can be obtained by using the universal flame ionisation detector, and if deuterium oxide is used as the eluent, by linking the separation to NMR spectroscopy and mass spectrometry [9].

References 1.

2. 3.

4.

5. 6. 7. 8. 9.

R.M. Smith (Ed.), Retention and Selectivity in Liquid Chromatography. Prediction, Standardization and Phase Comparisons, Journal of Chromatography Library Series, Vol. 57, Elsevier, Amsterdam, 1995, 462 pp. R.M. Smith, Review-Functional group contributions to the retention of analytes in reversed-phase high-performance liquid chromatography, J. Chromatogr. A, 656 (1993) 381–415. R.M. Smith, Simon J. Bale, S.G. Westcott and M. Martin-Smith, Modification of the high-performance liquid chromatographic retention of β-diketones by the inclusion of metal ions in the mobile phase, Analyst, 115 (1990) 1517–1523. R.M. Smith, J.P. Westlake, R. Gill and M.D. Osselton, Retention reproducibility of basic drugs in high-performance liquid chromatography on a silica column with a methanol — high pH buffer eluent. Changes in selectivity with the age of the stationary phase, J. Chromatogr., 592 (1992) 85–92. R.M. Smith (Ed.), Supercritical-Fluid Chromatography, RSC Chromatography Monographs No. 1, Royal Society of Chemistry, London, 1988, 238 pp. R.M. Smith, Sample preparation perspectives: Supercritical-fluid extraction of natural products, LC– GC, 12 (1995) 930–939; LC–GC Int., 9 (1996) 8–15. A.G. McKillop, R.M. Smith, R.C. Rowe and S.A.C. Wren, The modelling and prediction of migration rates in capillary electrophoresis: Separation of alkylpyridines, Anal. Chem., 71 (1999) 497–504. R.M. Smith, R.J. Burgess, O. Chienthavorn and J.R. Stuttard, Superheated water: a new look at chromatographic eluents for reversed-phase liquid chromatography, LC–GC Int., 12 (1999) 30–36. R.M. Smith, O. Chienthavorn, I.D. Wilson, B. Wright and S.D. Taylor, Superheated- heavy water as the eluent for HPLC–NMR and HPLC–NMR–MS, Anal. Chem. Released on www on September 18, Anal. Chem., 71 (1999) 4493–4497.

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D.63. Lloyd R. Snyder Lloyd Robert Snyder was born in 1931, in Sacramento, CA. He received his B.Sc. and Ph.D. degrees in chemistry from the University of California at Berkeley in 1952 and 1954. His subsequent career in industry has included the following assignments: Shell Oil Co., Houston, TX (1954–1956); Technicolor Inc., Burbank, CA (1956–1957); Union Oil Co. of California, Brea, CA (1957–1971); Technicon Corp., Tarrytown, NY (1971– 1982); Lloyd R. Snyder Inc., Yorktown Heights, NY (1982– 1985). Since 1984, he has been Vice President of Research at LC Resources Inc., Walnut Creek, CA. He was also an Editor of the Journal of Chromatography (1987–2000), was an Adjunct Professor of Chemistry at Pace University (1980–1986), and taught beginning and advanced courses on HPLC as an American Chemical Society short course instructor with Jack Kirkland (1971–1995). L.R. Snyder is the author or co-author of 300 research publications, 9 patents and 7 books. The latter include “Principles of Adsorption Chromatography”, Dekker (1968); “An Introduction to Separation Science”, Wiley-Interscience (1973, with B. Karger, Cs. Horva´th); “Introduction to Modern Liquid Chromatography”, Wiley-Interscience (1974, with J. Kirkland); “Introduction to Modern Liquid Chromatography”, Wiley-Interscience, 2nd edn. (1979, with J. Kirkland); “Practical HPLC Method Development”, Wiley-Interscience (1988, with J. Glajch and J. Kirkland); “Troubleshooting LC Systems”, Humana Press (1989, with J. Dolan); “Practical HPLC Method Development”, 2nd edn., Wiley-Interscience (1997, with J. Glajch and J. Kirkland). L.R. Snyder’s work in chromatography has been recognized by a number of awards: American Chemical Society Award in Petroleum Chemistry (1970); the Stephen Dal Nogare Award in Chromatography (1976); Chromatography Memorial Medal, Scientific Council of the Academy of Sciences of the USSR (1980); the American Chemical Society National Award in Chromatography (1984); Pittsburgh Society Award in Analytical Chemistry (1984); L.S. Palmer Award in Chromatography, Minnesota Chromatography Forum (1985); the A.J.P. Martin Award, (British) Chromatographic Society (1989); Northeast Regional Chromatography Discussion Group National Chromatography Award (1991); American Chemical Society Orange County Section Service Through Chemistry Award (1993); the Eastern Analytical Symposium Award in Separation Science (1994); California Separation Science Society Award for Distinguished Contributions in Separation Science (1996); Anachem Award (Association of Analytical Chemists) (1998). His primary work has been in the field of liquid chromatography (1957 to present). Since 1966, he has contributed to the development, theory and application of HPLC. See Chapter 5B, a, b, d, h

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63.I. A SEARCH FOR SIMPLE LC MODELS Lloyd Robert Snyder 26 Silverwood Ct., Orinda, CA 94563, USA

My career in chromatography began in 1954 with an introduction to the then new technique of gas chromatography. Since 1957, I have worked in the field of liquid chromatography, with primary emphasis in the following areas.

63.I.1. Retention vs. structure The possible prediction of LC retention continues to fascinate many chromatographers. However, most work that has addressed this question has been restricted to a narrow range of compound types and separation conditions, with limited practical application. In 1957, I began a comprehensive study of separations by adsorption chromatography (i.e., normal-phase chromatography, NPC), especially of compounds likely to be present in higher boiling petroleum fractions. The aim of this project was the design of a theoretical model that would allow predictable compound-type separations for subsequent analysis of the resulting fractions by high-resolution mass spectrometry and other techniques. This in turn required reliable predictions of separation as a function of compound structure, mobile phase and LC mode (NPC or ion exchange). Work on a theory of NPC received most of my attention, resulting in the comprehensive treatment described in my 1968 book (“Principles of Adsorption Chromatography”, Dekker). An important discovery from this investigation of NPC was the phenomenon of solute or solvent localization — the tendency of polar molecules to attach to polar sites on the adsorbent surface. In the absence of an understanding of localization, any real understanding of retention in adsorption chromatography was doomed to failure. A second important observation was that solute retention in NPC is in many cases dominated by interactions of solvent and solute with the stationary phase, to the extent that corresponding mobile phase interactions can often be ignored. The latter NPC model in combination with a similar (but lesser) effort directed toward ion exchange allowed the design of a multi-step procedure, which resulted in the desired compound class separation of petroleum fractions boiling between 200 and 550ºC. This procedure (see Fig. 1), developed with the help of my associates, Ev Howard and Bruce Buell, allowed the first complete analysis of the nitrogen and oxygen compounds present in a petroleum sample [1]. The separation scheme of Fig. 1 involves three different adsorbents (silica, alumina, and charcoal) plus cation exchange. Because of our development of a quantitative theory of retention as a function of structure for the conditions of Fig. 1, it was possible to predict which compound types would be found in each of the dozens of fractions provided by this procedure. Not only did this knowledge contribute to the initial design of the scheme of Fig. 1, it also provided additional confirmation of structure of the compounds provisionally identified in each fraction by mass, UV and=or IR spectroscopy.

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Fig. 1. Simplified and abbreviated representation of a separation scheme used to characterize the nitrogen and oxygen compounds in high-boiling petroleum distillates. Resulting fractions could be analyzed qualitatively and quantitatively by mass, UV and IR spectrometry. Adapted from [1]. Fractions which have been separated by alumina and silica are designated by A and S, respectively. Thus, Ai S2 refers to fraction Ai from the alumina separation, and fraction 2 from the silica separation of fraction Ai .

63.I.2. High-performance liquid chromatography The automation and optimization of liquid chromatography for application to any sample, which led to what we now call HPLC, began in several laboratories during the 1960s and continued over the next 30 years. I have previously reviewed my own and others’ contributions to this development [2]. My initial research (1966–1971) involved the development of homemade equipment and columns, attempts to formulate a systematic approach to HPLC method development, and practical applications of the technique. Fig. 2 reviews some of this early work, with emphasis on the all-important performance of the column. Our first separations (1967) featured reasonable column performance but long run times; the next development (1968) emphasized much shorter run times (but smaller values of N), while the 1969 separation is an application of

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Fig. 2. NPC chromatograms from the author’s laboratory in the period 1967–1969. Top: six aromatic hydrocarbons; Bottom left: seven polycyclic aromatic hydrocarbons; Bottom right: hydrogenated quinoline sample. Adapted from [2].

‘real’ interest (hydrogenated quinoline sample) that was used to assay several hundred samples. Our experience was no doubt similar to that of other workers at that time that began with model compound studies, then advanced to practical applications of HPLC. My long association with Jack Kirkland began in the late 1960s, and in 1971, we presented our American Chemical Society short course (Introduction to Modern Liquid Chromatography) for the first time. Over the next 25 years, this course would undergo substantial change, as reflected in four books on HPLC (1974, 1979, 1988, and 1997) that were used as course texts. By the end of 1998, the short course had been presented to about 7000 students, with over 45,000 copies of these books sold by that time. The

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short course experience provided Jack and myself a unique opportunity to bounce ideas off our students, learn from their own experience, and combine the two inputs into a coherent description of HPLC and how this technique is best carried out. The stimulus of work in this new field (HPLC) also provoked a renewed interest in some topics from my earlier days in chromatography, notably gradient elution and selectivity (see following).

63.I.3. Gradient elution theory Gradient elution was introduced in the 1950s as an important technique for the separation of wide-retention-range samples, as well as for other applications. A practical understanding was lacking, however, of how gradient separations could be optimized in practice. An important contribution to our current knowledge of gradient elution practice was the introduction in 1964 of the concept of linear–solvent–strength (LSS) separation [3]. Thus, if gradient conditions are selected which provide a constant decrease in log k with time, it becomes possible to treat gradient and isocratic separation as essentially similar. This in turn makes method development for gradient elution no more difficult than for isocratic separation, as described in our recent book (Chapter 8 of “Practical HPLC Method Development”, 2nd edn., Wiley). The development and use of the LSS model from its initial conceptualization has been described in over 50 research and review articles from my group that are largely summarized in [4]. Dennis Saunders in 1970 and John Dolan from 1980 to the present time made major contributions to this development. Among the numerous applications of the LSS model, three areas deserve special notice: preparative chromatography, separations of large biomolecules, and computer simulation. Just as gradient and isocratic elution can be regarded as equivalent for the case of small-sample (non-overloaded) separations, the same is true for moderate column overloading (‘touching band’ separation). Most chromatographers would even today regard preparative HPLC in a gradient mode as very complex and therefore difficult to understand, predict or control. As a result of the application of the LSS model to this situation (see Chapter 13 of Practical HPLC Method Development, 2nd edn.), the development of preparative separations in a gradient mode is today a relatively straightforward task. A second important application of the LSS model is for the separation of macromolecules — especially proteins and large peptides. When compared to corresponding separations of molecules with molecular weights <1000, gradient separations of peptide and protein molecules (as well as synthetic polymers) larger than 10,000 Da exhibit some apparent peculiarities. This in turn led many workers to assume that the basic retention process differs for these two groups of molecules (large vs. small). For example, an ‘on–off’ model was popular for a time, in which it was assumed that a large molecule is totally retained until mobile phase of a certain ‘critical’ composition reaches the column inlet, at which point the sample molecule becomes totally unretained and then moves through the column at the speed of the mobile phase. In reality, it is now known [4,5] that the separation process for large and small molecules in gradient elution is basically the same (the ‘on–off’ model normally does not apply). The only difference

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is the greater change in retention for large molecules for a given change in mobile phase composition (%B), and this can be exactly compensated by the use of flatter gradients. Our present understanding of the true nature of large-molecule gradient elution allows a straightforward approach to method development for these separations (Chapters 8 and 11 of Practical HPLC Method Development, 2nd edn.). The LSS model has also facilitated the development of computer simulation as an important tool for HPLC method development (see below).

63.I.4. Selectivity optimization An ability to rationally control band spacing in the chromatogram is critical to effective method development. At the same time, optimizing selectivity has remained the biggest challenge to the working chromatographer. My own interest in this area began during studies cited above on retention in adsorption chromatography. At that time, we discovered that selectivity could be varied enormously by varying the choice of mobile phase solvents (see example of Table 1), but solvent selectivity was determined mainly by the localization of the solvent (more polar solvents localize more), and much less by differences in solvent functionality related to hydrogen-acceptor or dipole strength [6]. The latter observation was the opposite of conventional thinking at that time, but subsequent work has largely validated the concept (e.g., see Appendix III of Practical HPLC Method Development, 2nd edn.). Somewhat later, Jack Kirkland, Joe Glajch and I collaborated in an extension of the latter work to the design of an efficient procedure for optimizing solvent selectivity for NPC [7]. This work also resulted in a better understanding of localization and its practical consequences [8]. Separations by reversed-phase chromatography (RPC) are unaffected by solvent localization, but differences in solvent hydrogen bonding and dipole moment are known to play an important role in solvent selectivity. Prior to 1974, an important question was: how can common solvents be compared or classified in terms of hydrogen bonding and dipolarity effects? Building on experimental data reported by Lutz Rohrschneider, I proposed at that time a so-called solvent selectivity triangle or SST [9,10]. This concept evolved over the next 20 years (cf. 1974 and 1993 versions in Fig. 3), but beginning

TABLE 1 CHANGE IN SELECTIVITY IN ADSORPTION CHROMATOGRAPHY (NPC) BASED ON DIFFERENCES IN SOLVENT LOCALIZATION a Compound

1,3,5-Trinitrobenzene N,N-dimethyl-1-naphthylamide ÞD a

Value of k for indicated mobile phase 5=10=85 Acetonitrile=benzene=pentane (localizing)

Benzene (non-localizing)

5.9 7.1 1.2

0.3 88 290

Table 6.7 of Practical HPLC Method Development, 2nd edn. Alumina as column packing.

558 Fig. 3. Solvent selectivity triangle. 1974, Ref. [9]; 1993, Ref. [10]. Reprinted with permission. Groups I, II, etc. in triangle for 1974 comprise solvents of similar functionality; e.g., group I contains aliphatic alcohols, group II contains aliphatic amines and ethers.

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about 1979, it was widely applied for the purpose of understanding and optimizing separation selectivity (e.g., [11]). Several important concepts were inherent in the SST: (a) solvents with differing selectivity were located in different parts of the triangle; (b) the selectivity of any individual solvent can be approximately duplicated by some mixture of three solvents of very different selectivity; (c) optimum solvent selectivity could be obtained by the use of the latter three solvents for any sample. Solvent strength can be adjusted apart from selectivity, by the addition of a weaker solvent, e.g., hexane in NPC and water in RPC.

63.I.5. Computer simulation On the basis of various advances in the theory of chromatography, by the late 1970s, it became clear to several groups that computers might be used to predict and optimize HPLC separation. At this time, John Dolan, Russel Gant and I described a crude software program that on the basis of a few initial experiments was able to predict retention and resolution for changes in the mobile phase (%B), temperature, flow rate and column dimensions [12]. A few years later, John Dolan and I carried this idea a step further; we developed commercial software (DryLab ) for predicting separation and resolution by means of computer simulation. Since that time, DryLab has undergone continuing improvement ([13] and Chapter 10 of Practical HPLC Method Development, 2nd edn.) and is today the most widely used software for optimizing HPLC separation. In our latest version of DryLab (DryLab 2000), four experiments plus computer simulation can be used to optimize separation as a function of column temperature T and either gradient steepness tG or %B [13]. While the traditional view has been that the latter two variables are only marginally effective for changes in selectivity, it actually appears that this approach is as effective as other 2-D schemes for HPLC optimization [14]. Equally important, changes in temperature and either tG or %B appear to be more convenient and yield more robust final methods. It is interesting to note that both our first (1979 [12], and latest (1999 [14]) attempts at computer simulation [12] emphasize the same variables (T and %B) for optimizing selectivity — despite an almost universal preference for the use of other variables. DryLab 2000 is also the first commercial software that allows the user to simultaneously vary any two separation conditions (e.g., %B, pH, solvent mixtures, etc.) for any chromatographic procedure (e.g., capillary electrophoresis, gas chromatography, supercritical-fluid chromatography, etc.) [15]. Thus, the chromatographer is no longer constrained to a predetermined experimental design. In summarizing my involvement over the past 45 years with chromatography, and especially HPLC, I can best quote from my recent review of the history of HPLC [2]. “HPLC would not have been possible without innovations in equipment, the development of suitable columns, and an understanding of how best to use the technique. My own work since 1967 has dealt almost entirely with advancing our understanding of HPLC separation and in conveying this understanding to an audience of practical workers. This was perhaps the easiest of the tasks that needed to be addressed at the beginning of the HPLC era.”

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References 1. 2. 3. 4. 5.

6. 7. 8.

9. 10. 11.

12.

13.

14.

15.

L.R. Snyder, Petroleum nitrogen and oxygen compounds, Accts. Chem. Res., 3 (1970) 290. L.R. Snyder, Modern practice of liquid chromatography: Before and after 1971, J. Chem. Ed., 74 (1997) 37. L.R. Snyder, Linear elution adsorption chromatography. VII. Gradient elution theory, J. Chromatogr., 13 (1964) 415. L.R. Snyder and J.W. Dolan, The linear–solvent–strength model of gradient elution, Adv. Chromatogr., 38 (1998) 115. L.R. Snyder and M.A. Stadalius, HPLC separations of large molecules. A General Model, in Cs. Horva´th (Ed.), High Performance Liquid Chromatography. Advances and Perspectives, Vol. 4, Academic Press, New York, NY, 1986, p. 195. L.R. Snyder, Solvent selectivity in adsorption chromatography on alumina. Non-donor solvents and solutes, J. Chromatogr., 63 (1971) 15. J.L. Glajch, J.J. Kirkland and L.R. Snyder, Practical optimization of solvent selectivity in liquid–solid chromatography using a mixture-design statistical technique, J. Chromatogr., 239 (1982) 268. L.R. Snyder, Mobile phase effects in liquid–solid chromatography, in Cs. Horvath (Ed.), High-Performance Liquid Chromatography, Advances and Perspectives, Vol. 3, Academic Press, New York, NY, 1983, p. 157. L.R. Snyder, Classification of the solvent properties of common liquids, J. Chromatogr., 92 (1974) 223. L.R. Snyder, P.W. Carr and S.C. Rutan, Solvatochromically based solvent-selectivity triangle, J. Chromatogr., 656 (1993) 537. J.L. Glajch, J.J. Kirkland, K.M. Squire and J.M. Minor, Optimization of solvent strength and selectivity for reversed-phase liquid chromatography using an interactive mixture-design statistical technique, J. Chromatogr., 199 (1980) 57. J.R. Gant, J.W. Dolan and L.R. Snyder, A systematic approach to optimizing resolution in reversedphase liquid chromatography, with emphasis on the role of temperature, J. Chromatogr., 185 (1979) 153. J.W. Dolan, L.R. Snyder, N.M. Djordjevic, D.W. Hill, D.L. Saunders, L. Van Heukelem and T.J. Waeghe, A computer-assisted strategy for HPLC method development. I and II, J. Chromatogr. A, 803 (1998) 1, 33. J.W. Dolan, L.R. Snyder, N.M. Djordjevic, D.W. Hill, L. Van Heukelem and T.J. Waeghe, Reversedphase separation of complex samples by optimizing temperature and gradient time. I. Peak capacity considerations, J. Chromatogr. A, 857 (1999) 1. P. Haber, T. Baczek, R. Kaliszan, L.R. Snyder, J.W. Dolan and C.T. Wehr, Computer simulation for the simultaneous optimization of any two variables and any chromatographic procedure, J. Chromatogr. Sci., 38 (2000).

D.64. Shigeru Terabe Shigeru Terabe was born on October 21, 1940 in Toyokawa, Aichi, Japan. He received his Bachelor of Engineering (1963), Masters of Engineering (1965), and Doctor of Engineering (1973) degrees from Kyoto University. From 1965 to 1978, the Shionogi Research Laboratory employed him in Osaka, Japan. He joined the Department of Industrial Chemistry, Faculty of Engineering, Kyoto University in 1978 and was promoted to Associate Professor in 1984. In 1990, he joined the Faculty of Science, Himeji Institute of Technology as a Professor of ana-

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Professor Terabe separating polychlorobiphenyls by MEKC as an exchange scientist at Toxicological Branch of Center for Disease Control, Atlanta, GA (July 28, 1989).

lytical chemistry. He has been serving as the Dean of the Faculty of Science, Himeji Institute of Technology since April 1, 1998. S. Terabe received the 1994 Martin Gold Medal from The Chromatographic Society (UK), the 1995 Frederick Conference on Capillary Electrophoresis Award (USA), and in 1996 The Japan Society for Analytical Chemistry Award. He served as an advisory board member of Analytical Chemistry (1990–1992), and is one of the Editors of Journal Chromatography A (1994), and is a member of Editorial Advisory Boards of Journal of Microcolumn Separation, Chromatographia, Journal of Biochemistry Biophysical Methods, Journal of Pharmaceutical Biomedical Analysis, Journal of Capillary Electrophoresis, and Journal of High Resolution Chromatography. He organized the 6th International Symposium on High Performance Capillary Electrophoresis (1994, San Diego, CA, USA), and the 10th International Symposium on High Performance Capillary Electrophoresis and Related Microscale Techniques (1997, Kyoto). His research interests include the development of high-resolution separation methods with special emphasis on capillary electrophoresis (CE) including micellar electrokinetic chromatography (MEKC). His investigations are directed to MEKC since his invention of the technique in 1982. His present major topics are to interface MEKC with mass spectrometry, and to develop on-line concentration techniques of neutral analytes for MEKC. He has published 173 research or review articles, and 15 books or book chapters. S. Terabe is a pioneer in capillary electrophoresis. See Chapter 5B, a, e, l, p, r

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64.I. MICELLAR ELECTROKINETIC CHROMATOGRAPHY (MEKC) Shigeru Terabe Faculty of Science, Himeji Institute of Technology, Kamigori, Hyogo 678-1297, Japan

Micellar electrokinetic chromatography has made it possible to separate neutral analytes by electrophoresis.

64.I.1. Prelude to MEKC At the beginning of 1980, HPLC was already a widely accepted separation technique, but the separation efficiency or the theoretical plate number was much lower than that of capillary-gas chromatography (GC). The use of an open-tubular capillary column was considered to be a promising strategy of realizing high efficiency in LC and a few groups were working in that area. In early 1980, we started a project on capillary LC using 25-µm i.d. glass capillary columns with an octadecylsilylated inside surface. To perform this micro LC, the techniques developed by Professor Daido Ishii’s Group (Nagoya University) were very helpful. An open-tubular capillary of 25 µm i.d. was too wide to produce the expected high plate numbers. However, to use much narrower bore capillaries was impossible due to the lack of sensitivity of the available UV absorbance detector. The paper by Jorgenson and Lukacs [1] on capillary-zone electrophoresis (CZE) gave a strong impact on the chromatographic society, because of the high plate number reached 400,000 using a 75-µm i.d. ð 80 cm glass capillary. CZE is, however, not chromatography and its separation principle is based on differences in electrophoretic mobilities or charge to mass ratios. Therefore, CZE is not capable of separating neutral analytes. A few months preceding the publication of the paper by Jorgenson and Lukacs, Dr. T. Nakagawa, a retired colloid chemist, contributed a brief article in a bimonthly periodical distributed among the members of the Division of Colloid and Surface Chemistry, The Chemical Society of Japan, and suggested using a micelle in electrophoresis to separate neutral analytes based on micellar solubilization. He was interested in the possible principle of applying the micellar solubilization phenomenon to electrophoretic separation, but he was not aware of the development of this new electrophoretic technique. The paper of Jorgenson and Lukacs shocked me and reminded me of the suggestion by Dr. Nakagawa. I had a strong impression that it must be very interesting to try to use micelles in CZE.

64.I.2. Success in MEKC experiments It took more than one year since that time for me to do the first experiment on MEKC because of a deficiency of funds to buy a high-voltage power supply. Although Jorgenson and Lukacs employed a Pyrex glass capillary in their first experiments, flexible fused silica capillaries were available when we started capillary electrophoresis

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Fig. 1. First MEKC electropherogram recorded September 7, 1982 at Department of Industrial Chemistry, Kyoto University. Peak identification: 1, acetylacetone; 2, phenol; 3, mesityl oxide; 4, o-cresol; 5, m-cresol; 6, p-cresol. Conditions: capillary, 50 µm ð 134 cm (120 cm to the detector); running solution, 25 mM SDS in 50 mM phosphate buffer (pH 7.0); applied voltage, 25 kV; current, 22 µA; detection, 270 nm with a Jasco UVIDEC-II.

(CE) experiments. The result of the first MEKC experiment, which was performed at the Department of Industrial Chemistry, Kyoto University in 1982, was very exciting because some neutral compounds such as phenol and cresols were easily separated with up to 200,000 plates using a sodium dodecyl sulfate (SDS) solution at pH 7 (Fig. 1). The high efficiency separation was demonstrated by the complete separation of six isomers of xylenols, which were difficult to separate by reversed phase HPLC, under neutral conditions [2]. The separation principle of MEKC is based on the differential partitioning of the analyte between the micelle and the surrounding aqueous phase, and the differential migration of the micelle and the aqueous phase. When a voltage was applied between the two ends of the capillary, the ionic micelle is subject to both electrophoresis and electro-osmosis, whereas the bulk solution in the capillary migrates by electro-osmosis, generating a differential migration between the micelle and aqueous phase, as shown in Fig. 2. Neutral analytes introduced into the capillary are partitioned between the micelle and the aqueous phase and the migration velocity of the analyte depends on the fraction of the analytes incorporated into the micelle. Under neutral or alkaline conditions, the electro-osmotic flow is stronger than the electrophoretic migration of

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Fig. 2. Schematic illustration of the separation principle of MEKC. The micelle is assumed to be anionic; EOF, electro-osmotic flow; S, neutral analyte; small arrows, electrophoretic migration of the micelle.

the micelle in the opposite direction, and even anionic micelles migrate toward the cathode at a retarded velocity. The analyte that is incorporated at a higher level into the micelle migrates slower than that which is less incorporated. Thus, the principle is the same as that of chromatography: the micelle corresponds to the stationary phase and the surrounding aqueous phase to the mobile phase; the micelle is called the pseudo-stationary phase. Some fundamental equations to describe chromatographic parameters had to be modified in MEKC, because both chromatographic phases migrate and hence the migration time window is limited. However, most of the analyte behaviors were successfully explained by the chromatographic theory [3,4]. A primer booklet on MEKC was published in 1992 [5]. It was natural for a chromatographer to extend the micelle to other pseudo-stationary phases: cyclodextrins, ionic polymers, microemulsions, dendrimers, polymer surfactants, proteins, etc. The general name of electrokinetic chromatography (EKC) was given to the technique as a mode of CE [6]. Although the separation principle of EKC has been widely accepted, the border is not always clear between EKC and CZE using ionic modifiers.

64.I.3. Applications of MEKC Since MEKC is an appropriate method for the separation of small molecules that include both neutral and ionic, applications of MEKC were found mainly in the field of biomedical, pharmaceutical, agrochemical, and environmental analysis. The selectivity can be manipulated utilizing the techniques developed in HPLC: the choice of pseudo-stationary phases (surfactants), the concentration of the pseudo-stationary phase, the use of modifiers of the aqueous phase. The advantages of CE hold also in MEKC: minimum sample requirement, almost no consumption of organic solvents,

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minimum generation of wastes, low running costs, etc. A significant disadvantage of CE is relatively low concentration sensitivity because of the short path length of the UV absorbance detector. This problem is mostly solved by the use of on-line concentration techniques, or by the use of a high sensitivity detection method such as laser induced fluorescence. Many examples of applications of MEKC have been summarized in a review article [7].

64.I.4. Future prospects of MEKC Mass spectrometry (MS) is one of the most useful detection methods in analytical separation techniques including CE. However, the use of a relatively high concentration of a surfactant in MEKC causes several problems in MS detection. Most of the problems are solved by several techniques and in the near future, MEKC–MS can be a popular technique. Several new on-line concentration techniques increase the sensitivity more than 1000-fold, which enables MEKC to be a high sensitivity method comparable to GC. In late 1990, capillary electrochromatography (CEC) attracted great interest among chromatographers, probably because CEC is expected to be a micro high-resolution version of HPLC. However, it should be emphasized that MEKC is closer to the ideal format of chromatography than CEC from the viewpoint of column technology. It has been only 15 years since the birth of MEKC and new innovations will come in the 21st century.

64.I.5. Summary Micellar-electrokinetic chromatography developed by Terabe et al., has made it possible to separate neutral analytes by capillary electrophoresis (CE), which extends significantly the applicability of CE. Capillary electrophoresis including micellar-electrokinetic chromatography is an ultramicro scale analytical separation technique that can be employed in the microfabricated devices for single cell analysis. From the viewpoint of the column technology, micellar-electrokinetic chromatography is a type of ideal chromatographic technique because the micelle is homogenous in structure and of small size, and uniformly packed in the column.

References 1. 2. 3. 4.

J.W. Jorgenson and K.D. Lukacs, Zone electrophoresis in open-tubular glass capillaries, Anal. Chem., 53 (1981) 1298–1302. S. Terabe, K. Otsuka, K. Ichikawa, A. Tsuchiya and T. Ando, Electrokinetic separations with micellar solutions and open-tubular capillaries, Anal. Chem., 56 (1984) 111–113. S. Terabe, K. Otsuka and T. Ando, Electrokinetic chromatography with micellar solution and opentubular capillary, Anal. Chem., 57 (1985) 834–841. S. Terabe, K. Otsuka and T. Ando, Band broadening in electrokinetic chromatography with micellar solutions and open-tubular capillaries, Anal. Chem., 61 (1989) 251–260.

566 5. 6. 7.

Chapter 5 S. Terabe, Micellar Electrokinetic Chromatography, Beckman Instruments, Fullerton, 1992, pp. 46. S. Terabe, Electrokinetic chromatography: An interface between electrophoresis and chromatography, TrAC, Trends Anal. Chem., 8 (1989) 129–134. S. Terabe, N. Chen and K. Otsuka, Micellar electrokinetic chromatography, Adv. Electrophor., 7 (1994) 87–153.

D.65. Klaus K. Unger Klaus Unger was born on June 16, 1936 in Zwickau, Germany. After receiving his Ph.D. in Inorganic Chemistry and Analytical Chemistry at the Department of Chemistry of the Technical University of Darmstadt, Germany, 1965, Klaus K. Unger performed his habilitation thesis on the ‘Chemical Surface Modification of Silica’ at the same place in 1969. Since 1977, he is Professor of Chemistry at the Institute of Inorganic Chemistry and Analytical Chemistry of the Johannes Gutenberg-Universita¨t, Mainz, Germany. He was a Visiting Professor at the Northeastern University, Boston, MA, USA (1973), the National University of Singapore, Singapore (1982), the Monash University, Melbourne, Australia (1989), and the University of Lund, Lund, Sweden (1993–1994). Klaus Unger is the author of more than 330 scientific publications in material science and separation science. His research areas cover the synthesis of tailor-made adsorbents and catalysts, the chemical surface modification of porous and finely divided solids and the application of these materials in analytical, preparative and process high-performance liquid chromatography. His current research focuses on capillary electrochromatography and on multidimensional HPLC for protein analysis. Over 120 masters and doctoral (Ph.D.) students have received their advanced degrees under his direction. Klaus K. Unger is the author and co-author of several monographs: “Porous Silica”, Elsevier, Amsterdam, (1979); “Packings and Stationary Phases in Chromatography”, M. Dekker, New York, (1989); “Characterization of Porous Solids, Studies on Surface Science and Catalysis”, Elsevier, Amsterdam, Vol. 39 (1988), Vol. 62 (1991), Vol. 87 (1994); “A Practical Guide to HPLC”, GIT Verlag, Darmstadt (1999) and others. Klaus K. Unger is the recipient of the following awards: Pregl Medal of the ¨ sterreichische Gesellschaft fu¨r Analytische Chemie, Vienna, 1991, and the A.J.P. O Martin Award in Chromatography of the Royal Chromatographic Society, London, 1993, Humboldt-Research Award, Riksbankens Jubileumsfond, Stockholm, 1993, and American Chemical Society National Award in Chromatography, 1995, and Doctor honoris causa (Dr. h.c.) of the Kaunas University of Technology, Kaunas, Lithuania, 1999. See Chapter 5B, a, e, g, h, i, l

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65.I. THE IMPACT OF SILICA CHEMISTRY ON SEPARATION SCIENCE AND TECHNOLOGY: A PERSONAL VIEW SPANNING THREE DECADES Klaus K. Unger The Institute of Inorganic Chemistry and Analytical Chemistry, Johannes Gutenberg-Universita¨t, Duesberg weg 10–14, D55099 Mainz, Germany

65.I.1. Introduction to the field During my graduate studies in chemistry at the Technische Hochschule, Darmstadt, Germany, I became fascinated by the research on finely divided and porous materials as well as on surface chemistry at the famous Zintl-Institut fu¨r Anorganische Chemie und Physikalische Chemie. During my Ph.D. thesis from 1962–1965, I developed procedures to adjust and to tailor the pore size of silicas made by the classical sol–gel process. These materials were applied as packings for size-exclusion chromatography (SEC) of synthetic polymers and colloidal sols. The aim was to search for rigid SEC packings as an alternative to soft polysaccharides and to semirigid crosslinked polymer gels. In my habilitation thesis from 1965 to 1969, the focus of my research centered on chemical surface modification of silica with reactive silanes and the characterization of the adsorptive properties of surface modified silicas. Having experienced classical column-liquid chromatography with coarse silica in glass columns, I learned of the modern high-performance variant by contact with the late Istva´n Hala´sz, who was at this time lecturer at the Johann-Wolfgang-Goethe-Universita¨t Frankfurt=Main, Germany. Istva´n advised me in building HPLC equipment and I transferred my knowledge in chemical surface modification to his laboratory.

65.I.2. Breakthrough studies in silica bead manufacturing and surface modification of silica Although the theoretical concepts in HPLC were highly advanced at the beginning of 1970, and clear directives could be given to make a separation more efficient and faster, the introduction of the method was still hampered by the lack of technical achievements. One of the primary requirements was to employ microparticles of rigid silica of 5–10 µm particle size obtained by milling and size fractionation of larger particles. An appropriate sizing technology was, therefore, developed which enabled a cut to narrow particle size fractions by means of air elutriation by Alpine AG, Augsburg, Germany. Secondly, a technique was needed to pack these particles densely into stainless-steel columns with porous frits as endfittings. For this purpose the balanced density technique was applied which employed a suspension of the silica particles in a high-density liquid. This suspension was pumped into the column at high pressure and high flow rate using an autoclave as slurry reservoir. With these two achievements highly efficient HPLC columns were packed with silicas in their native form and operated under straight phase conditions.

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At the beginning of 1970, several research groups around the world attempted to manufacture spherical silica particles, such as J.J. Kirkland at DuPont de Nemors, USA, John Knox, Shandon Ltd., UK, and P. Myers, Phase Separations Ltd., UK. Spherical particles were assumed to generate a more stable and homogeneous column bed than irregular shaped ones and also to give a more favorable pressure drop vs. flow rate dependency than irregular ones. This hypothesis was widely evidenced experimentally. Nowadays the majority of silica packings in HPLC are spherical in shape. In 1973, we invented a process of making spherical silicas by a two step process [1]. In the first step tetraethoxysilane was partially hydrolyzed and condensed in an acidic ethanolic solution to polyethoxysiloxane (PES). In the second step, ethanol was removed and the PES was then completely hydrolyzed and condensed in a two-phase system containing ammonia solution of a pH of about 10 and an ethanolic PES solution. This process was performed under vigorous stirring. The PES was emulsified in the aqueous phase to liquid microdroplets which quickly solidified to silica hydrogel beads. The size of the beads was mainly determined by the kinematic viscosity of the PES and the speed of stirring using a specially designed stirrer. In this way silica beads were obtained with an average particle diameter between 5 and 20 µm. The pore diameter of the beads was varied between 5 and 15 nm with a specific surface area between 600 and 200 m2 =g. Still the batches had to be size classified into narrow cuts. Immediately the question arose on the ultimate minimum particle diameter in HPLC with respect to column efficiency, pressure drop and analysis time. Optimization studies by Knox [2] revealed that the optimum particle size should be about 3–4 µm. To verify these predictions we performed efficiency studies on columns packed with particles in the 1–10 µm size range [3]. Parallel to the manufacture of silicas the practical limitation in using native silicas in straight-phase chromatography became apparent: slow column equilibration, control of water content in n-hexane eluents. To circumvent these problems so-called reversed phase packings were developed. These materials were based on mesoporous silicas which were subjected to a reaction with n-octadecylchloro- or alkoxysilanes. In the period between 1975 and 1985, we studied in depth the silanization of the silica surface with respect to the kinetics, the surface stoichiometry, ligand density and surface coverage [4,5]. In the following period reversed phase chromatography with bonded n-octadecyl and n-octyl groups became the most widely applied packings in HPLC. In 1973, I took the unique chance to work for six months in the laboratory of Professor B.L. Karger, Northeastern University, Boston, Massachusetts, USA. During this visit I got to know most of the famous chromatographers in the USA, for example, J.J. Kirkland, L.R. Snyder, R.P.W. Scott and J.C. Giddings amongst others. In particular, J.J. Kirkland gave me a comprehensive introduction into the field of chromatography. In 1973, I met J.F.K. Huber at a symposium at Karlsruhe where I presented my first HPLC studies on spherical silica beads in the session on high pressure techniques. J.F.K. Huber became my strongest mentor over a period of twenty years and I would like to express my gratitude to him. Finally, in 1983 J. Knox spent about six months in my laboratories at Mainz where we worked on porous carbons as HPLC packings. With the late Go¨ran Schill I had an intensive cooperation over many years on ion pairing

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reagents. Thus, I had the unique privilege to meet most of the outstanding scientists in the field of separation, which ultimately inspired my work to a large extent. 65.I.3. The switch to high-resolution biopolymer separations At the beginning of 1980, fundamental studies were undertaken to utilize mesoporous and macroporous silicas with an appropriate bonding chemistry for interactive biopolymer separations. In 1983, an international conference series was established at Washington DC, USA named ISPPP (International Symposium on the Separation of Proteins, Polypeptides and Polynucleotides). In 2000, the 20th conference of this series will take place in Ljubljana, Slovenia. The proceedings of these symposia which were published as special issues in the Journal of Chromatography demonstrated the major achievements in this field (see Appendix S-6). Based on the rich experience in bonding chemistry, our group focused on the synthesis of size-exclusion packings, ion exchangers, hydrophobic interaction and affinity packings applied to protein and peptide separations [6]. From knowledge on the earlier work of Stoeber et al. [7], we succeeded in the synthesis of nonporous silica beads in the 1–2 µm size range. By conducting surface reactions we converted these beads into reversed phase, hydrophobic interaction, ion exchanger and affinity packings. With these packings applied in short columns of 30 mm length and 4 mm i.d., we were able to perform extremely fast separation of biopolymers. An example is given in Fig. 1

Fig. 1. Separation of a tryptic digest of hemoglobin A on a 30 ð 4:6 mm column packed with 1.5 µm MICRA non-porous silica packings surface modified with polystyrene under reversed phase gradient conditions: Gradient from 1% to 100% acetonitrile in water containing 0.1% TFA in 120 seconds according to the following program: time in sec, % acetonitrile 0.1%; 20=15%; 90=30%; 100=40%; 110=40%; 120=60%. Flow-rate: 2.5 ml=min, UV detection: 205 nm, Sample volume: 0.5 µl.

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[8]. A third area of active research was the coating of oxidic adsorbents with polymers as an alternative to silanization. This branch of activity was mainly initiated by my co-researcher Dr. A.A. Kurganov. He extended these studies in particular on packings based on Titania and Zirconia [9].

65.I.4. Novel ordered mesoporous silicas: the gateway to new adventures in separation science and technology Around 1985, the synthesis of silica-rich zeolites such as the MFI-type (ZSM-5, Silicalite I) attracted our interest. These zeolites were formed in the presence of structure-directing agents of so-called templates, e.g., tetraalkylammonium salts. Our objective was to understand the role of the template in the formation mechanism and to find the reaction conditions for template-free synthesis. In the following we successfully developed a process to manufacture template-free ZSM-5 up to pilot scale. In 1992, Mobil Oil researchers presented the synthesis of ordered mesoporous silicas of the M41S family, which was a breakthrough in large pore size zeolite chemistry. One of the most prominent members of this mesoporous silica families, MCM-41, was formed by the self-assembled, co-operative interactions of silicates and organic detergents such as long-chain n-alkylammonium salts to form periodic mesostructured phases. After removal of the template a unidimensional regular pore system was obtained with average pore diameters in the range between 2.5 and 4 nm. My co-researcher, F. Schu¨th, applied this concept of periodic inorganic=organic assemblies to the synthesis of other ordered mesoporous oxides [10]. Based on our experience with the synthesis of MCM-41 and the Stoeber reaction

Fig. 2. The merger of the synthesis of ordered mesoporous silica and the Stoeber reaction based on sol–gel chemistry.

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yielding micron- and submicron size nonporous silica-spheres, we combined both syntheses to one: converting tetraethoxysilane dissolved in an alcoholic ammonia solution in the presence of n-hexadecylamine as template into mesoporous micron size silica beads. In this way we were able to control the pore structural parameters as well as the particle size and size distribution of the silica beads (see Fig. 2) [11]. We have also been able to show that the synthesis concept is applicable to form beads of MCM-48 [12]. By choosing an appropriate polymeric template for the synthesis of MCM-48, we have recently shown that the average pore diameters of the spheres can be reproducibly adjusted over the full mesopore size range between 2 and 50 nm. It should be emphasized that the particles precipitate from a homogenous solution at room temperature. The particle size distribution is sufficiently narrow so that no sizing is required to use them as column packing material in chromatographic separations, e.g., high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). As demonstrated for the 1.5–2.0 µm nonporous bonded silicas in biopolymer separation, the 2 µm porous bonded silicas obtained by this procedure bear a high potential for fast HPLC separations.

65.I.5. Capillary electro-chromatography (CEC): challenges and opportunities for high throughout separations Pioneered by Pretorius, Jorgenson, Tsuda, Knox and others, CEC sparked remarkable interest as a novel separation method. CEC combines features of both HPLC and Capillary Electrophoresis. Due to the fact that the analytes are transported by the electroendoosmotic flow (EOF) to the cathode, no hydraulic flow is required. Consequently this allows one to use smaller particles than those applied in fast HPLC columns (dp ¾ 3 µm), which leads to much higher column efficiencies. Recently, we studied the column performance of CEC capillaries packed with 0.2, 0.5, 1.0, and 3.0 µm monodisperse silicas, respectively [13]. All columns showed the same linear dependency of the EOF vs. the field strength at otherwise constant conditions. Critical evaluation of the plate height vs. linear velocity plots indicated that the expected column performance (H ' dp ) was only obtained for columns packed with 1–2 µm particles. The performance of CEC columns with smaller particles was much worse. It was apparent that these columns were still operated at the descending part of the H vs. u curve where the axial diffusion is the dominant contribution to the total plate height. In order to fully use the potential of the submicron particles in CEC, the field strength must be increased to about 2000 V=cm. This can be achieved in two ways: increase of the electrical potential above 30 kV and shorten the column length to smaller than 100 mm. Both, however, cannot be accomplished with the existing commercial CEC equipment. Novel instrument design is thus required to apply short columns packed with submicron- or micron-size particles. This can serve as the basis for a separation technology applicable to high throughput screening [14].

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References 1. 2. 3.

4. 5. 6.

7. 8. 9.

10. 11.

12. 13. 14.

K.K. Unger, J. Schick-Kalb and K.F. Krebs, Preparation of porous silica spheres for column liquid chromatography, J. Chromatogr., 83 (1973) 5–9. J.H. Knox, Practical aspects of LC theory, J. Chromatogr. Sci., 15 (1977) 352–364. K.K. Unger and W. Messer, Comparative study on the performance of spherical and irregularly shaped silica and alumina supports having diameters in the 1 to 10 µm size range, J. Chromatogr., 149 (1978) 1–12. P. Roumeliotis and K.K. Unger, Structure and properties of n-alkyldimethylsilyl bonded silica reversed phase packings, J. Chromatogr., 149 (1978) 211–224. J.N. Kinkel and K.K. Unger, Role of solvent and base in the silanization reaction of silicas for reversed-phase high-performance liquid chromatography, J. Chromatogr., 316 (1984) 193–200. K.K. Unger, K.D. Lork and J. Wirth, Development of advanced silica-based packing materials, in M.T.W. Hearn (Ed.), The HPLC of Peptides, Proteins and Polynucleotides, VCH Publishers, New York, (1991) pp. 59–117. W. Stoeber, A. Fink and F. Bohn, Controlled growth of mono-disperse silica spheres in the micron size range, J. Colloid Interface Sci., 26 (1968) 62–75. T. Issaeva, A.A. Kurganov and K.K. Unger, Super high speed liquid chromatography of proteins and peptides on nonporous MICRA NPS RP packings, J. Chromatogr. A, 46 (1999) 13–23. A. Kurganov, U. Tru¨dinger, T. Issaeva and K.K. Unger, Native and modified alumnia, titania and zirconia in normal- and reversed-phase high performance liquid chromatography, Chromatographia, 42 (1996) 217–222. U. Ciesla, D. Demuth, R. Leon, P. Petroff, G. Stucky, K.K. Unger and F. Schu¨th, Surfactant based synthesis of oxidic catalysts and catalyst supports studies, Surface Sci. Catal., 91 (1995) 331. G. Buechel, M. Gruen, K.K. Unger, A. Matsumoto and K. Tsutsuni, Tailored syntheses of nanostructured silicas: control of particle morphology, particle size and pore size, J. Supramol. Sci., 5 (1998) 253–259. K. Schumacher, M. Gruen and K.K. Unger, Novel synthesis of spherical MCM-48, Microporous and Mesoporous Mater., 27 (1999) 201–206. S. Luetdke, Th. Adam, N. von Doehren and K.K. Unger, Towards the ultimate minimum particle diameter of silica packings in capillary electrochromatography, J. Chromatogr. A, 887 (2001) 339–346. K.K. Unger, D. Kumar, M. Gruen, G. Buechel, S. Luedtke, Th. Adam, K. Schumacher and S. Renker, Synthesis of spherical porous silicas in the micron and submicron size range — challenges and opportunites for miniaturized high-resolution chromatographic and electrokinetic separations, J. Chromatogr. A, 892 (2000) 47–55.

D.66. Irving W. Wainer Irving Wainer was born on March 27, 1944 in Detroit, Michigan. He received his B.Sc. degree in Chemistry from Wayne State University in 1965. He continued his graduate studies at Cornell University where he received his Ph.D. degree in Organic Chemistry in 1970. He completed his post-doctoral training in Molecular Biology at the Institute of Molecular Biology in Eugene, Oregon, from 1970 to 1973. He was employed as a Research Associate with the Department of Pharmacology at the Thomas Jefferson University in Philadelphia, Pennsylvania from 1973 to 1978. He joined the US Food and Drug Administration in 1978 as a Research Chemist.

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In 1986, he became the Director of Analytical Chemistry of the Pharmaceutical Division at St. Jude Children’s Research Hospital in Memphis, Tennessee. In 1990, he was appointed Professor of Chemistry with the Department of Oncology at McGill University in Montreal, Canada. In 1997, he joined the Department of Pharmacology at Georgetown University Medical Center at Washington, DC, where he is currently a Professor and Director of the Bioanalytical Center. I.W. Wainer is the Senior Editor of the Journal of Chromatography B and was the founding Editor of Chirality. In the years 1996 to 1998, he published 34 articles in refereed journals and 6 additional manuscripts have been accepted for publication, 1 book, 2 book chapters and 21 abstracts. His research interests include enzyme-based HPLC, enantioselective separations, and receptor independent enhancement of DNA replication by steroids. He received the 1992 A.J.P. Martin Medal from the Chromatographic Society. He is an Elected Fellow of the American Academy of Pharmaceutical Sciences and an Elected Member of the United States Pharmacopeial Convention Committee of Revision for 1995–2000. See Chapter 5B, a, e, h, i, q, r

66.I. ENANTIOSELECTIVE CHROMATOGRAPHY IN THE PHARMACEUTICAL AND PHARMACOLOGICAL SCIENCES Irving W. Wainer Georgetown University Medical Center, Division of Clinical Pharmacology, 3900 Reservoir Rd NW, Washington DC 20007–2197, USA

In the 1980s, one of the key questions facing the pharmaceutical industry and regulatory agencies was the elaboration of guidelines for the development and use of chiral drugs. Chromatography, particularly enantioselective liquid chromatography using HPLC chiral stationary phases, played a key role in this process. My involvement in this area began in 1978 when the US Food and Drug Administration (FDA) hired me with the responsibility to initiate a program to demonstrate that the enantiomeric purity of drugs could affect their therapeutic efficacy. A key goal of this program was the development of accurate, efficient and reproducible methods to separate the component enantiomers of racemic drugs and to determine their enantiomeric purity. This task was complicated by the fact that a FDA method had to meet a number of stringent requirements. First of all, the method had to be accurate and reliable. Second, the method had to utilize scientific equipment that was readily available, preferably commercially available, to the same extent and at the same quality throughout the world. Third, the method had to be easy, one that a chemist or technician could perform without any type of advanced training. And last, the method had to be relatively cheap. My group began its work with an investigation of the determination of the enantiomeric purity of bulk drug preparations using nuclear magnetic resonance spec-

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troscopy and chiral lanthanide shift reagents. A second method involved the chemical conversion of the component enantiomers of a racemate into diastereomers and their chromatographic separation. The former method involved expensive equipment and was relatively insensitive. The latter method was difficult to apply to the determination of trace amounts of one enantiomer in the presence of the other. In 1981, HPLC columns containing chiral stationary phases (HPLC-CSPs) became commercially available. My group obtained a number of these columns and studied their usefulness in the separation of the component enantiomers of racemic drugs and in the evaluation enantiomeric purity. We found that HPLC-CSPs separated the component enantiomers of racemic drugs in less time and at less expense than the other separation techniques we were investigating. In 1983, my laboratory published our first paper describing the application of HPLC chiral stationary phases to pharmaceutical analysis [1] and by 1999, we had published a total of 23 papers, 7 book chapters and 3 books in this area. As an example of the utility of HPLC-CSPs in pharmaceutical analysis, an HPLC-CSP method was developed to replace the existing regulatory method used to determine the enantiomeric purity of amphetamine preparations. Not only was the enantioselective HPLC method accurate and reproducible, it was faster and cheaper than the other available methods. Indeed, the HPLC-CSP method replaced one that required the use of ten tablets and took over a day to complete with a process that used a single tablet and took only one hour to complete. The method was then subjected to and validated by an inter-laboratory collaborative study, the results of which were published by my group in 1988 [2]. In addition to the analysis of the enantiomeric composition of chiral compounds as the bulk drug and as pharmaceutical formulations, my group at the FDA demonstrated that enantioselective chromatography on HPLC-CSPs could be used in pharmacokinetic and metabolic studies. In 1984, our first publication in this area reported the determination of ()- and (C)-propranolol in human serum [3]. This work continued after I left the FDA and has produced 28 papers, 5 book chapters and 3 books reporting the effect of chirality on the metabolism, disposition, efficacy and toxicity of clinically relevant drugs. By the mid-1980s the application of HPLC-CSPs to pharmaceutical and pharmacological sciences had advanced to the point where in 1988 we published the book “Drug Stereochemistry: Analytical Methods and Pharmacology”; a second edition was published in 1993 [4]. By 1988, there were over thirty commercially available HPLC-CSPs columns. The commercial availability of a wide variety of HPLC-CSPs columns afforded the opportunity to select a priori small group of HPLC-CSPs that would most likely afford efficient separation of the component enantiomers of any given racemic drug. By the time I left the FDA in 1986, my group had published papers describing the chromatographic separation of hundreds of different racemic drugs on HPLC-CSPs. Indeed, by the late 1980s hundreds of scientific publications describing the use of chiral HPLC to separate the enantiomers of racemic compounds were available. This body of scientific scholarship indicated that the use of chiral HPLC had become well known and widespread and provided information regarding how to use HPLC-CSPs to separate the component enantiomers of racemic compounds. Based upon these developments, my laboratory produced guides to the selection of HPLC-CSPs, in

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manuscript form in 1987, a comprehensive ‘how-to’ guide entitled “A Practical Guide to the Selection and use of HPLC Chiral Stationary Phases” in 1988 [5]. My ‘how to’ guide was divided into two sections. The first section was a classification and explanation of the chiral stationary phases used in the commercially available chiral HPLC columns. I classified these chiral stationary phases into five different types based on how they interacted with and separated the enantiomers of racemic compounds. ž Classified as Type I, the chiral stationary phases that chemically interacted with and separated racemic compounds primarily by attractive interactions, hydrogen bonding, pi–pi interactions and dipole stacking interactions. ž Classified as Type II, the chiral stationary phases that chemically interacted with and separated racemic compounds primarily through attractive interaction where inclusion complexes also played an important role. ž Classified as Type III, the chiral stationary phases that chemically interacted with and separated racemic compounds primarily by the formation of inclusion complexes, wherein the sample entered a chiral cavity within the chiral stationary phase. ž Classified as Type IV, the chiral stationary phases that chemically interacted with and separated racemic compounds primarily by forming coordination complexes with transition metal ions. ž Classified as Type V, the chiral stationary phases that were based upon immobilized proteins. These phases interacted with and separated racemic compounds primarily by forming complexes based upon combinations of hydrophobic and polar interactions. In addition to classifying the types of chiral stationary phases (CSPs), the first section of my ‘how-to’ guide provided an explanation of the general characteristics of each type of CSP, described the compounds that had been separated on that type of chiral stationary phase, and the appropriate mobile phases and mobile phase additive for use with that CSP. My ‘how-to’ guide then provided similar information regarding each of the 35 commercially available CSPs. This section of my guide also contained over 100 references such that the reader could go to the referenced scientific literature for even more information and finer detail regarding a particular CSP. The second section of my ‘how-to’ guide contained tables designed as an easy-to-use guide for selecting an appropriate chiral stationary phase for separating the component enantiomers of any given racemic compound. The tables were divided by the functional groups of the racemic compound to be separated and explained which chiral stationary phases would interact with and separate racemic compounds containing those functional groups. Using these tables, the reader only needed to know the molecular size, structure and functional groups of a racemic compound to select a small group of commercially available chiral HPLC columns that would most likely separate the component enantiomers of that compound. By the late 1980s, the field of chiral separations and the investigations into the impact of stereochemistry on pharmacological properties had matured to the point where there was a need for a journal devoted solely to stereochemistry. In 1989, the journal Chirality was launched and I was the Founding Editor-in-Chief. The lead article of the first issue of Chirality, authored by Dr. Wilson DeCamp of the FDA, was entitled ‘The FDA Perspective on the Development of Stereoisomers.’ This article confirmed

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Fig. 1. One of the initial enantioselective HPLC resolutions of the enantiomers of amphetamine achieved at the US Food and Drug Administration in 1983. The separation was accomplished on a 3,5-dintrobenzoylphenylglycine HPLC chiral stationary phase using a hexane–isopropanol mobile phase. Before chromatography, the amphetamine enantiomers were converted into benzoyl derivatives using benzoyl chloride. This assay eventually led to the first international collaborative study utilizing enantionselective chromatography in the regulatory control of the stereochemical purity of marketed pharmaceuticals.

that enantioselective chromatography on HPLC-CSPs had become an accepted and successful technique in pharmaceutical development and regulation. One of the key aspects related to the use of HPLC-CSPs is an understanding of the chiral recognition mechanisms operating on these columns. Our investigations into these mechanisms began with the first series of experiments we conducted in the early 1980s when we looked at the electronic and steric factors involved in the separation of α-methylphenethylamines such as amphetamine [1]. These studies have, over time, led to the development of chemometric analyses of the chiral recognition mechanism using molecular modeling techniques and the construction of quantitative structure– enantioselective retention relationships [6,7]. Recently, these studies have been expanded to include the use of multivariate regression and artificial neural networks,

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which allows for the prediction of the exact manner in which an enantioselective separation will occur on a particular specific HPLC-CSP, the magnitude of this separation and the order that the enantiomers will elute from the column [8]. Our investigations into the chiral recognition mechanisms operating on immobilized biopolymer-based HPLC-CSPs, have also demonstrated that these mechanisms reflect the naturally occurring ligand–biopolymer interactions. Thus, we have demonstrated that an HPLC stationary phase containing immobilized human serum albumin can be used to measure the extent of protein binding for a given compound as well as to identify the site on the protein at which that compound binds [9]. These studies have produced 30 publications and 3 book chapters and a series of new stationary phases containing one or more carrier proteins such as serum albumin (including human, rat, rabbit and mouse albumins), or enzymes such as chymotrypsin, lipase, β-glucuronidase, etc. Recently, our laboratory has also developed a series of HPLC stationary phases containing membrane-bound receptors [10]. In our view, these immobilized biopolymer chromatographic stationary phases can be used to mimic in vivo pharmacological and biochemical interactions and provide direct instead of inferential data. The use of dynamic equilibria obtained from these new chromatographic systems, rather than static equilibria obtained from standard ligand binding experiments, the reproducibility of chromatographic experiments and the high throughput provided by a chromatographic system should result in a new paradigm for studies in the biological and pharmacological sciences.

References 1.

2. 3.

4. 5. 6.

7.

8. 9.

I.W. Wainer and T.D. Doyle, Application of HPLC chiral stationary phases to pharmaceutical analysis: Electronic and steric effects in the resolution of α methylphenethylamine derivatives J. Chromatogr., 259 (1983) 465–472. M.C. Alembik and I.W. Wainer, Resolution and analysis of the enantiomers of amphetamine by LC on a chiral stationary phase: Collaborative study, J. Off. Anal. Chem., 71 (1988) 531–533. I.W. Wainer, T.D. Doyle, K.H. Donn and J.R. Powell, The direct determination of ()- and (C)-propranolol in human serum using a high-performance liquid chromatographic chiral stationary phase, J. Chromatogr., 306 (1984) 405–411. I.W. Wainer, (Ed.), Drug Stereochemistry, Analytical Methods and Pharmacology, 2nd Edition, Revised and Expanded, Marcel Dekker, Inc., New York, NY, 1993. I.W. Wainer, A Practical Guide to the Selection and Use of HPLC Chiral Stationary phases, J.T. Baker Chemical Company, Phillipsburg, NJ, 1988. R. Kaliszan, T.A.G. Noctor and I.W. Wainer, Stereochemical aspects of benzodiazepine binding to human serum albumin II: Quantitative relationship between structure and enantioselective retention in high performance liquid affinity chromatography, Molec. Pharmacol., 42 (1992) 512–517. T.D. Booth and I.W. Wainer, Investigation of the enantioselective separations of α-alkyl arylcarboxcylic acids on an amylose tris (3,5-dimethylphenylcarbamate) chiral stationary phase using quantitative structure-enantioselective retention relationships (QSERR): Identification of a conformationally driven chiral recognition mechanism, J. Chromatogr. A, 737 (1996) 157–169. T.D. Booth, K. Azzoui and I.W. Wainer, Prediction of chiral chromatographic separations using combined multivariate regression and neural networks, Anal. Chem., 69 (1997) 3879–3883. I.W. Wainer, Enantioselective high performance liquid affinity chromatography as a probe of ligand-biopolymer interactions: An overview of a different use for HPLC chiral stationary phases, J. Chromatogr. A, 666 (1994) 221–234.

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10. Y. Zhang, X. Xiao, K. Kellar and I.W. Wainer, Immobilized nicotinic receptor stationary phase for on-line liquid chromatographic determination of drug-receptor affinities. Anal. Biochem., 264 (1998) 2.

D.67. Harold F. Walton Harold F. Walton was born in 1912 in Cornwall, England. He married Sadie Goodman of Ithaca, New York in 1938 and they have three children: James Walton, Liz Louise Calie and Daniel Goodman Walton. He became a naturalized citizen of the USA in 1948. He received his education at Kingswood School, Bath, England, and a Doctor of Philosophy from Exeter College, Oxford, 1937, followed by a Post Doctoral Fellow at Princeton University, 1937. H.F. Walton was a Research Chemist at the Permutit Co., New Jersey from 1938–1940. He has held the following positions in academia and industry: Assistant Professor at Northwestern University, 1940 to 1946; Professor of Chemistry at the University of Colorado, 1947–date; retired in 1982 as Professor Emeritus, Chairman, University of Colorado Department of Chemistry, 1962 to 1966. He organized the first US–Japan conference on high-performance liquid chromatography in 1975. H.F. Walton was visiting Professor and Fullbright Grantee, University of Trujillo, Peru (1966–1967, 1970), and Honorary Professor, University of Trujillo and University of San Marcos, Lima, Peru. He has lectured in various countries, including Venezuela, France and Japan. His many honors include: The American Chemical Society, Colorado Section Award (1976), the Stephen Dal Nogare Award in Chromatography (1988), and the University of Colorado Medal for Distinguished Service in 1995. H.F. Walton is a pioneer in ligand-exchange chromatography of amphetamine drugs and amino sugars. He did the early research in the 1950s to 1980s and provided the ‘steps in the ladder’ for ion-exchange investigations and analytical chemistry. He is the author and co-author of over 130 scientific papers, reviews and books on ion-exchange chromatography in Analytical Chemistry [1964, 48], Ion-exchange [77,88], Ion-exchange and Liquid Column Chromatography [Fundamental Reviews in Anal. Chem., 105], and in his Chapter 5 in the 5th edition of Erich Heftmann’s book on chromatography [1992, 129] are best known. H.F. Walton has been recognized with many awards and honors, has written eight books, lectured widely and is a member of a number of professional societies. Instead of writing several pages on his most significant achievements, he chose to present his scientific activities as a series of events and publications with dates. He has two patents: (1) Harold Frederic Walton, ‘Methods of Regenerating Electrolytic Polishing Solutions’, US Patent 2,645,635 (1953). (2) Harold Frederic Walton, ‘Preparation of Organomercuric Hydroxides’, US Patent 2,871,252 (1959).

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He also has served as Consulting Editor of: Journal of Chromatography Symposium Volumes, Separation Science and Technology and Nouveau Journal de Chimie (retired, 1985). See Chapter 5B, a, c

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

H.F. Walton, Schweres Wasser und Diplogen, das neue Isotop des Wasserstoff, Geistige Arbeit (Berlin), 1 (1934) 7–6. H.F. Walton and J.H. Wolfeden, Temperature coefficient of the electrolytic separation of the hydrogen isotopes, Nature, 138 (1936) 468. A.J. Edwards, H.F. Walton, R.P. Bell and J.H. Wolfenden, The electrolytic separation of deuterium, J. Chem. Soc. (1936) 286. H.F. Walton and J.H. Wolfenden, The electrolytic separation factor of deuterium at very low concentrations, J. Chem. Soc. (1937) 1677. H.F. Walton, Notes on the manipulation of the Farkas microthermal conductivity analysis apparatus, Trans. Faraday Soc., 34 (1938) 450. H.F. Walton and J.H. Wolfenden The electrolytic separation of deuterium: Influence of temperature and current density, J. Chem. Soc., 34 (1938) 436. H.F. Walton, Ion-exchange between solids and solutions, J. Franklin Inst., 232 (1941) 371. H.F. Walton, Equilibria in a carbonaceous cation exchanger, J. Phys. Chem., 47 (1943) 371. R. Nelson, and H.F. Walton, Cation exchange at high pH, J. Phys. Chem., 48 (1944) 406. R. Kozak and H.F. Walton, Separation of metal ions by cation exchange, J. Phys. Chem., 49 (1945) 471. H.F. Walton, Ion-exchange, J. Chem. Ed., 23 (1946) 454. H.F. Walton, E.N. Hiebert and E.H. Sholtes, Quarternary ammonium salts as colloidal electrolytes, J. Colloid Sci., 1 (1946) 385. H.F. Walton, The activity of hydrochloric acid in solutions of 1-n-dodecanesulfonic acid, J. Am. Chem. Soc., 68 (1946) 1182. H.F. Walton, The activity of 1-n-dodecanesulfonic acid in aqueous solutions, J. Am. Chem. Soc., 68 (1946) 1180. H.F. Walton, The critical concentration of dodecanesulfonic acid, J. Am. Chem. Soc., 69 (1947) 469. H.F. Walton, Inorganic Preparations, Prentice-Hall, Inc., Englewood Cliffs, NJ, 1948. H.F. Walton, Ion-exchange equilibria; in F.C. Nachod,(Ed.), Ion-exchange, Academic Press, New York, NY, 1949. H.F. Walton, An undergraduate course in inorganic preparations, J. Chem. Soc., 27 (1950) 449. H.F. Walton, Ion-exchange, Sci. Am., 183 (1950) 48. H.F. Walton, Ion-exchange: The New Chemistry, (edited by Scientific American), Simon and Schuster, New York, NY, 1958. H.F. Walton, The anode layer in the electrolytic polishing of copper, J. Electrochem. Soc., 97 (1950) 219. H.F. Walton and A.A. Schilt, Ionization constant and rate of hydrolysis of succinimide, J. Am. Chem. Soc., 74 (1952) 4995. H.F. Walton, Principles and Methods of Chemical Analysis, Prentice-Hall, Inc., Englewood Cliffs, NJ, 1952. H.F. Walton, Trace elements: the needle in the haystack, Colo. Quart., 1 (1953) 314. H.F. Walton, Chelation, Sci. Am., 188 (1953) 68. H.F. Walton, Chelation, The New Chemistry (edited by Scientific American), Simon and Schuster, New York, NY, 1958. R.H. Stokes and H.F. Walton, Metal-amine complexes in ion-exchange, J. Am. Chem. Soc., 76 (1954) 3327.

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28. T.D. Waugh, H.F. Walton and J.A. Laswick, Ionization constants of some organomercuric hydroxides and halides, J. Phys. Chem., 59 (1955) 395. 29. H.F. Walton and H.A. Smith, Rapid gravimetric method for determination of mercury in organic compounds, Anal. Chem., 28 (1956) 406. 30. H.F. Walton, Ion-exchangers in organic chemistry; in C. Calmon and T.R.E. Kressman, (Eds.), Ion-exchangers in Organic Biochemistry, Interscience Pub. Inc., New York, NY, 1957. 31. H. Bloom and H.F. Walton, Chemical prospecting, Sci. Am., 197 (1957) 41. 32. H.F. Walton, Elementary Quantitative Analysis, Prentice-Hall, Inc., Englewood Cliffs, NJ, 1958. 33. H.F. Walton, Ion-exchange, Ann. Rev. Phys. Chem., 10 (1959) 123. 34. H.F. Walton and J.M. Martinez, Reactions of mercury (II) with a cation exchange resin, J. Phys. Chem., 63 (1959) 1318. 35. H.F. Walton, Thiocetamide as an analytical reagent, Arapaho Chemicals Inc., Boulder, Colorado, 1959. 36. H.F. Walton, Uranium geochemistry in the Rockies, Chem. Eng. News., 38 (1960) 118. 37. M.G. Suryaraman and H.F. Walton, Heterogeneity of ion-exchange resins, Science, 131 (1960) 118. 38. H.F. Walton, Principles and analytical applications of ion-exchange; in E. Heftmann, (Ed.), Chromatography, Reinhold Publishing Corp., New York, NY, 1961. 39. H. Bloom and H.F. Walton, Geochemical prospecting, Encyclopedia of Chemical Technology, 2nd Suppl. 1960. 40. S.R. Watkins and H.F. Walton, Absorption of amines in cation exchangers, Anal. Chem. Acta, 24 (1961) 334. 41. H.F. Walton, D.E. Jordan, S.R. Samedy and W.N. McKay, Cation exchange equilibria with divalent ions, J. Phys. Chem., 65 (1961) 1477. 42. H.F. Walton, Chemistry, Collier’s Encyclopedia, Ed. 5, Crowell-Collier Publications Co., New York, NY, 1962. 43. Leone Cockerell and H.F. Walton, Metal-amine complexes in ion-exchange. II. 2-aminoethanol and ethylenediamine complexes, J. Phys. Chem., 66 (1962) 75. 44. M.C. Suryaraman and H.F.Walton, Metal-amine complexes in ion-exchange. III. Diamine complexes of silver (I) and nickel (II), J. Phys. Chem., 66 (1962) 78. 45. H.F. Walton, Ion-exchange, in L. Meites, (Ed.), Handbook of Analytical Chemistry, McGraw-Hill, New York, NY, 1963. 46. H.F. Walton, Separations by distillation and evaporation, and ion-exchange methods of Analysis; in F.J. Welcher, (Ed.), Standard Method of Chemical Analysis, Van Nostrand, Princeton, NJ 1963. 47. H.F. Walton and J.J. Latterrell, Metal-amine complexes in ion-exchange. IV. Separation of amines by ligand exchange, Anal. Chem., 1963 (Symposium Volume), 356. 48. H.F. Walton, Ion-exchange chromatography, Anal. Chem., 36 (1964) 51R. 49. H.F. Walton, Undergraduate analysis at the University of Colorado, The Analyzer, Beckman Instruments Corp. (1964). 50. H.F. Walton, Principles and Methods of Chemical Analysis, Ed.2, Prentice-Hall, Inc., Englewood Cliffs, NJ, (1964). 51. H.F. Walton and J.J. Latterell, Separation of amines by ligand exchange, Part II. Anal. Chem. Acta, 32 (1965) 101. 52. H.F. Walton, Ion-exchange in analytical chemistry, J. Chem. Ed., 442 (1965). 53. H.F. Walton, The humanistic values of science, Colorado Quart. XIII 3 (1965) 215, Reprinted in the Colorado Alumnus and Chemistry. 54. H.F. Walton and K. Shimomura, Refractometric column monitoring in ion-exchange chromatography of carboxylic acids, Anal. Chem., 37 (1965) 1012. 55. H.F. Walton, Sister A.G. Hill and R. Sedgeley, Separation of amines by ligand exchange, Part III, Anal. Chem. Acta, 33 (1965) 84. 56. H.F. Walton, Experiments in inorganic paper chromatography, J. Chem. Ed., 42 (1965) 477. 57. H.F. Walton, Ion-exchange chromatography, Anal. Chem., 38 (1966) 79R. 58. H.F. Walton, K. Shimomura and L. Dickson, Separation of amines by ligand exchange. Part IV. Ligand exchange with cellulosic and chelating exchangers, Anal. Chem. Acta, 37 (1967) 102. 59. H.F. Walton and D.T. Ireland, Ionization of diketocyclobutenediol and its metal complexes, J. Phys.

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Chem., 71 (1967) 751. 60. H.F. Walton and H.S. Sherry, The ion-exchange properties of zeolites. II. Ion-exchange in the synthetic zeolite Linde 4A, J. Phys. Chem., 71 (1967) 1475. 61. H.F. Walton, I. Gamalinda and H.S. Sherry, Equilibrium and water uptake in barium-hydrogen and related ion-exchange systems, J. Phys. Chem., 81 (1967) 1622. 62. H.F. Walton, Principles of ion-exchange; in E. Heftmann, (Ed.), Chromatography, Ed. 2, Reinhold Publishing Co., New York, NY, 1967. 63. H.F. Walton, Techniques and applications of ion-exchange chromatography; in E. Heftmann, (Ed.) Chromatography, Ed. 2, Reinhold Publishing Co., New York, NY, 1967. 64. H.F. Walton, Ion-exchange chromatography, Anal. Chem. Ann. Rev., 40 (1968) 51R. 65. H.F. Walton and K. Shimomura, Thin-layer chromatography of amines by ligand exchange, Sep. Sci., 3 (1968) 493. 66. H.F. Walton, Chromatography; in The Encyclopedia Americana, Americana Corp., New York, NY, 1967. 67. H.F. Walton, Chemical analysis; in The Encyclopedia Americana, Americana Corp., New York, NY, 1967. 68. F. Hilgeman, K. Shimomura and H.F. Walton, Ion-exchange chromatography of metal ions with ethanolamine eluents, Sep. Sci., 4 (1969) 111. 69. P.H. Tedesco and H.F. Walton, Metal complexes of ‘squaric acid’ (diketocyclobutenediol) in aqueous solution, Inorg. Chem., 8 (1969) 932. 70. H.F. Walton, Teaching in Peru, Colorado Quart., 17 (1969) 281; Exchange, 4 (1969) 31. 71. H.F. Walton, Ion-exchange, Anal. Chem. Ann. Rev., 42 (1970) 86R. 72. H.F. Walton and W. Riemann III, Ion-exchange in Analytical Chemistry, Pergamon Press, Oxford, UK, 1970. 73. H. Grays and H.F. Walton, Ion-exchange separation of silver and lead, Sep. Sci., 4 (1970) 463. 74. H.F. Walton, The most enjoyable course I ever taught, J. Chem. Ed., 48 (1971) 461. 75. H.F. Walton, Ion-exchange chromatography; in R.E. Wainerdi and E.A. Uken, (Eds.), Modern Methods of Geochemical Analysis, Plenum Press, New York, NY 1971. 76. H.F. Walton, Principios y Me´todos de analisi quimico, Spanish translation of Principles and Methods of Chemical Analysis item no. 50. Trans. by J. Dominquez; publ. by Editorial Reverte´ Me´xicana, 1970. 77. H.F. Walton, Ion-exchange, Anal. Chem., 44 (1972) 256. 78. C.M. de Hernandez and H.F. Walton, Ligand-exchange chromatography of amphetamine drugs, Anal. Chem., 44 (1972) 890. 79. V. Shaw and H.F. Walton, Chromatography of alcohols and sugars on Fe(III)-loaded cation exchange resin, J. Chromatogr., 68 (1972) 267. 80. K. Shimomura, T. Jung Hsu and H.F. Walton, Liquid-exchange chromatography of aziridines and ethanol amines, Anal. Chem., 45 (1973) 501. 81. H.F. Walton, Our universities are not as bad as theirs, Colorado Quart., 22 (1973) 29. 82. H.F. Walton, Analisi chimica, Encyclopedia della Chimica, Vol. I. 83. E. Murgia, P. Richards and H.F. Walton, Liquid chromatography of xanthines, analgesic drugs and coffee, J. Chromatogr., 87 (1973) 523. 84. H.F. Walton, Ligand-exchange chromatography; in J. Marinsky and Y. Marcus, (Eds.), Ion-exchange and Solvent Extraction, Vol. 4, Chapter 2, M. Dekker, Inc., New York, NY, (1973). 85. H.F. Walton, A simple recording titrator, J. Chem. Ed., 50 (1973) 795. 86. P. Larson, E. Murgia, T. Jung Hsu and H.F. Walton, Liquid chromatography of analgesic drugs on ion-exchange resins, Anal. Chem., 45 (1973) 2306. 87. H.F. Walton and J. Reyes, Modern Chemical Analysis and Instrumentation, M. Dekker, Inc., New York, NY, 1973. 88. H.F. Walton, Ion-exchange, Anal. Chem., 46 (1974) 398R. 89. H.F. Walton, Liquid chromatography of organic compounds on ion-exchange resins, J. Chromatogr., 102 (1974) 57. 90. J.D. Navratil, E. Murgia and H.F. Walton, Liquid-exchange chromatography of amino sugars, Anal. Chem., 47 (1975) 122.

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91. E. Murgia and H.F. Walton, Ligand-exchange chromatography of alkaloids, J. Chromatog., 104 (1975) 417. 92. H.F. Walton, Principles of ion-exchange and techniques and applications of ion-exchange chromatography, Chapters 12 and 13; in E. Heftmann (Ed.), Chromatography, Ed. 3, Van Nostrand Reinhold, New York, NY, 1975. 93. H.F. Walton, Ion-exchange analysis in soil chemistry and Ion-exchange analysis in water pollution, Chapter 10 and 15; in R.L. Grob, (Ed.), Chromatographic Analysis of the Environment, M. Dekker, Inc., New York, NY, 1975. 94. J.D. Navratil and H.F. Walton, Ligand-exchange chromatography of diamines and polyamines, Anal. Chem., 47 (1975) 2443. 95. H.F. Walton, Chromatography of non-ionic organic compounds on ion-exchange resins, Sep. Purif. Methods, 4 (1975) 189. 96. H.F. Walton (Ed.), Benchmark papers in analytical chemistry, Vol. 1, Ion-Exchange Chromatography, Dowden, Hutchinson and Ross, Inc., New York, NY 1976. 97. H.F. Walton, Ion-exchange and liquid-column chromatography, Anal. Chem., 48 (1976) 52R. 98. J.D. Navratil and H.F. Walton, Ligand-exchange chromatography, Amer. Lab., Jan. 1976, 69. 99. D.M. Ordemann and H.R. Walton, Liquid chromatography of aromatic hydrocarbons on ion-exchange resins, Anal. Chem., 48 (1976) 1728. 100. T. Hanai and H.F. Walton, Chromatography of chlorinated biphenyls on an ion-exchange resin, Anal. Chem., 49 (1977) 764. 101. T. Hanai and H.F. Walton, Liquid chromatography of chlorinated biphenyls on pyrolytically deposited carbon, Anal. Chem., 49 (1977) 1954. 102. J.D. Navratil and H.F. Walton, Ion-exchange and liquid chromatography, Chemistry, 50 (July 1977) 18. 103. J.D. Navratil, R.E. Sievers and H.F. Walton, Open-pore polyurethane columns for collection and preconcentration of polynuclear aromatic hydrocarbons from water, Anal. Chem., 49 (1977) 2260. 104. R.E. Sievers, R.M. Barkley, G.A. Eiceman, R.H. Shapiro, H.F. Walton, K.J. Kolonko and L.R. Field, Environmental trace analysis of organics in water by glass capillary column chromatography, J. Chromatogr., 142 (1977) 745. 105. H.F. Walton, Ion-exchange and liquid-column chromatography, Anal. Chem., Fundamental Reviews issue, 50 (1978) 36R. 106. T. Hanai, H.F. Walton, J.D. Navratil and D. Warren, Liquid chromatography of polar aromatic compounds on cation exchange resins and porous polymer gels, J. Chromatogr., 155 (1978) 261. 107. H.F. Walton and G.A. Eiceman, Analisis cromatgrafico de compuestos organicos en aguas potables y de desecho, Bull. Chem. Soc. Peru, 44 (1978) 1. 108. W.R. Chappell, C.C. Solomons, H.F. Walton and W.L. Weston, Health effects of consumption of renovated water: Chemistry and cytotoxicity, US Environmental Protection Agency, Report EPA-600=1-79-014, 1979. 109. H.F. Walton and G.A. Eiceman, Trace organic analysis of wastewater by liquid chromatography, National Bur. of Standards Spec. Pub. No. 519; in Trace Organic Analysis: A New Frontier in Analytical Chemistry, 1979. 110. H.F. Walton, Chemical analysis and toxicity of nonvolatile organic compounds in wastewater, Amer. Water Works Assoc., Water Reuse Symp. 1979, p. 2239. 111. G.R. Aiken, E.M. Thurman, R.L. Malcolm and H.F. Walton, Comparison of XAD macroporous resins for the concentration of fulvic acid from aqueous solution, Anal. Chem., 51 (1979) 1799. 112. H.F. Walton, G.A. Eiceman and J.L. Otto, Chromatography of xanthines on ion-exchange resins, J. Chromatogr., 180 (1979) 145–156. 113. K. Aramaki, T. Hanai and H.F. Walton, Liquid chromatography of alkaloids, drugs and nitrogen heterocycles on a porous polymer, Anal. Chem., 52 (1980) 1963. 114. H.F. Walton, Ion-exchange and liquid-column chromatography, Anal. Chem., 52 (1980) 15R. 115. H.F. Walton and J.D. Navratil, Ligand-exchange chromatography, Chap. 5, 65–92; in N.N. Li, (Ed.) Recent Developments in Separation Science, Volume VI (1981), CRC Press, Boca Raton, FL. 116. A. Hernandez and H.F. Walton, Liquid chromatography of chlorinated biphenylols, J. Chromatogr., 242 (1982) 346–348.

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117. J. Otto, C.M. De Hernandez and H.F. Walton, Chromatography of aromatic acids on lanthanum-loaded ion-exchange resins, J. Chromatogr., 247 (1982) 91–101. 118. H.F. Walton, Ion-exchange, article in McGraw-Hill Encyclopedia of Science and Technology, Vol. 5, New York, NY, 1982, pp. 326–330. 119. D.S. Dieter and H.F. Walton, Counter-ion effects in ion-exchange partition chromatography, Anal. Chem., 55 (1983) 2109–2112. 120. H.F. Walton, Ion-exchange chromatography, Chap. 7, in E. Heftmann (Ed.), Chromatography and Electrophoretic Methods, Vol. 1, Elsevier Scientific Publications, Inc., Amsterdam, The Netherlands, 1983, pp. 225–255; J. Chromatog. Library Series. 121. H.F. Walton, Liquid chromatography analysis in water pollution, Chap. 7, in R.L. Grob (Ed.), Chromatographic Analysis of the Environment, Ed. 2, M. Dekker, Inc., New York, NY, 1983, pp. 293–296. 122. H.F. Walton, Ion-exchange methods in environmental analysis, Chapter 15, in R.L. Grob (Ed.), Chromatographic Analysis of the Environment, Ed. 2, M. Dekker, Inc., New York, NY, 1983, pp. 627–650. 123. H.F. Walton, Counter-ion effects in partition chromatography, Sep. Sci. Technol., 19 (1984) 849–855. 124. H.F. Walton, Counter-ion effects in partition chromatography: Carbohydrates, amino acids and hydroxy acids, J. Chromatogr., 332 (1985) 203–209. 125. H.F. Walton, Audio course on ion-exchange chromatography, with tapes and manual, American Chemical Society, Washington, DC, 1987. 126. V.A. Davankov, J.D. Navratil and H.F. Walton, Ligand Exchange Chromatography, CRC Press, Boca Raton, Fl. 1988 (also translated into Russian). 127. H.F. Walton, Solventes para espectroscopia de absorcio´n ato´mica, Boletin de la Soc. Quim. del Peru, 54 (1988) 253. 128. H.F. Walton and R.D. Rocklin, Ion-exchange in Analytical Chemistry, CRC Press, Boca Raton, FL, 1990. 129. H.F. Walton, Chapter 5, Ion-exchange chromatography, in E. Heftmann (Ed.), Chromatography, Ed. 5, Elsevier Publishing Co., Amsterdam, The Netherlands, 1992, pp. A227–A265. 130. H.F. Walton, The Curie–Becquerel story, J. Chem. Ed., 69 (1992) 10–15. 131. H.F. Walton, Sesenta an˜os de desarrollo de la quı´mica analı´tica, (Sixty Years of Development of Analytical Chemistry), Bol. Soc. Quim. Peru, 59 (1993) 215–221.

D.68. Phillip C. Wankat Phillip C. Wankat, born July 11, 1944 in Oak Park, IL. He is the Clifton L. Lovell Distinguished Professor of Chemical Engineering at Purdue University. He was Head of Freshman Engineering from 1987 to 1995, and Interim Director of Continuing Engineering Education in 1996. He has published over 100 technical articles, 16 book chapters, 35 articles on engineering education and 4 books. He earned his B.Sc. in Chemical Engineering from Purdue University in 1966, his Ph.D. in Chemical Engineering from Princeton in 1970, and a M.Sc. in Ed. in Counseling and Personnel Services from Purdue in 1982. He has been faculty member at Purdue since 1970. His research is in the general area of separation techniques including adsorption, chromatography, distillation, extraction, ion exchange, membranes, and simultaneous fermentation and separation. P.C. Wankat is noted for the development of novel processing methods in separations. He pioneered the idea of intensification of adsorption processes and he has been heavily involved in the development of novel simultaneous fermentation=separation processes

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for production of ethanol, butanol and lactic acid from whey. He was Editor-in-Chief of “Separation and Purification Methods” from 1989 to 1997 and is Associate Editor of “Chemical Engineering Education”. P.C. Wankat has won a variety of teaching and counseling awards from Purdue University plus the national teaching and research awards listed below. He frequently presents teaching improvement workshops at national meetings and at universities. He has been most interested in teaching separations, teaching new engineering professors and Ph.D. students how to teach, and developing new teaching methods for graduate and undergraduate students in engineering. Honors and awards American Society for Engineering Education, Chemical Engineering Division, Union Carbide Lectureship Award, 1997; Fellow, American Institute for Chemical Engineers, 1997; American Chemical Society Award in Separations Science and Technology, 1994; Chemical Manufacturer’s Association Catalyst Award, 1993; American Society for Engineering Education Awards: Fellow (1991), Chester F. Carlson (1990), George Westinghouse (1984), Western Electric (1984), Dow Outstanding Young Faculty Award (1980). Major publications ž “Large Scale Adsorption and Chromatography”, two vols., CRC Press, Boca Raton, FL. 1986, 361 pages. ž “Equilibrium-Staged Separations”, Elsevier, NY, 1988, as of Sept. 1992, PrenticeHall; 707 pages plus a separate 252 page Solution Manual. ž “Rate-Controlled Separations”, Elsevier, London, England, 1990; 873 pages plus a separate 134 page “Solution Manual” (1991); now published by Kluwer, Amsterdam. ž “Teaching Engineering” (with F.S. Oreovicz), McGraw-Hill, NY, 1993. See Chapter 5B, a, b, c, g, n

68.I. INTERACTIONS IN RESEARCH ON LARGE-SCALE CHROMATOGRAPHY AND ADSORPTION Phillip C. Wankat School of Chemical Engineering, 1283 CHME Bldg., West LaFayette, IN 47907–1283, USA

I was not trained in chromatography as either an undergraduate or graduate student, which is, unfortunately, typical of the education of chemical engineers. I learned about chromatography as an Assistant Professor from Professor C. Judson King’s textbook “Separation Processes”, and by auditing a course on chemical separations at Purdue taught by Professor L.B. (Buck) Rogers. I later audited this chemical separations course a second time when taught by Professor Fred Regnier, and a third time when taught by

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Professor Peter Kissinger. These were three very different courses, although the title and main topic were the same. My contributions have been at the borders between large-scale (or preparative) chromatography and adsorption. The research has included both theory and experiment. Most of this research has appeared in the chemical engineering literature. I also have presented chromatographic ideas to chemical engineers in books (Wankat, 1986, 1990) [1,2]. My major contribution took ideas from chromatography and applied them to ion exchange and adsorption. These ideas were then reapplied to chromatography. I was long puzzled by why liquid chromatography used very small particles with high pressure drops for analytical systems, but in commercial adsorption and ion exchange separations, large particles with small pressure drops were used. Preparative chromatography was somewhere in-between these two extremes. I thought that the answer would be that the pressure had to be kept low in the commercial processes, but I had not seen a convincing argument. However, since I was sure I would find that the large-scale design was correct, I hesitated to spend the time to explore this question. During 1983, while I was on sabbatical at Laboratoire des Sciences du Genie Chimique (LSGC) and Ecole Nationale Superieure des Industries Chimiques (ENSIC), in Nancy, France at the kind invitation of Dr. Daniel Tondeur, I had time to delve into this question. Before starting, I believed that this would be a somewhat futile exercise, but I wanted to understand the difference. I made a number of simplifying assumptions and within half a day had convinced myself that the analytical chromatographers were essentially correct and the engineers were wrong. Commercial adsorbers, large-scale chromatographs, and ion exchange systems should use small particles packed into short columns using fast cycles. For the initial analysis, I assumed that pore diffusion controlled, and that velocity was kept constant. These are reasonable assumptions for many liquid systems. Under these conditions, it is easy to show that the separation, throughput and pressure drop will all be constant if the ratios L=dp2 , tcycle =L, and tfeed =tcycle are kept constant. Thus, if particle diameter dp is decreased by a factor of 2, the column length L, the cycle time tcycle , and the feed time tfeed should all be decreased by a factor of 4. This simple analysis was used by Wankat (1986) for adsorption, pressure swing adsorption and chromatography. The much more detailed Thomas solution was also used to show that the scaling procedure worked (Wankat, 1986) [1]. Since pore diffusion does not always control, more detailed scaling procedures, which permit changes in velocity, were developed and applied to adsorption, chromatography and ion exchange in a number of papers. Wankat and Koo [3] present a detailed analysis for both linear and nonlinear isocratic elution chromatography. A simplified version of the scaling procedure was included in the textbook by Wankat [2], which also showed that extra column volumes need to be reduced by the same factor as L. These procedures are now classified in the broad category of intensification methods, and a number of other authors have developed similar procedures. Intensification methods have made some inroads in the design of large-scale sorption equipment despite continued resistance by many design engineers. Intensified equipment has less margin for error and must be designed more carefully. Surprisingly, since the ideas

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first came from chromatography, displacement chromatographers have not adopted the use of small particles. The scaling procedures developed for adsorption also work for displacement chromatography [4]. A second area of interest which intertwines adsorption and large-scale chromatography has been the development of new processing schemes. Chemical engineers are taught that the archetype of separation cascade is the continuous, steady state, counter-current system, which contacts two phases flowing counter-currently to each other. This idea is largely based on the tremendous importance of distillation in the chemical and petroleum industries. The engineers and scientists at Universal Oil Products (UOP) realized that flowing solids counter-currently to a fluid without significant axial mixing was extremely difficult. They borrowed the idea of simulating counter-current motion first used in the Shank’s system for leaching and applied it to fractionation by adsorption or chromatography. This simulated moving bed system (SMB) is now a major separation method for the separation of hydrocarbons, sugars, and pharmaceuticals. I borrowed some of these ideas and applied them to chromatography. The first moving feed port ideas (Wankat, 1986) eventually proved to be less useful than they appeared at first. However, moving withdrawal chromatography, a column switching technique, increases the productivity of chromatography columns especially when there are very slow components. The column is built as a series of segments with inlet and outlet ports between each section. Difficult separations are achieved using the full length of the column. Fast and slow components, which separate easily, are removed from the system as soon as they are separated. This drastically reduces the waiting period between the feed pulses, which increases the throughput of feed. The system also reduces the inventory of packing needed and reduces solvent consumption. The initial research on moving withdrawal chromatography was theoretical (e.g., Wankat, 1986) and was then followed by experimental research (Agosto et al., 1989) who also showed that the method can be employed with gradient elution [5]. For many years I have been interested in pressure swing adsorption (PSA), which is a process used on a very large scale for purifying hydrogen, drying compressed air, and separating oxygen and nitrogen from air. My research in chromatography has become intermixed with the PSA research. One of the first efforts was to combine elution chromatography with PSA (Suh et al., 1987) [6]. A pressurized gas consisting of a weakly adsorbed carrier gas and several solutes is fed to the column at high pressure. The solutes are adsorbed which produces pure, high-pressure carrier gas. This carrier gas is then expanded to a low pressure and used to separate the solutes by elution chromatography. Thus, the method can be thought of as a type of gas elution chromatography, which produces its own carrier gas. The solutes are both separated (the chromatography part) and concentrated (the PSA part). The above procedure does separate the solutes, but they are all recovered as dilute streams in carrier gas. There is often a need in industry to separate concentrated gas mixtures. Addition of large amounts of carrier gas is undesirable. For concentrated systems, displacement chromatography is a useful prototype. We first did experiments with gas displacement chromatography (Simms et al., 1996) [7]. Although this procedure worked quite well, the need to add both a nonadsorbed or weakly adsorbed carrier gas and a strongly adsorbed gas displacer would make the method too expensive for

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Fig. 1. Displacement chromatography using components of the feed as the presaturant and the displacer. Regeneration was done by purge=thermal method. Feed: 49.1% methane, 29.2% ethane and 21.7% propane.; presaturant: methane; desorbent: propane; feed time D 8 min, p D 40 psig, T D 23ºC, Union Carbide ˚ as adsorbent. (Simms et al., 1996). Reprinted with permission from Chemical zeolite molecular sieve 5 A Engineering Science. Copyright 1996, Elsevier Science Ltd.

large-scale use. From the previous research in chromatographic PSA, we realized that we could generate the carrier gas from the least adsorbed component in the gas stream. Since theories of displacement chromatography do not prohibit it, we hypothesized that the displacer could be the most strongly adsorbed component in the gas stream. The experimental confirmation of this is shown in Fig. 1 (Simm et al., 1996). In commercial operations, the use of components of the feed for the weakly adsorbed solvent and for the desorbent has the major advantage that no mass separating agents need to be added. It can also sidestep the issue of finding a suitable displacer. This idea can be readily translated to liquid displacement chromatography. Economically removing the strongly adsorbed displacer is under study. Acknowledgements This research was made possible by the diligent work of my graduate students, collaboration with Drs. Daniel Tondeur, Linda Wang and Michael Ladisch, and support from a number of National Science Foundation grants. References 1.

P.C. Wankat, Large Scale Adsorption and Chromatography, Vols. 1 and 2, CRC Press, Boca Raton, FL, 1986. [Out of print. Copies are available from the author].

588 2. 3. 4. 5. 6. 7.

Chapter 5 P.C. Wankat, Rate-Controlled Separations, Kluwer, Amsterdam, 1990, chapters 7 and 8, pp. 288–451. P.C. Wankat and Y.M. Koo, Scaling rules for isocratic elution chromatography, AIChE J., 34 (1988) 1006–1019. P.C. Wankat, Scaling rules and intensification of liquid chromatography: Extension to gradient elution and displacement chromatography, Prep. Chromatogr., 1 (1992) 303–322. M. Agosto, N.H.L. Wang and P.C. Wankat, Moving withdrawal liquid chromatography of amino acids, Ind. Engr. Chem. Res., 28 (1989) 1358–1364. S.S. Suh, G. Netherton and P.C. Wankat; in A.I. Liapis (Eds.), Fundamentals of Adsorption 1987, 527–536 pp. C. Simms, B. Armumgam and P.C. Wankat, Modified displacement chromatography cycles for gas systems, Chem. Engr. Sci., 51 (1996) 701–711.

D.69. Ian David Wilson Ian David Wilson was born in England, in the county of Shropshire, on the 2nd of May 1953. Thanks to the efforts of some inspirational science teachers, he became particularly interested in chemistry and biology and from school went on to the University of Manchester Institute of Science and Technology (UMIST) where he graduated in biochemistry in 1974. This was followed by postgraduate study on the subject of mitochondrial biogenesis in the Department of Biochemistry at UMIST leading to a M.Sc. in 1975. He then undertook a further period of research in the Department of Chemistry at the University of Keele under the direction of E.D. Morgan on the gas chromatography of insect steroid hormones leading to the award of a Ph.D. in 1978. This was followed by a short postdoctoral period at University College Medical School in London on the development of HPLC-based analytical methods for the thiol-containing drug penicillamine. Since then he has worked in the pharmaceutical industry, initially with Hoechst Pharmaceuticals at Milton Keynes and currently AstraZeneca Pharmaceuticals (formerly ICI and then Zeneca) at Alderley Park in Cheshire. He is interested in most aspects of chromatography and is the author and co-author of over 200 publications and edited works in this area. He is an Editor of the Journal of Planar Chromatography and The Encyclopaedia of Analytical Science and a member of the Editorial Advisory Boards of a number of journals including Chromatographia and the Journal of Chromatography (B). I.D. Wilson was awarded the SAC silver medal by the Analytical Division of the Royal Society of Chemistry in 1989, the Silver Jubilee medal of the Chromatographic Society in 1994, and the Analytical Separations Methods medal of the Royal Society of Chemistry for 1996. He was also the winner of the inaugural Desty Memorial lecture, given in London in 1996. A past chairman of the Chromatographic Society, he is currently a Visiting Professor at Sheffield Hallam University and also the Royal Holloway (University of London). In 1999, he was awarded the degree of D.Sc. from the University of Keele. He is interested in all types of separations, but sample preparation, HPLC and TLC

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are particular favorites. Current interests are on the application of HPLC–NMR and HPLC–NMR–MS, HPTLC–MS–MS, solid-phase extraction using molecular imprinted polymers and electrically driven TLC. See Chapter 5B, b, d, h, k, p, r

69.I. THE FASCINATION OF SEPARATIONS Ian David Wilson Drugs Kinetics Group, Safety of Medicines Department, AstraZeneca Pharmaceuticals, Mereside Alderly Park, Macclesfield, Cheshire SK10 4TG, UK

In retrospect, I think that I can trace my interest in separations in general, and chromatography in particular, back to my secondary school where I had the opportunity to explore the wonders of paper chromatography using inks, dyes and exotic substances, such as amino acids — thanks to a Shandon Southern chromatography kit. It must have made quite an impression, as I still have those chromatograms in my possession, carefully filed away in the original ring binder. If I let my mind drift just a little, I can still smell the pyridine that seemed to be the ubiquitous major component of all the solvent systems that we used. This sort of experience decided me on a career in science. On leaving school in 1971, I went to the University of Manchester Institute of science and Technology (UMIST), where, in 1974, I obtained a degree in biochemistry. I stayed on in the Biochemistry Department at UMIST to do research (leading to an M.Sc. in 1975) on the mitochondrial DNA of yeast with Dr. Tony Luha. This led to my next exposure to chromatographic techniques. The purification of the isolated DNA involved long and tedious separations on large columns of Sephadex and homemade hydroxyapatite. This research also led to my first experiences with trying to repeat literature techniques only to find that this is not always as straightforward as perhaps it should be! However, my first real experience of ‘modern’ high-resolution chromatographic methods came when I moved to the University of Keele to work towards a Ph.D. under the direction of Professor E.D. Morgan. This research involved the gas chromatographic analysis of a group of insect developmental hormones, the ecdysteroids, in the desert locust Schistocerca gregaria. Following a lengthy solvent extraction scheme, the analytes, in this case ecdysone and 20-hydroxyecdysone, were silylated to make them volatile. Then, following a short TLC step to remove interferences, the derivatives were analyzed using packed-column gas chromatography on a Pye 104 gas chromatograph equipped with an electron capture detector (ECD). Detection using the ECD was possible because these polar, polyhydroxylated steroids contained, somewhat fortuitously, a conjugated en-one group that allowed them to capture electrons. Colin Poole had developed the method, now Professor Colin Poole, during his doctoral studies and was remarkably sensitive, being capable of detecting ng=g of these hormones. The method was technically demanding and was rapidly supplanted by the then recently introduced technique of HPLC (in its infancy at the time). However, for all its technical difficulty the sensitivity

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Fig. 1. Packed-column gas chromatography with electron capture detection of the silyl ethers of (1) ecdysone and (2) 20-hydroxyecdysone extracted from whole 5th instar larvae of (A) Locusta migratoria and (B) Schistocerca gregaria at the time of the peak concentration of 2. These chromatograms are typical of those used to provide the data given in ref [1]. Redrawn from I.D. Wilson, Ph.D. Thesis, University of Keele, 1979.

of GC–ECD has only recently been equaled by HPLC–MS-based techniques. Using this methodology we were able to follow the changes in the titre of these hormones throughout the life cycle of the insect from egg to adult [1]. Although I finished my doctoral research some 2 decades ago, I have maintained an interest in natural products and the ecdysteroids, and have been very fortunate in being able to continue working with Professor Morgan and his research group ever since. Unfortunately upon completing my doctoral studies, it became increasingly obvious that there was no real market for developmental insect physiologists, even ones that could use a gas chromatograph, and that a change of direction was required. In 1978, I therefore took a postdoctoral position with Professor A.E.M. Mclean in the Laboratory of Toxicology and Pharmacokinetics at University College in London. The aim of the research was to develop an analytical method for the anti-arthritis drug, penicillamine, in body fluids. Penicillamine is a sulfhydryl-containing amino acid (dimethylcysteine). Developing a sensitive and specific analytical technique for this analyte posed a great many challenges not the least of which were chemical instability, lack of a suitable chromophore and potential interference from a range of endogenous materials. In the end we devised a method involving HPLC with post column reaction with the thiol-specific Ellman’s reagent [2]. The method was simple and effective, but not especially sensitive, and electrochemical detection has since proved to be much more suited to this type of work. However, before I was really able to try the method, I was offered the opportunity by Drs. Joseph Chamberlain and Dennis Dell of joining the Analytical Department of Hoechst Pharmaceuticals at Milton Keynes. Given the poor prospects of an academic career at the time, this was too good an opportunity to waste, and I had no hesitation in accepting. It was an eye opening experience and I was (and still am) very impressed with the sheer professionalism of industrial analysts.

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I spent a very happy decade in Hoechst moving latterly to the Drug Metabolism Group, where I learned how to isolate radiolabelled metabolites from the urine and feces of a range of species. It is difficult to describe (and much more difficult to explain) the excitement and satisfaction that follows from painstakingly isolating and purifying a few micrograms of a drug metabolite, of hitherto unknown structure, from a refractory biological matrix, and then see the structure slowly unfold under spectroscopic interrogation. During this time, whilst trying to develop a RP-HPLC method for the separation of an acidic drug and its ester glucuronide metabolite, we, together with a number of other groups, stumbled across the phenomenon of transacylating ester drug glucuronides [3]. Thus, with acidic drugs, a very common metabolic reaction is the production of ester glucuronides. This both inactivates the drug and converts it into a form in which it is more easily excreted. However, under certain conditions of pH, the biosynthetic β-1-D-glucuronides undergo internal rearrangement to give the 2,3 and 4-O-acyl derivatives. As such these metabolites are weakly reactive nucleophiles that can form adducts to protein and have been suggested as the cause of certain types of adverse drug reaction and toxicity. Perhaps surprisingly these isomeric glucuronides can be very well separated by RP-HPLC. Although this phenomenon is now quite well known, this area of drug metabolism continues to provide us with much stimulating research; we attempt to understand the factors that govern the rate, and thus the reactivity of these glucuronides, thus to develop less reactive, and therefore safer, medicines. Whilst at Hoechst, I also acquired a continuing interest in TLC, and particularly the use of reversed-phase and ion-pair TLC. Both of these techniques are now well established, but at the time reversed-phase TLC with bonded layers had only been recently introduced. More recently this interest in planar chromatography has led us to explore the use of TLC, with both on- and off-line MS and MS=MS, and most recently, electrically driven TLC. The strong emphasis on sensitive and specific methods of analysis for drugs and their metabolites in biological samples resulted in an abiding interest in bioanalysis, and is one of the reasons for my involvement in the series of meetings established at the University of Surrey by Dr. Eric Reid, known as ‘The Bioanalytical Forum’. This meeting, in many ways still unique, provides a opportunity for bioanalysts to discuss both the advances and pitfalls of new techniques. Also at Hoechst, I became interested in the then Chromatography Discussion Group, now The Chromatographic Society, through which I have had the opportunity to become much more involved in the world of separation science through attending and helping to organize meetings on a range of topics. From Hoechst, I joined the Pharmaceuticals Division of Imperial Chemical Industries (ICI) in 1986, moving from Milton Keynes to their research site at Alderley Park in Cheshire. Following its demerger from the parent company, ICI Pharmaceuticals became Zeneca Pharmaceuticals, and at the time of writing has just become AstraZeneca Pharmaceuticals. Initially the work for my new employer involved the same sort of investigations of drug metabolism for compounds in development that I had performed at Hoechst. However, over time this evolved into providing metabolic and pharmacokinetic support for the various ongoing drug discovery programs. Currently the work of my unit

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centers on the provision of quantitative HPLC–MS–MS, and qualitative MS and NMR, to support both drug discovery and development. At Zeneca, in addition to continuing my interest in TLC and metabolite isolation, I have become interested in trying to better understand the mechanisms involved in solid-phase extraction (SPE), particularly of basic compounds. Indeed our group was one of the first to point out the likely mixed mode mechanism for the extraction of basic compounds based on an ionic interaction with free silanols [4]. With further work it became clear that this was often a major contributor to the extraction of basic compounds, and as such was a major source of batch to batch variation in silica-based SPE cartridges. More recently, our research in SPE has concentrated on investigating the potential of molecular imprinted polymers (MIPs). MIPs are highly crosslinked polymers made in the presence of the target analyte ‘template’. Removal of the template leaves cavities in the polymer that can then ‘recognize’ the analyte offering the possibility of providing highly specific extractions. The use of such ‘smart’ materials is still at a very early stage in its development, and is not without problems; it represents a very promising area for the development of sample preparation methodologies. As a result of a long-standing collaboration with Professor Jeremy Nicholson of Imperial College on the use of NMR in bioanalysis, we have been active in exploring new applications of the newly emerging technique of HPLC–NMR. Our first attempts at the use of this technique [5] on the identification of ibuprofen metabolites in a crude extract of human urine were remarkably successful, enabling us to do in a few hours work that would previously entailed weeks of effort. Our subsequent experiences have all tended to confirm the initial impression that HPLC–NMR is indeed a very effective, and surprisingly sensitive, tool for metabolite identification. As such it has enabled us, in many instances, to dispense with the need for prior isolation from the biological

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Fig. 2. Examples of the data that result from the type of on-flow HPLC–NMR–MS experiments described in Ref. [6]. The mass spectra in the upper part of the figure are for acetaminophen glucuronide (A), and acetaminophen sulphate (B), which eluted at 7.5 and 8.5 minutes, respectively. The pseudo-two dimensional on-flow HPLC–NMR chromatogram (C) given in the lower part of the figure also shows the presence of hippuric acid and phenylacetylglutamine (13.2 and 13.6 minutes, respectively).

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matrix prior to identification. HPLC–NMR has also proved to be especially useful in the investigation of the study of the transacylating glucuronides discussed above, enabling the identification of the rearrangement products separated on the column, and allowing a detailed study of the kinetics of the various reactions to be performed. More recently, we have taken the opportunity to experiment with systems where HPLC with UV detection is linked simultaneously to both NMR and MS in a single HPLC–NMR–MS (MS) set-up [6]. This combination of separation and spectroscopic detection has also proved to be very fruitful for metabolite and natural product identification. Our latest research in the linking of spectroscopic detectors to liquid chromatography has involved building a prototype HPLC–UV–NMR–MS–IR system to explore the limits to hyphenation. Perhaps not surprisingly these limits have much less to do with linking the instrumentation and a lot to do with an appropriate choice of solvent systems. With the sensitivity of NMR spectrometers constantly increasing, and the development of capillary systems under active investigation by groups in Europe and the USA, the future of this area seems to be assured as an exciting area in which to be working. Overall I have been very lucky in the work that I have chosen to do, and in the colleagues with whom I have been able to collaborate. Whether any of the work that we have performed is of any lasting value is impossible to say, but it was certainly interesting at the time.

References 1. 2.

3. 4.

5.

6.

I.D. Wilson and E.D. Morgan, Variations in ecdysteroid levels in 5th instar larvae of Schistocerca gregaria in gregarious and solitary phases, J. Insect Physiol., 24 (1978) 751–756. D. Beales, R. Finch, A.E.M. Mclean, M. Smith and I.D. Wilson, Determination of penicillamine and other thiols by combined high-performance liquid chromatography and post column reaction with Ellman’s reagent: application to human urine, J. Chromatogr., 226 (1981) 498–503. H.P.A. Illing and I.D. Wilson, pH dependant formation of β-glucuronidase resistant conjugates from the biosynthetic ester glucuronide of Isoxepac, Biochem. Pharmacol., 30 (1981) 3381–3384. R.J. Ruane and I.D. Wilson, The use of C18 bonded silica in the solid-phase extraction of basic drugs — possible role for ionic interactions with residual silanols, J. Pharm. Biomed. Anal., 5 (1987) 7233–7270. M. Spraul, M. Hofmann, P. Dvortsack, J.K. Nicholson and I.D. Wilson, High-performance liquid chromatography coupled to high-field proton nuclear magnetic resonance spectroscopy: Application to the urinary metabolites of ibuprofen, Anal. Chem., 65 (1993) 327–330. J.P. Shockor, S.E. Unger, I.D. Wilson, J.D. Foxall, J.K. Nicholson and J.C. Lindon, Combined HPLC, NMR spectroscopy and ion-trap mass spectrometry with application to the detection and characterization of xenobiotic and endogenous metabolites in human urine, Anal. Chem., 68 (1996) 4431–4435.

D.70. Edward S. Yeung Edward Yeung was born on February 17, 1948 in Hong Kong. He received his A.B. degree in chemistry from Cornell University in 1968 and his Ph.D. in Chemistry

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from the University of California at Berkeley in 1972. Since then, he has been on the chemistry faculty at Iowa State University, where he is currently Distinguished Professor in Liberal Arts and Sciences. His research interests span both spectroscopy and chromatography. He has published in areas such as nonlinear spectroscopy, laser-based detectors for liquid chromatography, capillary electrophoresis, trace-gas monitoring methods, singlecell analysis, DNA sequencing, and data treatment procedures in chemical measurements. He is an Associate Editor of Analytical Chemistry. He served on the Editorial Advisory Board of Progress in Analytical Spectroscopy, Journal of Capillary Electrophoresis, Mikrochimica Acta, Spectrochimica Acta Part A, Journal of Microcolumn Separations, Electrophoresis, and Journal of High Resolution Chromatography. He was awarded an Alfred P. Sloan Fellowship in 1974, and was appointed an Honorary Professor of Zhengzhou University and of Zhongshan University, PRC, in 1983 and in 1995, respectively, and was elected Fellow of the American Association for the Advancement of Science in 1992. He received the ACS Division of Analytical Chemistry Award in Chemical Instrumentation in 1987, R&D 100 Awards in 1989, 1991 and 1997, the Lester W. Strock Award in 1990, the Pittsburgh Analytical Chemistry Award in 1993, the L.S. Palmer Award in 1994, the ACS Fisher Award in Analytical Chemistry in 1994, the Frederick Conference on Capillary Electrophoresis Award in 1997, the Eastern Analytical Symposium Award in 1998, and the National ACS Award in Chromatography in 2002. See Chapter 5B, a, b, f, h, k, l, q, r

70.I. LASER-BASED DETECTION FOR CHROMATOGRAPHY Edward S. Yeung Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, IA 50011, USA

70.I.1. Introduction Analytical chromatography includes separation, detection and measurement. Indeed, the ‘chroma-’ in chromatography was derived from the color of the eluted bands that are visible in the classical glass columns. Work in our laboratory was initially stimulated by the need for more sensitive and more selective detectors for the miniaturized versions of liquid chromatography (LC) [1]. Subsequently, we designed specific separation protocols in order to take advantage of the unique features of certain types of optical detectors [2]. With the advent of capillary electrophoresis (CE) and the onset of the Human Genome Project, detection has been vaulted into the limelight in the development of analytical separations [3].

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70.I.2. Laser-induced fluorescence (LIF) detection The advantage of measuring a small increase in signal on top of a low background in fluorescence, as compared to measuring a small difference between two large signals in absorption, is well known. Fluorescence detection in LC in commercial systems has only shown marginal gains in detectability over absorption detection. This is a result of the reasonable absorption pathlength (1 cm) available in analytical scale LC (4.6 mm i.d. columns) and the inability to efficiently couple conventional fluorescence excitation light sources into a small flow cell. When lasers are used to excite fluorescence, detectability is significantly enhanced due to the increase in signal levels and the reduction in stray light. The enhancement is even more pronounced as one moves towards microcolumn LC (1 mm i.d. or 300 µm i.d.), open-tubular LC (10 µm i.d.), and CE (50–75 µm i.d.), where absorption pathlengths are severely limited. Recent work has shown that tens of molecules injected into a CE column are sufficient to generate a chromatogram, and that even single molecules can be monitored in electrophoresis [4]. In addition to impressive detection limits, LIF also provides additional selectivity. This is because not all absorbing compounds fluoresce. The combination of excitation wavelength and emission wavelength can often bring discrimination. An extreme case of selective detection is that of the resolution of polycyclic aromatic hydrocarbons in crude oil by a combination of one- and two-photon fluorescence detection [5]. As analytical chromatography gained importance in biological and in clinical applications, sensitive detection became the key issue due to the limited sample sizes and low concentrations. One of the most widely practiced separations in the next millennium promises to be DNA sequencing or genetic typing in capillary tubes. The availability of LIF detection is essential to reading at high speeds and in multiple capillaries when only attomole (1018 mole) amounts of each fragment are present per band [3]. Lasers have also breathed new life into known detection modes, such as the native fluorescence of many biomolecules. Such weak emission has previously been thought to be inadequate for practical measurements. However, since chemical derivatization is not needed, one has the opportunity of following biological events in real time and obtaining reliable quantitative information. Studies in single cells have been successful when native LIF is combined with CE or microcolumn LC [3]. These experiments require UV excitation, typically at 275 nm or 305 nm. The large-frame lasers that provide these excitation wavelengths are presently available only in a few analytical research laboratories. Advances in lasers as a result of the growth in communications technology should make these UV lasers accessible in small packages and at low cost for incorporation into the next generation of commercial instruments.

70.I.3. Optical rotation detector Chirality is an interesting molecular property that usually signifies life. A detection mode utilizing this property has broad applications in the pharmaceutical and food industries. Polarimeters are available when sample volumes are large and when concen-

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trations are high. It is however not trivial to adapt commercial polarimeters for detection in LC. We recognized that, analogous to fluorescence detection, detectability is limited by background fluctuations and not by the lack of signal. When light passes through cross polarizers, small optical imperfections in the crystal lead to stray light that can be confused with the signal. To overcome this problem, we selected specific areas of a given calcite crystal that have minimal defects for constructing a sensitive polarimeter. Calcite polarizers are typically 1 cm ð 1 cm in cross section. The use of a laser beam that is 1 mm to 2 mm in diameter facilitates the selection of these ‘sweet spots’ in the crystals. Stray light can be thus reduced by three orders of magnitude. The small beam size also fits well into a miniaturized flow cell for connection to the LC column. The construction of the flow cell was not straightforward. The channel must be as narrow as possible to minimize the detection volume, but must be as long as possible to maximize the signal. We found that a 5–10 cm channel at 1 mm diameter worked well for analytical scale LC. The cell windows are additional sources of stray light because they also contain scattering centers and strain in homogeneities. The simplest solution is to use very thin microscope coverslips attached by using a flexible glue. Additional improvements were possible by modulation at high frequencies, where laser fluctuations are drastically reduced. In the end, we were able to achieve a sensitivity of a few microdegrees in a 10-cm path [6]. The micropolarimeter is not amenable to further miniaturization to interface with e.g., CE. The extremely small peak volumes do not allow sufficient optical pathlength for measurement. Instead, we were able to take advantage of circular dichroism associated with chiral compounds for detection. There, we combined the high sensitivity of LIF and the polarization purity of lasers, to record optical activity as fluorescence-detected circular dichroism. Even pg levels of material can be detected after CE [7].

70.I.4. Indirect detection With the success of achieving unprecedented baseline stability when lasers are used as the light source in various optical detectors, we were able to explore other detection concepts. In indirect detection, the LC eluent (or buffer in CE) produces a large but constant response at the detector. When analytes are present, the background response will be decreased due to displacement or replacement of the eluent components. A stable background signal leads to good detectability of the analyte. We successfully demonstrated indirect detection based on fluorescence, refractive index, absorption, and optical rotation [3]. Since indirect detection is based on properties of the eluent and in general not the analyte, universal response can be expected. This has led to a concept of quantitation without using standards in LC [2]. For unknown samples, e.g., in a processing stream, one can survey the amount of contaminants that are present by this method. The tedious tasks of identifying a contaminant, making up standards and then quantifying it, can thus be limited to only those components that are above a certain threshold. While the concentration detectability of indirect detection is typically worse than

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that associated with direct detection, the mass detectability can be quite impressive. In the study of biological cells, chemical derivatization to label analytes with strong fluorophors is usually difficult because of the small sample volumes. Indirect fluorescence detection has been successfully used to detect simple anions and cations at the low femtomole range for single red blood cells that are 90 fl in volume [3]. Separation was by CE and the instrumentation is identical to that used for LIF detection.

70.I.5. Summary Laser-based detectors have provided substantial improvements in detection power over conventional detectors, sometimes by several orders of magnitude. This opens up entirely novel applications of chromatography, especially in the area of biology. Lasers are not as intimidating as they once were, since even the uninitiated knows about laser printers and laser disc players. They are already being incorporated into commercial instruments, such as CE systems. The DNA sequencer, either in the current slab-gel format or in the up-and-coming multiple CE format, relies on a laser that is not even obvious to the user. We can expect that when diode lasers with suitable wavelengths become available in the future, they will gradually replace the tungsten or mercury light bulbs in standard chromatographic detectors. The added benefits are lower power consumption and higher stability.

References 1. 2. 3. 4. 5. 6. 7.

E.S. Yeung, Advances in optical detectors for micro HPLC, in F.J. Yang, (Ed.), Microbore Column Chromatography, M. Dekker, New York, NY 1988, pp. 117–143. R.E. Synovec and E.S. Yeung, Quantitative analysis without analyte identification by refractive index detection, Anal. Chem., 55 (1983) 1599–1603. E.S. Yeung, Optical detectors for capillary electrophoresis, Adv. Chromatogr., 35 (1995) 1–51. Q. Xue and E.S. Yeung, Differences in the chemical reactivity of individual molecules of an enzyme, Nature, 373 (1995) 681–683. M.J. Sepaniak and E.S. Yeung, High performance liquid chromatographic studies of coal liquids by laser-based detectors, J. Chromatogr., 211 (1981) 95–102. E.S. Yeung, Polarimetric detectors, in E.S. Yeung, (Ed.), Detectors for Liquid Chromatography, J. Wiley and Sons; New York, NY 1986, pp. 204–207. P.L. Christensen and E.S. Yeung, Fluorescence detected circular dichroism for on-column detection in capillary electrophoresis, Anal. Chem., 61 (1989) 1344–1347.

E. SUMMARY: IF MIKHAIL TSWETT WERE ALIVE TODAY Would solitary M.S. Tswett be at home in a chromatography symposium of 500 to 1000 scientists, or a national conference of 10,000 to 30,000 scientists? Would M.S. Tswett like to meet the scientists who have described their research in this chapter, or confer with the Nobel Awardees in Chapters 2 and S-9? We, the Editors, believe his answer would be yes, yes, and yes!

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To contrast the chromatography of the 1900–1930s with that of today — 2001 — as presented in this book, M.S. Tswett would find that chromatography has had: ž An amazing increase in sensitivity, precision, accuracy and quantitation. ž A widespread application to diverse molecules — colored and colorless, small and large, organic and inorganic, ionic and non-polar. ž A strong penetration into many areas of science and technology. ž Contributed to major advances in instrumentation, detection, and use of hyphenated techniques. ž An on going enhancement in automation, computer recording=calculation and interpretation of analytical data. ž Provided a significant variety of research tools in the new areas of genomics and proteomics. ž Contributed to major advances in research, education and training in the separation sciences for universities, institutes and corporations. The research chromatographers know usually all of these features and are contributing daily to the forward movement of this new dicipline of science. Scientists are the drivers with a sense of curiosity, a spirit of inquiry, and a search for understanding, and need public support to explore for answers to complex problems in health, agriculture, and the environment. To summarize, Chromatography and the larger Separation Sciences have undergone a series of major paradigm shifts with significant impact and contributions to the advancement of the sciences.

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Chromatography around the World Charles W. Gehrke a , Robert L. Wixom b and Ernst Bayer c a Department

of Biochemistry and the Experiment Station Chemical Laboratories, College of Agriculture, University of Missouri, Columbia, MO 65212, USA b Department of Biochemistry, University of Missouri, Columbia, MO 65212, USA c Institut fu ¨ r Organische Chemie, Universita¨t Tu¨bingen Research Center, Auf der Morgenstelle 18, 72076 Tu¨bingen, Germany

CONTENTS 6A. Chromatography in Japan A. B.

C.

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Kiyokatsu Jinno . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.1. Research activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Short description of curriculum vitae of Japanese investigators in chromatography . . . . . B.1. Shoji Hara . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.2. Hiroyuki Hatano . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.3. Nobuo Ikekawa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.4. Daido Ishii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.5. Hiroshi Miyazaki . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.6. Tsuneo Okuyama . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.7. Shigeru Terabe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Developments of chromatographic techniques in the last 50 years in Japan — Kiyokatsu Jinno C.1. Present chromatographic societies . . . . . . . . . . . . . . . . . . . . . . C.2. Gas chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . C.3. Liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . C.4. Capillary electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . C.5. Other chromatographic techniques . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6B. Chromatography in Russia in the 20th century A. B. C.

Prominent Russian chromatographers: V.G. Berezkin, V.A. Davankov, B.V. lev, K.I. Sakodynskii, A.A. Zhukhovitskii . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.V. Kiselev and his School . . . . . . . . . . . . . . . . . . . . .

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Chapter 6 A.A. Zhukhovitskii and his School . . . . . . . . . . . . . . . . . . The activities of M.I. Yanovskii . . . . . . . . . . . . . . . . . . . M.S. Vigdergauz and his School . . . . . . . . . . . . . . . . . . . K.I. Sakodynskii and his School . . . . . . . . . . . . . . . . . . . Precipitation chromatography . . . . . . . . . . . . . . . . . . . . Headspace (vapor phase) analysis . . . . . . . . . . . . . . . . . . Activities of V.A. Davankov . . . . . . . . . . . . . . . . . . . . . Biopolymer systems . . . . . . . . . . . . . . . . . . . . . . . . K.1. Critical chromatography of polymers . . . . . . . . . . . . . K.2. Exclusion chromatography . . . . . . . . . . . . . . . . . K.3. Ion chromatography . . . . . . . . . . . . . . . . . . . . Development of planar chromatography . . . . . . . . . . . . . . . . Chemically bonded stationary phases . . . . . . . . . . . . . . . . . Circular chromatography . . . . . . . . . . . . . . . . . . . . . . The activities of V.G. Berezkin . . . . . . . . . . . . . . . . . . . . Industrial on-line analysis . . . . . . . . . . . . . . . . . . . . . . Advances in chromatography detection . . . . . . . . . . . . . . . . Reaction chromato-mass spectrometry . . . . . . . . . . . . . . . . Identification by a combination of GC=MS and the use of retention indices Determination of trace inorganic impurities . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Yukui Zhang and Guowang Xu . . . . . . . . . . . . . . . . . . . . . . Chromatography in China in the 20th century — Yukui Zhang and Guowang Xu B.1. Journals, societies and conferences . . . . . . . . . . . . . . . . B.2. Chromatography-related instruments . . . . . . . . . . . . . . . B.3. Stationary phases and columns . . . . . . . . . . . . . . . . . . B.4. Theoretical research and expert system of chromatography . . . . . B.5. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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6D. Development of chromatography in Latin America A. B. C. D. E. F. G.

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6E. Chromatography in The Netherlands (University of Amsterdam) A. B. C.

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Chromatography at the University of Amsterdam: three-and-a-half decades of discovery — Hans Poppe, Peter J. Schoenmakers, Robert Tijssen . . . . . . . . . . . . . . . . . . . D.1. Historical and personal remarks on the relation of the three University of Amsterdam Awardees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D.2. Present interests: separation of macromolecules . . . . . . . . . . . . . . . . D.3. Discussion on the present research approach . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Bonzai tree.

6A. Chromatography in Japan Kiyokatsu Jinno School of Materials Science, Toyohashi University of Technology, Toyohashi 441-8580, Japan

A. KIYOKATSU JINNO Kiyokatsu Jinno was born on April 4, 1945 in Japan. His current affiliation is Professor and Chair of the Chemometric Center, School of Materials Sciences, Toyohashi University of Technology. He received the B.Sc. and M.Sc. Degrees from Nagoya University in 1968 and 1970, respectively, and a Ph.D. in Engineering and Radioanalytical Chemistry from Nagoya University in 1973. Jinno’s professional experiences have been as a researcher at Toshiba Research and Development Center, a faculty member at Toyohashi University (1978), and as Visiting Professor at the University

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of California, Irvine (1981), University of California, Riverside (1984), Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, and Department of Chemistry, University of Helsinki (1991). His international activities include service as an associate editor and editorial board member of: Journal of Microcolumn Separations (USA); Current Protocols in the Field of Analytical Chemistry (USA); Trends in Analytical Chemistry (The Netherlands); Journal of Chromatographic Science (USA); Journal of High Resolution Chromatography (Germany); Chromatographia (Germany); Journal of Liquid Chromatography and Related Technologies (USA), Polycyclic Aromatic Compounds (USA); Journal of Capillary Electrophoresis (USA); LCžGC (USA); LCžGC International (UK); LCžGC Asia–Pacific; The Analyst and Analytical Communications (UK); Journal of Chromatography A and Analytica Chimica Acta (The Netherlands); Chromatography (Japan); An International Forum for Publication of Research Dealing with Advances in Chromatography, Electrophoresis and Related Separation Methods (Hungary); Analytical Chemistry (1993–1995, USA); Journal of Pharmaceutical and Biomedical Analysis (1993–1996, UK). Jinno is also a committee member of the International Symposium on Capillary Chromatography, IUPAC Commission on Separation Methods in Analytical Chemistry V3 and on the Technical Advisory Board for PharmAnalysis Europe. He is also a member of The Chemical Society of Japan, The Japanese Society for Analytical Chemistry and the American Chemical Society. He has published 240 original research papers, 40 reviews, and chapter contributions and books and made 75 invited presentations at international meetings.

A.1. Research activities (1) Retention mechanism in reversed-phase LC. (2) Retention prediction and computerized system for optimization and peak identification, database construction for LC [15]. (3) Design, synthesis and evaluation of novel stationary phases for liquid phase separations, especially focusing on molecular shape recognition mechanism [16]. Novel phases include orthogonal orientation and parallel orientation to silica gel surfaces such as liquid–crystal phase and C60 fullerene phases [17]. (4) Hyphenated techniques such as LC–FTIR, LC–ICP, SFC–FTIR, SFC–ICP, etc. [18]. (5) Sample preparation with low solvent consumption such as SPME and its hyphenation to LC (SPME–LC) and CE (SPME–CE) [19]. (6) Sample preparation using supercritical fluids such as CO2 and hot water. (7) Instrumentation in capillary liquid phase separations, column technology, detection and injection problems (microcolumn LC, CE, CEC, etc.). (8) Application of above developed methods for environmental analysis, forensic and drug analysis, clinical analysis and other practical applications.

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(9) Quantitative structure–activity relationships for carcinogenic compounds such as polycyclic aromatic hydrocarbons [15]. (10) Other spectroscopic investigations related to the above developments. See Chapter 5B, a, d, e, h, o, p, s

B. SHORT DESCRIPTION OF CURRICULUM VITAE OF JAPANESE INVESTIGATORS IN CHROMATOGRAPHY B.1. Shoji Hara Birth date: Education:

Awards:

January 5, 1927. B.Sc. in Pharmaceutical Science in 1950 from University of Tokyo. Ph.D. in 1960 from University of Tokyo. Positions held: Instructor, Tokyo College of Pharmacy from 1958–1960. Professor, Tokyo College of Pharmacy from 1960 to 1997. Research field: Fundamentals and application of thin-layer chromatography. Fundamental studies on chromatographic retention and its mechanism. Over 200 publications and 30 books on organic and analytical chemistry, especially chromatography. M.S. Tswett, Chromatography Award, 1986. See Chapter 5B, a, b, d, h

B.2. Hiroyuki Hatano Birth date:

September 27, 1924 (deceased on January 25, 1998). Education: B.Sc. in 1947 from Kyoto University. Ph.D. in 1958 from Kyoto University. Positions held: Research Assistant, Kyoto University from 1947 to 1949. Assistant Professor, Kyoto University from 1949 to 1955. Instructor, Kobe University from 1955 to 1958. Instructor, Kyoto University from 1958 to 1963. Professor, Kyoto University from 1963 to 1988.

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Professor, Kanagawa Dental College and Emeritus Professor, Kyoto University from 1988 to 1998. Spectroscopic and spectrometric instrumentation and the coupling with chromatography. Scanning tunneling microscopy and scanning optical fluorescence microscopy. Computer chemometrics. Chemical and biological sensors, the application to biochemistry and biophysical chemistry. Radiation chemistry and biochemistry. Over 450 publications. M.S. Tswett, Chromatography Award, 1982. See Chapter 5B, a, f, h, k, p, s

B.3. Nobuo Ikekawa Birth date: Education:

Positions held:

Research field:

Awards:

October 12, 1926. B.Sc. in Pharmaceutical Science in 1951 from University of Tokyo. Ph.D. in 1959 from University of Tokyo. Research Associate at Institute of Applied Microbiology, University of Tokyo from 1955 to 1961. Lecturer in the above institute from 1961 to 1969. Associate Professor at the Department of Chemistry, Tokyo Institute of Technology

from 1969 to 1981. Professor in the above department from 1981 to 1986. President of Niigata College of Pharmacy from 1986 to present. Biological substances such as steroids and vitamins. GC, LC and GC–MS for such substances. Over 200 publications. M.S. Tswett, Chromatography Award, 1982. See Chapter 5B, a, d, h, k, p, r

B.4. Daido Ishii Birth date: Education:

November 23, 1926. B. Eng. in 1950 from Nagoya University. Ph.D. in 1962 from Nagoya University.

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Positions held:

Awards:

Research Associate, Nagoya University from 1950 to 1962. Associate Professor, Nagoya University from 1962 to 1965. Professor, Nagoya University from 1965 to 1990. Professor, Kumamoto Institute of Technology and Emeritus Professor, Nagoya University from 1990 to present. Research field: Development of microcolumns for LC. New detection systems for LC and downsizing of flow analysis systems unified chromatography. Over 200 publications. M.S. Tswett, Chromatography Award, 1987. M.J.E. Golay, 1990. See Chapter 5B, a, d, f, h, l, o

B.5. Hiroshi Miyazaki Birth date: Education:

Research field:

Awards:

June 1, 1929. B.Sc. in Pharmaceutical Science in 1950 from Tokyo College of Pharmacy. Ph.D. in 1976 from Tohoku University. Positions held: Lecturer at the Pharmaceutical Institute, Tohoku University from 1974 to 1978. Lecturer at the Science Institute, Tokyo Metropolitan University from 1978 to 1979. Vice Director of the research laboratories of the pharmaceutical division, Nippon Kayaku Co., Ltd. from 1982 to 1983. General manager in the above company from 1983 to 1984. Director of the corporate planning office in the above company from 1984 to 1988. Professor of School of Medicine, Showa University from 1988 to 1992. Professor of Niigata College of Pharmacy from 1997 to present. Gas chromatography, mass spectrometry, capillary electrophoresis and isotachophoresis for the analysis of biologically important substances. Over 150 publications. M.S. Tswett, Chromatography Award, 1986. See Chapter 5B, a, b, d, h, e

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B.6. Tsuneo Okuyama Birth date: Education:

Research field:

Awards:

May 10, 1928. B.S. in 1953 from Osaka University. Ph.D. in 1959 from Osaka University. Positions held: Instructor, Tokyo Metropolitan University from 1958 to 1959. Assistant Professor, Tokyo Metropolitan University from 1959 to 1973. Professor, Tokyo Metropolitan University from 1973 to 1991. Professor, Tokyo Dental College and Emeritus Professor, Tokyo. Metropolitan University from 1991 to present. President, Protein Technos Institute from 1992 to present. Development of two-dimensional electrophoresis and its applications for the analysis of plasma proteins. The use of isotachophoresis, isoelectric focusing and HPLC in the proteins and peptides analysis. Over 250 publications and a number of books. M.S. Tswett, Chromatography Award, 1988. See Chapter 5B, a, h, l, r

B.7. Shigeru Terabe Birth date: Education:

Research field:

Awards:

October 21, 1940. B. Eng. in 1963 from Kyoto University. M. Eng. in 1965 from Kyoto University. Ph.D. in 1973 from Kyoto University. Positions held: Research Associate at Faculty of Engineering, Kyoto University from 1978 to 1984. Associate Professor in the above department from 1984 to 1990. Professor at the Faculty of Science, Himeji Institute of Technology from 1990 to present. Dean of the Faculty of Science in the above institute from 1998 to present. Development of high-resolution separation methods with special emphasis on capillary electrophoresis including micellar electrokinetic chromatography (MEKC). Over 170 publications and 15 books or book chapters. A.J.P. Martin, 1995. M.J.E. Golay, 1999. See Chapter 5B, a, d, h, k, l

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C. DEVELOPMENTS OF CHROMATOGRAPHIC TECHNIQUES IN THE LAST 50 YEARS IN JAPAN — KIYOKATSU JINNO

As major developments of chromatographic techniques have been made in the US and in European countries in the analytical chemistry community, many Japanese scientists in different research fields had also studied these powerful separation techniques and the Japanese successful history in chromatography has been made by their pioneering research efforts. At this moment, we have several scientific societies that are devoted to chromatography and related separation sciences; the history of these societies can show the whole history of Japanese activities and contributions to the development of chromatographic techniques. Therefore this chapter will first describe the present situation of Japanese chromatographic societies and then go into the details of the history of the developments in chromatography in Japan.

C.1. Present chromatographic societies Japan has several major societies in which chemistry is focused. The largest and the core one is The Chemical Society of Japan (CSJ). However, the structure of this society is somewhat different from that of the American Chemical Society (ACS). ACS has over 30 divisions in which the many different fields of chemistry are focused and discussed. CSJ does not have such a clear divisional structure and this produces the existence of many other smaller societies in which the divisional targets can be discussed and focused. The Japan Society for Analytical Chemistry (JSAC) is one that is centered on analytical chemistry. As Japanese believe that chromatography is one of the techniques in instrumental analytical chemistry, this society has the right to organize scientific meetings to discuss chromatographic techniques. They have several discussion groups under the JSAC structure and for chromatographic and separation sciences they have three main discussion groups: Discussion Group on Gas Chromatography (GC Kondankai), Discussion Group on Liquid Chromatography (LC Kondankai), and Discussion Group on Electrophoresis (Denkieido Kondankai); all are active to progress the technological developments in these separation science fields. Kondankai means in Japanese a discussion meeting or group. In addition to these discussion groups under the JSAC organization centralized in Tokyo, we have had also other societies just focusing on chromatographic separation sciences, which were started by several great scientists who had their interests in chromatography in different local areas, such as the Research Group on Automatic Liquid Chromatography in Japan (Ekikuro Kenkyukai) and the HPLC Discussion Group (HPLC Danwakai) in Nagoya. About ten years ago a new society centering only on chromatography was started, which is the Society for Chromatographic Sciences (Kuromato Kagakukai). At present, this one is the core society for separation sciences in Japan. To explain the Japanese history in chromatography, the above history of each society was mentioned. In addition, the contributions of the leading scientists who are also international award winners will next be introduced. To make a clear organization in

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this chapter, chromatographic and separation techniques are divided as GC, LC, CE and other techniques. See Chapter 5B, b, d, e, f, g, i, m, p, q, s. Expertise for group

C.2. Gas chromatography Just five years after James and Martin introduced the partition concept into gas chromatography in 1952 [1], the first GC instrument made in Japan was commercially introduced by Shimadzu Scientific Instruments Company in 1957 under the model name GC-1A. At nearly the same time, Hitachi, which is another major company in Japan in analytical instrumentation, had also started to commercialize the KGL-1 gas chromatograph for the Japanese market. The GC-1A had a TCD (thermal conductivity detector) available at that time. Many conventional gas chromatographs were commercially introduced after these two systems and instruments with multi-step temperature programming were marketed in 1963. The Shimadzu GC-1C was a unique instrument, since it had multi-functions such as four detectors, automatic fraction collection, a pyrolysis device and a pre-cut device. In the 1960s, new highly sensitive detection systems became commercially available as the FID (flame ionization detector), AID (argon ionization detector) and ECD (electron capture detector). Without any time lag from the modern worldwide developments in GC, the instrumentation from Shimadzu had traced the world trends very well; in 1959, they already had a FID system and even FPD (flame photometric detector) in 1971. Especially ECD and FPD were very useful for the analysis of pesticides such as PCBs and other environmentally hazardous compounds. After those inventions and developments, the gas chromatographs became much smaller in size, with much higher performance to analyze many samples simultaneously and computerized as it is today. Several Japanese scientists started to use Shimadzu and Hitachi GC instruments after their introduction and at the same time these scientists realized the need to organize small meetings to introduce this useful technique to other people, and to discuss with each other the results from their research works, the recent trends in this technique and the future of this technique. This was the origin of The GC Discussion Group in JSAC that started in September, 1958, by several key persons who were working in the Tokyo Industrial Institute. This group has been continuously organizing the meetings and seminars on GC technology from that time for over 40 years; they celebrated the 200th discussion meeting in 1995 in Tokyo. In this GC Discussion Group, the GC Data Committee (GCDC) was organized to collect data and to make the database for GC analysis — the work continues to the present. With the development of electronic devices and other related technology, the GC systems have also changed very much as compared to the original model GC-1A. However, the basic concept has not changed and the applicability of this technique has expanded with time. Fig. 6A.1 shows the photo of the most recent group members in this discussion group who are active in GC technology in Japan. These people are from both academic life and the industry, and they are eager to promote GC technology by organizing seminars,

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Fig. 6A.1. Current members of GC Discussion Group in JSAC.

discussion meetings and also short courses for the practice of GC instrumentation with the help of JSAC. Very recently, the demand for this discussion group has increased due to environmental concerns, especially focusing on the analysis of dioxin and PCB-related compounds, which would produce unexpected biological functions like hormonal dysfunction in the human body. Since GC and GC–MS are the most powerful and promising tools to carry out research on these compounds, this discussion group has organized many meetings with invited speakers from different fields as environmental sciences. International invited speakers are also present in their regular meetings and made this group’s potential high. Drinking water control is another important issue in the Japanese communities and the purge and trap method with GC is believed to be the best method to analyze water samples. The basic ideas on the regulation for water quality control have been discussed and this group made some decisions. Leaders from academic life and industry have supported efforts to encourage others in scientific and practical ways during these developments in order to advance Japanese GC technology. Nobuo Ikekawa, who was in the Chemistry Department of Tokyo Institute of Technology and now the President of Niigata College of Pharmacy, is one of many outstanding academic scientists. He studied GC separation techniques and hyphenation between GC and MS for biological materials as vitamins, steroids and hormones. His scientific establishments were of very high standard and promoted the Japanese GC community to become an important part in the world in chromatographic research and development. However, his contribution to the Japanese GC community was even more important by organizing twice major international symposia on chromatography in 1982 in Tokyo, and in 1986 in Chiba, which were part of the Symposia Series on Advances in Chromatography. Those two symposia held in Japan permitted many Japanese chromatographers to meet and talk with many foreign scientists. This excitement then ignited further efforts of Japanese chromatographers to go international by improving on their ways of English communication and making presentations at other symposia held in various countries. From this standpoint, Ikekawa’s contribution to the Japanese GC community should be highly appreciated. To enhance his efforts in organizing the international meetings,

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some other people have assisted him greatly. One of them is Hiroshi Miyazaki who was a scientist at Nippon Kayaku, which is a pharmaceutical company. He is now Professor at Niigata College of Pharmacy. He learned the basic concept of hyphenated GC techniques, especially GC–MS, from E.C. Horning at Baylor University in Houston, Texas, and carried out outstanding work in this field. These two scientists, who are both Tswett Award winners, had a key role in the Japanese GC community. Their pioneering works and efforts have made the community mature and dynamic and the GC community in Japan is now one of the groups leading the world-trends in this field.

C.3. Liquid chromatography Within the population of Japanese scientists in separation sciences, one may find that the number of people who are working in the field of LC is higher than in GC. This ratio between GC and LC is a little different from those numbers in other countries in the world. Japanese chromatographers like LC more than GC, and research and development in LC technology are more popular than those in GC are. Every year we have an annual meeting organized by JSAC and many scientific papers are presented. The number of presentations in LC is always larger than in GC, and the number of participants in each lecture room is also so much different; it is always larger for the LC lectures than that for the GC lectures. Naturally, if you think that the applicability of LC covers much larger areas than GC, then the number of researchers involved in this field should be larger than in GC. However, in a number of other countries, in the US and in Europe, this trend is not true, and where GC technology seems to be stronger and much more popular than the LC technology. However, we have to look at the Japanese LC history in order to understand this situation. The first LC research in Japan was conducted by Hiroyuki Hatano, who was at the Chemistry Department of Kyoto University in the 1950s. In 1959, he cooperated with people from Hitachi to produce the automatic amino acid analyzer Hitachi KLA-2 based on the LC technique [2]. This was the first Japanese instrument which had the function of liquid chromatography and it was followed by the first liquid chromatograph model, the KLF-1 [3]. Then, in 1960, Hatano organized a research group on automatic liquid chromatography in Japan (Ekikuro Kenkyukai), and after the first meeting, he held the meetings twice yearly until his retirement from Kyoto University in 1988: one in the summer in the form of a seminar and another in the winter as the place for presentations of scientific papers. Many inventions and technical developments made by the Japanese in LC and related separation science fields were first presented and published at this winter meeting series. Such inventions include microcolumn LC technology, coined by Daido Ishii, who was at the Department of Applied Chemistry, Nagoya University (he is now Professor at the Kumamoto Institute of Technology), and micellar electrokinetic chromatography (MEKC) proposed by Shigeru Terabe, who was working at Kyoto University and now is in the Chemistry Department of the Himeji Institute of Technology. Some other worldwide, well-known chromatographers have established their research using the knowledge provided in this series of meetings.

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The contributions of Hatano to the Japanese LC community is very significant and widespread. He also organized several international symposia on liquid chromatography and chromatographic techniques in Japan. Naturally, Hatano was also assisted by many young promising scientists in this field in his efforts to organize these meetings, who were introduced by him to carry out research activities. Hatano was the first person to combine the LC separation technique directly with the ESR (electron spin-resonance) method and with ion mobility spectroscopic detection in chromatography, the so-called plasma chromatography technique, with the collaboration of Herb Hill of the Department of Chemistry, Washington State University [5]. Karasek from the University of Waterloo, Canada, and Hatano were pioneers in calling the attention to the scientific community on the environmental impact of dioxin and PCB-related compounds. In this field, Hatano organized the first international meeting on dioxin and PCB in 1994. Unfortunately, the Japanese community lost him on January of 1998. The photo shown in Fig. 6A.2 was made in December 1998, in Kyoto, when a small memorial party for Hatano was held. Over 100 people attended this memorial. The most important contribution to the worldwide LC community by a Japanese scientist is primarily the invention of microcolumn LC by Daido Ishii in 1976 [6], actually the first information was made in the JASCO Report in Japanese, which is the information magazine issued by the Japan Spectroscopic Instruments Company, JASCO [7]. He proposed the usefulness of microbore packed columns for LC separations, in which he used a packed 0.5 mm i.d. Teflon tubing as the column material. Almost simultaneously, Scott and Kucera in the US had also proposed 1 mm i.d. microbore packed columns in 1976 [8]. After the first proposal, Ishii, whose achievements were recognized with the Tswett and Golay Awards (Chapter 2), carried out extensive studies to promote this technique with much smaller diameter, open-tubular columns, hyphenated with MS, especially using FAB (fast atom bombardment) ionization and FTIR, and other related developments. His efforts had a large impact on the LC world and Japan became an important country to be watched for their scientific contributions to microcolumn technology. Instrumentation for microcolumn LC had also been made. JASCO first exhibited their Familic-100 microcolumn LC system at the Pittsburgh Conference in 1979. Ishii then started to organize an HPLC discussion group in Nagoya until his retirement from Nagoya University in 1990 to promote his methods to other applications. In order to expand such an exotic separation technique to practical problems and to carry out research works in more basic matters, he organized international meetings in addition to presenting his achievements at many international meetings held in other countries. The most important such meeting was the Japan–US joint seminar held in the East–West Center of the University of Hawaii in August 1982. This seminar was financially supported by JSPS (Japan Society for the Promotion of Sciences) on the Japanese side and NSF (National Science Foundation) on the USA side. The organizer from the US was Milos Novotny, of the Department of Chemistry of Indiana University, who is also a pioneer in microcolumn LC techniques. The memorial picture shown in Fig. 6A.3 was taken at that seminar. You can find many dear faces in this picture who were from Japan, the US and several other countries. The papers presented at this Hawaii meeting have been collected in book form that was published by Elsevier [9]. This meeting has contributed tremendously to the

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Fig. 6A.2. Picture taken at Professor Hatano’s memorial party on December 12, 1998 in Kyoto.

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Fig. 6A.3. Memorial picture taken at University of Hawaii in August, 1982, when the US–Japan joint seminar on microcolumn separations was held.

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further developments of microcolumn LC and miniaturization in separation techniques in the whole world. If someone should ask for the most contributory meeting to the chromatographic community, this Japan–US joint seminar should be cited as one of few such meetings in the 20th century. An amazing fact is that computer-chip technology is now permitting separation systems based on such miniaturization. Ishii has also organized international symposia in Japan. The first was held in 1986; this was the 7th International Symposium on Capillary Chromatography as part of the symposium series held until then at Hindelang and Riva del Garda; it was the first time this symposium series moved out of Europe. Again in 1990, he organized the 12th symposium, in Kobe, Japan. These symposia made the Japanese chromatographic community realize the importance of miniaturization in separation systems. Ishii also proposed the unified chromatography approach in which GC, LC and SFC (supercritical fluid chromatography) should be done with one single analytical instrument which permits one to change the column temperature from very low to high, at the same time changing the pressure to the column [10]. Several other key scientists had an important role in the development of Japanese LC technology in the last 30 years. Shoji Hara, who had been at the Tokyo College of Pharmacy, has worked in the field of pharmaceutical separation and also carried out many basic studies of LC separations. Before entering the LC field, he was active in TLC (thin layer chromatography), developing a number of different types of adsorbents and solvent systems for TLC and extended the science to LC applications [11]. Hara studied the basic retention mechanism between the stationary phase, the mobile phase solvents, and the solutes in molecular-level interactions and found that such a concept could apply to the separation of chiral isomers. He succeeded in synthesizing novel stationary phases which can separate stereoisomers and he also used computers to interpret the retention mechanism. The most modern approach in separation sciences had already been started by his research group about 20 years earlier; it is amazing how he could foresee the future scientific trends. Tsuneo Okuyama, at the Chemistry Department of Tokyo Metropolitan University, is also such a person, who carried out ‘cutting edge’ chromatographic research and development. His major field is biochemistry and his research was mainly the structure of proteins and their functions. In order to resolve the structure of proteins that have unique functions in biological systems, he used LC and electrophoretic techniques such as the slab-gel method. Naturally, we have nowadays the most useful high-resolution separation techniques based on electrophoresis as CE (capillary electrophoresis), which he also studied for a long time. He was the key person in the period of 1980 to 1990 in the Japanese LC community. He was the chairman of the LC Discussion Group in JSAC and just after the 1989 International Symposium on Chromatography held by that organization in Tokyo, he realized the need to establish one major Japanese society in chromatography and separation sciences. Okuyama had tried to make a unified society from the original LC discussion group in JSAC, the HPLC discussion group in Nagoya, and the LC discussion group in Kyoto, and finally very recently his successor was able to unify several LC societies into the Society for Chromatographic Sciences. Therefore, Okuyama’s contributions to the Japanese LC community is a harmonic unification of the independent, competitive societies, in addition to his scientific achievements for which

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he was recognized by the Tswett Award at the International Symposium on Advances in Chromatography, held in Minneapolis in 1988. The Society for Chromatographic Sciences now has over 700 members and its journal, Chromatography, contains original papers and conference abstracts. This society will continue to encourage research and development in chromatographic areas especially focusing on LC, but also with emphasis on GC and other chromatographic techniques.

C.4. Capillary electrophoresis After the Japan–US joint seminar on microcolumn separation techniques in Hawaii in 1982, Hatano organized a post-conference meeting in Kyoto in a setting of this beautiful Japanese old-capital city. Naturally, visitors would like to sightsee Kyoto, especially the old temples and ancient architectures, but from the old time, Kyoto has always been the center of Japanese culture and the scientific level is also quite high. To introduce such an environment in Kyoto, Hatano brought several people from Honolulu to Kyoto by plane and held a small meeting on microcolumn separations again there for many Japanese attendees. From several outstanding separation scientists from the USA who joined in this post-conference meeting, J. Jorgensen was also present for the first time. His talk on his latest achievements on capillary zone electrophoresis (CZE) resulted in great excitement by the audience in Kyoto. His results clearly indicated that CZE could give very high theoretical plate numbers in shorter or comparable analysis time to LC [12]. It seemed to be the very powerful separation technique for the near future, and LC could be replaced by CZE for non-volatile organic compounds that cannot be analyzed by GC. However, CZE can only analyze charged compounds since in CZE migration of the solutes is made by electrophoretic mobility in addition to the electroosmotic mobility. As common sense would say, it was considered that many neutral uncharged compounds would not be suitable as the probe for the separation. The Japanese audience in Kyoto included a young professor who was intending to improve the low resolution of LC to be of higher efficiency, such as for GC. This was Shigeru Terabe. He found the time to meet and discuss the electromigration separation techniques with Jorgensen, who was the pioneer of the CZE technique at that time. In their conversation Terabe briefly mentioned the possibility to separate uncharged, neutral compounds with a very similar approach to CZE; Jim Jorgensen seemed to be shocked about what Terabe told him because up to then no one considered such a possibility in CZE. The technique he mentioned is ‘micellar electrokinetic capillary chromatography’ (MEKC or MECC), which later became a powerful and popular analytical technique in electromigration methods [13]. Such an opportunity to discuss MEKC and CZE between Jorgensen and Terabe helped to decide the future of the electromigration separation methods. However, an important matter that should be mentioned here is the opportunity given by Hatano’s idea to organize the post-conference after the Hawaii meeting. Although the MEKC method has been invented and developed by Terabe and his colleagues at Kyoto University [7], more advancements had been made by several US scientists such as Barry Karger at Northeastern University in Boston. Karger’s research was enhanced

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by Terabe, who spent several months in his laboratory in 1985. The opportunity for Karger and Terabe to meet was made by Hatano when he organized an International Symposium on Chromatography in Kyoto in January 1985. The meeting was held in the northern part of Kyoto City where there was a heavy snowfall in the morning of the second day, and the scenery there was beautiful. Karger invited Terabe to the Barnett Institute in Boston for that summer in order to advance the activity in CE of his research group. Terabe collaborated with Karger and a new postdoctoral fellow, Aharon Cohen. The founder of the symposia series, called International Symposium on High-Performance Capillary Electrophoresis (HPCE), is Karger, and the reason why he has such a prominent position is that he has an excellent eye to see the future trends in CE technology and he could bring Terabe and other outstanding scientists to his laboratory in Boston to further develop the technique. Capillary gel electrophoresis was one such method made by his scientific talent. In the early 80s Terabe had been thinking on how to improve the efficiency of LC to be equal to that of GC, and he found the unique approach proposed by Ishii by microcolumn LC but with open-tubular columns. However, according to the theoretical calculations, LC requires the column inner diameter to be comparable to the particle size of the packing materials, about 3–5 µm. Such small diameter open-tubular capillary columns were not considered to be useful for LC separations, although Jorgensen’s group and Ishii’s group were continuing the developments. Changing the basic approach from LC to CZE, Terabe had the idea of a micelle from the article about micellar solubilization phenomena written by a retired Japanese colloid chemist; Jim Jorgensen’s paper on CZE in Analytical Chemistry [12] prompted him to apply such a micelle technique to CZE [13]. He found that MEKC separation with a 50 µm i.d. fused-silica capillary column for phenolic compounds gave 200,000 plates with sodium dodecyl sulfate (SDS) solution in 1982. After this invention, many scientists applied this MEKC approach to practical analytical problems and basic studies. CZE and MEKC now compose the separation technique called CE (capillary electrophoresis). Terabe’s invention of MEKC produced a new branch of chromatography, CEC (capillary electrochromatography), since under the high voltage one can expect chromatographic interactions between the sample solutes and some stationary phase-like materials, such as micelles in MEKC and bonded phases in CEC. Based on this discovery, Terabe was awarded the 1994 A.J.P. Martin gold medal by the Chromatography Society in the UK. In Japan when Jorgensen and Terabe introduced the capillary technique in electrophoresis, many chemists and biochemists were using slab-gel electrophoresis and also isotachophoresis (ITP). However, this invention led many to move into the capillary field, especially using a fused-silica type narrow-bore capillary tube as the separation column under applied high voltage. CE instruments imported from the US and Europe and home made set-ups have produced a number of new, original scientific achievements in this field by the Japanese scientists. The discussion group on electrophoresis in JSAC has been organizing an annual meeting on this topic and many papers have been presented. The 10th International Symposium on Capillary Electrophoresis was held in Kyoto in July 1996, with many scientists from abroad attending the conference. Such an occasion led also the Japanese CE people to be more productive to enhance the capability of this exotic separation technique. In addition to these contributions of

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Terabe in the developments on MEKC, he also introduced a low-cost power supply system useful for the CE experiments by many academic people who did not have enough budgets to do such experiments in their laboratory. The power supply is a Matsusada Precision HCZE-30, which can apply up to 30 kV to the capillary with a cost of JPY 300,000 (about $2500) and has enough capability to permit many basic experiments without using CE instruments valued up to $40,000 to $60,000. This practical type of contribution is also highly acknowledged in the advancements of CE research, since many scientists could work in this field using low capital consumption.

C.5. Other chromatographic techniques Other chromatographic techniques that should be mentioned here are SFC and TLC. When capillary SFC was introduced by Milos V. Novotny and Milton L. Lee at Indiana University in the late 70s [14], it was believed that other chromatographic techniques such as GC and LC would be replaced by SFC, since SFC was considered to be so powerful as to give high resolution and faster analysis time without using expensive carriers (expensive gases or solvents). However, investigators very soon realized that this dream is a dream, since many limitations of SFC have been found when applied to practical analytical problems. It is obvious that SFC is a complementary technique to GC and LC and it is appropriate to choose the suitable method to solve each analytical problem. One cannot say which chromatographic technique is the most powerful and the best; one can only say which chromatographic technique is the most suitable to solve a particular analytical problem. In Japan SFC has also been developed just as in the USA and Europe, but mainly by industrial guidance. JASCO is one of the companies which produce and market SFE=SFC instruments at this time; historically they had started in the supercritical fluid business before any other companies in the world, except Lee Scientific Co. in Utah, which was founded by Milton Lee of Brigham Young University. While Lee Scientific used capillary SFC combined with SFE, and used CO2 based supercritical fluids, JASCO started their own project in the SFC=SFE field by conventional LC columns (4.6 mm i.d.) as the separation medium, so-called packed column SFC, and relatively large-scale extraction processes using a CO2 based medium. The photodiode array detection system used in HPLC was first applied to the detection in SFC by JASCO and they are still producing and selling the system JASCO SFE Super-200, the successor of the original Super-100. It is an interesting story that the photodiode array detector for SFC by JASCO was a discrete type and had 32 diodes on the chip rather than the typical CCD type diode array detector. After JASCO started in the SFE=SFC business, JEOL, which is another spectroscopic instrument company for mass spectrometers and NMR spectrometers, also started in the field of SFE=SFC. JEOL started to develop a new cascade pumping system that was amenable to supply the mobile phase in microliters per minute to packed columns as the separation medium, different from JASCO’s approach; this pumping system worked very well. However, the cost of the pumping system was not suitable for the typical users who wanted to use that system in SFC and thus for economical reasons, JEOL cut the further development on SFC. At that time, JEOL had studied the possibility of FFF (Field Flow Fractionation),

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which had been proposed by Calvin Giddings of the University of Utah, but the development was interrupted because of their low profitability in the SFC business. It is also interesting to note that although Hitachi carried out some investigations on SFC in the 1960s before Lee’s results had been publicized, they too, did not have any interest in this separation technique. Another large instrumentation company, Shimadzu, had an interest in the SFE=SFC business and had at one time tried to sell in Japan the instrument made by Suprex (Pittsburgh, PA), but very soon they also discontinued any business in SFE=SFC. The reason may be caused by the low activity and poor potential of SFC in separation sciences as compared to GC and LC. It is also interesting that Shimadzu does not market any CE instruments, although they were involved earlier in isotachophoresis. Maybe Shimadzu had some foresight to see the near future and they did not want to spend time and money on techniques that would not be fruitful in analytical instrumentation. In this regard, we may think that the CE business will not have any fruitful time in the future. The author knows that Shimadzu has placed considerable effort in developing a chip-type CE system; this may be the way for the CE technology in the near future as many people have been proposing this possibility as the micro-total analytical systems (µTAS). Anyway, in other chromatographic techniques, except GC and LC, we do not have valuable development and advancement in Japan. TLC is the easiest chromatographic technique, which does not require expensive instrumentation. Hara has been active in this field, developing new stationary phases which can separate and give resolution for chiral isomers based on his idea of molecular interaction in the chromatographic separation process. TLC is useful for this type of investigation because it is easy to use, evaluate and change the separation conditions for optimization of separations. After the evaluation was completed by TLC, Hara changed direction to stationary phases and the concept to LC separations using conventional size LC columns and preparative scale separations. He also tried to use microbore columns for chiral separations. TLC is still a popular method in the organic synthesis laboratory, where small size plates are used to monitor the reaction processes and check the products before making precise measurements by IR and NMR used for confirmation. Therefore, though only limited technical and scientific developments have been carried out in TLC, the method is still being used very widely, together with the other chromatographic techniques.

D. CONCLUSIONS In this summary, we have concentrated on the developments in the last 20 years and dealt only briefly with the early chromatographic development in Japan. This discussion emphasized particularly the activities of those Japanese scientists, who were recognized by bestowing on them several international awards, most notably the Tswett, Martin and Golay Awards. This overemphasis of more recent activities may have produced an unfair description of early Japanese technical advancement. However, even this short description may show the readers the efforts and results of Japanese chromatographers in developing these powerful separation techniques.

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To somebody who will read this chapter 25 or so years from now, it hopefully will demonstrate the contribution of Japanese scientists to this very important field. By then, the latest chip technology will be developed and chromatography will be quite different than it is today. Hopefully, the thoughts of our readers will again be directed to the future of chromatography, which will be fun even then, just as it had been for our generation.

REFERENCES See Chapter S-13 Supplement on Chem Web Preprint server (http:==www.chemweb. com=preprint=).

Brown bear.

6B. Chromatography in Russia in the 20th century Viktor G. Berezkin Institute of Petrochemical Synthesis, Russian Academy of Sciences, Leninskii pr., 29 Moscow 117912, Russia

A. PROMINENT RUSSIAN CHROMATOGRAPHERS Viktor Grigor’evich Berezkin, author of this chapter was born in 1931, in Moscow. He graduated from the Chemistry Department of Moscow State University. He joined

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the A.V. Topchiev Institute of Petrochemical Synthesis of the Academy of Sciences in 1958 and has been associated there ever since, advancing to the position of the head of the Chromatography Laboratory. He received his doctorate in 1961, the Doctor of Sciences degree in 1968, and the rank of professor in 1973. Berezkin carried out important investigations in almost every branch of chromatography and dealt with both the theoretical and practical aspects of the techniques. Under his leadership 42 doctoral degrees and some Doctor of Sciences degrees were awarded. He has received the Russian M.S. Tswett Medal and the State Prize of the Russian Federation. He is the author of 350 publications, 100 patents and 16 monographs, some of which were also published in English, Hungarian, Polish and Bulgarian editions. V.G. Berezkin’s scientific activities cover various aspects of both gas and thin-layer chromatography. See Chapter 5B, a, b, d, h Vadim Aleksandrovich Davankov was born in 1937 in Moscow. He started his studies at the Mendeleev Institute of Chemical Technology, in Moscow and continued them at the Technical University of Dresden, Germany, graduating in 1962. After returning to Moscow he joined the Institute of ElementOrganic Compounds of the Academy of Sciences advancing to the position of the Deputy Director of the Institute. He received his doctorate in 1966 and his Doctor of Sciences degree in 1975; both of his theses were devoted to the chromatographic separation of enantiomers. The main fields of activities of Davankov had been chiral ligand-exchange chromatography and the separation of enantiomers. More recently he became involved in the development of the artificial kidney and is working on this project in conjunction with a number of American hospitals and other organizations. Davankov has served as president of the Scientific Council on Chromatography of the Russian Academy of Sciences. He has received the Russian M.S. Tswett Medal and State Prize of the Russian Federation. He is the author of 350 publications and a number of patents. His book on Ligand Exchange Chromatography (co-authored with J. Navratil and H. Waltin) published in 1988 in the United States is the only monograph dealing with this field. See Chapter 5B, a, h, q Boris Veniaminovich Ioffe (1921–1997) was born in Perm and graduated in 1942 from the Chemical Faculty of Sverdlovsk State University. During the war he has served in the army. From 1945 on he had been associated with the Chemistry Department of the State University of Leningrad (St. Petersburg) advancing to professor. In the 1970s he organized the Gas Chromatography Laboratory at the University and became its head.

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He also excelled in teaching: 30 doctoral degrees and five Doctor of Science degrees were received under his direction. Ioffe’s main field of research was both synthetic and theoretical organic chemistry and the physico-chemical applications of gas chromatography. In the former field he is noted for advances in the chemistry of amines and hydrazine derivatives, while in GC he pioneered in headspace analysis and in environmental studies. He authored and co-authored 300 publications, a number of books (of which the one on headspace–gas chromatography was also published in English edition in the U.S.) and received ten patents. See Chapter 5B, a, d Andrei Vladimirovich Kiselev (1908–1984) was born in Moscow. He graduated in 1930 from Moscow Technical University. From 1943 on he had been associated with the Chemistry Department of Moscow State University, since 1951 as a full professor. He received his doctorate in 1939 and his Doctor of Sciences degree in 1950. In the 1940s he organized the Laboratory of Adsorption in the Department and became its head; since 1960 the scope of the laboratory was extended to encompass both adsorption and chromatography. From 1946 on he was also associated with the Institute of Physical Chemistry of the Academy of Sciences where, in 1960, he organized the Laboratory of Surface Chemistry. Kiselev was internationally recognized as one of the most important scientists in the field of adsorption and surface studies; under his direction 128 doctoral degrees were awarded, many also to foreign students. He authored 960 publications and 17 monographs, some of which were also published in foreign editions in the U.S., France, Germany, Japan, Israel, Poland and Czechoslovakia. He also received 30 patents. Kiselev’s achievements were recognized by the title of the Honored Scientist of the Russian Federated Socialist Republic and by a number of awards such as the Russian M.V. Lomonosov and D.I. Mendeleev Prizes, the Silver Medal of the French Chemical Society, the Kopernik Medal of Torin University (Poland), as well as the M.S. Tswett Chromatography Award of the International Symposium on Advances in Chromatography; he was also an honorary member of the British Faraday Society. See Chapter 5B, a, b Karl Ivanovich Sakodynskii (1930–1996) was born in Novorossiisk and graduated in 1954 from the Mendeleev Institute of Chemical Technology in Moscow. He received his doctorate in 1957 and his Doctor of Sciences degree in 1973; he has advanced to the rank of professor in 1976. From 1957 on he had been associated with the Karpov Institute of Physical Chemistry where he advanced to the position of the assistant scientific director

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of the Institute. In 1983 his group was transferred to the All-Union Research Institute of Chemical Reagents where he became the head of the Chromatography Department. The main fields of activities of Sakodynskii were preparative chromatography and the development of novel organic polymeric stationary phases. His studies on the life and activities of M.S. Tswett, the inventor of chromatography, are also noteworthy. Sakodynskii was also very active in promoting the cooperation of scientists between the east and west. He has served as the vice president of the All-Union Scientific Council of Chromatography of the Academy of Sciences and as the president of the Russian Association of Chromatographers. Under his leadership 52 doctoral degrees and 2 Doctor of Science degrees were awarded. Sakodynskii received the title of the Honored Scientist and the State Prize of the Russian Federation and the M.S. Tswett Chromatography Award of the International Symposium on Advances in Chromatography. He was the author of 350 publications, four monographs and received 100 patents. See Chapter 5B, a, b, e, j Aleksander Abramovich Zhukhovitskii (1908–1990) was born in Rostov-on-the-Don and graduated from the Donskoi Polytechnic Institute in 1930 as a chemical engineer. He received his doctorate in 1933 and his Doctor of Sciences degree in 1937. Between 1939 and 1948 he had been associated with the Karpov Institute of Physical Chemistry, in Moscow, where he advanced to the position of assistant scientific director of the Institute. In 1948 he was invited to join the Steel and Alloys Institute as head of the Physical Chemistry Department and he remained at this Institute for the rest of his life. Prior to 1975 he had also been associated with the All-Union Research Institute for Geological Prospecting of Petroleum and from 1975 on with the All-Union Scientific Research and Design Institute of Automation and Control Systems in the Oil and Gas Industry. Zhukhovitskii pioneered in gas chromatography in the USSR together with N.M. Turkel’taub; he started in this field as early as in 1951. During his long career he developed a number of unique gas chromatographic methods and had also been involved in the development and construction of a number of gas chromatographs produced in the Soviet Union. In 1962, together with Turkel’taub, he wrote a monograph on gas chromatography which has been the basic textbook for many thousand chromatographers in the Soviet Union. Zhukhovitskii was the author and co-author of 300 publications, five books and received 50 patents for his inventions. In 1968, he received the title of Honored Scientist of the Russian Federated Socialist Republic and in 1977 the M.S. Tswett Chromatography Award of the International Symposium on the Advances in Chromatography. See Chapter 5B, a, b, d

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B. INTRODUCTION The twentieth century has been very fruitful for Russian chromatography. In March 1903, M.S. Tswett announced the discovery of chromatography in his lecture “On a novel class of adsorption phenomena and their use in biochemical analysis” presented at the meeting of the Biology Division of the Warsaw Naturalists’ Association. In subsequent studies and especially in his book “Chromophylls in the Plant and Animal World” [1], M.S. Tswett pointed out the main directions of further development of chromatography, which were realized in the latter half of the 20th century. For example, he suggested displacement chromatography, noted the advantages of using fine particle size sorbents, organic adsorbents, application of step gradient for elution of compounds, and of small columns (2–3 mm in diameter, 20–30 mm in length), etc. [2]. As with humor M. Twain wrote: “A classic is something that everybody wants to have read and nobody wants to read.” The next great success of Russian chromatography was the discovery of thin-layer chromatography (TLC) by Izmailov and Shraiber [3]. They first described this technique in 1938 in a study on ‘Spot Chromatography Method of Analysis, and its Use in Pharmacy’ [4]. The development of chromatography and introduction of chromatographic techniques to industry, medicine and science in the USSR began in the latter half of the 20th century. When considering the achievements of chromatography in the former Soviet Union (FSU), in our opinion, it is necessary to make some preliminary remarks: ž We should point out that in our present review, we only consider the most important developments carried out in the territory of the former Soviet Union. Because of the limited contacts, which existed for decades with western scientists, many of these achievements are unknown to the readers of this book. Our review does not cover the field of ion-exchange chromatography. ž We do not cover development, which was carried out in the present-day Baltic States and in the Caucasian republics. ž Mainly the subjects of the achievements organize this review. However, in the case of the most important Russian chromatographers who created whole schools with many students and followers, we discuss separately the activities of their groups. ž In compiling this exhaustive review, we were helped by many colleagues whose cooperation is greatly appreciated.

C. A.V. KISELEV AND HIS SCHOOL A.V. Kiselev (1908–1984) had been one of the foremost and best known Russian chromatographers. His school made a great contribution to the development of adsorption chromatography in Russia and all over the world. Kiselev displayed his talents as a scientist and as a teacher during the school’s formation and its scientific activity. The following known chromatographers belong to Kiselev’s school: K.D. Shcherbakova, L.D. Belyakova, R.S. Petrova, Ya.I. Yashin, A.A. Lopatkin, Yu.S. Nikitin, V.Ya. Davydov, D.P. Poshkus, Yu.A. Elltekov, etc.

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The main focus of the school’s activity dealt with the application of chromatographic methods for the study of adsorption and adsorbents, for the interpretation of adsorption and the prediction of chromatographic phenomena and preparation of adsorbents for chromatography. Monographs by Kiselev and his coworkers [5–10] favored the formation of a modern concept of adsorption chromatography as an important scientific direction and stimulated its further development. When considering the contribution made by Kiselev’s school to the development of adsorption chromatography, it is necessary to note the following important accomplishments: ž Theoretical and experimental studies of adsorption and chromatographic phenomena. The scheme below suggested by Kiselev reflects its content. The movement in the direction of A reflects the study (and theoretical prediction) of sorption equilibrium in chromatography (for example, the development of molecular statistical adsorption theory on graphitized carbon black and zeolites [10]). But movement in the direction of B is the solution of an inverse task, namely the study of the sorbate’s structure based on the data from adsorption equilibrium and the corresponding theoretical calculation of the chromatographic sorption equilibrium. Kiselev called this approach ‘chromatoscopy’ [11].

ž

ž

ž ž

Many scientists paid their attention to chromatoscopy as a new concept for the study of the structure of molecules. In 1980 C.S.G. Phillips said in the discussion after the lecture of Kiselev and Poshkus (Faraday Symposium) that: “The real potential of chromatography in this area has yet to be properly tapped, and it is therefore particularly valuable to have this bolder concept explored at this meeting in the most stimulating paper by Kiselev and Poshkus, in which they demonstrate how surfaces may be used to reveal remarkable details of the structure of simple molecules”. A general approach (1969) for the retention interpretation of various adsorbents. New classification of adsorbents by geometrical structure, and by the chemical nature of the surface is an important result of this work [5]. Adsorbents such as graphitized carbon black [12], porous glass [13], Silochrom [14], barium sulphate [15] and others were proposed and introduced into the analytical practice. Methods proposed for the chemical and geometrical modification of adsorbents [16– 19]. Capillary adsorption chromatography was first realized independently (1961) in the Soviet Union by V.I. Kalmanovskii, A.V. Kiselev and co-workers [20], and by Mohnke and Saffert in Germany [21].

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It is necessary to note that even after Kiselev’s death, his school is still actively working today.

D. A.A. ZHUKHOVITSKII AND HIS SCHOOL A.A. Zhukhovitskii’s (1908–1990) school had been one of the leading Russian chromatographic schools. Their studies have made a great contribution to the development of chromatography in Russia and all over the world [22]. In the 1940s, Zhukhovitskii was one of the pioneering investigators of the exact theory of sorption dynamics [23,24]. Ideas of this investigations were used in the development of the technique of chromatography without a carrier gas. In the 50s, together with N.M. Turkel’taub (1915–1965), he proposed a new direction of using a moving temperature field in chromatography. The application of the moving temperature field with a negative gradient was of most importance in the practice [25–27]. In the 70s and 80s, Zhukhovitskii’s principal concern was the development of high-concentration chromatography. In this period, the following new chromatographic variants were proposed: chromatography of vapors at concentrations close to saturation [28,29], and chromadistillation [30,31] which can be considered to be a distillation process at the condition of chromatography; chromadistillation is limited when a more volatile component is applied to the column before separation. Note that the great scientific heritage of Zhukhovitskii does not yet appear to be studied. In my opinion a number of further achievements in chromatography will be related to the development of his original ideas.

E. THE ACTIVITIES OF M.I. YANOVSKII M.I. Yanovskii (1916–1990) worked with the well-known researcher of catalysis, S.Z. Roginskii. His studies dealt with the application of chromatography for catalysis [32]. He developed a number of original chromatographic techniques for the study of adsorption and kinetics of catalytic reactions; among the techniques it is worth noting the technique for reversible catalytic reactions. The main characteristic of the technique is the combination of catalytic transformation with continuous chromatographic separation of the reaction products. This allows shifting the equilibrium of the reversible reaction and, in some cases, increasing the yield of the target products. Yanovskii hypothesized that biochemical reactions, at the isothermal conditions of the living organism, can also take place in chromatographic systems.

F. M.S. VIGDERGAUZ AND HIS SCHOOL M.S. Vigdergauz (1934–1993) and his school centered their attention on the fundamentals of gas chromatography [33–37]. He contributed significantly to the development of gas chromatography of hydrocarbons [37], qualitative gas chromatography [36], and

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application of non-ideal eluents in gas chromatography [36]. Vigdergauz first showed that at high pressures (more than 20 atm) retention indices are linearly dependent on the pressure [38]. He proposed original chromatographic systems containing two coupled columns with a controlled pneumatic restrictor placed between the columns. A change in the restrictor’s value leads to a variation in the selectivity of chromatographic system [39]. Also, he proposed the application of multiple internal standards. This allowed for a significant increase in the accuracy of qualitative measurements [40]. He also paid great attention to the natural chromatographic process and the problem of defining chromatography as a branch of science [41].

G. K.I. SAKODYNSKII AND HIS SCHOOL K.I. Sakodynskii (1930–1996) and his co-workers studied polymeric organic sorbents, developed new sorbents of this type and extended the fields of their applications. With Panina, Sakodynskii wrote the first monograph on such materials, discussing in detail various questions related to their characteristics, preparation and applications. The most interesting results were obtained in the study of the following sorbents [43–50]: organic sorbents containing surface polar groups, formed during direct reactions of nitration, amination, phosphorylation, etc.; sorbents based on copolymers of N-vinyl-9 (5)-methylpyrralizole and ethylene glycol dimethylacrylate, copolymers of 2,5-methylvinylpyridine, glycidylmethacrylate, and divinylbenzene; macroporous sulfocationates, containing elements of the 4th period of the Periodic Table; a special selectivity on the retention of nitrogen oxides and hydrocarbons was noted for the Cu2C cation form; polymeric complexes of Ag, Cu, and Hg (on the basis of vinyl derivatives of pyridines) having a high selectivity to sorption of halogen-, sulphur-, and nitrogencontaining compounds; polymers with maximum operating temperatures of 320ºC and even 450ºC. Sakodynskii, Yudina and co-workers paid great attention to the development of various types of stationary liquid phases [51]. This is the second main direction of Sakodynskii’s school. The polysiloxane “lestosil” [52] is of special interest as a thermostable phase with reproducible properties:

where R D CH3 or C6 H5 ; a D 0–0.33; x D 6–60; y D 10–350; and n D 10,000–30,000.

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Sakodynskii and his co-workers also carried out very extensive investigations on the use of gas chromatography for preparative purposes: on the proper conditions for injecting large-volume samples, on the relationships permitting the establishment of the proper conditions for large-diameter overloaded columns, and on the development of columns necessary for the preparative separation and production of larger amounts of highly pure compounds. The result of these activities was the design, construction, and establishment of a preparative-scale laboratory pilot plant, and production-size units. In the ‘Etalon’ instruments 10 cm diameter columns were used which were then enlarged in the pilot plant type units to 50 cm diameter columns, with a throughput of about one kilogram per cycle. They were even successful in the establishment of industrial systems having columns of 15–200 cm in diameter [51]. Such industrial units have been in operation in the Soviet Union for many years. In 1971, a plant utilizing columns of 50 cm in diameter was installed at Donetsk and used for the isolation of thiophene from crude benzene. Later a plant using a column of 120 cm in diameter was established at a factory producing fine reagents in Shostka and used for the production of toluene of 99.9% purity (production capacity: 1200 metric tons per year). Also, at a phenol factory in Makeevka a plant was established for the isolation of indole with 96% purity, from an original fraction containing only 3.5–4.5%, with a production capacity of 200 tons=year. In 1972, Sakodynskii, with Volkov, summarized their results on preparative gas chromatography in a monograph [52]. Finally, it should be noted that Sakodynskii studied in detail the life and scientific activities of Tswett, the founder of chromatography, and published a number of papers on this subject of which the series published in the Journal of Chromatography is the most important [53].

H. PRECIPITATION CHROMATOGRAPHY In 1948, E.N. Gapon suggested precipitation (sedimentation) chromatography, based on the formation of precipitates and their migration through the column. This is the single example of a special variant of liquid chromatography, where the in situ formed new phases (precipitates) move along the chromatographic packing. This variant, like other variants, of liquid chromatography was realized in both column and thin layer versions. Precipitation chromatography is used for the analysis of inorganic (for example, cations of transition elements, rare earth, and dispersed elements, halide and thiocyanate anions) and organic compounds associated with the precipitant or eluent; the precipitates have different solubilities. The method can also be applied to the determination of compound solubility in various media. K.M. Olshanova and V.D. Kopylova contributed to the development of this method [54,55].

I. HEADSPACE (VAPOR PHASE) ANALYSIS B.V. Ioffe (1921–1977), and A.G. Vitenberg made an outstanding contribution to headspace analysis coupled with gas chromatography. Ioffe used the term “vapor

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phase analysis” instead of headspace analysis [56–60], and defined the technique as a “combination of methods for getting information on the nature, composition, and condition of liquid and solid components by the analysis of the gas phase in contact with them.” This definition does not limit the operation mode (static or dynamic) or conditions (equilibrium or non-equilibrium), and also does not restrict the technique to a combination with gas chromatography: the gas phase can also be analyzed by spectral or chemical methods. Ioffe and Vitenberg placed their efforts on the development of equilibrium vapor phase analysis. Their activities were carried out at Leningrad (St. Petersburg) University and dealt with the development of vapor phase analysis and its formation as a separate branch of chromatography and analytical chemistry. The theory of vapor phase analysis was presented on the basis of the rules of static and dynamic gas extraction of volatiles and semi-volatiles and on the concept of equilibrium concentration.

J. ACTIVITIES OF V.A. DAVANKOV V.A. Davankov’s school has been involved in crucial new developments. Davankov proposed ligand-exchange chromatography on chiral complexing sorbents for the separation of optical isomers. The implication of coordination chemistry of ions, for example Cu2C , into the chromatographic process is a peculiarity of ligand-exchange chromatography. Due to the linking in their coordination sphere, these ions provide close steric interaction of the sorbent’s chiral ligand with the enantiomers to be separated [61,62]. In liquid chromatography this technique was first applied for the quantitative separation

Fig. 6B.1. Chromatographic separation of 0.5 g D,L-proline. Experimental conditions: 475 ð 9 mm column, polystyrene containing L-proline in Cu2C form (0.3–0.5 mm); eluent: water, then 1 M NH4 OH. Polarimetric detection by 6.2 ml fractions.

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of racemates of different classes of compounds, such as amino acids (see Fig. 6B.1), hydroxy acids, and amino alcohols. In the scientific literature this technique is called ‘Davankov’s concept’. Enantioselective (chiral) liquid chromatography was developed on the basis of this concept. Now ligand-exchange chromatography is in common use and American firms continue to introduce the ‘Davankov columns’. Davankov’s concept of super cross-linked polymeric materials is of scientific importance. These materials have unique properties and an ability to swell in any liquid medium independently of their thermodynamic affinity to polymerize, and an ability to significantly exhibit reversible deformation. In co-operation with M.P. Tsuryupa, he synthesized a new generation of neutral polymeric sorbents based on cross-linked styrenes which are unrivalled among the available sorbents [63,64]. The proposed sorbents have a unique sorption capacity for compounds in water and air media. In co-operation with the English firm ‘Purolife’, the industrial production of these sorbents was developed. These sorbents are now in common use in the industrial sorption process of sugar syrup bleaching, removal of soil, disposal of waste gases and waters, in analytical chemistry for the concentration of organic trace impurities from water and air by solid-phase extraction, and in high-performance liquid chromatography as a prospective hydrophobic neutral sorbent compatible with any aqueous and organic eluents. Recently, Davankov published a number of important studies on gas chromatography (see e.g., [65]).

K. BIOPOLYMER SYSTEMS G.V. Samsonov [66,67] contributed to the industrial techniques for the removal of biologically active compounds on polymer sorbents. Also he obtained interesting results for biopolymer systems (cross-linked polyelectrolytes, biologically active compounds). This leads to production of new drugs of directional action. He developed the fundamentals of the production process of obtaining pure antibiotics, enzymes, hormones, nucleotides, etc., as a result of his theoretical and experimental studies on the dynamics of the chromatographic process. He also developed a suggested theory of sorption dynamics and fundamentals of the thermodynamic theory of the interaction of complex organic ions with the cross-linked polyelectrolytes. The polyelectrolytes are characterized by a high selectivity and a high rate of mass-transfer. The results of his studies are in common use in Russia and abroad.

K.1. Critical chromatography of polymers In the 80s, the Russian scientists A.V. Gorshkov and V.V. Evreinov, in Moscow, and A.A. Gorbunov and A.M. Skortsov, in St. Petersburg, developed a new, important, and universal technique for the study of the structure and topology of macromolecules. They called it the ‘critical chromatography of polymers’. When considering the adsorption of a macromolecule as a phase transition taking place in a system of monomers, the existence of a special, critical mode of separation lying at the boundary of exclusion and adsorption processes could be predicted and theoretically justified. This critical mode

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was first realized by Gorshkov and Evreinov in 1983, and then applied to the separation of polymers in accordance with the number of end-groups as well as their topology. In a constant system at the point of phase transition, the separation of macromolecules due to their molecular masses (or sizes) disappears. The loss of separation offers strong possibilities for the investigation of the primary structure and topology of the macromolecule. The separation of polymers in a critical mode obeys simple and universal laws. In a framework of the gaussian model these laws could be found even analytically. Utilizing the concept of a ‘macromolecule with broken links’, one can take into account the real chemical nature of the polymer, solvent, and adsorbent. This approach coupled with the theory of polymer adsorption has been considered as the basis of the unified mechanism of polymer chromatography. The ‘theoretical chromatograph’, constructed in this way, gives the possibility to quantitatively predict the character of the separation of polymers in the critical as well as in the adsorption and exclusion modes. At this time, ‘critical chromatography’ is the only physical method of investigation working exactly at the point of phase transition. This method embodies all of the important features of the critical state of matter. Realization of ‘critical chromatography’ permitted the solution of some of the questions of polymer chemistry, e.g., the determination of the number and types of macromolecules and groups, separation of linear and cyclic macromolecules, investigation of molecular-mass distribution of blocks in copolymers, as well as the character of sequence of different monomers in random heteropolymers. The basic idea of the method as well as the concrete practical approaches are discussed in the book by Entelis, Evreinov, and Gorshkov [68]. In the former USSR this method was applied to a variety of problems mainly for the study of the polymerization process of reactive oligomers. A typical example of the separation of macromolecules in accordance with the types of their structures is shown in Fig. 6B.2. An important

Fig. 6B.2. Separation of (1) linear (MW D 20,500), and (2) ring-shaped (MW D 25,000) polystyrene macromolecules having one and the same size in the critical mode of polymer chromatography. Column, silica; mobile phase, cyclohexane=THF (90=10%, vol.); temperature 30ºC. The shadowed region corresponds to the linear precursor in the cyclic sample.

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contribution to the method has been made by B.G. Belen’kii (Institute of High Molecular Compounds, St. Petersburg), V.V. Gur’yaniva, and T.N. Prudskova (Scientific and Research Institute of Polymers), I.V. Blagodatskikh (Institute of Elemento-Organic Compounds), and Ya.I. Estrin (Institute of Chemical Physics in Chernogolovka). Since the beginning of the 90s this concept has been successfully developed by H. Pasch (Deutsches Kunststoff Institut, Darmstadt, Germany) and D. Berek (Polymer Institute of the Slovak Academy of Sciences).

K.2. Exclusion chromatography B.V. Mchedlishvili and his school developed new concepts of the exclusion chromatography of suspensions (latexes, viruses, bacteriophages, cells) [69–73]. He pioneered the development of the physico-chemical fundamentals of exclusion chromatography of viruses [72–76], by studying the adsorption of virus particles on modified macro-porous silica, the penetration process of virions and hard colloid particles into the pores of such solid supports. The main parameters of exclusion chromatographic separation of virus suspensions (diameter, pore-specific volume, size of silica particles, eluent flow rate) were investigated and optimized [77–81]. The universal dependence of the distribution coefficient (Kd ) of the virus on the ratio .½/ of its average size and the pore size of the solid support was established [72]: K d D .1 2½/0:5 Mchedlishvili proved the principal non-equilibrium process of exclusion chromatography of viruses (nearly in the whole eluent velocity range, from 103 up to 102 cm=min) [81]. At these conditions the separation coefficient (for zones of virus particles and protein mixtures) depends only on physico-chemical parameters (distribution coefficient, diffusion coefficient of viruses and proteins) and in general does not depend on other parameters of the dynamic system. The non-equilibrium techniques of exclusion chromatography of viruses are fundamental for the industry in obtaining pure virus preparations: diagnostic preparation for AIDS virus (see Fig. 6B.3), vaccines for influenza and rabies and tick encephalitis [77,79,81]. The investigation and separation of large particles is important in the development of practical applications of chromatography. It is interesting to note this in the studies by V.V. Pomazanov, K.I. Sakodynskii, and co-workers [82,83] on the separation of microorganisms. Polyphase systems including colloid systems, and suspensions were used as the moving phase in these studies.

K.3. Ion chromatography O.A. Shipgun carried out important investigations in the field of selective sorbents for ion chromatography in the Laboratory of Chromatography, within the Analytical Chemistry Division of Moscow State University. In this respect polyelectrolyte and zwitterionic sorbents were of special interest. An approach, which is based on the fact

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Fig. 6B.3. Exclusion chromatogram of a suspension culture of AIDS virus (100 ml): (the dependence of the virus and protein impurities content of the sample on the eluent volume, Ve . Column 100 ð 5 cm, macroporous glass MPS-2000 V-GKh, modified by polyvinylpyrrolidone (pore size 0.2 µm, pore volume 2 cm3 =g). 1-virus zone, 2-zone of protein impurities. Relative units of virus activity (left ordinate), protein content (right ordinate) are shown along the axis of ordinates.

that an anion-exchange layer may be bound at the surface of a cation-exchanger by electrostatic interaction of reversibly charged functional groups, was used for producing polyelectrolyte anion-exchangers. Water-soluble polymers containing positively charged nitrogen atoms in the backbone and side chains were used in synthesizing pellicular sorbents [84–86]. Sulfonated silica served as the matrix. The prepared anion-exchangers show perfect selectivity and efficiency up to 15,000 theoretical plates per meter (see Fig. 6B.4). It was found that the selectivity of the produced sorbents strongly depends on the structure of the polymers used. Sorbents based on poly (4-vinyl) pyridinium bromide showed the highest selectivity in separating anionic complexes of transition metals with EDTA [85]. Sorbents based on aliphatic ionenes are suitable for the simultaneous determination of weakly and strongly retained anions [86]. Aromatic ionenes (viologens) are suitable for the selective determination of aromatic acids by additional interactions of the aromatic radicals in the molecules of the analyte and the polymer. A number of silica-based ion exchangers containing amino groups attached to the silica surface have been synthesized and investigated as possible stationary phases for ion-exchange chromatography. Depending on the structure of the bonded layer these ion exchangers can behave as selective anion- and cation-exchangers. The main feature is their unique selectivity allowing simultaneous isocratic separation of alkaliand alkaline-earth metal ions, single-run separation, and determination of common (nitrate, dihydrophosphate, chloride, bromide, nitrate, etc.) anions, and such strongly retained bulky anions as iodide, perchloride, thiocyanate as well as sulphate. The simultaneous single-column separation and determination of both anions and cations is also possible [87,88]. The special structure of a bonded layer provides a better efficiency in comparison with common silica-based ion exchangers. The possibility of regulation of ion-exchange capacity for such adsorbents makes it possible to use very dilute eluents

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Fig. 6B.4. Chromatograms of mixtures of weakly and strongly retained anions. Column: 3 ð 50 mm. Detector: UV, ½ D 254 mm. Flow rate 1.0 ml=min. Sorbent: Silasorb-S modified by 4–6 Ionene. Eluent: 2 mM benzoic acid, pH 5.5. Sorbent: Silasorb-S modified by 6–10 Ionene. Eluent: 0.3 mM potassium hydrogen phthalate, pH 7.0. Peaks: 1, iodate; 2, formate; 3, chloride; 4, nitrite; 5, nitrate; 6, iodide; 7, sulfate; 8, thiocyanate; 9, perchlorate.

that in combination with conductometric detection provides better limits of detection [89].

L. DEVELOPMENT OF PLANAR CHROMATOGRAPHY Tswett already pointed out that the particle diameter is an important parameter in chromatography, influencing its efficiency. Along this line, the separation efficiency in thin-layer chromatography was significantly increased when fine particle adsorbents were used as proposed by B.G. Belen’kii, E.S. Gankina, and V.V. Nesterov in 1967 [90]. They developed the technique by optimization of the mathematical model of

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TLC [91]. It seems likely that this study is the second example of optimization in chromatography after Hamilton’s work published in 1963 [92]. This optimized technique of high-performance TLC (HPTLC) using silica gel 5 µm fraction and 5 cm ð 5 cm TLC plates for the analysis of the phenylthiohydantoin, dinitrophenyl, and 1-dimethylamino-5-sulfonaphthyl derivatives of amino acids was in common use in the former USSR and now used in Russia for protein studies. In particular, all the derivatives of primary structures of proteins were determined in Russia in the 70s and 80s by this technique. Thus, HPTLC is in common use in Russia. The idea of an increase in efficiency (and other separation characteristics) as a result of a decrease in the average diameter of the sorbent particles was used for further development of TLC and elaboration of HPTLC [93]. In Russia, M.P. Volynets is the first to apply classic TLC for qualitative inorganic analysis and radiochemical studies [94,95]. She and co-workers proposed a technique for the detection and identification of compounds in chromatographic zones in situ by local X-ray fluorescent microanalysis [96] and elaborated TLC application for radioactive samples [97]. The publications on the separation and determination of actinide elements in various states of oxidation were not available in the scientific literature. M.P. Volynets and co-workers studied the behavior of americium and plutonium of different valences [98,99]. Thin-layer chromatography can be realized as a form of eluent column chromatography as well [100]. In this case a detector (for example, optical) is placed directly onto the plate. The flow of mobile phase moves continuously along the plate. The mobile phase evaporates after the detector. A.M. Woronstsov made a great contribution in the development of this technique [101,102]. He designed a simple and inexpensive device for continuous flow TLC and developed its practical application for environmental analysis [101,102]. The metrological characteristics of the device are essentially better than in classic TLC. This direction is of practical importance. It allows the development of very simple and inexpensive liquid planar chromatographs which can be used for environmental control, medicine and etc.

M. CHEMICALLY BONDED STATIONARY PHASES Lisichkin’s school has been actively involved in this research at Moscow State University for the past 20 years. They investigated the modification of the solid support, and the development of new original sorbents. In the late 70s, sorbents for liquid chromatography on the basis of silica modified with alkylsilanes with various carbon chain lengths in bonded groups were made and studied. Also new methods of synthesis were developed. These methods allowed bonding organic compounds of nearly all chemical classes on the surface of the solid support and to obtain ion exchange and affinity sorbents of different polarity (nonpolar, low, medium, and highly polar). In the studies related to modified silica various methods of synthesis and examples of their practical application for liquid chromatography were considered [103,104]. Great attention was paid to ion-exchange sorbents on the basis of modified silica, and the general rules of the sorption of transition

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Fig. 6B.5. Determination of drugs in blood plasma. Column: 150 ð 4 mm; sorbent: internal surface ODS, external surface-shield of cross-linked albumin globules and reduced aldehyde groups; eluent: 0.1 M sodium acetate, buffer solution (pH 6.85)–acetonitrile (90 : l0), 1.0 ml=min, UV-detector, ½ D 254 nm. 1, plasma proteins; 2, aspirin C theobromine; 3, Phenobarbital; 4, caffeine; 5, papaverine; 6, phenacetin.

metals on the modified silica gels were studied. The acidic–basic properties of silica modified by gamma aminopropyl triethoxysilane were considered and their synthesis, application and characteristics were described. The separation of oligonucleotides with unit numbers from 1 to 22 on silica ammonium anionite is of particular interest. The peaks of the individual oligonucleotides were equidistantly eluted from the column, with a total separation time of less than 1 hour. Isomeric cresols, xylenols, and phenol were completely separated on a column filled with silica modified by (CH2 )10 CN groups, then silanized by trimethylchlorosilane [105]. With Zorbax CN and Spherisorb CN sorbents, the separation fails. Silica modified by antibiotic polypeptides (for example, bacitracin-Silochrom) showed good results in affinity chromatography for obtaining the neutral proteinase. A preparative column with gramicidin-Silochrom was used for the preparative production of purified human thrombin [106]. Note that the method of polypeptide fixing on the silica surface is of both theoretical interest and practical importance. The chromatographic properties of the sorbent depend on the modifier molecule connected to the surface through one or two anchor groups [107]. The application of chromato-membrane methods in gas chromatography led to the development of chromato-membrane vapor phase analysis [115]. The chromatomembrane technique allows new solutions of pre-concentration and isolation of polar impurities present in air with subsequent determination by ion chromatography [116]; for the determination of petroleum products and phenols in water and for the continuous fluorimetric determination of phenols and petroleum products [117].

N. CIRCULAR CHROMATOGRAPHY Circular chromatography was originally discovered by A.J.P. Martin [118]. This important scientific development was promising as an improvement of the separation of

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Fig. 6B.6. Application of a circular chromatograph for the separation of deuterium-substituted benzenes. The schematic of the system: 1.2, regulator; 2, gauge; 4, capillary columns; 5, multiport valve; 6, controlled pneumatic resistor; 7, detector; 8, split injector. Chromatogram of deuterated derivatives of benzene obtained on a capillary column (37.5 m length ð 0.28 mm I.D.) coated with squalane, at 150ºC: 1, C6 D6 ; 2, C6 H3 D3 ; 3, C6 H5 D; 4, C6 H6 .

compounds with close properties (for example, compounds of various isotopic composition, optical isomers, etc.), and of the preparative separation characterized by improved economics (economy of sorbents and the moving phase). Chizkov and co-workers contributed significantly to the development of circular chromatography [119–121]. They proposed and developed a two-cycle variant of circular chromatography using two columns. The scheme of this technique and a chromatogram of deuterated benzenes is presented in Fig. 6B.6. A separation of deuterium-substituted benzenes obtained after 16 semicyles (column switching), corresponds to a total column length of 1200 m and a total efficiency of 5,000,000 theoretical plates. It would be almost impossible to realize single standard columns with such characteristics. Also Chizkov and co-workers [122] proposed the original ‘impulse hydrodynamic’ technique. In this case the stage of column switching is attended with a simultaneous compression of the chromatographic zones circulated in the system. This leads to an essential increase in the total separation efficiency. They established the theory for the proposed technique, and developed original systems for its realization; they also investigated the main prospects of the technique in both gas and liquid chromatography [123–125].

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O. THE ACTIVITIES OF V.G. BEREZKIN Activity of V.G. Berezkin and his chromatographic school (including his pupils and followers) is primarily focussed on the development of fundamentals of gas and planar chromatography and also on the elaboration of new chromatographic methods. During his earlier work he was one of the originators of the ‘polyphase sorbent’ concept in gas–liquid chromatography (GLC). In accordance with this concept, a compound to be separated is sorbed by both the macrofilm of the stationary liquid phase and on two interfaces (gas=liquid and liquid=solid support). However, pure GLC had not been realized as yet. Based on this concept he suggested in 1968 [126] a general equation for the net retention volume, ‘Berezkin’s equation’, which includes terms describing the influence of the various interfaces in the column (gas=liquid, liquid=solid, gas=surface between the gas and liquid phases). Also he developed an equation describing the dependence of the relative retention on the liquid phase loading in both packed and capillary columns, and strictly speaking he demonstrated that the relative retention values are not chromatographic constants of the analyte. This concept, its theory, the experimental methodology, and practical applications were presented in a monograph [127]. Recently, he showed the importance of this concept for capillary GLC in a review [128]. Berezkin also developed a new concept on the role of the carrier gas in traditional capillary gas chromatography [129–131]. Contrary to the widespread concept on the independence of the relative retention values of the analyte on the nature and pressure of the carrier gas, it has been theoretically and experimentally shown that the carrier gas has a significant influence on relative retention under the conditions of routine analytical gas chromatography, even when the carrier gas pressure does not exceed 5 atm. For traditionally used carrier gases (He, H2 , N2 , and CO2 ), the theory was elaborated and linear equations were obtained describing the dependence of relative retention on the average column pressure. It was shown that the limiting value of relative retention at carrier gas pressures approaching zero should be considered as the true chromatographic constant and not the relative retention at average pressure as believed earlier. In this respect the retention factor, the relative retention time, and the Kovats’ retention index all represent ‘relative retention’ and they are not chromatographic constants. Berezkin has also shown both theoretically and experimentally that the mobile phase in gas chromatography could have a significant influence on the resolution of a critical peak pair to be separated and in all these processes the influence of the surface between phases has great importance. Berezkin also proposed acidic–basic gas chromatography, a new variant of gas chromatography. This technique is based on the chemical interaction of an acidic carrier gas (e.g., carbon dioxide) and a basic analyte (e.g., amine), or a basic carrier gas (e.g., ammonia) and acidic analytes (e.g., organic acids). The interaction results in an increase in the retention of the analytes. The new concept on the role of the carrier gas and the new relationships describing the influence of the carrier gas on retention permits the selection of the carrier gas to improve separation in a manner similar to the selection of the mobile phase in liquid chromatography. Berezkin and co-workers also proposed a new type of TLC plates, with the adsorption

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layer covered by a polymer film [132–133]. Elution of the analytes on the plates with a closed sorbent layer was considered both theoretically and experimentally under the conditions of both classic and overpressure TLC. V.G. Berezkin paid great attention to the systematization, summarization and recognition of new directions of chromatography. He is the author of some monographs [127,134–137]. Some of the monographs were pioneering (for example, the monographs on reaction gas chromatography, capillary gas adsorption chromatography and on gas–liquid–solid chromatography).

P. INDUSTRIAL ON-LINE ANALYSIS V.N. Lipavskii contributed to the development of industrial on-line analysis by gas chromatography [138]. This is usually performed dynamically, by continuously varying actual samples, as compared with static analysis in a laboratory. The general characteristics of an on-line analysis are: (1) a second on-line analysis of the same sample is impossible; (2) it is impossible to use a classic internal standard in the measurement; and (3) the stage of sample preparation is a part of the technique performed at temporal analysis intervals. These characteristics influence the establishment of the gas chromatographic method for industrial analysis. But the original requirements for method evaluation still exist. Information must be obtained during analysis, the measurement error of each component has to be known and the time lag must be considered and all these can lead to sacrificing the stability of automatic control. The studies performed allow obtaining the dynamic model of an industrial on-line chromatograph as a part of the control system and to formulate requirements for its construction [139]. The proposed dynamic model takes into consideration both the cycle type of the chromatograph and the time lag of the information as a result of the time of analysis and the dynamic properties of sample preparation. The problem of quantitative accuracy of on-line chromatographic measurements can be solved using the above listed characteristics of industrial analysis. The developed system is based on using an external standard. In this case, the standard compound is injected onto the column during analysis. This shortens the analysis time and decreases the incidental component of error introduced by other factors. Both the components to be measured or other compounds can be used as the standard [140]. The system utilized a specially developed sample introduction system. The control compound can be injected on the column either during each analysis cycle or by a special signal given by the operator. Except for calibration and check of the chromatograph, the system allows diagnostics of the total chromatographic system. An analogous methodology was independently proposed by C.L. Guillemin called a “reference standard” [141].

Q. ADVANCES IN CHROMATOGRAPHY DETECTION I.A. Revelskii carried out a number of interesting studies in the field of detection and determination of impurities and developed a new technique for highly sensitive

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mass-spectrometric detection. It is based on mass spectrometry with photoionization at atmospheric pressure. This technique permits one to register the mass-spectra of compounds containing only a single ion (molecular or quasi-molecular), and to determine more appropriately the number of components present in mixtures compared to other known techniques, and to identify them [142,143]. The new methodology based on a combination of a very rapid determination of the total content of P-, S-, halogen and N-containing organic compounds in various media permits the screening of samples for the most dangerous toxicants. It is based on gas chromatography, gas chromatography=mass spectrometry, and reaction gas chromatography=mass spectrometry [144]. Revelskii and co-workers also proved the possibility of using ionization detectors without previous calibration [145]. For example, traces of P-, S-, halogen and N-containing organic compounds could be determined by microcoulometry in aqueous and organic solutions without any previous calibration [144]. V.I. Kalmanovskii made a significant contribution to the development of the theory of detection in gas chromatography and to the development of new detection techniques. He developed a classification of chromatographic detectors by concentration and flow. This classification is based on the type of conversion of the analyte molecules during detection [146]; this classification was published four years before the analogous publication of Hala´sz [147]. In 1971, Kalmanovskii and V.A. Sheshenin proposed a new variant of the electron capture detector with a constant recombination rate [148]; the first similar foreign study was published in 1980 [149]. In 1971, Kalmanovskii and co-workers proposed a new variant of thermionic detection where the source of vapors of the alkali halides and the ionization source were separated [150]; an analogous foreign publication was in 1974 [151]. In metrology chromatographic measurements, Kalmanovskii also introduced the concept of a chromatograph as an individual calibrated measurement system, the quantitative characteristics of which were independent of its application conditions, the analyzed compounds and the used calibration methods [152].

R. REACTION CHROMATO-MASS SPECTROMETRY In the 80s, Russian scientists, V.G. Zaikin and A.I. Mikaya [153] formulated the principles of a new technique which they called “reaction chromato-mass spectrometry”. The method includes the chemical modification of the compound analyzed in the combined GC–MS system, before, within, or after the chromatographic column. A chemical transformation takes place in a special gas-phase microreactor (reactions of hydrogenation, dehydration, and hydrogenolysis) or in the immediate region of the chromatographic column (exchange alkylation, reaction of acylation, basic or acidic H=D exchange). In some studies hydrogen and deuterium were used as both the carrier gas and reagent. Intensive studies on the technique allowed finding a number of new possibilities for structure-analytical purposes. Thus, post-column reactions allowed obtaining chemical transformation of each sample component separated by gas–liquid chromatography into derivatives providing more structural information by mass-spectra.

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Deuteration by reaction GC–MS (deuterohydration, deutero-exchange) provides new possibilities because the mass-spectra of the deuterated products allow an indication of the structure of the compounds (position of double bond, nature and position of functional groups) with great confidence. This approach was suggested for the study of ions in chemical transformation in the gas state. Using a precolumn microreactor and deuterated substances provides a powerful means to study the mechanism of heterocatalytic reactions. The method was effectively used for the study of alcohols, ketones, carboxylic acids, amines, thiacyclanes, stereo-isomeric compounds, etc. The technique serves as the basis for the development of very rapid and highly effective techniques for the solution of various structure-analytical tasks of organic or physical chemistry, etc. The results obtained were also summarized in a monograph [153] and review publications [154,155]. Foreign researchers noted and developed the same concepts [156–159].

S. IDENTIFICATION BY A COMBINATION OF GC/MS AND THE USE OF RETENTION INDICES Gas-chromatography=mass spectrometry (GC=MS) is the generally accepted method for the analysis of complex mixtures of volatile and semi-volatile organic compounds. The most reliable and unambiguous results of GC=MS identification imply the application of at least two non-correlated analytical parameters: the use of standard mass-spectra and of the GC retention indices (RI) on standard phases. I.G. Zenkevich proposed a method including the additional treatment of the MS information for the evaluation of a homologous series of unknown analytes and based on a so-called ion series mass-spectra [160]. The application of GC retention indices may be organized in two manners. First, the formation of an exhaustive RI database for various analyte groups is possible (see [161]) as the example of an RI database for low-boiling compounds on the polymeric sorbent Porapak Q. This approach unites both the experimentally determined and pre-calculated RI values. The new proposed RI precalculation algorithm is based on the semi-logarithmic relationships between the RI and the boiling point (BP) of the analyte and taxonomical parameters, reflecting the position of a compound in a corresponding series (within a homologous series, this may be the molecular weight or the number of carbon atoms, but within congener groups it must be, i.e., the number of heteroatoms, etc.); a,b,c, are correlation coefficients: log RI D a log BP C b A C c Similar correlation equations may be recommended not only for single, but also for two and multidimensional taxonomical groups of organic compounds [162,163]. An alternative way for the identification of unknown compounds includes a preliminary evaluation of any structural fragments in the molecules on the basis of MS data with a subsequent artificial replacement of these fragments by more simple groups, and an RI library search for such structural analogues. This approach in GC–MS identification has been successfully tested for the series of benzyl, benzoyl, dialkylaminoalkyl, and chloro-substituted compounds [164].

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T. DETERMINATION OF TRACE INORGANIC IMPURITIES The gas chromatographic analysis of volatile unstable compounds is a difficult problem and deals with limited possibilities of chromatographic methods and the determination of limiting low concentration of impurities [165]. V.A. Krylov successfully developed new GC techniques for the determination of impurities in such a complicated field. He developed a thermodynamic method for the determination of impurity ‘affiliation’ either to the breakdown products formed during the chromatographic analysis or to the original impurity of the compound. Great attention in impurity analysis was paid to the purity of carrier gases used. Techniques for carrier gases purification (by water up to <107 %, by O2 , CO2 , N2 < 107 % to 108 %) and suppression of residual activity of chromatographic columns were developed. Using quartz capillary column this group pioneered in developing high sensitive analysis of inorganic chlorides and metallo-organic compounds. This technique is characterized by detection limits of 106 to 109 % [166,167].

U. SUMMARY In summary, it may be said that the important changes in policy and economics in Russia undoubtedly will serve to a greater integration of chromatography. In this connection I would like to remind the words of the great Russian writer A.P. Chekhov: “There is no national science like there is no national arithmetic”.

REFERENCES See Chapter S-13 on the ChemWeb Preprint server (http:==www.chemweb.com= preprint=))

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Chinese dragon.

6C. Chromatography in China Yukui Zhang and Guowang Xu National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116011, China

A. YUKUI ZHANG AND GUOWANG XU Yukui Zhang, born at Baoding, Hebei Province on September 13, 1942, graduated from Nankai University in 1965. Since then he has worked at the Dalian Institute of Chemical Physics (DICP), Chinese Academy of Sciences. He obtained the positions of Research Assistant (1978) and Associate Professor (1986), became a Research Professor of chemistry, and Director of the Chromatography Department. He became the Director of the National Chromatographic Research and Analysis (R and A) Center in 1992. From 1992 to 1998, he was the Vice-Director of the scientific committee of DICP, and from 1994 to 1998 he was Vice-Director of DICP. His research interests are in GC, HPLC, CZE, CEC and intelligent separation systems. He is also very active in organizing national and international scientific symposia on chromatography and analytical chemistry. He is the President of the Chinese Chromatographic Society, and President of the

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Chromatographic Society of Liaoning Province. In 1983, he worked at the University of Tu¨bingen, Germany and in 1992, at the American Environmental Protection Agency (EPA) in Research Triangle Park, N.C. He has published more than 200 papers, received 4 patents, co-edited the books HPLC and Its Expert System (1993), Ion-Pair HPLC (1994), and High Performance Liquid Chromatography (1998). He has received the first class Award of Science and Technology from Liaoning Province, and Advancement Awards of Science and Technology from the Chinese Academy of Sciences. He has been invited to give plenary lectures at many international conferences. He has also been evaluated as one of the excellent advisors of students for the M.S. and Ph.D. degrees in the Chinese Academy of Sciences. See Chapter 5B, d, h, l Guowang Xu, born in Sheng county, Zhejiang Province on November 19, 1963, graduated from the Department of Chemical Engineering of Zhejiang Institute of Engineering, China. In September 1984, he became a graduate student in the Chromatography Department of Dalian Institute of Chemical Physics (DICP), Chinese Academy of Sciences. From July 1987, he had been associated with Lu earning his Ph.D. degree in June 1991. Subsequently, he has joined the National Chromatographic Research and Analysis (R and A) Center and has been Head of the GC group since 1992 and an Associate Professor since June 1994. From October 1995 to September 1997, G. Xu worked at the Universita¨t Medizinische Klinik, in Tu¨bingen, Germany under a fellowship from the Max-Planck-Institut. In November 1997 he was promoted to Professor, and since May 1999 he is a Ph.D. advisor at DICP. He is the General Secretary of the Chinese Chromatographic Society. His main research field is in clinical capillary electrophoresis, environmental analysis and chromatography-related method development and applications, especially in the fields of petroleum and environment analysis. Together with Zhang, he has been responsible for the contracts with the EPA, USA. G. Xu has published and presented about 100 papers in journals and conferences and is the co-author of two books. He has received 2 patents. See Chapter 5B, a, d, h, l, r, s

B. CHROMATOGRAPHY IN CHINA IN THE 20TH CENTURY — YUKUI ZHANG AND GUOWANG XU B.1. Journals, societies and conferences Since the early 1950s, Chinese scientists have been involved in chromatographic research and analysis. Now, there are more than 50,000 practicing chromatographers covering every discipline within the area of chromatography and related techniques

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in China. In 1985, the Chinese Society of Chromatography was established, which is presided over by the National Chromatographic Research and Analysis (R and A) Center in Dalian. There are more than 21 divisions of this society in the different provinces and coastal cities. More than 700 papers on chromatography and related techniques are published annually in the scientific literature; about 10% of them are published in international journals. Most of the Chinese chromatography-related papers are published in the following Chinese periodicals: ž Sepu (Chinese Journal of Chromatography), founded in 1984, by the Chinese Society of Chemistry; ž Fenxi Huaxue (Chinese Journal of Analytical Chemistry), founded in 1973; ž Fenxi Ceshi Xuebao (Chinese Journal of Instrumental Analysis), founded in 1982; ž Acta Chimica Sinica (Chemistry in China), founded in 1933; ž Fenxi Kexue Xuebao (Journal of Analytical Science), founded in 1985; ž Scientia Sinica (Sciences in China), founded in 1952; ž Yaowu Fenxi Zazhi (Chinese Journal of Pharmaceutical Analysis), founded in 1981. The Chinese Society of Chemistry, the Chinese Association on Instrumental Analysis, the Chinese Society of Chromatography and the Chinese Academy of Sciences sponsor these periodicals. Many national and international symposia or seminars on chromatography and related techniques are held in China. The most important ones are: ž National Symposium on Analytical Chemistry (every two years); ž National Conference on Chromatography (every two years); ž Beijing Conference and Exhibition on Instrumental Analysis (BCEIA) (every two years); ž National Symposium on Biomedical Chromatography (every two years); ž National Conference on Capillary Electrophoresis (every two years); ž The Asian Conference on Analytical Sciences (every two years); ž The Asia–Pacific International Symposium on Capillary Electrophoresis and Related Micro-scale Techniques (every two years); ž The International Symposium of Worldwide Chinese Scholars on Analytical Chemistry (every three years). Since 1998, five of the above-mentioned symposia have been held (see Table 6C.1) and BCEIA VIII has been be held in October 1999. They reflect the status of chromatography and related techniques in China. The National Conference and Exhibition on Chromatography (NCEC) has been the biggest one in the chromatography-related field in China (Table 6C.2). The Chinese Society of Chemistry, the Chinese Association of Instrumental Analysis and the Chinese Society of Chromatography have sponsored it. It is held in different cities and organized biannually by the National Chromatographic Center, and has the aim of promoting academic exchange among the scientists in the country. The 12th NCEC was held on May 17–20, 1999; 408 papers and 15 plenary lectures on high-performance liquid chromatography (HPLC), gas chromatography (GC), ion chromatography (IC), capillary electrophoresis (CE), thin-layer chromatography (TLC) and related techniques as well as instruments were given. Forty-seven companies related to chromatographic instruments and accessories exhibited their products at this meeting. Some of them are famous international companies such as Beckman, Wa-

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TABLE 6C.1 IMPORTANT CHROMATOGRAPHY-RELATED SYMPOSIA HELD IN CHINA IN 1998 AND 1999 Symposium

Date

Place

No. of papers on chromatography

13th National Conference on Chromatography 9th Beijing Conference and Exhibition on Instrumental Analysis (BCEIA) 5th Sino-German Symposium on Chromatography 1st China–Japan Joint Seminar on Separation Sciences 12th National Conference on Chromatography 8th Beijing Conference and Exhibition on Instrumental Analysis (BCEIA) 3rd National Conference on Capillary Electrophoresis 5th Asian Conference on Analytical Sciences 2nd Asia–Pacific International Symposium on Capillary Electrophoresis and Related Micro-scale Techniques 3rd International Symposium of Worldwide Chinese Scholars on Analytical Chemistry

April, 2001 Oct., 2001

Tai’An Beijing

333 91

Dec., 2000 July, 2000 May, 1999 Oct., 1999

Dalian Dalian Hangzhou Beijing

41 28 423 80

Oct., 1998

Dalian

129

May, 1999 Oct., 1998

Xiamen Dalian

122 169

Dec., 1998

Hong Kong

133

TABLE 6C.2 NATIONAL CONFERENCES AND EXHIBITIONS ON CHROMATOGRAPHY HELD IN CHINA (NCEC) Symp.

Date

Place

No. of papers on chromatography

Symp.

Date

Place

No. of papers on chromatography

1st 2nd 3rd 4th 5th 6th

Oct., 1961 Aug., 1965 Mar., 1979 Jun., 1983 Oct., 1985 Oct., 1987

Dalian Lanzhou Dalian Shanghai Chengdu Shanghai

45 111 144 250 384 361

7th 8th 9th 10th 11th 12th

Oct., 1989 Jul., 1991 Jul., 1993 Apr., 1995 Aug., 1997 May, 1999

Beijing Dalian Qindao Nanjing Dalian Hangzhou

365 325 311 435 390 408

ters, Hewlett-Packard (H-P), Shimadzu, Varian, Perkin-Elmer, etc. Thirty-six Chinese factories or companies showed their instruments and accessories in this meeting. The International Beijing Conference and Exhibition on Instrumental Analysis (BCEIA) similar to PITTCON in the USA, is the most important meeting in China. The conference is sponsored by the State Science and Technology Ministry, and organized biannually by the China Association of Instrumental Analysis since 1985. There are six academic societies that arrange one division of the conference, from which we can get the latest information about the development of instrumental analysis in China. Also, its aim is to promote academic exchanges among the scientists of all countries and also trade cooperation between Chinese and foreign partners in this field. BCEIA has been the largest and the most influential international conference and exhibition on instrumental analysis ever held in China, enjoying an international status

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TABLE 6C.3 CONFERENCE STATISTICS OF PREVIOUS BCEIA 1 No. of

BCEIA 1985

BCEIA 1987

BCEIA 1989

BCEIA 1991

BCEIA 1993

BCEIA 1995

BCEIA 1997

Countries OPs DPs Papers

15 180 460 633

25 126 508 695

20 82 345 447

25 102 308 531

25 134 308 544

23 142 244 472

21 174 379 553

1

OPs: overseas participants; DPs: domestic participants.

TABLE 6C.4 EXHIBITION STATISTICS OF PREVIOUS BCEIA 1 No. of

BCEIA 1985

BCEIA 1987

BCEIA 1989

BCEIA 1991

BCEIA 1993

BCEIA 1995

BCEIA 1997

Countries OCs DCs Visitors

13 109 13 >12000

16 149 19 >14000

13 100 24 >12000

15 107 36 >14000

15 121 42 >14000

14 70 64 >16000

15 66 63 >18000

1

OCs: oversea companies; DCs: domestic companies.

in China similar to that of the Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectroscopy (PITTCON) in the United States. Meanwhile, a long-term friendly relationship and cooperation between BCEIA and PITTCON has been established (see Table 6C.3). Since 1985, seven BCEIA Conferences were held in Beijing except BCEIA’97, which was held in Shanghai. The eighth BCEIA’99 was held on October 25–28 (Conference), and October 25–29 (Exhibition), 1999, in Beijing, the capital of China. Tables 6C.3 and 6C.4 give the statistics of previous BCEIA. Related fields are electron microscopy, mass spectrometry, spectroscopy, chromatography, magnetic resonance and electroanalytical chemistry. Theories, new methods and techniques on instrumental analysis, research and development of instrumentation and their applications in chemistry, physics, biology, medicine, environmental science, life sciences and other basic sciences are covered. In the meantime, from BCEIA’91 on, the conference has had a plenary session with specially invited lectures by internationally prominent scientists on applications of multiple instrumental analysis to current focal problems. For example, E. Bayer from Germany, Edward S. Yeung from the USA, R.R. Ernst from Switzerland, and H. Hashimoto from Japan have been invited to give a plenary lecture. These lectures given by prominent scientists shorten the distances between China and the world and allow Chinese scientists to learn the ‘state of the art’ of key subjects. During the conferences the large variety of exhibitions of scientific instruments has been a great success. More and more people are interested in the ‘state of the art’ of analytical instruments and products from other countries. Many instruments are sold during the exhibitions. Technical seminars have always been welcome. Beginning

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with BCEIA’89, the conference has presented the BCEIA gold award for home-made analytical instruments to promote the development of home-made instruments. The BCEIA proceedings are in English. Interested readers should contact the BCEIA office at the Internet address http:==www.bceia.org Since opening the door of the country in 1978, scientific cooperation and exchange between China and foreign countries have been increasing. More than 200 Chinese chromatographers go annually abroad to attend international scientific conferences, visit research centers and to study as Ph.D. students. Many foreign scientists active in chromatography also visit China. Foreign countries with close connections with China are the USA, Germany, Japan, Sweden, the Netherlands, Italy, etc. In China, every factory tends to have its own analytical center, as do certain companies, for example, SINOPEC (Sino-Petrochemical Engineering Company). Ministries such as the Public Security Ministry and the Nucleic Ministry also have their own research and analytical center. Different grade environmental centers, testing centers for import and export goods, and forensic analytical centers are located all over the country. There are a total of 21 analytical centers belonging to different provinces and coastal cities. With the support of the United Nations, more than 12 analytical centers have been established at universities. Among such centers, the State Science and Technology Ministry of China supports 13 as national analytical centers. The National Chromatographic R and A Center in Dalian is one of them. Total investment for such centers is estimated at more than 1 billion US dollars in the last few years. Chromatography and related techniques are being applied widely in these centers.

B.2. Chromatography-related instruments Before 1958, Chinese chromatographers used home-made equipment. At the beginning of 1950s, the GC detectors used were the volume detector and the platinum-wire combustion–titration detector, but they were useful only for permanent gases and gaseous hydrocarbons. In liquid chromatography, indicators were used for identification of the separated compounds. In paper chromatography either an indicator or UV was used for detection. In later research on gas–liquid chromatography, Chinese scientists built platinum-wire thermal conductivity detectors (TCD), high-sensitivity thermal-sensitive electric resistance detectors and tungsten-wire TCD for packed GC, and the FID detector in capillary GC. At the beginning of the 1960s, GC instruments were produced in Chinese factories with the cooperation of the Dalian Institute of Chemical Physics (DICP), Chinese Academy of Sciences. In the mid-1960s, a large-scale preparative gas chromatograph column with an I.D. of 3–4 cm was developed and used successfully; it could be used to prepare 0.5 kg of chromatographic-purity reagent every day. More than 100 chromatographic reagents were prepared with this machine. At the end of the 1960s, applications-specific chromatographs were developed. A series of detectors were also developed and produced: the thermal-ionization detector, helium-ionization detector, cross-sectional ionization detector, electron-capture detector, flame-photometric detector, microwave plasma detector and the nitrogen–phosphor detector. In the meantime, eight factories for producing GC instruments were established.

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In the 1970s, GC–MS was developed. Two models of HPLC, SY-01 and 150 with UV (254 nm) detector were also produced at a small scale. Since 1978, Chinese scientific research increased significantly. Now many types of chromatography-related instruments are produced in China. For example, W.L. Yu developed gas chromatographic atmospheric-pressure helium microwave plasma emission spectrometry (GC–MES) in 1985, and GC microwave-induced plasma atomic emission spectrometry (GC–MIP) in 1991. The model 2030 HPLC instrument developed in Dalian received the BCEIA gold award in 1991. Here, we review the state of the art on Chinese chromatography-related instruments based on the 12th NCEC. GC is a mature technique by any standard. Therefore, China has more institutions that can produce GC instruments than HPLC instruments. Some new models of GC instruments were displayed at the 12th NCEC, for example, SP-2000 from the Beijing Analytical Instrument Factory, GC-9900 from the Shanghai Kechuang Chromatography Instruments Company, GC 7890II from the Shanghai Techcomp (a Hong Kong– Shanghai joint venture) and a micro-GC instrument with solid-state TCD (SSD) produced by DICP. Besides the general-purpose gas chromatographs, a number of applications-specific analyzers were developed based on GC, e.g., analyzers for natural gas, coal gas, refinery gas, liquefied petroleum gas, distilled spirit, mine gas, transformer oil and analyzer of hydrocarbons in liquid oxygen. On-line industrial GC has also been produced, for example, at the Nanjing Factory of Analytical Instruments. Compared with the many GC companies, only four companies produce HPLC and ion-exchange chromatography (IEC) instruments. The largest is the Dalian Elite Scientific Instrument Co., Ltd. Two models of HPLC including isocratic and gradient operation are provided. In Chinese laboratories, people can find very advanced CE instruments from Beckman, Hewlett Packard, Bio-Rad, etc.; however, only a limited number of institutes and universities can build CE instruments. Most of them are research prototypes. The Beijing Institute of New Technology and Hebei University have a better commercial capacity. Some GC and LC instruments and column types made in China are summarized in Tables 6C.5 and 6C.6. All of them are lower cost systems, without auto-injector and cryogenic port. Automation, reliability and product stability are the key areas for Chinese instruments to be improved in the future. Chromatographic workstations are attracting more and more attention in China. More than 20 institutions have this kind of product. Several years ago, the price of a computer was very high, but now it has been significantly reduced. Compared with the data processor for chromatography, the chromatographic workstation can be used more conveniently and has more functions. It is clear that the chromatographic workstation will gradually take the place of data processors. All of the workstations developed in China are based on a Chinese word processor system. Different from other workstations, GCLAB 5.0 developed in Dalian is connected with a ‘living’ chromatogram base [1]; the users can search 500 typical chromatograms to obtain the key references. Besides GCLAB, the National Chromatographic R and A. Center also developed the DL-800 and WDL-95 workstations especially for HPLC.

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As the oldest national ultra-pure gas base, DICP has developed a series of purification tubes to remove the impurity in the gases. The purifiers produced can be used to remove oxygen and water with a clear color change, methane, hydrocarbons and nitrogen. Besides individual tubes, the specific purification machines can also be tailor-made. In China, many companies produce hydrogen, nitrogen or air generators, and a generator with combined production of hydrogen, nitrogen and air also exists. The analytical laboratories are beginning to say goodbye to gas cylinders. Typical purity of nitrogen produced by such a generator is oxygen less than 2–3 ppm, and dew point below 56 to 70ºC. The typical purity of hydrogen is 99.999%. Typical flow rate is 0–300 ml=min for hydrogen and nitrogen, 0–3000 ml=min for air. The working pressure of most generators is 0–0.4 MPa. At the present time, it is estimated that more than 10,000 HPLC and 50,000– 70,000 GC instruments are being used in China and about 3000 new chromatographs are required each year. For that, Hewlett-Packard has built a factory in Shanghai to produce the HP 4890D and 1490 GC, and the Shimadzu (Suzhou) Instruments Manufacturing Co., Ltd. was established in 1998 to produce the GC-14C. The Chinese government, in its 9th five-year plan, has granted a large budget to advance and improve homemade analytical instruments. It can be expected that, in the coming years, Chinese chromatography-related instruments will have a significant development.

B.3. Stationary phases and columns The research on gas adsorption chromatography in China began in 1953. P.C. Lu and his colleagues investigated the pretreatment conditions of home-made active carbon, silica and aluminum oxide, and separated the petroleum gases and natural gases. Later, 5A and 13X molecular sieves were prepared successfully at DICP and the level of gas analysis was advanced [1]. In the late 1960s, porous organic polymer beads (GDX), similar to Porapaks and the Chromosorb Century Series, and carbon molecular sieve (TDX) were developed by the Beijing Institute of Chemistry, Chinese Academy of Sciences, and produced by the Tianjin 2nd Chemical Reagent Factory. Graphitized carbon black has been produced since 1983 at the Jilin Carbon Factory with the cooperation of the Jilin Institute of Chemical Engineering. Now different grades of active carbon, fine and wide porous silica, and α and γ-aluminum oxide are produced in the various provinces. Molecular sieves A, X and Y, as well as GDX and TDX are produced in Shanghai, Tianjin and Dalian. Research on gas–liquid chromatography started in China in 1956. Based on the red diatomite support 5701 developed by DICP, red diatomite support 6201 (similar to Chromosorb P) has been produced in Dalian since 1965. In the meantime, red diatomite support 201, white diatomite supports 101 and 102 (similar to Celite 545 and Chromosorb W), were also prepared in Shanghai. Today various supports are produced in China. Capillary gas chromatography began in China in 1959. At the beginning, B.L. Zhu and J.Q. Ding at DICP used a plastic tube to prepare capillary columns. Several years later, some institutes in Beijing, Lanzhou and Shanghai began to study capillary chromatography. After a machine to produce glass capillary tubes was constructed in 1962 at

652

TABLE 6C.5 GAS CHROMATOGRAPHIC INSTRUMENTS MADE IN CHINA (from ref. [17]) Producers

Beijing Analytical Instrument Factory

Shanghai Analytical Instrument General Factory

Lunan Chemical Industrial Instrument Factory

SP-3700 SP-6000 SP-3400 SP-3420 SP-3800 SP-2000 SP-2304A SP-2307 SP-2308 SP-2305 SQ-204 SQ-203 SQ-206 GC102 GC-102N GC102NJ GC102GD 1102 GC112 GC122 SP-502 SP-9800 SP-9802 SP-6800 SP-2000 SP-501N DH-910 ZT-960 7890F 7890II

Column types

Chromatographs

capillary

fast GC

* * * * * *

* * * * *

packed * * * * * * * * * *

* *

Detectors portable

other

1

2 3 4

* * *

* * * * * * * * *

specified

5

TCD

FID

ECD

FPD

TSD

* * * * * *

* * * * * *

* * * * * * *

* * * * * *

* * * * * *

* * *

* * * * * * * * * * *

*

*

*

*

* * *

* *

*

*

* * * *

6 * * * * *

5

7 * * * * *

* * * * * * * * * *

8 9 * *

* * * * * * * * * *

* *

NPD

other

* *

* *

Chapter 6

Shanghai Techcomp Instrument Co., Ltd.

Products

Producers

Products

Shanghai Kechuang Chromatographic Instrument Corporation

GC900 GC910 88 GC-9900 GC-920 GC-950 GC-960 GC-4000 GC-4006 GC-4007 GC-4008 GC-4009 GC-4010 9750 9710 SC-2000 SC-200 8800 8810 8820 GS-101 GS-1 GS-5 KF-2100

Shanghai Haixin Chromatographic Instrument Factory Dongxi Electronic Institute

Wenling Fuli Analytical Instrument Co., Ltd. Chongqing Chuanyi Co., Ltd. Shanghai Rex Chuangyi Instrument Co., Ltd. Dalian Elite Scientific Instrument CO., Ltd. Dalian Institute of Chemical Physics

Column types

Chromatographs

capillary

packed

fast GC

*

* * * * * * * *

* * * * * *

* * * * * * *

*

specified

Detectors portable

other

TCD

FID

ECD

FPD

5 5 5

* * * * * *

* *

* *

* *

* * * * * * * * * * * * * *

* *

* *

*

*

* * * *

10 7 11 12 12 * * * * * * * * * *

* 5 5

* * * * * * *

TSD

NPD

other

Chromatography around the World

TABLE 6C.5 (continued)

* * * *

* *

*

* * *

13 14 *

15

653

Footnotes: 1 D a series of applications-specific analyzers, including natural gas analyzer, coal gas analyzer, refinery gas analyzer, liquefied petroleum gas analyzer, distilled spirit analyzer, mine gas analyzer, transformer oil analyzer; and analyzer of hydrocarbons in liquid oxygen; 2 D a new concept GC; 3 D simple GC; 4 D transformer oil analyzer; 5 D a series of GC instruments; 6 D distilled spirit analyzer; 7 D transformer oil analyzer; 8 D oil-well analyzer; 9 D organic carbon analyzer; 10 D natural gas analyzer; 11 D coal gas analyzer; 12 D sanitary hygienic sample analyzer; 13 D hydrocarbon analyzer in air separator; 14 D on-line hydrocarbon analyzer in air separator; 15 D solid state TCD. Asterisk means similar in content to ‘1999 International Chromatography Guide’ in J. Chromatogr. Sci., 37 (1999).

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TABLE 6C.6 LIQUID CHROMATOGRAPHIC INSTRUMENTS MADE IN CHINA (from ref. [17]) Producers

Products

Detectors

Dalian Elite Scientific Instrument Co., Ltd. Beijing Analytical Instrument Factory

P200 P200II SY-5000 SY-5300A BFS-2010 LC-5500 DX-100T * EP-100 * EP-1000 *

Pumps

electrochemical fluorescence UV=VIS other piston diaphragm other

Dongxi Electronic Institute, Beijing Beijing Epoch

*

* * * * * *

* * * * * * * *

1 1

Asterisk means similar in content to ‘1999 International Chromatography Guide’ in J. Chromatogr. Sci., 37 (1999). 1 D ion chromatograph.

Fig. 6C.1. Gas chromatogram of Polywax 655. Column: 6 m ð 0.53 mm fused silica; carrier gas: He; column temperature: 40–430ºC at 6ºC=min; injection mode: on-column.

DICP, a great advance was made in capillary chromatography. Before 1966, the columns prepared were mainly wall-coated open tubular columns with squalane and Apiezon grease as the stationary phase. During the Cultural Revolution, the research was stopped. After 1972, the research was started again. Now, Chinese scientists have been able to use both the static and dynamic methods to produce columns with different polarities including coated or cross-linked WCOT columns, porous-layer open tubular columns (PLOT), support-coated open tubular columns (SCOT), packed capillary columns, etc. Many groups [2,3] produce GC columns. Z.F. Lue developed a simple method for the preparation of deactivated and thermal glass SCOT column which can be used isothermally at 300ºC. The bleeding of this column is comparable to that of the fused-silica columns. W.Z. Lu’s group [4] at the Beijing Institute of Petroleum Processing prepared a series of capillary columns to separate crude petroleum oils and products (i.e., stable alumina column, cross-linked, silanol-terminated methyl polysiloxane WCOT and 13X PLOT stainless-steel capillary columns for high-temperature gas chromatography operated at over 400ºC) (Fig. 6C.1). Similarly, J. Wu’s group at the Beijing Scientific

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Institute prepared aluminum-coated capillary columns for Petroleum Exploration and Development. R.N. Fu’s group [5] at Beijing University of Technology prepared a series of columns with special selectivity to separate isomers based on cyclodextrin derivatives, mesomorphic ‘polysiloxane polymers and crown ether polysiloxane stationary phases. C.Y. Wu’s group [6] at Wuhan University studied a series of specially selective columns with crown ether polysiloxane stationary phase; they used similar techniques and the sol-gel technique to prepare solid phase micro-extraction fiber. The columns produced at DICP have been applied in various fields and solved many practical problems in China. Compared with other institutions, the Lanzhou Institute of Chemical Physics (LICP), Chinese Academy of Sciences, can prepare more types of PLOT columns including molecular sieve 5A, aluminum oxide, carbon molecular sieve and different polar organic polymer porous-layer open tubular capillary (OPPLOT A, Q, R and S) columns [3]. In the meantime, several groups including L.M. Zhou’s group at DICP [7], and Q.Y. Ou’s group at LICP developed chiral columns made from amide types and cyclodextrin derivatives [3]. The price of these columns made in China is lower than of similar columns from Supelco, SGE and Alltech. Crude fused-silica capillary tubes were prepared in 1983 at the Beijing Institute of Petroleum Processing, and now are produced by the Hebei Yongnian Optical Conductive Fiber Plant. Different from the Western countries, industrial laboratories in China are using more packed columns; some analytical standards of environmental samples and chemicals are based on the use of packed gas chromatographic columns. Various HPLC silica-based packings of the YWG and YQG series have been developed at DICP in the late 1970s, and produced at the Tianjin 2nd Chemical Reagent Factory. Some new packings have been studied or are being developed by different groups. X.D. Geng, developed packings for high-performance hydrophobic interaction chromatography (1991). J.D. Wang’s group at DICP studied the bonding of C8 and C18 groups to 2.1 µm mono-disperse non-porous silica for the preparation of novel hydrophobic packings. G.Q. Liu’s group at the Institute of Chemistry, Chinese Academy of Sciences, has developed a series of HPLC packings, especially for biomolecules. Besides the imported columns, LC columns produced by the Dalian Elite Scientific Instruments Co., Ltd., occupy most of the Chinese market. Several years ago, the Younite Unimicro Technologies Co., Ltd. was organized. It is a USA–China joint venture and specializes in producing packed capillary columns for CEC. Although the research on capillary electrophoresis and electrochromatography is very high in China, the market for electrochromatography is very small.

B.4. Theoretical research and expert system of chromatography With the development of the chromatographic technique, the theory of column chromatography has been studied extensively to achieve high-performance, high-selectivity chromatographic columns and optimum separation conditions. In the late 1950s, J.Q. Ding and B.L. Zhu at DICP gave not only the chromatographic profile equation including retention time, longitudinal dispersing and mass transfer rate based on dynamics, but they also proved the result with the electric simulation.

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The relationship of the chromatographic peak zone width and retention time was also discussed. P.C. Lu and his colleagues have made a systematic study of the chromatographic theory since the late 1950s [8]. To evaluate the resolution of two peaks, in 1957 Lu suggested using the ratio of the concentration maximum with the smaller peak’s height of two peaks, to the concentration at the intersecting point of the two peaks as the resolution criterion. Since the early 1950s, the standard method for two separated peaks, resolution is calculated by dividing the difference of the retention times with the width of the two peaks at half height. It has been pointed out that for different concentrations of the components, the required resolution should be different depending on the analytical requirements. In the early 1960s, Lu studied high-speed gas chromatography. A theoretical consideration on the selection of optimum operation condition for high-speed gas chromatography was reported and applied to the separation of gaseous hydrocarbon mixtures. The theory of column chromatography for multi-component separation was given in 1979. In the meantime, based on these theoretical considerations, a series of high-performance gas chromatography and liquid chromatography columns have been developed, for example, small- bore 2 mm HPLC columns with 60,000 plates per meter. The result on these theoretical studies have been systemically summarized in two books [8,9]. Since the late 1970s, Lu’s interest has been in intelligent chromatography, especially in expert systems (ESC) [10–13], because more and more complicated samples are today being analyzed, especially in the field of life and environmental sciences. To analyze these samples, ESC is helpful. The research for ESC has been done by more than 30 persons in P.C. Lu’s Center between 1979 and 1995. Lu has proposed that such an expert system must be based on a strong theoretical foundation. It can be used to optimize the methods including not only finding out the optimum mode, column system and operating condition, but also its optimum quantitative and qualitative analytical methods. In this case, the kinetic theory of chromatography is not only used to develop a high-performance column system, but also to predict the whole chromatogram for further developing a fitting curve method for intelligent optimization and quantitation. Y.K. Zhang and associates observed the linear relationship between the standard deviation of a Gaussian constituent (σ), and τ (τ D time constant for exponential decay) in the EMG equation, and the retention time in 1980. H.F. Zhou developed a statistical mathematical way to solve the differential equation with all variables including extracolumn effects, and established an equation with only two parameters to predict those asymmetrical peaks. Y.K. Zhang and associates studied curve fitting of the chromatogram based on the EMG equation (exponential modified Gaussian equation). Lin and co-workers developed an algorithm for saving the chromatograms by storing retention time, peak height and the EMG parameters. For optimization, Lu pointed out that one must include the recommendation of the optimum method, then the column system, and finally the optimum operation conditions. The optimum operational conditions must be stepwise. Linear programming is only a limiting case for the analysis of homologues. For that, Lin Binsheng studied the optimization and identification in any kind of multi-step temperature-programmed

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gas chromatography. X.M. Zhang and co-workers studied an optimization strategy with stepwise temperature programming [13], and G.W. Xu suggested an intelligent column temperature optimization method; both of these have been used to analyze environmental samples [14]. In HPLC, H.F. Zhou suggested the ‘moving overlapping resolution maps’ method. X.M. Liang and co-workers developed an intelligent method to optimize the buffers. With the help of a computer, the user can simulate the moving trace of the components in the column. After the operation conditions are optimized, the remaining problem is to predict the retention factor of the analyzed compounds on the mode and column system used. Since the 1960s, Lu has developed the thermodynamic theory of chromatography by using statistical thermodynamics and established the relationship between macro-property (retention capacity) and micro-property (molecular structure parameters of the solute and column system). H.F. Zhou and associates studied the effects of the molecular structure on the parameters of the fundamental retention equation. For qualitative analysis with such high-resolution chromatographic methods, more precise retention data are needed. A series of new methods have been developed to solve this problem. For example, the correction of the compressibility of the gas phase along the column was studied, especially during changing the temperature; the correction of delay was considered to catch the peaks during the changing of liquid mobile phase, and retention values were predicted directly from the reference data obtained by high-resolution gas chromatography. H.C. Li and C.Z. Dai used a computer in 1981 to calculate and identify the retention indices of lower boiling hydrocarbons. Curvers and associates (18) described the procedures which allow the calculation of the retention temperatures, and from these, the accurate programmed retention indices; within certain limits, then the initial oven temperature and programming rate can be chosen freely. Based on Curvers’ method, Y.F. Guan and his group developed a method to predict the retention index in temperature programmed gas chromatography by using the isocratic retention index at two different temperatures. N. Chen described a method for peak identification from the interaction index c, which was derived from the fundamental retention equation log k 0 D a C cC B in reversed-phase high-performance liquid chromatography to quantitatively describe the difference between the solute–strong solvent and solute– weak solvent interaction. The expert system of gas chromatography has been used by G.W. Xu’s group to optimize the analytical method for 70 toxic compounds in air in cooperation with the EPA in the USA, and to suggest the column systems for industrial gas chromatographs used in ethylene plants [15]. An expert system for high-performance liquid chromatography has been used by X.M. Liang’s group to store the chromatogram and analyze the 209 PCB congeners, 136 PCDD and PCDF congeners in air, water. In the meantime, a unified comprehensive and intelligent analytical method (UCIAM) is being developed at this laboratory to analyze the quality control of traditional Chinese medicines and to establish a scientific foundation for protocols. Besides DICP, many groups have made major contributions. For example, J.D. Hou at Zhejiang University has achieved very good results in the optimization of the HPLC operational conditions, the quantitative structure retention relationship and the determination of the thermodynamic parameters by gas–liquid chromatography [16].

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B.5. Applications Chromatography was developed in China with the goals for scientific research and production. In the 1950s, the main subject was the analysis of petroleum products. In the 1960s, the applications of chromatography in chemical product analysis were developed greatly; high-speed and trace analysis were the significant achievements. Tang’s group at DICP developed a series of methods to analyze the impurities in ultra-pure gases by preconcentration gas chromatography with the helium ionization detector. Since 1978, Chinese chromatography has been advanced greatly in various fields. Among the analysts active in chromatography, five are members of the Chinese Academy of Sciences: P.C. Lu at DICP, T.H. Zhou at the Beijing Institute of Material Medicine, W.Z. Lu at the Beijing Institute of Petroleum Processing, X.B. Xu at the Beijing Research Center for Eco-environment Science and Y.Z. Chen at Lanzhou University. P.C. Lu’s main interests are in developing chromatographic theory, new methods and new techniques, especially the expert system on chromatography. T.H. Zhou developed a series of pharmaceutical analytical methods and built the first monitoring center of doping control analysis of athletes’ biological fluids in China. W.Z. Lu developed a series of methods and techniques to solve the tasks of petroleum product analysis. Y.Z. Chen has been engaged in the investigation of natural products and traditional Chinese medicines. Finally, X.B. Xu has been involved in the various studies on occurrence and distribution of pollutants in the environment. Because of the limited space in this chapter, the readers are referred to the proceedings of chromatography-related conferences held in China and Chinese journals on analytical chemistry to learn more about the detailed work of Chinese scientists. Primarily, the papers presented at the 12th NCEC (1999 conference) were on fundamentals and stationary phase column development in GC (28 papers) and HPLC (16 papers). Most of the papers on applications (153 papers) were in the fields of: (a) petroleum, chemical industry and light industry, (b) plant, food and agriculture, (c) clinical, drug and biochemistry, and (d) environmental pesticides. There were a number of papers on TLC (22 papers), IC (8 papers) and CE=CEC (22 papers). In capillary electrophoretic (CE) research, China is one of the pioneering countries. In the early 1980s, A. Zhu, at the Institute of Chemistry, Chinese Academy of Sciences, first introduced CE into China. Since that time, and especially in the 1990s, CE has undergone extensive development in this country, in addition to the many achievements made by overseas Chinese scientists. Three national conferences on CE and an Asia– Pacific international symposium on capillary electrophoresis and related micro-scale techniques have been held in China, organized jointly by DICP and the Institute of Chemistry of the Chinese Academy of Sciences. Now, Chinese scientists have achieved good results not only in the CE method development, but also in the applications of CE in biochemistry, clinical chemistry, pharmaceutics, etc. Of the papers presented at the 3rd NCCE (1998 conference) 13 papers were on fundamentals, 8 papers on novel methods, columns and buffers, 5 papers on detection and instrumentation and 11 papers on chiral separation. The 20 applied papers covered clinical chemistry, traditional Chinese medicines, pharmaceutical sciences and food and plant analyses. A recent view of China’s Dalian Institute may be found in R. Stevenson, The World of Separation Science, Amer. Lab. News Ed. 31 (1999) pp. 4–8.

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REFERENCES See Chapter S-13 on the Chem Web Preprint server (http:==www.chemweb.com= preprint=)

Toucan bird.

6D. Development of chromatography in Latin America Fernando Mauro Lanc¸as University of Sa˜o Paulo, Institute of Chemistry at Sa˜o Carlos, 13560-970 Sa˜o Carlos (SP), Brazil

A. INTRODUCTION Although intensively used in Latin America since its early discovery, the chromatographic techniques started in the beginning of the 1980s to receive the desired attention from the local scientific Institutions [1]. Before this period, a few islands of progress in the chromatographic techniques were observed (but usually not documented) in Latin America. Part of this phenomenon is due to the fact that since the invention of gas chromatography in the 1950s, most of the early chromatographic applications occurred in

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the industrial field rather than in academia. A good example supporting this assumption can be found in the Brazilian Petroleum Industry, particularly at PETROBRAS, a huge petrochemical complex spread in several areas over the country, for whose development chromatography was an essential tool in the prospect quality control, refining processing, or development of new applications [2]. Hundreds of GCs were installed in the several plants and branches of this petrochemical complex, and these have been (and still are) the moving force in assuring the quality of the chemical feedstock derived from crude oil. A similar situation happened in Venezuela where INTEVEP, the major domestic petrochemical complex [3] was responsible for most developments and applications of gas chromatography in the industry. Unfortunately, since the beginning of the development of the industries in Latin America, the researchers working in such areas had no incentives (and in many cases not even allowed) to publish their results in the open scientific literature. As a result, a huge volume of precious scientific and technological information was lost, since several of these early investigators retired from their work and started new businesses or dedicated the rest of their life to non-technical activities. It should also be pointed out that until very recently patents were not considered to be an important issue from both points of view: the investigators and the companies where they work. Again, many important scientific and technological advances achieved in the industry were never published or patented and, as a consequence, were lost along the time. This situation started to change with the organization of the Latin American Congress in Chromatography (Congresso Latino-Americano de Cromatografia, COLACRO) in Rio de Janeiro, Brazil, March 1986, chaired by F.M. Lanc¸as from the University of Sa˜o Paulo, Brazil [4]. From its beginning, this meeting helped in the promotion=diffusion of the chromatographic techniques within the Latin American continent and the interaction of Latin American scientists and technicians with those from other continents. COLACRO was created to be a truly International Symposium series from the beginning, although the indication of ‘Latin America’ in this title might suggest a more restricted meeting. A very important COLACRO issue has been to break down the above-described cultural paradigm through a strong interaction with the industries. In this sense, the major sponsor of the first COLACRO was PETROBRAS, a Brazilian petrochemical establishment [5]. To ensure the diffusion of our ideas aiming to obtain the participation of all Latin American countries in the meeting, a Latin American Committee on Chromatography (Comiteˆ Latino-Americano de Cromatografia, CLAC) was formed with two representatives from each Latin American country. One representative is appointed from the academic environment (through local chemical societies, chromatography divisions, chromatography discussion groups, etc.), while the other member is selected from non-academic institutions (usually from industries), thus forming a Latin American network. Since its foundation, F.M. Lanc¸as from Brazil, has been appointed as the general chairman of this organization, which organizes and is responsible for each COLACRO meeting. Through all of these precautions, a considerable participation of scientists from the industrial area has been observed [6–12] with their major contributions being later published in the open literature, particularly in the Journal of High Resolution Chromatography (HRC). A comparison between the number of papers published in international journals (particularly but not restricted to HRC)

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before and after COLACRO (see, e.g., the Editorial published in HRC covering this subject) [13] will show the success of the symposium series with respect to the number of papers now published and the papers coming from non-academic environments. To ensure the scientific and technological quality of the contributed papers (posters) as well as invited papers (plenary lectures), an International Scientific Committee was formed which includes F.M. Lanc¸as (chairman, Brazil), Karl Cramers (Netherlands), Harold McNair (USA) and Pat Sandra (Belgium). More than 2000 contributions (posters) and 150 invited plenary lectures were accepted and presented during the seven symposia organized up to now [6–12]. The invited speakers, most of them listed in this book as pioneers in their fields [4,14–18] brought a unique contribution to separation science through their experience. Contributed posters allowed an interactive exchange of experience among Latin American scientists and with their colleagues from other parts of the world. In this sense, the original goals of COLACRO have been fulfilled. Another important issue of COLACRO was to recognize those scientists who were pioneers in their research field and whose contributions were important to spreading the chromatographic techniques worldwide. With this purpose the COLACRO Medal was instituted; since the first meeting, it should be pointed out that the COLACRO Medal is not restricted to Latin American scientists; in fact the first award was given to Harold M. McNair from Virginia Polytechnic Institute and State University, USA, in 1986 [5]. Although not only the COLACRO medallists have been involved with the development of chromatography and related techniques in Latin America, these medallists are among the pioneers of the development of these techniques in Latin America. Since the meeting is held biannually in different countries, the local committee of the host country is responsible for selecting the name of up to three candidates and submit them to the Permanent Scientific Committee with their corresponding curriculum vitae and a short description of the reason for their nomination. This committee will then make the final decision about the awardees, thus reflecting a truly peer-reviewed decision by an international board of scientists. As a consequence, the selected nominees are among the best scientists in separation science in their countries and present a good reflection of the situation in that particular subject. The next chapter for the development of chromatography in Latin America continues to be written. The COLACRO (COLACRO VIII) met in Buenos Aires, Argentina, in April 2000. We hoped to continue to spread the chromatographic and related techniques worldwide and promote the interaction among scientist dealing with separation sciences in Latin America and in the rest of the world. Table 6D.1 displays some interesting features of the several COLACRO meetings.

B. ACKNOWLEDGMENTS The Fundac¸a˜o de Apoio a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and all COLACRO sponsors for grants that allowed to start and maintain the meeting all these years are acknowledged. Without their generous support our ideas could have never been fulfilled.

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TABLE 6D.1 SELECTED FEATURES OF COLACRO MEETINGS Edition: Country 1 Year Posters Participants 2 1 2

I

II

III

IV

V

VI

VII

VIII

Bra 1986 170 400

Arg 1988 145 350

Bra 1990 131 380

Mex 1992 87 300

Chi 1994 139 380

Ven 1996 204 420

Bra 1998 360 600

Arg 2000 –

Bra D Brazil; Arg D Argentine; Mex D Me´xico; Chi D Chile; Ven D Venezuela. Estimated number of participants.

C. FERNANDO MAURO LANC¸AS University of Sa˜o Paulo, Institute of Chemistry at Sa˜o Carlos, 13560-970 Sa˜o Carlos (SP), Brazil. Phone: C55-16 273 9983; Fax: C55-16 273 9984; E-mail: [email protected]

Fernando Mauro Lanc¸as obtained a B.Sc. degree in biological sciences in 1972 and a B.Sc. degree in chemistry in 1974. After experience as a high school teacher, he enrolled in a M.Sc. program at the University of Campinas, Brazil, receiving his degree in 1978. Subsequently, he obtained a Ph.D. degree from the same university, in the area of analytical chemistry (use of separation methods for the analysis of radio-labelled species produced in nuclear reactors and gamma-ray irradiation sources). The program was completed in 1981; in the same year he started a postdoctoral fellowship with Harold H. McNair at Virginia Polytechnic Institute and State University in Blacksburg, USA, on the use of chromatographic techniques for the analysis of alternative liquid fuels. Upon his return to Brazil in 1984, F.M. Lanc¸as organized the Sa˜o Paulo State Chromatography Discussion Group and the First Brazilian Symposium on Chromatography. In 1986, he organized and chaired the First Latin American Congress on Chromatography (COLACRO), held in Rio de Janeiro, Brazil, that became the most important forum in separation sciences in Latin America. In 1988, during the meeting in Buenos Aires, Argentina, F.M. Lanc¸as was awarded with the COLACRO Medal. Since 1977, F.M. Lanc¸as has been associated with the University of Sa˜o Paulo, Brazil, as a Professor of Chemistry and Head of the Chemistry Department. He published more than 150 papers in peer-reviewed journals, two books, and has supervised more than 70 graduate theses. He has been serving on the editorial board of several international journals. F.M. Lanc¸as’ major scientific interest at this time is focused on the development and application of all modes of microcolumn separation techniques, sample preparation, hyphenated techniques and unified chromatography. His early educational interest was in the social sciences where he took an undergraduate course (not concluded) with emphasis on philosophy. This was followed by a B.Sc. degree in physical and biological sciences (concluded in 1972) and by a B.S. degree in

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chemistry, concluded in 1974, before obtaining M.Sc. and Ph.D. degrees in 1978 and 1982, respectively, both from the State University of Campinas (UNICAMP), Brazil, in the field of analytical chemistry. F.M. Lanc¸as’ research interest started during his adolescence, when he was fascinated by the many wonderful applications of nuclear energy, and worrying about the wrong use of the nuclear reactors. At the age of 18, he started a series of seminars in Brazil discussing the beneficial and malefic uses of nuclear power. This area was later selected to pursue his M.Sc. and Ph.D. degrees, both on the use of analytical chemistry with an emphasis on radiochemistry. During his master’s thesis, a study of the kinetics and annealing mechanisms of radioactive Cr-51 produced in nuclear reactors on neutron irradiation of coordination compounds led to prove the stepwise mechanism of those reactions. Extensive separation of all fragments produced on neutron irradiation using ion-exchange chromatography and high-voltage electrophoresis gave support to the proposed mechanisms and recombination model [19]. In his research leading to the Ph.D. degree, radio-labelled Cr-51 was used to dope solid-phase coordination compounds in order to investigate the transference of the activity from the doping species to the host compound. Again, chromatography was extensively used in this work to isolate the several species produced after the developed doping process, thus showing that the recombination mechanisms of doping hot species was similar to those of the radio-labelled species produced in the nuclear reactor [20]. After receiving his Ph.D., F.M. Lanc¸as spent two years (1982–1983) as a postdoctoral fellow at Virginia Tech at Blacksburg, USA, with H.M. McNair. At that time his major interest was on the use of high-resolution chromatographic techniques in the analysis of alternative fuels (coal, biomass, shale oil), a project already started in Brazil. Upon his return to Brazil, F.M. Lanc¸as’ activities in furthering chromatography in Latin America included (1) the organization and chairmanship of the First National Symposium on Chromatography, promoted by the Brazilian Chemical Society (SBQ) in 1984, (2) the organization of the Sa˜o Paulo state Chromatography Discussion Group (in 1984), in which he served as its first president, (3) and the foundation of several Chromatography Discussion Groups in Brazil and other Latin American countries between 1984 and 1988. During this time he organized the first Symposium on Chromatography at the National Meeting on Analytical Chemistry and the First Latin American Congress on Chromatography, COLACRO, which was held in Rio de Janeiro, Brazil, 1986. Also, he started the Laboratory of Chromatography at the University of Sa˜o Paulo, Institute of Chemistry at Sa˜o Carlos, Brazil. Since 1984, F.M. Lanc¸as has concentrated his research efforts on the development of instrumentation and applications of sample preparation and high-resolution chromatographic techniques to the study of complex samples, particularly those related to social problems including environmental concerns [21], food [22], public health [23], and alternative fuels [24]. He has been involved with the development and preparation of high-resolution capillary columns for gas, liquid, supercritical fluid chromatography and electrodriven separation methods [25], including: electrochromatography; the development of special instrumentation for supercritical fluid extraction (SFE) and pressurized solvent extraction (PSE) [26]; instrumentation for the conversion (liquefaction, pyrolysis and extraction) and analysis of alternative fuels [27]; the use of mass spectrometry as a

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detector for GC, HPLC and SFC [28]. Concerning the applications of these techniques, his interest has been focused on environmental analysis (organic micropollutants in water, soil and air), food (pesticides, nitrosamines, PCBs, PAHs, nitro-PAHs), public health (chiral pharmaceutical compounds, phytotherapeutic compounds), and alternative fuels (characterization of the products formed upon conversion processes). More recently, F.M. Lanc¸as developed several on-line hyphenated systems for sample preparation, clean up, concentration (enrichment) and analyte determination. One of these systems serves as an interface for on-line coupling of supercritical fluid extraction to electrodriven separation methods such us capillary zone electrophoresis (SFE–CZE) [29]. Among others, this system allows the extraction of pesticides from food followed by a sample clean up and concentration steps in the interface before being on-line transferred to the electrophoretic system for analyte determination. Another high-priority field of research is unified chromatography. This project involves the development of an instrument and accessories to allow the use of gas chromatography, liquid chromatography, and supercritical-fluid chromatography and related techniques (such as subcritical fluid chromatography, enhanced fluidity chromatography, high-density gas chromatography) in the same instrument [30]. Since 1977 F.M. Lanc¸as has been associated with the University of Sa˜o Paulo, Institute of Chemistry of Sa˜o Carlos were he founded the Laboratory of Chromatography and is now Professor of Chemistry. He has published close to 150 scientific papers, two books, several chapters in international books, and supervised more than 70-graduate thesis and 10 undergraduate thesis. He has presented invited courses and lectures in different locations among several countries including Argentina, Brazil, Chile, Colombia, Cuba, Indonesia, Italy, Mexico, the United States and Venezuela. F.M. Lanc¸as is serving or has served until recently as a member of the editorial board of several journals including the Journal of High Resolution Chromatography, Journal of Microcolumn Separations, Journal of Capillary Electrophoresis, Fuel Science and Technology International and Energy Sources and Pesticides. F.M. Lanc¸as is member of several scientific associations, including the Brazilian Chemical Society, the Brazilian Society for the Advancement of the Sciences, the New York Academy of Science, the American Chemical Society and the Latin American Committee on Chromatography. See Chapter 5B, d, h, k, l, o, s

D. CLYDE N. CARDUCCI Clyde N. Carducci is Consultant Professor of the University of Buenos Aires (UBA), and Professor of Analytical Chemistry in the Faculty of Pharmacy and Biochemistry of the University. She has been elected Vice-President of the Argentine National Academy of Pharmacy and Biochemistry of Argentina and is presently a member of the Committee of Analytical Chemistry of Pharmaceuticals for the National Argentinean Pharmacopoeia. Since 1986, she has been a member of the Latin-American Committee of Chromatography and Related Techniques (COLACRO), and since 1983 has been

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active in the Chromatography Division of the Argentine Chemical Association. For two years C.N. Carducci has been a member of the Latin American and Caribbean Section of the AOAC International from Argentina. In March 1998, she was awarded the COLACRO Medal during the Seventh Latin-American Congress on Chromatography and Related Techniques. In December 1997 she received an award for her meritorious career during the Third LatinAmerican Symposium on Biomedical, Biopharmaceutical and Industrial Application of Capillary Electrophoresis. She won the prize of the Faculty of Pharmacy and Biochemistry of the University of Buenos Aires for her doctoral thesis. She was recognized for her analytical work on chromatography related to biochemical and pharmaceutical areas with two prizes awarded by the Foundation of the Faculty of Pharmacy and Biochemistry and by the National Academy of Pharmacy and Biochemistry, Buenos Aires. C.N. Carducci is associated with the Facultad de Farmacia y Bioquimica of the University of Buenos Aires, Argentina. She has been working during the 80s on the development of HPLC and sample preparation methods applied to the analysis of biochemical endogenous compounds, such as bile acids to achieve a better understanding of their physiopathology [31]. She also has original contributions on HPLC methodologies aimed at pharmaceutical laboratories for research and quality control [32]. Sample preparation with a preconcentration step, interaction of drugs with different stationary phases and extracolumn effects were also fields of research of her interest [33,34]. In the beginning of the 90s, she started to work on capillary electrophoresis applied to the analysis of drugs and pharmaceuticals [35,36]. Emphasis was focused on the evaluation of impurities and on in vitro dissolution test for pharmaceutical dosage forms with very low levels of drugs, some of which had not yet been codified. Special aims such as validation of the new proposed methods and cross-validation with HPLC were considered. These contributions pointed to a goal for the improvement of analytical methodologies employed in biochemical and pharmaceutical laboratories. Her academic and scientific activities allowed her to organize a group of specialists in the chromatography techniques with sound analytical knowledge. Many postgraduate courses have been given at several universities, institutes and associations aimed to promote these techniques in the research and industrial fields of the pharmaceutical and biochemical areas. Working at the Chromatography Division of the Argentine Chemical Association, she participated in the organization of numerous courses related to fundamentals and applications in the chromatographic separation field. She was involved in the organization of the COLACRO symposia held in Buenos Aires in 1988 and in Buenos Aires in 2000. See Chapter 5B, h, l, r

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E. REMOLO CIOLA Remolo Ciola was born in Roncegno, Trento, Italy and later became a Brazilian citizen. Educated at the University of Sa˜o Paulo (BS degree in Chemistry, 1948), he obtained his Master’s Degree under Robert L. Burwell at the Chemistry Department, Northwestern University, USA (1958), and a Ph.D. at the University of Sa˜o Paulo under Heinrich Rheinboldt (1961). R. Ciola held several academic and industrial research positions. From 1951 to 1958 he was Assistant Professor of Chemistry at the Instituto Tecnolo´gico da Aerona´utica (Aerospace Institute of Technology) in Sa˜o Jose´ dos Campos, Brazil. This was followed by a long period (1958–1975) as Scientific Director of the Refinery Research Center in Capuava (SP), Brazil. From 1961 to 1997 R. Ciola was the Scientific Director of Instrumentos Cientificos C.G. Ltda., a private company mainly dedicated to building and marketing chromatographic instrumentation and accessories, located in Sa˜o Paulo. In the academic world, R. Ciola was Professor of Chemistry at the Institute of Chemistry, University of Sa˜o Paulo (1971–1994), Professor of Industrial Organic Chemistry at the Instituto Maua´ de Tecnologia (1975–1979), and private consultant (1969–1971) on catalysis for the Petrobras Research Center, Rio de Janeiro, Brazil. During his academic and industrial career R. Ciola was honored with several distinctions, including the Heinrich Rheinboldt Award (1978), the Esso Award of the Brazilian Chemical Association (1973), the Jabuti Award in Sciences from the Camara Brasileira do Livro (1982) for his book ‘Basic Catalysis’, and the COLACRO Medal in 1990. He is the author of several scientific papers and seven books (in Portuguese). His major research interests are in catalysis, polymer chemistry, petroleum, inorganic catalysis, essential oils and chromatography. In the early fifties, immediately after the feasibility of gas chromatography as an analytical tool was demonstrated, R. Ciola pioneered the development of the first gas chromatograph and chromatographic columns in Latin America. At that time he was quite involved with petrochemical companies analyzing the products formed upon the catalytic hydrogenation of hydrocarbons and alcohols. These activities in petroleum chemistry started a new research line mainly in gas chromatography involving the characterization of all volatile feeds as a control technique and in process evaluation. All studies on petrochemical catalytic processes were carried out using an in-house built gas chromatograph, coupled on-line to catalytic reactors. This system was widely used in the analysis of several petrochemical processes, including the oxidation products of propylene to acrolein and acrylonitrile, toluene to benzoic acid, naphthalene and o-xylene to phthalic acid, benzene to maleic acid and p-xylene to terephthalic acid and to p-methyl benzoic acid, and naphthalene produced by the catalytic hydrogenolysis of petroleum fractions. R. Ciola also successfully used gas chromatography in studying the kinetics of many homogeneous and heterogeneous catalytic reactions, in the evaluation of mass transport mechanisms in several catalysts pores, and as a special technique to determine the

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specific surface area of catalysts, as well as the number of acidic or metallic active centers per gram of catalyst. Based upon his large experience in the industrial applications of gas chromatography, particularly in the petrochemical area, as well as in his research work as Associate Professor at the University of Sa˜o Paulo, R. Ciola started a successful local company named Instrumentos Cientı´ficos C.G. (C. from Ciola and G. from his partner Gregori). The company designed and manufactured several series of gas and liquid chromatographs and supplied thousands of instruments to industrial and research institutions. Several analytical problems brought by the users resulted in the further development of special methodologies involving both packed and capillary columns, catalytic reactors, multicolumns separations for special samples, as well as in the development of custom-made columns and special detectors. In this aspect, hundreds of chromatographic methods for amino acids, pesticides, hormones, pharmaceutical and drugs developed for the final users, including laboratories at hospitals and the police, and at chemical, petrochemical and petroleum companies. With the aim of producing pure analytical standards (99.99% purity) to be used in methods developments and kinetics studies, R. Ciola developed and utilized preparativescale gas chromatography. Another research interest of R. Ciola was in the gas production by high-pressure hydrogenation of Brazilian shale oil, controlled by a specially designed chromatographic system. For compound identification, the retention index of several series of compounds and their correlation under several different columns and experimental conditions were developed. Another area of fruitful research in which R. Ciola was involved was in the synthesis of chemically bonded stationary phases for gas and liquid chromatography, as well as for adsorption studies. In addition to gas and liquid chromatography R. Ciola was also involved in the development and use of supercritical fluid chromatography and extraction in the analysis of essential oils and fats [37], and the use of high-temperature capillary gas chromatography for the analysis of fats with PTV sample introduction [38]. While at the University of Sa˜o Paulo, R. Ciola (recently retired) was teaching several graduate courses on chromatographic techniques and catalysis, and supervised several master and Ph.D. Theses in both research areas. He has written seven books covering chromatography, basic catalysis and general chemistry, all in Portuguese; those on Basic GC and Basic HPLC [39,40] became very popular as textbooks in Brazilian universities. See Chapter 5B, d, f, h, j, o

F. ARMANDO MANJARREZ MORENO Armando Manjarrez Moreno obtained his degree in chemistry (‘Licenciatura Degree’) from the National School in Chemical Sciences, Universidad Nacional Autonoma de Mexico in 1952. He finished and defended his Ph.D. in 1957 at the same institution,

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which was followed by a postdoctoral study in the field of natural products at Columbia University, New York City, USA. A.M. Manjarrez developed an intensive research program at the Universidad Nacional Autonoma de Mexico (UNAM) from 1952 to 1970, where he was responsible for training a whole generation of Mexican chromatographers. From 1967 to 1998, A.M. Manjarrez held several positions at the Mexican Institute of Petroleo (Instituto Mexicano del Petroleo) including Head of the Analytical Chemistry Division, Vice-Director of the Basic Processes Investigation, Vice-Director for Professional Development and Manager of the Investigation Division. He is author or co-author of more than 75 papers and holds 17 patents on chemical products, processes and catalysts. A.M. Manjarrez received several distinctions during his career including the National Award in Chemistry from the Mexican Society of Chemistry, the COLACRO Medal from the Latin American Committee of Chromatography, and the ‘Academic Merit Award’ from the Universidad Nacional Autonoma de Mexico. He holds a position as a national investigator of the highest academic level according to the Mexican system, being also a member of the national evaluation committee that evaluates the researchers and their projects in the areas of engineering and technology. Presently, A.M. Manjarrez is general coordinator of the courses and projects in the Environmental Program of the Universidad Nacional Autonoma de Mexico. The scientific career of A.M. Manjarrez covering close to 50 years of intensive work can be divided in three different but complementary steps. The first step corresponds to close to 20 years at the Institute of Chemistry in the Universidad Autonoma de Mexico in the area of natural products, where he finished his undergraduate studies in 1952 and the Ph.D. in 1957, both in Chemistry. In this institution in the mid-50s, A.M. Manjarrez was first introduced to the chromatographic techniques through a colleague who had the opportunity to study this technique at the California Institute of Technology under Zechmeister. In 1958, A.M. Manjarrez was a postdoctoral fellow at Columbia University in New York City, in the field of natural products; during this period he had a chance to work with the incipient technique then named vapor-phase chromatography. Upon returning to Mexico he studied the chemical nature of the essential oils of several species from the Mexican flora; this work allowed him to apply the recently learned methods as well as the development of several methodologies and new instrumental set-ups since at that time commercial gas chromatographs were not available in Mexico [41,42,43]. The second step of A.M. Manjarrez’ professional career started in 1967 with a sabbatical visit to the Mexican Institute of Petroleum (Instituto Mexicano del Petro´leo, IMP); this experience ended with his permanent move to the IMP and his re-location as a part time professor at the Faculty of Chemistry, in Mexico City, UNAM. During his 32 years working at the IMP, A.M. Manjarrez has occupied several managerial positions, including Head of the Analytical Chemistry Division of IMP where he had the opportunity to develop and apply analytical methodologies, particularly involving gas chromatography, for the solution of numerous problems related to the petrochemical

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industry. Most of his contributions in this area can be found in the petroleum chemistry technical literature [44,45]. At the same time, as a Professor at the Faculty of Chemistry, UNAM, A.M. Manjarrez dedicated part of his time to the supervision of more than fifty undergraduate as well as graduate students contributing to the education of three generations of analytical chemists, most of them in the field of chromatography. The third and actual step of A.M. Manjarrez career started at the beginning of 1999 with his full time enrollment with the University Program for the Environment at UNAM (PUMA: Programa Universitario de Medio Ambiente de la UNAM). This program investigated the several problems affecting the quality of the air, soil and water at the Metropolitan Zone of the Valle de Mexico; thus he is having an opportunity to develop and apply several chromatographic-based methodologies to environmental problems. In summary, during his close to 50 years successful scientific career mainly dedicated to the development and application of the chromatographic techniques, A.M. Manjarrez has contributed to both the academic and industrial areas, and is now active in the solution of the environmental problems of his native country using chromatographic techniques. See Chapter 5B, d, h, j, r, s

G. JOAQUIN LUBKOWITZ Joaquin Lubkowitz was educated in Venezuela were he had an important role in the development and application of several chromatographic methodologies to the petroleum industry. During this time, he was responsible for training several chromatographers now involved with academic and industrial institutions in Venezuela. J. Lubkowitz immigrated to the USA where he is still quite involved with the chromatographic techniques. He is now Director of Separation Systems, Inc., in Florida, USA. See Chapter 5B, d, h

REFERENCES See Chapter S-13 Chromatography Around the World for supplementary information for Latin America (http:==www.chemweb.com=preprint=).

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Windmill.

6E. Chromatography in The Netherlands (University of Amsterdam) Robert Tijssen University of Amsterdam, Institute for Chemical Engineering, Workgroup Polymer Analysis, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands

A. HANS POPPE Hans Poppe was born in Amsterdam (NL) on November 18, 1937. From 1955 to 1962, he studied chemistry at the University of Amsterdam (UvA), where in 1962 he mastered with specializations in organic chemistry, but also chemical physics and analytical chemistry. His Ph.D. research was carried out under the supervision of Gerrit den Boef, while W. van Tongeren was his Ph.D. advisor in March 1965, with a thesis on Photometric Determinations of Cobalt. His further career has been solely in academia. Since 1962, he was employed in the Laboratory for Analytical Chemistry of the University of Amsterdam.

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From 1970 to 1971 he was active in a group on Electro-Analytical Chemistry and Optical Methods, directed by his earlier supervisor, G. den Boef. Since 1971 he became active in the field of separation methods in a group in the same laboratory directed by Josef F.K. Huber until 1974. In that year he took over the direction of this group when J.F.K. Huber left for a position in Vienna. H. Poppe became Full Professor in Analytical Chemistry at the University of Amsterdam in 1983, and was Director of the Laboratory of Analytical Chemistry until 1998, when this department was taken up by the Institute for Chemical Engineering of the Faculty of Chemistry. Among the professional activities carried out by H. Poppe, were that of Chairman of the Working Party on Analytical Chemistry of the Dutch Organization for the Advancement of Pure Research, Chemistry Division SON (1985–1988 and 1991– 1994). He also acted as Chairman of the scientific and organization committee of the 11th International Symposium on Column Liquid Chromatography, ‘HPLC 1987’, Amsterdam, 1987. He is member of the editorial advisory boards of the Journal of Chromatography, LC–GC Magazine, Chromatographia, Analytical Abstracts, Journal of Microcolumn Separations, Electrophoresis, and since 1986, a member of the permanent scientific committee of the symposium series ‘International Symposium on Column Liquid Chromatography’. At the 20th International Symposium on Capillary Chromatography which was held May 26–29, 1998 in Riva del Garda (Italy), the Golay Award was presented to H. Poppe for his contributions to the basic understanding of microseparations. H. Poppe was very much involved in the development of HPLC: among others (a.o.), the input of his group was in the field of detection, fundamental studies on phase systems and column technology. His group was one of the first to use HPLC technology (small particles [1]), also for fast gas chromatography. On the occasion of his ‘Desty Memorial Lecture for Innovation in Separation Science’, H. Poppe was recently awarded the prestigious (and named after the Nobel laureate) A.J.P. Martin Gold Medal 1999 of the Chromatographic Society. This was awarded to him on October 18, 1999 in London by Keith Bartle. More recently his research topics (see selected references [1–10]) include capillary zone electrophoresis (CZE) [7,9], hydrodynamic chromatography (HDC) [3], field flow fractionation (FFF) [6], and open-tubular liquid chromatography (OTLC) [8]. Characteristic of his work is the fundamental approach, based on sound physico-chemical and chemical engineering principles. His PC-‘animations’ of separation mechanisms have aroused a lot of enthusiasm, offering a clear insight of the fundamentals not only by his students but also to the scientific community. The well-known yearly international summer schools on HPLC and CZE in Amsterdam originated by him and his coworkers Johan Kraak and Wim Th. Kok, firmly established the sound principles of Poppe’s school to literally hundreds of separation scientists in the field. Poppe co-authored some 185 publications, and has served as a mentor for 30 Ph.Ds.

B. PETER J. SCHOENMAKERS Peter J. Schoenmakers was born on June 17, 1954, in Schiedam (The Netherlands), his professional life consists of two parts. (i) Industrial occupation: Principal Research

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Chemist, in the Analytical-Separations Department of the Shell Research and Technology Centre, Amsterdam (formerly known as Koninklijke Shell Laboratory Amsterdam, KSLA). (ii) Academic occupation: Professor in Analytical Chemistry (specialization in polymer analysis) at the University of Amsterdam since 1998. P.J. Schoenmakers studied chemical engineering and analytical chemistry at the Technical University of Delft, The Netherlands (degree 1977, partly under the guidance of his present colleague Robert Tijssen). He also studied at the Northeastern University Boston, USA, with Barry L. Karger (1978). In 1981, he earned a Ph.D. in Analytical Chemistry from Leo de Galan in Delft with a thesis on reversed-phase liquid chromatography. Among the other subjects he investigated while in Delft were the application of solubility parameters in chromatography, the theory of gradient elution and, in the final year, the optimization of chromatographic selectivity. Research on the latter subject has been conducted in Delft ever since, and formed the basis of his book on “Optimization of Chromatographic Selectivity, a Guide to Method Development” (see selected references [11–19], ref. [14]) that has been translated also into Russian. His industrial occupation started in 1983 at the Philips Research Laboratories, Eindhoven, The Netherlands, with research topics on supercritical fluid chromatography [13,16,17], open-tubular liquid chromatography, optimization and chemometrics [14], method development, and expert systems for chemical analysis [15]. From 1986 to 1991 he participated in a large European co-operation project (ESPRIT P1570) on on-line chromatography–FTIR spectroscopy, application of neural networks in FTIR, and pH effects in reversed-phase LC. P.J. Schoenmakers received the 1989 Silver Jubilee Medal of the Chromatographic Society, in recognition of his important contributions to separation science. In 1992, he moved from Eindhoven to Amsterdam to the Shell laboratory to become leader of the Chromatography Group with a specialization in separation of complex mixtures. In 1996–1997, he worked at Shell-Westhollow Technology Center in Houston, TX (USA) as an exchange scientist and covered a variety of subjects in the areas of gas and liquid chromatography. In 1997, he was appointed as Principal Research Chemist, in the Analytical-Separations Department. Topics of his research include novel and complex separation methods, while he is responsible for all research projects in chromatography and mass spectrometry, including academic cooperation projects. In the latter role, he initiated many extra-mural research projects also at the (UvA) with R. Tijssen and H. Poppe. Among these projects, one was aimed at establishing superior methods for the separation of synthetic polymers (his present occupancy) and a project on comprehensive two-dimensional gas chromatography [18] with Jan Beens (UvA) and the late J.B. Phillips (University of Southern Illinois, USA). P.J. Schoenmakers is co-author of more than 75 papers on chromatography and other areas within analytical chemistry. He is also co-author of several patents on instrumentation and software for chromatography and FTIR spectroscopy. He is contributing author and co-editor of several other books, among which the recent Handbook of HPLC (1998) should be mentioned [19]. P.J. Schoenmakers is on 10 editorial and advisory boards. He is active in many symposium organizations and organized a number of one-day and two-day symposia on specialist aspects of chromatography and chemometrics in The Netherlands and in the

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United Kingdom. He also was the organizer of the 1990 National Chemistry Olympics in the Netherlands.

C. ROBERT TIJSSEN Born on D-day 1944, June 6, in Appelbeck (Germany), Robert Tijssen’s profession is currently (part-time) Professor in Analytical Chemistry, specialization in separation of macromolecules, at the University of Amsterdam (UvA), since 1993. After receiving his chemical engineering B.Sc. from the University of Amsterdam (1966) he was employed as a technical assistant (1966–1973) at the Technical University Delft (TUD), Department of Analytical Chemistry, under the direction of Pieter Karsten and Frits Vorstenburg. During this period, he also studied chemical engineering, and when P. Karsten retired Leo de Galan, his later Ph.D. advisor, took over the leadership. In 1973, Tijssen obtained his M.Sc. in Analytical Chemistry (cum laude). With two rather theoretical papers published as early as 1970 and [with Robertus T. Wittebrood] 1972 (see selected references) [20–30], it was already clear that basic research in physical transport phenomena, and later thermodynamics, was his main interest. Between 1973 and 1975, he was employed as scientific coworker at the Delft Technical University, became workgroup leader of the Chromatography Department, and lectured on instrumental analysis, mainly on GC and LC. His industrial occupation started in 1975, when he was invited to work at the Koninklijke Shell Laboratorium Amsterdam as a Research Chemist=Physicist and later workgroup leader in the Analytical Group, sections Physical Separations and Organic Chemical Analysis. In this position he worked part of 1988 as a visiting scientist at the Shell-Westhollow Technology Center in Houston, TX, USA. In 1979, he obtained his Ph.D. in Analytical Chemistry at the Technical University Delft under Leo de Galan (analytical chemistry) and C.J. Hogendoorn (physical transport-phenomena) with F.J. Zuiderweg (ex-KSLA) as one of his opponents. The subject: ‘Axial Dispersion in Helically Coiled Columns for Chromatography’ reflects his interest in basic mechanisms, which has continued to the present. In September 1982, during the 14th International Symposium on Chromatography in London, R. Tijssen was awarded one of the two first Silver Jubilee Medals, founded by the Chromatography Discussion Group. The medal was awarded for his contributions to chromatography, notably his work on column technology and the mechanisms behind dispersion of sample zones, as well as for his studies on the selectivity of chromatographic phase systems. This interest in fundamentals led to an invitation as a staff-lecturer at the NATO Advanced Study Institute: ‘Theoretical Advancement in Chromatography and Related Separation Techniques’, Ferrara (Italy), 1991 [27]. On July 1, 1993, Robert Tijssen was called to become Bijzonder Hoogleraar (Special Professor) at the University of Amsterdam, in the group headed by H. Poppe, with special assignment to the

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separation of macromolecules. In this position he was the co-advisor of four Ph.Ds and presently guides three more, on subjects such as field-flow fractionation (two Ph.Ds), and hydrodynamic chromatography (one Ph.D.). It concerns the separation-by-flow of macromolecules in micromachined channels on a silicon waver (‘chip’) with the smallest dimension of 1–0.1 µm. The maximum length of the channel is about 10 cm. Next to teaching at the University of Amsterdam, R. Tijssen is also active in EEG-Educational Programs (TEMPUS). R. Tijssen is the co-author of 70 papers. He has contributed chapters to several books, and has delivered 38 invited lectures, among which were several plenary and keynote lectures. Among the other professional duties he is a member of a number of editorial boards and also occupies the position of secretary (since 1990) and chairman (since 1994) of the Workgroup Separation Methods of the Royal Dutch Chemical Society.

D. CHROMATOGRAPHY AT THE UNIVERSITY OF AMSTERDAM: THREE-AND-A-HALF DECADES OF DISCOVERY — HANS POPPE, PETER J. SCHOENMAKERS, ROBERT TIJSSEN University of Amsterdam, Institute for Chemical Engineering, Workgroup Polymer Analysis, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands

D.1. Historical and personal remarks on the relation of the three University of Amsterdam Awardees Amsterdam (The Netherlands) belongs to the locations where chromatography was coming ‘alive’ long before the start of the Journal of Chromatography (1958) and right after its ‘rebirth’ in the 1930–1940s by Lederer, Hesse, James, Martin and Synge to name just a few important pioneers. It is not generally known that W.J. van Dijck and A. Klinkenberg of the Bataafse Petroleum Maatschappij (better known by its later name ‘Shell’) at the Koninklijke Shell Laboratory in Amsterdam, (KSLA) around 1940 pioneered a technique which they called ‘Extraction in a Percolator’, a precursor of liquid–liquid chromatography (LLC). Yet, the famous 1941 Martin and Synge publication preceded (and thus halted) the Shell patent application in 1941, because: “they had no problems they were immediately able to solve with the aid of the new technique while we had a problem : : : ” to cite Synge’s 1952 Nobel Lecture. Although internally reported in Shell (in 1945 by A. Klinkenberg) as a comprehensive theory for LLC, it lasted until 1956 that the equally famous van Deemter–Zuiderweg– Klinkenberg paper on the rate-theory of chromatography appeared. Publication was postponed, mainly because the subject was considered too difficult for the reader audience at that time (!). This patronizing behavior of editors-in-chief strikes again later in this story. Meanwhile, during the 1945–1955 period within KSLA gas chromatography was experimentally developed by Henk Boer, F. van de Craats, A.I.M. Keulemans, A.

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Kwantes and G.W.A. Rijnders. Keulemans in particular followed up Martin’s work with great vigor, and published his renowned book on gas chromatography in 1957. He was named professor at the new University of Technology in Eindhoven, and the University of Amsterdam had to wait some eight more years before chromatography became ‘alive and kicking’. It was in Eindhoven, in 1960, that J.F.K. Huber was invited by Keulemans to work with him on GC. Huber took advantage of the presence of M.J.E. Golay and later A.J.P. Martin as visiting scientists in Eindhoven. Huber started his research in LC in 1963, became senior scientist at the University of Amsterdam in 1965, accepted there an Associate Professorship in Separation Science in 1969, and became Full Professor in 1972 in the Laboratory for Analytical Chemistry. It was there, that he worked together with H. Poppe in 1970 to 1974, among other things on micro-particulate columns in LC and GC. When Huber left Amsterdam to join the University of Vienna, Hans Poppe took over the leadership of the separation science group, initially as the workgroup leader. In 1983, he became professor and director of the whole Analytical Department, until 1998. Since his 60th birthday, which was celebrated with an International ‘Pop ’60’ Symposium in Amsterdam, he wishes to be left alone to exercise his hobby of chasing ghost peaks in capillary zone electrophoresis. Meanwhile, since the early sixties, research on chromatographic separation methods was proceeding at the borders of the Amsterdam harbor (the IJ, or sometimes mistakenly described as the Y) at KSLA. Sie and Rijnders (1967) refined the theory behind the efficiency of chromatography, also carrying out chromatography with non-inert gases as the carrier at higher pressures and thus exploring the field of supercritical fluid chromatography. It was at that time that Robert Tijssen as a graduate student worked and studied at the Technical University of Delft (TUD) (NL). Tijssen 5B, a, b, d, f, g, h, k, l, p Poppe 5B, a, b, d, f, g, h, k, l Schoenmakers 5B, a, b, d, f, g, h, o Tijssen was trying to master the tricks played by his (second and equally!) stubborn all-glass (and all-leaking!) Jana´k gas chromatograph. They never became friends but the interest in separations was firmly born. It was the interest in other physical phenomena, such as the naturally meandering flow in the Gulfstream, that brought him the idea to use analogous radial (so-called secondary) flows in more or less tightly coiled and thus curved tubular columns for chromatography, in order to enhance cross-sectional mass transfer (rather than heat transfer). In 1970, he published the approximate theory that describes and predicts the resulting reduced peak broadening, and this work was extended in 1972 [20], with the aid of the great theoretical and mathematical abilities of a brilliant companion student, Robert T. Wittebrood (presently at Shell Canada). Not only in theoretical problems (most readily solved in the restful atmosphere of a bathroom) but also in experiments, the two students enjoyed working together. For example, they (together with their colleague Hugo A.H. Billiet, still at TUD) went to two consecutive International Symposia on Chromatography in 1972 (Montreux,

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Switzerland and Saarbru¨cken, Germany) in one car, and camping in one tent! Also during the symposia they camped, e.g., near the magnificent (but polluted) Lake of Geneva, knee-high in heaps of falling leaves, which half-buried the tent. Equally romantic was their camp in the high regions of the hills in the Vogesen near Saarbru¨cken, where it really froze during the nights and their beards literally turned into ice sculptures. Of course, all this was for reasons of money shortage in the first place when the Montreux symposium fee outran their student budgets (to be honest, they bought only one entrance ticket : : : ). There are vivid recollections, also by Peter Schoenmakers, of experiments by the two Rob’s during nighttime, which bewildered the Personnel Organization, but really contributed to science. Only then, they were able to precisely stretch out straight glass capillaries in the passages of the laboratory. Columns were drawn (and sometimes coated) at the spot with lengths up to 50 meters, the length of the building. Injection of gas samples was performed by one of them manually (in less than several hundreds of milliseconds, requiring a very fast finger indeed) at one end, while the other manned the detection and recording devices. Contact was made by babyphone connection, not only necessary to keep each other awake by telling jokes, but also by determining the exact time to switch on the very fast galvanometric recorder only seconds before elution of the peaks. Peaks could be very sharp and fast, and so the recorder was set at high photometric paper speeds of about 1 m=s (for several seconds only, of course!). The results were very interesting in that they showed distinct deviations from the infallible axial dispersion ‘law’ after Golay, and that for even perfectly straight tube flow. The latter was confirmed later in Tijssen’s thesis based on more careful experiments at KSLA. These results could certainly point toward interfacial resistance effects as an additional mechanism of peak broadening [24,26]. The matter is still unresolved and requires further thought. During the 1970 Dublin Symposium on Gas Chromatography, Rijnders invited Tijssen to spend the required graduation research period at KSLA in his group. As the interest of both parties was highly directed towards the mechanisms behind peakor zone broadening, the collaboration became a success, and in 1975 Tijssen joined KSLA as a Research Scientist. At the same time he obtained a Ph.D. project at the TUD in analytical chemistry (thesis in 1979) on the subject: “Axial Dispersion in Helically Coiled Open Columns for Chromatography”. In this work he expressed his interests in mass transfer phenomena (induced by the magnificent Feynman-type of lectures of Wiero J. Beek), which occupies him up to the present day. These transport phenomena are indeed so vital for the efficient operation of separation methods, as was shown by van Deemter, Zuiderweg and Klinkenberg in the early fifties. Further, it came as a revelation that the molecular interactions that determine transport phenomena such as diffusion, also play an equally important role in the partitioning process, and thus in the selectivity of chromatographic separations (e.g., expressed in terms of solubility parameters [21], from which not only partitioning but also diffusion may be predicted). In short, transport phenomena were fun, and it was no coincidence that F.J. Zuiderweg was on the promotion committee as an opponent. At the time that Tijssen lectured instrumental analysis in Delft (1973–1975), P.J. Schoenmakers was his graduate student also showing a high interest in the mechanisms

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behind the selectivity of partition chromatography. Tijssen, like many others at that time, was using the concept of solubility parameters after Scatchard and Hildebrand (‘like dissolves like’). Together with Hugo Billiet and later also with Leo de Galan (presently at Unilever Research), they developed a predictive model for the selectivity of chromatographic separations based on an extended solubility parameter model (1976) [21]. The model was successful in predicting trends and offering systematic rules for predicting optimum selectivity in GC, high-performance LC, reversed-phase LC [11,12], supercritical-fluid chromatography [13,14,16], and more recently (Billiet) in capillary-zone electrophoresis and ion chromatography. The subject of selectivity, notably in chemically bonded grafted layers on supports for reversed-phase LC, was kept alive in the Delft laboratory over the years, and received new impetus when the success of the Self Consistent Field approach by Scheutjens and Fleer of the Agricultural University of Wageningen (NL) became apparent world-wide in physical and colloid chemistry (since the early 1980s). It really was a “flexible theory for hard systems” as Jan Scheutjens († 1992) once phrased it so elegantly; it brought main insight in the secrets of RPLC selectivity, and was reviewed by Tijssen, Schoenmakers, Bo¨hmer, Koopal and Billiet in 1990–1993 [28]. Currently the solubility parameter concept is again being picked up and used in the present workgroup to predict trends in thermal diffusion coefficients of polymers in solution, needed for optimization of thermal field flow fractionation (Ph.D. subject of Edwin P.C Mes, see Chapter S-14 on Future Chromatographers). Over the years, the subject of selectivity went, of course, not unnoticed in the University of Amsterdam Laboratory of Hans Poppe; he devoted much time to a fundamental treatise on the subject (adsorption isotherms, etc., 1975–1993) [4,5] before his main interest, together with one of his close coworkers, Wim Th. Kok, went to electroosmosis, electrophoresis and electrochromatography. During the period of 1975–1994, Tijssen developed at Shell, first under the guidance of M.E. van Kreveld, and later as a workgroup leader himself, many new applications and methods in the field of separation science and flow injection analysis. For the first several years his research director was Jan J. van Deemter and, again no coincidence, Tijssen focused on column technology and mechanisms behind dispersion of sample zones, initially as a research scientist. He carried out basic research in the fields of mass transport but also thermodynamics, which resulted in the development of new analytical chromatographic separation and flow injection analysis (FIA) techniques: high-speed microcapillary LC [22–24], direct-inlet open-tubular LC=mass spectrometry [24], high-speed gas chromatography [26], hydrodynamic chromatography [25,27], hyphenated chromatography (a.o. LC–GC, several detectors, comprehensive GC ð GC) [17], and fast and efficient microreactors for FIA [23]. These techniques found industrial applications in projects such as enhanced oil recovery, polymers and process analysis. Early in his Shell-career, the same fate that van Deemter et al. met 20 years earlier, viz. the reluctance of editors to accept so-called ‘theoretical’ papers, struck once more. At the occasion of the Amsterdam 1977 12th Symposium on Advances in Chromatography, Tijssen tried to get a paper published on the earlier mentioned zone-dispersion phenomena in straight and coiled columns. A well-known editor-in-chief of an equally well-known journal, told him boldly that he would not publish the paper, because he

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could not understand it, and thus (this is historical) his readers could not understand it either. Jan van Deemter, still in charge as the Shell Research Director in the field, maybe reliving his own problems in the fifties, then radically forbid that the lecture by Tijssen on the same subject be given at the Conference. The gentleman he always was, he visited the Conference to address the poor symposium organizer in person. The result was that the lecture disappeared from the program, but that also the interest of the audience was aroused. As a direct consequence, Tijssen talked for one hour with Calvin Giddings on the heart of the matter of the manuscript, and which was accepted without any change in Giddings’ own journal, some months later [22]. Tijssen was very much impressed and inspired by Giddings’ work and when he wrote a review on zone-spreading [29], he dedicated it to the memory of Giddings († 1996). Around that same time (1975–1985), the advent of flow injection analysis (FIA) occurred, mainly through the enthusiasm of Scandinavian groups in Copenhagen and Lund. In these groups, continuous flow analysis, which makes use of gas-segmented liquid flow in coiled tubes to maintain a certain zone integrity was, for obvious reasons, replaced by the real continuous non-segmented liquid flow that is common in LC. The zone widths observed indeed could be maintained within practical limits (even below values predicted by Taylor dispersion), but this fact was erroneously interpreted as stemming from turbulence of the flow. Tijssen took the standpoint that FIA is a ‘simple’ form of chromatography, concerning transport of unretained solutes in uncoated systems only. As a result zone dispersion phenomena in tubular FIA reactors (and not to forget in the connecting lines and other extra-column devices) could be described in the same way as in chromatographic and chemical engineering systems. Certainly the objective conditions for turbulence in FIA systems were absent, and rather consideration of, again, secondary flow could save the phenomena. In his 1979 thesis, Tijssen explained this and in plenary lectures at the two following International Symposia on Flow Injection Analysis (Amsterdam, 1980 [23], and Lund, 1982) a lively debate was held on the two opposite points of view. The outcome was clear: it is dispersion rather than dilution that causes the changes in concentration during (laminar!) transport in FIA systems. Hans Poppe and his friend and co-worker Johan C. Kraak, also present at the FIA conferences, heartily joined this discussion (and added fun to it), in support of the chemical engineering approach. In 1988, Tijssen worked in Houston (Texas) as an exchange scientist at Shell Development Co. on coupled LC–GC techniques for complex industrial mixtures. This work was later extended by Jan J. Blomberg (SRTCA) and Jan Beens [18], formerly colleagues at KSLA. J. Beens, retiring early from KSLA, was in 1994–1998 at the University of Amsterdam, where he became one of Tijssen’s first ‘own’ Ph.Ds. In Beens’ words, Rob made his life pleasantly free by acting as a ‘remote promotor with remote control via e-mail’. Indeed, special and part-time professorship transforms the supposed ‘chair’ into a special ‘stool’ and certainly into an ‘e-mail professorship’. With the help of his collaborators Nico van den Hoed, Joop Bleumer, Jaap Bos and Jan van der Does, new techniques such as hydrodynamic chromatography (HDC) in microcapillary columns, (1983) [25,27] were originated. In 1980, looking for an alternative to size-exclusion chromatography (SEC) within Shell, a new size separation technique was born. The concept of separation-by-flow was proposed and theoretically

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Fig. 6E.1. Historical (never published) first separations of polystyrenes and toluene by microcapillary HDC [J. (Joop) Bleumer and Robert Tijssen, KSLA (1980)]. The original handwriting is of RT. The handdrawn lower peaks (M1 , M2 ) indicate where elution was expected (at that time for the indicated masses (M1 D 9.7–106 , M2 D 9.9–107 ). The four sigma-values inscribed are (from top to bottom): measured for toluene, estimated by Taylor–Golay for toluene, measured for PS 3.7 MDa, and estimated by Taylor–Golay for PS 3.8 MDa.

founded by DiMarzio and Gutmann in 1969–1975, and the first practical separations were shown by Small in 1974, using packed columns with nonporous particles of about 18 µm in diameter. It was in Tijssen’s group at KSLA that the first experiments, separating large polystyrenes from each other and from toluene, was demonstrated in open microcapillary tubes. Fig. 6E.1 shows the non-published very first differences in residence times of different sized (large) molecules in a 10 µm ID fused-silica capillary. At that time this ID size was the lowest that could be made, but the separation-by-flow principle was readily visible for molar masses above 1 M. It lasted until 1983 [25], when smaller macromolecules (molar masses in the range down to 104 ) could also be separated by this technique (then called hydrodynamic chromatography) in even smaller-sized microcapillaries. Ever since that time, the world record of the smallest column diameter used in practice, viz. 1.2 µm, has not yet been broken.

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Poppe and coworkers (Johan Kraak, Rob Oostervink and Gerrit Stegeman, presently at GEP (NL)) correctly argued (1987–1994) that the technique was much better off in practice using packed columns. This, of course, is based on the fact that the interstices between packing particles can be considered to form a whole bundle of microcapillaries, so that detection is far easier. They readily demonstrated this by experiment, using Klaus K. Unger’s expertise in preparing the required small non-porous packing particles [3]. Although there was a common line of thinking in the two research groups, and occasional mutual support (such as in the case of FIA), there was surprising little cooperation in the research activities of Shell and the University of Amsterdam, close neighbors located at the opposite borders of the Amsterdam harbor (IJ!). At best there were occasional meetings by surprise between Poppe and Tijssen on the public ferryboat across the harbor (the KSLA-Laboratory and Poppe’s home are both at the northern border of the harbor). Their common interest in the concept of separation-by-flow (HDC), but also in SEC and FFF [6], finally brought both parties together, and in 1993 Tijssen was appointed a professor in separation of macromolecules at the University of Amsterdam. In 1995, he left Shell to be followed up by P.J. Schoenmakers, to choose for a pure academic setting. The atmosphere in H. Poppe’s well-established group at the University of Amsterdam was not only scientifically stimulating, but also, as a result of Poppe’s famous sense of humor and critical mind, refreshing. Poppe’s lifetime coworker and friend Johan Kraak (presently retired) as well as AYS (angry young scientist) Wim Kok contributed very much to the critical and realistic but sometimes also hilarious atmosphere in the group. Not only during the summer courses organized by this team (with famous smoked-eel (‘paling’) parties), but also for entertaining guests and sometimes even for visiting symposia (e.g., the 1993 Hamburg Symposium, together with John Knox), Poppe often acted as the host and captain of his large wooden sailing ship. During the ‘Pop ’60’ celebration Symposium in 1997 at the University of Amsterdam, John Knox treated his friend Poppe with a theory on how to anchor his ship safely during stormy weather. After obtaining his Ph.D. in 1981, Schoenmakers left Delft for Philips Research Laboratories in Eindhoven (NL) in 1983, where he mainly investigated supercritical fluid chromatography [13,16,17]. The collaboration with Tijssen got a new momentum when during the 1991 Riva Symposium on Capillary Chromatography the roots were planted for a changeover from Philips to Shell. After three years of collaboration and sharing a half-persons room (better described as ‘cell’) together, when Tijssen left for the University of Amsterdam, Schoenmakers took over the separations group at Shell Amsterdam (then, for organizational reasons, alas, renamed from KSLA into Shell Research and Technology Centre Amsterdam). Soon thereafter, the foreseeable retirement of Hans Poppe from the University of Amsterdam, made it necessary to find a successor, and it was decided that P.J. Schoenmakers was the best choice for that. It so happened that Schoenmakers was installed as a professor in July 1998, and once again, he became the roommate of Tijssen for almost a year (this time in a large three-persons room). Presently the three Awardees at the University of Amsterdam are working together, though in separate rooms.

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D.2. Present interests: separation of macromolecules The interest in separations of macromolecules logically grew at Shell, where a large workload on polymer characterization was carried out, mainly by gel permeation (GPC) or better size-exclusion chromatography (SEC). The characterization of polymeric substances is a key step in the development of new materials, products and the associated processes, and as a result, chromatographic separations are increasingly pervasive in polymer analysis. This insight found footage in the Faculty of Chemistry of the University of Amsterdam, and considering the coming retirement of Hans Poppe, it was decided that his successor, Peter Schoenmakers, should follow the road of polymer analysis, and the mission statement of the original separations group was recently redefined into one for a Research Group for Polymer Analysis. The objective of the new group is to generate novel, or greatly improved techniques and methods for the analysis of synthetic polymers. The main focus is still on polymer separations, as separations are essential to characterize the many distributions (molecular mass, functionality, chemical composition, etc.) that are present in synthetic polymers. Together with other groups, work on other characterization methods, such as mass spectrometry and Raman spectroscopy is carried out. Ways to obtain molecular-mass distributions of polymers are studied that compare favorably with conventional methods in one or more of the following respects: ž greater accuracy and precision, ž high-resolution separations (avoid adsorption, degradation and deformation), ž absolute measurements, ž use of special detection devices, ž experiments on a smaller scale and on smaller, cheaper instruments requiring smaller (cheaper, better controlled) columns and much lower amounts of (toxic, expensive) solvents as well as much smaller amounts of precious samples, ž shorter analysis times, ž applicable to very-high-molecular-mass (×106 ) molecules and to small particles, ž either high-pressure pumps or high-voltage power supplies are used to drive these systems, ž also better ways to obtain chemical composition distributions and functionality (end-group) distributions by liquid chromatography are investigated. D.3. Discussion on the present research approach Especially, polymer characterization in terms of molecular mass (M) and its distribution (MMD) is required, as these quantities are strongly connected to process variables and product properties. SEC, presently a relatively comfortable method, is the main one in routine quality control of polymeric products. Still, there are difficulties in the application of SEC (as a single technique) to be summarized as follows. ž The separation is fundamentally according to size (exclusion from solid walls), and not to mass. This results in unreliable calibration, notably when the conformational behavior is unknown such as for new polymers, copolymers, branched polymers, use of ‘exotic’ solvents.

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ž SEC is rather slow and inefficient, the more so for larger molecules, and corrections to compensate for the limited resolving power of SEC cannot be easily effected in practice. ž The volume scale is rather large, leading to wasteful use of large amounts of expensive, often environmentally hazardous and toxic solvents, which makes it a method of large running costs. ž The SEC method breaks down for very large molecules (M × 106 , degradation) or particles, because of the lack of suitable porous column packings, or with more or less labile aggregates such as micelles and vesicles. ž There is a growing awareness that MMD information, however important, is insufficient for obtaining a satisfactory correlation between analytical separations data and relevant characteristics of the product (absolute rather than relative M, chemical structure, functionality, branching etc.). Yet, SEC is the main method for quality control of polymeric products because of the narrow focus on the single property of size, this has been replaced by using SEC in a multidimensional configuration, by coupling it to other separation techniques [HPLC, GC, temperature-rise elution fractionation (TREF), critical chromatography (CC), gradient LC of polymers (GREF), thermal FFF (ThFFF) for structure-based separations or to various detection techniques (viscometry, light scattering (MALLS), infra-red (FTIR), magnetic resonance (NMR), mass spectrometry (MALDI)]. Although these coupled techniques are extremely powerful, the above-mentioned limitations, inherent to SEC, persist. Alternative size-based separation methods (such as FFF and HDC) have been developed, but the field of polymer separations is highly conservative (methods often date back to the 1970s). Despite the great economic importance of the SEC-methods, developments in this field have not kept pace with the progress made, e.g., in pharmaceutical and biochemical analysis. Here, the dimensions of the separation systems have been greatly reduced by miniaturization, while also electromigration is largely used. Both aspects lead to substantial benefits, especially concerning limited solvent consumption and high separation efficiencies. With a view to the latter aspects, the University of Amsterdam group believes that there is room for improvement of SEC, using miniaturization as the key development, and electromigration as an essential tool. Admittedly, this is not always as easy as in other LC methods, due to the inherently slow diffusion of polymers, extra-column band broadening is harder to avoid, and the more difficult generation of electro-osmotic flow in organic solvents needed for dissolving polymers. In order to improve SEC by miniaturization alone, in a pilot study smaller-diameter columns than standard and down to 2 mm ID were used. Indeed this resulted in the predicted solvent savings. This is, however, partly compensated by a loss in efficiency, probably by inevitable extra-column effects. As a result, the combination of miniaturization and electromigration may be essential, as plate heights are very much improved to the same levels as in wide-bore pressure-driven standard columns, by using electro-osmotic pumping of the solvent (DMF), rather than pressure drive [30] (see Fig. 6E.2). Also, because the particle size has been reduced from the classical 20 µm down to 3 and 5 µm, the analysis time is largely reduced, and the goal of fast (‘flash’) SEC (EDSEC or SEEC) for process analysis comes into view.

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Fig. 6E.2. Comparison of separation performance of pressure- and electrically driven SEC in the same packed capillary column for different PS samples (masses indicated). Capillary ID 100 µm; length 30 cm, ˚ (a˚ngstro¨m); mobile phase DMF C 1 mM LiBr C 1 mM SDS. T D particle size 5 µm; pore size 300 A relative retention time of polymers with respect to that of toluene (ca. 8 min).

Preliminary results obtained with electrodriven-SEC (by Edward Venema, see Chapter S-14 on Future Chromatographers, one of Tijssen’s first Ph.Ds, and presently at GEP, NL) and CE in porous-particle packed columns (Remco Stol) indicate that intraparticle mass transfer is positively influenced by electrodrive. The retention window is reduced, however, and careful choice of experimental conditions is needed to obtain a positive net-improvement of the SEC-separation [30]. However useful, such improvements of SEC itself, given that a combination with the various detectors is problematic in miniaturized systems, still gives only limited information, it cannot distinguish between copolymers and mixtures of polymers, and cannot handle particulate materials, very large macromolecules and polymeric aggregates. In this respect, new techniques such as FFF are promising. Cross- or hollow-fiber flow-FFF (HF5) for example, yields absolute particle size- and density information. The technique would probably gain in popularity if it could be designed in a tubular type of column, which is handled like a standard LC-column. For that purpose M.Sc. Michel van Bruijnsvoort carried out investigations on flow FFF in ceramic hollow fibers, which are resistant to organic solvents. The results show promise for size information on polymeric materials, colloids and polyelectrolytes. Thermal FFF on the other hand, gives extremely useful information on both size and chemical nature of the analytes (MMD and CCD). Recently, the University of Amsterdam group showed successful coupling of ThFFF with MALLS, SEC and HDC. The success of ThFFF as a separation method very much depends on the existence and magnitude of the thermal diffusion effect originated by the imposed and strong

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thermal gradient. For that purpose a preliminary theoretical model was designed, based on a Flory–Huggins lattice-model for polymer solutions. The model predicts from first principles that the thermal diffusion of polymers is independent of molecular mass (as observed), and depends very much on the physico-chemical nature of the monomer, while the difference of interaction energies (characterized by their solubility parameters) between the monomer and the solvent largely determines the magnitude of the effect. As a result, trends in thermal diffusion (positive, negative and zero diffusion coefficients in various solvents) and temperature dependence of the effect, are qualitatively well described. Quantitative prediction awaits further improvements, using more realistic polymer–solvent models (e.g., Scheutjens–Fleer theory). HDC in packed columns with small nonporous particles of 1 µm is comparable to SEC, but is much faster and far less problematic in calibration (absolute and even universal), solvent consumption (HPLC-type of separation columns) and bandspreading [3,27,29]. Because there are still a number of drawbacks, electrically driven HDC was considered, obviously seeking even better efficiencies. It turned out, however, that the required low ionic strengths (<25 µmol=l) could not reliably be maintained in practice (Edward Venema, 1998). Earlier research (1983–1986) of Tijssen’s group at KSLA proved that HDC can also be carried out in open capillaries of µm (ID) size (microcapillary HDC) but the detection problems are then virtually insurmountable. Presently at the University of Amsterdam it is believed that micro-machining techniques open up quite new and superior methods for the characterization of polymers, particles, etc. In an NWO-STW-sponsored collaboration with the MESA Institute at the University of Twente (Enschede, NL), the aim to use rectangular micro-machined channels (heights between 0.1 and 2 µm, widths up to 10 mm, and lengths of up to 10 cm) has been shown to be feasible (Emil Chmela). Later implementation of integrated injection and detection (‘lab on a chip’) is planned, and directed to obtain HDC-separation with the following properties: ž mass range from well below 104 towards above 107 Da; ž very low solvent consumption; ž integrated ‘columns’ and extra-column injection- and detection devices, potentially all embedded in an elevated temperature device; ž absolute calibration based on first principles; ž low back pressure (<50 bar) combined with short analysis times (<1 min); ž minimum shear deformation and degradation; and ž for channel heights < 0.3 µm, we envisage reptational migration of large macromolecules. Where special size separation effects not based on the hydrodynamic radius of the whole molecule, but rather on segment size or the persistence length occur (notably for copolymers this would offer quite novel and useful separation effects). This new technique might, even before its actual birth, be named Reptational Hydrodynamic Chromatography (RHDC). In total, the subject of separation-by-flow (or HDC) is far from matured. On these and other aspects, Tijssen is presently preparing a monograph on hydrodynamic chromatography. The University of Amsterdam group has a great deal of knowledge and experience on capillary electro-chromatography (CEC) and electrophoresis (CE); Hans Poppe,

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Wim Kok and Remco Stol are active in this field. CEC offers the potential of highly efficient and relatively rapid separations. It offers an, often attractive, alternative to liquid chromatography. In the special case of polymers, electrodriven SEC is being investigated as an alternative to conventional SEC (see above). Because gradient-elution CEC has not yet been developed, CEC does not yet offer an alternative to gradient-elution LC of polymers. CEC may be attractive for the analysis of low-molecular components (monomers, oligomers, additives, etc.), if UV detection can be used. CE is a technique suitable for separating charged components. Few synthetic polymers are charged in solutions, but CE may still analyze them if they are made to interact with charged species, such as ionic surfactants. Just like small molecules, polymers can also be separated by liquid chromatography. Polar (normal-phase) or non-polar (reversed-phase) stationary phases can be used. Most commonly, gradients are used to separate polymers by liquid chromatography (gradient LC of polymers). Alternatively, isocratic (constant composition) conditions can be used and it may be possible to obtain conditions where polymers are eluted based on molecular structure, independent of their molecular mass. In that case, polymers may for example be separated according to end-groups (critical chromatography, CC). The University of Amsterdam-group research interests include a better understanding of these methods, leading to develop methods more systematically and much more rapidly. One tool to achieve this is computer simulation. Another is working on the combination of gradient LC with FTIR spectroscopy and mass spectrometry.

REFERENCES Selected references of the collected works of Poppe, Schoenmakers and Tijssen are listed in chronological order (see Chapter S-13 on the Chem Web Preprint server (http:==www.chemweb.com=preprint=))

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Overview: Chromatography — A New Discipline of Science Charles W. Gehrke a , Robert L. Wixom b and Ernst Bayer c a Department

of Biochemistry and the Experiment Station Chemical Laboratories, College of Agriculture, University of Missouri, Columbia, MO 65212, USA b Department of Biochemistry, University of Missouri, Columbia, MO 65212, USA c Institut fu ¨ r Organische Chemie, Universita¨t Tu¨bingen Research Center, Auf der Morgenstelle 18, 72076 Tu¨bingen, Germany

CONTENTS A. B. C.

Introduction . . . . . . . . . . . Attributes of modern chromatography Chromatography in the near future . References . . . . . . . . . . .

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A. INTRODUCTION Throughout this treatise Chromatography: A Century of Discovery, chromatography has been described as ‘a branch of science’. This is consistent with many other general commentaries. However, with the passage of the 20th century, it behooves us to assess the nature of chromatography for the 21st century. Chromatography as a ‘scientific discipline’ was first proposed by Berezkin [1] who considered the evolution of the word “Chromatography” and took into account the basic ideas of the definition suggested by M.S. Tswett. He presents the shortcomings of the official definitions and describes the new generalized definition that would give an adequate characterization of chromatography [1]. The scientific method proceeds from questions (sometimes intuition) to facts, experiments and=or observations, and then to a tentative hypothesis that can be tested or investigated. The world is understandable. Science goes beyond the evidence to explain what happens, to explore how, to advance why and thus lead to new hypotheses. Scientific experiments must have controls, be rational, avoid human or instrumental bias, be precise and be objective. Science moves frequently from observation to measurement

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and then goes on to produce new knowledge, to build models and to make predictions. The interpretation of scientific evidence may change, when new observation(s) challenge current theory(s). Chromatography, at this year 2001, fulfills each of these characteristics. The overall body of science, being so large, is divided into scientific fields or content disciplines, that facilitate specialization, coherence, communication and further advances of knowledge. Some branches or divisions of science are alive and grow for a period and then undergo senescence and whither away. Chromatography has grown and flourished as documented in this bound volume and the Supplement on the Internet. Consequently, longevity of a subject is necessary, but not sufficient to acquire a new status, such as becoming a discipline of science. From a historical viewpoint, chromatography has developed in the pre-1960 decades from areas of natural product chemistry, analytical chemistry, biochemistry and physical chemistry, and subsequently these earlier major components have undergone integration to become the present modern chromatography. For our review, some examples of the development and transformation in overall science include: Earlier names

Present names

Natural Science

!

Anatomy Physiology Physiological Chemistry Agricultural Chemistry Bacteriology Biochemistry C Microbiology C Genetics

! ! ! ! ! !

Biology C Chemistry C Geology C Physics C Astronomy, etc. Histology Physiological Chemistry (obsolete) Biochemistry Biochemistry Microbiology Molecular Biology

The word ‘discipline’ in English has several dictionary meanings, but in academia or in science, discipline refers to an organized field of study or knowledge, such as history, biology, psychology or chemistry. Thus, modern chromatography is similar to other sciences — chemistry, biology, or other present-day sciences — in having the following attributes.

B. ATTRIBUTES OF MODERN CHROMATOGRAPHY ž An organized field of study, such as GLC, HPLC, chiral chromatography, etc. ž A major and broad research undertaking leading to publications (see references throughout this book). ž A theoretical base that leads to methods or applications (see Chapters 1, 4, 5, 6 and S-11). ž A sense of direction and consistency within the discipline (see Chapters 1, 2, 3, 4, 5, 6, S-9, S-11 and S-14).

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ž A series of focused professional journals (Appendix 2 has at least 9 such research journals). ž A body of reviews, books, treatises and handbooks (see references at the end of the 15 chapters and in Appendices 4, 5 and 7). ž A comprehensive cluster of active professional societies (see Chapters 2 and S-8) with frequent meetings, seminars and=or conferences (see Appendix 6). ž A strong core of excellent leaders (see Chapters 1, 2, 4, 5 and 6), a large group of highly trained scientists (such as all the readers of the chromatography journals), and a rising generation of new leaders (see S-14). ž A key group of educators for chromatography in major universities with the charge to seek new horizons and continuous renewal, and to transmit this body of accumulated knowledge to students at several levels (see Chapters 4, 5, 6, S-9, S-11 and S-15). ž A sophisticated set of instruments (or tools) and methods for the research presented (see Chapters 4, 5, 6, S-9, S-10 and S-11). ž A source of funds for research — government agencies, research institutes, scientific industries or private foundations. ž A broad outreach to the other areas of sciences and society (see Chapters 1, 3, 4, 5, 6, S-8, S-9, S-11 and S-15). Each of the above criteria, when expressed alone or in combinations of two or three, is necessary but not sufficient to be a scientific discipline; an active modern scientific discipline requires all of these factors and attributes. Chromatography has each of these assets as described in detail in the stated chapters. Yes, chromatography has been considered in the past as a method (or a set of skills) for separation, identification, measurement and elucidation of structure of both small and complex molecules, but with the required theoretical base for modern chromatography, it became a branch of science. Now with the above 12 key characteristics chromatography becomes a scientific discipline. The evidence for this conclusion may be found in the preceding Chapters 1 to 7 and in the subsequent Chapters S-7 to S-15 plus the Appendices on the Internet — ChemWeb Preprint server (http:==www.chemweb.com=preprint=). To conclude, the Editors recommend that hereafter, chromatography merits the designation as A Discipline of Science.

C. CHROMATOGRAPHY IN THE NEAR FUTURE Two recent publications enhance the attributes of modern chromatography presented in our Overview (Chapter 7). Z. Deyl and F. Sˇvec have edited a new book (2001), entitled “Capillary Electrochromatography” (CEC) [2], which describes how CEC “is a rapidly emerging technique that adds a new dimension to current separation science. The major ‘news’ in this method is the hydrodynamic flow of the eluting liquid, which is typical of HPLC and is replaced by a flow driven by electro-endoosmosis. This increases the selection of available separation mechanisms: : : ” that are described in the ten chapters of this book. A combination of established processes (IEC, RPLC, etc.) with electromigration methods are now possible.

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The six articles in “Today’s Chemists at Work” by ACS Editors review LC, GC, IC and IEC, HPLC, affinity chromatography plus TLC, HPTLC and HPPLC, SFE and hyphenated methods (compare the relevant subsections of our Chapters 1, 4, 5, 6 plus S-9 and S-10). C.T. Vogelson writes that “most single column chromatographic techniques have reached their theoretical limits of utility”. The hyphenated methods with IR, MS or NMR “continue to develop and are regularly featured for their novelty and utility”. The Editors of “Chromatography – A Century of Discovery”, note that the Ryans’ Chapters are complementary to our book but have only several book references; for in-depth-journal or book references, refer to our cited chapters and Appendices in “Chromatography – A Century of Discovery” and on the Internet (http:==www.chemweb.com=preprint=).

REFERENCES 1. 2. 3.

V.G. Berezkin, Chromatography as a Scientific Discipline, Translated from Zavodskaya Laboratoriya Diagnostika Materialov, Industrial Laboratory, 65 (8) (1999) 2–11. Z. Deyl and F. Sˇvec (Eds.), Capillary Electrochromatography, Vol. 62 in J. Chromatogr. Library Series, Elsevier, Amsterdam, 2001, 429 pp. J.F. Ryan, (Eds.) Liquid Chromatography, Today’s Chemist at Work, Vol. 10, No. 9, pp. 1–88 (September 2001); this is the third set of reviews prepared by the editors of the journals of the American Chemical Society.

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1. QUOTATIONS In Chapter 1: Quote from J.C. Giddings from Unified Separation Science (1991), Wiley-Interscience, P. Murphy. Table 1.1 from J.C. Giddings – Unified Separation Science (1991), p. 232, Wiley-Interscience, P. Murphy.

2. PHOTOGRAPHS/GRAPHICS p. II: Graphic by Sammae Heard, University of Missouri – “Chromatography – The Bridge to the Sciences and Technology” – copyrighted. p. XII: Photograph of C.W. Gehrke and R.L. Wixom – by University of Missouri Medical Photography. Chapter 1: Photograph of M.S. Tswett – courtesy of V. Berezekin. Photograph of Towns Important in Tswett’s Life – “75 Years of Chromatography – A Historical Dialogue”, L.S. Ettre and A. Zlatkis (1979), p. 485, Elsevier Science. Photograph of L.S. Palmer – courtesy of the University of Minnesota Archives. Pittcon Photographs – courtesy of R.L. Wixom. Graphics by Sammae Heard, University of Missouri – copyrighted. Chapter 4: Fig. 1 – Photograph of C.W. Gehrke, Centennial President of AOAC (1984) – courtesy of C.W. Gehrke. Fig. 2 – Photograph of University of Missouri Chromatography Team – courtesy of C.W. Gehrke. Fig. 10 – Photograph of University of Missouri Research Team at NASA Ames Research Center – courtesy of C.W. Gehrke. Fig. 20 – Photograph of Clean Room for Apollo 12 Analysis – courtesy of C.W. Gehrke. Fig. 21 – Photograph of Laboratory of Chemical Evolution, University of Maryland – courtesy of C.W. Gehrke.

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Chapter 5: Photographs supplied by Authors of all contributions – no permission needed. Photograph of G. Blobel – supplied by Author. Graphics by Sammae Heard, University of Missouri – copyrighted. Photograph of A.J. Oparin and C. Ponnamperuma in the book “A Lunar Based Analytical Laboratory”, p. XIII, A. Deepak Publishing, Inc., Hampton, VA, USA. Photograph of Cs. Horva´th – Barrk K. Citt, photographers. Photograph of K. Unger – Studio Hirch 6843 Darmstadt, Germany – no permission needed. Chapter 6: Graphics by Corrine Barbour, University of Missouri – copyrighted.

3. PUBLISHED FIGURES In Chapters 4, 5 and 6: V. Berezkin – Figs. 1, 2, 3 and 5 with permission to reproduce from Russian Journals. Fig. 1 – Davankov, V.A. et al., Doklady (1970) 94; Fig. 2 – Blagodatskikh, I.V., et al., Polymer Science, Ser. A. 39 (10) (1997) 113S; Fig. 3 – Zhdanov, V. M. et al., Mol. Genet. Mikrobiology. Virusol. 10 (1984) 37; Fig. 5. Serdan, A.A. et al., Zhurnal fizicheskoi khimii, 65 (10) (1991) 2638. Fig. 4 from Pirogov, A.V., et al., J. Chromatogr. A. (850) (1999) 53; and Fig. 6 from Chizhkov, V. P., Talanta 34 (1) (1987) 227, Elsevier Science. P. Brown – Figs. 1 and 3 – “Chromatography and Modification of Nucleosides”, C.W. Gehrke, Ed.(1990), Vol. 45C, pp. C151 and C166, Elsevier Science. Fig. 1 also published in J. Chromatogr. 126 (1979) 725–736, Elsevier Science; Fig. 2 – J. Chromatogr. 126 (1979) 737–750; Fig. 3 – Hartwick et al., J. Chromatogr. 126, (1979) 659–676; Fig. 4 – Zakaria et al., J. Chromatogr., 179 (1979) 109–117, Elsevier Science; Fig. 5 – “Computers and Biomedical Research”, H.A. Scoble, vol. 16 (1983) pp. 300–310, Academic Press. M. Beroza – Figs. 3, 4, and 5 – M. Beroza, J. Chromatogr. Sci. 13 (1975) 314–321, Preston Publications. K. Cramers – Fig. 1 – K.A. Kramers et al., J. Chromatogr. A 856 (1999) 315–329, and Fig. 2 – A.J.J. van Es and K.A. Cramers from J. Chromatogr. 477 (1989) 39–47, Elsevier Science. H. Engelhardt – Figs. original – no permission needed. P. Flodin – P. Flodin, J. Chromatogr. 5 (1961) 102, Elsevier Science. J. Fritz – Fig. 1 – J. Li, W. Ding and J.S. Fritz, Abstract no. 1178, PittCon 99, Orlando, FL, USA. C. Gehrke – Book – “75 Years of Chromatography – A Historical Dialogue”, L.S. Ettre and A. Zlatkis (1979), p. 75–86, Elsevier Science. Fig. 3 – “Amino Acid Analysis by GC”, Vol. I, p. 36, CRC press; Figs. 4, 5 and 6 – courtesy of C.W. Gehrke; Fig. 7 – Vol. I, p. 39; Figs. 11, 12 – courtesy of C.W. Gehrke; Figs. 13, 14, 15 – Vol. II, pp. 155, 156, 162; Fig. 16 – courtesy of C.W.Gehrke; Figs. 17, 18 – Vol. II, pp. 156–159; Fig. 19 – Vol. I, pp. 78, 79; Figs. 20, 21 and 23 – courtesy of C.W. Gehrke; Figs. 22 – Vol. II, p. 148. A. Guttman – Fig. original – no permission needed. J. Hermansson – Figs. 1, 2, 3A, 3B and 4. H. Hill – Fig. 2 – C. Wu et al., Anal. Chem. 72 (2) (2000) 391–395, Am. Chem. Soc. C. Horva´th – Fig. 3 – “75 Years of Chromatography – A Historical Dialogue”, L.S. Ettre and A. Zlatkis (1979), Elsevier Science; and Fig. 5 – I. Molna´r, Chromatographia 11 (1978) 260–264, Chromatographia. J. Jana´k – Fig. – Chromatography of fatty acids, J. Chromatogr. 21 (1966) 207, Elsevier Science.

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B. Karger – Fig. 2 – O. Salas-Salono, Anal. Chem. 70 (1998) 3996–4003, and RPLC behavior of RNase A, from X.-M. Lu et al., J. Chromatogr. 359 (1986) 19–29, Elsevier Science. J. King – Fig. 1 – J.W. King, J. Chromatogr. 28 (1990) 9–14, Elsevier Science; and Fig. 2 – J.W. King, JAOAC 81 (1998) 9–17, AOAC International [Ref. 12]. J. Kirkland – For Fig. 5, restriction fragments – Original from Hewlett Packard – no permission needed. E. Klesper – Figs. 2, 3 and 6 – Original – no permission needed; and Figs. 1, 4, 5, 7, and 8 – all published earlier but stated with permission. J. Knox – Fig. 3 – J. Knox and H.P. Scott, J. Chromatogr. 282 (1983) 297–313, Elsevier Science; and Fig. 2 – J.H. Knox and I.H. Grant, Chromatographia 32 (1991) 317–328, Elsevier Science. D. Martire – Ph.D. Thesis of Guiterrez and Osonubi (1998) – no permission needed. H. Miyazaki – 2 Figs. – no permission needed. E. Morgan – Fig. 1 is published in M.F. All et al., Biochemical Systematics and Ecology (A Pergamon Journal) 16 (1998) 647–654, Elsevier Science. Fig. 2 – A.T. Affygale and E.D. Morgan, Anal. Chem. 58 (1986) 3054. Copyright 1986, Am. Chem. Soc. J. Pora´th – Figs. 1A and 1B – J. Chromatog. A 732 (1996) 261–269, Elsevier Science. J. Pawliszyn – From blanket permission of all Figs. from “Solid Phase Microextraction, Theory and Practice” (1997), Wiley-VCH, New York. M. Prost – Figs. 1 and 2 – Original – courtesy of M. Prost, Spiral Co., Dijon, France. R. Smith – Fig. 1 – A.G. McKillop et al., J. Chromatogr. A 730 (1996) 321–328, Elsevier Science. P. Sandra – Figs. – Original – no permission needed. G. Schomburg – Fig. 2 – USA patent, G. Schomburg. R. Scott – Fig. 1. C. Simpson – Figs. 1–3 – Original – no permission needed. L. Snyder – Figs. 1 and 2 – no permission needed. S. Terabe – Figs. 1 and 2 – unpublished data – no permission needed. R. Tijssen – Fig. 1 – unpublished – no permission needed. K. Unger – Fig. 1 – I. Issaeva et al., J. Chromatogr. A 46 (1999) 13–23, Elsevier Science. P. Wankat – Fig. 1 – C. Simms et al., Chem. Eng. Sci. 51 (5) (1996) 701–711 with approval. C. Welch – Figs. – no permission needed. I. Wainer – Fig. 1 – Original – no permission needed. I. Wilson – Fig. 2 – J.P. Shochor et al., Anal. Chem. 68 (1996) 4431–4435, Am. Chem. Soc. G. Xu – Fig. 1 – Unpublished – no permission needed.

695

Author/Scientist Index with additional biographical information

INTRODUCTION The columns in this Index on the following pages contribute to the integration of the book. Along with the chromatographer’s name may be found their country at the time of their award (see Country Codes below), deceased (indicated by a superscript ‘d’) and those present in the earlier biographical article in L.S. Ettre and A. Zlatkis’ book, “75 years of Chromatography – A Historical Dialogue” (1979) (indicated by a superscript ‘e’). The investigator’s research or subject area is indicated in column 2, and follows the ‘Subject Code’ listed below. Many of these Authors are also found in the Supplement on the Internet and are listed in the Author Index on the Internet. Subject code or seminal concepts for Author Index (same as Table 1.2, Chapter 1.G): a. b. c. d. e. f. g. h. i. j. k. l. m. n. o. p. q. r. s.

Theoretical contributions to chromatography. Classical adsorption=partition chromatography (plus PC and TLC). Ion-exchange chromatography. Gas chromatography=capillary-gas chromatography. Support or bonded-phase chemistry. Detectors in chromatography. Size-exclusion chromatography. High-performance liquid chromatography. Affinity chromatography=bioaffinity chromatography=biosensors. Petroleum chromatography. Instrumentation in chromatography. Electrophoresis=capillary electrophoresis=capillary electrochromatography. Ion chromatography. Synthetic and biological membrane separations and other techniques. Supercritical-fluid chromatography=extraction. Hyphenated=coupled=tandem techniques in chromatography. Chiral chromatography=pharmaceutical separations. Biomedical sciences=chromatography. Environmental sciences=chromatography.

696

Author=scientist index

Note: All living Awardees and several other chromatographers (Chapter 2, Tables 2.1 to 2.12) were invited to contribute an article on their research careers for Chapters 5 or 6; 125 scientists did so and may be found in the following Index. Some researchers declined for various reasons, and hence their page reference is solely for Chapter 2; their Subject Area below – for some, but not all – is derived from published biographical sources (American Men and Women of Science, Who’s Who, International Who’s Who, etc). Such insertions may not be sufficiently detailed or current. The Subject Areas are based on what was written in the Author’s respective chapters, or published biographical information; in general, they are representative of their research work. Some of the following page references in the Author and=or Scientist Index are brief in this text, mainly due to coverage of a century of chromatographic leaders. Also, for brevity, this Author Index has concentrated mainly on senior authors or principal investigators. However, these brief comments will enable the reader to find, if desired, a fuller description of their lives and their research areas. Many of the scientists in this Index, and some others are also found in the Supplement on the Internet at www.chemweb.com=preprint=. COUNTRY CODES FOR AWARDEES IN THIS AUTHOR=SCIENTIST INDEX* Abbrev.

Country

Abbrev.

Country

A AR B BR CAN CH CZ D E ES F FI H I

Austria Argentina Belgium Brazil Canada Switzerland Czechoslovakia (Czech Republic) Germany (Federal Republic) Spain Estonia France Finland Hungary Italy

IS JAP MEX N NL P PRC RSA R S SU UK USA

Israel Japan Mexico Norway The Netherlands Poland China Republic of South Africa Russia Sweden Soviet Union United Kingdom United States of America

* Country at the time of their Award.

AUTHOR/ SCIENTIST INDEX – Plus key information A Adams, B. A. (UK) Adlard, E. R. (UK) e Albaiges, J. (E) Albert, C. (D) ˚ . (S) Albertsson, P. A Armstrong, D. W. (USA)

c d, f, j, s

b a, b, d, e, h, q, r

17 23, 49, 45 50 50 42, 45, 51, 109–123

Author=scientist index

697

B Bartle, K.D. (UK) Bauman, W.C. (UK) Bayer, E. (D) Beems, J. (NL) Belfort, G. (USA) Bell, L. (USA) d Berezkin, V.G. (SU/R) Bergstrom, W.B. (USA) Beroza, M. (USA) Berridge, J.C. (USA) Billiet, H.A.H. (NL) Binsheng L. (PRC) Bleumer, J. (NL) Blomberg, J.J. (NL) Boer, H. (NL) Bos, J. (NL) Bowers, L.D. (USA) Bradshaw, J.S. (USA) Brenner, M.L. (USA) Brinkman, U.A.Th. (NL) Broge, J.M. (USA) Broughton, P.B. (UK) Brown, P.R. (USA) Bruner, F. (I) d

o c d, e, k, q d, h, p n a, b, d, h d, k, s d b g, h d, h, p d, k g h c, d, n p

a, h, l, r d, e, p, s

50 18 XV–XVII, 42, 45, 46, 47, 80, 123–134 678 44 52 621–643 42 42, 135–142 50 657, 677 656 678 678 29, 674 678 52 44, 257–263 52 45 52 44 44, 47, 48, 144–154 47

C Cabrera, K. (D) Camilleri, P. (UK) Carducci, C. (AR) Carr, P.W. (USA) Chen, N. (PRC) Chen, Y.Z. (PRC) Chester, T.L. (USA) Chizkov, V.P. (SU) Ciola, R. (BR) Claesson, S. (S) d Clark, B.J. (UK) Cohn, W.E. (USA) d Colin, H. (F/USA) Consden, R. (UK) d Cram, S.P. (USA) Cramers, C.A. (NL) Cramers, K.A. (NL) Cremer, E. (A) d,e Curvers, J. (NL)

e, h h, l, r d, h, I d r a, o b d, f, h, j, o j c d d see below a, d, e, i, l, m, p a, b, d, f d

50 51, 664–665 42, 48, 51, 52 657 658 49, 154–161 638 51, 666–667 23, 57 50 18, 42 50 15 48 46, 47, 49, 51, 162–168 12, 23, 46 657

D Dai, C.Z. (PRC) Dal Nogare, S. (USA) d,e Dandeneau, R.D. (USA)

d a, d, d

657 42, 47 47

698 Davankov, V.A. (SU/R) Dawkins, J.V. (UK) Day, D.T. (USA) d Desty, D.H. (UK) d,e DeVries, J.W. (USA) Deyl, Z. (CZ) Dhe´re´, C. (CH) d Dijkstra, G. (NL) Dimick, K.P. (USA) d Ding, J.Q. (PRC) Drake, E. (USA)

Author=scientist index a, h, q a, e, g b d, j, k c, s l b d, k a, b

622, 630–631 168–171 22 23, 45, 46 52 25 9 29 48 655 22

E Eglinton, G. (UK) Engelhardt, H. (D) Engler, C. (D) d Ettre, L.S. (USA) e

d, h, p a, e, h, k, n, o, s b, j a, d, e, h, k

Erveinov, V.V. (SU) Evans, M.B. (UK)

g a, d, e, i, m, r

45 45, 48, 171–177 23 3, 25, 27, 41–42, 45–47, 49, 52, 55–68, 178–183 631–632 183–189

F Fair, J.R. (USA) Flodin, P.G.M. (S) e Freeburg, J.A. (USA) Freiser, H. (USA) Fritz, J.S. (USA) Fu, R.N. (PRC)

a, c, g, l, r

c, g, h, l, s b

44 46, 368–375 52 44 42, 48, 52, 375–385 655

G Games, D.E. (UK) Gehrke, C.W. (USA) d,e Gehrke, C.W. & Associates Geng, X.D. (PRC) Giddings, J.C. (USA) d,e Gilpin, J.E. (USA) d Glueckauf, E. (UK) d,e Golay, M.J.E. (USA) d,e Goodall, D.M. (UK) Goppelsroeder, F. (D) d Gorbunov, A.A. (SU) Gordon, A.H. (UK) d Gorshkov, A.V. (SU) Grant, D.W. (UK) Grob, Konrad (CH) Grob, Robert L. (USA) Guan, Y.F. (PRC) Guiochon, G. (F/USA) Guttman, A. (USA)

a, d, e, h, k, r, s h a, b, d, h j a, c, d a, d, f, h, k g b g b g d, j d, e, p b, d, h, s d a, b, e, h, p a, f, l, q, r

45 XI–XV,42, 44, 48, 69–97 69–97 655 2, 25, 42, 44–46, 48, 190–191, 678 23 18, 20 13, 42, 44, 46, 48 50 7 631 15 631 29 47, 50 48, 51, 192 657 42, 44–46, 48, 193–199 200–205

Author=scientist index

699

H Hagen, D.F. (USA) Hais, I.M. (CZ) d Hala´sz, I. (D) d Hall, N.F. (USA) Hancock, W.S. (USA) Hara, S. (JAP) Hatano, H. (JAP) d Hawthorne, S.B. (USA) Heftmann, E. (USA) e Helfferich, F.G. (USA) Henis, J.M.S. (USA) Hermansson, J. (S) Hesse, G.E. (D) d,e Hill, H.H. (USA) Hjerte´n, S. (S) Holmes, E.L. (UK) Horning, E.C. (USA) d,e Horning, M.G. (USA) e Horva´th, Cs. (USA) e Horwitz, E.P. (USA) Hou, J.D. (PRC) Huber, J.F.K. (A) d,e

b, f b, d, e, h b h a, b, d, h b, h, k, p, s a, o, p, s b, r a, c c, i, n I, r, s b, d, q d, f, h, k, m, o, p a, e, h, i, l, m, n, s c d, f, k, p, r d, f, p, r, a, b, d, e, h, l, r, t b, h h a, e, h,k, p

52 25 46 15 45 46, 605, 616, 620 46, 605–606, 612–613, 617 49, 205–212 29 42, 212–217 44 50, 218–225 12, 23, 46 49, 226–230 42, 231–236 17 42, 46, 48 29, 46 25, 42, 44–48, 51, 236–248 44 657 42, 45, 48, 248–250, 675

I Ikekawa, N. (JAP) Ioffe, B.V. (SU/R) d Issaeva, T. (D) Ishii, D. (JAP) Issaq, H.J. (USA) Ismailov, N.A. (SU) d Izatt, R.M. (USA)

a, d, h, k, p, r a, d e, h a, d, e, f, h, k, o, r b c, d, n

46, 605, 611 622–623, 629–630 46, 47, 250–257, 606–607, 612 51 15 44, 256–263

J James, A.T. (UK) e Jana´k, J. (CZ) e Jellum, E. (N) Jennings, W.G. (USA) Jinno,K. (JAP) Johnson, D.C. (USA) Johnson, J.F. (USA) d Johnson, P. (USA) Jones, C.E.R. (UK) Jorgenson, J.W. (USA)

a, d, r a, b, d, h, s, a, b, c, d, g, h, i, p, r a, d, e a, d, e, h, o p, s

a, d, e, f, h, k, l, p, r, s

12, 23, 28–29, 46 12, 23, 25, 46, 263–270 45, 46, 49, 270–277 45, 47, 49, 52, 277–283 47, 603–621 52 42 52 45 42, 45, 47–48, 51, 283–290, 617–618

a, b, d, s, a, d, f a, b, d, h, l, p, r

52 29, 45, 48, 290–300 641 44, 46, 48, 51, 300–309, 617–618

d, j,

K Kahlil, S.K.W. (USA) Kaiser, R.E. (D) e Kalmanovskii, V.I. (SU) Karger, B.L. (USA)

700 Karmen, A. (USA) e Karrer, P, (CH) d Keulemans, A.I.M. (NL) d,e Khym, J.–X. (USA) King, C.J. (USA) King, J.W. (UK) Kirchner, J.G. (USA) d,e Kirkland, J.J. (USA) e Kiselev, A.V. (SU/R) d,e Kiselev & School (SU/R) Kitchner, J.A. (UK) Klesper, E. (D) Klinkenberg, A. (NL) Knapman, C.H.E. (UK) Kok, W. (NL) Knox, J.H. (UK) Kova´ts, E. sz. (CH) e Kraak, J. (NL) Kra¨nzlin, G. (D) d Kraus, K.A. (USA) d Kressman, T.R.E. (USA) Kreveld, M.E. (NL) Krylov, V.A (SU) Kuhn, R. (D) d Kuhn, T.S. (USA) Kuhr, W.G. (USA) Kunin, R. (USA) Kuo, K. (USA) Kwantes, A. (NL)

Author=scientist index d, f b b c a, d, h, o, s b a, d, e, g, h, k a, b c a, d, h, o a, d l a, d, e g b c c d d b

29, 46 10 11, 46, 674 18 44 49, 309–316 15–16, 29 42, 45, 48, 51, 316–329 46, 623, 652–627 625–627 18 329–246 674 45 677, 680, 685 42, 45–48, 346–357, 680 29, 45, 46 680 8 18–19, 42 18

c a, d, h d

643 9–10 3 50 18 70, 78, 80 675

d, h, k, l, o, s b b a, d, h, j, l, o, p d n d d, f h a, d, f, h, k, o r, s d b d, f, h, k, p, r e e a, e, h, q a, d, f, k, r, s b a, b, j d, h d

51, 659–670 9–10, 25, 46 25, 29 42, 45–49, 51, 357–364, 619 657 44 657 29, 45–46 50 50, 364–368 640 9 46 636 655 42 42, 46, 48, 385–389 651, 656, 658 654, 658 51, 669 654

L Lanc¸as, F.M. (BR) Lederer, E. (E) d,e Lederer, M. (I) e Lee, M.L. (USA) Li, H.C. (PRC) Li, N.N. (USA) Liang, X.M. (PRC) Liberti, A. (I) e Lindner, W. (A) Lingeman, H. (NL) Lipavskii, V.N. (SU) Lippmaa, T. (ES) d Lipsky, S.R. (USA) d,e Lisichkin, G.K. (SU) Liu, G.Q. (PRC) Lochmu¨ller, C.H. (USA) Lovelock, J.E. (UK) e Lu, P.C. (PRC) Lu, W.Z. (PRC) Lubkowitz, J. (USA) Lue, Z. F. (PRC)

Author=scientist index

701

M Macek, K. (CZ) Manjarrez, A. (MEX) Marriott, P. (AUST) Markell, C.G. (USA) Markides, K.E. (S) Martin, A.J.P. (UK) e Martin, M. (F) Martire, D.E. (USA) Mchedlishvili, B.V. (SU) McNair, H.M. (USA) Meinhard, J.E. (USA) Merrifield, R.B. (USA) Michaels, A.S. (USA) Miyazaki, H. (JAP) Moore, S. (USA) d,e Moreno, A.M. (MEX) Morgan, E.D. (UK) Mosbach, R. (S) Myers, R.J. (USA)

a, b, d, e, i, s d, h, j, r, s

a, d, e, f, h, l, o, q a, b, d, a, h, r a, b, d, h, o g d, h, o b b, c, h n a, d, e, l, p, r, s b, c, k d, h, j, r, s d, f, k, s g c

25, 46, 390–396 51, 667–669 50 52 50, 396–399 12, 15, 23, 41–43, 46, 57–58 50, 399–406 48, 51, 406–411 633 49, 51, 661 15 412–413 44 46, 413–419, 607, 612 21, 41–42, 77, 79 667–669 50, 419–424 45 18

d a, b, d, f, h, l, o, p, r, s

50 50 42, 44–49, 51, 424–434, 613, 619

N Nicholson, J.K. (UK) Nickless, G. (UK) Novotny, M.V. (USA) O Okuyama, T. (JAP) Olson, K.N. (USA) Oostervink, R. (NL) Oparin,A.I. (SU) d Ou, Q.Y. (PRC)

a, h, l, r g b

47, 608, 616 52 680 72 655

P Palmer, L.S. (USA) d,e Partridge, S.M. (UK) Patton, H.W. (USA) Pawliszyn, J. (CAN) Phillips, C.S.G. (UK) e Phillips, John B. (USA) d Pierson, S. (USA) Pirkle, W.H. (USA) Poole, C.F. (UK/USA) Ponnamperuma, C. (USA) d Poppe, H. (NL) Porath, J. O. (S) e Pretorius, V. (RSA) d,e Price, S. M. (USA) Prost, M. (FR) Purnell, J. H.(UK) d

b c d, f k, n, p a, d, f, j d, p a, q, r a, d, e, l, o, p, s c a, b, d, f, g, h, k, l a, e, g, i, r b, d„ f a, d, e, h, k, p, r, s, a, c

7–9, 33, 51–52 18 29 50, 434–440 28, 29, 31, 45, 58 46 52 42, 45, 48, 51, 440–452 46, 50, 453–459 70–72, 95 45, 47, 670–671, 674–685 46, 459–470 46 52 470–474 46

702

Author=scientist index

R Ray, N.H. (UK) e Regnier, F. E. (USA) Reineccius, G.A. (USA) Revelskii, I.S. (SU) Rijks, J.A. (NL) Rijnders, G.W.A. (NL) Riley, C.M. ((USA) Rogers, L.B. (USA) d Rogowski, W.F. (P) d Rohrschneider, L. (D) e Runge, F.F. (D) d

d, f, j e, h d p d, h, k, l d, o d, h b d b

29 42, 45, 48, 51 52 640–641 48, 475–477 48, 675–676 50 42, 48 9 29 7

S Sackett, P.H. (USA) Saelzer, R. (Chile) Sakodynksii, K.I. (SU/R) d,e Samsonov, G.V. (SU) Samuelson, O. (S) Sandra, P. (B) Schoenmakers, P.J. (NL) Schomburg, G. (D) e Schwartz, R.D. (USA) Scott, C.D. (USA) e Scott, R.P.W. (UK/USA) e Shipgun, O.A. (SU) Shraiber, M.S. (SU) d,e Sie, S.T. (NL) Sievers, R.E. (USA) Simmonds, P. (UK) Simpson, C.F. (UK) Sjo¨vall, J.B. (S) Skortsov, A.M. (SU) Small, H. (USA) Smith, N.W. (UK) Smith, R.M. (UK) Snyder, L.R. (USA) e Spedding, F.H. (USA) d Stol, R. (NL) Stahl, E. (D) d,e Stalling, D. L. (USA) Stegeman, G. (NL) Stein, W.H. (USA) d,e Stock, R. (UK) Strain, H. H. (USA) Stross, F.H. (USA) e Synge, R.L.M. (UK) d,e

a, b, e, j c c a, d, e, h, l, p, q, s a, b, d, f, g, h, o a, b, d, e, f, i d, f, g, j, h, r a, d, f, h, k, p, q c b, c o a, d, e, f, k, o, s a, d, e, f, h, l, q, r, s c, d, e, g„ h, p, r c c, g, m a, b, e, h, l, o a, b, d, h c l b, p, s, r c, d, h g b, c, k, p, r

52 51 46, 623–624, 628–629, 633 631–633 19–20 45, 47, 48, 51, 488–495 50, 671–685 45–49, 495–505 29 29 42, 45–46, 506–511 633–635 15 675 46, 49, 511–521 50 50, 521–533 46, 533–541 631 42, 48, 541–546 50 50, 546–551 42, 45, 48, 51–52, 552–560 19

b d, f, j a, b, r

16, 42 74–96 680 21, 41–42, 77, 79 45 42 29 12, 41, 57

d, e, h, r a, d, e, k, l

250–257 45, 47, 250–257, 560–566, 608, 612, 617

T Takeuchi, T. (JAP) Terabe, S. (JAP)

Author=scientist index Teranishi, R. (USA) d,e Tijssen, R. (NL) Tiselius, A.W.K. (S) d,e Tompkins, E.R. (USA) d Tswett, M.S. (R) d,e Tuey, G.A.P. (UK) d Tyssen, R. (NL)

703 d a, b, d, e, f, g, h, k, l, p a, b, c, g, l, r c b See Tijssen, R.

29 670–685 41 18–19 4–6, 30, 32–34, 44, 625 45 50

U Unger, K. K. (D) USSR Medallists (SU)

a, e, g, h, i, l

42, 45, 566–572 53–54

h d a, d g g b b a,d b

683 674 29, 677–678 678 678 674 51 9 627–628 636

a, e, h, i, q, r a, c e a, b, c, g, n, a, q r b, d, h, k, p r b d b, c, d, r b b b

45, 572–578 48, 578–583 655 44, 583–588 440–452 50, 588–594 9–10 675 XV, 1–38 636 655 654

a, d, h, l, r, s s

648–658 658

b a, b, f, h, k, l, q, r d

627 52, 594–498 650

p

641

V Van Bruijnsvoort, M. (NL) Van de Craats, F. (NL) Van Deemter, J.J. (NL) e Van den Hoed, N. (NL) Van der Does, J. (NL) Van Dijck, W.J. (NL) Vega, M. (Chile) Vegezzi, G (CH) d Vigdergauz, M.S. (SU/R) d Volynets, M.P. (SU) W Wainer, I.W. (CAN) Walton, H.F. (USA) Wang, J.D. (PRC) Wankat, P.C. (USA) Welch, C.J. (USA) Wilson, I.D. (UK) Winterstein, A. (CH) d Wittebrood, R.T. (CAN) Wixom, R.L. (USA) Woronstov, A.M. (SU) Wu, C.Y. (PRC) Wu, J. (PRC) X Xu, Guowang (PRC) Xu, X.B. (PRC) Y Yonovskii, M.I. (SU) d Yeung, E.S. (USA) Yu, W.L. (PRC) Z Zaikin, K.G. (SU)

704 Zechmeister, L. (H, USA) d,e Zenkevich, I.G. (SU) Zerenner, E.H. (USA) Zhang, X.M. (PRC) Zhang, Y. (PRC) Zhou, L.M. (PRC) Zhou, T.H. (PRC) Zhu, A. (PRC) Zhu, B.L. (PRC) Zhukhovitskii, A.A. (SU/R) d,e Zlatkis, A. (USA) d, e Zuiderweg, F.J. (NL) Zumwalt, R.W. (USA)

Author=scientist index b p a d, h, l a, b, h, r q l a, b a, b, d d, f, j, k, p, q, r, s a, d c, d, h

10–11, 42 642 47 657 644–658 655–658 658 658 651, 655 46, 624, 627 23, 42, 45–46 78–96

705

Subject Index This Subject Index covers the many areas in the printed volume. The key words in Chapters S-8 to S-15 in the Supplement are essentially the same as those in the printed volume, and they may be found in the Supplement’s Subject Index on the Internet; both should be examined for full coverage. The major headings in bold type below provide a degree of interaction for the Book and Supplement. Since chromatography appears in many places of the subcategories, it is abbreviated below to ‘C’.

A Automated chemistry 72 Automation in ‘C’ 365, 485 Awardees in Chromatography: Awards in ‘C’ 39–54 Identification of recipients 39–54 Peer review 30 Requirements 29–30 B Biographies of Chromatographers 601–685 See Author Indexes Biomarkers 97, 270 Biomass to oil 133 Biotechnology 243

99–599,

C Catalytic transformation 627 Cell, single, analysis 433 Chlorofluorocarbons 488 Coherence 214–216 Chromatography, general attributes: Branch of Science 30–32, 687 Builders of ‘C’ 40–56 ‘C’ as a bridge 30–31 Discipline of science 7, 30–31, 687–688 Discovery process 31 Driving forces 24

History, early of ‘C’ 2–38, 687–688 Milestones in ‘C’ 28 New discipline in ‘C’ 689 Seminal concepts 26, 108 Separation of sciences 27 See Authors w=‘a’ Subject Code Chromatography, literature of: Bibliography 23 Journals, abstract 25 Journals, original 518 Patterns in scientific literature 23–26 See Subject Index of Supplement for other references Chromatography, serial books by publisher See Subject Index for Supplement Chromatography, main variants of: See also Subject Index for similar headings in Supplement Adsorption ‘C’ 4–11, 194, 583–587, 623, 625–627 See Authors w=‘b’ subject code Affinity ‘C’ 463 (or bioaffinity ‘C’ or bioselective ‘C’) See Authors w=‘s’ Subject Code Chiral ‘C’ 111–122, 218, 221–224, 441– 452, 573, 575, 596–598, 616, 622, 630–631 See Authors w=‘q’ subject code Gas–liquid ‘C’ (including capillary GC) 12–15, 125–131, 178–183, 184, 192, 263–265, 277–282, 290–291, 359, 414, 420, 454, 476, 478, 499, 515, 590,

706 610–612, 623, 624, 629, 639–640, 646, 650–655, 666, 674–676 See Authors w=‘q’ Subject Code High-performance liquid ‘C’ 144–145, 155–161, 171, 184, 192, 231, 237–247, 274, 316–318, 378, 400, 430, 500, 522, 552–560, 554, 583, 645, 646, 650, 657, 665, 671 See Authors w=‘h’ Subject Code Ion-exchange ‘C’ 17–21, 57, 534, 578–583, 633, 646 See Authors w=‘c’ Subject Code Liquid–liquid partition ‘C’ 11–12, 349, 548, 674, 684 Paper ‘C’ 15, 390 See Authors w=‘b’ Subject Code Planar ‘C’ 15–17, 635–636 See Authors w=‘b’ Subject Code Size-exclusion ‘C’ 354, 368, 372, 373, 460– 463, 633, 681, 682–683 See Authors w=‘g’ Subject Code Supercritical-fluid ‘C’ (SFC) 155–161, 207, 309–316, 329–344, 358, 363, 396–398, 424, 434, 457, 486, 514, 547, 619, 663 See Authors w=‘o’ Subject Code Thin-layer ‘C’ (TLC) 15–17, 172, 393, 588, 616, 619–620, 625, 636, 639, 646, 667 See Authors w=‘b’ Subject Code Chromatography, other forms: Carbon-skeleton ‘C’ 136 Cascade-mode multiaffinity ‘C’ (CSMAC) 468 Circular ‘C’ 637–638 Computer chip technology 616 Displacement ‘C’ 587, 625 Electron donor affinity ‘C’ 465 Expert systems 656–658, 672 Fast gas ‘C’ 167, 248, 475, 671 Field-flow fractionation (FFF) 321, 403, 671, 674, 683 Gel-permeation ‘C’ (GPC) 460, 681 Head space analysis ‘C’ 629 High-performance TLC 689 High speed gas ‘C’ 656, 677 Hydrodynamic ‘C’ 545, 671, 674, 677–680, 684 Hydrophobic-interaction ‘C’ 466 Hyphenated ‘C’ 307, 604, 606, 609, 662, 664, 677 Immobilized metal affinity ‘C’ 213, 466, 515 Intelligent ‘C’ 465 Inverse gas ‘C’ 185

Subject index Ion ‘C’ 259–262, 375, 377, 541–543 Ligand exchange ‘C’ 117, 578 Multi-dimensional ‘C’ 198, 307, 672 Open-tubular gas ‘C’ 178–182, 654 Open-tubular liquid ‘C’ 252, 613, 671–672, 677 Plasma ‘C’ 613 Polyelectrolytes 631 Precipitation ‘C’ 629 Preparative ‘C’ 196, 354, 624, 629, 667 Process ‘C’ 257 Reaction chromato-mass spectrometry 641– 642 Reversed phase ‘C’ 242, 326, 604 Thermal field flow fractionation (ThFFF) 323 Turbulent gas ‘C’ 165 Vapor phase analysis ‘C’ 629, 668 Unified ‘C’ 662 Chromatography, relation to other science areas: Analytical biochemistry 74–76 Analytical chemistry 141 Biotechnology 243 Clinical chemistry 658 Pharmacology 219 Public health 663 Chromatography, specific features: Agarose derivatives 464 Aluminum oxides 651 Anion exchange resins 17, 578 Bonded phases 383 Capillary columns 280–282, 676 Cation exchange resins 17–20 Chelating resins 463 Chiral stationary phases (CSP) 443–447 Column design=development 75, 197, 396 Column selection 351 Computer simulation 559 Computer chip technology (microfabricaton) 200, 202, 616 Coupled techniques 200, 202 See hyphenated techniques Critical ‘C’(of polymers) 631–633 Cyclodextrins 111, 118, 655 Derivatization 13, 83–85, 455 Detectors 75, 386, 517, 596–598, 606, 619, 640–641, 649–651 Dextrans, cross-linked 371–373 See SEC Electron exchange resins 21 Elution theory 556, 672 Fused silica columns 280

Subject index Glycopeptides=proteins 142, 218 Headspace analysis (vapor phase analysis) 629, 668 Hydrophobic gels 466 Hyphenated techniques=coupled columns 272–273, 284, 593, 606, 641–642, 650, 664 Impurities, inorganic 643 Lab-on-a-chip 200, 202, 616 Microcolumns in ‘C’ 64, 251–252, 612, 613, 622, 662 Molecular sieving 460 Multi-dimensional ‘C’ 366 Non-porous packings 328 Phases, π complex 119 Phenol-formaldehyde resins 17 Polydimethylsiloxane sorption 361–363, 628, 654–655 Potato starch columns 21, 369 Porous packings 320, 328 Rate theory=equation 674 Retention mechanisms 550, 553, 643 Sample preparation 206–211, 534, 588, 604, 662 Selectivity 547, 557, 617 Sephadex ‘C’ 368, 370, 461 Silica chemistry ligands, surface 498, 566, 567, 570, 634, 636–637, 655–657 Stationary phase and modification 173–174, 319–321, 624, 628, 651 Sulfonated styrene polymers 17 Support phases See Authors w=‘e’ Subject Code Tandem techniques See Authors w=‘p’ Subject Code Unified ‘C’ 159–161, 253, 255, 607 D Detectors in ‘C’ 365–366, 388, 507, 514, 595– 596, 671 Dextran gels (Sephadex) See SEC Dioxin crisis 488–495 Drugs, and drug metabolism 219, 414, 549, 574, 591 E Electrophoresis: Capillary electrophoresis (CE) 175, 231, 274, 284, 287, 424, 501, 523, 571, 607–608, 616–618, 620, 646, 650, 658, 665, 671, 684 Capillary electro‘C’ 245, 487, 504, 663, 684

707 Capillary isotachophoresis (CEC) 184–185, 608 High performance capillary electrophoresis (HPCE) 432–433, 618 Micellar electro‘C’ 501 Micellar electrokinetic capillary ‘C’ (MEKC) 561–563, 608, 612, 617–618 Slab gel electrophoresis 616 Two-dimensional electrophoresis 608 Zone electrophoresis See CE See Authors w=‘l’ Subject Code Environmental Sciences, ‘C’ in: Dioxins, PCBs, waste clean-up; water pollutants, PCDDs, and PCDFs 74–76, 132, 488–495, 514–515, 561, 645, 663–664, 669 See Authors w=‘s’ Subject Code F Food and Drug Administration

111

G Gaia hypothesis 387 Genomics 599 See Subject Index of Supplement H Hereditary diseases, ‘C’: Abnormal hemoglobins 569 Inborn errors of metabolism 271–274 Molecular diseases 271–274 Metabolic profiling 271–274 See Authors w=‘r’ Subject Code History of ‘C’ Precursors of ‘C’ 7 Pioneers of ‘C’ 2–23 Hormone, ‘C’ of: Corticosterone 470–473 See Authors w=‘r’ Subject Code I Industry, ‘C’ in 277, 292, 471–472, 629, 640, 667, 673 Instrumentation in ‘C’ 294–295, 476 See Authors w=‘k’ subject Code K Keele Microreactor

423

708

Subject index

L Lab-on-a-chip

200, 202, 616

M Macromolecular separation See SEC Manhattan Project 17–20 Mass spectrometry 472–473, 491–493 See also hyphenated techniques Metabolic processes, ‘C’ of: Biochemical markers 271–272 Metabolic profiling 271–272 Protein targeting 142–143 Protein trafficking=transport 142–143 Signal peptides 142–143 See additional items in Supplement on the Internet Metabolites, studied by ‘C’: Amino acids 15, 76–77, 268 Bile acids 534–534, 665 Cholesterol 538 DNA 201–202, 204, 306, 327 Enantiomers 111–112, 221–224, 383, 573, 622, 631 Food flavors and aromas 278 Lipids=fatty acids 78, 268, 483 Nucleosides, ribo- and deoxyribo- 144–154 Porphyrins 330 Squalene 185 Steroids=sterols 534–535, 611 Wine 125 See other metabolites in Supplement on the Internet Mass spectrometry 226–230, 253, 414–415, 472–473 Metallurgical Project 19 Methods of Analysis, AOAC Int 14, 135 Microfabricated devices 201–203 Milestones in ‘C’ 201–203 Minerals, purification=waste removal 260 N Nanotechnology 433 Natural Products, ‘C’ of: Attractants 136–141, 421 Carotenoids 7–8 Chlorophyll 4–9 Hydrocarbons 113, 348, 357, 361–362 Natural products 658 Petroleum 21–23, 265, 645, 658, 660, 663– 668 Pheromones, sex 136–141, 421

Plant pigments 4–11 Rubber 187–189 Nature of science: Biographies of scientists 109–599 Bridges in science=technology 8, 30–31 Disciplines in science 7, 687–689 Driving forces 23–25, 687–688 Flow of thought 3, 12 History of science 3–38, 687–688 Milestones in ‘C’ 27–28 Peer review, in science 30 Scientific disciplines 3, 687–688 Scientific literature for ‘C’ 23–25, 27 Scientific method 687–689 Seminal concepts in ‘C’ 26–27, 108–109 Sequence of scientific discoveries 3 See other related areas in the Supplement on the Internet Nobel Awards, related to ‘C’: Nobel laureates=lectures 40–54 Nuclear magnetic resonance (NMR) See hyphenated techniques O Oak Ridge National Laboratory 18–19 Organic synthesis, ‘C’ in: Achiral synthesis 110–113 Amino acids, Strecker Rx 93 Chiral synthesis 110–113 Origin of life 71 P Peer review, nature of 29–30 Peptides, structure=‘C’ of: Signal peptides 142–143 See also Protein chromatography Permissions 691–693 Pesticides 74, 658 Petroleum, ‘C’ of: Alternative fuels 663 Analyses, petroleum 21–23, 645, 658, 666– 667 Early history 21–23 Filtration hypothesis 23 Sources=biogenesis 21 Pioneers in ‘C’ 21–23 Polymers, synthetic ‘C’ 330, 632–672, 681– 685 Professional Societies Organizations and their symposia: All-Union Scientific Council on Chromatograpr, Acad. Sci. USSR 52–54

Subject index Assoc. Off. Anal. Chem. (AOAC) 72–73, 135 Am. Chem. Soc. 42–44, 57 Chem. Soc. Japan 609 Chinese Acad. Sci. 646 Chinese Assoc. Instrum. Anal. 646 Chinese Soc. Chem. 646 Chromatographic Forum of the Delaware Valley 47–48, 65–68 Chromatographic Soc. (UK) 43–45, 49–50, 58–59 Congresso Latino Americano de Cromatografia (COLACRO) 50–51, 56, 64–65, 660–661 Danube Symposium on ‘C’ 56, 63–64, 249 Eastern Analytical Symp. 49–51 Faraday Soc. Discussion 58 French Soc. G.A.M.S. 60, 249 Gesellschaft Deutschen Chemiker (and ‘C’ Group) 134 Int. Beijing Conf. and exhibition on Instrum. Anal. (BCEIA) 646–648 Int. Symposia on Adv. in ‘C’ 44–46, 56, 60–61 Int. Symp. on Capillary ‘C’ 44–47, 56–59, 62–63 Int. Symposia on HPLC 56, 61–62 Instrument Soc. of Amer. 51, 59 Japan Soc. for Anal. Sci. 609 Minn. ‘C’ Forum 51–52 New York Acad. Sci. 57 Nobel Foundation 41–42 Pittsburgh Conf. Anal. Chem. and Appl. Spectro. (PittCon) 25, 49, 56–57, 65–68 Role(s) of Professional Soc. 55–65 Soc. Anal. Chem. of Pittsburgh 25, 48–49 Symposia=Conferences 55–68 Proteins, structure, metabolism and=or ‘C’ of: See Index of Supplement on the Internet for key words Polymers=biopolymers ‘C’ 113, 569, 681–633, 672 Proteomics 599

709 R Radioactive fission products 17–26, 260 Radioactive Isotopes 17–20, 606 Rare earths 17–20 S Sedimentation field flow fractionation (SFFF) 322 Seminal concepts in ‘C’ 26, 108 Sensors, chem. or boil. 606 Separation methods 233–235, 289, 301–303, 478–487 Sephadex 534 Solid phase extraction 259, 376, 435 Solid phase synthesis 42 Sructure analysis, O3 136 Subcritical water extraction 211, 551 Space Sciences, ‘C’ in: Contamination question 94 Lifemolecules in lunar soil 79–85 Microanalysis of amino acids 83–86 Moon rocks, analysis of 83–94, 240, 513– 514 Murchison meteorite 95 See Authors w=‘r’ Subject Code Supercritical fluid extraction (SFE) 206–211 Symposia on chromatography See description in Chapter 3 and Appendix 6ABCD See also terms under Professional Societies T Theoretical approach in ‘C’ 190–191, 193–199, 353, 399–402, 407–409, 526, 623, 626– 627, 657, 673 Thermodynamics 197, 406 V Vitamin, ‘C’ of: ‘p-Aminobenzoic acid’

611

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