Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction

JOURNAL OF CHROMATOGRAPHY UBRARY- volume 53

hyphenated techniques in supercritical fluid ch rorna tograph y and extraction

This Page Intentionally Left Blank

JOURNAL OF CHROMATOGRAPHY LIBRARY-volume

53

hyphenated techniques in supercritical fluid chroma tograph y and extraction edited b y

School of Materials Science, Toyohashi University of Technology, Toyo hashi, Japan

ELSEVIER Amsterdam -London

-New York-Tokyo

1992

ELSEVIER SCIENCE PUBLISHERS B.V. Sara Burgerhartstraat 25 P.O. Box 21 1,1000 AE Amsterdam,The Netherlands

ISBN 0-444-88794-6

0 1992 Elsevier Science Publishers B.V. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, withoutthe prior written permission of the publisher, Elsevier Science Publishers B.V., Copyright & Permissions Department, P.O. Box 521,1000 A M Amsterdam,The Netherlands. Special regulations for readers in the U.S.A. -This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made i n the U.S.A. All other copyright questions, including photocopying outside of the U.S.A., should be referred to the copyright owner, Elsevier Science Publishers B.V., unless otherwise specified. 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. This book is printed on acid-free paper. Printed in the Netherlands

V

Contents Preface

vii

Contributors

ix

Chapter 1 General Detection Problems in SFC by Herbert H.HiII and David A.Atkinson

1

Chapter 2

Chapter 3 Chapter 4

Fourier Transform Ion Mobility Spectrometry for Detection after SFC by Herbert H.Hill and Edward E.Tarver Advances in Capillary SFC-MS by J.David Pinkston and Donald J.Bowling

25

Advances in Semi Micro Packed Column SFC and Its Hyphenation by Makoto Takeuchi and Toshinori Saito

47

Chapter 5 Flow Cell SFC/FT-IR by Larry T.Taylor and Elizabeth M.Calvey Chapter 6

Chapter 7

SFC/FT-IR Measurements Involving Elimination of the Mobile Phase by Peter RGriffiths, Kelly LNorton and Anthony S.Bonanno Practical Applications of SFC-FTIR by Keith D.Bartle, Anthony AClifford and Mark W.Raynor

Chapter 8 Recycle Supercritical Fluid Chromatography On-line Photodiode-Array Multiwavelength UV/VIS Spect ro rnetry/lR Spectrometry /Gas chromatography by Muneo Saito and Yoshio Yamauchi Chapter 9

9

Inductively Coupled Plasma Atomic Emission Spectrometric Detection in Supercritical Fluid Chromatography by Kiyokatsu Jinno

65

83

103

129

151

Chapter 10 Microwave Plasma Detection in SFC by Debra R.Luffer and Milos V.Novotny

171

Chapter 11 Multidimensional SFE and SFC by Joseph M.Levy and Mehdi Ashraf-Khorassani

197

vi

Chapter 12 Advances in Analytical Supercritical Fluid Extraction (SFE) by Steven B.Hawthorne, David J.Miller and John J. Langenfeld

225

Chapter 13 Introduction of Directly Coupled SFE/GC Analysis by Tsuneaki Maeda and Toshiyuki Hobo

255

Chapter 14 SFE, SFE/GC and SFE/SFC: Instrumentation and Applications by Marja-Liisa Riekkola, Pekka Manninen and Kan Hartonen

275

Chapter 15 Computer Enhanced Hyphenation in Chromatography - Present and Future by Eldon R.Baumeister and Charles L.Wilkins

305

Subject Index

319

vii

Preface Although pioneering work on supercritical fluid chromatography (SFC) by Klesper, Corwin and Turner (J.Org.Chem., 27,700,1962) clearly demonstrated the potential of this approach in separating thermally labile substances, it has experienced a rather slow growth and limited acceptance as an analytical tool because of the technological difficulties in handling supercritical fluids in chromatographic systems. Interest in SFC has recently been revived, however, because the previous technical limitations have been overcome through the availability of high-pressure instrumentation (solvent pump and sample injection systems) originally developed for high-performance liquid chromatography (LC). SFC has several advantages compared to gas chromatography (GC) and LC. As is well known, the physical properties of supercritical fluids are intermediate between those of gases and liquids. Solute diffusivities are about 100 times higher in a supercritical fluid than in the corresponding liquid phase while the viscosities are similar to those in the gas phase. Furthermore, the greater density of supercritical fluids as compared with gases imbues the mobile phase with solvating powers which can readily be controlled by the application of pressure. As a result, these unique properties should enable greatly enhanced chromatographic efficiency as compared to LC (although not as high as in GC), shorter analysis times than in LC and the possibility of separating high-molecular-weight and thermally labile compounds that cannot,be separated by GC. Focusing on detection in SFC, even though most GC and LC detectors have been used, including flame ionization, UV-absorption, fluorescence and refractive index detectors, a need for identifying the eluted components still exists just as in the case of LC. The high degree of compound selectivity possible through the combination of chromatography with detectors providing structural information is of great value for the identification of the components of complex mixtures. This has been the driving force behind the development of effective hyphenated techniques. In this instance, the properties of dilute supercritical fluid solutions allow the application of both gas and condensed detection methods for chromatography, which are very difficult in LC. Therefore SFC is a promising separation technique which has a higher potential than LC in hyphenated techniques. The ideal detector for all chromatographic separation techniques is a mass spectrometer (MS), because of its higher sensitivity as compared to other detectors and its specificity in identifying unknown compounds or for confirming the presence of suspected compounds. The gas-like properties of supercritical fluids favor the combination of SFC with MS, just as in GC-MS which has already proved to be a great success. The main reason for this success in GC-MS is that the effluent is compatible with classical gas phase ionization methods. However, in LC, scientists have not yet developed an instrument compatible in usefulness to a gas chromatograph-mass spectrometer (GC-MS) because of the high density of the solvents used as the mobile phase. The intermediate properties of

viii

supercritical fluid which are between those of gases and liquids allow a much easier interfacing when combining SFC with MS. An alternative possibility is infrared (IR) spectroscopy. This permits the identification of certain functional groups in the compounds. In addition, except for optical isomers, no two compounds having different structures have the same IR spectra. The sensitivity provided by IR instruments is poorer than that achieved by MS,yet as regards to the available information, IR spectroscopy is more often complementary than competitive to MS. Also several properties of the supercritical fluids provide more convenient and effective ways for interfacing chromatography to IR as opposed to LC-IR interfacing. Atomic spectroscopy is also a candidate for the construction of the hyphenated techniques in SFC. For example, inductively coupled plasma emission spectroscopy (ICP) or microwave induced plasma emission spectroscopy can be combined with SFC to realize element specific detection. Chromatography-chromatographyhyphenation will be more important in the near future in order to obtain higher resolution by multidimensional separations. SFC should have the important role in this area, because the characteristics of the fluids provide a good possibility for combining two chromatographic techniques without severe problems. Combining supercritical fluid extraction (SFE) techniques with different types of chromatography will especially play a key-role in the near future for practical analytical chemistry. A computer-assisted approach in hyphenated techniques in SFC might be more important than developing the interfaces because the interpretation of data supplied from the analytical systems valuable in determining the usefulness of the techniques. In this multi-authored book, the latest developments in the above mentioned hyphenated techniques using supercritical fluids are introduced and reviewed by outstanding experts from all over the world, especially focusing on relatively non-popular type hyphenations because the editor wants to avoid the contents overlapping to those in several other publications on SFC. The editor would like to sincerely thank each of the contributors who submitted their work for inclusion in this volume.

Kiyokatsu Jinno Toyohashi, JAPAN

ix

Contributors David A.Atkinson Department of Chemistry, Washington State University, Pullman, WA 99164-4630, USA. Mehdi Ashraf-Khorassani Suprex Corporation, Pittsburgh, PA 15238, USA. Keith D.Bartle School of Chemistry, University of Leeds, Leeds LS2 9JT, UK. Eldon R.Baumeister Department of Chemistry, University of California, Riverside, CA 92521, USA. Anthony S.Bonanno Department of Chemistry, University of Idaho, Moscow, ID 83843, USA. Donald J.Bowling The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, OH 45239-8707, USA. Elizabeth M.Calvey Food and Drug Administration, Contaminants Chemistry, Washington D.C. 20204, USA. Anthony A.Clifford 9JT, UK.

Division of

School of Chemistry, University of Leeds, Leeds LS2

Peter R.Griffiths Department of Chemistry, University of Idaho, Moscow, ID 83843, USA. Kari Hartonen Finland.

Department of Chemistry, University of Helsinki, Helsinki,

Steven B.Hawthorne Energy and Environmental Research Center, University of North Dakota, Grand Forks, ND 58202, USA. Herbert H.HiII Department of Chemistry, Washington State University, Pullman, WA 99164-4630, USA. Toshiyuki Hobo Department of Chemistry, Tokyo Metropolitan University, Hachioji 192, Japan. Kiyokatsu Jinno School of Materials Science, Toyohashi University of Technology, Toyohashi 441, JAPAN. John J.Langenfeld Energy and Environmental Research Center, University of North Dakota, Grand Forks, ND 58202, USA. Joseph M.Levy Suprex Corporation, Pittsburgh, PA 15238, USA.

X

Debra R.Luffer 47405, USA. Tsuneyakl Japan.

Department of Chemistry, Indiana University, Bloomington, IN

Maeda

Research Center, DKK Corporation, Musashino 180, Department of Chemistry, University of Helsinki, Helsinki,

Pekka Manninen Finalnd.

David J.Miller Energy and Environmental Research Center, University of North Dakota, Grand Forks, ND 58202, USA. Kelly L.Norton 83843, USA.

Department of Chemistry, University of Idaho, Moscow, ID

Milos V.Novotny IN 47405, USA.

Department of Chemistry, Indiana University, Bloomington,

J.David Pinkston The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, OH 45239-8707, USA. Mark W.Raynor

Carlo Erba Strumentazione, Radano, Milan, Italy.

Marja-Liisa Riekkola Helsinki, Finland.

Department of Chemistry, University of Helsinki,

Muneo Saito JASCO Corporation, Hachioji 192, Japan. Toshinori Saito Technical Group, JEOL MOLEH Co., Ltd., Akishima 196, Japan. Makoto Takeuchi

Basic Research Division, JEOL Ltd., Akishima 196, Japan.

Edward E.Tarver Department of Chemistry, Washington State University, Pullman, WA 99164-4630, USA. Larry T.Taylor Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0212, USA. Charles L.Wilkins Department of Chemistry, University of California, Riverside, CA 92521, USA. Yoshio Yamauchi

JASCO Corporation, Hachioji 192, Japan.

K. Jinno (Ed.), Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction Journal of Chromatograph Library Series, Vol. 53 0 1992 Elsevier Science F h i s h e r s B.V. Ail rights resewed.

Chapter 1 GENERAL DETECTION PROBLEMS IN SFC Herbert €IBill . and David A. Atkinson

Department of Chemistry Washington State University Pullman WA, 99164-4630

INTRODUCTION Hyphenated t e c h n i q u e s i n a n a l y t i c a l c h e m i s t r y h a v e e v o l v e d a s c o m b i n a t i o n s o f two o r more u n r e l a t e d a n a l y t i c a l methods h a v e b e e n i n t e r f a c e d t o p r o v i d e two s e t s of d a t a a b o u t t h e same s a m p l e . I n t h e most common c a s e , ( t h e o n e d i s c u s s e d e x c l u s i v e l y i n t h i s b o o k ) t h e f i r s t s t a g e of a h y p h e n a t e d s y s t e m s e r v e s as a s e p a r a t i o n s t e p w h i l e t h e s e c o n d s t a g e provides s p e c t r a l information about t h e s e p a r a t e d components. By s i m u l t a n e o u s l y o b t a i n i n g two s e t s o f d a t a ( s e p a r a t i o n d a t a a n d s p e c t r a l d a t a ) on t h e same s a m p l e , h y p h e n a t e d a n a l y t i c a l t e c h n i q u e s h a v e become p o w e r f u l t o o l s of a n a l y s i s , p r o v i d i n g much more u s e f u l i n f o r m a t i o n t h a n t h a t o b t a i n e d b y o p e r a t i n g t h e two t e c h n i q u e s i n d e p e n d e n t l y . When t h e s e p a r a t i o n s t a g e o f a h y p h e n a t e d s y s t e m i s S F C , s p e c i a l i n t e r f a c i n g .problems e x i s t b e t w e e n t h e SFC a n d t h e s p e c t r o m e t e r d u e t o t h e u n i q u e p r o p e r t i e s of s u p e r c r i t i c a l f l u i d s . Phase changes, v a r y i n g sample i n t r o d u c t i o n r a t e s , mobile phase c o m p a t i b i l i t y , mobile phase e l i m i n a t i o n , i n t e g r i t y o f t h e SFC s e p a r a t i o n , a n d ambiguous d e t e c t o r t e r m i n o l o g y a r e a l l p r o b l e m s w i t h SFC h y p h e n a t e d a n a l y t i c a l m e t h o d s . T h i s s e c t i o n d i s c u s s e s t h e g e n e r a l n a t u r e of these p r o b l e m s , w h i l e t h e i n d i v i d u a l t o p i c s w i l l d i s c u s s i n more d e t a i l how t h e s e p r o b l e m s a r e s o l v e d f o r e a c h s p e c i f i c technique. THE PHASE CHANGE U n f o r t u n a t e l y t h e r e a r e few d e t e c t o r s which o p e r a t e u n d e r s u p e r c r i t i c a l c o n d i t i o n s . Most d e t e c t i o n m e t h o d s which a r e c u r r e n t l y u s e d w i t h SFC o p e r a t e e i t h e r u n d e r l i q u i d , g a s , o r vacuum c o n d i t i o n s . T h i s means t h a t as t h e sample t r a v e l s from t h e c h r o m a t o g r a p h t o t h e d e t e c t o r , i t must u n d e r g o a p h a s e change from a s u p e r c r i t i c a l f l u i d t o a l i q u i d o r g a s b e f o r e it can be d e t e c t e d .

L

Figure 1 provides a classification diagram of spectral detectors used after SFC. Each detector is divided into three classes: the cell design, the response mechanism, and the pressure under which the detector operates. As can be seen from the figure, spectral detectors for SFC fall into categories of ion detectors and optical detectors and are operated at high, ambient, or low pressures. For high pressure operation, detection occurs at pressures similar to those used for the supercritical separation and the detector cell must be of a lTclosed"design in order to maintain the pressure in the detector. For ambient and low pressure detectors, the cell design is usually rTopen"to aid in the elimination of the mobile phase as it decompresses to form a gas.

. MS

SJS

FT-'IMS

MES Classification Diagram of Spectral Detectors Used i n SFC: Fourier Transform Infrared Spectrometry (FTIR), Supersonic Jet Spectrometry (SJS) , Microwave Plasma Emission Spectrometry (MES), Mass Spectrometry (MS) and Fourier Transform Ion Mobility Spectrometry (FTIMS)

.

Cell Design. Phase changes in closed cells are not particularly difficult to achieve since the change is normally from a supercritical fluid to a liquid state. The detector simply must be cooled below the critical temperature of the mobile phase. For most SFC systems, room temperature will produce a liquid of the mobile phase at supercritical pressures. Decompression of the mobile phase is accomplished relatively easily after the analytical data have been obtained. The primary problems associated with closed detectors are in design. Closed cells must be capable of withstanding high pressures while providing

3

windows t o monitor o p t i c a l e v e n t s , I n a d d i t i o n , c e l l volume m u s t be kept a s s m a l l a s p o s s i b l e t o p r e v e n t band broadening of t h e chromatographic p e a k s . I n open c e l l d e t e c t o r s , t h e phase change o c c u r s b e f o r e d e t e c t i o n . P r e s s u r e i s r a p i d l y reduced from s u p e r c r i t i c a l f l u i d c o n d i t i o n s o f t h e column t o ambient o r vacuum c o n d i t i o n s o f t h e d e t e c t o r c e l l . To accomplish t h i s r a p i d decompression w i t h o u t sample decomposition, mobile p h a s e c l u s t e r i n g , sample p r e c i p i t a t i o n , o r p l u g g i n g of t h e system, s p e c i a l l y d e s i g n e d r e s t r i c t o r s m u s t be p l a c e d i n t h e flow p a t h between t h e column and t h e d e t e c t o r .

Restrictors. There a r e f i v e commonly used r e s t r i c t o r s f o r SFC: t h e l i n e a r o r F j e l d s t e d r e s t r i c t o r ( Z ) , t h e tapered o r C h e s t e r r e s t r i c t o r (3), t h e i n t e g r a l o r G u t h r i e r e s t r i c t o r (4), t h e p i n h o l e o r S m i t h r e s t r i c t o r ( 5 ) and t h e f r i t o r R i c h t e r r e s t r i c t o r (6). Each of t h e s e r e s t r i c t o r s h a s s p e c i a l advantages which warrant t h e i r u s e i n s p e c i f i c circumstances. Figure 2 provides schematic c r o s s - s e c t i o n a l diagrams of t h e s e f i v e r e s t r i c t o r s .

Figure 2:

Cross-sectional schematic diagrams of SFC restrictors used in hyphenated techniques.

The F j e l d s t e d r e s t r i c t o r ( F i g u r e 2 a ) i s t h e s i m p l e s t i n d e s i g n and t h e f i r s t t y p e used w i t h c a p i l l a r y SFC. I t c o n s i s t s of a s h o r t c a p i l l a r y t u b e ( 1 0 t o 25 cm), u s u a l l y uncoated s i l i c a , w i t h an i n t e r n a l d i a m e t e r which i s s i g n i f i c a n t l y reduced o v e r t h a t o f t h e column. For example, t h e i n t e r n a l d i a m e t e r of t h e column might be 1 0 0 pm and t h a t o f t h e r e s t r i c t o r might be 1 0 pm. Advantages o f F j e l d s t e d r e s t r i c t o r s a r e t h a t they a r e inexpensive, easy t o r e p l a c e , and do not p l u g a s e a s i l y a s some of t h e o t h e r r e s i s t o r s . The primary d i s a d v a n t a g e i s t h a t decompression o c c u r s o v e r

4 As a r e s u l t , t h e s o l v a t i n g power of t h e mobile phase d e c r e a s e s t h r o u g h o u t t h e l e n g t h o f t h e r e s t r i c t o r making it d i f f i c u l t t o t r a n s f e r h i g h e r molecular weight compounds t o t h e d e t e c t o r . I n a d d i t i o n , c o o l i n g from t h e decompression c a u s e s c o n d e n s a t i o n o f s o l v e n t and sample molecules which produces c l u s t e r p a r t i c l e s t h a t can p e r t u r b d e t e c t o r r e s p o n s e .

t h e e n t i r e l e n g t h of t h e t u b e .

To reduce t h e e f f e c t of s o l v e n t c l u s t e r i n g , a r e s t r i c t o r can be t a p e r e d a t t h e end down t o an i n t e r n a l d i a m e t e r of 1 t o 3 pm. T h i s C h e s t e r r e s t r i c t o r ( F i g u r e 2b) can be produced manually from a flame i n t h e l a b o r a t o r y , b u t f o r

reproducibility, robotically pulled r e s t r i c t o r s are recommended ( 7 ) . S t i l l , i n t h i s d e s i g n , decompression o c c u r s over s e v e r a l cm of t h e t a p e r e d s e c t i o n and t h e t h i n t a p e r e d s e c t i o n i s extremely f r a g i l e . With p r a c t i c e and p e r s e v e r a n c e , t h e G u t h r i e r e s t r i c t o r ( F i g u r e 2c) can a l s o be made i n t h e l a b o r a t o r y . I t is c o n s t r u c t e d b y c a r e f u l l y h e a t i n g t h e end of a f u s e d s i l i c a c a p i l l a r y u n t i l it j u s t c l o s e s . Then t h e c l o s e d end is

p o l i s h e d u n t i l it is reopened by a s m a l l c r a c k shaped o r i f i c e . The size of t h i s o r i f i c e is a d j u s t e d t h r o u g h p o l i s h i n g u n t i l t h e d e s i r e d flow r a t e t h r o u g h t h e r e s t r i c t o r is achieved. With t h e G u t h r i e r e s t r i c t o r , p r e s s u r e is dropped over a much s h o r t e d d i s t a n c e t h a n w i t h e i t h e r t h e F j e l d s t e d o r C h e s t e r r e s t r i c t o r s . T h u s , compounds w i t h lower v o l a t i l i t i e s s t a y i n s u p e r c r i t i c a l s o l u t i o n u n t i l t h e y a r e r a p i d l y decompressed and d i s p e r s e d i n t h e g a s p h a s e . The u l t i m a t e r e s t r i c t o r d e s i g n f o r n e a r l y i n s t a n t a n e o u s decompression i s t h e S m i t h r e s t r i c t o r ( F i g u r e 2d) which c o n s i s t s of a l a s e r d r i l l e d " p i n h o l e " o r i f i c e i n a t h i n metal f o i l . Used p r i m a r i l y f o r i n t e r f a c i n g SFC s y s t e m s w i t h vacuum systems, t h e S m i t h r e s t r i c t o r i s d i f f i c u l t t o c o n s t r u c t and i n s t a l l . T h e primary problem w i t h b o t h t h e S m i t h and t h e G u t h r i e r e s t r i c t o r s i s p l u g g i n g . Small p a r t i c l e s e n t r a i n e d i n t h e mobile phase o r n o n - v o l a t i l e components which have p r e c i p i t a t e d r e a d i l y p l u g t h e r e s t r i c t o r . When plugged, t h e r e s t r i c t o r m u s t be removed, cleaned o r replaced. compromise between t h e S m i t h o r Guthrie r e s t r i c t o r and t h e F j e l d s t e d o r C h e s t e r r e s t r i c t o r is t h e R i c h t e r r e s t r i c t o r ( 6 ) . The R i c h t e r r e s t r i c t o r ( F i g u r e 2e) resembles a porous f r i t i n s i d e a f u s e d s i l i c a c a p i l l a r y . . The p o r o s i t y of t h e f r i t p r o v i d e s m u l t i p l e p a t h s and reduces t h e frequency of plugging w h i l e p r o v i d i n g a r e l a t i v e l y s h o r t decompression zone. A

5 RESOLUTION INTEGRITY

When interfacing any detection method to a separation process such as SFC, the integrity of the separation must be maintained. The resolution between two components which is gained during a highly efficient separation can be significantly degraded by a poorly designed interface. Transfer-line broadening, stagnant-volume broadening, detector-cell broadening, and electronic broadening are all mechanisms which contribute to overall postseparation peak broadening ( 7 )

.

Transfer-line broadening occurs when the eluents must be transferred from the column to the: detector. Longitudinal diffusion and radial diffusion can contribute to broadening effects in supercritical fluids. Since longitudinal diffusion is decreased and radial diffusion in increased by increasing the linear flow velocity of the supercritical mobile phase in the transfer line, there should exist an optimal flow velocity for transferring eluents from the column to the detector. Stagnant-volume refers to that volume in a transfer line or detector cell which is not swept by the mobile phase. Thus solutes which have become trapped in a stagnant volume must rely on diffusion to reenter the flowing mobile phase stream. Stagnant volume can be reduced by careful attention to plumbing of the interface to in3ure that all portions of the postcolumn region are swept by the mobile phase. While peak broadening due to stagnant volumes, longitudinal diffusion, and radial diffusion can occur in the detector, the major contribution to peak broadening is simply the volume of the detector. The detected volume of a sample (V,) is equal to the volume of the solute which passes through the detector (V,) plus the cell volume of the detector (V,).

Thus the detected volume is larger than the actual volume by the cell volume. Finally, chromatographic peaks can be broadened electronically if response times are too slow. The following equation shows how to estimate the percent band broadening which will occur as a function of the electronic response time.

where % S is the percent contribution to band broadening by the electronics of the system, tE is the response time of

6

the electronics, N is the number of theoretical plates for the separation and tR is the retention time of the solute. a general rule, no more than 5% peak broadening should occur after separation is complete. This implies that the detector volume should be less than one-twentieth of the volume of a separated solute zone. With good detector designs, transfer line, stagnant volume, and electronic broadening can be eliminated. As

MOBILE PHASE COWPATIBILITY

Perhaps the most difficult problem associated with SFC detection is compatibility with the mobile phase. Carbon dioxide has emerged as one of the principal mobile phases used in SFC not only because of its convenient critical parameters and non-toxicity but also because of its compatibility with both flame ionization and W absorption detection. Nevertheless, the use of modifiers with C 0 2 to increase polarity is severely limited by the detection method employed. For example, flame ionization cannot be used when C02 is modified with methanol and W absorption cannot be used when C02 is modified with compounds containing chromaphors. Even when pure carbon dioxide is used, interferences occur with other detection systems. With IR detection, for example, C02 obscures broad areas of the usable spectra and these obscured areas vary as a function of pressure. Exotic mobile phases such as supercritical xenon can be used to avoid interferences with on-line IR detection methods or off-line methods must be employed to eliminate the solvent from the solute prior to detection. Whether pure or modified mobile phases are used for SFC separation, it should be clear that the response of the detector to the mobile phase is more important in the selection of a mobile phase than are the separation characteristics of the the phase. No evaluation of detection methods for SFC can be complete without a detailed discussion of mobile phase compatibility. DETECTOR EVALUATION

Finally, comparative evaluation of hyphenated detection methods for SFC can present a problem since such a wide variety of analytical methods can be interfaced with SFC. Each analytical method of detection has a particular set of information, called Figures of Merit IFOM), which characterize the analytical merit of the technique.

For qualitative analysis, hyphenated techniques usually employ a detection method in which spectral information can

7

be obtained for each component that has been separated by SFC. Optical and ion spectroscopies are normally employed although electrochemical 01: additional separation methods may a l s o be used following SFC to obtain qualitative information about the separated components. Optical methods discussed in this book include FTIR, ICP and microwave plasma emission spectrometry. I o n methods include ion mobility and mass spectrometry. When these optical and ion techniques are operated in the spectral mode, resolution of the spectra and minimum quantity required of a component to produce a meaningful spectra are important qualitative FOMs to report. I n addition, information on sampling time, scan time and spectral noise should be reported. These spectrometers can also be operated in a monitoring mode in which one wavelength or one ion species i s continuously monitored to produce a continuous tracing of the developing chromatogram. In this mode, quantitative information about the components is possible.

Figure 3 :

Quantitative Figures of Merit f o r SFC Detectors (Reprinted from R e f . 7 w i t h permission).

8 FOMs f o r q u a n t i t a t i v e a n a l y s i s a r e b e s t d i s c u s s e d i n terms of an a n a l y t i c a l c a l i b r a t i o n g r a p h shown i n F i g u r e 3 .

Important q u a n t i t a t i v e FOMs t o r e p o r t from t h e d a t a produced from a c a l i b r a t i o n graph such a s t h i s i n c l u d e t h e root-means q u a r e n o i s e of t h e b a s e l i n e , t h e s l o p e o f t h e l i n e which i s t h e s e n s i t i v i t y of t h e d e t e c t o r , t h e l i n e a r and dynamic range of t h e d e t e c t o r , and f i n a l l y t h e d e t e c t i o n l i m i t of t h e d e t e c t o r , which i s t h e mass flow r a t e o r c o n c e n t r a t i o n of t h e s o l u t e r e q u i r e d t o produce a s i g n a l which i s 3 t i m e s t h e rms n o i s e . LITERATURE CITED

1. 2.

3. I

4. 5.

6. 7.

Novotny, S . R . S p r i n g s t o n , P . A . Peaden, J . C . F j e l d s t e d , and M . L . Lee, Anal. Chem., 53, 4 0 7 A ( 1 9 8 J . C . F j e l d s t e d , R . C . Kong, and M . L . Lee, J. Chromatogr. 279, 4 4 9 ( 1 9 8 3 ) , T . L . C h e s t e r , D . P . I n n i s , and G. D . Owens, Anal.

M.

-

.

Chem 57, 2 2 4 3 ( 1 9 8 5 ) E . J . G u t h r i e and H . E . Schwartz, J . Chromatogr. Sc 24, 2 3 6 ( 1 9 8 6 ) . R . D . S m i t h and H , R . Udseth, Anal. Chem., 55, 2266 (1983). H. Cortes; C. D . P f e i f f e r , B . E . R i c h t e r and T . S . Stevens. U.S. P a t e n t # 4 . 7 9 3 , 9 2 0 . 1 9 8 7 . H . H. H i l l and M . M . G a i l a g h e r , ’ J . M . S . , 114 (1990).

.

2(3),

K. Jinno (Ed.), Hy henated Techniques in Supercritical FluifChromatographyand Extraction Journal of Chromatography Libraty Series, Vol. 53 0 1992 Elsevier Science Publishers B.V. All rights resewed.

9

Chapter 2 FOURIER TRANSFORM ION MOBILITY SPECTROMETRY FOR DETECTION AFTER SFC Herbert H. Hill and Edward E. Tarver Department of Chemistry Washington State University Pullman WA, 99164-4630

One of the simplest of the hyphenated techniques is Fourier transform ion mobility spectrometery (FTIMS). It is the only analytical method in which qualitative information can be obtained from ionization processes at atmosphere pressure. Based on gas phase atmospheric pressure electrophoretic separation of ions, IMS serves as a complementary technique to UV/Vis absorption and fluorescence methods of detection. Typical ion mobility spectra after SFC can be seen in Figure 1. Current in nanoamperes (nA) is plotted as a function of the arrival time in milliseconds (ms) of the ion at the collecting electrode. Gas phase ions migrating through an electric field in opposition to the flow of an inert drift gas such as nitrogen at a characteristic velocity. This velocity is determined by the charge on the ion (q), the kinetic energy of the gas (kT), the gas phase diffusion coefficient of the migrating ion (D) and the strength of the electric field (E). v = (q/kT)DE For a given temperature and pressure the velocity/electric field ratio is constant and becomes a qualitative indicator of the ion called the gas phase mobility of the ion (K). v/E = (q/kT)D = K Experimentally mobility is calculated from drift time (t) in the following manner. K = d/tE

10

a.

I

I

0

10

20

30

40

so

Tima (rnsec)

Figure 1:

Ion Mobility Spectra of Spiramycin after SFC. A) SFC mobile phase was pure carbon dioxide. B) SFC mobile phase was chlorodifluoromethane. In both spectra the ion mobility drift gas was nitrogen. (ref. 1)

where d i s t h e d i s t a n c e t h e i o n t r a v e l e d i n c e n t i m e t e r s , t i s t h e a r r i v a l t i m e i n seconds, and E i s t h e electric f i e l d i n volts/centimeter. Normal o p e r a t i n g c o n d i t i o n s f o r a n a l y t i c a l i o n m o b i l i t y s p e c t r o m e t r y r a n g e from room t e m p e r a t u r e t o 3 0 0 C a n d f r o m 7 0 0 t o 800 t o r r . Since K v a r i e s w i t h b o t h temperature and pressure, mobility i s usually corrected t o standard temperature a n d p r e s s u r e a n d c a l l e d t h e r e d u c e d m o b i l i t y c o n s t a n t (KO).

KO = K (273/T) (P/760)

11

DETECTOR DESIGN

The d e t e c t o r d e s i g n w h i c h h a s b e e n u s e d most o f t e n f o r i o n m o b i l i t y d e t e c t i o n a f t e r s u p e r c r i t i c a l f l u i d chromatography i s a u n i d i r e c t i o n a l flow d e s i g n w i t h sample i n t r o d u c t i o n from t h e SFC column t h r o u g h a r e s t r i c t o r l o c a t e d o r t h o g o n a l t o t h e d r i f t flow of t h e s p e c t r o m e t e r and i n f r o n t of t h e i o n i z a t i o n r e g i o n . A d e t a i l e d d r a w i n g o f t h i s s p e c t r o m e t e r is shown i n F i g u r e 2 .

Sra,nlesa-Steel D ~ I Ring I~

L__

40cm

..~.--.---.._....__~ Oven Bare

Figure 2:

Schematic cross-section of the ion mobility detector. (ref. 4)

The d r i f t r e g i o n o f t h e s p e c t r o m e t e r , where t h e d r i f t t i m e s o f t h e i o n s a r e m e a s u r e d , i s bounded b y two i o n g a t e s . These g a t e s are o f t h e Bradbury t y p e which c o n s i s t o f p a r a l l e l w i r e s ( 2 ) . The g a t e i s c l o s e d when a v o l t a g e i s p l a c e d b e t w e e n e a c h w i r e a n d i s open when e a c h g a t e w i r e i s a t t h e p o t e n t i a l o f t h e d r i f t f i e l d a t t h a t point i n t h e d r i f t tube. F o u r i e r t r a n s f o r m i o n m o b i l i t y s p e c t r a a r e o b t a i n e d when b o t h t h e e n t r a n c e and e x i t g a t e s a r e opened and c l o s e d s i m u l t a n e o u s l y a n d t h e f r e q u e n c y a t which t h e y a r e o p e n e d a n d I o n s which a r e c l o s e d i s v a r i e d from 1 0 t o 2 0 , 0 0 0 Hz. t r a v e l i n g through t h e spectrometer a t a constant average v e l o c i t y , come i n a n d o u t o f p h a s e w i t h t h e p u l s i n g o f t h e e x i t gate. The r e s u l t i n g f r e q u e n c y d i s p e r s i v e i n t e r f e r o g r a m is

12

converted to a time dispersive ion mobility spectrum by Fourier transformation. Figure (3a) illustrates the simultaneous square wave entrance and exit gate functions. Figure (3b) shows the positive half of the corresponding gating correlation function &(t,v). Where t is the drift time of the ion and V is the frequency at which the gates are pulsed open. Since the gate connection functions are equal, &(t,v) is an autocorrelation function. The maximum value of &(t,v) is 0.5, the fraction of time that the entrance gate is open. The correlation function requresents the filtering action of the gates. A s the frequency (V) is scanned the only ions that reach the collector with full intensity are those with drift times of 0, l/V, 2/V, 3/v etc. Ions with do not reach the detector at drift times of 1/2v, 3/2v, 5/2v all. The signal, S(v) detected is a function of a compound mobility spectrum, m(t), and the gate correlation function which determines the fraction of ions reaching the collector

...

...

S(V) = m(t) &(t,v) The maximum value of S(v) is equal to 0.5 1, where I, total ion current when both gates are open.

is the

If m(t) consists of a single ion with draft time t, then S(V) is also a triangle wave with maxima and minima: S(V),,

= 0.5 . I

v = 0, l/t,

2 / t , 3/t

...

v = 1/2t, 3/2t, 5/2t.. . I f m(t) consists of a number of ions of different drift times, then S(v) will be the sum of interfering triangle waves. In practice, diffusion processes in the drift tube smooth these triangle waves to sine-like waves such that Fourier transformation becomes a reasonable approximation of the real time domain ion mobility spectrum.

Other designs which have been used with SFC include the bidirectional flow design (3) and a unidirectional flow design in which the sample was introduced axially and behind the ionization region (4). Neither of these designs have proved to be very useful for SFC. In the bidirectional design, C02 contaminated the drift region of the spectrometer. In the design where the sample was axially introduced, it appeared that the sample was not being introduced efficiently into the ionization region. With the orthogonal sample introduction design, as shown in Figure 2, the entire sample was swept through the ionization region of the spectrometer providing maximum sensitivity.

13

1111 (I11

Transit Time

Figure 3 :

Typical entrance and e x i t g a t e f u n c t i o n s , FT mode. B) Typical g a t i n g c o r r e l a t i o n function, FT mode. ( r e f . 23)

A)

A l t h o u g h i o n i z a t i o n f o r IMS c a n be a c h i e v e d by p h o t o i o n i t i o n ( 5 ) o r c o r o n a i o n i z a t i o n ( 6 ) , o n l y b e t a i o n i z a t i o n from '%i s o u r c e s h a s b e e n u s e d w i t h SFC. I n t h e i n s t r u m e n t shown i n F i g u r e 2 , a 15 m ' l l i C u r r i ( c ) 63Ni s o u r c e w a s u s e d t o p r o d u c e a f l u x o f 1 X 10' CW/Vs b e t a p a r t i c l e s w i t h a n e n e r g y r a n g e o f 0 t o 67 K e V ( w ) . I n n i t r o g e n , t h e s e b e t a p a r t i c l e s produce an When s m a l l q u a n t i t i e s average of 1 X l o 6 c w i o n p a i r s / s e c o n d . of water a r e p r e s e n t i n t h e n i t r o g e n d r i f t g a s , c h a r g e t r a n s f e r , p r o t o n t r a n s f e r and c l u s t e r r e a c t i o n s o c c u r t o p r o d u c e h y d r a t e d c l u s t e r s o f t h e common p o s i t i v e r e a c t a n t i o n s of N O ' , N H q f f a n d H30'. I n t h e n e g a t i v e 1 0 0 mode when 0 2 i s p r e s e n t i n t h e d r i f t gas, n e g a t i v e l y charged r e a c t a n t ions a r e w a t e r c l u s t e r s o f 02-. I f O2 and o t h e r e l e c t r o n c a p t u r i n g compounds a r e n o t p r e s e n t i n t h e d r i f t g a s t h e n t h e r e a c t i v e c h a r g e d s p e c i e s i n t h e n e g a t i v e mode a r e f r e e e l e c t r o n s a t thermal e n e r g i e s o f about 0 . 0 2 t o 0.05 e V . A n a l y t i c a l r e s p o n s e i n t h e IMS d e p e n d s on i o n - m o l e c u l e r e a c t i o n s o f these p o s i t i v e o r n e g a t i v e r e a c t a n t i o n s w i t h a n a l y t e m o l e c u l e s i n t r o d u c e d i n t o t h e d e t e c t o r from t h e c h r o m a t o g r a p h i c e f f l u e n t . The r e s u l t a n t p r o d u c t i o n s formed from these r e a c t i o n s e n t e r t h e d r i f t r e g i o n of t h e s p e c t r o m e t e r where t h e y a c h i e v e c h a r a c t e r i s t i c d r i f t v e l o c i t i e s i n t h e e l e c t r i c f i e l d and t h e i r a r r i v a l t i m e s i n m s a r e monitored a t the collector electrode. F o r p o s i t i v e p r o d u c t i o n s , s e n s i t i v i t y i n t h e IMS i s a f u n c t i o n of proton a f f i n i t i e s of t h e a n a l y t e while t h e formation of n e g a t i v e p r o d u c t i o n s i n t h e IMS d e p e n d s on t h e e l e c t r o n a f f i n i t y o f t h e a n a l y t e . Compounds w i t h h i g h p r o t o n a f f i n i t i e s such a s amines, s u l f i d e s , phosphates, e t c p r o v id e e x c e l l e n t

14

responses in the positive ion collection mode of the IMS while electronegative compounds such as halogenated compounds, highly oxygenated and nitro- containing compounds produce sensitive responses in the negative ion mode. Selective responses can be achieved if the ionization region is doped with specific compounds which alter the reactant ion species and subsequent ion-molecule reactions. For example, acetone can be added to the ionization region to produce ( a ~ e t o n e ) ~ Hpositive + reactant ions which react selectively with organophosphosphorus compounds ( 7 , 8 ) and methylenechloride can be added to the ionization region to produce chloride negative reactant ions which enhance ion-molecule reactions with nitro compounds ( 9 , l O ) . Because the response of the detector is so sensitive to the types of reactant ion species present in the ionization region, it is important to pay particular attention to the mobile phase and how it affects reactant ion formation and ion-molecule reactions. MOBILE PHASE COMPATIBILITY

described above, the typical ion mobility spectrometer has two gas flows entering the instrument: the drift gas and the sample gas. The drift gas enters the spectrometer at a relatively high volume flow rate (0.6 to 1.5 L/min) and consists of an inert gas such as nitrogen or air through which the velocity of the ions are measured. The sample gas is that gas which contains the analyte and transfers it into the spectrometer. When the spectrometer is interfaced to a chromatograph, this sample gas is normally the mobile phase used for the chromatographic separation. Thus, for supercritical fluid chromatography the most common mobile phase is carbon dioxide, but other phases are also important. The following is a discussion of various supercritical fluid mobile phases and the effects of these gases on ion mobility spectrometry.

As

Carbon Dioxide. Early investigations of I M S with carbon dioxide focused on the drift region. It was reasoned that if carbon dioxide was used as the drift gas as well and the sample introduction gas problems associated with introducing large volumes of C02 from the supercritical fluid chromatograph would be minimized. In the earliest experiments with C 0 2 it was concluded that C02 formed such large clusters with ions that the mobility of the ion cluster was independent of the core ion species (11). It was found however that with large molecular ions as the core ions, differences in mobility did occur in C02 drift gas. When the drift gas was heated to 220 C, separation patterns for a series of methyl esters was similar to that obtained when nitrogen was used as the drift gas (12). However, drift times in C02 were significantly longer than those in nitrogen. For example, the drift time for methyl stearate was found to be 30.2 ms in C02 compared to 23.4 ms in N2. Although longer drift times permitted more time for peak

15

b r o a d e n i n g t h r o u g h d i f f u s i o n and r e d u c e d t h e a n a l y t i c a l r e s p o n s e o f t h e s i g n a l , i o n m o b i l i t y s p e c t r a from c h r o m a t o g r a p h i c p e a k s w e r e o b t a i n e d a f t e r SFC f o r a s e r i e s of t e s t compound which c o u l d n o t be d e t e c t e d by UV/VIS m e t h o d s (13). Because o f t h e r e d u c e d s e n s i t i v i t y w i t h C02 d r i f t gas, t h e s t a n d a r d d r i f t g a s e s u s e d i n IMS, n i t r o g e n a n d a i r w e r e The e v a l u a t e d when C 0 2 was u s e d f o r s a m p l e i n t r o d u c t i o n . problem w i t h t h i s approach i s i l l u s t r a t e d i n F i g u r e 4 .

REACTANT I ON5

WITHOUT

0

Figure 4:

5

10 DRIFT TIME

CO,

IS

20 (ms)

Ion mobility spectra (obtained with a bidirectional flow spectrometer) of reactant ions with and without carbon dioxide. The lower mobility spectrum show the normal pattern observed for nitrogen drift gas. The upper tracing demonstrates the shift in the reactant ion pattern when small quantities of carbon dioxide are introduced into the spectrometer through the chromatographic column. With unidirectional flow spectrometers, reactant ions are not influenced by small quantities of carbon dioxides. (ref. 13)

The l o w e r i o n m o b i l i t y s p e c t r u m shows t h e n o r m a l p a t t e r n o b s e r v e d f o r n i t r o g e n d r i f t g a s . The u p p e r t r a c i n g demonstrates t h e s h i f t i n t h e r e a c t a n t ion p a t t e r n t h a t o c c u r r e d when s m a l l q u a n t i t i e s o f C 0 2 w e r e i n t r o d u c e d i n t o t h e s p e c t r o m e t e r t h r o u g h t h e c h r o m a t o g r a p h i c column. Moreover, t h e

pattern of the reactant ions was dependent on the rate of introduction of the CO . When SFC is temperature or pressure programmed, the flow o$ CO2 into the spectrometer changes and thus the reactant ions can be expected to change during the course of an SFC separation. The problem was solved with a specially designed unidirectional flow ion mobility spectrometer in which the gas exit of the IMS was placed at the rear of the spectrometer rather than in the center (14). This geometry, shown in Figure 2, prevented CO2 and the sample from mixing with the nitrogen in the drift region of the spectrometer. With this design, reactant ions remained constant throughout the chromatographic run. With the stability of the reactant ions assured, the ion mobility spectrometer could be used as a chromatographic detector in which selective and non-selective ion mobility monitoring could be accomplished by monitoring specific drift time windows or the entire product ion region respectively. Modified Carbon Dioxide.

Pure C02 is limited as a mobile phase by its non-polar character. As the range of supercritical fluid chromatography is expanded to the separation of polar compounds, the addition of small quantities of polar, but C02 soluble compounds have been used to increase the elution power of CO2. These modifiers have included compounds such as acetone, acetonitrile, freons, methylenechloride and, most commonly, methanol. One major difficulty with the addition of modifiers to C02 is compatibility with detection methods. Although one of the most sensitive and useful detectors for SFC is the flame ionization detector, it cannot be used when ionizable organic modifiers have been added to the C02 mobile phase. UV/Vis detection is precluded by modifiers that absorb light in this region of the spectrum. When ion mobility detection is used after SFC, the addition of modifiers to the mobile phase can modify the reactant ions in the spectrometer and change the response characteristics of detection. However, initial investigations of C 0 2 modified mobile phases with ion mobility detection have indicated that when the proper detector geometry is employed, ion mobility detection may work well with modifiers (15). Morrissey and Hill (16) first demonstrated IMS as a detection method with CH30H as a modifier for CO2 (Figure 5). Huang, Markides and Lee (15) have also used CH30H, as well as CH3CN, modified C02 after capillary chromatographic separations while Morrissey and Widmer (17) investigated IMS after packed column SFC with methanol modified C02.

17

f

0.

Figure 5:

Separation of polydimethylsilicone in A) pure carbon dioxide and B) carbon dioxide modified with 5% (v/v) methanol. (ref. 16)

Freons. As a class of compounds, chlorofluro-hydrocarbons vary in solvent strength, polarity, and critical parameters such that, except for environmental concerns, they appear to be an ideal mobile phase system for SFC. The primary reason they have not been used more extensively is that they respond in the FID. Initial investigations have indicated that freons can be used with the positive mode of the IMS (18) (See Figure 6). In the negative mode, it is expected that some interferences may occur although no investigation of feron response in the IMS has been completed.

18

a.

'1 b

3

Figure 6:

- -

&miv w

Iikil

* r&m(.I*

.

..

m

u

Separation of di- and triglycerides in A) pure carbon dioxide and B) chlorodifluoromethane. (ref. 1)

APPLICATIONS TO SFC

As an SFC detection method, ion mobility spectrometry has several unique advantages which include the following: 1) nonselective, FID-like, responses for most compounds, 2 ) selective, ECD-like, responses for electro-negative compounds, 3) selective detection of compounds not containing heteroatoms, 4) detection of compounds not containing chromaphors and 5) the detection of compounds contained in mobile phases which have been modified with organic modifiers. Examples of these five applications are given below: 1) Non-selective detection of the IMD has been demonstrated with the universal (FID-like) detection of a variety of drugs including steroids such as estrone, progesterone, and testrosterone, opiates such as hydromophone, codeine, and morphine and benzodiazopinones such as diazepam, nitrazepam and flurazepam (19). In addition to these drugs, compounds such as

.

19

triton X-100 (14), -114 and -305 (17), a variety of benzoates (13), and polystyrene 800 (14) have been detected. 2) The ECD detection mode of the IMD after SFC has been applied to the determination of 2,4-dichloro-phenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (20), Arachlor 1248 and 1260, captan from a bean extract and a series of pesticides as shown in Figure 7 (21).

- 1

I

(15

AT

Figure 7:

I

i

L 175

M

Negative ion mobility detection of chlorinated pesticides after SFC. Peak number 1) Chloropicrin, 2) BHC, 3) Lindane, 4) Heptachlor, 5) Endosulfan, 6) Dieldrin, 7) DDE, 8) Endrin, 9) DDT. (ref. 21)

3) Selective detection of non-heterocontaining compounds has been accomplished for individual oligamers (see Figure 8) in polymeric compounds (14), and cholesteral in commercial fish oil.

20

d.

3

C.

b.

Figure 8:

Triton X-100 separation with aelective and non-selective ion mobility detection. A) Selective response for tenth component. Drift times monitored: 23.95-24.15ms. 8 ) Selective response for fifth component. Drift times monitored: 19.15-19.35ms. C) Selective response for third component. Drift times monitored: 17.42-17.52ms. D) Non-selective response produced by monitoring drift times between llms and 30ms. (ref. 20)

4) Application to compounds without chromaphors include straight-chain methyl esters ranging in molecular weight from 88 to 466 (12) lipids in cabbage seed oil (1) and polydimethy1 si 1 icones ( 2 2 )

.

21

5) Investigations of compounds separated with modified C02 or non C 0 2 mobile phases are just beginning. Initial applications have included polydimethylsilicone (22), pyrene and other polycyclic aromatic hydrocarbons ( 2 5 ) , dinaphthylamine (15), macrobicylic crown ethers (15), benzoquinone (17), and benzophenone (17) using CH3CN and CH3OH modified C02 With freon a s a mobile phase the separation and I M D detection'of several diand triglycerides and the antibiotic, spiramycin (1).

complete list of compounds which have been detected by I M D after SFC is provided in Table I along with appropriate references.

A

TABLE I COMPOUNDS DETECTED BY SPC-IMS

OPIATES (19) codiene hydromorphone morphine

LIPIDS (1) dilaurin dimyristin trimyristin tripslmitolein tripalmitin triolein tristearin triarachidin tribehenen

BENZODIAZOPINONES (19 ) flurazepam nitrazepam diazepam

ANTIBIOTICS (11 erythromycin rafampicin spiramycin

PCLYCYCLIC AROMATIC HYDROCARBONS napthalene (21) dimethylnapthalene (31) acenapthalene (21) phenanthiene (21) fluoranthrene (21) pycene (15,211 dinapthylamine (15) anthracene (15) 1-Phenylphenanchrene (15) benzo[aIpyrene (15) benzo [a]chrysene (151

BENZOATES (13) n-hexyl benzoate n-amyl benzoate iso-my1 benzoate n-butyl benzoate n-propyl benzoate ethyl benzoate methyl benzoate

STEROIDS (19) progesterone estrone testosterone cholesterol (1)

METHYL ESTERS methyl melissate (121 methyl stearata 112,13) methyl laurate (12.13) methyl caprate (12,13) methyl caprylate 1121 methyl capsoate (12) methyl butyrate (12) methyl propionate (12) methyl myristate l12,13)

AGRICHEMICALS (20,21) lindane BHC heptachlor endosulfan dieldrin DDE CHCLESTERYL ESTERS (1) endrin cholesteryl acetate DDT cholesteryl n-butyl ether caotan cholestervl crotonate 2,b-Dichlorophenoxyacetic acid 2.4,s-trichlorophenoxyacetic acid cholesteryl octonate cholesteryl n-decylate cholesteryl laurate POLYMERS cholesteryl myristate triton-X-100 (14) cholesteryl palmitate t riton-X-114 ( 17) cholesteryl stearate rriron-X-305 (17) cholesreryl aracidate polystyrene 800 (14) cholesteryl behenate dow corning 200 122) polydimethylsilicone ( 2 2 bacon grease extract (1) KETONES (17) cabbage seed oil extract (1) benzoquinone commercial fish oil extract (1) benzophenone ~~

CONCLUSION

The multifarious nature of Ion Mobility Spectrometry makes it difficult to discuss concisely the analytical figures of merit (FOM) for all operating conditions. In addition, as discussed earlier, multiple mobile phase options may also alter response characteristics. Table I1 provides a summary of minimum detectable amounts which have been measured to date. It should be noted that these measurements were made with prototype instruments under unoptimized operating conditions. In general, ion mobility spectrometry should be able to exceed these values for well optimized instruments. TABLE I 1 DETECTION LIMITS FOR IMD MOBILE SAMPLE Cholesteryl Acetate (1) Cholesteryl n-butyl ether ( 1 ) Cholesteryl hexanoate ( 1 ) Cholesterol ( 1 ) Tristearin ( 1 1 Dimyristin ( 1 ) Trirnyristin ( 1 ) Pyrene ( 1 5 ) Pyrene ( 1 5 ) Dinapthylamine 15) Dinapthylamine 1 5 ) Benzoquinone ( 1 )

PllASE

MINIMUM DETECTABLE AMOUNT

co2

0.012 ng

CO2

co2 CO2 C02

CO2 co2

co2 C02tCHjCN ( 5 % ) CO2 C02 tCH3CN (5% ) C02+methanol ( - 8 % )

0.013 0.016 0.036 0.040 0.050

ng ng ng ng

ng

0 . 1 0 8 ng 0.1 ng 2 . 1 ng 0 . 1 ng 0 . 3 ng

2 .O ng

2.9 3.0 2.5 9.3 4.48 9.57 14.9 4.94 1.04 3.71 1.11 1.85

x 10-14 moles x 10-14 moles x 10 - 14 moles x 10-14 moles x 10-14 moles x 10-l4 moles x 10-14 moles x 10-13 moles x 10-13 moles x 10-13 moles x 10-12 m o es ~ x 10-11 moles

Currently, however, a fully optimized FTIMS instrument for SFC as not been produced. Questions concerning detector design and engineering as well as fundamental questions of ion mobility and chemical reactions remain unanswered: What is the best way to heat the restrictor without introducing noise to the spectrometer? Can ion mobility resolution be enhanced? Where is the optimum location within the ionization region to introduce the sample? How does drift gas temperature affect sensitivity? And, most importantly, how does the ion molecule chemistry of mobile phases affect response? Nevertheless, the current state of the art of FTIMS offers considerable advantage for detection after SFC. Detection limits, mobile phase compatibility, and response versatility exceed most inexpensive detection methods available for SFC today. Moreover, ion mobility data can be used to suggest unknown identities. When matched with chromatographic and ion mobility data from standards, confirmation of identifications can be achieved.

23 LITERATURE CITED M. A. Morrissey, Ph.D. Thesis, Washington State University, (1988).

Bradbury, N.E., Neilson, R.A. Phys. Rev.,

49,

388 (1936)

Carr, T.W., Editor, Plasma Chromatography, Plenam Press NY (1984) Bairn, M.A., Hill, H.H., Anal. Chem. 54, 38 (1982)

Bairn, M. A., Eatherton, R. L., Hill, H. H., Anal Chem 1761 (1983)

55,

C. B. Shumate, Ph.D. Thesis, Washington State University, (1989) Spangler, G.E., Campbell, D.N., Carrico, J.P. Pittsburgh Conf., Anal. Chem. Appl. Spectrom. Atlantic City, N.J. (1983) Blyth, D . A . , Proceeding of the International Symposium Against Chemical Warfare Agents, Stockholm (1983) Proctor, C.J., Todd, J.F.J., Anal Chem

3, 1794 (1984)

Spangler, G.E., Carrico, J.P., Campbell, D.N., J. Test Eval. 13, 234 (1985) Ellis, H. W., Pai, R. Y., Gatland, I. R., McDaniel, E. W., Wernlund, R., and Cohen, M. J., J. Chem. Phys. 64, 3935 (1976) Rokushika, S., Hatano, H., and Hill, H. H., Anal. Chem. 58, 361 (1986)

Rokushika, S., Hatano, H., and Hill, H. H., Anal. Chem. 59, 8 (1987)

Eatherton, R. L., Morrissey, M. A . , Siems, W. F. and Hill, H. H., J. High Res. Chrom. & Chrom. Com., 9 , 154 (1986). Huang, M. X., Makides, K. E., Lee, M. L., Chromatographia 31, No. 3/4 (1991)

Hill, H. H., Morrissey, M. A., Modern Supercritical Fluid Chromatography, White, G. M., Editor, Huethig Publ. (1988) Morrissey, M. A., Widemer, H. M., Manuscript in Press (1991) Klesper, E., Turner, (1962)

-, Corwin, A., J. Org . Chem., 27 700

24

19)

Eatherton, R. L., Morrissey, M. A., Hill, H. H., Anal. Chem. 60, 2240 (1988)

20)

Morrissey, M. A., Hill, H. H., J. of Chrom. Sci. 27 (1989)

21)

Tarver, E. E., Hill, H. H., Manuscript in Preparation (1991)

22)

Morrissey, M. A., Siems, W. F., Hill, H. H., J. Chromatr. 505, 215 (1990)

23)

Knorr, F. J., Eatherton, F. L., Siems, W. F., Hill, H. H., Anal. Chem. 2, 402 (1985)

K. Jinno (Ed.), Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction Journal of Chromatography Library Series, Vol. 53 0 1992 Elsevier Science Publishers B.V. All rights

reserved.

25

Chapter 3

ADVANCES IN CAPILTARY SFC-PIS

J. David Pinkston and Donald J . Bowling The Procter & Gamble Company, Miami Valley Laboratories, P.O. Box 398707, Cincinnati, Ohio, U.S.A. INTRODUCTION The mass spectrometer is arguably the most informative detector which may be linked to a chromatographic method of separation. This is evidenced by the widespread acceptance and use of gas chromatography-mass spectrometry (GC-MS), and by the great efforts expended toward linking liquid chromatography and mass spectrometry (LC-MS). It is therefore not surprising that reports of capillary SFC-MS [1,2] closely followed the first report of capillary SFC [ 3 ] . This review of recent advances in SFC-MS is divided into two chapters, the first covering capillary (wall-coated-open-tubular)-column SFC-MS and the second packed-column SFC-MS. This division is natural, since the choice of column type (i.e., effluent flow rate) dictates which interface, which method of ionization, and even, in certain cases, which type of mass spectrometer may provide the best results. Recent advances in capillary SFC-MS are reviewed in this chapter. This review will focus primarily on recent advances in this laboratory. However, the challenges encountered by the authors are typical of those encountered by other workers in the field of capillary SFC-MS. The review will first examine the use of cryopumping to enhance the performance of capillary SFC-MS at high SFC pressures. Next, an investigation of the nature of "electron ionization" in capillary SFC-MS using C02 as mobile phase will be presented. Finally, the ability of capillary SFC-MS to provide "real-wor1d"answerswill be illustrated with two recent applications to the characterization of mixtures. The first involves a fatty-alcohol reaction mixture, and will focus on the match between the SFC-MS reconstructed-total-ion-current(RTIC) chromatogram and the SFC-flame ionization detection (FID) chromatogram, and on the "conventional" nature of the methane chemical ionization (CI) spectra provided by the SFC-MS instrument. While the first application is rather typical of many applications of SFC-MS, the second involves a purely inorganic mixture, selenium sulfide, and is clearly atypical. This latter application demonstrates the flexibility and wide applicability of capillary SFC-MS in the characterization of nonpolar mixtures.

EXPERIMENTAL SECTION SFC-FID The SFC-FID instrument was a Lee Scientific Model 622 SFC/GC (Dionex, Lee Scientific Division, Salt Lake City, UT, USA). The column was a 10-m long, 50-um i.d., SB-Methyl-100column with a 0.5-um film thickness (Dionex, Lee Scientific). The mobile phase was SFC-grade GO2 (Scott Specialty gases, Plumsteadville, PA, USA). The injector loop volume was 0.2 uL, and the time-split injection time was 30 ms. The flow restrictor was a thin-walled tapered restrictor produced as previously described [ 4 ] . For the octadecanol-reaction-mixtureresidue, the FID and column-oven temperatures were held at 350°C and 100°C, respectively. The mobile-phase pressure was held at 110 atm for 5 min upon injection and then increased at a rate of 5 atm/min. SFC-W The S F C - W runs of the selenium sulfide compounds were performed on a custom-built instrument similar to those previously described [ 4 ] . Briefly, the instrument consisted of a 250-mL, pressure-controlled syringe pump, a room-temperature, internal-loop injector, a column oven, and a W-absorbance detector. The injector loop volume was 0.06 uL, and the entire volume of the loop was injected directly on-column. The 10-m long, 50-um i.d., SB-Methyl-100column (Dionex, Lee Scientific) had a film thickness of 0.25 um. The column was linked directly to the injection valve. The detector end of the column was linked to a 50-um i.d., blank-fused-silicatransfer line. The transfer line led out of the column oven to a commercially available, cooled, capillary flow cell for W detection (Dionex, Lee Scientific). The circulating bath cooling the cell block was held between 5°C and 10°C. The mobile phase was thus in the liquid state during detection. This minimized baseline drift due to variation in the refractive index during pressure-programmed SFC runs. The ultraviolet absorbance detector was a Model WIS-204 (Linear Instruments, Reno, Nevada, USA). Flow restriction was provided by a heated, thin-walled tapered capillary which was linked to the outlet of the W flow cell. For the selenium sulfide runs, W absorbance was measured at 285 nm. The SFC pressure was held at 80 atm for 5 min upon injection and was then increased at a rate of 10 atm/min. Other chromatographic conditions are listed in the appropriate section of text. SFC-MS

The SFC-MS system used for this work has been described previously [5-71. The SFC portion of the SFC-MS instrument is a custom-built unit similar to the one described above. A 10-m long, 50-um i.d., SB-Methyl-100 column with a 0.25-um film thickness (Dionex, Lee Scientific) was used. The injector loop volume was 0.1 uL. The mass spectrometer was a TSQ-70 triple quadrupole mass spectrometer (Finnigan-MAT,San Jose, CA, USA) with a mass range of 4000 Da/unit charge. The ion source and analyzer regions of the mass spectrometer are differentially pumped by two 330-L/s turbomolecular pumps. Finnigan's direct-fluid-introductioninterface was used.

27 For the octadecanol-reaction-mixtureresidue, the mass spectrometer was operated in the positive methane chemical ionization mode. The electron current was 200 uA and the electron energy was 200 eV. The column oven and interface probe stem were held at 100°C while the interface probe tip was held at 350°C. The ion source temperature was 150°C, while the analyzer manifold temperature was 70°C. The first and second quadrupoles (91 and 42) were operated in the rf-only mode, while Q3 was scanned from 92 Da/unit charge to 1000 Da/unit charge at 1 scan/s. The electron multiplier was set at -1400 V, while the conversion dynode was held at -15 kV. The electrometer was set at lo-' A/V. Other chromatographic and mass spectrometric conditions are listed above in the SFC-FID section or are included in the appropriate section of text. For the selenium sulfide mixtures, the column-oven and interface-probe-stemtemperatures were set at 60°C, while the interface-probe tip was held at 200°C. One percent NH3 in CH4 was used as the reagent gas during the negative chemical ionization runs. Q1 was scanned from 90 Da/unit charge to 1090 Da/unit charge at a rate of 1 scan/s while 42 and 93 were operated in the rf-only mode. The electron multiplier was held at -1500 V, while the conversion dynode was set at -20 kV. For the electron ionization runs, 43 was used as the mass analyzing quadrupole and was scanned from 90 Da/unit charge to 690 Da/unit charge every 0.5 s . Other chromatographic and mass spectrometric conditions are listed in the S F C - W section or are included in the appropriate section of text. Sample Preparation Decafluorotriphenylphosphine (DFTPP) was received from William Budde (U.S.E.P.A., Cincinnati, OH, USA) and was used in the investigation of the nature of electron ionization in SFC-MS without further purification. Solutions of the octadecanol-reaction-mixtureresidue were made in dichloromethane (American Burdick & Jackson, Muskegon, MI, USA) at a concentration of 1% (w/v) for the SFC-FID run and 2% for the SFC-MS run. The selenium sulfide samples were dissolved in CS2 (Fisher ACS Grade, Fisher Scientific, Pittsburgh, PA, USA) at a concentration of 1% (w/v). CRYOPUMPING FOR IMPROVED PERFORMANCE IN CAPILUIRY SFC-MS The direct-fluid-introductioninterface [l] has been used for most capillary SFC-MS. In this interface, the entire effluent from the capillary column is introduced into the ion source ionization volume through a flow restrictor. (The capillary column is directly linked to a flow restrictor using a zero-dead-volumeunion or the flow restrictor is formed at the end of the column. Typical capillary-column restrictors are fixed restrictors. Thus, mobile-phase flow rate usually increases as the SFC pressure increases.) At typical flow rates for the commonly-used 50-um-i.d.capillary columns and at SFC pressures below 300 to 350 atm, the vacuum systems of most commercial, differentially-pumpedmass spectrometers are adequate to maintain a sufficiently high vacuum such that performance is not compromised, However, at pressures above 300 to 350 atm, the performance of the TSQ-70 drops off rapidly due to the influence of the high-mobile-phasepressure on the ionization process, due

28 to collisional effects in the ion source and analyzer regions, or due to a combination of both of these effects. This drop in performance typically consists of a change in the mass spectra (e.g., more fragmentation) and a drop in the signal-to-noiseratio (S/N). Such effects have been observed in other laboratories [8,9]. In our early SFC-MS work, this drop in performance above 300 to 350 atm was usually not a problem since the upper-pressure limit of our SFC systems was 400 atm. However, the drop in performance became a real limitation after the upper-pressure limit of many of our SFC systems was increased to 560 atm [lo]. Operating under the assumption that at least part of the problem was due to collisional effects stemming from inadequate pumping capacity, and based upon earlier success with cryopumping in SFC-MS [ll], we decided to investigate the use of simple, liquid-nitrogen-cooledcryopumps in capillary SFC-MS. Three cryopumps were utilized for these investigations. Initially, two were placed in the ion-source-manifoldregion, while the third was placed in the analyzer-manifoldregion above the first quadrupole mass filter, as shown in Figure 1.

.q

Finnigan TSQ-70

330 L/s Turbo

330 L/s Turbo

Figure 1. Schematic diagram of the SFC-MS instrument with the three cryopumps in their initial configuration. Cryopumping made a dramatic improvement in capillary SFC-MS performance above 300 to 350 atm [12]. A number of analytes were initially used to probe the performance of the system with and without cryopumping. One was the tert-butyldimethylsilyl (TBDMS) derivative of a pure alkyl-ethoxy carboxylate (AEC) standard (structure: CH3-(CH2)n-(OCH2CH2)e-O-CH COOTBDMS where n=ll and e=8). 2The SFC-MS response for this analyte was almost unaffected by cryopumping when it eluted at 214 atm in the NH3 CI mode. However, when this standard eluted at an SFC pressure of 550 atm, the response with

29 cryopumping was approximately 6 times the response obtained without cryopumping . Among the mixtures used to evaluate the utility of cryopumping was the trimethylsilyl (TMS) derivative of a mixture of poly(ethy1ene glycol) standards (PEG-M) [12]. This PEG-M mixture contains standards with average molecular weights ranging from 300 to 1450, and thus covers a wide molecular-weight range. During the pressure-programmed SFC-MS run, cryopumping resulted in an increase in the intensity of the ammonium adduct ions of the PEG-M oligomers above the 19th oligomer (elution pressure of 304 atm). With cryopumping, the abundance o f the ammonium adduct ion of the 38th oligomer (elution pressure of 426 atm) was approximately twice that obtained without cryopumping. The cryopumps are easy to use. They are filled with liquid nitrogen before a series of runs is performed. The cryopumps are "topped off" three to four times during the course of a day. The condensed C02 is pumped from the instrument as the cryocooled surfaces warm overnight. We have recently optimized the arrangement of the three cryopumps. In the most effective configuration, one cryopump is located in the ion-source-manifoldregion, while two cryopumps are placed in the analyzer-manifold area. One of the latter pair is placed above the first quadrupole mass filter (91) while the other is placed over 43. Details of this optimization study will be reported in a future publication. It is now clear that cryopumping consistently improves SFC-MS performance above 300 atm. We have become increasingly confident in our ability to deliver reliable mass spectrometric data over the full pressure range of our SFC systems. In fact, since we began using cryopumping we have not yet encountered an analyte which can be eluted and detected by capillary SFC-FID which cannot likewise be eluted and detected by SFC-MS. The use of the cryopumps is so straightforward and their benefits so clear that we now use cryopumping on a routine basis for all our SFC-MS work. STUDY OF "ELECTRON IONIZATION" IN CAPILLARY SFC-MS

Electron ionization (EI) is performed by bombardment of gas-phase analytes by energetic electrons. Electron energies commonly used may range from just above the ionization potential of the analyte (8-12 eV for most organic species) to well over 200 eV, but 70 eV is considered standard. The pressure in the ionization region is generally maintained at a level where a significant amount of ion-molecule reactions doesn't occur in the interval between ionization and mass analysis. Electron ionization spectra are generally easier to reproduce than the spectra obtained using other types of ionization. This has facilitated the assembly of large libraries of EI spectra. These libraries are used by automated-library-searchingroutines. In addition, EI-fragmentation pathways have been studied for many years and are rich in structural information. The EI-fragmentationpatterns of truly unknown species are often useful for structural elucidation. Early in the development of SFC-MS, it was suggested that EI spectra could be obtained of less-volatile compounds by EI SFC-MS [13]. Many

researchers have, in fact, produced EI or EI-like spectra. One approach has been the use of "transport" type interfaces, such as the moving belt [14-161 or the particle beam [17,18] interfaces. These transport interfaces, especially the moving belt, allow the chromatograph and the mass spectrometer to operate independently. They produce unquestionably EI spectra and are especially well suited for applications involving relatively volatile compounds. A second approach has been the direct introduction of the supercritical

effluent into an EI or C1 ion source [8,19-251. Charge-exchange spectra are obtained when a CI (enclosed) ion source is used, no additional reagent gas is employed, and the primary ions produced from the mobile phase do not react with the analyte by ion transfer or association (as in the case of such common SFC mobile phases as C02 or N20). It is not clear to what degree traditional electron ionization and charge-exchange (CE) ionization contribute to the spectra obtained in EI SFC-MS when a more open, EI ion source is used. This is especially true since the mobile-phase pressure is generally programmed during most SFC-MS runs, while the flow restrictor is fixed. Thus the pressure of C02 in the ion soi,rcevaries over a wide range during a typical run. The predominant ionization mechanism may thus change over the course of a run. We decided to investigate these possibilities by acquiring "EI" spectra of a well-accepted EI standard, decafluorotriphenylphosphine (DFTPP) [26,27], at a variety of mobile-phase pressures. The DFTPP was introduced into the ion source by direct-insertionprobe at 46°C. The mobile phase, C02, was introduced through the standard direct-fluid-introductionSFC-MS interface (Finnigan-MAT). The mass spectrometer was scanned from m/z 90 to m/z 540 at a scan rate of 4 s/scan. We began by acquiring a standard, 70-eV EI spectrum of DFTPP using an YE+07

-I

0 0

Figure 2 . EI spectrum of DFTPP with no C02 introduced into the ion source.

31

I

#a40

441.9

I 0 9

8

80

)tE+07

I

i

M+'

(143.0

167.9

0

I

4 m/z

Figure 3. C02-charge-exchange spectrum of DFTPP using the "CI" ionization volume. "EI-solid-probe"ionization volume. (The TSQ-70 has interchangeable EI and CI ionization volumes which fit into the ion source.) This spectrum is shown in Figure 2. This spectrum matches the established EI ion abundance criteria for DFTPP [26]. Figure 3 shows the CO charge-exchange spectrum of DFTPP. The latter spectrum was acquired 2- a "CI-solid-probe"ionization volume with a mobile phase pressure of using 306 atm. The analyzer pressure under these conditions was 2.8 x 10-5 torr (uncorrected), corresponding to the upper limit of analyzer pressure generally encountered during CI operation. (No cryopumping was used during this phase of the experiment.) The C02-mobile-phasepressure was held at this level to insure that only C02-charge-exchange ionization would occur during acquisition of the spectrum shown in Figure 3 . The spectra shown in Figures 2 and 3 do, in fact, differ dramatically. While the molecular ion, at mass-to-chargeratio (m/z) 442, is the base peak in both spectra, the EI spectrum exhibits much more fragmentation than does the CE spectrum. (Note that the Y-axis has been expanded by a factor of 40 in the region below m/z 250 in Figure 3.) In charge-exchange ionization, a well-defined amount of internal energy is deposited in the ionized species. This amount of internal energy corresponds to the difference between the ionization potential of the molecule being ionized and the recombination energy of the charge-exchange reagenf ion. In the , CO charge-exchange plasma, the major reagent ions are CO' CO? , and the ionized C02 dimer. The recombination energy for is 14.0eV, that for C02+ is 13.8 eV, and the GO recombination energy of the dimer is lower than that of CO2" by the binding energy of the dimeric species [28]. On the other hand, a wide range of internal energies, ranging from near 0 eV to near (70 eV - IPM) (where IPM is the ionization potential o f the species undergoing

32 ionization), is deposited into species ionized by traditional EI. We have not found measured values for the ionization potential of DFTPP or for the appearance potentials of the DFTPP fragment ions. However, based upon the tabulated values for molecules of similar structure [ 2 9 , 3 0 ] , we estimate that the ionization potential of DFTPP lies between 8 . 5 eV and 10 eV. It's not unreasonable to assume that the appearance potentials of many of the EI fragment ions of DFTPP lie above the maximum recombination energy available in C02 CE ( 1 4 . 0 eV). This may explain the lack of fragmentation in the C02 CE spectrum (Figure 3 ) . The lack of fragmentation may also be partially due to collisional stabilization of the DFTPP molecular ions produced in the high pressure C02-CE plasma. The EI-solid-probe ionization volume was then inserted into the ion source. Decafluorotriphenylphosphine was again introduced by direct probe. Spectra were acquired as the SFC-mobile-phasepressure was raised in 50-100atm steps to simulate the effect of increasing C02 pressure in the ionization region during an SFC-MS EI run. The experiment was performed both with and without cryopumping. The spectra acquired at an SFC-mobile-phasepressure of 100 atm, both with and without cryopumping, were virtually identical to the conventional EI spectrum, shown in Figure 2.

As the SFC pressure was increased, the appearance of the spectra underwent some change. However, the spectra always appeared more EI-like than CE-like. For example, Figure 4 shows the EI spectrum of DFTPP collected at the highest SFC-mobile-phasepressure employed, 550 atm, with cryopumping. The spectrum exhibits a drop in the relative abundance of the molecular ion. The relative abundance of some of the fragment ions also differ from what is observed in the conventional EI spectrum, shown in Figure 2. For example, the ions at m/z 1 6 8 , 1 8 4 , and 354 have a greater relative abundance in both the C02-CE spectrum (Figure 3 ) and in the EI spectrum at an SFC pressure of 550 atm (Figure 4 ) than in the traditional EI spectrum (Figure 2). Some collision-induceddissociation (CID) is also occurring in the spectra acquired at high pressure. This is shown by the small peak at m/z 346 present in the spectra shown in Figures 3 and 4 . This ion is not present in the normal EI spectrum or in the unimolecular decomposition spectrum [26]. However, despite these differences, the EI spectrum collected at an SFC-mobile-phasepressure of 550 atm has much more the appearance of the EI spectrum than that of the CE spectrum, with its lack of fragmentation. Is the fragmentation observed in the EI spectra collected at an SFC pressure of 550 atm simply a result of CID of CE-produced ions? Probably not, based upon a comparison of the conditions used during the acquisition of the spectra shown in Figures 3 and 4 . The C02-CE spectrum (Figure 3 ) was collected without cryopumping at a re1 tively high SFC-mobile-phasepressure and analyzer pressure 2.8 x lo-' torr) . In contrast, the analyzer pressure was only 7 . 5 x lo-' torr during the acquisition of the EI spectrum collected at 550 atm with cryopumping (Figure 4 ) . Were the fragmentation due to CID of CE-produced ions, we would expect more fragmentation during the acquisition of the spectrum collected with the higher analyzer pressure, In fact, however, this spectrum (Figure 3 ) exhibits the lowest level of fragmentation.

33 Figure 5 shows the EI spectrum collected at an SFC-mobile-phasepressure of 550 atm without cryopumping. The spectrum has the overall appearance of the spectrum collected with cryopumping, but with a much lower S/N. This is not unexpected, given the cryopumping results discussed in the previous section. I

1 s . 0

19

1

e

NE+02

442.0

"1

8

80

40-

-al s m

16?. 0 254.0 20-

107.0

0100

I"'

200

300

400

500

m/z Figure 4 . EI spectrum of DFTPP with cryopumping, using the "EI" ionization volume at an SFC pressure of 550 atm. +01 1

I

lM

.-

-

I

'

I-

.L

m Q)

M

20

04.6 352.3

0 1e 0

200

300

400

m/z Figure 5 . EI spectrum of DFTPP without cryopumping, using the "EI" ionization volume at an SFC pressure of 550 atm.

SO0

34 These data lead us to conclude that, even at an SFC-mobile-phasepressure of 550 atm, traditional electron ionization appears to be a major ionization mechanism in EI SFC-MS under the conditions described here. Future experiments are planned to further probe the nature of the ionization mechanism in EI SFC-MS.

APPLICATIONS OF CAPILTARY SFC-MS Two applications of SFC-MS will be presented here. The first is typical of many applications of SFC involving relatively nonpolar, organic materials. This application illustrates the excellent correspondence between the SFC-FID and the reconstructed-total-ion-current(RTIC) chromatograms obtained with cryopumping. The second application is far less typical o f traditional applications of SFC. It involves a purely inorganic mixture of selenium sulfides. SFC-MS Characterization of an Octadecanol-Reaction-MixtureResidue A semi-solid residue was generated during a reaction involving octadecanol. A battery of analytical tests was used to characterize the

residue in order to better understand the reaction and its possible side-products. Among these tests were GC-MS and SFC with FID detection. A series of peaks appeared in the SFC-FID chromatogram that had not been detected during the GC-MS run. We were asked to identify these peaks by SFC-MS. Providing an RTIC chromatogram which matches the original SFC-FID chromatogram greatly facilitates this type of peak identification. To do s o , it's often helpful to match the chromatographic conditions (column type and length, sample size, flow rate, etc . . . ) used in the SFC-FID run. Cryopumping, as discussed earlier in this chapter, has enhanced our ability to match these chromatographic conditions over the full SFC-pressure range. Figure 6 compares the SFC-FID and the methane-CI, SFC-MS chromatograms of the octadecanol-reaction-mixtureresidue. The SFC-MS run was performed with cryopumping. Peak-to-peak correspondence between the chromatograms is apparent. The first two peaks are residual octadecanol and a low level of eicosanol. Figure 7 shows the CH4 CI spectrum of octadecanol. This spectrum is typical of the CH CI spectra of fatty alcohols [31]. The spectrum is dominated by the fM-11' ion at m/z 269 resulting from hydride abstraction, and the [M-17]+ ion at m/z 253 resulting from the loss of water from the protonated molecule. A significant level of alkyl and alkenyl fragment ions from the alkyl chain are also present. The next family of peaks are high molecular weight alcohols, ranging in carbon-chain length from 30 through 38, which result from condensation of two fatty alcohols. Figure 8 is the CH4 CI spectrum of the 36-carbon alcohol. As in the spectrum of octadecanol (Figure 7), the spectrum is dominated by the [M-17]+ ion at m/z 505, resulting from l o s s of water from the protonated molecule. Unlike the spectrum of octadecanol, the peak at m/z 521, corresponding to hydride abstraction, has a relative abundance of less than 5 % . This has been postulated to indicate

35 300

6

b

1

2

0 Retention Time

0 Y

(An)

RxC

10-

a-

b-

4-

1-

Scan Number

Figure 6 . SFC-FID chromatogram (top) and CH4 chemical ionization SFC-MS RTIC chromatogram (bottom) of the octadecanol-reaction-mixtureresidue.

1°7

W -&?-

*

80-

-

60-

h

[M-OH]'

I/]

5

4

G

$

.CI

4

4097.1 125.1

36 structures which do not carry a hydrogen on the carbon which is in the alpha position relative to the hydroxyl group [31]. However, this is probably not the case here, since the ions at m/z 491 and at m/z 477 correspond in mass to the loss of -CH20H and of -(C2H4)-OH. The third family of peaks are fatty-fatty esters, ranging in carbon number from 30 through 38. Low levels of fatty acids present in the reaction mixture led to the formation of these esters. They elute in the same retention-time range as do the condensation alcohols. Figure 9 is an 1.

100-

6

NEt06

[M-OH]+

h

H

w 80-

*8

1

h

[M-CHzOH] +

60-

4

491 6,

La

U

-0

0

-

100-

cM-l1+

n

R

h

+

[M+ 11

88-

%

v)

4

.G

U

60I 20;. 2

Figure 9. CH4 CI SFC-MS spectrum of the 30-carbon fatty-fatty ester.

37 example of one of the CH4-CI spectra of these esters. The spectrum corresponds to the 30-carbon ester, consisting primarily of rhe ester of dodecanoic acid with octadecanol. A s in the spectrum of octadecanol, the SFC-MS CH4 CI spectrum of this ester is typical of a traditional CH4 CI spectrum of a fatty-fatty ester [31]. The major ions in the spectrum correspond in mass to the protonated molecule (m/z 453), the [M-l]+ ion at m/z 451 resulting from hydride abstraction, the C18H31 alkyl fragment at m/z 253, the C18H35 alkenyl fragment at m/z 51, the protonated, 12-carbon acid moiety at m/z 201, and the C12H230 fragment at m/z 183. Ions at m/z 481 and m/z 493 correspond to the ethyl (29 Da) and the C3H5+ (41 Da) adducts, respectively, while the ions at m/z 437 and at m/z 423 correspond to the loss of a methyl and of an ethyl group. The spectra of the esters correspond primarily to esters of octadecanol with fatty acids ranging in carbon-chainlength from 12 through 20. The last group of peaks, which was not detected in the earlier GC-MS run, corresponds to esters of the high-molecular-weightcondensation alcohols described above with the fatty acids present at low levels in the reaction mixture. All the species having the same total carbon number elute in the same peak, regardless of their alcohol and acid chain lengths. However, as in the spectra of the fatty-fatty esters discussed above, the methane CI spectra of these higher molecular weight esters provide information on the alcohol and acid moieties of the isomers. For example, Figure 10

Figure 10. CH4 CI SFC-MS spectrum of the 54-carbon condensation alcohol-fatty acid ester.

shows the SFC-MS CH4 CI spectrum of the 54-carbon ester. The base peak is the ion resulting from hydride abstraction, at m/z 787. The protonated molecule is far less intense than in the spectrum of the lower molecular weight fatty-fatty ester shown in Figure 9. The primary structure present in this peak is the ester of the 36-carbon condensation alcohol with the 18-carbon fatty acid. This information is derived from the fragment ions at m/z 505 and m/z 521 in the spectrum shown in Figure 10. Thus SFC-MS was used to identify the major peaks in the SFC-FID chromatogram of the octadecanol-reaction-mixtureresidue. The peak-to-peak correspondence between the SFC-FID and the SFC-MS chromatograms, as well as the "classical" nature of the CH4 CI spectra, greatly facilitated this task. This information has been of considerable value in understanding and optimizing this particular reaction. SFC-HS Characterization of Selenium Sulfide Mixtures

Selenium sulfides have been used for many years as active components in anti-dandruff shampoos and treatments. These compounds are usually prepared by melting and stirring elemental selenium and sulfur. The resulting mixtures are quite complex. The most abundant and stable species produced in these melts are 8-membered ring structures of the formula SenS8-,, where n is equal to 0 through 8 [32]. The selenium atoms are most often adjacent to each other in structures containing more than one selenium. Little is known about which structures are, in fact, the active components of the mixture. Unfortunately, the analytical characterization of selenium sulfide mixtures is, at best, difficult. The primary tools used in this characterization have been 77Se nuclear magnetic resonance spectroscopy (NMR) [32-341, Raman spectroscopy [35-381,MS [39-411, and high performance liquid chromatography (HPLC) [35,42]. The conventional (solution) NMR and HPLC methods suffer from low solubilities of the selenium sulfides in most organic solvents and mobile phases, especially with regard to the higher-selenium-contentspecies. This often results in rapid column fouling, poor linearity and reproducibility, and increase in the column back-pressure in the HPLC approach. Mass spectrometric studies of selenium sulfide crystals have generally been accomplished using direct-insertion-probeintroduction. The positive ions produced by electron ionization with electron energies of 40 eV [39], 11 eV [40],and 70 eV [40,41]have been studied. The 70-eV EI spectra were dominated by fragment ions. Most samples studied by MS were subjected to purification through single or multiple recrystalizations. Yet many authors recognized that the samples introduced on the direct probe were still mixtures of selenium sulfide compounds [41]. Though the ratio of S to Se in the melt was varied from 7 to 1 in one report, the spectra of the crystals isolated from the melts by dissolution and recrystalization were very similar [41]. The spectra differed in the relative abundances of fragments and presumed molecular ions, indicating that the samples contained varying ratios of a variety of Se-S compounds.

39 Supercritical fluid chromatography has been successful in separating many nonpolar mixtures. The selenium sulfides are nonpolar, so we decided to pursue the possibility that SFC and SFC-MS might provide alternative methods for the separation and characterization of these mixtures. As expected, we found that the selenium sulfides are not detected by FID. They do, however, absorb strongly in the W (250-300 nm). We therefore used SFC with UV absorbance detection at 285 nm for our initial separations. Figure 11 shows the S F C - W chromatogram of a selenium sulfide mixture resulting from a melt of 1 part Se to 2 parts S (mole

1-

- 1 -

1

30

24

20

I

I

I

I

I

12 8 4 16 Retention Time (min)

0

Figure 11. S F C - W chromatogram of the mixture resulting from a 1:2 melt of Se and S (mole basis). basis) run at a column-oven temperature of 80°C. Selenium sulfide compounds are known to decompose upon heating [ 3 2 ] . We found that the peak ratios in the chromatogram were relatively unaffected by increasing the column-oven temperature from 40°C to 60°C and then on to 80°C. However, raising the oven temperature to 120°C resulted in a chromatogram which exhibited only broad humps. All subsequent runs were performed at an oven temperature of 60°C. Both positive electron ionization and negative chemical ionization were used to characterize the selenium sulfide mixtures. Negative CI produced the strongest reconstructed-total-ion-currenttraces. Figure 12 is a typical RTIC chromatogram of one of the selenium sulfide mixtures. The chromatograms were similar to those produced using W detection, showing a solvent front followed by a series of peaks with decreasing intensities. Unfortunately, the negative CI spectra were dominated by fragment ions. No molecular weight information was obtained. However, positive EI provided both molecular and fragment ions. Figure 13 is the EI spectrum

40

of the first peak in the multiplet from a typical run acquired with an electron energy of 20 eV. The spectrum corresponds to the spectrum of sulfur, S s . The next peak in the multiplet is SeS,. The 20-eV EI spectrum of this peak is shown in Figure 1 4 . The isotope pattern of a single Se atom is clearly present in the Se-containing ions. The possible structures of the fragment ions are indicated on the spectrum. Most of the fragment ions in this spectrum correspond i n mass to structures %

100

RIC

FEt07 4.711

80

60

40

Scan Number Figure 12. Negative CI RTIC chromatogram of a SeS mixture similar to the one described in Figure 11. 127.

-

100.

I

e

b

n

w

is4

80-

h

e 4 In

9

15

60-

.8

4

s5

C

c (

0

> 3

-62

XEt04

40-

(d

2 0-

s3

r

I 0-

I.

,

I

I.

257.6

19t.7

129.7

15. 9

I

,

,

,;6

I

; ;,

,

, 299

250

259.9 ,

,

I;

300

m/z Figure 13. Positive EI spectrum acquired at 20 eV of the first peak in the SeS chromatogram, S 8 .

41 incorporating the selenium atom. The charge thus appears to be preferentially retained on the Se-containing fragment upon fragmentation. The spectra of the subsequent chromatographic peaks correspond to Se2S6 and Se3S5. Thus the peaks in the chromatogram all correspond to 8-membered ring structures of sulfur and of selenium sulfides. These 8-memberedrings are known to be the major components of selenium sulfide mixtures [32]. The elution time increases

lB01

-

1

a,

.-P

%E+03

M+'

175.9

40-

SeS4

1 7 3 2

4

143.0

(d

207.6 205.3

100

239.6

150

-05.6

7

451.5

-2

299. 6

235.0

200

250

4

r I

303.7

SeS3

0 350

300

2

Figure 14. Positive EI spectrum acquired at 20 eV of the second peak in the SeS chromatogram, SeS7. l i

XE+04

8

SeS3

11 . 8

SeS

s4

M+'

127.8 1 7 3 2

SeS4

I

303.6

239.7 159.7

A

351.5

150

100

200

250

3Q0

350

m/z

Figure 15. Positive EI spectrum acquired at 150 eV of the second peak in the SeS chromatogram, SeS7.

42

with increasing degree of selenium incorporation. During the course of this investigation, EI SFC-MS runs were performed with electron energies ranging from 20 eV to 150 eV. The 150-eV EI spectra of the SeS and Se2S6 species are shown in Figures 15 and 16, respectively. 7As expected, the relative abundance of the molecular ion decreased while those of the fragment ions increased as the electron energy was raised. This is evident in a comparison of Figures 14 and 15. In contrast to the spectra of SeS7, the 150-eV EI spectrum of Se2S6 contains a number of fragment ions of significant relative abundance corresponding to structures which contain no Se.

m/z Figure 16. Positive EI spectrum acquired at 150 eV of the third peak in the SeS chromatogram, Se2S6. The extinction coefficients of the individual SenS(8-n species have not been determined. However, if one makes the assumption that the extinction coefficients are similar, one can compare the ratios of the individual selenium sulfide species detected by S F C - W to the ratios of these species detected by other analytical methods. The ratios of the hig r Se-content species to the SeS component are lower by S F C - W than by "Se nuclear magnetic resonance [ i 3 ] . The solubility of the selenium sulfides in organic solvents drops as the selenium content increases. Correspondingly, the higher-Se-content species may have lower solubilities in supercritical GO2, and may not be efficiently eluted from the chromatographic column. An organic modifier added to the mobile phase might increase the solubilities of the higher-Se-contentspecies.

CONCLUSION Simple, liquid-N2-cooledcryopumps have significantly improved the performance of our SFC-MS system at SFC pressures between approximately 300 and 560 atm, the upper limit of our SFC pump. This improved

43 performance has made applications of capillary SFC-MS using the direct-fluid-introductioninterface increasingly straightforward. The use of the cryopumps is so simple, and their benefits so clear, that we now routinely use cryopumping for all SFC-MS work in which the SFC pressure approaches or exceeds 350 atm. The applications described above clearly demonstrate that SFC-MS is applicable to both organic and purely inorganic, nonpolar mixtures. The CI spectra generated by the present system appear to be conventional CI spectra, as demonstrated in the application to the octadecanol-reaction-mixtureresidue. Even at the highest SFC pressure attainable in our system, 560 atm, the electron ionization spectrum of DFTPP has more EI character than C02-charge-exchangecharacter. Despite the advances in capillary SFC-MS achieved over the past decade, many areas for future research remain. Supercritical fluid chromatography instruments with upper pressure limits beyond 560 atm loom on the horizon. These high pressure instruments will present new interfacing challenges for on-line SFC-MS. Another area for research will be the investigation of high performance, yet less expensive mass analyzers for SFC-MS, such as the ion trap mass spectrometer [44,45] and the time-of-flightmass spectrometer [46]. Another area is the exploration of alternate ionization methods, such as atmospheric pressure ionization [47]. Alternate or modified mobile phases also hold promise for the characterization of more polar and/or higher molecular weight mixtures. We look forward to the coming decade! ACKNOWLEDGMENTS

The authors thank T.L. Chester for useful discussions and for reviewing the manuscript. Thanks to J . Hurst for his assistance in conducting the cryopumping experiments and to W.L. Budde €or provided the DFTPP sample. The authors also acknowledge the contributions of D.L. DuVal, A.L. Guy, R.S. Honkonen, M. Quijano, and C.D. Sazavsky to this work. REFERENCES

1 R.D. Smith, J.C. Fjeldsted and M.L. Lee, J. Chromatogr., 247 (1982) 231. 2 R.D. Smith, W.D. Felix, J.C. Fjeldsted and M.L. Lee, Anal. Chem., 54 (1982) 1883. 3 M. Novotny, S.R. Springston, P.A. Peaden, J.C. Fjeldsted and M.L. Lee, Anal. Chem., 53 (1981) 407A.

4 T.L. Chester, D.P. Innis and G.D. Owens, Anal. Chem., 57 (1985) 2243. 5 J.D. Pinkston, D.J. Bowling and T.E. Delaney, J. Chromatogr., 474

(1989) 97. 6 J.D. Pinkston, T.E. Delaney and D.J. Bowling, J. Microcol. Sep., 2

(1990) 181.

44 7 D.J. Bowling and J.D. Pinkston, Presented at the 38th ASMS Conference on Mass Spectrometry and Allied Topics, June 3-8, 1990, Tucson, AZ, U.S.A. 8 S.D. Zaugg, S.J. Deluca, G.U. Holzer and K.J. Voorhees, HRC CC, J . High Resolut. Chromatogr. Chromatogr. Commun., 10 (1987) 100.

9 H.T. Kalinoski and L.O. Hargiss, J. Chromatogr., 505 (1990) 199. 10 T.L. Chester, D.J. Bowling, D.P. Innis and J.D. Pinkston, Anal. Chem., 62 (1990) 1299.

11 G.D. Owens, L.J. Burkes, J.D. Pinkston, T. Keough, J.R. S i m s and M.P. Lacey, ACS Symp. Ser., 366 (1988) 191.

12 D.J. Bowling and J.D. Pinkston, unpublished data. 13 L.G. Randall and A.L. Wahrhaftig, Anal. Chem., 50 (1978) 1703. 14 A.J. Berry, D.E. Games and J.R. Perkins, Anal. Proc. (London), 23 (1986) 451.

15 A . J . Berry, D.E. Games and J.R. Perkins, J . Chromatogr., 363 (1986) 147. 16 E.R. Verheij, G.F. La Vos, W.M.A. Niessen, U.R. Tjaden and J . van der Greef, J. Chromatogr., 474 (1989) 275.

17 J.D. Henion, P.O. Edlund, E.D. Lee and L. McLaughlin, Presented at the 36th ASMS Conference on Mass Spectrometry and Allied Topics, June 5-10, 1988, San Fransisco, CA, U.S.A. 18 P.O. Edlund and J.D. Henion, J. Chromatogr. Sci., 27 (1989) 274. 19 R.D. Smith, H.R. Udseth and H.T. Kalinoski, Anal. Chem., 56 (1984) 2971. 20 G. Holzer, S . Deluca and K.J. Voorhees, HRC CC, J. High Resolut. Chromatogr. Chromatogr. Comun., 8 (1985) 528. 21 J . Cousin and P.J. Arpino, J . Chromatogr., 398 (1987) 125. 22 E.D. Lee, S. Hsu and J.D. Henion, Anal. Chem., 60 (1988) 1990. 23 E.C. Huang, B.J. Jackson, K.E. Markides and M.L. Lee, Anal. Chem., 60 (1988) 2715. 24 H. Kallio, P. Laakso, R. Huopalahti, R.R. Linko and P. Oksman, Anal. Chem., 61 (1989) 698. 25 H.T. Kalinoski and L.O. Hargiss, J. Chromatogr., 474 (1989) 69.

45 26 Y. Tondeur, W.J. Niederhut, J.E. Campana, R.K. Mitchum, G.W. Sovocool and J.R. Donnelly, Biomed. Environ. Mass Spectrom., 15 (1988) 429. 27 J.W. Eichelberger, L.E. Harris and W.L. Budde, Anal. Chem., 47 (1975) 995. 28 A.G. Harrison, Chemical Ionization Mass Spectrometry, CRC Press, Boca Raton, 1983. 29 H.M. Rosenstock, K. Draxl, B.W. Steiner and J.T. Herron, J . Phys. Chem. Ref. Data, 6 (1977) 1-1. 30 R.W. Kiser, Introduction to Mass Spectrometry and Its Applications, Prentice-Hall,Englewood Cliffs, 1965. 31 M.S.B. Munson and F.H. Field, J. h e r . Chem. SOC., 88 (1966) 2621. 32 R.S. Laitinen, Acta Chem. Scand., A41 (1987) 361. 33 R.S. Laitinen and T.A. Pakkanen, J . Chem. SOC., Chem. Commun., (1986) 1381. 34 R.S. Laitinen and T.A. Pakkanen, Inorg. Chem., 26 (1987) 2598. 35 R. Laitinen, N. Rautenberg, J. Steidel and R . Steudel, 2. Anorg. Allg Chem., 486 (1982) 116. 36 H.H. Eysel and S. Sunder, Inorg. Chem., 18 (1979) 2626. 37 R. Laitinen, J. Steidel and R. Steudel, Acta Chem. Scand., A34 (1980) 687. 38 R. Laitinen and R. Steudel, J. Mol. Struct., 68 (1980) 19. 39 R. Cooper and J.V. Culka, J. Inorg. Nucl. Chem., 29 (1967) 1217.

40 C.R. Ailwood and P.E. Fielding, Aust. J. Chem., 22 (1969) 2301.

41 R. Laitinen, L. Niinisto and R. Steudel, Acta Chem. Scand., A33 (1979) 737. 42 R. Steudel and E.-M. Strauss, 2. Naturforsch., 38b (1983) 719. 43 R.S. Honkonen, personal communication.

44 J.F.J. Todd, I.C. Mulchreest, A . J . Berry, D.E. Games and R.D. Smith, Rapid Corn. Mass Spectrom., 2 (1988) 5 5 . 45 J.D. Pinkston, T.E. Delaney, K. Morand and R.G. Cooks, unpublished data. 46 J.F. Holland, C.G. Enke, J . Allison, J.T. Stults, J.D. Pinkston, B. Newcome and J.T. Watson, Anal. Chem., 55 (1983) 9978.

46 47 J.F. Anacleto, F. Benoit, R.K. Boyd, S. Pleasance, M.A. Quilliam, L. Ramaley, P.G. Sim, Presented at the 39th ASMS Conference on Mass Spectrometry and Allied Topics, May 19-24, 1991, Nashville, TN, U . S . A .

K. Jinno (Ed.), Hy henated Techniques in Supercritical FluifChromatography and Extraction Journal of Chromatography Library Series, Vol. 53 0 1992 Elsevier Science Publishers B.V. All rights reserved.

47

Chapter 4

Advances in Semi Micro Packed Column SFC and Its Hyphenation Makoto Takeuchi' and Toshinori Saito'

' Basic Research Division, JEOL Ltd. Akishima, Musashino 3-1-2, Tokyo 196, Japan 'Techunical Group, JEOL MOLEH Co. Ltd. Akishima, Musashino 3-1-2, Tokyo 196, Japan

INTRODUCTION Using a semi-micro packed column and a UV detector, binary fluids of carbon dioxide and an organic solvent were successfully used as eluent on SFC, for analytical separation of low molecular weight polymers, polymer additives and many other industrially valuable compounds. [1,2] However, samples which have no functional group with absorption in the range of ultraviolet and visible light, for example, hydrocarbons, pol yethers, fluorocarbons, silicones, polyols, organic metals and so on, have been out of examination in detection. For these compounds, use of a RI detector was attempted since it was used widely in GPC or reversed phase HPLC, but it could not be used on SFC a s a detector before the decompression stage, because of a failure to keep the durability to pressures exceeding 100 kglcm' in our experience. The flame ionization detector has been widely used so far in capillary SFC, in which indiscriminative property required the use of carbon dioxide without a modifier. This was also true in packed column SFC. This fact leads to the development of a very inert packing material free from adsorption of polar samples. Although considerable advancement has been recently attained, but it is still not enough for elution of polar compounds. In this sense, packed column SFC has not yet been comparable to capillary SFC although the advantages such as high sample loadability and fractionation capability for analytical preparative purposes are existing. Polar modifiers such as alcohol are very effective for deactivation of the site of adsorption on the stationary phase; moreover, it has superior effectiveness to accelerate the elution and sometimes to enhance the selectivity on separation of polar samples. For such polar compounds with no response in a UV detector, the hyphenated use of an evaporative light scattering detector ( ELSD ) seems very promising. Direct on-line coupling of SFC to FAB/MS is also very promising to detect a polar sample [3,4] which has no response in a UV detector, if the eluate is effectively ionized and M/Z of the ions exist in the scanning mass range provided. In addition to this fact, it has a precious value for resolving the problem for identification of separated peak components.

4a

EXPERIMENTAL

Apparatus The basic construction of the SFC used was the standard assembly of JSF-8800 (JEOL MOLEH) [l]shown in Fig. 1. The hyphenation of ELSD and FAB/MS interface is also shown schematically. Two pumping systems, whose unique function is the quantitative delivery at a given pressure and temperature, independently of the compressibility of the liquids used [2], were equipped for delivering liquefied COz and an organic solvent used as modifier, respectively.

Heat

gas

coo1er

/ Pressure reservoir CAP-PRO2 500 m l

Back press. controller

oven

Pressure reservoir CAP-PRO1 70 ml

Fig. 1 Flow diagram of semi micro packed column SFC (JSF-8800) and its hyphenation to ELSD or FAB/MS interface. For delivery of rinse solventsto a back pressure control unit (CAP-PBOl), a large pressure reservoir (CAP-PROS) of which capacity was 500 ml was provided for a one day work. A sampling valve (Rheodyne 7520) with a 1 ,ul volume was used for semi micro packed columns (SFPAK ODs-5S25 1.7 mm X 250 mm, and ODs-5S50 1.7 mm X 500 mm , both packed with 5 ,um ODs, JEOL MOLEH). The column temperature was kept constant or swept downwards by the linear programming equipped with HP 5890 GC oven in the range from 40°C to 250°C . A variable wavelength (195 370 nm) UV detector (CAP-UVO1) with a high-pressure semi micro cell ( CAP-CCO1 1 ,ul volume, 5 mm light path, JEOL MOLEH) was connected with a 0.1 mm i.d. X 500 mm length stainless steel tube from the outlet of the column to UV cell provided with a cooling system. Pressure regulation was attained by a specially designed temperature-controlled low-dead-volume release valve provided with a supplementary solvent line [ 3 ] ,which was built in a back pressure controller (CAP-BPO1).

-

49 The function of the release valve is to carry out decompression, while keeping the back pressure of the column at a pre-set value during elution. Regardless of using temperature programming or composition programming of binary fluid, it is primarily independent of the flow rate of eluent, while a restrictor used frequently with capillary SFC to produce the pressure or the fluid density required is dependent on the flow rate. The hyphenation to the evaporative light scattering detector (ELSD)[Varex Mark IIA] or to Fast Atom Bombardment Mass Spectrometer (JMS-SX100, -AX505, LX-1000, JEOL) is performed after decompression by the release valve. [4]

Materials The carbon dioxide employed ( four nine grade, Showa Tansan) was purified by means of a CAP-GCOI gas purification unit (JEOL MOLEH). Chromatography grade methanol, ethanol, tetrahydrofuran (THF) (Wako) were used as modifiers and rinse solvents without further purification. The packing materials were offered mainly from Nomura Chemicals Co. Ltd., Chemical Inspection & Testing Institute, Japan and Shiseido. As the test samples, the Triton X-100 was purchased from Sigma Chemical Co. Ltd., alcoholethylate and its UV-labeled compounds were offered from some chemical company, and a series of PPG oligomers were offered by courtesy of Osaka Research Lab., Chemical Products Division, Takeda Chemical Industries, Ltd.

BINARY FLUID AND ITS PHYSICAL PROPERTIES A binary fluid of C 0 2 and methanol or ethanol which belongs to Type I ( miscible with each other in both phases of liquid and gas at any composition) is most frequently used. Therefore, the physical pi~pertiessuch as the viscosity and density of the binary fluid at a given composition, temperature and pressure have to be known before beginning an experiment of SFC or SFE. The changes of critical temperature and critical pressure of binary fluid of CO, and an organic solvent were given by Smith et al [5], and Kuppers et al [6]. For example, the critical temperature changes monotonously from 32 "C of pure CO, to 240.5 "C of pure CH, OH. It increases along with the increase of composition of CH,OH, while the critical pressure increases from 73 kg/cm2 of pure C02up to 160 kg/cm2 at about equal composition, then decreases to 79 kg/cm2 of pure CH30H. Therefore, if the pressure is kept above the maximum critical value, the binary fluid can be kept in mono phase at any composition and any temperatures. The maximum critical pressure of C02-C2HsOH seems to be quite similar to the value of 160 kg/cm2.

50

EXPERIMENTAL ESTIMATION OF VISCOSITY OF THE BINARY FLUID The pressure drops through the column in which the binary fluid was flowing at the constant mass flow rate were observed by the following procedure: The two pump systems provided were used in the "unlocked mode" when the flow rates of C02and a modifier solvent were changed for obtaining the required composition and constant total flow rate. The built-in feed-back mechanism which worked for obtaining the quantitative flow rate is completed by changing the plunger stroke just to meet the volume required to compensate the compression loss. The details were described elsewhere. [7, 81 The column temperature was changed by stepwise, i.e., 5O"c, 75"c, lOO"C, 125"C, 150 "C, 175"C, 2OO0c, 225"c, and 250°C. The back pressure was adjusted manually to less than 2 0 0 f 1 kg/cm2 and left for a while until the entire thermal equilibrium was attained, then a pressure drop was observed by the top pressure minus the back pressure. After measurement at 250"C, the temperature was come down to the initial 50°C and at the same time the next composition was set. After several minutes for attaining the iso-cratic and iso-bark conditions, measurement was performed again in the same procedure described above. ~

.

_

_

_

_

_

.

--

~

~

-

k c k press. set]

7 -

p:tmin for temp. seI + k m i n for F.R. sed

7

1 DP -

:

differential press. of pump

BP : Back Press. TP : Top Perss.

51

Fig. 2 shows iso-thermal plots of the observed pressure drops through the column against the compositions at the pressure 200 k 1 kg/cm2. As far as the mass flow can be kept constant, the pressure drops through the column at given conditions (composition, temperature, pressure) represent the product of the viscosity and linear velocity of the fluid in the column at the given conditions. On the other hand, the linear velocity of the fluid in the column can be observed by measuring the retention time of a nonretained sample while changing the composition and temperature as the same way as the above.

kg/cm2 80 70

-

60 /

,,i I

50 -

40 30 C

C

CO, 300 CH,OH 0

275

25

250 50

225 15

200 100

175 125

125 175

300

Composition X

Fig. 2 Iso-thermal plots of the observed pressure drops AP through the column against the composition. I n the column the binary fluid of CO,CH,OH was flowing at constant mass flow rate of 300 ,uI/min, at 200 1 kglcm2.

*

52 As the mass flow is the product of the linear velocity and the density at the given conditions, the composition and temperature dependence of the retention time shows the density profile of the binary fluid in a column at the given conditions, as shown in Fig. 3 . It is of interest that the minimum product of linear velocity and viscosity of the binary fluid exist not in pure C02, but in a mixture containing some CH30H in CO2. This phenomenon was observed on other binary fluids such as C02-C2HsOH, C02-CH2CI2 and C02 -1,4dioxane. And it is suggested in Fig. 2 that the concentration of CH3OH can be changed from 0 to 50% at moderate temperature with a little increase in pressure drop. And the temperature dependence of the density of a binary fluid shows a very large change and an almost linear characteristic in a wide range of compositions, as shown in Fig. 3. This means that the linear temperature programming is effective for eluting oligomers with equal space separation.

/

/

/ / / / ,.

,

1-I

25

CH,OH

25

50

..

100

150

200

250

300

Composition X

Fig. 3 Density profile of the binary fluid CO,-CH,OH in the column estimated by observing of retention time of nonretained peak at a given temperature and composition. All these measurements were performed at the pressure of 200 k I kgicm’.

53

HYPHENATION BETWEEN ELSD AND SEMI MICRO PACKED COLUMN SFC The light scattering detector has been used so far mainly for liquid chromatography to analyze triglycerides, phospholipids, sugars, steroids, fatty acids and oligomers of surfactants as ethoxylate alcohol. [ 9 -131 Recent attempts to use ELSD on SFC by means of direct coupling ELSD to the SFC column have been reported. [14, 151 In our experiment, hyphenation was done easily by connecting the outlet of JSF-8800 to the inlet of the ELSD with 1/16” 0.d. stainless tubing. The ELSD, Varex Mark IIA, was used without further modification. For making proper aerosol, it was necessary to increase the flow rate of a supplemental rinse solvent which had been set around 100ul/min in normal run with a UV detector, to about 500 fil/min. The other instrumental parameters such as air flow of 2 l/min , temperatures of 90 100 “C were set around the value as the standard for a detector. Usually, the standard eluent flow rate, 300pI/min of liquefied CO2 becomes to 0.1 l/min of gas flow after decompression. To prevent the solvent used from spreading out with the exhaust gas, a supplemental cooled trapping device was introduced, as shown in Fig. 4.

-

cold finger /

-1ooc

250 rn viral

Fig. 4 Schematic illustration of cooled-trap unit to prevent the solvent being spread out by exhaust gas of ELSD.

54

Fig. 5 shows the separation of a synthetic oligomer of nonionic surfactant (Triton X-loo), using the binary fluid of CO2 and C&OH (300 ,ul/min: 50 yI/min). Temperature gradient elution from 130°C by a rate of -1.5"C /min was performed at aback pressure of 175 kg/cmz. In Fig. 5, A is UV response at 220 nm, B is ELSD response at air flow 40 mm (-2 I/min) with nebulizer temperature of 90 "C and rinse solvent (THF) flow rate of 500 pl/min. The observed time difference between two detectors was about 19 sec, and additional dispersion after the UV cell was not so significant that the resolutions of both ELSD and UVD were almost the same practically. The lowest molecular weight components of the oligomer were not observed or weakly observed in ELSD, but residual major components were observed with weight average distribution rather than number average distribution in the UV detector. This information obtained by UV-ELSD might be valuable for discussing whether the structure of additive unit of oligomer has UV-active function group or not.

1

EL^^

__1

I

1

u

UV D

I

0

I0

n

20

min

Fig. 5 Temperature gradient elution of nonionic surfactant, Triton X-100, by UV-ELSD hyphenation system. The column was 1.7 mm i.d. x 500 mm length packed with ODs-5. The other experimental conditions are shown in the text.

55 Using the same sample and Lhe same eluting conditions, the peak height of three selected components in which number of EO (ethoxy, CH2CH20-) was n = 4, 9 and 14, was observed at six different nebulizer temperatures at 50, 60, 70, 80, 90 and 100°C , as shown in Fig. 6. The optimum temperature for each component was slightly different. It seems that the higher the molecular weight, the higher is the optimum temperature for each component. The nebulizer temperature dependence of the response in ELSD was examined using a surfactant ( polyoxyethylene glycol monododecyl ether CLoH13-0[CH2CH20-] nH) as a sample by gradient elution of 0.1% acetic acid aqueous solution and acetonitrile in the reversed phase HPLC. [13] The results indicated a fairly large change of response depending on the temperature and the number of sequences of oxyethylene unit, in comparison to the result observed on the similar compound CI3H2,-0[CH2CH20-] nH by temperature gradient elution on SFC, as shown above.

I

n = 9

-Y

m

a,

PI

50

60

70

80

90

1000~

Fig. 6 Peak height changes of n = 4, n 9 and n = 14 of Triton X-100 oligomers against the nebulizer temperatures. The optimum temperature for each component is slightly different each other. It increases along with molecular weight.

This difference may come from the dependence of the optimum nebulization temperature on the boiling temperature of the solvent used, while in gradient elution the composition of the solvents with different boiling temperatures is changed. On the contrary, the temperature gradient in SFC is not changed the eluent composition. Even if modifier concentration gradient is used, composition change is very small because of the negligible amount of a modifier against the rinse solvent; therefore the composition of the fluid into the ELSD remains almost constant.

56 Fig. 7 shows the separation of oligomer of alcoholethoxylate ( average number of EO n = 7 11) observed with ELSD. This sample can be observed with UV detector, as shown in Fig. 8, after treatment with 3,s dinitro-benzoyl-chloride in order to derive it to be UV-labeled compounds. The results in Fig. 7 and Fig. 8 do not indicate a significant difference; however, ELSD is much convenient because no label treatment and blank test are required to confirm the residual reagent or its impurities which are essential in UV detection.

-

Fig. 8

Fig. 7

I

I

0

4

10

20

rnin

30

Fig. 7 The separation of oligomers of alcoholpolyethoxylate ( average number of EO n = 7 11) observed by ELSD. Column: 1.7 X 500 mm, Eluent: C 0 2 300 ,ul/min, Modifier: C,H,OH 50 plhnin, Temperature: 130°C with -2°C /min rate, Back Pressure.: 175 kglcni', Nebulizer Temp.: 90 "C , Air Flow: 45 rnm, Rinse Solvent: THF 500 plimin.

-

0

10

20

30

rnin

Fig. 8 The separation of alcohol-polyethoxylate UV-derivative observed by UV detector. Elution conditions were the same as in Fig. 7.

57 Farther applications of the UV-ELSD system were tried for the analysis of polyoxypropyleneglycol oligomer ( PPG ) prepared by using different polymerization initiators as shown in Fig. 9-A propylene glycol, B tolylenediamine and C mixture of glycerol and sugar. The same samples were measured by the UV- FAB/MS system, as will be described later. As shown in Fig. 9, the sample B shows a response on both UV and ELSD, making it possible to know the detectability of low-molecular-weight components. On the other hand, samples A and C have no response on UV, but on ELSD their responses are clearly observed. All these samples are mixtures of polyoxypropylene unit of n = 1 3. The detectability of sugar compounds on ELSD was remarkable.

-

1

b

5

I0

1'5 H, (OCHCH, CH, -) nOH H (OCHCH, CH, -) nOH t H , (OCHCH, CH, -) nOH

z

20 min

-5

0

10

15 min

Fig. 9 The separation of polyoxypropylene glycol (PPG) prepared by polymerization using different initiators; A propylene glycol, B tolylenediamine, C mixture of glycerol and sugar. Column: 1.7 X 250 mm ODs-5, Eluent: CO, 300 pl/rnin, Modifier: C,H,OH 50 3 150 p h i n per 30 min, Temperature: 15Ooc, Nebulizer Temp.:90 , Air Flow: 45 mm, Rinse Solvent: THF 500 pI/min.

"c

58 An application of this system for separation of larger molecular weight compound is shown in Fig. 10. The sample was polyoxypropylene glycol oligomer (PPG), having a molecular weight of 3000 and polymerized by glycerol as an initiator. The elution was done by modifier gradient at 15Ooc. The detector response at the beginning of elution was magnified 10 times for observing small peaks of oligomer. More than 55 peaks were observed and molecular weight distribution was clearly characterized.

H, (OCHCH, CH, -) nOH H (OCHCH, CH, -) nOH Hz (OCHCH, CH, -) nOH

I

-

Fig. 10 The separation of polyoxypropylene glycol (PPG) polymerized by using glycerol as initiator, observed with ELSD. The detector response at the beginning of elution was magnified 10 times. Column: 1.7 x 500 mm ODs-5, Eluent: 300 pl/min, Modifier: 75 + 150 pI/min per30 min, Temperature: 150 "C , Back Press.: 185 kgicm'. The other parameters were the same as in Fig. 9.

59

HYPHENATION BETWEEN FAB/MS AND SEMI MICRO PACKED COLUMN SFC Coupling of SFC to FAB/MS was achieved by means of an LC-FAB/MS frit interface already been available [16, 171, as shown in Fig. 11. A mixture of methanol and THF containing 1% glycerol was used as a rinse solvent, which was added just after the decompression stage of the release valve, to transport the eluate to the frit interface via a splitter. In this splitter gaseous C 0 2 and excess solution were removed through 1/16” 0.d. stainless steel tubing and components of interest were conducted to the M S spectrometer, along fused silica capillary tubing (60 p m i.d. X 1 m). The correct operation of the splitter was critically dependent on the position of the fused silica capillary in the 1 / 16” stainless steel tubing when correctly positioned condensation created a localized flow into the capillary to allow compounds of interest to be collected from gaseous C02and evaporating solvent. Under the optimum condition where the flow rate of the rinse solvent is set around 300 pllimin, no bubbles are formed in the capillary. Then the eluate are ionized continuously together with matrix fed from the capillary. While if bubbles are formed in the capillary, the ion concentrations of interest and matrix are decreased to make a change on mass chromatogram becauase of discontinuous feeding due to form a bubble.

Air,

ELSD

Fig. 1 1 Schematic presentation of the release valve and the connecting arrangement of it to ELSD or to FAB/MS interface. Rinse solvent was supplied from A position. For ELSD about 500 ,ul/min of THF was supplied, while for FABMS about 300 pllmin of the mixture solvent of THF and CH,OH containing 1%

glycerol was supplied.

60 Fig. 12 shows a chromatogram obtained by SFC with a UV-FAB/MS hyphenation system for the sample of tolylenediamine PPG derivatives mentioned before. A is a UV response and B is a MS chromatogram monitored at M/Z = 355 ( protonated molecular ion of n = 1 for all four possible sites ) and successive series ions produced by adding oxypropylene group of M = 58.

CII 3 ,(CllZCII 0 )a-II

b

a

A

I V

\

x

1.3

Fig. 12 The separation of tolylenediarnine polyoxypropylene glycol (PPG) oligorner observed with SFC-FABIMS. A is UV response, B is MS chromatogram of ions shown in the figure. Column: 1.7 x 250 mm ODs-5. Eluent: C 0 2 300 ,uI/min, Modifier: C,H,OH 50 ,uI/min, Temperature: I20 "C -3 "C/min, Back Pressure; 154 kg/cm2.

61 Fig. 13 shows three MS spectra of the peaks a, b, and c marked in the UV chromatogram in Fig. 12. In addition to the protonated molecular ions, moderate numbers of fragment ions were observed, which might be useful for the assignment of the molecular structure.

413

b

471

Fig. 13 The mass spectra of the eluting components a, b, and c marked on the chromatogram shown in Fig.12. Protonated molecular ions and their fragment ions were observed.

62 Fig. 14 shows a MS chromatogram and MS spectral change of glycerol PPG derivative which has no response in UV. The protonated molecular ion of n = 1 for all three possible sites, M/Z = 267 and successive series-ions produced by adding oxypropylene group, MW = 58 were observed. The samples used for measurement of glycerol PPG derivatives include the corresponding PPG derivatives of sugar. In the first run of UV-FAB/MS, however, the corresponding ions were not found. At that moment, it was not clear whether the cause of the failure to detect ions of sugar derivatives was SFC eluting condition or the ionization process or the detecting mass range. It is possible to optimize the detection in SFC-FAB/MS by selection of matrix, adjustment of accelerating voltage of ions and setting of scanning mass range after confirmation of the peaks for a sugar PPG derivative in a chromatogram with ELSD.

557 ___1

499

*I5

44 I

"4.5

383

12.0

1.1 1.8

z

Im

IIS 7

a

50

0-

-

b 51)

217

3111

Fig. 14 The mass chromatogram and mass spectra of glycerol polyoxypropylene glycol (PPG) oligomer. Column: 1.7 x 250 mm ODS-5, Eluent: C 0 2300 ~ V r n i n Modifier: , CH,OH 50 ,uI/min, Temperature: 120 "C - 3 C /min, Back Press.: 153 kg/cm*. Acc.Volt. of mass spectrometer:3 kV.

CONCLUSION The hyphenation between ELSD and semi micro packed column SFC using the binary fluid of COz and a polar organic solvent was quite useful for analytical separation of polymers, polymer additives and many other industrially valuable compounds. This system is also valuable for optimizing elution conditions prior to doing for SFC-FABIMS work.

REFERENCES M. Takeuchi and T. Saito. J. Chromatogr. 515 (1990) 629 - 438. F. P. Schmitze and E. Klesper. J. Supercritical Fluids 3 (1990) 29 - 48. M. Takeuchi and T. Saito. J. High Resol. Chromatogr. 14 (1991) 347 - 351. K. Matsuura, M. Takeuchi, K. Nojirna and T. Kobayashi. Rapid Communications in Mass Spectrometry 4 (1990) 381. [5] R. D. Smith, B. W. Wreight and H. T. Kalinoski. Yoshioka et al (Edrs) Progress in HPLC Vol. 4, VSP, Utrecht, Tokyo (1989) 383. [6] S. Kuppers, D. Leyendecker and F. P. Schmitz and E. Klesper. J. Supercrit Fluids 3 (1990) 121 - 126. [7] T. Saito and M. Takeuchi. JEOL NEWS 23A 2 (1987) 43 - 47. [8] T. Saito, M. Takeuchi. Yoshioka et al (Edrs), Progress in HPLC Vol. 4, VSP, Utrecht, Tokyo (1989) 25 - 51. [9] A. Stalyhwo, H. Colin and G. Guiochon. Anal. Chem. 57 (1985) 1342. [lo] N. Sotirhos, C. Thormgren and B. Herstof. J. Chromatogr. 331 (1985) 313. [ l l ] R. Macrae and J. Dick. J. Chromatogr. 210 (1981) 138. [12] P. A. Asmus and J. B. Landis. J. Chromatogr. 316 (1984) 461. [13] Y. Mengerink, H. C. J. DeMan and Sj. VanDerWal. J. Chromatogr. 522 (1991) 593 604. P. Carraud, D. Thiebaut, M. Caude, R. Rosset, M. Lafosse and M. Dreux. [14] J. Chromatogrsphic Science 25 (1987) 395 - 398. [15] D. Nizery, D. Thiebaut, M. Caude and R. Rosset, M. Lafoss and M. Dreux. J. Chromatogr. 467 (1989) 49 - 60. [l] [2] [3] [4]

This Page Intentionally Left Blank

K. Jinno (Ed.), Hyphenated Techniques in Supercritical fluid Chromatography and Extraction

Journal of Chromatograph Library Series, Vol. 53 0 1992 Elsevier Science Jublishers B.V. All rights reserved.

65

Chapter 5

L a r r y T. Taylor* V i r g i n i a P o l y t e c h n i c I n s t i t u t e and S t a t e U n i v e r s i t y Department of Chemistry 24061-0212 Blacksburg, VA E l i z a b e t h M. Calvey Food and Drug A d m i n i s t r a t i o n D i v i s i o n of Contaminants Chemistry 20204 Washington, DC

1"ICTION

Both c o n v e n t i o n a l high performance l i q u i d chromatography (HPLC) and g a s chromatography ( G C ) d e t e c t o r s have proven t o be c o m p a t i b l e w i t h s u p e r c r i t i c a l f l u i d chromatography (SFC). For example, f l a m e i o n i z a t i o n , e l e c t r o n c a p t u r e , f l a m e p h o t o m e t r i c , t h e r m i o n i c , u l t r a v i o l e t ( U V ) and f l u o r e s c e n c e d e t e c t i o n [ 1 , 2 1 have a l l s u c c e s s f u l l y been d e m o n s t r a t e d . Various e f f o r t s have been made t o c o u p l e s p e c t r o m e t r i c d e t e c t o r s w i t h chromatographic s y s t e m s t o g a i n more s p e c i f i c i n f o r m a t i o n r e g a r d i n g t h e f u l l i d e n t i f i c a t i o n of e l u t i n g components. Fourier transform i n f r a r e d (FT-IR) [ 3 , 4 1 , a t o m i c e m i s s i o n [ S I , n u c l e a r magnetic r e s o n a n c e and mass [61 s p e c t r o m e t e r s have been i n t e r f a c e d t o s u p e r c r i t i c a l f l u i d chromatographs w i t h v a r y i n g d e g r e e s of s u c c e s s d u r i n g t h e 1980s. The FT-IR d e t e c t o r i s c o n s t r a i n e d by two major problems: mid-IR a b s o r p t i o n by most c h r o m a t o g r a p h i c a l l y c o m p a t i b l e mobile p h a s e s and r e l a t i v e l y low FT-IR s e n s i t i v i t y compared t o s e v e r a l o t h e r more T o minimize t h e s e problems, v a r i o u s i n g e n i o u s established detectors. i n t e r f a c e d e s i g n s have been e x p l o r e d . These d e s i g n s a p p e a r t o v a r y g r e a t l y , b u t t h e y can be c l a s s i f i e d by two approaches: solvent e l i m i n a t i o n coupled w i t h t r a n s m i s s i o n or r e f l e c t a n c e I R , and f l o w c e l l coupled w i t h t r a n s m i s s i o n o r a t t e n u a t e d t o t a l r e f l e c t a n c e I R . Each approach h a s a unique s e t of c h a r a c t e r i s t i c s t h a t makes it a t t r a c t i v e for certain applications. The f o c u s of t h i s c h a p t e r i s t o d e s c r i b e the s t a t e as i t r e l a t e s t o t h e flow c e l l i n t e r f a c e . o f - t h e - a r t o f SFC/FT-IR

Early S t u d i e s

The flow c e l l approach i n v o l v e s c o n n e c t i n g a h i g h p r e s s u r e flowt h r o u g h c e l l a t t h e end of t h e column and p o s i t i o n i n g t h e flow c e l l i n t h e FT-IR beam so t h a t t h e column e f f l u e n t i s m o n i t o r e d i n r e a l t i m e a s i t flows t h r o u g h t h e c e l l . The I R s i g n a l i s d i r e c t l y p r o p o r t i o n a l t o t h e amount of sample i n t h e c h r o m a t o g r a p h i c peak t h a t i s f l o w i n g t h r o u g h t h e c e l l a t any r e s p e c t i v e t i m e . The f l o w c e l l must be a b l e t o w i t h s t a n d h i g h p r e s s u r e , have I R t r a n s p a r e n t windows and e x h i b i t a volume c o n s i d e r a b l y

smaller than the peak volume. The ideal cell dimensions from both a chromatographic and spectrometric point-of-view are not compatible; therefore, a compromise is necessary to achieve the hyphenated experiment. A number of variables must be considered such as the type of column (open tubular vs packed) used, the IR transparency of the mobile phase and the limit of detection desired. Initial flow cell SFC/FT-IR studies were performed isobarically with 4.6 mm i.d. packed columns where peak volumes in excess of 40 pL were common. The flow cells employed were those developed for HPLC/UV wherein the windows were fitted with IR transparent material such as calcium fluoride or zinc selenide. Pathlengths ranged from 5 to 10 mm with cell volumes of 4 to 8 uL. Cell window materials could withstand pressures as high as 280 atm [7-101. From a spectrometric perspective, the largest pathlength would be desirable, since that would increase the signal-tonoise ratio of the spectra obtained. However, to maintain the same cell volume, the cell diameter must be decreased which leads to a drop in energy throughput and optical efficiency. Furthermore, an increase in pathlength results in a decrease in the width of the IR windows afforded by the mobile phase. Various detection limits have been reported in the literature for this flow cell approach. Jordan and Taylor [ 9 ] reported a detection limit study employing N-methylaniline as the analyte and C02 as the mobile phase under isobaric conditions. The separation was performed on a 5-!.~m phenyl derivatized silica column (25 cm x 4.6 mm, i.d.1 at an average pressure of 3000 psi and an oven temperature of 6OoC. The IR band of interest was the For each spectrum, 4 scans/file (scan aromatic C=C stretch at 1608 cm-'. time, 0.45 s/scan) were collected, and co-addition was performed over 1.370 of the Gram-Schmidt reconstructed peak. The same number of scans of background spectra (C02 only between 1650 and 1550 cm-'1 were co-added and an average peak-to-peak noise value was found over the different injected quantities. The injected minimum detectable quantity (IMDQ) of N-methylaniline with a 5-mm pathlength cell was found to be 470 ng (i.e., three times noise peak-to-peak, 3 x N 1. Identical injections using a 10-mm cell yielded an IMDQ of 360 ng.'-8orin and co-workers [ l o ] reported detection limits of 250 ng and 70 ng f o r benzonitrile and methyl benzoate, respectively, using CO, as the mobile phase. A 10 pm octadecyl bondedphase silica packed column (15 cm x 4.6 mm, i.d.1 was employed at 40'C. The detection limit was defined as twice the signal-to-noise ratio via monitoring the aromatic ring stretching vibration of benzonitrile and the carbonyl stretch of methyl benzoate. Figure 1 shows an example of an analytical scale packed column SFC/FT-IR analysis of phenols, alcohols and esters on a bonded phase, phenyl derivatized silica HPLC packed column using supercritical CO, [71. Sequential FT-IR/UV (254 nm) detection was employed. Injection of approximately 30 p q of each component yielded the Gram-Schmidt reconstructed and UV chromatographic traces. The solvent for the seven components was CC1, which eluted first as a minor component with UV, but as a major component with FT-IR detection. Chromatographic peak shapes were comparable for the two modes of detection. A nearly complete IR spectrum was obtained for each eluting component in real time. Wieboldt and Smith [ l l ] published results of the analysis of volatile citrus oil components using a HP 1082B liquid chromatograph modified for SFC. The system was limited to isobaric conditions and the W flow-cell

67

2. Z.6-DJ-TERT-BUlYLPHENOL 3 . 0 - N I TAOPHEN,OL 0 € N Z Y L flCf!?RTE 5 . BENZYL 0ENZOFITE 6 . THENOL 7 . ?HEN'IL F)CETFlTf: 8 . BENZTL FlLCOHOL

't.

0

m

c

B

I

E

lllUTEt

MOBILE PMASE

Figure 1.

-

C02

SFC/UV (254 nm) trace and Gram Schmidt reconstructed chromatogram of seven component mixture. Mobile phase is isobaric COP. Reprinted with permission from Reference 7 . Copyright 1985 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH.

was modified for IR detection by replacing the standard quartz windows with ZnSe windows. Figure 2 shows the Gram-Schmidt Reconstruction (GSR) of a 0.5 UL aliquot of a citrus oil test mixture chromatographed on a PRP1 analytical column (4.6 mm, i.d. x 15 cm; 5 Um dp) at 5OoC. The column head pressure was 1750 psi; the column back pressure was maintained at

68

6.00 $60

Figure 2.

i.43

9.88

960

RETENTION T I k (UIN) $60 sso $60

4.31

4.7s

f . 1.9_

h.84 960

Gram-Schmidt reconstructed chromatogram of citrus oil test mixture. Mobile phase: COP; flow rate: 1 mL/min; isobaric: column head pressure = 1 2 5 0 psi, back pressure maintained at 1400 psi; injection: 0.5 LIL; column: 4 mm, i.d. X 1 5 cm, 5 pm dp PRP-1 column; oven temperature: 50°C. Reprinted with permission of Reference 11. Copyright 1988 American Chemical Society

.

1400 psi; and the C02 flow rate was 1 mL/min. Figure 3 shows the spectra from the leading and trailing edges of the starred chromatographic peak in Figure 2 along with library reference spectra. This application is an important example of the value of resolving components spectrometrically when their chromatographic separation is not optimized. While these early studies demonstrated the feasibility of flow cell SFC/FT-IR, they were very restrictive insofar as the chromatography was concerned. Specifically, density programming gave rise to steeply sloping baselines and open tubular capillary columns yielded peak volumes much too small for the existing flow cells. Wieboldt et al. [12-141 have been successful in addressing both areas as will be discussed in the next section.

69

A

'ih

3500

3600

2$00

2600

,500

1600

400

LYIVENUMBER

rbW

3$00

SdW

2900

ZbOO

I500

lb00

400

MVEWMBEA

Figure 3.

O n - l i n e FT-IR s p e c t r a of ( A ) l e a d i n g e d g e and ( B ) t r a i l i n g e d g e of s t a r r e d c h r o m a t o g r a p h i c peak i n F i g u r e 2 w i t h t h e best matched s p e c t r a l s e a r c h l i b r a r y r e f e r e n c e s p e c t r a . conditions: 8 vL f l o w c e l l , 8 c m - I r e s o l u t i o n , 8 s c a n s co-added p e r f i l e , 2 . 2 7 sec t i m e r e s o l u t i o n between f i l e s . Reprinted with p e r m i s s i o n of R e f e r e n c e 11. C o p y r i g h t 1 9 8 8 American C h e m i c a l Society.

70 W i e b o l d t e t a l . 1 1 2 1 have d e s c r i b e d t h e r e q u i r e m e n t s f o r an o p t i m i z e d f l o w c e l l d e s i g n f o r c a p i l l a r y SFC. T h i s same c e l l d e s i g n i s a p p l i c a b l e t o m i c r o p a c k e d column SFC and i n terms of c h r o m a t o g r a p h i c p e r f o r m a n c e s h o u l d p e r f o r m b e t t e r b e c a u s e p e a k volumes and c e l l volumes a r e more compatible. The d i m e n s i o n s of t h e f l o w c e l l a r e 0.60 mm i . d . x 5 mm p a t h l e n g t h , which p r o v i d e a c e l l volume of 1.4 vL. The t r a n s f e r l i n e s from t h e c h r o m a t o g r a p h i c column a n d t o t h e r e s t r i c t o r were made from f u s e d The f l o w c e l l w a s d e s i g n e d w i t h a c e l l s i l i c a ( 0 . 5 m X 50 vm i.d.1. volume f i v e times a s g r e a t a s t h e t h e o r e t i c a l l y a l l o w a b l e d e t e c t o r c e l l volume f o r a 2 0 m X 100 vm i . d . c a p i l l a r y column, w i t h a p l a t e h e i g h t of 0.6 times t h e i n t e r n a l column d i a m e t e r and a k' v a l u e o f 1. The o p t i c s of t h e d e t e c t o r system d i c t a t e d t h e c e l l d i a m e t e r , t h e r e f o r e , any changes i n t h e c e l l volume c o u l d o c c u r o n l y a t t h e e x p e n s e o f d e t e c t o r p a t h l e n g t h a n d s e n s i t i v i t y ( i . e . , s h o r t e r p a t h l e n g t h , less s e n s i t i v i t y ; l o n g e r p a t h l e n g t h , less t h r o u g h p u t ) s i n c e t h e o p t i c a l p a t h l e n g t h i s d e p e n d e n t on t h e m o b i l e phase. The f l o w c e l l was o p t i m i z e d f o r SF-CO, b e c a u s e it i s t h e most w i d e l y u s e d m o b i l e p h a s e f o r SFC. A s a r e s u l t o f t h e i n c r e a s e d a b s o r p t i o n o f t h e F e r m i b a n d s i n C 0 2 , a 5-mm p a t h l e n g t h w a s f o u n d t o b e The f l o w t h e maximum p r a c t i c a l l e n g t h when w o r k i n g a t h i g h d e n s i t i e s . c e l l d e s i g n h a s a s e p a r a t e t e m p e r a t u r e c o n t r o l f o r t h e t r a n s f e r l i n e and t h e f l o w c e l l . T h e s e areas were i n d e p e n d e n t l y h e a t e d b e c a u s e a n improvement i n peak s h a p e was e x p e c t e d when t h e f l o w c e l l w a s a t a lower t e m p e r a t u r e d u e t o peak c o m p r e s s i o n a s t h e d e n s i t y o f t h e c a r r i e r f l u i d increases within t h e cell. T h i s t y p e o f i n t e r f a c e a n d method of d a t a t r e a t m e n t i s d e m o n s t r a t e d by t h e s e p a r a t i o n o f a m e t h y l e n e c h l o r i d e m i x t u r e of f o u r p e s t i c i d e s ( i . e . A l d i c a r b , methomyl, c a p t a n , a n d phenmedipham) o n a p o l y ( m e t h y l s i 1 o x a n e ) open t u b u l a r c a p i l l a r y column (10 m X 100 urn) w i s h d e n s i t y programming a t 100°C [ 1 2 ] . FT-IR s p e c t r a were r e c o r d e d a t 8 c m resolution with 8 scans co-added p e r f i l e . F i g u r e 5 shows t h e chromatogram g e n e r a t e d from a p p r o x i m a t e l y 50 ng o f e a c h component i n j e c t e d and r e c o n s t r u c t e d f r o m t h e t o t a l I R response. Figure 6 is t h e I R spectrum of Aldicarb, t h e f i r s t component e l u t e d . S e v e r a l c h e m i c a l f e a t u r e s a r e i m m e d i a t e l y a p p a r e n t from t h e spectrum. The s t r o n g band a t 1762 cm i s c a u s e d by t h e - c a r b o n y l C=O stretch. indicates The p r e s e n c e o f t h e C-0 s t r e t c h i n g band a t 1217 c m an ester f u n c t i o n a l i t y . The band a t 3460 cm-' i s d e f i n i s i v e e v i d e n c e f o r a s e c o n d a r y N-H s t r e t c h . The a d d i t i o n a l band a t 1507 c m indicates that The two b l a n k p o r t i o n s of t h e t h e n i t r o g e n i s p a r t o f a n a m i d e group. s p e c t r u m are t h e r e g i o n s i n w h i c h t h e s u p e r c r i t i c a l C 0 2 m o b i l e p h a s e absorbs a l l t h e a v a i l a b l e I R energy. T h i s f l o w c e l l SFC/FT-IR i n t e r f a c e h a s been u s e d w i t h a v a r i e t y o f mixtures. F i v e s t e r o i d s were s e p a r a t e d [15] u s i n g a c y a n o p r o p y l p o l y s i l o x a n e open t u b u l a r c a p i l l a r y column. S e q u e n t i a l flame i o n i z a t i o n d e t e c t i o n a f t e r p a s s a g e t h r o u g h t h e FT-IR f l o w c e l l y i e l d e d s i m i l a r chromatographic traces f o r t h i s mixture ( F i g u r e 7 ) . Peracetylated n i t r o g e n d e r i v a t i v e s of s e v e n m o n o s a c c h a r i d e s h a v e b e e n s e p a r a t e d o n b o t h 1.0 mm i . d . packed [ 1 6 ] and 100 p m i . d . open t u b u l a r columns 1171. M u l t i p l e d e r i v a t i v e s f o r e a c h m o n o s a c c h a r i d e were d i s t i n g u i s h a b l e by t h e v a r i o u s c a r b o n y l s t r e t c h i n g v i b r a t i o n a l modes i n t h e o n - l i n e I R s p e c t r a . B e f o r e t h i s s t u d y GC c o u p l e d w i t h f l a m e i o n i z a t i o n d e t e c t i o n o f t h e d e r i v a t i v e m i x t u r e had s u g g e s t e d a s i n g l e p r o d u c t p e r m o n o s a c c h a r i d e . Mass s p e c t r o m e t r i c d a t a (SFC/MS) were needed t o c o n f i r m t h e p r e s e n c e of t h e n i t r i l e and oxime m o i e t i e s b e c a u s e t h e n i t r i l e s t r e t c h was masked by

'

'

'

'

71 Capillary Spc Carbon d i o x i d e i s a s u i t a b l e m o b i l e p h a s e f o r SFC/FT-IR b e c a u s e o f Only t h e r e g i o n s f r o m 3475 t o 3850 cm-' f r o m 2040 t o its I R transparency. 2575 c m - ' and below 800 cm-' are c o m p l e t e l y l o s t b e c a u s e o f s t r o n g a b s o r p t i o n by C O a . I n f o r m a t i o n , however, i s p o t e n t i a l l y l o s t or r e d u c e d where i n c r e a s e d a b s o r p t i o n by C o 2 i s c a u s e d by between 1 2 0 0 a n d 1400 c m - ' , The Fermi r e s o n a n c e whose m a g n i t u d e i s a f u n c t i o n o f C02 d e n s i t y . i n c r e a s e i n t h e a b s o r p t i v i t y of t h i s r e g i o n t h a t o c c u r s a s d e n s i t y i n c r e a s e s c a u s e s s e v e r e b a s e l i n e d r i f t which may mask s o l u t e p e a k s . W i e b o l d t and Hanna [141 overcame t h i s u n d e s i r a b l e b a s e l i n e r i s e i n s u p e r c r i t i c a l f l u i d chromatograms by u s i n g Gram-Schmidt o r t h o g o n a l i z a t i o n A s shown i n F i g u r e 4 , t h e a d d i t i o n of w i t h a n augmented b a s i s v e c t o r s e t . a v e c t o r from t h e h i g h d e n s i t y r e g i o n o f t h e chromatogram o f a p a r a f f i n wax m i x t u r e d e c o n v o l u t e s t h e c h r o m a t o g r a p h i c p e a k s from t h e b a s e l i n e d r i f t c a u s e d by t h e d e n s i t y program.

1 F i g u r e 4.

A

Compensation f o r c a r b o n d i o x i d e d e n s i t y g r a d i e n t i n SFC/FT-IR ( A ) Gram-Schmidt r e c o n s t r u c t e d of p a r a f f i n mix tu re. chromatogram u s i n g 10 b a s i s v e c t o r s from s t a r t of r u n ; ( B ) same d a t a w i t h a n a d d i t i o n a l b a s i s v e c t o r t a k e n f r o m f i l e 900 (29.12 min.) added t o t h e b a s i s set. R e p r i n t e d w i t h p e r m i s s i o n from Ref. 11. C o p y r i g h t 1987 American C h e m i c a l S o c i e t y .

72

20.00 30.00 RETENTION TIME (YIN)

rZ1

Figure 5.

i 99

370 ss7 $36 MTFI POINTS

615

+o.

00

Ids,

Separation of a carbamate pesticide mixture by SFC/FT-IR. Mobile phase: C02; linear velocity, -1.4 cm/s; density program: 6.0 min. hold at 0.180 g/mL, then to 0.360 g/mL at 0.010 g/mL/min., then to 0.600 g/mL at 0.040 g/mL/min, followed by 10.0 min. hold; injection: 200 nL; split ratio: 22:l; column: 1 0 m x 100 lJm SB-Methyl-100 capillary column; Oven temperature: 100°C. Peaks: A = aldicarb, B = methomyl, C = captan, D = phenmedipham. Reprinted with permission from Ref. 13. Copyright 1989 American Chemical Society.

73

6H3 ALDICARB 3600

Figure 6 .

3ioo

2B00 2;oo WAVENUMBER

zboo

IkOO

l~oo-Boo

O n - l i n e SFC/FT-IR s p e c t r u m o f a l d i c a r b ( p e a k A i n F i g u r e 5 ) . Conditions: 8 cm-' r e s o l u t i o n ; 8 s c a n s coadded p e r f i l e ; 1 2 f i l e s coadded. R e p r i n t e d w i t h p e r m i s s i o n f r o m Ref. 1 3 . C o p y r i g h t 1989 American Chemical S o c i e t y .

C 0 2 a b s o r b a n c e and t h e C = N s t r e t c h ( o x i m e ) w a s t o o weak t o b e o b s e r v e d a t t h e l e v e l s analyzed. The u t i l i t y o f t h i s f l o w c e l l i n t e r f a c e h a s b e e n shown a l s o by t h e s e p a r a t i o n of p y r e t h r i n s [ 1 2 ] , u r e a s , b e n z a m i d e s , s u l f o n a m i d e s (181, a n d p r o p e l l a n t components [191 a n d t h e a n a l y s i s of t o b a c c o e x t r a c t s [ 3 1. Raynor e t a l . [ 2 0 1 h a v e more r e c e n t l y d e s c r i b e d t h e d e v e l o p m e n t o f a f l o w c e l l i n t e r f a c e for u s e w i t h a 50 pm i . d . o p e n t u b u l a r column. T y p i c a l peak volumes were e s t i m a t e d t o b e a p p r o x i m a t e l y 0 . 4 pL a t a s u p e r c r i t i c a l f l u i d f l o w r a t e of 1 . 5 uL/min. A n i n n e r d i a m e t e r o f 0.5 mm and a p a t h l e n g t h of 4 mm y i e l d e d a c e l l volume of 0 . 8 U L which was deemed t o b e p r a c t i c a l i n l i g h t of b o t h m e c h a n i c a l a n d s p e c t r o m e t r i c c o n s t r a i n t s . ZnSe windows ( 2 mm t h i c k ) were employed i n t h e c e l l d e s i g n w h i c h c o u l d b e p r e s s u r i z e d u p t o 300 a t m o s p h e r e s w i t h o u t f a i l u r e . In contrast t o W i e b o l d t ' s d e s i g n t h e c e l l w a s n o t t h e r m o s t a t e d . Upon c o m p a r i n g t h e FT-IR and f l a m e i o n i z a t i o n d e t e c t o r ( F I D ) r e s p o n s e it became o b v i o u s t h a t t h e band b r o a d e n i n g e f f e c t s of t h e f l o w c e l l w e r e s e r i o u s l y d e g r a d i n g t h e peak s h a p e s of t h e e l u t i n g components when g a s e o u s f l o w r a t e s of l e s s t h a n 3

74

A

I

” . Ln Ln

5

P 0 Y)

0

” *

Y

Y

1

1.8

‘I

16.8

IS.)

11.)

TIUE

IS0

100

PRESSURE

n too

tl.,

ri.8

UIN

4 0

488

ATY

B

150

400

440

440

Pn€SSune. atm.

Figure 7.

Separation of model steroid mixture ( A ) by SFC/FT-IR and ( B ) by SFC/FID (post FT-IR). Separation performed on SB-cyanopropyl25 column ( 1 0 m x 100 vm, i.d.1 at 60°C with 100% COz. S = CH2C12, 1 = progesterone, 2 = testosterone, 3 = 17hydroxyprogesterone, 4 = 11-deoxycortisol, 5 = corticosterone. Reprinted with permission from Ref. 15. Copyright 1988 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH.

75 mL/min were b e i n g u s e d , w h i c h i s d o u b l e t h e f l o w r a t e t y p i c a l l y u s e d f o r a c c e p t a b l e column e f f i c i e n c i e s . I n o r d e r t o remedy t h i s s i t u a t i o n a makeup f l u i d l i n e h a v i n g t h e same d i m e n s i o n s a s t h e column w a s i n s t a l l e d j u s t before t h e flow cell. Therefore, a restrictor a d j u s t e d t o give a gaseous f l o w r a t e of 3.5 ml/min r e s u l t s i n a column f l o w r a t e o f 1 . 7 5 ml/min. P h e n y l - e t h o x y - a c r y l a t e o l i g o m e r s were a n a l y z e d s u c c e s s f u l l y e m p l o y i n g this interface. F i g u r e 8 shows t h e FID r e s p o n s e of the o l i g o m e r separa t i o n a t 65OC w i t h p r e s s u r e programming f r o m 100 a t m t o 250 a t m a t 2 . 5 atm/min a f t e r a n i n i t i a l 1 5 min i s o b a r i c p e r i o d . I n a d d i t i o n t o the major o l i g o m e r s , a s e r i e s o f smaller p e a k s were f o u n d t o e l u t e between them.

2

3

1

F i g u r e 8.

L

SFC/FID chromatogram of p h e n y l - e t h o x y - a c r y l a t e o l i g o m e r s w i t h Conditions: 1 0 m x 50 !Jm SBFT-IR f l o w c e l l i n l i n e . B i p h e n y l - 3 0 ( 0 . 2 5 Urn f i l m ) open t u b u l a r column; C O a m o b i l e Reprinted with permission p h a s e ; 50 m s e c t i m e - s p l i t i n j e c t i o n . from R e f e r e n c e 20. C o p y r i g h t 1989 A s t e r P u b l i s h i n g Corp.

76 The GSR o b t a i n e d d u r i n g t h e same r u n i s shown i n F i g u r e 9. A proposed s t r u c t u r e o f t h e main o l i g o m e r s i s shown a b o v e t h e chromatogram i n F i g u r e 8. The I R s p e c t r a m e a s u r e d f r o m p e a k s 1, 3 and 5 ( F i g u r e 9 ) i l l u s t r a t e t h e t y p e of chemical i n f o r m a t i o n t h a t can b e o b t a i n e d from t h e ( C = O ) and 1190 cm-' (C-0) s e p a r a t e d o l i g o m e r s . A b s o r p t i o n s a t 1 7 3 5 cm-' i n each of t h e s p e c t r a are d u e t o t h e p res e nce of an ester i n t h e chai n. Absorptions due t o t h e p r e s e n c e of a r o m a t i c r i n g s i n t h e chai n o c c u r a t 1 6 1 3 cm-' a n d 1511 cm-'. The m a j o r C-0 a b s o r p t i o n of t h e e t h e r g r o u p o c c u r s a t 1150 cm-'. The r a t i o of t h e i n t e n s i t i e s of t h i s band a n d t h e e s t e r C=O band i n c r e a s e s as t h e c h a i n l e n g t h i n c r e a s e s a n d c a n b e u s e d t o d i f f e r e n t i a t e between o l i g o m e r s .

,0170 ,0138

10

0 0

20 225

30

40 450

50

Oligomer peak 1

60 Time min 675 Dala pints

,0125 ,0102

0010

i 1.

0013 !-

4cQ

F i g u r e 9.

O n - l i n e SFC/FT-IR chromatogram of p h e n y l - e t h o x y a c r y l a t e o l i g o m e r s w i t h c o r r e s p o n d i n g IR s p e c t r a of p e a k s 1, 3 a n d 5. Copyright 1989 R e p r i n t e d w i t h p e r m i s s i o n from R e f e r e n c e 20. A s t e r P u b l i s h i n g Corp.

77 Detectability

Major considerations regarding a flow cell SFC/FT-IR interface are the minimum identification limit (MIL), defined as I211 the quantity of compound required for identification by spectral interpretation o r computer search and the injected minimum detectable quantity (IMDQ), defined as [221 the quantity of material which must be injected onto the column of choice to yield an IR response three times the noise level. In contrast to solvent elimination methods, in which the number of scans can be increased to reduce spectral noise, flow cell methods provide a finite number of scans per peak since spectral acquisition is performed in real time. Maximum sensitivity will not be realized if data are taken only at the peak maximum (or at a fixed time), since peaks with higher k' values will be broader, and a smaller fraction of the total analyte will be sampled. Consequently, in order to achieve maximum sensitivity for all analytes, a method to optimize S/N of the IR spectrum generated in the flow-cell experiment must be adopted. Both from a theoretical treatment [231 and from experimental data [201 it has been shown that the maximum S/N is obtained when +1.37 standard deviations of the chromatographic peak area (-75%) are sampled. Certain spectroscopic and molecular parameters also affect detectability. For example, if the molar absorptivity of the vibrational mode is very large, detection limits will be significantly lower, provided the noise level is constant. Given fixed molar absorptivities lower frequency modes will give higher S/N than higher frequency modes. Shah and co-workers [15] have indicated that the IMDQ is approximately 2 ng f o r a strongly absorbing compound such as caffeine. Caffeine was eluted with 100% COP from a 25% cyanopropyl polysiloxane open tubular column in approximately 17.5 min with a k' of 0.97. Various quantities of caffeine (250, 50, 25, 5 and 2.5 ng) were injected and the absorbance of the intense carbonyl peak (from the on-line FT-IR spectrum) was correlated with the amount injected. For each injection, 12 files ( 4 8 scans) were coadded across the caffeine chromatographic peak to acquire the IR spectrum of greatest S/N. An S / N (peak-to-peak f50 cm-' from the reference peak) of greater than three was obtained for as little as 2.5 ng injected. Figure 10 illustrates a portion of the on-line FT-IR spectrum generated under these conditions. With spectral detectors such as FT-IR, identification limits may be more useful than detection limits. Wieboldt et al. [12] determined that the MIL for methyl palmitate was 10 ng on column (10 m x 100 pm, 0.50 prn film of polydimethylsiloxane).

Other Mobile Phases

Practically all SFWFT-IR ivestigations have dealt with 100% CO,. Novotny and French [24] studied supercritical xenon with a 20 m x 150 pm fused silica open tubular column with a 1 pL volume flow cell (1 mm pathlength). They found that xenon was an attractive mobile phase because it was completely transparent in the IR region but also had similar solvating properties to supercritical CO,. Infrared spectra of dimethylterephthalate [20] separated with supercritical C02 and xenon are shown in Figure 11. Xenon affords several advantages which include the following: (a) no spectral subtraction of the background is required:

78

m 0)

0 0 (0

b

0 0

r.

Ln W O

uo 2 ' c m

gx

:: (I:'

m 4-,

0 0 0 0 0

Figure 10.

On-line SFC/FT-IR spectrum of caffeine (2.5 ng injected). 12 SFC conditions: SBcoadded files, 4 scans/file, 1 file/sec. cyanopropyl-25 column (10 m x 100 vm, i.d.1 at 6OoC with 100% CO,; linear pressure programming (100-175 atm/l5 min., 175-400 atm/5 min.). Reprinted with permission from Reference 15. Copyright 1988 Friedr. Vieweg L Sohn Verlagsgesellschaft mbH.

(b) OH-stretching vibrations in the 3500 cm-' region and aromatic CHdeformations below 800 cm-' are detected; and, (c) spectra measured in xenon more closely match available library spectra than spectra obtained in CO,. As far as other fluids are concerned, supercritical nitrous oxide has been shown to be unsuitable for flow cell SFC/FT-IR [25]; whereas, supercrktical sulfur hexafluoride affords IR transparency solely in the region (i.e. CH-stretch). Certain fluorocarbons possess rather 3000 cm moderate critical pressures and temperatures. The IR windows of CO, and Freon 23 (trifluorochloromethane) complement each other. The use of CO, as the mobile phase precludes examination of the IR region where 0-H vibrational modes are expected to appear (aproximately 3600 cm ' ) . This region can be monitored effectively by the use of a supercritical Freon 23 mobile phase. Due to the lower solvent strength of Freon 23, an increase

'

79

b

.n 0.

N

1

F i g u r e 11.

O n - l i n e FT-IR s p e c t r a of d i m e t h y l t e r e p h t h a l a t e m e a s u r e d i n R e p r i n t e d w i t h p e r m i s s i o n o f K. D. ( a ) xenon a n d ( b ) CO,. Bartle.

i n p r e s s u r e w a s n e e d e d t o e l u t e t h e compounds i n a s h o r t t i m e p e r i o d : however, t h e c h r o m a t o g r a p h i c r e s o l u t i o n o b t a i n e d w a s c o m p a r a b l e t o p r e v i o u s s e p a r a t i o n s o b t a i n e d w i t h s u p e r c r i t i c a l C02. A c o m p a r i s o n of I R s p e c t r a f r o m s e p a r a t i o n s 1261 of f r e e f a t t y a c i d s ( F F A S ) i n b o t h CO, a n d F r e o n 2 3 m o b i l e p h a s e s i s shown i n F i g u r e 1 2 . T h e s e o n - l i n e s p e c t r a d e m o n s t r a t e t h e c o m p l e m e n t a r y n a t u r e o f t h e r e s p e c t i v e mobile p h a s e s .

80 In contrast to the FFA spectrum obtained in COz, the 0 - H stretching band obtained in Freon 23 is fairly intense, and thus the compounds are easily identified as acids, not esters. The remainder of the IR region is practically obscured by Freon 23 absorbance except f o r the carbonyl stretching region where again the doublet suggestive of a dimer-monomer mixture is found. Since the 0-H stretching mode for the dimer is expected to be broadened and shifted, the intensity of the sharp band at 3500 cm-' should be a qualitative measure of the monomer content.

FREON 23

c02

r

1

4000

Figure 12.

3370

1

1

1

I400 2740 2110 WAVtWUMMllS

1

860

Comparison of IR spectra of an FFA measured in supercritical COz and supercritical Freon 23. Reprinted with permission of Reference 26. Copyright 1986 Preston Publications.

The addition of polar modifiers to increase the solvent strength of or to deactivate the stationary phase reduces the applicability of online FT-IR for obtaining identifiable spectra. Wieboldt et al. [12] have reported that 1% hexanol blocks large regions of the mid-IR spectrum. Jordan and Taylor [91 showed that with a cell of 5-mm pathlength, the addition of as little as 0.2% methanol reduced she accessible IR windows to 3400-2900 cm-’, 2800-2600 cm-’, 2100-1500 cm and 1200-1100 cm Morin and co-workers 1271, using a cell of 10-mm pathlength and 8-uL volume, studied the IH transparency of modified CO, by adding various polar solvents under subcritical conditions. Although the addition of polar solvents caused a severe l o s s of available IR windows, specific frequencies could still be selectively monitored. For example, the carbonyl and carbon-carbon double bond stretching regions always remained transparent with methanol and acetonitrile as modifiers. The use of LD,CN as-a modifier permitted monitoring of the C-H stretching region (2900-3100 cm ’ ) , and when less than 9% CD,CN was added, the aliphatic CH2 and CH3 bending region (1600-1400 cm-’) could also be monitored. Since the introduction of the first commercially available SFC system in the early 1980s, the development of the hyphenated flow cell SFC/FT-IR The technique has attained a high level of sophistication [ 2 8 1 . advantages of the flow cell approach include the following: a) the entire effluent stream is monitored so all sample components are detected intact; bl other detectors such as the mass spectrometer may be placed in series after the FT-IR detection; and c) the interface is mechanically simple. The disadvantages of the flow cell approach include the following: a ) separate spectral libraries may be required for various mobile phases becasuse complete spectra are not feasible; b) usable mobile phases are limited when identifiable spectra are desired; and c) lower sensitivity can be expected in most cases. COz

’.

Acknowledgment E. M. Calvey gratefully acknowledges a long-term training appointment from the U. S . Department of Health and Human Services. The financial assistance of the U. S . Environmental Protection Agency is deeply appreciated.

REFERENCES

1 D. J. Bornhop and J. G . Wangsgaard, J. Chromatogr. Sci., 27 (1989) 293. 2 B. E. Richter, D. J. Bornhop, J. T. Swanson, J. G . Wangsgaard and M. R. Andersen, J. Chromatogr. Sci., 27 (1989) 303. 3 L. T. Taylor and E. M. Calvey, Chem. Rev., 89 ( 1 9 8 9 ) 321. 4 K. Jinno, Chromatographia, 23 (1987) 55. 5 K. A. Forbes, J. F. Vecchiarelli, P. C. Vaden and R. M. Barnes, Anal. Chem., 62 (1990) 2033. 6 R. D. Smith and H. R. Udseth, Anal. Chem., 59 ( 1 9 8 7 ) 13; E. C. Huang, T. Wachs, J. J. Conboy and J. D. Henion, Anal. Chem. 6 2 (1990) 713a. 7 K. H. Shafer and P. R. Griffiths, Anal. Chem., 55 (1983) 1939.

82 C. Johnson, J. W. Jordan, L. T. Taylor and D. W. Vidrine, Chromatographia, 2 0 ( 1 9 8 5 ) 717; M. E. Hughes and J. L. Fasching, J. Chromatogr. Sci., 2 4 ( 1 9 8 6 ) 5 3 5 . 9 J. W. Jordan and L. T. Taylor, J. Chromatogr. Sci., 2 4 ( 1 9 8 6 ) 82. 10 P. Morin, M. Caude, H. Richard and R . Rossett, Chromatographia, 2 1 8

( 1 9 8 6 ) 523.

11 R. C. Wieboldt and J. A. Smith, A.C.S.

Symposium Series, 3 6 6 ( 1 9 8 8 1

229. 12

R. C. Wieboldt, G. E. Adams and D. W. Later, Anal. Chem., 6 0 ( 1 9 8 8 )

2422. 1 3 R . C. Wieboldt, Nicolet FT-IR Application Note AN-8705, March 1 9 8 7 . 1 4 R . C. Wieboldt and D. A. Hanna, Anal. Chem., 5 9 ( 1 9 8 7 ) 1255. 1 5 S. Shah, M. Ashraf-Khorassani and L. T. Taylor, Chromatographia, 2 5 ( 1 9 8 8 ) 631. 1 6 E. M. Calvey, L. T. Taylor and J. K. Palmer, High Resolut. Chromatogr. Chromatogr. Comm., 11 ( 1 9 8 8 ) 739. 1 7 E. M. Calvey, J. A. G. Roach, L. T. Taylor and J. K. Palmer, J. Microcol. Sep., 1 ( 1 9 8 9 ) 294. 1 8 S . Shah and L. T. Taylor, J. High Resolut. Chromatogr., 1 2 ( 1 9 8 9 ) 5 9 9 . 1 9 M. Ashraf-Khorassani and L. T. Taylor, Anal. Chem., 6 1 ( 1 9 8 9 ) 145. 20 M. W. Raynor, A. A. Clifford, K. D. Bartle, C. Reyner, A. Williams and B. W. Cook, J. Microcol. Sep., 1 ( 1 9 8 9 ) 101. 2 1 K. H. Shafer, S. L. Pentoney and P. R. Griffiths, Anal. Chem., 5 8 ( 1 9 8 6 ) 58. 22 P. R. Griffiths, In Fourier Transform Infrared Spectroscopy, J. R. Ferraro and L. J. Basile (eds.), Academic Press, New York, 1 9 7 8 , V o l . 1, 143. 23 C. C. Johnson and L. T. Taylor, Anal. Chem., 5 6 ( 1 9 8 4 ) 2642. 2 4 S . B. French and M. Novotny, Anal. Chem., 5 8 ( 1 9 8 6 ) 1 6 4 . 25 M. Ashraf-Khorassani, L. T. Taylor and P. Zimmerman, Anal. Chem., 6 2 ( 1 9 9 0 ) 1177. 26 J. W. Hellgeth, J. W. Jordan, L. T. Taylor and M. Ashraf-Khorassani, J. Chromatogr. Sci., 24 ( 1 9 8 6 1 183. 27 P. Morin, M. Caude and R . Rosset, J. Chromatogr, 407 ( 1 9 8 7 ) 8 7 . 2 8 K. D. Bartle, M. hi. Raynor, A. A. Clifford, I. L. Davies, J. P .

Kithinji, G. F. Shilstone, J. M. Chalmers and B. W. Cook, J. Chromatogr. Sci, 27 ( 1 9 8 9 ) 283.

K. Jinno (Ed.), Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction Journal of Chromatography Library Series, Vol. 53 0 1992 Elsevier Science Publishers B.V. All rights resewed.

83

Chapter 6 SFCIFT-IR MEASUREMENTS INVOLVING ELIMINATION OF THE MOBILE PHASE Peter R. Griffiths, Kelly L. Norton and Anthony S. Bonanno Department of Chemistry, University of Idaho, Moscow, ID 83843, U.S.A, INTRODUCTION Two distinct ways of implementing the interface between chromatographs and Fourier transform infrared (FT-IR) spectrometers have been described. In the first the column effluent is passed through a suitable flow-cell while in the second the mobile phase is continuously eliminated and the eluites are deposited on an appropriate substrate prior to the measurement of the infrared spectrum. Both of these approaches have been applied to the SFC/FT-IR interface.

DRAWBACKS OF SFC/lT-IR USING FLOW-CELLS The feasibility of measuring the infrared spectra of species separated by capillary SFC as the eluates pass through a high-pressure flow-cell has been demonstrated by several groups [l-1 11 and is discussed in this book in the chapter by Taylor [12]. Although spectra of injected quantities of 10 to 100 ng can be obtained, on-line SFC/FT-IR performed in this way has several disadvantages, most importantly that certain regions of the spectrum are lost because of absorption by the mobile phase (unless xenon is used [4]). The fraction of the spectrum that is lost depends on the complexity, polarity and symmetry of the molecules comprising the mobile phase. The most common mobile phase used for SFC is, of course, carbon dioxide. This molecule is so simple that it might be expected that only a very small percentage of the infrared spectrum will be lost because of absorption by the mobile phase. A detailed look at the spectrum of CO,, however, shows that this is not the case. CO, has two infrared-active fundamental vibrational modes, the antisymmetric stretching band that is centered at approximately 2350 cm-' (v,) and the bending mode near 668 cm-' (vJ. The symmetry-forbidden symmetric stretch (v,) is centered at about 1390 cm-'. Although one might predict that only those regions around the two strongly infrared-active bands will be obscured, the use of long pathlength flow cells increases the absorption by weak bands in the CO, spectrum. Most of the weak bands are overtones and combinations, but it is even possible that the symmetric stretching fundamental would become weakly allowed if the CO, molecule is slightly bent at high pressure. The intensity of v, is enhanced by Fermi resonance with 2v, [13], so that two bands at 1390 and 1285 cm" are observed in the spectrum of liquid or supercritical carbon dioxide. The intensity of this doublet appears to increase with density in a nonlinear manner [I] (see Figure l), lending some credence to the argument that the 0 - C - 0 bond of CO, is slightly bent at the densities required for SFC. These two modes also interact with v, to generate a remarkably intense doublet, (v, + v,) and (v, 2 4 , that obscures another 200 cm"-wide region of the spectrum around 3600 cm-'. The intensity of this doublet can be seen by examining Figure 2. As a result, at least three regions of the spectrum are lost in SFCIFT-IR spectra obtained using a flow-cell, so that the characteristic vibrational modes of several important functional groups cannot be observed. These include the 0 - H stretch (3800 - 3500 cm-'), C = N stretch (2200 - 2100 cm"), C-C1 stretch (800 600 cm-I), and those aromatic C-H out-of-plane bending modes that absorb below 750 cm-'. (The latter vibrational modes are particularly important for determining the substitution patterns of aryl rings.) In addition, the doublet due to v1 and 2v3, results in a pressure dependent loss of signal-to-

+

84

noise ratio (SNR) in the spectral region between 1400 and 1280 cm-’ so that spectra may become unacceptably noisy in this region when the pressure of C02 exceeds 250 atm. This already marginal situation deteriorates when SFC separations are carried out using a packed column, as it is common practice for organic modifiers to be added to C02 to enhance the

1201 W

4400

4000

I

3600

3300

2800

2400

2000

1600

12CO

WAVENUMBERS

Figure 1. Transmittance spectrum of supercritical C02 at 1200 psi and 50°C measured in a 1-mm cell. (Reproduced from Ref. 1 by permission of the American Chemical Society, O1983.)

900 PSI

-

4

ll0OPSI

1300 PSI

1 5 0 0 PSI

1700 PSI

7J”

1

c(

2600

2200

1800

1400

WAVENUMBERS

Figure 2. The effect of increasing pressure on the transmittance spectrum of supercritical C02 at 50°C in a 1-mm cell. (Reproduced from Ref. 1 by permission of the American Chemical Society, O1983.)

85 elution of polar compounds. For example, if methanol is added to C Q at a concentration of 5%, most of the useful infrared spectrum is lost [14]. Because these cosolvents are generally polar andhave many more vibrational modes than CO,, they usually obscure a larger fraction of the spectrum. The transmittance can sometimes be increased to a useful level by reducing the pathlength of the cell substantially, but this compromises sensitivity due to the decreased optical thickness of each solute. Even when SFCIFT-IR spectra are measured with an adequate SNR, another problem arises in that the position of absorption bands in the solute spectra vary slightly with the pressure and temperature of the mobile phase [15]. As a result, spectral searching of SFCIFT-IR spectra obtained using a flow-cell will only yield unambiguous results if the reference spectrum is measured with the analyte dissolved in the same fluid, at same pressure and temperature, that is used for the analytical separation.

MOBILE PHASE ELIMINATION Background The problems discussed above may be circumvented by eliminating the mobile phase before measuring the spectra of the eluites. This task is greatly simplified by the fact that most SFC mobile phases are gaseous at STP and can, therefore, be pumped away immediately after emerging from the restrictor. It then becomes simply a matter of trapping the solutes in such a way that their spectra can be readily measured. This approach was first demonstrated by Shafer ef al. [14], who separated mixtures of quinones on a I-mm i.d. silica packed column using 2% CH,OH:98% CO, as the mobile phase. Each eluite was deposited on a moving glass plate on which a layer of powdered KCl had been laid down from a methanol slurry. The substrate was then moved into the focus of the beam from an FTIR spectrometer and the diffuse reflectance @R) spectrum of each analyte was measured. Later work by the same research group [ 16-18] and others [ 19-20] showed that it was not necessary to use a powdered substrate and that superior results could be obtained simply by depositing the eluites on a moving ZnSe substrate and measuring their transmittance spectra. Theory

The key to obtaining the maximum sensitivity in this work is to deposit the analytes in such a manner that they occupy as small an area as possible in order to build up their thickness (and hence their absorbance). Pentoney ef al. [16,17] showed that it was possible to deposit the eluites from a capillary SFC column in a spot less than 150 pm in diameter. Spectra of each of the analytes trapped in this manner could then be measured using an FT-IR microscope. Similar "direct deposition" @D) techniques have been developed for GCIFT-IR [ 18,21-241 and HPLCIFT-IR [25341 measurements. A simplistic (but only partially accurate) way of understanding why the analyte should be deposited in such a small area can be seen by examining the Beer-Lambert law relating the absorbance, A@), of a pure material at any wavenumber, Y, to its absorptivity at that wavenumber, a(v) (cm-') and optical thickness, b (cm): A ( v ) = a(v)b

Dl

A given quantity of analyte, q (grams), deposited as a cylinder of cross-sectional area, A, (cm') and height, b will occupy a volume, V (cm3), given by:

86

V

= q/p =

A,b

where p is the density (g ~ m - ~ By ) . substituting for b in eq. [ 11: A(v)

=

~

4v)q A, p

,

it can be readily seen that the absorbance varies inversely with the sample area. The problem with this approach is that it is necessary to stop down the beam to a diameter equal to the diameter of the deposit in order to accrue any benefit from this gain in absorbance. It might be thought that the energy loss (and hence the increase in noise on the baseline of a ratiorecorded spectrum) introduced by vignetting the beam will exactly offset the increase in absorbance gained by reducing the diameter of the deposit. By a careful analysis of the parameters governing the signal-to-noise ratio of spectra measured on a Fourier transform spectrometer, however, a way of achieving an increase in sensitivity of a DD SFC/FT-IR measurement by reducing the diameter of the deposit can be derived. The SNR of an infrared spectrum measured using a Fourier transform spectrometer is given in theory by the equation [4]: SNR =

U J T ) A v D' 8 tyr

C

141

ALP

where UJT) is the spectral energy density of the source (in units of W / sr cm2 cm"), Av is the resolution of the measurement (cm-I), D' is the specific detectivity of the detector (W-' H z " ~cm), 0 is the optical throughput (cm' sr), t is the measurement time (sec), A, is the detector area (cm'), and is the efficiency of the optics. The noise on the baseline of a spectrum plotted in absorbance is therefore:

Noise =

ln(10) AAP Uv(T) Av D' 8

tlP

C

Thus the SNR of the spectrum of a cylindrical deposit of a compound from an SFC is simply the ratio of equations [31 and 151: SNR =

a(v) q UJT) Av D' 8 tin

[GI

ln(10) A,UL A, P

The limiting optical throughput, 8,of this measurement is determined by the sampie area, A,, and the solid angle subtended by the beam condensing optics at the sample. If the solid angle of the beam at the sample is 0, steradians (sr):

e = n, A,. then the signal-to-noise ratio of a DD SFC/FT-IR spectrum is:

171

87

Since A, is not seen in equation [8], it would appear that the SNR of the measurement is independent of sample area, contradicting the conclusion drawn earlier in this section. However, for optimal microsampling, the solid angle of the beam should be equally large at the sample and at the detector. If the focal lengths of the mirrors focusing the beam on the sample and the detector are identical, as they often are in practice, the area of the detector must be equal to the area of the sample in a throughput-matched system. The sensitivity of the measurement can therefore be optimized through the use of a detector that is no larger than the sample, provided that all the radiation transmitted by the sample is focused on the detector. Since we have seen that the sample can be deposited as a 100-pm diameter spot, a detector that is 100-pm in linear dimension should therefore be installed for optimum SNR. Implementation of DD SFC/Fl'-IR It may well be asked how the various parameters in equation [8] affect the practice of DD SFCIFT-IR. The effects of several of them are readily understood. Even inexperienced workers will recognize that increasing the amount of sample deposited or the efficiency of the optics will result in greater SNR. The SNR will, of course, be improved by increasing the value of any parameter in the numerator of equation [8] or decreasing the value of the parameters in its denominator. However, while the effect of changing some of the other parameters appearing in this equation may appear to be obvious, a few practical warnings should be issued. For example, from equation (81 i[ would appear that the source temperature should be as high as possible, since U , r ) is largely governed by the Planck equation. Most infrared sources operate at temperatures between 1400 and 1700 K; emission profiles of a blackbody source at these two temperatures are shown in Figure 3. The advantage of operating at the higher temperature is obvious from this figure, but the downside to high temperature operation is that the source will burn out

0.003

0.002

0.001

0

0

2000

W av enu m b e r

'

4000

6000

[ c m- )

Figure 3. Spectral energy density from a blackbody source at temperatures of 1400 and 1700 K.

88 Table 1: Spectral range and sensitivity of MCT detectors.

DIE Narrow-range

Low wavenumber cutoff 5

750 cnil

D' 3 x 1O1O

Intermediate-range

650 - 600 c d

2 x loLo

Wide-range

= 450 cm"

sx

109

sooner. As a result, most manufacturers of FT-IR spectrometers do not recommend that users of their instruments adjust the source to improve the SNR. Measurements should also be carried out at a resolution that is as low as possible (large Av). If, however, the Av is larger than about one-half of the full-width at half height (FWHH) of the narrowest band in the spectrum, the measured absorbance will be significantly less than its true value. Since bandwidths in the spectra of organic compounds can vary greatly from band to band, the relative intensities of bands can change if the spectrum is not measured at adequate resolution. This can in turn affect the success of spectral library searching. From equation [8], it is apparent that the detector should be as sensitive as possible (high D3. Most standard FT-IR spectrometers are equipped with a deuterated triglycine sulfate (DTGS) pyroelectric bolometer that operates at room temperature. The D' of pyroelectric bolometers is quite low, however, and much better sensitivity is attained if the DTGS detector is replaced by a liquid-nitrogen-cooled mercury cadmium telluride (MCT) photoconductive detector. Several types of MCT detectors are commercially available; they are usually classified as narrow-range, intermediate-range, and wide-range. The spectral range and sensitivity of these detectors are summarized in Table 1. It can be seen that the greatest sensitivity is achieved with a narrow-range MCT detector, although the spectrum can not be observed below about 750 cm". This excludes the observation of several very important bands, including the C-CI stretch and some of the aromatic CH out-of-plane bending modes. Since there are few characteristic bands in the infrared spectra of organic compounds between 600 and 450 cm-', the sixfold loss in SNR found by switching to a widerange MCT detector seems too great a price to pay for the increased spectral range. However, if an intermediate-range MCT detector is employed, the loss in sensitivity with respect to the narrowrange detector is quite small and the important bands absorbing between 800 and 600 cm" can be observerd. The next parameter in equation [8] that should be considered is the solid angle of the beam, Q,. For a given optical throughput, the greater is Q,, the smaller is the area of the beam at its focus, and hence the greater is the advantage of depositing the SFC eluites in a small area. In most standard FT-IR spectrometers, it is common to use short focal-length paraboloidal or ellipsoidal mirrors to reduce the beam diameter at the detector to about 1 mm. Microscope objectives increase n, and thus create an even smaller focus, especially when small samples are being observed. As noted above, most of the early DD SFC/FT-IR measurements were performed by depositing the eluites as small spots on a suitable window which was transferred to an FT-IR microscope after the chromatography had been completed. Measurements of this type will be referred to in this chapter as osf-Z-linemeasurements [35] (see Figure 4). The optics required for on-line DD SFCIFT-IR are analogous to those of a rudimentary microscope, although they naturally lack much of the flexibility that is built into a good general-purpose FT-IR microscope. Measurements made when the eluites pass through the focused infrared beam immediately after deposition, will be referred to as on-line measurements (see Figure 5).

89

Figure 4. Schematic representation of off-line SFCIFT-IR. After deposition of the eluites onto a moving ZnSe substrate, the window is moved to the focus of a stand-alone FT-IR microscope where the spectrum of each spot is measured with the plate stationary.

Figure 5. Schematic representation of on-line SFC/FT-IR. Each eluite passes through the IR beam a few seconds after deposition.

90 The final parameter in equation [8] that is of practical importance for DD SFC/FT-IR is the measurement time, t. In any hyphenated technique, the spectral measurement should ideally be made in a time that is no longer than the FWHH of the chromatographic peak. This is equally true for DD SFCIFT-IR measurements made in real time, i.e. where the deposited spots are passed through the infrared beam immediately after deposition. However, if the SNR of the spectrum measured in this way is not high enough to permit an unambiguous identification of the analyte, the spot may be returned to the beam after the chromatography is complete so that the spectrum may be remeasured with extended signal-averaging. This capability was shown recently for DD GC/FT-IR measurements by Bourne el al. [24]. In this report, the FWHH of the narrowest GC peaks was about 4 seconds. Real-time spectra obtained when 50 pg of several analytes were injected into the chromatograph were barely above the detection limit, and yet useful spectra could be obtained after only one-minute’s post-run signal averaging (an approximately five-fold improvement in SNR). Because SFC peaks are usually wider than GC peaks, the advantage of post-run signal-averaging is not as great for DD SFC/FT-IR as it is for DD GCIFT-IR measurements. For example, if the FWHH of an SFC peak is 20 s, interferograms must be co-added for more than 30 minutes to achieve an improvement in SNR by an order of magnitude. For real-time GC/FT-IR spectra measured using a light-pipe, it was shown that the optimum compromise between maintenance of chromatographic resolution and achieving the maximum SNR occurs when the measurement time is equal to the FWHH of the GC peak [36]. The situation is little different in DD SFCIFT-IR. If an eluite is deposited as a spot (with an approximately Gaussian profile) with a FWHH of W pm, and the FWHH of a chromatographic peak is tl,, sec, the substrate should be moved at a velocity of W/tllZpm/s to yield the greatest SNR without significant loss of chromatographic resolution. A typical speed for translation of the window for DD SFC/FT-IR is therefore about 300 pmlmin. Thus for a separation taking one hour to complete, the entire chromatogram may be deposited in a length of little less than 2 cm. Assuming that the center of one trace should be separated from the center of an adjacent trace by 200 pm, it is possible (in principle, at least) to characterize almost 200 chromatograms on a single 20 x 50 mm window. OFF-LINE DD SFC/Fl’-IR MEASUREMENTS After the original experiments of Shafer er al. [14] for which the substrate was powdered KCI and diffuse reflectance spectra were measured, most DD SFC/FT-IR measurements have been accomplished by simply depositing the eluites on a ZnSe window. Most early depositions were performed with the substrate stationary [16,37-391. These experiments allowed the shape and dimensions of the deposit to be characterized using a standard FT-IR microscope, and demonstrated that it was possible to deposit eluites from a capillary SFC column as spots approximately 100-pm in diameter. After the initial work showing the feasibility of off-line capillary SFC/FT-IR measurements using a stationary substrate [37], Bartle’s group at the University of Leeds, in collaboration with scientists working at the ICI Research Laboratories, demonstrated the feasibility of characterizing antioxidants used as polyolefin additives by a similar technique [38] (see figures 6 and 7). In this study, the deposition step was carried out at the University of Leeds and the spectra were measured at an ICI laboratory located about 100 miles away. Besides showing that high quality SFC/FT-IR spectra of large molecules could be obtained using the direct deposition approach, Bartle’s work demonstrated that the vapor pressure of many SFC eluites is sufficiently low that the deposits last for several days on the substrate without being lost through vaporization. Because so many DD SFC/FT-IR measurements reported in the literature to date have involved the use of capillary SFC separations, the feasibility of applying similar methodology to packed column separations using modified CO, as the mobile phase has been questioned. This doubt is indeed justified in certain instances as it is a difficult matter to deposit eluites from conventional

91

n

12

18

1

b 150

lb

2b

150

3b

200

5b Time (minl 250 Pressure l a f m ) 4'0

6b

8b

70

60

350

300

Figure 6. Supercritical fluid chromatogram of 21 common polymer additives separated on a 10 m x 50 pm i.d. fused-silica capillary column, with a cross-linked polydimethylsiloxane stationary phase at 140°C. The CO, mobile phase was programmed from 150 to 350 atm at 3 atmlmin after an initial 12 minute isobaric period. (Reproduced from Ref. 38 by permission of the American

+GE

3590

3iao

2j70

2360

I350

iho

ii30

i20

310

WRVENUMBERS

Figure 7. (above) Off-line SFCm-IR spectra ot lrganox 1010 (Peak No. 21 in Figure 6 ) deposited on a stationary KBr window. (below) Reference spectrum of Irganox 1010 deposited on a KBr window. (Reproduced from Ref. 38 by permission of the American Chemical Society, "1988.)

B: C02/0.1% H&I

Time (min) Pressure (psi) (atm)

0

2000 136

5

3000 204

10 4000 272

15 50005500 340 374

20

25

Figure 8. Supercritical fluid chromatograms of three sulfanilamides (sulfisoxazole, sulfapyrazine, and methoxybenzoylsulfamethazine) separated on a Deltabond Cyano packed column (1 x 50 mm), measured with UV detection at 271 nm. (A) 100% CO,, (El) CO,/O.l% H,O, (C) C0,/2% CH,OH programmed from 2000 to 5500 psi at 200 psilmin at a temperature of 70°C. For (A) and (B) each component was injected separately and the chromatograms were superimposed for clarity. 1 mm, the volume flow rate is reduced by a factor of (4.6)’, i.e. 20, without changing the linear (4.6-mm i.d.) columns as small spots on any substrate because the high flow rate of gas from the restrictor disrupts the deposition process. By reducing the diameter of the column from 4.6 mm tovelocity in the column. This flow rate is much more amenable to DD SFC/FT-IR. Recently, we have demonstrated the feasibility of depositing sulfanilamides that have been separated on 1-mm i.d. packed columns using both pure and modified CO, and some typical results are illustrated below. Sulfanilamides are strongly polar materials that are insoluble in hexane and only sparingly soluble in water. When the separation of three of these sulfanilamides was attempted by capillary SFC on 5%-phenyl, 95%-methyl polysiloxane and 30%-biphenyl, 70%-methyl polysiloxane columns using CO, as the mobile phase, the peaks eluted as very broad (FWHH > 5 minutes), tailing peaks, raising the possibility that the poor SFC peak shape was caused by the low solubility of each compound in CO,. When the separation was carried out on a packed column (Deltabond-CN, Keystone Scientific), the peak widths were still excessive, see Figure 8A, again possibly indicating the low solubility of the analytes in supercritical CO,. Addition of 0.1 % of water to the CO, resulted in a dramatic sharpening of the peaks, as shown in Figure 8B, presumably due to deactivation of sites on the stationary phase that had not been end-capped during the preparation of the column. The water appeared to be strongly bound to the active sites; switching back to a mobile phase of pure CO, did not result in a significant change in peak shape, even after fluid was passed through the column for several hours. Even better peak shapes can be achieved with 2%-CH,OH, 98%-CO,, as can be seen in Figure 8C. The retention of all three components is decreased by the addition of methanol, suggesting a greater interaction between the sulfanilamides and the methanol cosolvent than between these analytes and water. DD SFCIFT-IR spectra of the three sulfanilamides were measured in an off-line mode. The restrictor was held about 100 p n above the surface of a ZnSe window that was translated at a speed

93

120 ng from SFC

A

4000

3000

2000

1000

Wavenumber Figure 9. (A) Off line SFC/FT-IR spectrum of 120 ng of sulfisoxazole (peak 1 in Figure 8B) depositied from C02/0.1% H,O onto a moving ZnSe window. (J3) Reference spectrum of sulfisoxazole prepared as KBr disk. of 200 pmlmin; the window was not cooled and the interface was not evacuated. After the chromatography was complete, the substrate was transferred to an FT-IR microscope. High quality transmission spectra were obtained with the window stationary (see Figure 9A). It has been noted Table 2: The results of searching the DD SFCIFT-XR spectrum of sulfisoxazole against the Georgia State Crime Laboratory Library.

Hit #

Comuound

1

*

2

*

3 4

* *

5

*

6 7

*

8 9 10

*

Sulfisoxazole in KBr Sulfamethoxamle in KBr Sulfisoxazole in KBr Sulfacetamide in KBr Sulfabenzamide in KBr Cannabigerol in KBr Sulfaquinoxaline in KBr Metaproterenol Sulfate in KBr Polythiazide in KBr Metaproterenol in KBr indicates library entries that are sulfanilamides

lIQl 0.410 0.427 0.438 0.482 0.510 0.537 0.537 0.549 0.549 0.549

94

that GCIFT-IR spectra obtained by direct deposition match well with the corresponding reference spectra in standard condensed phase databases [24]. It therefore comes as no surprise that the same is true for DD SFC/FT-IR. When the sulfanilamide spectra were searched against the Georgia State Crime Laboratory library of infrared spectra of commonly abused drugs, the correct match was obtained. The results of searching the sulfisoxazole spectrum shown in Figure 9A are reproduced in Table 2, and the reference spectrum is shown in Figure 9B. In this table, HQI refers to the hit quality index, which is proportional to the Euclidean distance between the spectrum of the analyte and each reference spectrum. The smaller the HQI, the better the spectral match. It can be seen that the reference spectrum of sulfisoxazole is indeed the best match and that all of the top "hits" were other sulfanilamides. In summary, off-line DD SFCIFT-IR spectra of components eluting from either packed or capillary SFC columns can be measured using the techniques described in this section. Any common mobile phase can be used for the separation. Spectra of all peaks in the chromatogram can be obtained by moving the window under the restrictor at a slow, constant velocity. The greatest sensitivity is always attained when the eluites are deposited as spots of very small diameter (< 100 pm) and when spectra are measured using a conventional FT-IR microscope. If the widths of the peaks increase for the more strongly retained components, it is possible to program the speed at which the window is translated so that the spot size remains approximately constant.

ON-LINEDD SFC/FT-IR MEASUREMENTS The logical development of this approach is an on-line measurement in which the deposited eluites are moved continuously through the infrared beam. A DD GC/FT-IR interface by which spectra can be acquired in real-time was recently described by Bourne el al. [24]; this instrument is marketed by the Digilab Division of Bio-Rad Laboratories as the Tracer. The optics of this system are shown in Figure 10. The beam from a rapid-scanning FT-IR spectrometer is focused onto a ZnSe window by a short focal-length ellipsoidal mirror. The transmitted beam is collected by a

Figure 10. Optical diagram of Bio-Rad Tracer interface modified for SFC/FT-IR.

95

Gram-Schmidt

Functional Group (1800-1680 crn-l)

I

20

I

I

30

I

40

I

50

60

Time (minutes)

Figure 11. (A) Flame ionization chromatogram of silicone grease from contaminated valve separated on a 10-m x 100-pm biphenyl column using CO, programmed from 2000 to 4000 psi after a 5minute hold. @) Gram-Schmidt reconstructed chromatogram of an injection of 60 nL of a 0.025% solution of acenaphthenequinone in CH,Cl, separated in the presence of a silicone grease contaminant. (C) Reconstructed chromatogram obtained by integrating the absorbance of each spectrum between 1800 and 1680 cm". Schwartzchild microscope objective and focused at an adjustable aperture by a field lens. The size of this aperture determines the effective area of the beam at the ZnSe substrate. The fraction of the beam that is transmitted by the aperture stop is refocused onto the element of an MCT detector by a second Schwartzchild objective. Since the sample diameter is 100 pm and the two microscope objectives are identical, the size of the detector element must be equal to the s u e of the deposited spot, i.e. 100 pm, to achieve the maximum sensitivity. We have modified the prototype version of the Tracer for DD SFC/FT-IR measurements. The modifications were relatively simple. The Tracer employs 50-fim i.d. fused-silica tubing to transfer GC effluent from the column to the window. For SFC this transfer line is replaced by a restrictor which acts as both transfer line and deposition tip. An integral restrictor [40] was used for all data shown in this chapter. The substrate could not be cooled with liquid nitrogen since CO, emerging from the restrictor would condense on its surface. For the data shown later in this section, the window was cooled to -10°C using a dry-icelmethanol mixture. In light of the increased width of SFC peaks, the speed at which the substrate was moved was reduced from the value of 1200 pm/min used in the GCIFT-IR interface to 100 pm/min. In previous work from this laboratory, acenaphthenequinone (AQ) has proven to be a useful probe for GC/FT-IR, SFCIFT-IR and HPLCIFT-IR both because of its low volatility and because of the sharpness of the absorption bands in its spectrum. In an attempt to illustrate some of the capabilities of DD SFCIFT-IR, a series of experiments was carried out using AQ as the probe molecule. In the first experiment, 60 nL of a solution 0.0025% of AQ in CH,Cl, was injected onto a 100-pm i.d. biphenyl column and eluted with CO, under a linear pressure program. The eluites emerging from the restrictor were condensed on a cooled ZnSe plate and passed through the infrared beam after a delay of 4 min. The chromatogram shown in Figure 11B was reconstructed from interferograms measured throughout the separation using the Gram-Schmidt vector orthogonalization

96

1

4000

I

I

3000

I

I

2000

I

I

1000

Wavenumber (cm -'I

Figure 12. (A) Spectrum characteristic of a silicone taken at the point marked I on Figure 11B; the expanded spectrum shows the presence of a weak combination band absorbing between 1700 and 1750 cm-'. (B) Spectrum taken at point I1 on Figure 11B; sharp absorption bands due to acenaphthenequinone are superimposed on the silicone spectrum. algorithm [41]. Examination of the spectrum measured at the point marked I on Figure 11B (see Figure 12A) clearly indicates that a silicone of some type had been deposited at this point. Similar spectra at many other times during this run indicated that every peak in the Gram-Schmidt chromatogram was caused by a polydimethyl siloxane oligomer. Apparently these oligomers had accumulated on the column prior to the injection of the analyte and had been separated along with the injected sample. We later found that they originated from a valve in the chromatograph that had been contaminated with silicone grease. The Gram-Schmidt chromatogram clearly shows the elution of two series of peaks that may be assigned to the linear and cyclic polydimethyl siloxane oligomers [20]. The chromatogram shown in Figure 11B demonstrates that the resolution of a capillary supercritical fluid chromatogram is retained by the DD SFC/FT-IR interface. Chromatograms that are reconstructed using the Gram-Schmidt technique indicate the presence of any component that has been deposited on the plate. (In this respect Gram-Schmidt chromatograms are analogous to total ion current chromatograms in GUMS.) More selectivity can be achieved by integrating the absorbance in selected spectral regions. The chromatogram shown in Figure 11C was computed by integrating the absorbance between 1800 and 1650 cm-', and should therefore indicate those positions on the plate when a compound containing a C = O group is present. The single, relatively broad peak seen in this chromatogram at a retention time of 52 minutes indicates the elution of the acenaphthenequinone probe. This point is marked I1 on Figure 11B. The spectrum measured at this time (see Figure 12B) shows the C = O band and several other sharp bands assignable to AQ superimposed on the spectrum of another silicone oligomer. It is interesting to note

97

i

1

AQ

I

I

I

5

I

I

25

15

Time (minutes) igure 13. Gram-Schmidt reconstructed chromatogram obtained after injecting 60 nL of a 0 . 0 2 9 solution of acenaphthenequinone in CH,CI, separated under isobaric conditions (3500 psi). (A different restrictor was used for this chromatogram than for Figure 11.)

0.3

0.2

0.1

0.0

2000

1600

1200

aoo

Wavenumber (cm-l ) Figure 14. (A) On-line DD SFC/FT-IR spectrum obtained at the peak marked AQ in Figure 1 (- 12.5 ng of acenaphthenequinone). @) On-line DD SFC/FT-IR spectrum of the third peak in Figure 13 (marked contaminant in AQ).

98

Acenaphthenequinone Functional Group

( 1 800 - 1680 c m - l)

I

I

5

10

I

I

15

20

Time (minutes)

'igure 15. Gram-Schmidt and functional-group reconstructed chromatograms obtained after injecting 60 nL of a 0.0025% (w:v) solution of acenaphthenequinone. The first peak is due to a nonvolatile impurity in the CO, mobile phase.

Ace napht henequinone 0.0

0.0

0.0

I

4000

I

I

3000

I

2000

I

I

1000

Wavenumber (cm-')

Figure 16. On-line DD SFCIFT-IR spectrum of the acenaphthenequinone peak in Figure 15, approximately 1 ng of AQ was injected for this spectrum.

99 the presence of several peaks of very low amplitude near the baseline of the functional group chromatogram. These peaks are actually caused by a weak combination band in the spectrum of the silicone oligomers (see box in Figure 12A) and demonstrate the exceptionally high sensitivity of DD SFC/FT-IR. After changing SFC chromatographs to the Computer Chemical Systems 7000 SFC chromatograph, the same solution of AQ in CH,CI, was again injected into the chromatograph. This time the functional group chromatogram indicated the presence of one major and two minor components, see Figure 13. The first minor peak was often observed in our chromatograms and appears to be due to a nonvolatile long-chain ester impurity in the CO, that accumulates at the head of the column as the pressure is returned to its initial value. The AQ spectrum, for which 12 ng had been injected, is shown as the upper trace in Figure 14, and the spectrum of the second minor peak to elute is shown as the lower trace. The unusually high wavenumber of the C=O stretching band in this spectrum (1775 cm-') and the fact that this band is one component of a doublet indicates coupling between the two carbonyl groups of a relatively unstrained aromatic anhydride. Apparently, when the solution of AQ had been standing while the chromatographs were switched, the AQ had been oxidized in the manner shown schematically below.

Since 12 ng of AQ were nominally injected, and because the spectrum of the anhydride required a 12x ordinate expansion to plot it on the same scale as the AQ spectrum, we assume that this compound was present in the injected solution at a level of approximately 1 ng. To demonstrate the high sensitivity of DD SFCIFT-IR measurements, a solution containing 1 ng of AQ was then injected. The reconstructed chromatograms, which again indicate the presence of the ester impurity as well as the peak due to the AQ probe, are shown in Figure 15. The spectrum from AQ peak is reproduced in Figure 16. It should be noted that the spectra shown in Figures 12, 14 and 16 were all measured in real time, i.e., as the deposited spots passed continuously through the infrared beam. The SNR of all the spectra was so high that returning the sample to the beam for post-run signalaveraging was unnecessary. SUMMARY The data shown in this chapter were designed to illustrate several important points concerning DD SFC/FT-IR. Firstly, the technique is equally applicable to separations effected on capillary or 1-mm i.d. packed columns using modified or unmodified mobile phases. Secondly, on-line data can be obtained after making quite minor modifications to a commercial DD GC/FT-IR interface. Finally, the minimum quantity of any analyte that can be identified in real time using this technique is a few hundred picograms for strongly absorbing compounds such as acenaphthenequinone. The minimum identifiable quantity will probably increase to about 1 or 2 ng for very weak infrared absorbers such as polycyclic aromatic hydrocarbons. We believe that DD SFC/FT-IR will prove to be an important tool for chemists who are involved in the analysis of samples containing trace amounts of organic compounds that are too nonvolatile to be separable by gas chromatography.

100

ACKNOWLEDGEMENT This work was supported in part by Grant No. DE-FG22-87PC79907 from the U.S. Department of Energy and Grant No. R-814441-01-1 from the U.S. Environmental Protection Agency. 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. 28. 29. 30. 31. 32. 33. 34. 35.

K.H. Shafer and P.R. Griffiths, Anal. Chem., 55, 1939 (1983). S.V.Olesik, S. B. French and R. Novotny, Chromatographia, 18, 489 (1984). C.C. Johnson, J.W. Jordan, L.T. Taylor, and D.W. Vidrine, Chromarographia, 20, 717 (1985). S.B. French and R. Novotny, Anal. Chem., 58, 164 (1986). M.E. Hughes and J.L. Fasehing, J . Chromarogr. Sci., 24, 535 (1985). J.W. Jordan, C.C. Johnson and L.T. Taylor, J. Chromatogr. Sci., 24, 82 (1986). R.J. Skelton, C.C. Johnson and L.T. Taylor, Chromatographiu, 21, 3 (1986). R.C. Wiebolt, G.E. Adams and D.W. Lites, And. Chem., 60, 2422 (1988). S. Shah, M. Ashraf-Khorassani and L.T. Taylor, Chromatographia, 25, 631 (1989). M.W. Raynor, A.A. Clifford, K.D. Bartle, C. Reyne, A. Williams and B.W. Cook, J. Microcol. Sep., 1, 101 (1989). M. Ashraf-Khorassani and L.T. Taylor, HRC, 12, 40 (1989). L.T. Taylor, Chapter 4 in "Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction" (Kiyokatsu Jinno, ed.) Elsevier Publishing Co., Amsterdam. E. Fermi, Z. Physik, 71, 250 (1931). K.H. Shafer, S.L. Pentoney and P.R. Griffiths, Anal. Chem., 58, 58 (1986). Ph. Morin, B. Beccard, M. Caude and R. Rosset, HRC, 11, 697 (1988). S.L. Pentoney, K.H. Shafer and P.R. Griffiths, J. Chromatogr. Sci., 24, 230 (1986). S.L. Pentoney, K.H. Shafer, P.R. Griffiths and R. Fuoco, HRC, 9, 124 (1986). P.R. Griffiths, S.L. Pentoney, G.L. Pariente and K.L. Norton, Mikrochim. Acfa (W'ien), I11 47 (1987). C. Fujimoto, Y. Hirata and K. Jinno, J . Chromarogr., 323, 47 (1985). K. Jinno, Chromatographia, 23, 55 (1987). K.H. Shafer, P.R. Griffiths and R. Fuoco, HRC, 9, 124 (1986). R. Fuoco, K.H. Shafer and P.R. Griffiths, Anal. Chem., 58, 3249 (1986). A.M. Haefner, K.L. Norton, P.R. Griffiths, S. Bourne and R. Curbelo, Anal. Chem., 60, 247 (1988). S. Bourne, A.M. Haefner, K.L. Norton and P.R. Griffiths, Anal. Chem., 62,2447 (1990). K. JiMO, C. Fujimoto, HRC, 4, 532 (1981). K. Jinno, C. Fujimoto, D. Ishii, J. Chromatogr., 239, 625 (1982). K. Jinno, C. Fujimoto, Y. Hirata, Appl. Spectrosc., 36, 67 (1983). K. Jinno, C. Fujimoto, Y . Hirata, J. Chromatogr., 258, 81 (1983). C. Fujimoto, T. Oosuka, K. Jinno, Anal. Chim. Acra, 178, 159 (1985). J.J. Gagel and K. Biemann, Anal. Chem., 59, 1266 (1987). J.J. Gagel and K. Biemann, Anal. Chem., 58, 2184 (1986). R.M. Robertson, J.A. de Haseth, J.D. Kiek and R.F. Browne, Appl. Spectrosc., 42, 1365 (19881. R.M.'Robertson, J.A. de Haseth and R.F. Browne, Appl. Spectrosc., 44, 8 (1990). A.J. Lange, P.R. Griffiths and D.J.J. Fraser, Anal. Chem., in press (1991). P.R. Griffiths, HRC, 14, 73 (1991).

101

36. 37. 38. 39. 40. 41.

P.R. Griffiths, Appl. Spectrosc., 31, 284 (1977). M.W. Raynor, I.L. Davies, K.D. Bartle, A.A. Clifford, A. Williams, J.M. Chalmers and B.W. Cook, HRC, 11, 766 (1988). M.W. Raynor, K.D.Bartle, I.L Davies, A. Williams, A.A. Clifford, J.M. Chalmers and B.W. Cook, Anal. Chem., 60, 427 (1988). K.D. Bartle, M.W. Raynor, A.A. Clifford, I.L. Davies, J.D. Kithingi, G.F. Shilstone, J.M. Chalmers and B.W. Cook, J. Chromatogr. Sci., 27, 283 (1989). E.J. Guthrie and H.E. Schwartz, J . Chromatogr. Sci., 24,236 (1986). J.A. de Haseth and T.L.Isenhour, Anal. Chem., 49, 1977 (1977).

This Page Intentionally Left Blank

K. Jinno (Ed.), Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction Journal of Chromatography Library Series, Vol. 53 0 1992 Elsevier Science Publishers B.V. All rights resewed.

103

Chapter 7

PRACTICAL APPLICATIONS OF SFC-FTIR Keith D Bartle and Anthony A Clifford, School of Chemistry, University of Leeds, LEEDS, LS2 9JT, UK and Mark W Raynor, Carlo Erba Strumentazione, Rodano, MILAN, ITALY INTRODUCTION It is a truism that the power of any separation method is greatly increased when it is coupled to a spectrometric identification technique. The ready availability and decreasing real costs of FTIR spectrometers have led to their increasingly frequent use as chromatographic detectors. FTIR is a powerful and highly specific detection technique. Since absorption bands can quite easily be assigned to individual functional groups in organic molecules, the technique is especially useful for the identification of unknown analytes and can also be employed as a chemically specific detector. The possibility of reconstruction of the chromatogram by the Gram-Schmidt procedure to give universal and non-destructive detection, along with IR spectra of separated constituents in a form readily suitable for library searching, pattern recognition and expert systems, together represent a powerful incentive for the use of FTIR. The development of flow-cell F"IR detection in SFC mirrors that of GC-FTIR, although the smaller internal diameters of SFC columns, resulting from slower diffusion in supercritical mobile phases and the IR absorption of CO, have led to rather different cell designs. SFC-FTIR does not suffer, however, from the solvent interferences which have hindered the development of HPLC-FTIR, and consequently both flow-cell and solvent elimination interfaces have been employed for SFC-FTIR. Since SFC analytes are non-volatile, mobile phases are easily eliminated, so that FTIR

104

spectra of separated peaks may be readily recorded. A wide range of sample types have been analysed by SFC-FTIR, with emphasis on those areas where SFC analysis is strong - the separation and identification of mixtures of thermolabile, reactive, and high molecular weight compounds: polymers and polymer additives; foods, flavours, and essential oils; hydrocarbons and fuels; drugs and steroids; pesticides; and propellants.

COUPLING SFC TO FTIR Solvent elimination methods Since SFC is unique among high-resolution separation methods in that separated, generally non-volatile components are presented in a readily eliminated stationary phase, the procedure of collecting peaks in, or on, an IR-compatible medium and allowing the solvent to evaporate before recording spectra is inexpensive and relatively straightforward.") A chromatographic peak is deposited from the end of the restrictor, which is connected to the end of the column by a heated transfer line, on to a small area of IR-transparent support. The mobile phases is allowed to evaporate, and the support is positioned in the IR beam. Spectra are collected from as many scans as are required to obtain spectra with adequate signal-to-noise ( S / N ) ratio. Griffiths has listed") as many as six different procedures whereby lTIR spectra may be recorded after solvent elimination. The flow from the end of the restrictor can be split to permit simultaneous detection by another means and thus permit identification of the deposition period. The mobile phase elimination procedure has the advantage of simplicity and high sensitivity. Spectra can be compared with condensed-phase libraries. There is considerable scope for the use of modified CO, mobile phases. The procedure can be made continuous so that reconstructed chromatograms are available.

105

On-line detection A high pressure flow cell may be connected at the end of the SFC column to allow on-line dete~tion.'"~)IR-transparent windows are incorporated in the cell, which is positioned in the focused beam of the spectrometer so that the column effluent is monitored continuously. A flow restrictor is located after the flow cell and can be introduced into another detector such as an FID. The flow cell design in capillary SFC-FTIR is an inevitable compromise between the chromatographic and spectroscopic requirement^.'^) The small SFC column diameters lead to maximum cell volumes of 100 nl to avoid resolution however, the requirements of IR sensitivity and radiation beam dimensions lead to minimum volumes in the region of 1 pl.

-

The band broadening which resulted from these cell dimensions was halved by employing make-up liquid at the same flow rate as that through a capillary column (Figure 1). The detection limit for satisfactory IR spectroscopy was hence d~ubled'~) but this disadvantage was overcome by a stopped-flow pro~edure.'~)The flow of mobile phase is stopped so that the analyte is held under liquid conditions in the flow cell where FTIR scans are recorded, and there is a substantial increase in IR spectrum intensity (Figure

2).

SENSITIVITY IN SFC-FTIR The cell volumes in on-line SFC-FTIR are inevitably large compared with those employed in SFC with UV detection because of the constraints imposed by spectroscopic considerations. For a cell with volume 800 nl transmitting 20% of available beam inten~ity'~'spectra with better than 10 (Sm) ratio could be recorded from the 16 scans possible during elution of an SFC peak from analyte masses of 10 ng for compounds with intense IR absorptions (e.g. esters) to 100 ng for poor IR absorbers such as aromatic hydrocarbons. Similar S/N values have been observed for a slightly larger flowcell; a S/N ratio of at least 3:l was observed for the C=O absorption of 2.5 ng caffeine."' The stopped-flow procedure permits a reduction of these quantities by roughly a factor of three.(4)

106 Injection valve

FID

Zero dead volume butt-connector

Syringe Pump

Flow cell in microbeam unit

Capillary columns: 1. Analytical column 2. Make-up fluid capillary

Figure 1. Instrumental components of on-line capillary SFC-FTIR. (Reproduced with permission from ref. 3).

7

On/off valve

Injection valve

FID

Zero dead volume

Syringe

Capillary columns: 1. Analytical column 2. Make-up fluid capillary

Flow cell in microbeam unit

3. By-pass capillary

Schematic of stopped-flow capillary SFC-FTIR system (Reproduced with Figure 2. permission from ref. 4).

107

The sensitivity of measurements made by solvent elimination procedures is greater, by a factor of between 2 and 4, in comparison with conventional flow cell work. Subnanogram detection levels are clearly possible in SFCFTIR.

APPLICATIONS Polymers, oligomers and polymer additives Fujimoto et a1@)and Pentoney et at7) showed early on the value of the solvent elimination method in the analysis of polymers. The FTIR spectra of dimethylsiloxane oligomers were recorded after separation on a capillary column with C0,.(7) The compatibility of a solvent elimination interface with modified mobile phases was demonstrated; styrene and methyl phenyl siloxane oligomers were separated on a packed capillary column with 10% ethanol in hexane.@) SFC-FTIR is a particularly good technique for characterising chemical additives in polymers.'" Many of these compounds, which include slip agents, plasticisers, UV absorbers, light stabilisers and antioxidants, are thermally labile or have a high molecular mass, which makes them difficult to analyse by other methods, particularly GC. Figure 3 shows the SFC-FID chromatogram of two polymer additives which were Soxhlet-extracted from a commercial polypropylene sample. At the same time, spots (- 200 nm in diameter) associated with these peaks were collected at the solvent elimination interface on a KBr disc and analysed in the FTIR microscope. A thousand spectra were measured with a resolution of 4 cm-' and accumulated in 4 minutes. The IR spectra obtained from the two spots (Figs 4 and 5) show the quality of the spectra that can be obtained with this approach. Chemical features are immediately apparent from the spectra. For example the IR spectra in Fig 4 has absorptions which are characteristic of the amide group at 3400 cm-' (N- H) and 1670 cm-I (C=O). The strong bands at 2800-3000 cm-' indicate strong aliphatic C-H stretching and a small amount of unsaturation is indicated by the absorption at 3100 ern-'. With this type of information it is simple to perfom a library search of reference spectra and obtain positive identification for the compound.'') As a result the

108

8

0 150

10 150

20

30 200

40 50 Time I min )

250 Pressure i a t m t

60

300

70

80

90

350

Figure 3. Capillary SFC-FID chromatogram of polymer additives: peak 1, Erucamide; peak 2, Irganox 1010.

Figure 4. in Figure 3.

FTIR spectrum of Irganox 1010recorded after solvent elimination in run shown

109

Figure 5. in Figure 3.

FTIR spectrum of Erucamide recorded after solvent elimination in run shown

3

2 5

1

6

4

!J

i 0

10

20

30

40

50

60

035

043

051

059

067

075 Oenslty (gem-')

Time (set)

I

035

Figure 6. Capillary SFC-FTD chromatogram of 1Zhydroxysteric acid oligomers. (Reproduced with permission from ref. 10).

110

two polymer additives were identified as Erucamide, a slip agent added as a processing aid, and Irganox 1010 (Ciba Geigy), an antioxidant. Raynor et a1@)showed how twenty-one common polymer additives with a variety of chemical types and ranging in molecular weight from 228 to 1178 could be separated by SFC on a non-polar capillary column in a single chromatogram; and that good quality FTIR spectra could be obtained on sample quantities of the order of 100 ng deposited on KBr discs with solvent elimination. This procedure was applied during the separation of a mixture of products resulting from self-condensation polymerisation of 12-hydroxystearic acid which was achieved (Figure 6) on a capillary column coated with an oligoethyleneoxide substituted methyl polysiloxane (glyme) stationary phase with CO, mobile phase.'") The degree of polymerisation was indicated from the ratio of ester (1724 cm-') to acid (1700 cm-I) carbonyl absorptions in the spectra of the components of each peak. Accompanying the decrease in the 1700 cm-' absorption was the loss of intensity at 1270 cm-' (C-0 of COOH) and the increase of the 1210 cm-' (C-0-C) band (Figure 7). Flow cell SFC-FTIR has been successfully employed'*) in the study of UV curing coatings which are mixtures of reactive diluents, thermally labile photoinitiators and reactive oligomers. Make-up fluid was added to the column effluent prior to the 800 nl volume flow cell to reduce loss of chromatographic resolution; Figure 8B shows the GSR chromatograms. The FTIR spectra of peak 2 is consistent with an acrylate group (absorptions at 1740 cm-' (CO), 1185 and 1245 cm-' (CO) and 1075 cm-' (olefinic C-H). Comparisons with standards led to the attribution of this peak to a phenylacrylate monomer - the main reactive diluent. The FTIR spectrum of peak 6 shown in Figure 8D is fairly complex in the fingerprint region but has a carbonyl absorption band at 1680 cm-I, which indicates the presence of a ketone in conjugation with an aromatic ring. Further evidence of the aromatic ring is found at 1595 cm-' because of C-C ring vibrations. Because aromatic ketones such as benzophenone are used as photoinitiators in UV curing coatings, an FTIR spectral library of these compounds was consulted, and 2-methyl-(4-methylthiopheno)-2-morpholinopropan-1 -one (Irgacure 907, Ciba Geigy) was found to be the closest match. The FTIR spectra of peaks 7-13 are similar to those of a number of ethoxy acrylate oligomers. These differed mainly in the nature of aromatic C-H deformation absorbances below 800 cm-' and it was therefore necessary to use xenon as mobile phase.'") Comparisons of SFC chromatograms and FTIR spectra recorded in xenon

111

SPOT 1

6

rUOO

3610

3229

2939

2Y40 2050 WAVENUMBERS

1660

1270

a80

Figure 7. FTIR spectra of 12-hydroxystearic acid oligomers recorded after solvent elimination in run shown in Figure 6. (Reproduced with permission from ref. 10).

112

D

1670 - 1690 crn-1 Reconstruction

0 0

10

20 225

30

40 450

50

60 Tirne(rnin) 675 Data Points

Figure 8. On-line capillary SFC-FTIR analysis of UV curing coating. B, SFC-FTIR chromatogram; C, 1670-1690 cm-' reconstruction of SFC-FTIR chromatogram (detects aromatic ketone: and D, FTIR spectrum of photoinitiator detected in C. (Reproduced with permission from ref. 1).

113

with those from a number of oligomers suggested that the ethoxyacrylate series is derived from bisphenol. This mobile phase was also found to be necessary in the analysis of oligomeric aromatic isocyanates by SFC-FTIR,('2) because the major N=C=O stretching absorption at 2270 cm" is masked by the CO, band at 2137 - 2551 cm-' (Figure 9).

Foods, flavours, essential oils and other natural products The volatile compounds which make up flavours and aromas are often most suitably analysed by gas chromatography but SFCFTIR has nonetheless found applications to thermolabile substances. Thus Morin et a1 ~howed('~*'~) how SFC on a packed silica column with on-line FTlR allowed separation and identification of sesquiterpene hydrocarbons which may decompose under GC conditions. Structural information was preserved, such as the differentiation of methyl from methylene groups, and geminal or single methyl (single or twin bands near 1380 cm-'). The method was applied('3*15) to the identification of sesquiterpenes in a number of essential oils, including pepper oil, pre-fractionated by reverse phase HPLC. Figure 10 shows the identification of a a-farnesene and germacrene in ylang-ylang oil. Flavour constituents amenable to SFC found in grape and other fruit juices and in distilled bevorages such as whisky, and which include phenolic acids, may be separated without derivatization.(") Figure 11 shows the capillary column SFC chromatogram of a mixture of five flavour phenolic acids separated on a glyme column. Figure 11 is the IR spectrum of vanillic acid (peak 2) in Figure 12, recorded after solvent elimination. An FTIR flow cell was used('@in the separation and identification of nonpolar lipids by packed-column SFC as early as 1985, although detection limits were in the microgram range in this early study. Hellgeth et a1 ~eparated''~) free fatty acids from butter, soap, and soybean and coconut oil by packed column SFC with CO, and Freon 23 as mobile phases. Components were detected by on-line TTIR of the strong bands near 1700, 3000 and 3550 cm-'. Pentoney et a1 preferred(") a separation by capillary column SFC with solvent-elimination FTIR for the analysis of triglycerides from vegetable oils and shortening. The higher resolution afforded by capillary columns allowed Calvey et a1 to separate the constituents of refined and hydrogenated soybean Triglycerides were separated on a 25%

114

8

b

Gram-Schmldl

Reconstructed Chromatogram

a

I1

\

Infrared spectrum of fifth eluting isocyanate oligomer

RCiCNTION i I M E (MIII)

30

>lo

12%

312

+06

500

Note: major absorption at 2270cmd is detected in xenon but not in carbon dioxide due to the band from 2200 2250 cm-l.

O R T R POINTS

Figure 9. On-line capillary SFC-FTIR chromatogram of mixture of isocyanates, with FTIR spectrum of fifth eluting oligomer, xenon mobile phase. B

A

s45.477

6

-

7

- .409

c

I

‘C

I

$ .341-

5‘ 4

4000 3530

2590 2120 1650 Wavenumber (cm”)

3060

1180

710

1180

710

I

I

.001 0.19

2.50

4.81

Time (minl

4000 3530

3060 2590 2120 1650 Wavenumber (ern")

Figure 10. Packed-column on-line SFC-FTIR chromatogram, and FTIR spectra of B, afarnesene and C, germacrene from ylang-ylang oil. (Reproduced with permission from ref. 14).

115

OCH,

n

CH,CH,COOH

OCH,

$I OH

5

HOOC

0

10

20

Time (sec)

d5

0.58

0.73

Density ( g cm-’)

Capillary SFC-FID chromatogram of aromatic flavour acids. (Reproduced with Figure 11. permission from ref. 10).

LtOOO

3600

3200

2800

2 k m O O O WAVENUMBER

I600

I200

800

400

FTIR spectrum of vanillic acid recorded after solvent elimination in run shown Figure 12. in Figure 11. (Reproduced with permission from ref. 10).

116

cyanopropyl - 25% phenyl - 50% methylpolysiloxane column, and free fatty acids and oxidative products on a 50% cyanopropyl - 50% methylpolysiloxane column. FI'IR spectra recorded in a flow cell in-line with FID showed the triglyceride double bond isomerisation which occurred during hydrogenation, and the presence of hydroperoxides in oxidation products. SFC with flow cell FTIR has been used in the analysis of sugar derivatives to extend the range of GC. Peracetylated aldonitrile derivatives and byproducts from monosaccharides were identified although the C-N absorption were obscured by CO, bands.""

Drugs and steroids The analysis of an acetylated steroid as shown by Figs 13 and 14 demonstrate^(^*^') the potential applicability of SFC-FTIR microspectrometry in the pharmaceutical and biomedical fields. Again, a great deal of structural information is immediately apparent from the IR spectrum: the broad band at 3400 cm-' is due to 0 - H stretching: the absorptions at 2800-3000 cm-' indicate aliphatic C-H stretching; ester groups are indicated by C=O bending absorptions at 1745 and 1718 cm-'; the 900-1400 cm-' region of the spectrum is also quite characteristic of an ester due to the strong 1250 cm-' absorption. Griffiths et af'") recorded the spectra of a number of barbiturates after solvent elimination to probe differences from condensed-phase libraries. Shah et al separated@)five steroid hormones including progesterone, testosterone, and corticosterone on a capillary column with flow cell detection. Since the CO, masked the OH stretching absorptions, the carbonyl region was used for identification. Of course when solvent elimination is employed, the entire spectrum can be used for identification. This procedure has been found effective('321) in the identification of plant sterols related to ecdysone (Figure 15).

Fossil fuels and hydrocarbons Although many polycyclic aromatic compounds (PAC) are readily analysed by GC, these compounds have also often been used as components of test mixtures for SFC-FI'IR. Morin et a1 c~upled''~) a packed column SFC to FTIR via a 8 p1 volume flow cell for the analysis of a seven-component PAC

117

Figure 13. Capillary SFC-FTIR chromatogram of a mixture of steroids. (Reproduced with permission from ref. 9).

n FTR spectrum of acetylated steroid recorded after solvent elimination in run Figure 14. shown in Figure 11. (Reproduced with permission from ref. 9).

118

0

10

20

30

Time (rnin)

Figure 15. Capillary SFC-FTIR chromatogram and FI'IR spectra recorded after solvent elimination of plant ecdysteroids. (Reproduced with permission from ref. 1).

Figure 16.

Capillary SFC-FID chromatograms of coal tar pitch extracted with CO, at 100

119

mixture, while Pentoney et a1 demonstrated''8' capillary SFC-FTIR using a solvent elimination interface for the analysis of a five-component mixture of polyphenyls; PAC from lampblack were similarly studied.(23)Although PAC are weak IR absorbers, they may be identified at the ng level by this method since compact spots are deposited. In contrast with mass spectrometry, FTIR allows ready differentiation of isomers;(24)Figure 17 shows the FTIR spectra of two peaks from the chromatogram (Figure 16) of the fraction extracted by CO, at 200 atmospheres; comparison with the spectra of standards allows the 4-ring isomers benz[a]anthracene and chrysene to be identified. The same group showed"" how SFC with flow cell FTIR detections was less preferable than the solvent elimination method if CO, is the mobile phase, since absorptions below 800 cm-' are obscured. Supercritical xenon was found'") to be superior in this respect by revealing these bands (Figure 18). This mobile phase was also found to dissolve PAC more readily than CO, so that the molecular weight range of fuels accessible to CO, could be extended.

Propellants and munitions SFC has proven to be an excellent technique for the separation of propellant constituents. Model mixtures of nitrated diphenylamine and nitrated aniline were resolved on both packed and capillary column^.(^^^"' Peaks were identified and their purity assessed by on-line FTIR. Nitrosoanilines were observed to convert to nitroanilines during storage from a comparison of FTIR spectra.(*') Highly deactivated packings were necessary in the packed column work if unmodified CO, was used as mobile phase. Dichloromethane extracts of double-base propellant were analysed'%) by packed column SFC with on-line FTIR detection. Di-n-propyl adipate, triacetin, 2-nitrodiphenylamine and nitroglycerine were separated and detected. The FTIR responses for the strong IR-absorbing nitrated constituents were greater than for F D detection. Sensitivity was improved by injecting supercritical CO, extracts directly into the chromatograph and making use of the extracting fluids being the same as the mobile phase; in this way 'good' and 'bad' propellants could be distinguished from the signals of minor peaks (Figure 19). Coupled SFE-SFC-FTIR was similarly applied(28)to the analysis of candelila

120

FTIR spectrum of chrysene recorded after solvent elimination in run shown in Figure 17. Figure 14. (Reproduced with permission from ref. 24)

Absorptions below 80Ocni1 are detected

in xenon but not in carbon dioxide

n I

'+do0

3600

3200

Ed00

2400 ZOO0 wfiVENUM0ER

1600

l>OO

600

{OD

Figure 18. RIR spectrum of phenanthrene recorded during on-line capillary S F C - R R with xenon mobile phase.

121

wax which is used to facilitate the extrusion of munitions. Supercritical CO, extracts of crude, purified, and synthetic wax were separated by packedcolumn SFC; on-line FTIR detection allowed the non-hydrocarbon constituents (esters, alcohols, sterols and free acids) to be distinguished from the hydrocarbons. Bands at 1748 and 1761 cm-' were indicative of carbonyl in different functional groups. The absence of methine C-H stretching absorption confirmed that the hydrocarbons were unbranched.

Pesticides Wiebolt et a1 have demonstrated the analysis of arba am ate'^^' (model components) and pyrethrin pe~ticide'~''mixtures by capillary SFC with flowcell FTlR detection. The model compound mixture contained aldicarb, methomyl, captan and phenmedipham; even after subtraction of the CO, background spectrum the NH stretch absorption of aldicarb was still detected'29' (Fig. 20). The method was of particular use in the analysis(30'of pyrethrins, naturally occurring insecticides (Fig. 21) isolated from Chrysanthemum cinerarioefolium which are thermally degraded during GC analysis; SFC was carried out under much milder conditions. Fig. 21 is a chromatogram of the pyrethrin extract showing baseline resolution of the six components on a biphenyl polysiloxane column. FTIR spectra of cinerin I and 11, jasmolin I and II and pyrethrin I and I1 were obtained with a flowthrough cell and clearly showed the structural information necessary to distinguish between the compounds. For example, the carbonyl absorption of both pyretherins is at 1722 cm-', but there are marked differences in the C - 0 stretching region (Figure 22). Chlorinated pesticides and the residue from a still in which the herbicide 2,4,5-trichlorophenoxyaceticacid had been distilled were analy~ed'~') by SFC with solvent elimination detection. The latter study demonstrated the complementary nature of GC and SFC-FTR: the latest eluting peak in the GC chromatogram originated from the compound which gave rise to the earliest peak in the SFC chromatogram, which contained at least ten other peaks.

122

W

'P

.Q00 i I

20

,.,,

LJ

-

( . , , , . , , , . , , , , , rC

0.00

is. 00

$0. O O T O O

R E T E N T I O N TI ME (MINI 350.

880

ldl0

1340

1670

2600

DATFl POINTS Figure 19. On-line packed column SFC-FTlR chromatograms of supercritical CO, extracts of "good' and "bad" double-base propellants. (Reproduced with permission from ref. 26).

m4000

3600

3200

2800

2400

2bOO

1800

1200

I

800

Figure 20. FTIR spectrum of aldicarb recorded during on-line chromatography SFC-FTIR (Reproduced with permission from ref. 29).

123

3

6

I

i-

r 0

I

20 Time Iminl

10

0.18

0.34 0.38 Density l g / m l )

1

30

40 I

I

0.54

0.68

Figure 21. On-line capillary SFC-FTIR chromatogram c. pyrethrins. (Reproduced with permission from ref. 30).

'J'

YUUO

3600

3200

2800 2GOO 2600 WAVENUPlEER

TA I600

1200

600

Figure 22. FTIR spectra of pyrethrins recorded during run shown in Figure 21. :Reproduced with permission from ref. 30).

124

Miscellaneous practical applications

A variety of test mixtures have been analysed as examples of the application of SFC-FI’IR, with both flow-cell and solvent elimination interfaces. These are listed in Table 1, along with the mobile phase employed. The advantages of direct reaction monitoring by capillary SFC-FTIR were illustrated by a study of the decomposition of allyldiisopropylamine oxide in which flow-injection analysis was u~ed.‘~’’ TABLE 1 Applications of SFC-FTIR : test mixtures Analytes

Interface

Year, authors and reference

Anisole, acetophenone mitrobenzene Acetophenone Benzonitrile Methylbenzoate Aromatic aldehydes and phenols Quinones Dodecane, aromatic ketones and phenols Indoles Carbonyl compounds

Flow cell

1983, Shafer et a1

Flow cell Flow cell

1985, Johnson et a1 (34) 1986, Morin et a1 (35)

Flow cell

1986, French & Novotny(36)

Solvent elimination Solvent elimination (matrix isolation) Solvent elimination Flow cell

(33)

1986, Shafer et a1 (37) 1988, Raymer et a1 (38) 1988, Griffiths et a1 (39) 1988, Morin et a1 (40)

CONCLUSION The applications of SFC with FTIR detection are widespread throughout research and industrial chemistry. Both solvent-elimination and flow-cell procedures have proved useful in the analysis of a variety of mixtures of high molecular weight, reactive and thermolabile compounds. SFC-FTIR is a

125

mature technique which can take its place in the armoury of the analytical chemist.

REFERENCES 1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

K.D. Bartle, M.W. Raynor, A.A. Clifford, I.L. Davies, J.P. Kithinji, G.F. Shilstone, J.M. Chalmers and B.W. Cook, J. Chromatogr. Sci., 27, 283 (1989) and references therein. R. Fuoco, S.L. Pentoney & P.R. Griffiths, Analyt. Chem., 61, 2212 (1989) M.W. Raynor, A.A. Clifford, K.D. Bartle, C. Reyner, A. Williams and B.W. Cook, J. Microcolumn Sep., 1, 101 (1989) M.W. Raynor, K.D. Bartle, A.A. Clifford and B.W. Cook, J. Microcolumn Sep., 2, 458 (1990) S. Shah, M. Ashraf-Khorassani and L.T. Taylor, Chromatographia, 23, 631 (1988) C. Fujimoto, Y. Hirata, and K. Jinno, J. Chromatogr., 332, 47 (1985) S.L. Pentoney, K.H. Shafer, and P.R. Griffiths, J. Chromatogr. Sci., 24, 230 (1986) M.W. Raynor, K.D. Bartle, I.L. Davies, A. Williams, A.A. Clifford, J.M. Chalmers, and B.W. Cook, Analyt. Chem., 60, 427 (1988) M.W. Raynor, I.L. Davies, K.D. Bartle, A. Williams, J.M Chalmers, and B.W. Cook, Eur. Chrom. News, 1, (4), 18 (1987) M.W. Raynor, K.D. Bartle, A.A. Clifford, J.M. Chalmers, T. Katase, C.A. Rouse, K.E. Markides and M.L. Lee, J. Chromatogr., 505, 179 (1990) M.W. Raynor, G.F. Shilstone, A.A. Clifford, K.D. Bartle, M. Cleary and B.W. Cook, J. Microcolumn Sep., in the press M.W. Raynor, G.F. Shilstone, K.D. Bartle, M. Cleary and B.W. Cook, J. High Res. Chrom., 12, 300 (1989) P. Morin, M. Caude, H. Richard, & R. Rosset, Analusis, 15, 117 (1987) P. Morin, M. Pichard, H. Richard, M. Caude and R. Rossett, J. Chromatogr., 464, 125 (1989). M. Pichard, M. Caude, P. Morin, H. Richard, and R. Rosset, Analusis, 18, 167 (1990)

126

16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

M.E. Hughes and J.L. Fasching, J. Chromatogr. Sci. 23, 535 (1985) J.W. Hellgeth, J.W. Jordan, L.T. Taylor and M. Ashraf-Khorassani, J . Chromatogr. Sci., 24, 183 (1986) S.L. Pentoney, K.M. Shafer, P.R. Griffiths and R. Fuoco, J. High. Res. Chrom., 9, 169 (1980) E.M. Calvey, S.W. Page, and L.T. Taylor, Proc 7th Int Conf Fourier Transform Spectroscopy, p. 540 (1989) E.M. Calvey, L.T. Taylor and J.K. Palmer, J. High Res. Chrom., 11, 739 (1988) M.W. Raynor, Thesis, University of Leeds, UK (1989) K.L. Norton, A.M. Haefner, M. Makishima and P.R. Griffiths, Proc 7th Int. Con$ Fourier Transform Spectroscopy, p. 455 (1989) H. Makishima, Thesis, University of California, Riverside (1989) M.W. Raynor, I.L. Davies, K.D. Bartle, A.A. Clifford and A. Williams, J. High Res. Chrom., 11, 766 (1988) M. Ashraf-Khorassani & L.T. Taylor, J. High Res. Chrom., 12, 40, (1989) M. Ashraf-Khorassani and L.T. Taylor, Analyt. Chem., 61, 145 (1989) B. Beccard and 0. Barres, Spectra 2000, 145, 53 (1990) M. Ashraf-Khorassani and L.T. Taylor, LC-GC, 8, 314 (1990) J.W. Later, D.J. Bornhop, E.D. Lee, J.D. Henion and R.C. Wieboldt, LC-GC, 5, 805 (1987) R.C. Wieboldt, K.D. Kempfert, D.W. Later and E.R. Campbell, J. High Res. Chrom., 12, 106 (1989) P.R. Griffiths, S.L. Pentoney, A. Giorgetti and K.H. Shafer, Analyt. Chem., 58, 1349A (1986). S.V. Olesik, S.B. French and M.V. Novotny, Analyt. Chem., 58, 2256 (1986) K.H. Shafer and P.R. Griffiths, Analyt. Chem., 55, 1939 (1983) C.C. Johnson, J.W. Jordan, L.T. Taylor and D.W. Vidrine, Chromatographia, 20, 7 17 (1985) P. Morin, M. Caude, H. Richard and R. Rosset, Chromatographia, 21, 523 (1986) S.B. French and M.V. Novotny, Analyt. Chem., 58, 164 (1986) K.H. Shafer, S.L. Pentoney, and P.R. Griffiths, Analyt. Chem., 58, 58 (1986)

127

38. 39. 40.

J.H. Raymer, M.A. Mosely, E.D. Pellizzari and G.R. Velez, J. High Res. Chrom., 11, 209 (1988) P.R. Griffiths, S.L. Pentoney, G.L. Pariente and K.L. Norton, Mikrochim Acta (Wien) HI, 47 (1988) P. Morin, B. Beccard, M. Caude and R. Rosset J . High Res. Chrom., 11, 697 (1988).

This Page Intentionally Left Blank

K. Jinno (Ed.), Hyphenated Techniques in Supercritical fluid Chromatography and Extraction Journal of Chromatography Library Series, Vol. 53

0 1992 Elsevier Science Publishers B.V. All rights reserved

129

Chapter 8 Recycle Supercritical Fluid Chromatography - On-line Photodiode-array Multiwavelength UV/VIS Spectrometry/ IR Spectrometry/ Gas chromatography Muneo Saito and Yoshio Yamauchi JASCO Corporation, 2967-5 Ishikawa-cho, Hachioji, Tokyo 192 Japan

INTRODUCTION Photodiode-array (PDA) multiwavelength detection is now widely used in HPLC (highperformance liquid chromatography) as a common technique. Therefore, HPLC - PDA multiwavelength detection may not be regarded as a hyphenated technique. However, application of a PDA multiwavelength detector to SFC is still restricted 11-31, and the number of reports on the recycle technique for fractionation in SFC is very limited [4,5]. In multiwavelength detection, there are many ways methods available for treating after-run data, including spectrum picking at a peak's rising, top and falling points, graphical representation of three-dimensional spectralkhromatographic data, e.g. , contour plots, three-dimensional chromatograms, etc., as well as on-line three-dimensional chromatogram monitoring [2,6,7]. In this regard, the authors consider PDA multiwavelength detection in preparative SFC (prep-SFC) to be one of hyphenated technique: chromatography spectrometry hyphenation. This chapter describes the use of a PDA multiwavelength detector as a real-time monitoring and after-run purity check system in prep-SFC with recycle operation. Also, the qualitative and quantitative analyses of fractionated components by IR (Infrared Spectrometry) and GC (Gas Chromatography) are discussed. Preparative SFC and PDA Multiwavelength Detection Prep-SFC (preparative SFC) has several advantages over prep-LC (preparative LC) such as [3,5,8-121: 1) easier separation of solutes from the mobile phase at low temperatures and in oxygenfree environments, 2) easier retention control simply by changing the density and temperature, and 3) faster separation. Prep-SFC with carbon dioxide offers very easy separation of the solutes from the mobile phase because low-pressure or low-density carbon dioxide has no solvating power. The solutes can be readily separated merely by reducing the pressure without elevating the fluid temperature. Furthermore, in this process, adiabatic expansion of the fluid lowers the temperature of both the fluid itself and the solutes [5-71.

130

A major technique used to control retention in prep-LC involves changing the solvent strength by varying the mobile phase composition. However, in prep-SFC, the solvent strength can be changed readily by varying the pressure and temperature. A small amount of polar modifier, generally less than lo%, can also be used. This amount of solvent can be readily removed in prep-SFC while in prep-LC a greater amount of the solvent is necessary to be evaporated.

Recycle Chromatography in PrepSFC The recycle technique is often used in prep-LC, as it offers the higher plate number by passing the sample solutes through the column repeatedly. If the same technique is utilized in prep-SFC, it could offer more advantages in addition to the above points and extend the application range of SFC as the author suggested previously [3,4]. Schoenmakers and Uunk [13] showed that, in SFC, a high column pressure drop causes a density gradually decreases along the column toward outlet, resulting in reduced efficiency. Therefore, the obtainable plate number is not in proportion to the column length. In this regard, the recycle operation of SFC is very favorable, because a higher efficiency can be achieved since the column back-pressure is maintained at the same value for a single column regardless of the number of recycles. Multiwavelength Detection in PrepSFC Multiwavelength detectors are now commercially available from several sources. Such a detector is normally used in combination with a personal computer and offers real-time three-dimensional monitoring and after-run processing of spectrometric/chromatographic data. Real-time monitoring is very useful for fractionation in prep-SFC, because the operator can monitor spectra of solutes at the instance as they are eluted from the column. This function is not available by using a conventional single wavelength detector. Infrared Spectrometry A hyphenation technique of SFC - IR is discussed elsewhere in this book. This chapter describes the qualitative analysis of the fractionated components by the off-line IR measurement using a common diffuse reflectance method for qualitative analyses. Gas Chromatography A hyphenation technique of SFC - GC is also discussed elsewhere in this book. For the quantitative analysis of the fractionated components, this chapter describes the off-line SFCGC.

EXPERIMENTAL Evaluation of Experimental Apparatus Before applying a practical sample to the recycle prep-SFC system, the authors evaluated the system by separating isomers of dibutylphthalate. In the evaluation, a mixture of di-nand di-iso-butylphthalates was recycled until baseline separation had been achieved.

131

Apparatus. Figure 1 shows the recycle prep-SFC system constructed for the experiment. Liquid carbon dioxide from cylinder (3) and modifier solvent (4) are delivered respectively by pumps (1) and (2) to the recycling flow line in the constant flow rate mode. The fluid pressure is controlled by back-pressure regulator (3,feeding back an excessive amount of mobile phase to the inlet of pump (1) or wasting from the outlet of the regulator via switching valve (6) when a modifier solvent is used. The technical details of the back-pressure regulator used are previously reported by the authors [14]. In the recycling flow line, in-line pump (8), injector (9), separation column (lo), Photodiode-array UV detector (11) and switching valve (12) are placed in series and connected in a closed loop. Back-pressure regulator (13) is connected to switching valve (12) for collection and/or wasting of peak components. By means of this arrangement, the pressure and the modifier concentration of the fluid delivered to the recycling flow line is always kept constant, even while a peak component is being collected in the following way. When the recirculating mobile phase is consumed for peak collection, the mobile phase is automatically compensated by reducing the amount of fluid fed back to pump (1) or passed to waste from back-pressure regulator ( 5 ) , maintaining the pressure constant in the recycling flow line. In order to reduce the fluid consumption, the flow rates of the pumps can be lowered while recycling is performed without collection. ......................................

.....................................................................

..........................

I'

.......................................................................

l

I

J.

haste

4 .................

Collection

Figure 1. Hydraulic diagram of recycle prep-SFC. Components; 1 = CO, pump, 2 = modifier pump, 3 = liquid CO,, 4 = modifier solvent, 5 = back-pressure regulator, 5' = pressure transducer for controlling 5, 6 = switching valve, 7 = pre-heat coil, 8 = in-line pump, 9 = injector, 10 = separation column, 11 = photodiode-array UV detector, 12 = switching valve, 13 = back-pressure regulator for collection, 13' = pressure transducer for controlling 13, 14 = air circulating oven.

132

Chemicals and column. Carbon dioxide purchased from Toyoko Kagaku, Kawasaki, Japan, was used as the mobile phase. Ethanol, HPLC grade was mixed with carbon dioxide as a modifier solvent. A test mixture containing di-n- and di-iso-butylphthalates was used for the demonstration of separation by recycle SFC. As a test solute for measuring the column efficiency, di-noctylphthalate was used. All chemicals were purchased from Wako Pure Chemicals, Osaka, Japan, except for carbon dioxide. A JASCO SuperMegaPak SIL column, 10 mm I.D. x 250 mm length was used. The packing material was 5pm silica gel. Evaluation Results Separation of isomers of dibutylphthalate by recycle SFC. Figure 2 shows the recycle chromatogram of the test mixture containing I mg each of di-n- and di-iso-butylphthalates and its contour plot. SFC conditions were: column inlet pressure, 26.7 MPa; column outlet pressure, 25.0 MPa; control pressure of pumps (1) and (2), 25.0 MPa; in-line pump flow rate, cu. 6 glmin; modifier solvent flowrate, 0.15 mllmin, and temperature, 40°C. CRCOUTl

.3201

Aug.

02 1988, 0 9 : 4 6

............. ..,,...,........ ............

.4

Figure 2. Recycle chromatogram and contour plot of mixture of di-n- and di-iso-phthalates. Detection: UV at 245 nm. The top diagram shows the contour plot.

As shown, the recycle separation has been performed successfully and symmetric peaks are obtained even after twelve time recycling, and the resolution increases with the number of recycles. The contour plot shown at the top in Fig. 2 dramatically indicates that the

133

above two compounds are baseline separated after ten time recycling, while the first peak without recycling shows very poor separation and looks almost like a fused peak.

1***** Jun. 18.

600

(

been c h r o m a t o p r a m (Data

igee is:e9 0 . 8 soo )

Gain LOW

e.ee

nuto

- CDOP 52.85 ZOPO

(

.3201 SJun. 2 1 1080. 0 8 5 1 mrn ) e.ee m r n )

1.28

nnu

i e

Figure 3. Recycle chromatogram of 1 mg of di-n-octylphthalate. Detection: UV at 245 nm.

Eficiency versus number of recycles. Di-n-octylphthalate, which gave a capacity factor (k') of ca. 1 under the same conditions as given above, was subjected to recycle SFC for calculating the column efficiency. Figure 3 shows the recycle chromatogram of di-noctylphthalate. As shown, recycle operation was successfully performed and a symmetric peak was obtained even after ten time recycling. The relationship between the number of recycles and the number of plates N is shown in Fig. 4. A plate number was calculated by using the peak area and height, assuming that the concentration distribution is Gaussian. It is remarkable that 39.2 x lo3 plates have been obtained with a column pressure drop ( A P) of only 1.7 MPa after ten time recycling, whereas the first cycle or elution without recycling offered only 6.1 x lo3 plates with the same pressure drop. The relationship is not linear, but of convex shape as shown in the figure. This means, assuming variances are additive, that there is some effect which makes the peak width wider as the solute resident time in the system becomes longer, other than the extra-column band broadening caused by instrumental variances. In order for the in-line-pump to deliver the mobile phase, the pump is placed outside the column oven. This means that the fluid temperature could be less than the critical temperature. It is possible that carbon dioxide and modifier solvent (ethanol) exist in the liquid state rather than the supercritical state. If so, diffusivity of the solute becomes slower

134

40-

0

0

0

30

-

8

h

,'

m

0 I

,'

v

u1

v

20-

1

0

8

c m

-

,'0

a

10

-

$'

4'

5

10

No, o f Recycles Figure 4. Relationship between the number of recycles and the number of plates.

As demonstrated, recycle SFC has been successfully performed. It offers a high column efficiency with a minimum pressure drop across the column, e.g., cu. 4 x lo4 plates and 1.7 MPa. These results encouraged us to apply this technique to a practical application described in the next section.

Practical Application to Isolation of Tocopherols from wheat Germ Oil Materials Wheat germ oil used was extracted and tocopherols were enriched by the method described in the previous report [ 5 ] . The tocopherol concentration of the oil was determined to be about 4 % by HPLC analysis. Carbon dioxide was purchased from Toyoko Kagaku, Kawasaki, Japan, and was used as the mobile phase. Ethanol, HPLC grade, was mixed with carbon dioxide as a modifier solvent. HPLC quantitation-grade a- and 8-tocopherol standards (purity 98% by

135 HPLC)(Eizai Co., Ltd., Tokyo, Japan) were used as the standard samples for the UV and IR analyses. KBr powder used in the diffuse-reflectance IR spectrum measurement was from JASCO, Hachioji, Tokyo. Chloroform, special reagent grade, was used for dissolving samples for the IR measurements. All the chemicals, unless otherwise specified, were purchased from Wako Pure Chemicals, Osaka, Japan. Two JASCO SuperMegaPak SIL columns, 10 mm I.D. x 250 mm length were used for the SFC separations. The packing material was 5pm silica gel. Apparatus Recycle SFC system. The hydraulic system of the recycle SFC is similar to the one described in the previous Section (Fig. 1). The flow diagram is as shown in Fig. 5. As can be seen, an additional column (10') is placed in between switching valve (13) and in-line pump (8). The column serves to give additional retention for components having weak retention. This is very important for the practical application of recycle SFC, because capacity factors (k') of peak components may be in the range from 0 to 10 when natural products are dealt. This means that migration velocities of components in a column varies by the same factors. As a result, components with smaller capacity factors (k') often pass through the column several times before components with greater capacity factors (k') are eluted from the column, resulting in re-mixing of separated components. Therefore, some means to retain the components with smaller (k') is required to operate recycle SFC successfully. Liquid carbon dioxide from cylinder (3) and modifier solvent (4)are delivered respectively by pumps (1) and (2) to the recycling flow line in the constant flow rate mode. The fluid pressure is controlled by back-pressure regulator (5), feeding back an excessive amount of mobile phase to the inlet of pump (1) or wasting from the outlet of the regulator via switching valve (6) when a modifier solvent is used. In the recycling flow line, in-line pump (8), injector (9), separation column (lo), photodiode-array UV detector (1 1) and switching valve (12) and second column (10') are placed in series and connected in a closed loop. Back-pressure regulator (13) is connected to switching valve (12) for collection (fractionation) and/or wasting of peak components. By means of this arrangement, the components having smaller k' values can be retained in column (10') whereas the target or unwanted peak components are passed to the backpressure regulator (13), preventing the components with smaller k' from being re-mixed and from coming out of column (10) together with the target components. The significance of this arrangement is that the fluid flow is completely stopped though the pressure is maintained by the fluid delivery system even while the target is being collected. JASCO Model IR-700 Grating Infrared Spectrophotometer was used with DR (diffuse reflectance) accessory (Model DR-8 1) for the measurement of IR spectra of the fractionated substances and standard tocopherols. Hewlett-Packard 5890A gas chromatograph was used with a flame ionization detector (FID). A J&W DB-5 capillary column, 15 m x 0.25 mm I.D. with 5 % diphenyl/ 95 % dimethylpolysiloxane phase (O.lpm thickness) was used for the examination of the purities of fractionated tocopherols.

136 .................................................................

......................................

3

4

3 .....

m

5

i? X'

13

................................

......... ] 3 '

c Waste

Collection

Figure 5. Hydraulic diagram of recycle prep-SFC. Components; 1 = CO, pump, 2 = modifier pump, 3 = liquid CO,, 4 = modifier solvent, 5 = back-pressure regulator, 5' = pressure transducer for controlling 5, 6 = switching valve, 7 = pre-heat coil, 8 = in-line pump, 9 = injector, 10 = first separation column, 10' = second separation column, 11 = photodiode-array UV detector, 12 = switching valve, 13 = back-pressure regulator for collection, 13' = pressure transducer for controlling 13, 14 = air circulating oven.

RESULTS AND DISCUSSION About 20 g of wheat germ powder was subjected to SFE/prep-SFC in order to prepare wheat germ oil, and the tocopherol-rich fraction was collected. a b l e 1 shows the mass balance in the process.

a b l e 1. Mass Balance in SFE/prep-SFC Process Wheat germ before extraction Wheat germ after extraction Total extracts Collected oil Tocopherol rich fraction *Water content in wheat germ (10 %) Recovered extract *The amount of water was obtained by weighing the and the value agreed with the supplier's value.

21.72 g 17.65 4.07 1.78 0.20 2.17 4.15 (102%) material before and after extraction,

137

Fractionation of a-and 8-tocopherols About 200 mg of tocopherol rich portion of the oil were fractionated as reported previously [5] and 98 mg of the fraction was subjected to recycle SFC. Figure 6 shows the chromatogram and contour plot obtained in recycle operation. The carbon dioxide flowrate was 5.0 mL/min and the modifier flowrate was first 0.20 mL/min then changed to 0.15 mL/min, the in-line pump flow-rate was 5.0 mL/min. The first back-pressure was set at 25MPa and the second at 20MPa.

a

'

I I

I I

I l l

I

W

IR'WI

R

IW1

R

F'

R

II IF'

R

I

Figure 6. Recycle chromatogram and contour plot of wheat germ oil containing tocopherols. The axis and characters below the time axis represent the time frames for wasting, shown as W, recycling as R, and fractionation as F. A represents wasted portion of the peak of triglycerides, B contains tocopherols, and C unknown lipids.

138 As shown in the chromatogram monitored at 220 nm, there appears a broad peak having small peaks superimposed on it. The broad peak is of triglycerides, which are the main constituents of the oil, and the first and second peaks on the tailing part of the peak are assumed to be of a- and &tocopherols, because they have UV absorption maxima at cu. 290 nm and have similar retention times to those obtained in preliminary experiments under the same conditions without recycling. The procedure for recycling and fractionation was as follows; 1) The first portion, triglycerides, eluting in the period time 0 to 6.5 rnin shown as A, was wasted with increased modifier flow rate, 0.20 mL/min. 2) The second portion shown as B containing a- and R-tocopherols were passed to the second column to be retained. 3) While the tocopherols were retained in the second column, the third portion shown as C was passed to waste. 4) Then, only the B portion was recycled. A peak appeared at 42 rnin are of triglycerides, which were now completely separated from tocopherols peaks at 62 and 71 min, and the peak of triglycerides at 42 rnin was wasted. The contour plot showed that a-tocopherol peak at 71 rnin contained no co-eluted compounds, consequently it was fractionated. 8-tocopherol peak at 84 rnin still had some co-eluted compounds and was therefore recycled again. The peak at 109 min showed that the concomitants were separated from the main component, i.e., R-tocopherol, and consequently, it was fractionated. Collected amounts were measured to be about 1.0 mg for both tocopherols.

Peak assignment and purity estimation by UV spectral data Peak assignment by UV spectral data. Figures 7A and 8A show the UV spectra of a- and R-tocopherols in the first cycle, i.e. without recycling, taken at 13.51 and 15.35min, respectively. Figures 7B and 8B show the spectra after recycling taken at 108.9 and 69.51min. Figures 7C and 8C are the spectra of the standard tocopherols obtained in a separate experiment. As can be seen, the spectra A are severely distorted in the region below 230nm by co-eluted compounds and steep rises toward shorter wavelengths are seen in both spectra. R-tocopherol was well purified after one additional cycle and the spectrum at 69.51min shows a clear inflection point at 220nm, which is also seen in the standard spectrum shown in Fig. 8C. On the other hand, a-tocopherol required two more cycles to be separated from co-eluted compounds that have relatively strong UV absorption at 210nm. The spectrum at 108.29min also exhibits a clear shoulder as shown in Fig. 7B.

139

Figure 7. UV spectra of a-tocopherol peak in the first cycle A , in the last cycle B (fractionated), and the standard C.

140

Figure 8. UV spectra of 8-tocopherol peak in the first cycle A, in the last cycle B (fractionated), and the standard C.

141

Purity check by ratio chromatogram. In order to check the purities of the fractions, the peaks monitored just before fractionation were examined closely by using the data processor of the photodiode-array UV detector. Figures 9A and 9B show normal chromatograms at 230 and 295nm, and ratio chromatograms between the above wavelengths, 230/295 over the time ranges from 107 to 11lmin for a-tocopherol and from 68 to 7lmin for J3-tocopherol, respectively. The ratio chromatogram shown in Fig. 9A exhibits a very rectangular shape. This means that the ratio of the UV absorptions at 230nm by 295nm remains constant. The absorption at 230nm potentially represents tocopherols, fatty acids and their esters, and the absorption at 295nm represents only tocopherols. Therefore, spectrometrically, this suggests that the UV spectrum along the time axis over the peak does not change its shape but only the intensity changes, indicating the absence of components having different UV spectra. The ratio chromatogram in Fig.9B also shows a rectangular shape. However, it is slightly convex at the top, indicating that there is a small amount of other compounds which give rise to a higher absorption at 230nm at the middle part of the peak. This peak may include B-tocopherol as the main component and a minor impurity, possibly free fatty acids and/or their esters. For reference, a ratio chromatogram obtained in the first cycle under the same data processing conditions is shown in Fig. 10. Tocopherols are considered to be eluted in the time range from 12 to 17min. However, the ratio chromatogram in this range is very unstable and wavy, as a result of co-eluted compounds.

Estimation of tocopherol contents in peaks. The amount of the tocopherols included in the aboifc peaks were calculated by the following procedure. Necessary values are listed in Tdble 2.

Tmble 2. Values used in the calculation of amounts of tocopherols.

Mol. weight Mol. absorp. MW E a-tocopherol 430.7 32.6 x lo3 (292nm) B-tocopherol 416.3 37.2 x lo3 (296nm)

Peak area 24.80 AU x sec 7.33 AU x sec

Peak height 0.430 AU 0.202 AU

Molecular weights (MW) and molar absorption coefficients ( E ) are taken from the literature [16]. Other values were obtained from the experimental results. The amounts of a- and B-tocopherols included in the peaks were calculated by using the above peak areas, peak heights, molar absorption coefficients, optical path length (0.5cm) and the flow rate (S.OmL/min) set for the in-line pump, assuming that the concentration distributions of the peaks are Gaussian. Obtained values are 4.9mg for a-tocopherol and 1.3mg for B-tocopherol. The above calculation was based on the assumption that the absorption coefficients in the liquid solvent (ethanol) were similar to those in supercritical carbon dioxide having a similar density. The total amount of tocopherols, 6.2mg, is in fair

142

agreement to the amount of tocopherols in the injected oil, which is, about 4mg. The amounts of fractionated tocopherols were weighed to be about lmg for both a- and Rtocopherols after eliminating ethanol, used as a modifier, by evaporation. These values do not agree with the above values, and only about 1/3 to 1/2 of the tocopherols in the peaks were recovered. The reason for this disagreement is considered to be that a part of the tocopherols was taken out by ethanol mist in carbon dioxide vented to the waste line.

8.00

R.*lO

0.01

LI

B

.

0.01 I

1

Figure 9. Ratio chromatograms (230nm/295nm) of a-tocopherol peak A and R-tocopherol peak B.

143

1.88

nau

I

L

Figure 10. Ratio chromatogram of the tocopherol rich portion of the first cycle.

Identification by IR spectra In order to confirm the pc ak assignment by UV spectra, IR spectra of the fractionated and the standard tocopherols were measured by employing the diffuse reflectance (DR) method. Figures 11A and 12A show the measured IR spectra of fractionated a- and B-tocopherols, respectively. Figures 11B and 12B are the spectra of the standard ones. These spectra are background compensated by subtracting the blank spectrum from the measured ones. As shown, the measured spectra are almost identical to those of the standards. The characteristic IR absorption bands are listed in Table 3 [17].

lkble 3. Characteristic IR absorption bands of a- and B-tocopherols. OH

a-tocopherol B-tocopherol

CH

3310 2930 3320 2940

C=C CH3, CH2 Aroma.

C-0 aryl

C-0 CH alkyl

1620 1460 1375 1270 1160 850cm-' 1595 1458 1373 1229 1158 860

144

All the absorption bands appeared in the standard and in the measured spectra except for those due to the hydroxyl (OH) group. This may be due to the over-compensation by the absorption by H,O in KBr and in the environment. Accordingly, fractionated substances are confirmed to-be a- and B-tocopherols. ? ?!!

E.

I

h

A

.

I . . . . I . . . . I . . . . 1

O

i

l

!

!

.

. .

I

B

,

.

.

.

1

.

,

. .

9

Figure 11. IR spectra of the fractionated a-tocopherol A and the standard B.

I

E

14

145

However, in addition to the characteristic bands, weak absorptions at about 1720cm.' are observed in all the spectra. This may be due to carbonyl (C=O) groups, which give rise to a strong band at 1900-1550cm-' by the stretching of the C = O bond. This suggests one of two things; 1) very small amounts of free fatty acids and/or their esters exist as impurities, or 2) a small portion of tocopherols was already oxidized.

at

,

,

1

,

(

,

,

1

,

I

,...

I , , , , ,

,

,

,

,

. ,

,

,

,

,

,

,

1

I

814

a

c

?

h

8

t

A

i

W

W

w_

1

Figure 12. IR spectra of the fractionated B-tocopherol A and the standard B.

814

146

Gas chromatographic analysis of fractionated tocopherols So far, the fractionated tocopherols were investigated by spectrometric methods, and the results were: 1) identification was achieved by IR spectrometry, 2) tocopherol purities are very high, 3) IR spectra suggest the presence of compounds with carbonyl group in their structures. Prior to the GLC analysis of the fractionated tocopherols, the standard mixture containing various triglycendes, and the SFE extracted wheat germ oil were separately injected to establish GC conditions that could elute these compounds. The conditions established were as: initial temperature = 250°C; temperature ramp rate = 10"C/min; initial time = Omin; final temperature = 350°C; final time = 8min; injector temperature = 300°C; detector (FID) temperature = 350°C.

I I

a-tocopherol

4

d

LA....-.

Figure 13. GLC chromatogram of the fractionated a-tocopherol.

147

Figure 13 shows the GLC chromatogram of the fractionated a-tocopherol. As can be seen, there is only one main peak and a series of very small peaks. The main peak is of atocopherol and small ones may be of fatty acid esters including mono- and di-glycerides. The IR spectrum shown in Fig. 11A supports this assumption. The purity of a-tocopherol is determined to be about 85% based on the area percentage, i.e., assuming that the response factors for the detector (FID) and the injection split ratios of a-tocopherol and impurities mentioned above are identical.

4

E-tocopherol

N

I

Figure 14. GLC chromatogram of the fractionated R-tocophero,.

148

Figure 14 shows a comparable chromatogram of 0-tocopherol. There is also only one main peak and a series of very small peaks. However, the retention times of the impurity peaks are very different from those in a-tocopherol. These may be fatty acid esters possibly mono- and diglycerides with higher polarities. The purity of 0-tocopherol is determined to be about 70% by area percentage, assuming the same conditions for atocopherol.

CONCLUSION With the aid of the PDA multiwavelength UV/VIS detector, isolation of tocopherols from wheat germ oil by recycle prep-SFC was successfully achieved. The primary qualitative analysis was performed by using the built-in after-run spectrometric/chromatographic data processing function of the detection system. Infrared spectrometry confirmed these results. The purities of a- and R-tocopherols were quantified by gas chromatography to be about 85 and 70%, respectively. These values can be improved by performing a few additional cycles. Recoveries during the fractionation of peaks are only 30 to 50%, which is not satisfactory. This was provably because the compounds were taken out by the ethanol mist in the vent line. In order to improve the recoveries, a cold trap may help. Since on-line three-dimensional chromatogram monitoring permitted examination of spectra of peak components as they were being separated on the column, the PDA detection system proved to be very useful for fractionation in recycle prep-SFC. As demonstrated, the method described in this Chapter is very suitable for the isolation of fat-soluble compounds from complex matrices. It is also suited for the separation and fractionation of compounds which have generally small a values, such as optical isomers, and therefore the separation cannot be improved by varying the mobile phase conditions, i.e., by a change in density, temperature or the addition of a modifier solvent. In such a case, the column length has a major effect on the resolution. Recycle prep-SFC offers a high efficiency, because the pressure drop A P is minimized, regardless of the number of recycles, this is virtually equivalent to using a long column with a minimum pressure drop.

Acknowledgement Permission from the following copyright owners to reproduce materials from previously published sources is acknowledged and appreciated: Dr. Alfred Heuthig Publishers, Heidelberg, Germany and Elsevier Science Publisher, Amsterdam, The Netherlands.

149

REFERENCES 1 K. Sugiyama, M. Saito, T. Hondo and M. Senda, J. Chromaatogr., 332(1985)107. 2 K. Jinno, T. Hoshino, T. Hondo, M. Saito and M. Senda, Anal. Chem., 58(1986))2629. 3 M. Saito, T. Hondo and Y. Yamauchi in R.M. Smith(ed), Supercritical Fluid Chroamtography, The Royal Society of Chemistry, London, 1988, pp.203-303. 4 M. Saito and Y. Yamauchi, High Resolu. Chromatogr. Commun., 11(1988)741. 5 M. Saito, Y. Yamauchi, K. Inomata and W. Kottkamp, J. Chromatogr. Sci., 27( 1989)79. 6 T. Hoshino, T. Hondo, M. Senda, M. Saito and S. Tohei, J. Chromatogr., 316(1984) 473. 7 T. Hoshino, T. Hondo, M. Senda, M. Saito and S. Tohei, J. Chromatogr., 332(1985)139. 8 E. Klesper, A.H. Corwin and D.A. Turner, J. Org. Chem., 27(1962)700. 9 R.E Jentoft and T.H. Gouw, Anal. Chem., 44(1972)681. 10 M. Perrut and P. Jusforgues, Entropie, 132(1986)3. 11 Y. Yamauchi and M. Saito, J. Chromatogr., 505(1990)237. 12 S. Higashidate, Y.Yamauchi and M. Saito, J. Chromatogr., 505(1990)295. 13 P.J. Schoenmakers and L.G.M. Uunk, Chromatogruphia, 24(1988)51. 14M. Saito, Y. Yamauchi, H. Kashiwazaki and M. Sugawara, Chromatographia, 25, 9, (1988)801. 15 M. Saito and YY. Yamauchi, J. Chromatogr., 505(1990)257. 16 Chemical Society of Japan, Kagaku Binran, 1975, pp.350-351. 17 J.F. Pennock in R.A. Morton (ed),Biochemistty of Quinones, Academic Press, London, 1965, p.67.

This Page Intentionally Left Blank

K. Jinno (Ed.), Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction Journal of Chromatography Library Series, Vol. 53 0 1992 Elsevier Science Publishers B.V. All rights reserved.

151

Chapter 9 INDUCTIVELY COUPLED PLASMA ATOMIC EMISSION SPECTROMETRIC DETECTION IN SUPERCRITICAL FLUID CHROMATOGRAPHY Kiyokatsu Jinno School of Materials Science Toyohashi University of Technology Toyohashi 441, JAPAN

Supercritical fluid chromatography (SFC) is rapidly growing technique as an alternative to gas chromatography (GC) and high performance liquid chromatography (HPLC) [l-31. Because supercritical fluids have properties intermediate between those of gases and liquids, the selectivity of SFC is resemble to that of GC or HPLC depending on the volatility and solubility of the compounds to be separated and also the operational temperature and pressure of the supercritical fluid. Popular GC and HPLC detectors such as ultraviolet absorption (UV)[4-6] and flame ionization (FLD)[7-10] can be useful in SFC. In addition to this advantage, information rich detectors such as mass spectrometers (MS) [ I 1-13] and Fourier transform infrared spectrometers (FTIR) [14-161 have been interfaced with SFC. However, none of these hyphenation provide elemental information on eluted components, and atomic emission spectrometric (AES) techniques are excellent candidates as information-rich SFC element selective detectors. Plasma based detectors are promising because they have low detection limits, high precision and/or accuracy, wide dynamic ranges, and capabilities for simultaneous multielement analysis. Complete chromatographic resolution is not required because of their elemental selectivity. Microwave induced plasmas (MIP) have been shown to be useful element selective detectors for GC [ 17-19] and a similar approach can be applied to capillary SFC. Near-infrared detection combined with MIP is a powerful and informative detector in capillary SFC for organo-chlorine and sulfur containing compounds [20,21]. A radio frequency plasma detector showed high sensitivity for pesticide analysis with capillary SFC [22]. Well established inductively coupled plasma (ICP) provides an alternative mode for chromatographic detection in SFC. HPLC-ICP interfacings involved either a spray-chamber system coupled with a cross-flow [23] or a concentric nebulizer [24], or alternatively "no-spray chamber" systems using either a cross-flow nebulizer [25] or a microconcentric nebulizer [26]. These interfaces are also expected to work well in SFC. Recently Olesik and Olesik [27] reported introduction of a supercritical fluid into an ICP-AES instrument. A quartz capillary tube with a 1-15 pm terminal restriction was inserted into the central tube of a conventional ICP-AES torch less than 10 mm below the discharge region. In this chapter, the author describes the results of the investigations on an interface for coupling SFC with ICP-AES of different design from those developed for HPLC-ICP and FIA (Flow Injection Analysis)-ICP.

INSTRUMENTATION A typical set-up of the instrumentation is shown in Figure 1 [28]. In this study, the mobile phase was pure carbon dioxide or carbon dioxide modified with polar solvents. In order to fill and pump the mobile phase smoothly, the pump head was cooled to

152

ca. -5°C by a methanolfwater mixture which was directed through a jacket by a circulating pump. A piece of stainless-steel (SS) tubing placed inside the jacket served as a precooling coil. Samples were injected with an injection valve with a 0.5 pL sample loop (at room temperature). The columns were a 25 cm x 1 mm i.d. S S tube and a 25 cm x 0.53 mm i.d. fused silica capillary packed with a diol bonded phase by the slurry technique. The column temperature was maintained somewhat above the critical temperature of the mobile phase used. For optimization experiments, an empty pseudocolumn, a 25 cm x 0.8 mm i.d. S S tube, was used. The columns were placed in an oven kept at a constant temperature. A 50 mm x 0.1 mm i.d. S S tube was used to connect the column to the injector. The column effluent was directed through a resmctor where direct nebulization into the plasma occured. Carbon dioxide used had a purity of ca. 99.9 % methanol and acetonimle (LC grade) were used as modifiers. Solvent mixing of the liquid carbon dioxide and the modifiers was accomplished "off-line" with a S S tank equipped with a stop valve and a short length of a connecting tube. First, a given amount of modifier was introduced into the tank through the connecting tube. The liquid carbon dioxide was then pumped into the tank, which was cooled by ice-water, with the pump. After disconnecting the tank from the pump, the two fluids were mixed by shaking the tank vigorously by hand. The tank was then connected to the pump to deliver the resultant mobile phase. The molar fraction of the modifier was calculated from the weights of the two fluids [29].

NEBULIZER

Figure 1. Schematic diagram of the SFC-ICP system. Components:a=mobilephase reservior, b=precooling coil, c=pump, d=injector, e=microcolumn, f=ICP-AES torch, g=slit, h=monochromator, i=resmctor, j=nebulizer gas inlet, k=flexible heater.

153 The column pressure was adjusted by setting the pressure of the pump, and thus the mobile phase flow rate was the independent variable. With this arrangement, the flow rate of the mobile phase through the column was calculated from the gas flow, which is the only measurable parameter. A inductively coupled plasma spectrometer equipped with a 1 m Czemy-Turner monochromator was used. In the series coupling study of SFC and ICP-AES, a UV detector was employed to monitor separations. The UV detector was inserted between the outlet of the column and the restrictor. A flow restriction is needed to achieve supercritical conditions throughout the column. For this purpose, the restrictor shown in Figure 2 was used. It consisted of a glass capillary (35 mm x 50 pm i.d. x 0.5 mm 0.d.) with a terminal restriction of ca. 5 pm. The restrictor was inserted into a SS tube (35.5 mm x 0.8 mm i d . x 1/16 inch 0.d.) to provide mechanical durability. The resmctor-tip design is similar to that described by Guthrie and Schwartz [30]. The outlet of the restrictor was placed within a laboratory-made ICP cross-flow type nebulizer. Thus the restrictor can serve as a sample transfer line and sample introduction tube to the ICP nebulizer, in addition to its original function. Two points appear crucial to the design of the SFC-ICP interface; minimization of extra column band-broadening and maximization of the efficiency with which effluent is introduced into the plasma. Because the interface or "nebulizer" investigated in this work has no spray-chamber, extra-column zone broadening is much less significant than with conventional ICP sample introduction systems, where a spray-chamber is used for nebulization. A flow restrictor, a nebulizer gas tube (0.25 mm i.d., 1/16 inch 0.d.) and a sample introduction tube (0.5 mm i.d., 1/16 inch 0.d.) were installed to the three-way union. In order to prevent icing by the Joule-Thompson effect that occurs when supercriticalcarbon dioxide expands to atmospheric pressure, the entire nebulizer was warmed with a flexible heater (k in Figure 1).

-

To Column

b

C

\

/

a

-

To Nebulizer

Figure 2. Schematic diagram of restrictor. Components:a=Pyrex glass capillary (35 mm x 50 pm i.d., 0.5 mm 0.d.) with an opening of ca. 5 pm, b=stainless steel tube (34.5 mm x 0.8 mm id., 1/16 in.o.d.), c=epoxy resin.

154

System Performance In SFC-ICP interfacing the signal intensity measured by ICP is strongly dependent on chromatographic conditions. Therefore, effects of the chromatographic conditions in SFC on the emission signal intensity has been investigated using the pseudocolumn under chromatographic conditions as like so-called supercritical FIA analysis. Solutions of ferrocene (Fe, 120 n&L) were injected into the column and the emission signal intensity of the 259.94 nm Fe I1 emission line was observed with the ICP-AES system. The nebulizer temperature has been changed at a column pressure of 160 atm and the obtained emission intensities for multiple injections of ferrocene solution are illustrated in Figure 3. With the nebulizer at room temperature, "spiking" was observed in the signals and reproducibility was poor (Figure 3A). The spiking is caused from intermittant freezing at the restrictor exit by Joule-Thompson effect. In contrast, warming the nebulizer to 40°C gave reproducible peaks without spiking(Figure 3B), and a relative standard deviation of 2.1 % from six replicate injections. Similar reproducibility at 50°C and 60°C to that at 40°C was found.

A

Figure 3. Reproducibility of emission signal intensity. Sample:0.5 pL of 0.4 pgIc1L ferrocene in dichloromethane, nebulizer temperature:(A) room temperature, (B) 40°C. Column pressure: 160 atm, temperature: 40°C.

155

The nebulizer Ar gas flow rate and the viewing height are also important parameters. Peak heights measured at various nebulizing gas flow rates and viewing heights have been measured and the results indicated that the nebulizing gas flow producing the highest net emission intensity is the optimum. The data also indicated that for greater viewing height the optimum gas flow rate slightly shifted to the lower values. At very low nebulizing gas flow rates, the amount of sample introduced is too small to give an adequate signal. At higher gas flow rates, the ICP plasma is cooled by the increase of the Ar gas loading and the conditions for dissociation and ionization become unfavorable. It is also reasonable to consider that the residence times of ions in the plasma become shorter as the flow rate increases. Column pressure is a most important parameter to control retention in SFC. Because the solubility of a solute is a function of the density of the supercritical mobile phase, the partition coefficients can be controlled by varying the pressure of the chromatographic system.The pressure was varied between 100 and 200 atm, andthe heights and areas of the ferrocene peaks were measured by the SFC-ICP system. The results are shown in Figure 4.

100 10

Column Pressure (otin) 120 140 160 180 2001220

15 C 0 2 Cns Fluw ( i i i l l n i l n )

20

Figure 4. Relative peak height and peak area vs. column pressure.

156 The carbon dioxide gas flow rate was calculated from the peak position by means of the FIA technique. The flow rate of the mobile phase was estimated to range from 26 to 43 pL/min as column pressure measured from 100 to 200 atm. While the peak heights increase with the flow rate, the peak area is centered about a constant value. This data indicate that the ICP works as a mass-sensitive detector. However, it is noteworthy that the peak height at 10 mL/min gas flow is less than half of that observed at 20 mL/min. This is because, at low flow rates, the solute band is more spread out as it passes through the column. Ferrocene solutions with concentrations ranging from 50 ng/FL to 2.5 pg/pL (i.e., Fe ranging from 7.5 ng to 375 ng) were injected into the pseudocolumn and the signal response was found to have a linearity over about two orders of magnitude; the detection limit was about 7.5 ng Fe at S/N=4. The linear range would be expected to be further expanded if ferrocene were soluble in dichloromethane at concentrations above 2.5 pg/pL. The chromatographic performance was evaluated for the 1 mm i.d. separation column. The effect of the nebulizing gas flow rate on the emission intensity of the 259.94 nm Fe line was measured at different viewing heights between 5 mm and 20 mm. The maximum signal intensity of the Fe component was observed at a viewing height of 10 mm and a nebulizing gas flow rate of 0.35 L/min. To demonstrate the potential of the ICP-AES detection system, a mixture of ferrocene derivatives was separated on the diol 1 mm i.d. column at 4OoC and 160 atm inlet pressure (flow rate was ca. 38 pL/min) under the above optimum condition. The response monitored at the 259.94 nm Fe emission line is shown in Figure 5B. which shows well-defined peaks. A UV chromatogram for the same separation is shown for comparison (Figure 5A). It appears that the chromatographicperformance is almost identical by both detection methods.

u n

I

ICP 259.94nm

I

b

uv 21onlll b

b

50

40

30

20

Tlme(min)

I

8

r

10

0

50

40

30

I

I

I

20

10

0

Ti me(inin)

Figure 5. Microcolumn separation of synthetic mixture of ferrocene. A : chromatogram monitored by UV at 210 nm, B : chromatogram monitored by ICP at 259.94 nm. Peak:a=ferrocene (0.4 pg/pL, 0.12 pg/pL Fe), b=acetylferrocene (1.5 pg/pL, 0.37 pg/pL Fe),c=benzoylferrocene (1.5 pg/pL, 0.29 pg/pL Fe).

157

Modifier Addition The solvent strength of supercritical carbon dioxide changes markedly on increasing the density of the fluid. An alternative way to increase the solvent strength of the mobile phase is to add small amounts of miscible polar organic solvents as modifiers ; this offers operational flexibility since the modifier concentrations are easily varied. To know the effect of modifier addition to the ICP signals, the 0.4 pg/pL ferrocene solution (Fe: 120 ng/pL) being successively injected into the pseudocolumn, where methanol and acetonitrile were used as the modifier. Obtained emission signals are shown in Figure 6, in which A and B indicate results with 8.0 mol % methanol and 7.3 mol % acetonitrile in carbon dioxide, respectively. The relative standard deviations of the data were 2.4 % and 3.3 %, respectively.

B

A

- . . ,,.

.

)-.

.

H 6111i 11

Figure 6. Reproducibility of the emission signal intensity. (A) mobile phase, 8.0 mol% methanol in carbon dioxide. (B)mobile phase,7.3mol% acetonitrile in carbon dioxide. (Reproduced with permission from reference 31. Copyright 1989 Aster Publishing )

158

The effect of sample gas flow rate on the emission intensity was studied while varying the viewing height from 5 to 15 mm above the turn of the load coil. The mobile phase was 8.0 mol % methanol in carbon dioxide. In Figure 7, the maximum emission intensity is seen at a viewing height of 10 mm and nebulizing gas flow rate of 0.35 L/min, which are the same as those for pure carbon dioxide. Addition1 experiments also indicated that modifiers such as methanol and acetonitrile give little or no change in the optimum nebulizing gas flow rate.

c

0

Nebulizer Gas Flow (L/tiiiti)

Figure 7. Relative peak height vs. nebulizing gas flow rate. Viewing height: a= 5 mm, b=10 mm, c=15 mm. (Reproduced with permission from reference 31. Copyright 1989 Aster Publishing)

159

A mixture of three ferrocene derivatives was separated on the 1 mm i.d. diol column with 2 mol % methanol in carbon dioxide (Figure 8). The addition of small amounts of modifier results in improved peak shape, reduced retention times, and enhanced separation compared with the chromatographic behavior found in using pure carbon dioxide mobile phase (Figure 5B).

b

I c I

20

I

10

c

'lime( niin)

1

Figure 8. Microcolumn SFC-ICP chromatogram of a svnthetic mixture of ferrocenes. Peaks:a=ferrocene,b=acetylferrocene, c=benzoylfemocene (ReDroduced with Demission from reference 31. Coiyright 1989 Aster Publishing)

160 Smaller I.D. Columns for SFC The advantage of changing modifiers in SFC-ICP is most significant when the analytes span a wide molecular weight/polarity range. Since the total effluent enters the ICP torch, quantitation should be straightforward. Also, it is expected that background signal changes due to organic modifiers will be small. However, knowledge of the loading limit of carbon dioxide into the SFC-ICP system is required for packed column SFC with conventional columns of which typical size is 4.6 mm i.d. x 250 mm long for HPLC separations. The loading limit has been examined to learn the highest mobile phase flow rate usable in this SFC-ICP system. Carbon dioxide was added to the ICP torch through the tee positioned behind the nebulizer. The ferrocene solution (Fe: 120 ng/jkL) was injected into the pseudocolumn at an inlet column pressure of 160 atm, and column temperature of 4OOC. The flow rate of gaseous carbon dioxide from the restrictor was ca. 26 mL/min. The emission intensity was constant in the gas flow range of 26-50 mL/min, at 55 mL/min it was 75 % of that in the 26-50 mL/min region, and the plasma was extinguished at 60 mL/min. Therefore, it was concluded that the upper loading limit of carbon dioxide is 50 mL/min, which corresponds to a supercritical fluid linear velocity of 4.0 mm/sec with a 1 mm i.d. microcolumn. It is noteworthy that ca. 40 min was required for the separation when the 1 mm i.d. column was used in conjunction with a linear velocity of 2.0 mm/sec as shown in Figure 5. For more rapid analysis the smaller i.d. columns are preferred. For example, when column diameter is reduced from 1 mm i.d. to 0.5 mm i.d., the linear velocity should be increased by a factor of 4. Hence the time required for separation should be decreased by a factor of 4. The loading limit of carbon dioxide into the ICP, 50 mL/min, corresponds to a supercritical fluid flow rate of 110 pL/min and a linear velocity of 16 mm/min with a 0.5 mm i.d. column. The effects of flow rate of the nebulizing gas and column pressure on peak height, peak area, linear dynamic range, reproducibility and detection limit were studied for the 0.5 mm i.d. column. To test reproducibility of the SFC-ICP system, 0.2 pg/pL ferrocene (Fe: 60 ng/pL) solution was injected into the column maintained at 160 atm. The response peaks obtained for the repeated injections gave a 1.8 % relative standard deviation of the peak height. The lowest detectable concentration at a 0.5 pL injection was 20 ndpL for ferrocene (Fe: 6 n&L) at a signal-to-noise ratio of 4. The similar optimum conditions with 0.5 mm i.d. columns to those with 1 mm i.d. columns were found for Ar nebulizing gas flow rate and the viewing height. The effect of the column pressure on the emission intensity was also investigated. The column pressure and the nebulizing gas flow rate were varied from 120 atm to 200 atm and from 0.2 L/min to 0.5 L/min, respectively. The pressure over the column was adjusted by controlling the pressure of the pump. When the pressure was vaned from 120 atm to 200 atm, the gas flow rate, which had been monitored independently at the exit of the restrictor, changed from 22 mL/min to 30 mL/min. This means that the mobile phase flow rate changed from 57 pL/min to 78 pL/min. It has been found that the maximum peak intensity is obtained using the nebulizing gas flow rate of 0.35 Wmin, regardless of column pressure. As discussed above, the ICP detector is mass sensitive, even if various parameters of SFC such as the column pressure, the column temperature and the modifier concentration, are changed. To investigate the effect of such parameters on the peak area, a 0.1 p d p L ferrocene solution (Fe 30 ndpL) was injected into the diol column and the column pressure was changed between 120 atm and 200 atm. The peak area was calculated from the resultant peaks (Figure 9(A)). Then, the column temperature was changed between 4OoC and 65OC while maintaining the column pressure at 160 atm (Figure 9(B)). In Figure 9(C), the effect of the modifier concentration (methanol in this case) on the peak area is also demonstrated, where methanol concentration was increased up to 15 mol %. As seen in Figure 9, the ICP detector serves as the mass-sensitive detector for SFC. Linear dynamic range was also examined. Ferrocene solutions containing from 10 ng to 1.25 pg were injected into the 0.5 mm i.d. diol column at a pressure of 160 atm. The peak height increases linearly from the detection limit, 10 ng ferrocene (Fe: 3 ng) to 2.5

161

pg ferrocene (Fe: 375 ng).

t

0

B

2

?

4

L 5 1 0 1 5 :

L . 140 180 Column Pressure (atm)

Loll

0

Column Temperature ("C)

Methanol Concentntion ( mol% )

Figure 9. A:Effect of the column pressure on the relative peak area at a column temperature of 40 "C. B:Effect of the column temperature on the relative peak area at a column pressure of 160 a m . C:Effect of methanol modifier concentration on the relative peak area at a pressure of 160 atm and the temperature of 55 "C for 1,2 and 3,65 "C for 4.(Reproduced with permission from reference 29. Copyright 1990 Aster Publishing). To demonstrate the potential of the 0.5 mm i.d. column a mixture of ferrocene derivatives was separated on the diol column (0.5 mm i.d. x 250 mm length) with carbon dioxide as the mobile phase [31]. The separation is completed within 8 min as demonstrated in Figure 10, much more rapidly than with the 1 mm i.d. column. The use of 2 mol % methanol modifier and the 0.5 mm i.d. column enables the analysis to be completed within 4 minutes with same loss in resolution. There is no doubt that higher velocities of the mobile phase can enhance the absolute detection limit of the ICP detector. As discussed above, in SFC, pressure is a very important parameter because of its ability to change the solvating properties of the mobile phase significantly and thus, pressure programming is a means of changing and extending the effective eluting power of the supercritical fluid mobile phase. Separation of four ferrocene derivatives was performed with step-wise pressure programming; column pressure was maintained at 100 atm and raised to 160 atm after 4 min. The chromatogram obtained is shown in Figure 11. At 100 a m , ferrocene and n-butylferrccene were eluted separately but acetylferrccene and benzoylferrocene were not eluted from the column. Separation of Organometallic Compounds SFC-ICP detection of metal acetylacetone (acac) complexes was performed to confirm its potentialal in metal selective monitoring. Wenclawiak et al. 1321 have described the separation of metal acac complexes by SFC. They used a conventional LC column packed with silica or octyl bonded silica and a binary solvent mixture of up to 30 mol % methanol or ethanol in carbon dioxide. Taylor et al. [33] reported the SFC separation of metal acac complexes, where conventional LC columns were used with carbon dioxide modified with 20 wt % methanol. They noted that two different chromatographic behaviors were observed, dependent on the specific metal coordinated to the acetylacetone ligand. Inert complexes probably associate through the outer coordination sphere, whereas labile complexes may interact with the stationary phase directly through the metal, after loss of one of the peripheral ligands. However, because they used a UV detector, the retention time was the only available information. Speciation of the eluates is

162

Figure 10. Microcolumn SFC-ICP chromatogram of a synthetic mixture of ferrocenes. mobile phase: carbon dioxide, 160 a m , 40 "C. detection: at 259.94 nm. Peak: a=ferrocene,b=acetylferrocene, c=benzoylferrocene (Reproduced with the permission from reference 29. Copyright 1990 Aster Publishing) 0

2

4

6

8

1

0

ICP(Fe 11,259.94nni)

Time (mln)

a

I

C

I

.

.

.

.

.

1 4 1 2 1 0 s 6 4 Tiiiie,iiiin

.

*

2

0

Figure 11. Stepwise pressure gradient separation of a synthetic mixture of ferrocenes. Peak:a=ferrocene, b=n-butylferrocene, c=acetylferrocene,d=benzoylferrocene. Pressure:O-4 min at 100 atm, then 160 atm.(Reproduced with permission from reference 31. Copyright 1989 Aster Publishing).

163 possible using element-specific detection. To demonstrate this approach, SFC, UV, and ICP were coupled in series. The metal acac complexes were injected into the column. A typical chromatogram for chromium and cobalt is shown in Figure 12. Other metal complexes, Al(acac)3, Cu(acac)2, Fe(acac)2, Mg(acac)2, Mn(acac)2 and Zn(acac)2 showed considerable tailing in the UV chromatograms, but no peaks obtained in the ICP chromatograms. The same results were observed when a solution of acetylacetone was injected into the column. These results suggest that the tailing peaks are due to the ligand liberated from the complex and thus it appears that Co(acac)2 and Cr(acac)3 are stable while the other metal acac complexes investigated decomposed under the SFC conditions used.

ICP

Cr 11, 228.6211111

a

I

L

C O 11, 267.7211111 Figure 12. Element-selective detection of

*saJ 30

20

I0

0

metal acac compounds. Peak:a=Cr(acac), b=Co(acac) Column: 1 mm i.d., mobile phase:2 mol% methanol in carbon dioxide, pressure:160 atm, temperature: 40 "C (Reproduced with permission from reference 3 1. Copyright 1989 Aster Publishing).

'I'iine(iiiit1)

In order to determine the stability of various acac complexes under supercritical conditions, UV spectra were measured using FIA combined with the UV multichannel detector [34]. Mobile phases in the FIA measurements were dichloromethane(A), supercritical carbon dioxide (B, 140 atm, 50°C) and carbon dioxide modified with 5 mol % methanol (C, 140 atm, 50°C). Some selected spectra are shown in Figure 13. Comparing UV spectra of acac complexes to that of the acac ligand under these three conditions, shows differences among the spectra which reflect the stability of the complexes. One can obtain the following information about the stability of acac complexes. In the dichloromethane environment only Co(acac)2 is unstable, but in the supercritical state all acac complexes except Al(acac)3, Co(acac)3, Cr(acac)3 and Cu(acac)2 are unstable. Those unstable acac complexes are probably decomposed at the high temperature of the supercritical measurements.

164

I I nc ;I c

B

t

vi

m 1

C

C

L

i

200

250

300 Wave Lenglli ( n n i )

I

J

350 Wave Leiigllr ( iiiii )

Figure 13-1. Typical UV spectra of the acac ligand and Co(acac)2 . A:in dichloromethane, B:supercritical carbon dioxide, C:5 mol % methanol with supercritical carbon dioxide.(Reproducedwith permission from reference 35. Copyright 1991 American Chemical Society).

165

Co(B CB c)3

Al(ncnc)3

A

250 Wnve Lcnglli ( iiiil )

35

250 300 Wnve Length ( niii )

350

Figure 13-2. Typical UV spectra of Al(acac)3 and Co(acac)g. A:in dichloromethane, B:supercritical carbon dioxide, C:5 mol % methanol with supercritical carbon dioxide.(Reproduced with permission from reference 35. Copyright 1991 American Chemical Society).

166

Cu(:1mc)2

Cr(ncnc)3

200

250 3w W n v e Lenglh ( nm )

350 200

250 300 Wave Leiigth ( mi )

3511

Figure 13-3. Typical UV spectra of Cr(acac)3 and Cu(acac)2. A:in dichloromethane, B:supercritical carbon dioxide, C:5 mol % methanol with supercritical carbon dioxide.(Reprodnced with permission from reference 35. Copyright 1991 American Chemical Society).

167

The ICP was then connected to the supercritical FIA system to determine whether the components which showed different UV spectra from those of the ligand were correctly assigned as the desired acac complexes. Emission signals were observed for Al, Co(III), Cr and Cu complexes. Other complexes which appeared to be unstable were also examined but emission signals of the metal species were not observed. The separation for those four stable complexes was examined with the SFC-UV-ICP system (Figure 14). Three main peaks appeared, although four components were injected into the system. Spectra of peaks a and b were similar to those of Cr(acac).j and Co(acac)3, respectively. Using ICP detection, two signals were obtained by monitoring the emission lines at 267.7 nm for Cr and 228.6 nm for Co. The retention times were consistent with peaks in the UV chromatogram. Therefore, one can assign the peaks due to Cr and Co complexes. Since the UV spectrum of the peak at 1.8 min in the UV chromatogram is similar to that of the ligand and no signals were observed at this retention time with ICP detection, it is apparent that Al(acac)3 and Cu(acac)%were not eluted as the complexes. When each A1 and Cu complex was injected into the system, it gave the peak at the retention time of the acac ligand

UV 275 nm A.U.

0

0

5

I5

10

MIN

Figure 14. SFC-UV multi-ICP chromatograms of a synthetic mixture of Al, Co, Cr and Cu acac complexes. Mobile phase: carbon dioxide modified with 5 mol % methanol at 140 atm and 50°C. (Reproduced with permission from reference 34. Copyright 1990 Huethig Verlag.)

168

and very similar UV spectrum. The results suggest that A1 and Cu complexes decomposed in the SFC system, and the metals are retained. A notable difference between supercritical FIA (where Al and Cu complexes gave clear signals in UV and ICP detection) and SFC, is an existence of the separation column. Interaction of the complexes with the column packing materials may cause their decomposition under the SFC conditions. Acac was added to the carbon dioxide 1methanol mobile phase, since it is expected that its presence would prevent liberation of the ligand from the complex. A mobile phase of 0.1 mol % acac and 4 mol % methanol in carbon dioxide was used, UV and ICP chromatograms of those complexes being shown in Figure 15. Peaks of each component are seen in the ICP chromatograms, while two peaks appeared in the UV chromatogram. UV spectra could not be obtained because of the high background caused by the absorption of the acac ligand, but the ICP detection indicates that the peak at 8 min in the UV chromatogram contains Al, Cr and Cu

UV 320nni

A.U.

0

320

ICP

I I .

0

5

10 hllN

t

0

5

267.1nm

, 0

10

MlN 324.8nm

b

5

10

hllN

0

5

10 hIIN

Figure 15. SFC-UV multi-ICP chromatograms of a synthetic mixture of Al, Co, Cr and Cu acac complexes. Mobile phase: carbon dioxide modified with acac 0.1 mol % and methanol 4.3 mol % at 140 atm and 5OOC. (Reproduced with permission from reference 34. Copyright 1990 Huethig Verlag).

169 complexes in this sequence and the peak at 10 min is due to the Co complex. This experiment indicated that A1 and Cu complexes are stable in the presence of acac ligand in the mobile phase. The other labile complexes which are unstable in supercritical conditions were not detected with either ICP or UV detectors with these SFC conditions using acac modified carbon dioxide mobile phase.

In conclusion, the SFC-ICP system is an effective tool for speciation of metal-containing compounds, even when the modifiers are added to the supercritical carbon dioxide mobile phase to control the retention of components. An advantage of microcolumn SFC-ICP hyphenation described in this chapter results from the significantly reduced flow rates of microcolumns compared with those of conventional columns, due to the reduced amount of effluents which enter the ICP. In SFC, two basic approaches are used most often to control retention: pressure programming and modifier addition. The results described above have clearly shown that for the SFC-ICP combination, both methods can be useful. The future of hyphenated technique between ICP and SFC will be as a supplementary technique, rather than in direct competition with GC-ICP, because different types of compounds can be separated by SFC and GC. However, information gathered from ICP detection may yield a more effective way to perform elemental analysis of separated components when the detection region is expanded to the near infrared region. The trend of hyphenation will support this future brightful.

ACKNOWLEDGMENTS The author would like to thank to the following people for the contribution to this work, C.Fujimoto, H.Yoshida and H.Mae in Toyohashi University of Technology. This work was supported in part by a Grant-in-Aid for Special Project Research from the Ministry of Education, Science and Culture of Japan under Grant number 61227013. REFERENCES M.Novotny, S.R.Springston, P.A.Peaden, J.C.Fjelstad and M.L.Lee, Anal.Chem., 53(1981)407A2. J.C.Fjelstad and M.L.Lee, Anal.Chem., 56(1984)619A. T.H.Goew and R.E.Jontoft, J.Chromatogr., 68( 1972)303. S.T.Sie and G.W.A.Rijnders, Sep.Sci., 2(1967)726. M.Novotny, W.Bertsch and A.Zlatkis, J.Chromatogr., 61( 1971)17. K.Jinno, T.Hoshino, T.Hondo, M.saito and M.Senda, AnaLChem., 58( 1986)2696. B.W.Wright and R.D.Smith, Chromatographia, 18(1984)542. R.C.Kong, C.W.Woolley, S.M.Fields and M.L.Lee, Chromatographia, 18(1984)362. S.Rokushika, K.P.Naikwadi, A.L.Jadhav and H.Hatano, Chromatographia, 22( 1986)209. S.B.Hawthorne and D.J.Miller, J.Chromatogr.Sci., 24( 1986)2S8. R.D.Smith, H.R.Udseth and B.W.Wright, J.Chromatogr.Sci., 24(1986)238. R.D.Smith, H.T.Kalinoski, H.R.Udseth and W.R.Wright, AnaLChem., 56( 1984)2476. J.B.Crowther and J.D.Henion, AnaLChem., 57( 1985)2711. K.H.Shafer and P.R.Griffiths, AnaLChem., 55(1983)1939. S.B.French and M.Novotny, Anal.Chem., 58(1986)164. C. C.Johnson, J.W.Jordan and L.T.Taylor, Chromatographia, 20(1985)717. C.I.M.Beenakker, Spectrochim.Acta,PartB, 32B( 1977)173. T.H.Risby, Y.Talmi, CRC Crit.Rev.Anal.Chem., 14(1983)231. J.J.Sullivan and B.D.Quimby, HRC., 12(1989)282.

170

L.J.Galante, H.Selby, D.R.Luffer, G.M.Hieftje and M.Novotny, AnaLChem., 60(1988)1370. D.R.Luffer, L.J.Galante, P.A.David, M.Novotny and G.M.Hieftje, Anal.Chem., 60(1988)1365. R.J.Shelton, Jr., P.B. Farnsworth, K.E. Markides and M.L.Lee, AnaLChem., 61(1989)1815. D.M.Fraley, D.A. Yates, S.E. Manahan, D. Stalling and J.Petty, Appl.Spectrosc., 35(1981)525. J.W.Carnahan, K.J. Mulligan and J.A.Caruso, Anal.Chim.Acta, 130 (1981)227. K.Jinno, S . Nakanishi and C.Fujimoto,Anal.Chem., 57( 1985)2229. K.E.Laurence, G.W. Rice and V.A.Fassel,Anal.Chem., 56( 1984)289. J.W.Olesik and S.V.Olesik, AnaLChem., 59(1987)796. C.Fujimoto, H.Yoshida and K.Jinno,J.Chromatogr., 411( 1987)213. K.Jinno, H.Yoshida and C.Fujimoto, J.Microcol.Sep., 2(1990)146. E.J.Guthrie and H.E.Schwartz,J.Chromatogr.Sci., 24( 1986)236. C.Fujimoto, H.Yoshida and K.Jinno, J.Microcol.Sep., l(1989) 19. B.Wenclawiak and F.Bickmann, Z.Anal.Chem., 320( 1987)261. M.Ashraf-Khorassani, J.W. Hellgeth and L.T.Taylor, Anal.Chem., 59(1987)2077. K.Jinno, H. Mae and C.Fujimoto,HRC., 13(1990)13. K.Jinno, H.Yoshida, H.Mae and C.Fujimoto, ACS Symposium series edited by P.C.Uden, Element Selectice Detection in Chromatography, American Chemical Society, Washington D.C., in press.

K. Jinno (Ed.), Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction Journal of Chromatography Library Series, Vol. 53 0 1992 Elsevier Science Publishers B.V. All rights reserved.

171

Chapter 10 MICROWAVE PLASMA DETECTION IN SFC

DEBRA R. LUFFER AND MILOS NOVOTNY Department of Chemistry, Indiana University, Bloomington, IN, 47405, U.S.A. INTRODUCTION The area of detection for SFC has been a very active one because the I1ultimatet1 detector has not yet been discovered. This device would be sensitive, universal, with a potential for excellent selectivity, and inexpensive. Because SFC possesses many characteristics common to both GC and LC, it is potentially compatible with most of the detection schemes pioneered for the latter two. The detection scope of SFC has been significantly expanded by the adaptation of GC-like detectors, primarily because of the advantages inherent to small-bore SFC, both in capillary and packed column systems (1,2). The major selective detection techniques will be described in this chapter, with special emphasis on the microwave-induced plasma detection scheme recently adapted for SFC. Many new detection schemes that have been introduced have attempted to fulfill the criteria of the so-called ltideallt SFC detector. An example of one such detector is the ion-mobility This detector operates in a time-ofdetector (IMD) ( 3 , 4 ) . flight mode, whereby ions created at atmospheric pressure are allowed to drift through a region of constant applied electric field. It was found that the C02 mobile phase could be efficiently eliminated from the detector, which improved the sensitivity. The detector has been considered l1selectiveltfor specific ions by tuning the arrival window to respond only at specific times. However, this detector is not selective in the normal sense, since prior knowledge of the sample components is mandated. The flame-based detectors have proven to be a key to universal detection in SFC, and it was deemed logical to try to adapt any successful model to the element-selective mode of detection. This approach has indeed become feasible since the flame-ionization detector (FID) has been modified for selective detection of various atoms. For example, a metal-selective FID has been recently described for SFC, based on the use of a hydrogen-atmosphere reducing flame (5) The technique appears useful for the detection of organometallic compounds containing a wide variety of metals, although the sensitivity and selectivity differ for each one. A further drawback to

172

this detection scheme is that there does not seem to be any manner of differentiating between different metals in a single sample. Another type of modification to the FID has yielded one of the most sought-after types of selective detector, which is a highly sensitive and completely specific oxygen detector. An example of the potential of this detector is in monitoring the prevalent organic oxygenated materials in petroleum products. The operation of the so-called 0-FID analyzer (6,7) consists of introduction of the effluent into a cracking microreactor, which converts any oxygenate into carbon monoxide. A second catalytic hydrogenation microreactor converts CO into methane, which is detected by the FID. This technique is limited to capillary GC at the present time, and is completely incompatible with even trace amounts of C02. Therefore, it would only be useful for SFC detection if mobile phases other than carbon dioxide could be used, such as NzO. Virtually all of the selective detectors described herein have been I1borrowed1lfrom GC and modified to accomodate the special requirements of capillary SFC. The key to successful adaptation seems to be the ability to tune the detector to specific atoms or functional groups in a highly selective manner. A good example of one such detector is the thermionic detector (TID), also known as the nitrogen-phosphorus detector (NPD), which can be employed for the selective determination of nitro-compounds ( 8 ) , nitrogen-containing compounds and phosphorus-containing compounds (9-12), with minimal modifications to the system. Recently, David and Novotny have shown that very sensitive and selective detection of pesticides and derivatized steroids and prostaglandins is feasible (10-12). In addition, a-ketoacids can be easily analyzed as their quinoxalinol derivatives and detected via the nitrogen-selective mode (9). The flame-photometric detector (FPD) is another example of a successful flame-based element-selective detector, utilizing a hydrogen-rich flame for the analysis of sulfur- and phosphorus-containing compounds (13-15). The chemiluminescent emission arising from the excited molecular S2 and HPO species can be monitored through the appropriate band filters. Since the detection limits of the phosphorus-mode of the thermionic detector are quite good, the FPD has been primarily utilized in the sulfur-mode for SFC. Recent work has evaluated the combination of SFC with FPD for sulfur-containing pesticides, organosulfur drugs, and coal-derived compounds (15). The sensitivity was found to be greater than that of SFC/FID, with only a slight increase in detection limit with increasing molecular weight of the sample. The major drawbacks include a squared dependence on sulfur concentration, and a limited dynamic range. An alternative method of selective sulfur detection for SFC is the sulfur chemiluminescence detector (SCD) (16,17) The sulfur response is obtained from the chemiluminescent radiation produced after a sulfur atom in the analyte reacts with F2 gas. This signal is derived from the resultant HF

.

173

vibrational overtone emission (16). In this case, the linear range is three decades and the signal appears to be independent of the mobile phase flow rate. However, the molar response has been found to be compound-dependent (17). Another selective detector that has been widely used in GC is the electron-capture detector ( E C D ) , which is generally successful for the determination of halogenated and nitrocontaining compounds. Recently, this detector has been coupled to capillary S F C without any modifications (18,19), and evaluated for the detection of chloro-, bromo-, and nitrocontaining compounds. A major drawback of all the detectors described above is that they are limited to certain specific elements, and therefore lack versatility. For example, an attempt was made to modify the T I D for selective detection of the elements B, C1, and S ( 2 0 ) , but with only limited success. Alternatively, organoselenium species have been found to respond in the S C D , although their response is about an order of magnitude less than the corresponding sulfur compound (17). Essentially, the use of at least two of the above-mentioned detectors will cover a fairly wide range of elements. At the present time there exists a need for a single detector that is capable of handling an unlimited number of elements. Of the many varied detection methods for chromatography that have been investigated, those based on atomic spectroscopy (21) have become quite attractive due to their technological availability, inherent selectivity, and multielement detection capability (22). In particular, plasma detectors possess several characteristics that make them even more appealing than flame-based photometric systems: these include improved sensitivity, a wide dynamic range, and fewer spectral interferences. In principle, the selectivity of plasma detectors should be adjustable to various elements simply by “dialing in” the wavelength by either scanning a monochromator, or by selecting an appropriate filter. Simultaneous multielement detection is simply achieved through the use of a multichannel detector, such as a photodiode array. Most importantly, with respect to chromatographic analyses, the plasma detector will often tolerate coelution from a column because it is virtually element-specific (22). Plasma sources are superior to flame-based detection methods because the higher plasma temperatures permit more complete atomization of the analyte. Also, some detectors, such as the FPD, suffer from non-linear response and problems with quantitation. Element-selective plasma-based detection should suffer from neither of these shortcomings, and should also provide the versatility lacking in a single-element detection technique. A radio-frequency plasma detector (RPD) based on the helium afterglow detector described by Fassel and co-workers (23) has been coupled with both GC (24) and S F C (25). The SFC detector has been utilized for non-metal selective detection of sulfur and chlorine the near-infrared spectral region. The helium RPD and this region of the spectrum were projected to be optimal for the sensitive and selective detection of many

174

non-metals. Early results indicate good detection limits (50300 pg/s), but the sensitivities were found to depend on the mass flow of mobile phase entering the detector (25). To date, except for the ICP and the RPD, the only plasma detector that has been coupled with an SFC system is a microwave-induced plasma (MIP) detector. At this point, the helium MIP will be introduced as a selective GC detector in order to set the stage for further discussions with respect to SFC. In 1965, the first article describing the use of the MIP as an elemental emission detector for GC was published by McCormack, Tong and Cooke (26), followed shortly thereafter by Bache and Lisk (27,28). Since then, more than 100 articles have been published on the subject, including some very complete and in formative reviews (29,30). In one publication alone (31), calibration curves and detection limits were reported for the following 29 elements: H, D, V, Nb, Cr, Mo, W, Mn, Fe, Ru, O s , Cot Nil Hg, B, All C, Si, Ge, Sn, Pb, P, As, S, F, C1, Br,, I, and Se. Various atomic and ionic lines in the UV and visible regions were monitored for this study, and the detection limits were typically in the sub-nanogram range. In addition, an oxygen-selective MIP has been described by Bradley and Carnahan (32), which is capable of an oxygen-to-carbon selectivity of 1000 by careful elimination of all oxygen sources. In general, helium MIP sources are typically produced by the application of 2450 MHz microwave radiation of 100 W or less to a flowing stream of helium. The plasma is considered to be self-sustaining as long as the supply of both gas and power are not interrupted. The original plasma sources could be sustained in argon at atmospheric or reduced pressure (5-50 Torr), and in helium at reduced pressure only (33). However, line emission was observed for all elements in the helium plasma, while F, C1, Br, N, and 0, only exhibited diatomic molecular spectra in the argon plasma. Hence, the helium plasma was preferred for non-metal selective detection. To overcome the need to operate the helium plasma at reduced pressure, the TMol0 cylindrical resonant cavity was introduced by Beenakker (34-36). The principal advantage of this configuration is the ability to view the plasma axially (end-on), rather than transversely through the wall of the easily devitrified quartz discharge tube (33). The microwave-induced plasma has been successfully employed as an element-specific detector for GC, and several reviews on the subject are available ( 2 2 , 2 9 , 3 7 , 3 8 ) . The Beenakker cavity may be the most widely used detector for GCMIP, but it is by no means the only one available. For example, there have been reports of a helium MIP generated between two electrodes at atmospheric pressure (39), and at low pressure (40). The obvious advantage of this configuration is the absence of the silica discharge tube, which is degraded during normal usage. In addition, there has been a description of a microwave plasma that is sustained by a surface-wave that is propagated along the length of a discharge tube (41). In this type of cavity, which has been called the Surfatron, the plasma itself is the excitation structure, unlike the Beenak-

175

ker cavity, which is a plasma contained in a waveguide ( 3 4 ) . The Surfatron derives its name due to the energy of the electric field being concentrated at the plasma-dielectric interface where the maximum wave amplitude is localized, and the suffix *#tron" , which implies *'means of sustainmentl', as in electron and neutron. The Surfatron has been found to be more reproducible, more stable, and easier to tune than the TMOI0This type of MIP source has been based discharge ( 4 1 - 4 5 ) . evaluated in-depth for its potential as a robust detector, by Hieftje and co-workers ( 4 6 - 4 8 ) . Table 1 summarizes the GCplasma detector sensitivities of various non-metals obtained with the Beenakker cavity ( 3 5 ) and with the Surfatron ( 4 2 ) . TABLE 1 GC detection limits with two different MIP detectors Detection Limit (pg/s) Beenakker Cavity" Surfatron Cavityb

E1ement C c1 Br S "From reference bFrom reference =See Table 3 .

0.4 7 5 25

2-5 2-4 7-12 C

35. 42.

The Surfatron plasma is relatively inexpensive to operate, and it produces an annular plasma in helium, which is believed to make it less likely to be extinguished by the high mass flow of mobile phase. This type of plasma can operate under low pressure or atmospheric conditions with argon or helium as the support gas, typically at relatively low flow These two rare gases differ mainly in rates ( < 3 0 0 mL/min). their ability to excite elements, such as the halogens, which have reasonably high ionization potentials. Although an argon plasma has higher excitation temperatures, a helium plasma provides higher ionization and excitation energies. In addition, the latter provides more complete molecular fragmentation because of its higher metastable states compared to the former ( 2 2 , 3 0 ) . The Surfatron MIP operates efficiently with low powers ( < 2 0 0 W) and the tuning adjustments are simple, which enables it to be operated with zero reflected power under a variety of experimental conditions ( 4 2 ) . The Surfatron has been developed and investigated by others as a detector for GC (42,44,49), with detection limits that are competitive with other GC detectors, but can be further improved by decreasing the diameter of the discharge tube and/or increasing the microwave power ( 4 2 , 5 0 ) . Recently, a thorough spectroscopic

176

investigation was performed on a He-C02 plasma, whereby detection limits of 6 . 3 ng were found for sulfur using the 545-nm line in the visible region (51). Based on these findings, it seemed reasonable that this detector could be adapted to SFC and thereby provide sensitive and selective detection of nonmetals when a helium plasma source is utilized. In addition, the use of a carbon or hydrogen wavelength channel would offer the potential of universal detection. This chapter discusses the coupling of the supercritical fluid chromatograph and microwave-induced plasma detector. The following section is devoted to a description of a heated interface between the chromatographic system and the plasma, which is the key to the success of this analytical technique. In addition, the SFC-MIP system will be evaluated for the selective detection of sulfur, chlorine, bromine, boron, and the universal detection of carbon. DEVELOPMENT OF THE HEATED INTERFACE Sample introduction can be crucial to the performance of the MIP, since a sample will diffuse through the entire discharge and sometimes around it completely, resting between the discharge and the wall of the quartz tube (30). With an ICP, the use of the small inner diameter central tube in the torch induces a high velocity of the sample aerosol, and forces it into a central channel of the plasma, where it is confined, because of the so-called "skin effect" (30). It is generally believed that a toroidal MIP would allow similar sample confinement and, consequently, maximum sample excitation. To date, the nearest approximation to a toroidal MIP is the annular plasma in helium, which is sustained by the Surfatron (52). However, the sample is not confined in the center of the plasma, as it is in the ICP; it diffuses into the annulus where is atomized and excited more efficiently. Hence, the key to efficient sample excitation is the utilization of a means of sample introduction into the lower energy region (center) of the plasma. Systematic studies of a discharge tube configuration consisting of concentric tubes have been performed (45) with the result that emission intensity is greatest if the plasma gas enters the plasma region solely by way of a tube of smaller inner diameter rather than the discharge tube itself. In addition, it was found that the smaller the inner diameter of this internal tube, the greater is the emission intensity. This is because the optimal conditions of excitation do not depend on gas flow rate, but on the speed of the gas as it exits the inner tube. For this reason, a "mixed-tubell configuration was employed for GC-Surfatron analyses (45), whereby the diameter of the delivery portion of the tube was less than the diameter where the plasma was sustained. From this point it became clear that a llgradualll junction might be ideal in order to enhance the turbulence that is suspected to be the origin of an emission intensity increase associated with this interface design (45).

177

Another factor to be considered in the design of the interface is the requirement that the SFC effluent must be heated as it exits the column. This is necessary to prevent the mobile phase from I1freezingt1 on the tip of the restrictor due to rapid decompression based on the Joule-Thomson effect. This condensation causes poor baseline stability and detector "spiking". Work with other SFC detectors has indicated that a restrictor temperature of 200°C is the minimum recommended temperature, whereas a temperature greater than 300°C assures efficient detection of high molecular weight compounds. It should be noted that simply inserting the restrictor into the plasma is not a suitable solution to the problem, as seen in other studies (25), because the temperature at the tip of the restrictor must be known, stable, and damage to the frit material is extremely likely if the restrictor is too close to the center of the plasma. The design of a successful interface must overcome the l*thermodynamicnightmare" associated with the Surfatron cavity configuration. Specifically, this involves a very hot plasma discharge contained within a quartz discharge tube that is itself contained within an ice-cold water-cooled cavity. The main consequence of this system is the very rapid dissipation of heat generated within the discharge tube, coupled with the significant cooling effect provided by the helium plasma gas. In other words, the goal was the construction of a rugged heating device of very small dimensions that could maintain and sustain a large amount of heat that is just as quickly dissipated. The successful design consists of a very small wire coil that is resistively heated (53). The entire assembly is shown schematically in Figure 1. The very small size of the device ( < 3 mm 0.d.) makes it useful in any type of application where space and accessibility are limiting factors. The coil consists of two very closely wound layers of copper-nickel wire that becomes a region of very high current density when heated. This wire possesses a 260°C maximum temperature enamel coating, thereby allowing the tightly wound coil to be resistively heated; however, the temperature range of the heating coil assembly must exceed 300°C in order to be effective. For this reason, the coil was conditioned with slow increments to the applied voltage, thus permitting the gradual formation of a protective oxide layer capable of sustaining the higher temperatures. The quartz tube insert that surrounds the heating coil and ceramic rod is in the shape of a nozzle. It was mentioned earlier that a gradual junction is necessary for the highsensitivity detection, as the plasma gas acts to llfocusll the effluent into the plasma. In order to test this hypothesis, two injections of a sulfur-containing compound in methanol were performed under identical conditions, except that in one case the nozzle was present and in the other case it was absent. Figure 2 A illustrates the effect of removing the nozzle; both the solvent and solute peaks are severely reduced in intensity, compared to those in Figure 2B.

178

Figure 1. Schematic diagram of the restrictor heating device (not to scale). ( A ) Quartz discharge tube, 4 mm i.d.; (B) Quartz nozzle insert, 3 mm i.d.; (C) Heating Coil, 1 cm long, and coated with high temperature cement: (D) Ceramic insulating tube with 4 holes; (E) Swagelok 1/4 in. nut with Teflon ferrule: (F) Swagelok 1/4 in. reducing union: (G) SGE 1/16 in. male nut silver-soldered to union (F); (H) S G E 1/16 in. female nut with Vespel ferrule; (I) Frit restrictor sleeved through and terminating at (C); (J) Fine gage thermocouple wires exiting (F); (K) Heater wires exiting (F); (L) 1/16 in. stainless steel tubing silver-soldered into (F). Reprinted with permission from reference 53.

Figure 2. Effect of the quartz nozzle on peak intensity for sulfur-selective detection of 2-chloro-benzothiazole (methanol solvent denoted by S ) ; ( A ) Without nozzle: (B) With nozzle. Reprinted with permission from reference 53.

179

Thermal stability of the coil was improved by coating it and the junction of the thermocouple with a high temperature cement. A typical coil that is being swept by 300 mL/min of helium easily attains 320°C. The high flow of mobile phase through the restrictor does not lower the temperature of the coil device, although the helium plasma gas flow cools the heating device substantially. Fortunately, the current density in the coil is great enough to allow the attainment of the targeted temperature without requiring an excessively large total current, i.e., less than 1.2 A . The temperature at the tip of the coil could be constantly monitored by reading a digital voltmeter connected to the wire leads from the thermocouple that are part of the heated coil assembly. The resistively heated wire coil placed approximately 2 cm from the plasma discharge does not disrupt or degrade the performance of the plasma, since the additional insulating effects of the quartz inner nozzle and the high temperature cement coating appear to be sufficient in preventing any problems. In effect, the plasma discharge seems to be stabilized when the heated wire assembly is present because of the elimination of condensation of mobile phase at the tip of the restrictor. The direct consequence of this is a less "noisy" introduction of the heated effluent into the plasma and hence, a more stable signal. NON-METAL SELECTIVE DETECTION All of the analytical work published to date on combined capillary SFC-MIP has utilized the Surfatron cavity configuration and comes from the laboratories of Novotny and coworkers ( 5 3 - 5 6 ) . However, there has been a recent advance in this area for the coupling of a packed column SFC system with a TMOIO cavity MIP detector, where the plasma was sustained in argon (57). The choice of argon was related to the fact that this study concerned the detection of iron in ferrocene and related compounds. Hence, the use of an IcP-type torch and high flow (1 L/min) of argon, in addition to a 2 L/min flow of auxillary argon, permitted methanol-modified carbon dioxide to be introduced directly into the plasma. The early studies on the capillary SFC-MIP system were concerned with the description and optimization of the system, whereas the later publications concentrated on the potential of the detector for general non-metal selective detection. This chapter will mirror this general format, beginning with a discussion of the optimization of the SFC-MIP system. This section will be followed by a discussion of the performance of the detector for the sensitive and selective detection of various non-metals, including a summary of detection limits and analytical figures of merit determined for the Surfatron plasma detector. The chapter will conclude with a discussion of current capabilities and limitations of this analytical technique, as well as projections for the future of SFC-MIP.

180

(i) Experimental Details and Optimization of the System A schematic of the entire apparatus is depicted in Figure The chromatographic system was a standard capillary SFC configuration. Several columns were used, depending on the application, and all had a 5 0 p m inner diameter and were approximately 10 m long. The most commonly used stationary phase was a non-polar methyl polysiloxane 0.2 pm film coated in the column. The gases that were used were carbon dioxide and nitrous oxide. The most important modification to the system worth noting was that the column was heated via a circulating water bath rather than in a conventional GC oven because of the sensitivity of the detector to temperatureinduced fluctuations in the carbon dioxide mobile phase flow. Each time the oven heating circuit switched on, a brief surge occurred in the flow of fluid through the column, which in turn resulted in a spike in the baseline (54). Other investigators have also noted this phenomenon (57). The solution involved the replacement of the oven by a circulating water bath. When it was necessary for the temperature of analysis to exceed 1OO"C, a heat-exchange fluid was substituted for the water, and the results were quite satisfactory (54). 3.

Microwave Power S'JPDlY

PM Tube ( 7 102)

Pump

u I

I

I

I

U

El Recorder

Figure 3 . Schematic diagram of the supercritical fluid chromatograph, spectroscopic system, and electronics. Reprinted with permission from reference 54.

181

The injections were performed with a standard HPLC valve equipped with a manully-actuated 60 nL rotor. All injections were splitless and were made at room temperature. Decompression of the mobile phase required for atmospheric detection was accomplished using either the integral restrictor described by Guthrie and Schwartz ( 5 8 ) , or the pre-fabricated frit restrictor. The spectroscopic detection was performed solely in the near-infrared region of the spectrum because the non-metal atomic emission lines were found to be less plagued by spectral interferences than the corresponding lines in the ultraviolet or visible regions. Some of these major interferences are from intense CO- and C2-band emission ( 5 9 ) , which limits the linearity of the response (30). Also, some of these atomic lines are more intense in the near-infrared spectral region, which results in a significant improvement in signal-to-noise ratio, as seen for selected bromine and chlorine atom lines ( 5 9 ) . The emission signal from the plasma was measured with an optical system designed specifically for the near-infrared spectral region, and this system has been described in detail elsewhere ( 5 4 , 5 5 ) . However, it is necessary to mention briefly the most significant components of the spectroscopic system. Firstly, it is important to note that the plasma is viewed axially (end-on), which minimizes interferences from the degradation of the quartz discharge tube. Secondly, the plasma itself is imaged onto the entrance slit of the monochromator by a glass lens since quartz does not transmit near-infrared radiation. The monochromator was specially adapted for the nearinfrared spectral region in two ways. Firstly, the holographic grating was blazed at 1 pm, which means that its maximum efficiency of diffraction occurs in the desired region. Secondly, the photomultiplier tube was chosen for maximum sensitivity between 600 and 1100 nm. The tube was mounted in a thermoelectrically-cooled housing in order to minimize noise interferences from dark current. An important parameter to be optimized is the applied microwave power since it is logical to assume that net emission intensity will increase with applied power: however, a compromise must be made with respect to the lifetime of the discharge tube and the limitations of the generator. The effect of power increase is two-fold. Firstly, it is especially significant with respect to the non-metals, such as sulfur, chlorine and bromine, since these elements possess only atom lines in the near-infrared region ( 5 9 ) and therefore cannot be depopulated to fill ionic states even as more power is coupled into the plasma. Secondly, it has been found that an increase in power will lengthen the plasma and thereby increase the electron density, which is an indirect measure of the concentration of energetic species capable of populating atomic and ionic states of an element. In addition, power is believed to increase rotational temperatures, which are indicative of the population of atomic and ionic states, and exci-

182

tation temperatures, which are indicative of the intensity of emission lines between excited states of an atom (45). Therefore, due to all these effects, an increase in power will result in an increase in the number of atoms of interest and thereby increase the sensitivity of element-selective detection. The effect of applied microwave power on emission intensity was investigated for the non-metals (54,60). It was found that the signal was relatively constant and optimal between 115 and 1 3 5 W. Moreover, baseline noise and discharge-tube devitrification accelerated at powers greater than approximately 135 W, regardless of the tuning adjustments. Based on these findings, an applied power of 120-125 W was selected as a suitable compromise for all subsequent experiments. The effect of helium flow rate on emission intensity is another crucial parameter that must also be optimized (54,55,60) The optimal value in this case was much more difficult to determine because of several convoluting factors, such as residence time of the analyte and stability of the plasma itself. For example, a low flow of plasma gas may increase residence time of analyte, yet at the same time the supercritical mobile phase may not be swept efficiently into the center of the plasma for complete atomization and excitation. Alternatively, a distinct advantage is incurred when a high gas flow is used due to the increased efficiency of excitation of certain elements that are not easily excited; however, if the gas flow is too high the plasma may be extremely distorted and unstable, making excitation less efficient. Initially, a compromise of 250 mL/min of helium gas was chosen: however, ultimately this value was optimized for each individual element based on their specific excitation energies and minimization of background interferences. The latter will be discussed in more detail below.

.

(ii) Analytical Performance of the system (a) Selectivitv and Sensitivitv Sulfur was the first element used to evaluate the performance of the detector in conjunction with SFC. The most striking example of the performance of the system is illustrated in Figure 4 , depicting the separation of four model sulfur-containing compounds in less than 15 minutes (54). It should be noted that these compounds could be easily handled by GC; however, this separation serves to illustrate the potential for sensitivity and selectivity offered by the combination of SFC with MIP detection. The negative deflection just before the thiophene peak in Figure 4 corresponds to the methylene chloride solvent elution. Analysis of the same mixture with FID could not resolve Peak 1 from the solvent, which is an excellent indication of the selectivity of the detector in conjunction with a carbon dioxide mobile phase.

183

4

2

Figure 4 . Separation of a mixture of model sulfur-containing polyaromatics and detection via the sulfur line; approximately 60 ng per component injected. [1= thiophene; 2= thianaphthene; 3 = dibenzothiophene; 4= 1,2-diphenylene sulfide]. Reprinted with permission from reference 54. Unfortunately, this chromatogram is afflicted with a severely decreasing baseline during the course of the pressure gradient. Basically, it is assumed that the increased mass flow rate of carbon dioxide is quenching the plasma. Several methods of signal-processing and background subtraction were attempted as a possible means of alleviating the severity of the baseline shift (54,60). Ultimately, a simpler and more efficient method of eliminating, or at least minimizing the baseline shift was discovered (53). This method involves the lltuningll of the helium gas flow rate until the baseline does

184

not shift with pressure increase. A similar observation has also been made with the RPD in conjunction with SFC (25). The major drawback to this approach is that a different optimum gas flow is required for each element, which is probably related to the fact that the spectral background near each line is unique. This effect is best exemplified by the separation illustrated in Figure 5, where the separation of three model compounds is performed on two different wavelength channels.

Figure 5. Effect of emission line intensity on optimum helium flow rate required for level baseline during pressure program of carbon dioxide at 130°C. [l = 2-chlorobenzothiazole; 2 = dibenzothiophene; 3 = 2-chlorophenothiazine]. (A) 100 mL/min; (B) 300 mL/min. Reprinted with permission from reference 53. In Figure 5A, an optimum helium flow rate of 180 mL/min was selected, which is compatible with the sulfur atom line at 921.3 nm. Alternatively, in Figure 5B, the 837.6 nm chlorine

185

line mandates a new optimum flow rate of 280 mL/min for the very same separation. A l s o notable from this figure is the absence of Peak 2 from Figure 5B, which is entirely consistent with the fact that this compound (dibenzothiophene) contains no chlorine atoms. This phenomenon is a clear illustration of the selectivity of the microwave plasma detector for the nonmetals in the near-infrared region of the spectrum. The concept of selectivity in the chromatographic sense can mean many things to different people. There exists a mathematical definition of selectivity used by several authors (23) that consists of the ratio of the concentration of carbon reference compound (e.g., octane) to analyte concentrations that yield a comparable signal, all at the analyte wavelength. There seems to be a flaw with this definition, however, since it is not a reasonable assumption that the background signal from the organic residue of a branched, cyclic, or aromatic compound can be accurately represented by the background due to a normal alkane of fixed chain-length. For example, the best analog for a compound such as dibenzothiophene might be fluorene, rather than a linear compound containing an equivalent number of carbon atoms. The reference organic compound selected for all selectivity studies by Novotny and co-workers was naphthalene, which was chosen because of its planarity and aromaticity. The molar selectivity ( S ) was calculated from: (signal per moles analyte element)

s

=

(1)

(signal per moles carbon in reference compound)

Table 2 summarizes the calculated selectivities for the various non-metals and two common mobile phases (53). TABLE 2 Molar Selectivity for Non-Metals as a Function of Mobile Phase

Mobile Phase co2

Element Line (nm) Molar Selectivity” ___-___-___-__--_--_---------------------s (1) C1(1) Br(I) Br(I)

837.6 827.2

325 100 110

889.8

165

921.3

185 50

45 20 >500

‘Calculated from the response of 2-chloro-benzothiazole or 5-bromo-indole over naphthalene.

186

Table 2 illustrates the fact that the selectivities are dependent not only on the element, but also on which emission line is monitored. A striking example of this difference can be noted f o r the case of the bromine lines in conjunction with a nitrous oxide mobile phase. The 827.2 nm emission line exhibits extremely poor selectivity over carbon, where the 889.8 nm line displays excellent selectivity under identical conditions. It is interesting to note that the difference is not nearly as striking with the carbon dioxide mobile phase. During the course of these investigations it was determined that many of the results obtained for a carbon dioxide mobile phase were in direct contrast with those obtained with the nitrous oxide mobile phase. A particularly interesting phenomenon that was noted involved the appearance of the solvent peak. It was seen earlier in Figure 4 that the solvent peak is evidenced by a negative deflection, when a carbon dioxide mobile phase is used. In the case of nitrous oxide, a positive peak is noted for the solvent, which is an indication of poorer selectivity. This assumption is supported by the data contained in Table 2, with the exception of the second bromine line. An unfortunate consequence of poor selectivity over carbon is that detection limits become uncertain when it cannot be determined how much of a signal is due to the analyte of interest, and how much is due to the signal contributed by the carbon background. This situation is strikingly illustrated in Figure 6, where three model compounds have been separated in conjunction with a nitrous oxide mobile phase and detected via four different wavelength channels (53). Compound 2 is dibenzothiophene, which contains only carbon, hydrogen, and sulfur, and therefore this compound should only produce a signal on the carbon channel (Figure 6A) and the sulfur channel (Figure 6D). It is clear that a peak is in evidence for both chlorine channels (Figures 6B and 6C), which is not an indication of chlorine content, but a consequence of the poor selectivity of the emission lines with respect to carbon signal, and this is probably related to strong CN-band emission that arises from the combination of organic material with the N20 mobile phase. It was mentioned earlier that the potential o f this detector as a universal detection scheme would be discussed. It is important to note that this detector could only serve as a universal detector when nitrous oxide is the mobile phase, for obvious reasons. Figure 6A reveals another reason why this mode is not promising since the sensitivity of each of the model compounds is decidedly poor, especially considering that 909.5 nm is the strongest carbon line in the near-infrared spectral region. I f Figure 6D is compared with Figure 6A, it is apparent that a single atom of sulfur in these molecules produces a greater response on the sulfur channel than the 12or 13-backbone carbon atoms on the carbon channel. Fortunately, there exist many other carbon lines in the other spectral regions that are much more intense, and this opens up the possibility of universal detection in the ultraviolet and visible regions.

187

1”

4 8 12 16 min 7

0

136

(C)

L 0

4

163

191nlrn

3

rrzL 6

12 min

i 3 6 7 7 a t r n

4

0

ik

136

4 a 12 w

0

136

156

8

156

12 min

lTIatm

min

177atm

Figure 6 . Selective detection of non-metals utilizing a nitrous oxide mobile phase and a column temperature of 130°C; solvent peak denoted by S. [l = 2-chloro-benzothiazole; 2 = dibenzothiophene; 3 = 2-chloro-phenothiazine; 4 = 5-bromo-indole]. ( A ) C(1) line at 909.5 nm and 100 mL/min He; (B) Cl(1) line at 912.1 nm and 100 mL/min He: (C) Cl(1) line at 837.6 nm and 80 mL/min He: (D) S(1) line at 921.3 nm and 100 mL/min He. Reprinted with permission from reference 53.

188

The detection limits for each of the non-metals were determined at the 95% confidence level (i.e.f signal-to-noise ratio of 2) ( 5 4 ) . The results were obtained using two mobile phase systems and the results are summarized in Table 3. TABLE 3 Sensitivity as a Function of Molecular Weight and Mobile Phase Mobile Phase co2

N20

Element Line (nm) Detection Limit (pg/s) ........................................... S(I) C1(I) Br(I) Br(I)

s (1) C1(1) C1(1) Br(1) Br(I) C(I)

921.3

73"

837.6

210

827.2 889.8

780 780

140b 250

921.3

26

85

837.6

110 97

400

912.1 827.2 889.8 909.5

550

300

520 550

2600

"Values on left obtained from either 2-chloro-benzothiazole or 5-bromo-indole. bValues on right obtained from 2-chloro-phenothiazine. Interestingly, in stark contrast with the selectivity results, the detection limits for nitrous oxide were generally superior to those for carbon dioxide. However, this may actually be related to the contribution of the carbon background signal caused by the formation of CN from the organic carbon and the N20 mobile phase. The other noticeable trend in Table 3 concerns the dependence of detection limit on molecular weight. This trend is consistent with results reported in other studies, such as SFC-RPD (25), and the effect is most likely a consequence of an increased amount of mobile phase entering the plasma (41,57). Therefore, if the plasma is quenched by mobile phase, it has less energy €or atomization and excitation of the analyte and the detection limit will suffer. At present this phenomenon is a major drawback to the general utility of the MIP as a versatile element-selective detector for capillary SFC. As an example of an application for the technique of SFC-MIP (53), the separation of four pesticides containing both sulfur and phosphorus is depicted in Figure 7. Figure 7A illustrates the sulfur-selective detection of these compounds utilizing SFC-MIP and the sulfur line. The sensitivity for each compound is dependent on the number of sulfur atoms in each molecule, which varies for each of the pesticides. The corresponding SFC separation of the very same compounds and detection via the thermionic detector in the phosphorus mode (9) is shown in Figure 7B.

189

4

0

7

14 min

145

195 mtm

I 95

Figure 7 . Comparison of separation and selective detection of four pesticides using a nitrous oxide mobile phase: peaks labeled with i represent sample impurities. [1= Phorate, 260 g/mole; 2= Di-Syston, 274 g/mole: 3 = Malathion, 330 g/mole; 4= Ethion, 384 g/mole]. (A) Surfatron MIP detection via the sulfur line (921.3 nm); (B) Themionic detection in the phosphorus mode. Figure 7B reprinted with permission from reference 9. (b) Boron-selective Detection All of the studies described up to this point concerned compounds that inherently contain one or more heteroatoms that can be targeted for selective detection; however, there are many compounds of biochemical interest that do not contain anything but carbon, hydrogen, and oxygen atoms. Very often, these molecules are difficult to separate by gas chromatogra-

190

phy because they contain polar functionalities, such as hydroxyl and/or amino groups, and thus are involatile. A common approach used to solve this problem is the derivatization of the polar groups with a suitable functional group that can impart greater volatility to the solutes of interest. One such application of this type of chemistry is the formation of cyclic boronate esters for compounds that contain 1,2- or 1,3diols (61-65). The GC-MIP analysis of steroidal carboranes has been successfully accomplished by Krull et al., whereby the system was tuned to the intense 248.8 nm boron atomic emission line (66). In addition, the detection limits for a boronic acid reagent analyzed by GC in conjunction with the Beenakker cavity were low (3.6 pg/s) with excellent selectivity (9.25 x lo3), and linearity over 2-3 decades (31). There has also been a publication of boronate ester formation of catechols in human urine extracts and subsequent separation by GC and detection by MIP (67). The preparation of these boronates is very simple, involving dissolution of the diol in an appropriate non-aqueous solvent, followed by the addition of a twofold excess of the boronic acid reagent at room temperature (67)

The results of the SFC-MIP analysis of cyclic boronate esters of compounds such as catechols, carbohydrates, and corticosteroids has been recently published by Luffer and Novotny (56). This particular study utilized the same system that was used in all the non-metal detection described above. However, the most intense boron emission line is located in the ultraviolet region of the spectrum. Consequently, several modifications were made to the system that permitted detection via the third order boron line at 749.4 nm (56). The most significant cost of this modification was a concomitant loss of sensitivity. The preliminary results obtained indicated that SFC-MIP shows some promise for the analysis of these particular biochemicals; however, as expected the sensitivities were poor in this region of the spectrum, although the selectivity was good. (c) Linearitv

Precision

The linearity for the Surfatron MIP in conjunction with GC has previously been reported to be three orders of magni-

tude (42). Studies on the SFC-MIP combination have similarly revealed linearity over three decades, with a roll-off noted at high concentrations (54). The relative standard deviation for a series of replicate injections ranged from 1 to 5%. The effective linear concentration range of this detector is I4, corresponding to an unsplit 60 nL injection volume. to (iii) Comparison with Other Selective Detectors

It would be instructive at this point to summarize the information detailed in this chapter by comparing the analytical figures of merit of the SFC-MIP detection system to those

191

of several other non-metal selective detectors mentioned in the Introduction. These include the thermionic detector, the electron-capture detector, the flame-photometric detector, and the sulfur chemiluminescence detector. The TID has demonstrated very low detection limits in the nitrogen and phosphorus modes, i.e. , < 2 pg/s and < 1 pg/s, respectively (9). In addition, due to the excellent selectivity of this detector in each respective mode, important biochemicals and their metabolites have been detected in physiological fluids after derivatization with an appropriate reagent containing nitrogen or phosphorus (10-12). The FPD has been shown to be capable of high selectivity and low detection limits (8 pg/s) for a large selection of organosulfur compounds (14). To date, there have been no reports on the SFC-FPD analysis of phosphorus-containing compounds, most likely because of the excellent results obtained with SFC-TID. By comparison, the relatively new SCD exhibits a selectivity of approximately 8000 for sulfur, and corresponding detection limits around 6 pg/s for small organosulfur compounds (16). However, the latter detector has a linear response to sulfur, unlike the FPD. The combination of ECD with capillary SFC has resulted in detection limits in the high femtogram range for chlorinated and brominated compounds, specifically in the area of pesticide detection (18,19). By contrast, the SFC-Surfatron MIP combination in the near-infrared spectral region demonstrates the greatest versatility of all these detectors because atomic emission detectors promise a theoretically limitless selection of elements, assuming the corresponding emission line is available and of suitable intensity. This versatility is the main advantage of the MIP over both the TID and ECD, whereby the latter two detectors are actually element-specific rather than truly element-selective. Unfortunately, the detection limits of the MIP do not yet approach those of either of these other two detectors. The MIP detector is superior to the FPD for the selective detection of sulfur because of the former's linearity of the response over 2-3 decades. With respect to the SCD, the two detectors are disadvantaged because of sensitivities that are somewhat dependent on the molecular weight of the solvent. In addition, like the SCD the Surfatron suffers from a shift in the baseline during a density-programmed run. PROSPECTS FOR THE FUTURE OF SFC-MIP The cost of assembling the detection scheme described in this chapter was approximately US $20,000. The addition of a photodiode array detector would add to this cost, but would provide the desirable feature of simultaneous multielement detection. At this point, the detector appears useful for the detection of low nanogram quantities of compounds containing the various non-metals described in this chapter. In addi-

192

tion, the area of chemical derivatization of biologically important compounds for the dual purpose of reducing polarity and introducing a suitable heteroatom for selective detection should receive considerably more attention in the future. It seems that further investigations of other plasma sources and other spectral regions would be worthwhile for the purpose of providing complementary results for a particular element that exhibits poor selectivity or sensitivity in the near-infrared region, such as boron, for example. On another note, an example of a new interesting approach that may fill an important niche is the combination of packed column SFC with the TMOIO cavity (57), mentioned above. The adaptation of this system to the detection of non-metals would be promising in conjunction with large scale separations of polar compounds requiring the use of modifiers. ACKNOWLEDGMENTS This research was supported by Grant No. CHE 8605935 from the National Science Foundation. REFERENCES 1.

M. Novotny, S. R. Springston, P. A. Peaden, J. C. Fjeldsted and M. L. Lee, Anal. Chem., 53 (1981) 407A.

2.

Y. Hirata, J. Chromatosr., 315 (1984) 39.

3.

R. L. Eatherton, M. A. Morrissey, W. F. Siems and H. H. Hill, Jr., J. Resolut. Chromatosr. Chromatour. Commun., 9 (1986) 154.

4.

m.,59

5.

M. A. Morrissey and H. H. Hill, Jr., 2. Hish Resolut. Chromatosr. Chromatosr. Commun., 11 (1988) 375.

6.

W. Schneider, J. Ch. Frohne and H. Bruderreck, J. Chromatoqr. , 245 (1982) 71.

7.

G. R. Verga, A. Sironi, W. Schneider and J. Ch. Frohne, J. Resolut. Chromatour. Chromatosr. Commun., 11

S. Rokushika, H. Hatano, and H. H. Hill, Jr., (1987) 8.

Anal.

(1988) 248. 8.

W. R. West and M. L. Lee, 2. Hish Resolut. Chromatosr. Chromatour. Commun., 9 (1986) 161.

9.

P. A. David and M. Novotny, Anal. Chem., 61 (1989) 2082.

10.

P. A. David and M. Novotny, J. Chromatosr., 452 (1988) 623.

193 11.

P. A. David and M. Novotny, J. Chromatoqr., 461 (1989) 111.

12.

P. A. David and M. Novotny, Anal. Chem., in press.

13.

m.,58

14. 15.

K. E. Markides, E. D. Lee, R. Bolick and M. L. Lee, S. V. Olesik, L. A. Pekay and E. A. Paliwoda,

m.,6 1

Anal.

(1986) 740.

Anal.

(1989) 58.

L. A. Pekay and

S.

V. Olesik, Anal. Chem., 61 (1989)

2616. 16.

D. J. Bornhop, B. J. Murphy and L. Krieger-Jones, Anal.

m.,6 1

(1989) 797.

17.

W. T. Foreman, C. L. Shellum, J. W. Birks and R. E. Sievers, 2. Chromatoqr., 465 (1989) 23.

18.

S. Kennedy and R. J. Wall, LC-GC, 6 (1988) 930.

19.

H. -C. K. Chang and L. T. Taylor, 2. Chromatoqr.

a. , 28

(1990) 29. 20.

P. A. David, Ph.D. Dissertation, Indiana University, March 1989.

21.

J. W. Carnahan, K. J. Mulligan and J. A. Caruso, Chim. Acta, 130 (1981) 227.

22.

P. C. Uden, Chromatoqraphv Forum, 1 (1986) 17.

23.

Anal.

G. W. Rice, A. P. D'Silva and V. A. Fassel, Spectrochim.

m, 40B

(1985) 1573.

24.

R. J. Skelton, Jr., K. E. Markides, P. B. Farnsworth, M. L. Lee and F. J. Yang, J. Hish Resolut. Chromatoqr. Chromatoqr. Commun., 11 (1988) 75.

25.

R. J. Skelton, Jr., P. B. Farnsworth, K. E. Markides and M. L. Lee, Anal. Chem., 6 1 (1989) 1815.

26.

A. J. McCormack,

S.

C. Tong and W. D. Cooke, Anal. Chem.,

37 (1965) 1470. 27.

C. A. Bache and D. J. Lisk, Anal. Chem., 37 (1965) 1477.

28.

C. A. Bache and D. J. Lisk, Anal. Chem., 38 (1966) 783.

29.

A. K. Mohammad and J. A . Caruso, &&. Chromatocrr., 26 (1987) Chapter 5.

194

30.

D. Deruaz and J.-M. Mermet, Analusis, 14 (1986) 107.

31.

S.

32.

C. Bradley and J. W. Carnahan, Anal. Chem. 60 (1988) 858.

33.

B. D. Quimby, P. C. Uden and R. M. Barnes, Anal. Chem., 50 (1978) 2112.

34.

C. I. M. Beenakker, Spectrochim. Acta Part B, 31B (1976) 483.

35.

C. I. M. Beenakker, Spectrochim. Acta Part B, 32B (1977) 173.

36.

A. Estes, P. C. Uden and R . M. Barnes, Anal. Chem., 53 (1981) 1829.

C. I. M. Beenakker and P. W. J. M. Boumans, Spectrochim. (1978) 53.

m, 33B

37.

T. H. Risby and Y. Talmi, CRC Crit. Rev. Anal. Chem., 14 (1983) 231.

38.

L. Ebdon, S. Hill and R. W. Ward, Analyst (London), 111 (1986) 1113.

39.

F. A. Huf and G. W. Jansen, Spectrochim. Acta Part B, 38B (1983) 1061.

40. 41. 42.

G. W. Jansen, F. A. Huf and H. J. de Jong, Spectrochim. (1985) 307.

m, 40B

J. Hubert, M. Moisan and A. Ricard, Spectrochim. 34B (1979) 1.

m,

G. Chevrier, T. Hanai, K. C. Tran and J. Hubert, 60 (1982) 898.

Can.

m.,

a.

43.

M. H. Abdallah, S. Coulombe, J.-M. Mermet and J. Hubert, Spectrochim. m, 37B (1982) 583.

44.

Y. Takigawa, T. Hanai and J. Hubert, 2. Hish Resolut. Chromatoqr. Chromatoqr. Commun., 9 (1986) 698.

45.

S.

46.

M. Selby and G. M. Hieftje, Spectrochim. A d , 42B (1987) 285.

47.

M. Selby, R. Rezaaiyaan and G. M. Hieftje, Spectrosc., 41 (1987) 749.

Coulombe, Ph.D. Dissertation, University of Montreal, 1983.

m.

195 48.

L. J. Galante, Ph.D. Dissertation, Indiana University, November 1987.

49.

T. Hanai, S. Coulombe, M. Moisan and J. Hubert, in R. M. Barnes (Editor), "Developments in Atomic Plasma Spectrochemical Analysis", Heyden, London, 1981, p. 337.

50.

W. R. McLean, Proc. SOC. Anal. Chem., 10 (1973) 144.

51.

B. Riviere, J.-M. Mermet and D. Deruaz, J. Anal. SDectrom., 3 (1988) 551.

52.

M. Selby and G. M. Hieftje, Spectrochim. Acta Part B, 42B

At.

(1987) 285. 53.

D. R. Luffer and M. Novotny, J. Chromatosr., 517 (1990) 477.

54.

D. R. Luffer, L. J. Galante, P. A. David, M. Novotny and G. M. Hieftje, Anal. Chem., 60 (1988) 1365.

55.

L. J. Galante, M. Selby, D. R. Luffer, G. M. Hieftje and M. Novotny, Anal. Chem. , 60 (1988) 1370.

56.

D. R. Luffer and M. Novotny, J. Microcol. S e p . , 3 (1991) 39.

57.

C. B. Motley, M. Ashraf-Khorassani and G. L. Long,

m.

SDectrosc., 43 (1989) 737. 58.

E. J. Guthrie and H. E. Schwartz, J. Chromatosr.

m., 24

(1986) 236. 59.

J. E. Freeman and G. M. Hieftje, Spectrochim. Acta Part B, 40B (1985) 475.

60.

D. R. Luffer, Ph.D. Dissertation, Indiana University, May 1990.

61.

C.

J. W. Brooks and J. Watson, J. Chem. Commun., (1967)

952. 62.

G. M. Anthony, C. J. W. Brooks, I. Maclean and I. Sangster, J. Chromatosr. M., 7 (1969) 623.

63.

C. J. W. Brooks and I. Maclean, J. Chromatosr.

m., 9

(1971) 18. 64. 65.

S. J. Gaskell, C. G. Edmonds and C. J. W. Brooks,

u., 9 (1976) 325.

Anal.

C. F. Poole and A. Zlatkis, J. Chromatoqr., 184 (1980) 99.

196 66.

I.

S.

Krull, S. W. Jordan, S. Kahl and S. B. Smith, Jr.,

2. Chromatosr. 67.

a., 20 (1982) 4 8 9 .

S. W. Jordan, I. S. Krull and S. B. Smith, Jr., (1981) 1131.

Lett., 15

Anal.

K. Jinno (Ed.), Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction Journal of Chromatograph Library Series, Vol. 53 0 1992 Elsevier Science Jublishers B.V. All rights resewed.

197

Chapter 11 MULTIDIMENSIONAL SFE AND SFC Joseph M. Levy and Mehdi Ashraf-Khorassani Suprex Corporation 125 William Pitt Way Pittsburgh, Pennsylvania, USA

15238

INTRODUCTION One of the distinct advantages of supercritical fluid extraction (SFE) is the ability to directly couple the extraction effluent from a sample matrix to an analytical chromatograph for quantitative or qualitative determinations. In the realm of analytical chemistry, the sample preparation step is often the most error prone step requiring arduous and sometimes lengthy procedures before the actual sample can be analyzed. SFE directly addresses sample preparation methodologies and provides analysts with the option to directly couple the sample preparation procedure to gas chromatography (GC) or supercritical fluid chromatography (SFC) to effectively achieve the analytical objectives. An added advantage is that the nature of the on-line interfacing of SFE to GC or SFC does not exclusively limit the use of GC or SFC to only SFE sample introduction. The flexibility exists whereby the analyst can use SFE in an off-line collection mode as well as on-line. In most cases, SFE as a sample preparation tool should be used in both the on-line and off-line modes. Off-line SFE gives the analyst the most capability for method development and analytical characterizations since the extraction effluent can be collected and then taken to any analytical instrument (i.e. GC, LC, SFC, MS, NMR, IR, W, etc.). After the initial method development for a sample type where initial characterizations have been performed, cnline SFE can be utilized to simplify the analytical tasks and reduce the methods to practice. A comparison of on-line and off-line SFE is described in Chapter 10. This chapter will describe the use of on-line SFE/GC and SFC in terms of theory of operation, interface mechanics, instrumentation and application examples from the literatsre and from our laboratory. In addition, examples of selectivity enhancements in SFE will be described. Also, an approach towards the automation of on-line SFE using sequential multiple vessels will be discussed.

198

ON-LINE INTERFACING MECHANICS

The on-line coupling of SFE to GC (SFE/GC) has the advantages of having no sample handling between the sample preparation step and the analytical determination step. A generalized scheme of on-line SFE is shown in Figure 1. A SFE system delivery pump compresses the primary extraction fluid (i.e. carbon dioxide) and solubilizes the analytes from a sample matrix which is contained in a heated extraction vessel (see Chapter 10 for instrumentation). These solubilized analytes are then transferred on-line to an analytical chromatograph (i.e. GC or SFC). The transfer line is used to control the volume of supercritical fluid that is flowing through the sample matrix. Depending on the analytical need, there is considerable flexibility obtainable when interfacing SFE on-line to a capillary GC. For most determinations, flame ionization and mass spectrometric detectors have been employed (1-17). However, it is also possible to utilize the more selective and sensitive nitrogen-phosphorous and electron capture detectors (11,17). In all of these cases, each detector has a very low to manageable response to C02 depending for the most part on the impurities present in the commercial supply of C02. Most of the published on-line SFE/GC applications have utilized capillary columns ranging from 0.20 mm internal diameter to 0.53 mm internal diameter and have encompassed the full range of GC stationary phase coatings (1-17). To date there have been no reports of SFE stripping off capillary column stationary phase coatings after on-line SFE interfacing.

ON-LINE SFE

I

I

SFE SYSTEM PUMP

I I

I

SUPERCRITICAL FLUID

SAMPLE MATRIX I

I

SOLUBILIZED ANALYTES

GClFlD GC/MS GC/SELECTIVE DETECTORS: NPD, ECD

SFC/FID

Figure 1:

Generalized Scheme of On-Line SFE

A s a means of sample introduction to GC, on-line SFE presents itself as an alternative to the conventional means of GC sample introduction such as headspace, purge and trap, thermal desorption, pyrolysis, and even conventional syringe injection. Figure 2 represents a comparison of on-line SFE/GC to conventional syringe injection into GC using eucalyptus leaves and fuel contaminated sediment sample matrices. The two modes of on-line SFE/GC; namely, split and on-column, were utilized. A s can be seen, the GC peak shapes and amplitudes were comparable f o r on-line SFE and conventional syringe GC sample introduction. A noticeable difference in the chromatograms was no evidence of a solvent peak for on-line SFE sample introduction since C 0 2 did not respond In comparison to to the GC flame ionization detector. headspace, purge and trap, thermal desorption and pyrolysis, SFE has the potential to encompass a wide range of volatile to even non-volatile analytes, depending on the sample matrix and the extraction conditions that solubilize the entire matrix (e.g. certain polymer matrices) and then introduce analytes into the GC that are well beyond the GC volatility range. FwCconmmlnad udlrnnt (b) spm

& ,

SF GC

C15

C’5H240

I

CM

JJ

I I

L

I

0

I0

J

M

Retention Ume (min)

Figure 2: Comparison of chromatographic peak shapes obtained using (a) on-column SFE/GC and (b) split SFE/GC with (a) conventional on-column and (b) split GC injections of methylene chloride extracts. Taken from references 10 and 12.

200 F i g u r e 3 shows a g e n e r a l i z e d s c h e z a t i c d i a g r a m o f a t y p i c a l o n - l i n e SFE i n t e r f a c e t o a G C . A t y p i c a l procedure f o r p e r f o r m i n g o n - l i n e SFE/GC i n v o l v e s f i r s t l o a d i n g ( u s u a l l y weighing o u t ) a sample m a t r i x i n t o an e x t r a c t i o n v e s s e l rangi n g i n w e i g h t f r o m low m i l l i g r a m t o 10-20 grams d e p e n d i n g on a n a l y t e s e n s i t i v i t i e s and a n a l y t i c a l o b j e c t i v e s . Figure 4 shows a schematic o f a t y p i c a l e x t r a c t i o n vessel s p e c i f i c a l l y These v e s s e l s a r e s t a i n l e s s s t e e l d e s i g n e d f o r u s e i n SFE. i n c o n s t r u c t i o n a n d v a r y i n terms o f t h e v o l u m e o f t h e v e s s e l body from 0 . 5 m i c r o l i t e r s t o a s l a r g e as 5 0 m i l l i l i t e r s i n volume. Each e n d o f t h e vessel c o n s i s t s o f removeable ( a n d d i s p o s a b l e ) f r i t s o r s c r e e n s ( 2 m i c r o n ) a n d s e a l s which a r e e i t h e r made o f PEEK m a t e r i a l o r m e t a l - t o - m e t a l s e a l s . The e n d s o f t h e vessels t e r m i n a t e i n e n d caps w i t h 1 / 1 6 i n c h fittings. A f t e r w e i g h i n g o u t a s a m p l e n a t r i x , t h e end c a p s o f t h e vessel a r e t i g h t e n e d w i t h a wrench ( c e r t a i n vessels c a n be h a n d - t i g h t e n e d t o a p a r t i c u l a r p r e s s u r e ) and p l a c e d A f t e r p r e s s u r e and thermal i n t o t h e e x t r a c t i o n oven. e q u i l i b r a t i o n f o r t h e c h a r g e d e x t r a c t i o n vessel i n t h e s t a t i c mode ( c l o s e d o u t l e t o f v e s s e l ) , an e l e c t r o n i c h i g h p r e s s u r e s w i t c h i n g valve c h a n g e s p o s i t i o n and s h i f t s t h e e x t r a c t i o n t o t h e dynamic mode ( o p e n e d o u t l e t o f t h e v e s s e l ) t r a n s f e r r i n g t h e e x t r a c t i o n e f f l u e n t t h r o u g h a heated t r a n s f e r l i n e (made of fused silica o r s t a i n l e s s steel) d i r e c t l y i n t o a c a p i l l a r y GC i n j e c t i o n p o r t a s shown i n F i g u r e 3 . T h e flow through t h e t r a n s f e r l i n e is r e g u l a t e d e x a c t l y b y c r i m p i n g t h e s t a i n l e s s steel l i n e o r u s i n g d i f f e r e n t i n n e r d i a m e t e r s o f f u s e d silica. The d e c o m p r e s s e d C02 g a s e o u s f l o w r a n g e s from 35 t o 300 m i l l i l i t e r s per m i n u t e d e p e n d i n g on t h e vessel v o i d volumes ( i . e . s a m p l e m a t r i x s i z e s ) . T o achieve h i g h l y e f f i c i e n t e x t r a c t i o n s , 3 t o 5 v o i d volumes o f s u p e r c r i t i c a l f l u i d n e e d t o be f l u s h e d t h r o u g h t h e c h a r g e d e x t r a c t i o n vessel. The d e c o m p r e s s e d f l o w s g o i n g i n t o t h e GC n e e d t o be s e t a t each e x t r a c t i o n p r e s s u r e s e t p o i n t t o a c h i e v e e f f i c i e n t extractions. Heated Transfer Line

--I I

I I I I I I

I

1 I I

PrepMaster Oven

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ---I

F i g u r e 3:

I I

I I I

GC Oven ~

---- - 1

G e n e r a l i z e d o n - l i n e SFE/GC s c h e m a t i c d i a g r a m

I I

201

\Retaining

Frits End Caps

Figure 4:

E x t r a c t i o n vessel b l o c k d i a g r a m

Two modes o f o n - l i n e SFE/GC e x i s t , namely s p l i t SFE/GC and on-column SFE/GC. Figure 5 is a p i c t o r i a l representation During o f what i s o c c u r r i n g d u r i n g SFE i n t r o d u c t i o n i n t o GC. s p l i t SFE/GC, t h e s o l u b i l i z e d a n a l y t e s e x i t t h e e x t r a c t i o n vessel t h r o u g h a s t a i n l e s s s t e e l (1/32 i n c h x 0 . 0 0 7 i n c h i n t e r n a l d i a m e t e r ) t r a n s f e r l i n e which i s i n s e r t e d d i r e c t l y t h r o u g h t h e s e p t u m and s e p t u m c a p o f a n u n m o d i f i e d s p l i t / splitless c a p i l l a r y injection p o r t . The s u p e r c r i t i c a l f l u i d s t a t e i s m a i n t a i n e d u n t i l it r e a c h e s t h e t i p o f t h e t r a n s f e r l i n e ( i . e . r e s t r i c t o r ) and d e c o m p r e s s e s d i r e c t l y i n s i d e t h e heated i n j e c t i o n p o r t . So t h e r e f o r e , i n t h e o r y , t h e a n a l y t e s a r e purposely n o t allowed t o f a l l o u t of s o l u t i o n u n t i l t h e y a r e c o m p l e t e l y t r a n s f e r r e d t o t h e GC i n j e c t i o n p o r t . The heat of t h e i n j e c t i o n p o r t a i d s i n minimizing t h e expansive c o o l i n g of t h e s u p e r c r i t i c a l f l u i d upon d e c o m p r e s s i o n . A f t e r decompression, t h e a n a l y t e s v a p o r i z e i n s i d e t h e h e a t e d i n j e c t i o n p o r t , mix w i t h t h e GC c a r r i e r gas, a r e homogenized i n s i d e t h e e x i s t i n g g l a s s s p l i t i n j e c t o n p o r t l i n e r , and g e t f l u s h e d o n t o t h e h e a d o f t h e GC c a p i l l a r y column. The e x c e s s decompressed g a s e o u s C02 ( a n d a n a l y t e s ) f l o w o u t o f t h e s p l i t v e n t d u r i n g t h e dynamic e x t r a c t i o n t r a n s f e r mode. This i s reproducible and p o t e n t i a l l y q u a n t i t a t i v e s i n c e t h e s p l i t r a t i o d o e s c h a n g e from r u n t o r u n . During on-column S F E / G C , t h e s o l u b i l i z e d a n a l y t e s e x i t t h e e x t r a c t i o n vessel t h r o u g h a f u s e d s i l i c a t r a n s f e r l i n e ( 1 0 - 5 0 micron i n t e r n a l d i a m e t e r ) w h i c h i s i n s e r t e d d i r e c t l y A l l of t h e i n t o an on-column c a p i l l a r y i n j e c t i o n p o r t , s o l u b i l i z e d a n a l y t e s and d e c o m p r e s s e d g a s e o u s CO2 e n t e r t h e GC c a p i l l a r y column, maximizing t h e s e n s i t i v i t y of an a n a l y s i s ( a n a l o g o u s t o on-column s y r i n g e GC i n j e c t i o n ) . F o r t h i s r e a s o n , however, t h e f u s e d s i l i c a t r a n s f e r l i n e n e e d s t o b e p h y s i c a l l y removed a f t e r t h e d y n a m i c e x t r a c t i o n t r a n s f e r mode s i n c e t h e d e c o m p r e s s e d C 0 2 e s s e n t i a l l y becomes t h e GC

202

Split SFE-GC

On-column SFE-GC

-extraction cell ____

on-column Injection port

Figure 5: Pictorial representation of on-line SFE/GC interfacing. Taken from reference 18. carrier gas and can extinguish the commonly used flame ionization detector. In split SFE/GC, the transfer line normally remains inserted in the injection port during the entire analytical run. The majority of the published applications have been accomplished using on-line split SFE/GC. In general, split SFE/GC is well suited for generalized method development and characterization of a variety o f different sample matrices (1-17)

.

For both on-line split and on-column SFE/GC, the stationary phase of the GC capillary column is responsible for focusing the extracted analytes. Depending on the volatility range of the respective analytes, additional cooling may be necessary to prevent the premature diffusion of the volatile analytes through the GC column and to achieve sharp chromatographic peak shapes. This can be accomplished by spot cryogenic cooling of the injection port or more commonly by cooling the entire GC oven. An example of this is shown in Figure 6 with the effect of the cryogenic trapping temperature on t h e S F E / G C characterization of rosemary spice. Sharper chromatographic peak shapes were

203 obtained the lower the setting o f the GC oven temperature for the earlier eluting (more volatile) species. Maintaining the GC oven temperature at -5OOC yielded poor extraction and transfer efficiencies because of the physical plugging of the restrictor (transfer line) inside the GC injection port. In practice, at temperatures below -5OoC, plugging of the transfer line occurs because of the physical freezing of the decompressed C 0 2 inside the GC capillary column (17). The duration of the dynamic extraction transfer mode is usually the same as the duration of the initial (cryogenically cooled) temperature of the GC oven. After the dynamic transfer, normal GC temperature programming is performed and the analytical GC results are obtained.

zsoc

-1ooc

Rotentlon T I M (mln)

Figure 6: Effect of cryogenic trapping temperature on the on-line SFE/GC/FID analysis of rosemary. Taken from reference 3.

204

SELECTIVITY IN ON-LINE SFE/GC A d i s t i n c t a d v a n t a g e compared t o c o n v e n t i o n a l l i q u i d e x t r a c t i o n s o f SFE i s t h e a b i l i t y t o t u n e t h e o p e r a t i o n a l e x t r a c t i o n parameters t o achieve t h e selective e x t r a c t i o n of c e r t a i n a n a l y t e s from a complex sample m a t r i x . An o b v i o u s a p p r o a c h i n c o n t r o l l i n g SFE s e l e c t i v i t y i s b y v a r y i n g e x t r a c t i o n t e m p e r a t u r e s and p r e s s u r e s . These p a r a m e t e r s d i r e c t l y c o n t r o l e x t r a c t i o n d e n s i t i e s which i n t u r n a f f e c t t h e t h r e s h o l d s o l u b i l i t i e s of s p e c i f i c a n a l y t e s . Certain classes of compounds, in theory, have distinct threshold solubilities. F i g u r e 7 p r e s e n t s an e x a m p l e o f t h e q u a l i t a t i v e SFE/GC c h a r a c t e r i z a t i o n o f p o l y n u c l e a r a r o m a t i c hydroc a r b o n f r a c t i o n s (PAH) a t d i f f e r e n t e x t r a c t i o n p r e s s u r e s . The p e a k s l a b e l e d A and B a r e t h e same r e s p e c t i v e components i n each f r a c t i o n f o r r e f e r e n c e . I n g o i n g from l o w t o h i g h SFE p r e s s u r e s , a n o t i c e a b l e d i f f e r e n c e , d e s p i t e some o v e r l a p , i s e v i d e n t i n t h e d i s t r i b u t i o n o f t h e c h r o m a t o g r a m s by r e t e n t i o n t i m e and peak amplitude, i n d i c a t i n g t h e p o t e n t i a l f o r A t 80 a t m o s p h e r e s , two o n - l i n e SFE c l a s s f r a c t i o n a t i o n s . r i n g , a l k y l a t e d two r i n g , t h r e e r i n g , a n d l o w e r a l k y l a t e d t h r e e r i n g PAHs were b e i n g e x t r a c t e d . A t 1 2 5 atmospheres, t h e e x t r a c t e d f r a c t i o n c o n s i s t e d of a l k y l a t e d t h r e e r i n g , f o u r r i n g a n d some a l k y l a t e d f o u r r i n g PAHs. A t t h e highest p r e s s u r e , 200 atmospheres, a l k y l a t e d f o u r r i n g and l a r g e r PAHs were b e i n g e x t r a c t e d ( 5 ) . The a b i l i t y t o t u n e s e l e c t i v i t i e s by v a r y i n g SFE d e n s i t i e s i s n o t o n l y d e p e n d e n t on t h e a n a l y t e s o f i n t e r e s t b u t a l s o on t h e sample m a t r i x i t s e l f . T h i s i s d u e t o t h e c o n t r i b u t i o n s o f o t h e r SFE mechanisms besides solubility, namely d i f f u s i o n a n d s a m p l e m a t r i x a d s o r p t i o n e f f e c t s (see c h a p t e r 1 0 ) .

A n o t h e r means of e n h a n c i n g SFE s e l e c t i v i t i e s and e f f i c i e n c i e s i s b y t h e u s e of m o d i f i e r s ( i . e . methanol, benzene, f o r m i c a c i d ) . These m o d i f i e r s e n h a n c e e x t r a c t i o n e f f i c i e n c i e s by a f f e c t i n g s o l u b i l i t i e s , d i f f u s i o n r a t e s , o r s u r f a c e a d s o r p t i o n d e p e n d i n g on t h e s a m p l e m a t r i x and t h e a n a l y t e s o f i n t e r e s t (see C h a p t e r 1 0 ) . I n SFE, m o d i f i e r s c a n be added t o t h e p r i m a r y s u p e r c r i t i c a l f l u i d by u s i n g d u a l s u p p l y pumps, p r e - m i x e d c o m m e r c i a l l y s u p p l i e d c y l i n d e r s , o r by u n i q u e l y a d d i n g a s p e c i f i c volume o f m o d i f i e r d i r e c t l y t o t h e e x t r a c t i o n vessel w i t h t h e sample m a t r i x ( 1 9 ) . I n onl i n e SFE/GC, d e p e n d i n g on t h e m o d i f i e r i d e n t i t y , t h e s p e c i f i c m o d i f i e r e l u t e s o n t h e GC a s a d i s c r e t e p e a k t o g e t h e r w i t h the extracted analytes. Depending on t h e m 2 d i f i e r c o n c e n t r a t i o n , a r e t e n t i o n g a p o r t h i c k f i l m c a p i l l a z y column may n e e d t o be employed t o p r o p e r l y e l u t e t h e l a r g e ( s o l v e n t - l i k e ) modifier peak. Some m o d i f i e r s s u c h a s f o r m i c a c i d , do n o t h a v e an a p p r e c i a b l e r e s p o n s e on c o n v e n t i o n a l f l a m e i o n i z a t i o n detectors. Table 1 l i s t s a n example o f t h e u s e o f m o d i f i e r s i n o n - l i n e SFE/GZ t o enhance t h e e x t r a c t i o n e f f i c i e n c y of selected a r o m a t i c a n a l y t e s from a s l u d g e / f l y a s h sample matrix. The p e r c e n t r e c o v e r i e s f o r t h e s e l e c t e d a r o m a t i c s

205

125 ttn,

I

200 8ml

A

0

10

II

30 30

20

30

50

40 Tim Imlnuteal

80

70

1

I

I

1

I

100

150

200

265

265

Temprstura

“XI

F i g u r e 7 : SFE/GC-FID c h a r a c t e r i z a t i o n o f p o l y n u c l e a r a r o m a t i c hydrocarbon f r a c t i o n s a t d i f f e r e n t p r e s s u r e s . T a k e n from reference 5.

206 were low when u s i n g o n l y s u p e r c r i t i c a l C 0 2 f o r SFE a n d were d i s t i n c t l y enhanced when t h e d i f f e r e n t m o d i f i e r s were a d d e d t o t h e sludge/fly ash i n t h e e x t r a c t i o n vessel. A period of s t a t i c equilibration a t t h e l i s t e d extraction conditions f o r 3 0 m i n u t e s was n e c e s s a r y a f t e r m o d i f i e r a d d i t i o n t o r e a l i z e t h e f u l l modifier e f f e c t . Moreover, by v a r y i n g o n l y t h e modifier identities (keeping t h e modifier concentrations c o n s t a n t a t 5 percent l e v e l s ) obvious d i f f e r e n c e s i n p e r c e n t r e c o v e r i e s were o b t a i n e d . S p e c i f i c a l l y , propylene carbonate and benzene achieved comparable e f f i c i e n c i e s a s opposed t o l o w e r e f f i c i e n c i e s b e i n g o b t a i n e d w i t h methanol m o d i f i e r f o r This could t h i s p a r t i c u l a r sample m a t r i x and a n a l y t e s e t . h a v e b e e n due t o t h e m u t u a l a t t r a c t i o n s between t h e e l e c t r o n c l o u d s of t h e m o d i f i e r s and t h e aromatic a n a l y t e s and t h e overcoming of s u r f a c e a c t i v a t i o n e n e r g i e s ( 1 9 ) .

Table 1 : Use of Modifiers in On-Line SFE/GC Percent Recovery Compound ethylbenzene cumene Pchloronaphthalene 1,2,4-trirnethylbenzene

Benzene 95 96 92 96

Methanol 80 79 82 84

Pro yiene Car onate

E

-2

96 96 93 96

74 72 66 71

SFE: 225 mg of sludge/ash, 375 atm, S 0 C , 10 minutes static, 7 minute dynamic, 50 ul modifier In 500 ul vessel

GC:

30 x 0.25 mm I.D. DB-WAX 30°C (7 mln) to 31OoC at 7°C/minute

F u r t h e r s e l e c t i v i t i e s c a n be e x p e r i e n c e d i n o n - l i n e SFE/GC by t h e u s e o f a l t e r n a t e s u p e r c r i t i c a l f l u i d s s u c h a s s u l f u r h e x a f l u o r i d e (SF 1 a n d n i t r o u s o x i d e (N20). The u s e o f t h e s e s u p e r c r i t i c a l f l u i d s i n o n - l i n e SFE h a s b e e n l i m i t e d c o m p a r e d t o CO u s e , b u t h a s d e m o n s t r a t e d c e r t a i n d i s t i n c t a d v a n t a g e s (8,1$,14,17) d u e t o t h e p h y s i c a l p r o p e r t i e s o f t h e supercritical fluids ( i . e . d i p o l e moment, p o l a r i t y , and surface tension). An e x a m p l e o f t h e u s e o f S F 6 compared t o C 0 2 f o r o n - l i n e SFE/GC i s shown i n F i g u r e 8 f o r t h e d e t e r m i n a t i o n of p o l l u t a n t s i n contaminated s o i l . With f l a m e i o n i z a t i o n GC d e t e c t i o n , a n i n i t i a l e x t r a c t i o n was p e r f o r m e d using supercritical SF6 on g a s o l i n e contaminated soil f o l l o w e d by a c o n s e c u t i v e e x t r a c t i o n u s i n g s u p e r c r i t i c a l C 0 2 . A s c a n be s e e n , t h e u s e o f SF for the initial extraction p r o v i d e d a predominance o f n - a l $ a n e s from t h e complex g a s o l i n e contaminated s o i l compared t o a l a t e r e x t r a c t i o n of a d d i t i o n a l a r o m a t i c species u s i n g C 0 2 .

207 8F6

TlyE (minutes)

F i g u r e 8: Consecutive e x t r a c t i o n s u s i n g SF6 first followed On-Line SFE/GC a n a l y s i s o f g a s o l i n e c o n t a m i n a t e d b y , C02. soil. SFE: 3 0 0 a t m , 60°C, 1 2 m i n u t e s , 3 0 0 m g . GC: 5 0 x 0 . 2 mm I . D . m e t h y l s i l i c o n e (PONA), -3OOC ( 1 2 m i n . ) 25OoC a t 7O~/rnin, D i f f e r e n t a d s o r b e n t s can a l s o be u s e d t o s e l e c t i v e l y i m m o b i l i z e c e r t a i n i n t e r f e r i n g a n a l y t e s from a complex sample matrix before a n a l y t i c a l determinations. A d s o r b e n t s , s u c h as c e l i t e , sodium s u l f a t e , magnesium s u l f a t e , f l o r i s i l , a l u m i n a , p o l y u r e t h a n e foam, a n d h y d r o m a t r i x (20,211 h a v e b e e n u s e d i n o n - l i n e SFE a p p l i c a t i o n s t o remove i n t e r f e r i n g a n a l y t e s a n d w a t e r from v a r i o u s m a t r i c e s . An o b v i o u s d i s a d v a n t a g e c o u l d be s i t u a t i o n s w e r e t a r g e t a n a l y t e s a r e i r r e v e r s i b l y bound t o t h e adsorbents d i s t o r t i n g a n a l y t i c a l r e s u l t s . Adsorbents h a v e b e e n u t i l i z e d by m i x i n g them w i t h p r o p o r t i o n a t e amounts o f sample m a t r i x b e f o r e i n t r o d u c t i o n i n t o a n e x t r a c t i o n vessel o r by u t i l i z i n g two e x t r a c t i o n v e s s e l s i n s e r i e s ( t h e f i r s t with t h e sample matrix, and t h e second with t h e

208 adsorbent before GC introduction). An example of the latter technique is shown in Figuze 9 with the on-line SFE/GC characterization of neat orange juice. Due to the high amount of water present in orange juice, an extraction vessel packed with molecular sieve adsorbent was placed between the sample matrix extraction vessel and the GC. As can be seen, a potentially quantitative characterization of the orange juice was achieved with a separation of limonene, terpenes, and sesquiterpene components.

Molecular Sieve Adsorbent

1‘5

25

35

TlyK (mlnut-)

Figure 9: Adsorbents in SFE. On-Line SFE/GC of orange juice. SFE: 400 atm, 6OoC, 15 minutes, 1.5 milliliters.

MULTIVESSEL ON-LINE SFE/GC An

approach to automating SFE sample introduction into

GC is by using valved multiple extraction vessels in sequence as is depicted in Figure 10. Basically, four vessels up to a maximum volume of 10 milliliters (limited by the supply syringe pump capacity in one fill cycle), are linked into a ten port, four position selector valve which controls the flow sequence of vessel one through vessel four. One vessel is extracted at a time with the static and dynamic extraction modes being controlled by a six port, two position switching valve. In the dynamic mode, the extraction effluent is transferred through the transfer line directly into the GC injection port. Immediately when this transfer is initiated, a ready signal is sent from the SFE and the GC run is started. The other vessels remain intact in the SFE oven until the GC run in complete and the sequence is then

209

re-initiated for the next extraction vessel. All of the extraction vessels are exposed to identical operational pressures and temperatures simultaneously, so leak-free vessels are mandatory to ensure quantitative repeatability.

-----------------------------

I I

L

I

Oven I _____---.---SFE ------------------

Figure 10: Multi-Vessel Configuration.

On-Line

Split

SFE/GC

System

example of the type of precision and repeatability that can be attained with a sequential multivessel on-line SFE/GC system is demonstrated in Figure 11 and Table 2. The test mixture consisted of naphthalene, phenanthrene, and pyrene in a solution of carbon disu1fi.de which was spiked onto four identical one milliliter vessels packed with glass wool. After establishing that the vessels and valving were leakfree, the on-line split SFE/GC-FID experiments were performed with each individual vessel in sequence. As can be seen from the raw area counts in Table 2, each of the respective PAHs demonstrated favorable relative standard deviations ( % RSD) It is important to note that these %RSDs represented the total on-line S F E / G C system repeatability including the extraction of the analytes, transfer of the extraction effluent to the GC, introduction of the analytes to the GC capillary column, GC analyte separation, and finally flame ionization detection of the analytes.

An

.

21 0

6

Naphthaletre

bhenanthrenc

'r

I

18.5

4

Pyrene

#

2

27.1

TIME (minutes)

F i g u r e 11: S e q u e n t i a l M u l t i v e s s e l On-Line SFE/GC o f PAH T e s t Mixture. SFE: 1000 ppm s o l u t i o n i n CS2 on g l a s s wool i n o n e m i l l i l i t e r e x t r a c t i o n vessel, 350 atm, 6OoC, 2 minutes 30 meter x 0 . 3 2 rnm I . D . s t a t i c , 1 0 m i n u t e s dynamic. GC: DB-5, 35OC ( 1 0 m i n u t e s ) t o 280°C a t 1S0C/minute.

21 1

Table 2: Sequential Vessel-to-Vessel Repeatability Using On-Line SFE/GC

Vessel #

Raw Area Counts Naphthalene 975999 1095281 1007597 1032128

mean standard devlatlon

=

% RSD

=4.92

=

1027751 50543

Phenanthrene 1171944 1278659 1226372 1176554

mean standard devlatlon % RSD

= = =

1213383 50011 4.12

Pvrene 1 2 3

1764219 1866492 1226372

mean standard devlatlon

= = =

Yo

RSD

1864604 81292 .__ 4.35

212

ON-LINE SFE/SFC

Supercritical fluid Chromatography (SFC) has been used widely as an analytical tool for the separation of relatively non-polar, thermally unstable and high molecular weight solutes. The physical properties of supercritical fluids have unique characteristics to solve many problems, which both gas chromatography (GC) and high performance liquid chromatography (HPLC) fail to solve. The coupling of SFE to SFC has been developed by several groups. The on-line modes of SFE/SFC have several distinct advantages that are beyond the scope of either technique when used separately. These advantages are (a) trace analysis capability, (b) preparation with minimal sample contamination, (c) higher reproducibility, (d) increased productivity, and (e) on-line automation of the sample preparation step with the chromatographic analysis step. In the on-line mode, a high pressure fluid extracts the sample matrix in a high pressure extraction cell and the extracted components are trapped or focused in a device prior to SFC analysis. SFE/SFC INTERFACE MECIIANICS

On-line SFE/SFC systems has been developed and investigated by different research groups. The arrangements in these hyphenated techniques usually involved several valves, one or two pumps and ovens. A system can be constructed very simply to perform qualitative analysis or can be arranged to perform both qualitative and quantitative analysis. A simple approach has been applied to perform on-line SFE/SFC in both static (after pressurization of the extraction cell, extraction is allowed without passing any flow of supercritical fluid through the cell) and dynamic (after pressurization of the extraction cell, supercritical fluid continuously flows through the outlet of the cell) modes. Figure 12 shows an on-line SFE/SFC system where one pump and two valves were used to perform both SFE and SFC. In this Figure (lZA), the extraction cell and sample loop are filled with the supercritical fluid which has appropriate density for the extraction of the sample. By switching the A valve to the second position (Figure 12B) , the extraction vessel and sample loop were bypassed while both remained under high pressure. During static extraction, the pump and column were equilibrated for chromatography. After a certain period of static extraction, the content of the sample loop which contained the extraction material was flushed by the supercritical mobile phase onto the SFC column (Figure 12C). It is important to mention here, that the same supercritical fluid was used for both extraction and chromatography. Because of similar reasons different size injection loops can be placed in the valve and different amounts of extracted sample could be injected to the SFC column, which usually depends on the concentration of the analytes of interest or on the sensitivity of detector (22). Similar methods have been used by other authors to perform on-line SFE/SFC in static mode

213

TO C O l u m

mWdor

Figure 12: S c h e m a t i c o f SFE/SFC s y s t e m i n e x t r a c t i o n chromatography modes u s i n g s i n g l e s y s t e m pump.

and

21 4 (23,24). Another method which has been used frequently for on-line SFE/SFC is the usage of the second supply pump to pressurize the extraction vessel and the sample. Meanwhile the first supply pump is used to solely obtain chromatographic separation. Applying the second pump for extraction usually creates less technical problems and more freedom since both modes of extraction can be achieved and also the chromatographic system is independent of the extraction system. Figure 13 shows an SFE/SFC system which is able to perform both modes of extraction (static and dynamic). In this style Pump A is used for extraction while Pump B is used for chromatography (25). Several systems were designed in a similar fashion which were able to perform dynamic and static extractions. ( 2 6 , 2 7 )

vent +-

Inlet A

-+ +

inlet B

Figure 13: Schematic of SFE/SFC system using dual supply pumps for extraction and chromatography.

215 O t h e r approaches t h a t have been u s e d t o perform o n - l i n e SFE/SFC have been s y s t e m s w i t h m u l t i p l e s w i t c h i n g v a l v e s t h a t p e r m i t t h e u s e r t o c o l l e c t t h e e x t r a c t e d sample i n a c o o l e d accumulator, w h i l e t h e same s u p p l y pump i s used t o s u p p l y s u p e r c r i t i c a l f l u i d mobile phase t o t h e chromatographic

system. I n t h e s e s y s t e m s e x t r a c t s a r e u s u a l l y f o c u s e d i n an i n t e r f a c e t r a p b e f o r e t h e chromatographic column, w h i c h i s c o o l e d down t o a c e r t a i n t e m p e r a t u r e . A f t e r e x t r a c t i o n and c o l l e c t i o n , t h e v a l v e s a r e s w i t c h e d and t h e c r y o c o o l e d t r a p t e m p e r a t u r e i n c r e a s e s u s i n g t h e oven h e a t o r a s e p a r a t e heater. By s w i t c h i n g t h e v a l v e s and h e a t i n g t h e c r y o c o o l e d trap, supercritical f l u i d c a r r i e s t h e extracted materials from t h e t r a p o n t o t h e a n a l y t i c a l column. F i g u r e 1 4 shows an o n - l i n e SFE/SFC system which u s e s a c r y o f o c u s i n g r e g i o n t o c o l l e c t the extracted material. In t h i s system the selector During t h i s v a l v e i s f i r s t p l a c e d t o t h e column p o s i t i o n . p e r i o d t h e t e m p e r a t u r e o f t h e cryocooled r e g i o n i s a d j u s t e d Next, t h e v a l v e i s s w i t c h e d t o t h e t o a d esi r ed l e v e l . e x t r a c t i o n v e s s e l where t h e s u p e r c r i t i c a l f l u i d w i t h t h e d e s i r e d d e n s i t y removes t h e e x t r a c t e d m a t e r i a l from t h e e x t r a c t i o n region t h r o u g h a r e s t r i c t o r i n s i d e t h e c r y o f o c u s e d region. During t h e decompression o f Cog, a t t h e t i p o f t h e r e s t r i c t o r , the extracted analytes are collected i n t h e cold t r a p . A f t e r e x t r a c t i o n and c o l l e c t i o n , t h e s e l e c t o r v a l v e i s s w i t c h e d t o t h e column p o s i t i o n and t h e s u p e r c r i t i c a l f l u i d moves t h e a n a l y t e s from t h e t r a p t o t h e column. Meanwhile t h e t r a p i s a l s o h e a t e d t o the n e c e s s a r y t e m p e r a t u r e t o h e l p move t h e a n a l y t e s o n t o t h e column. (28,291

U

CHROMATOGRAPHIC OVEN

SFE/SFC s y s t e m and c r y o g e n i c c o o l i n g t r a p w i t h Figure 1 4 : m u l t i p l e valves.

216

A n o t h e r s y s t e m which u s e s a c r y o f o c u s e d t r a p a c c u m u l a t o r t o p e r f o r m o n - l i n e h i g h p r e s s u r e SFE/SFC i s shown i n F i g u r e 15. The s y s t e m i s c o m p r i s e d o f t h r e e d i f f e r e n t v a l v e s ( t e n port/two p o s i t i o n , f i v e p o r t / f o u r p o s i t i o n and f o u r port/two p o s i t i o n s e l e c t o r v a l v e s ) a n d a z e r o - d e a d volume t e e . D u r i n g t h e e x t r a c t i o n p e r i o d , t h e m o b i l e p h a s e from t h e pump e n t e r s t h e tee. T u b i n g from one o u t l e t o f t h e t e e l e a d s t h e m o b i l e p h a s e t o t h e i n j e c t o r v a l v e f o r u s e o n l y i n c o n v e n t i o n a l SFC applications. Tubing from t h e o t h e r o u t l e t o f t h e t e e l e a d s t h e m o b i l e p h a s e t o t h e i n j e c t o r valve f o r u s e o n l y i n c o n v e n t i o n a l SFC a p p l i c a t i o n s . Tubing f r o m t h e o t h e r o u t l e t o f t h e t e e g o e s t h r o u g h t h e t e n p o r t valve t o t h e e x t r a c t i o n vessel and t h e n i n t o t h e f i v e p o r t s e l e c t o r v a l v e . From t h e t e n p o r t v a l v e , t h e e x t r a c t e d a n a l y t e s are p a s s e d t h r o u g h a r e s t r i c t o r i n t o t h e c r y o f o c u s e d t r a p , which i s c o o l e d t o t h e s p e c i f i e d e x t r a c t i o n t e m p e r a t u r e w i t h i n d u s t r i a l g r a d e , boned r y carbon d i o x i d e . A l l of t h e e x t r a c t e d m a t e r i a l i s t h e n c o l l e c t e d i n t h e cryofocused t r a p . The d e c o m p r e s s e d C 0 2 g a s from t h e t r a p i s t h e n v e n t e d t h r o u g h t h e t e n p o r t v a l v e i n t o t h e atmosphere oven. A f t e r t h e completion o f t h e e x t r a c t i o n , Upon r e a c h i n g t h e pump i s e q u i l i b r a t e d f o r c h r o m a t o g r a p h y . e q u i l i b r i u m , t h e t e n p o r t a n d f i v e p o r t s e l e c t o r v a l v e s are s w i t c h e d s i m u l t a n e o u s l y ( F i g u r e 15B). I n t h i s c o n f i g u r a t i o n , m o b i l e p h a s e passes t h r o u g h t h e tee, t h e i n j e c t i o n v a l v e , a n d t h e t e n p o r t valve i n t o t h e c r y o f o c u s e d t r a p , which i s t h e n b a l l i s t i c a l l y heated t o t h e s p e c i f i e d i n j e c t i o n temperature. A f t e r b a c k f l u s h i n g , t h e mobile phase carries t h e e x t r a c t e d components f r o m t h e t r a p b a c k t o t h e t e n p o r t v a l v e i n t o t h e c h r o m a t o g r a p h i c column. A d d i t i o n a l C 0 2 f l o w from t h e o t h e r o u t l e t of t h e t e e t o t h e cryofocused trap r e s t r i c t o r prevents b a c k f l u s h i n g of t h e e x t r a c t e d m a t e r i a l i n t o t h e r e s t r i c t o r .

(30) Obtaining q u a n t i t a t i v e r e s u l t s f r o m any a n a l y t i c a l methods h a s b e e n a c h a l l e n g i n g a r e a f o r a n a l y t i c a l c h e m i s t s . Most o f t h e SFE/SFC d e v i c e s d e s c r i b e d a b o v e a r e d e s i g n e d t o obtain q u a l i t a t i v e results. F o r example, t h e l a t e r d e s i g n by A s h r a f - K h o r a s s a n i e t . a l . (30), showed q u a n t i t a t i v e r e s u l t s f o r d i f f e r e n t h y d r o c a r b o n s t a n d a r d s u s i n g a n o n - l i n e SFE/SFC T h e r e s u l t s showed t h a t t h e amount o f m a t e r i a l system. e x t r a c t e d was d i r e c t l y p r o p o r t i o n a l t o t h e volume o f sample placed i n the extraction vessel. L a t e r t h e y showed t h e q u a n t i t a t i v e a n a l y s i s f o r d i f f e r e n t a d d i t i v e s i n low d e n s i t y p o l y e t h y l e n e (LDPE) ( 3 1 ) These a d d i t i v e s were e x t r a c t e d from l o w d e n s i t y p o l y e t h y l e n e , c o l l e c t e d i n t h e c r y o f o c u s e d t r a p and q u a n t i t a t e d u s i n g a m u l t i - l e v e l c a l i b r a t i o n c u r v e w i t h flame i o n i z a t i o n d e t e c t i o n .

.

The h e a t i n g and c o o l i n g o f t h e c r y o g e n i c c o l l e c t i o n r e g i o n a r e i m p o r t a n t f a c t o r s which n e e d t o b e c o n s i d e r e d . S i n c e a n a l y t e v a p o r p r e s s u r e s depend on t h e t e m p e r a t u r e , by varying t h e t r a p temperature e f f e c t i v e l y during e x t r a c t i o n a n d b a c k f l u s h i n g o n t o t h e column, o n e c a n o b t a i n b e t t e r e f f i c i e n c y on b o t h t r a p p i n g and b a c k f l u s h i n g o f a n a l y t e s . Ashraf-Khorassani, et .al. ( 3 0 ) have shown ( F i g u r e 1 6 ) t h e

217

b F & .......

,C...........l.,....................,..

Load Posttlon

m %

&

_--- ....

J

SELECTOR . . _ I _

......__......._....

-....--

...---.....

.

-.....

Inject Posltlon

F i g u r e 15: SFE/SFC s y s t e m a n d c r y o g e n i c t r a p w i t h m u l t i p l e v a l v e s i n c o l l e c t i o n a n d i n j e c t i o n modes.

effect of cooling and heating of the trap on the efficiency of collection and backflushing. Two different trap temperatures were applied to cryofocus different normal alkane standards (ClO-C60). At low temperatures (-5OoC), the collection efficiency was much higher (Figure 16A, 16B) for decane, compared to a higher temperature (-30°C) for the collection of same analytes (Figure 16C). Also the effect of the trap temperature on the backflushing of the analytes onto the chromatographic column after the extraction was demonstrated. Different trap temperatures were applied (-50°C and 180°C) upon backflushing the analytes. At lower trap temperatures (-50°C) , during backflushing, the higher molecular weight analytes with lower volatility were not transferred to the SFC column even at high pressures (450 atm) However at higher trap temperatures all of the analytes were transferred without any discrimination into the chromatographic column. Anderson et.al. (2 8 ) have shown similar results using SFE/SFC. They demonstrated by controlling the trap temperature, lower molecular weight compounds, such as limonene in grapefruit oil, were passed through the trap and higher molecular weight compounds (i.e. carrone) was trapped.

.

2

B

C

c I

a

10

1

15

C 0

*5

la

15

t

0

j

90

L 15

TIME, min.

Figure 16: Effect of cryogenic trapping temperature on SFE/SFC analysis of hydrocarbons using supercritical C 0 2 extraction and chromatography. Peaks: (1) CloI (2) C20r c ( 4 ) C40, (5) C5oi (6) CQO. Collection temperatures: Z ! ' C , (b) -5OoC, (C) -30 C, transfer temperatures: -5OoC, (B) 180°CI (C) 180°C.

the for (3) (A) (A)

219 A f u r t h e r d e m o n s t r a t i o n o f o n - l i n e SFE/SFC i s shown w i t h t h e a n a l y s i s of d i f f e r e n t a d d i t i v e s i n polymers. Figure 17 shows a chromatogram o f f i v e d i f f e r e n t a d d i t i v e s which a r e e x t r a c t e d from l o w d e n s i t y p o l y e t h y l e n e . For t h i s e x p e r i ment, a c e r t a i n amount o f p o l y m e r was placed i n t h e e x t r a c t i o n vessel, extracted (450 atm, 100°C), c o l l e c t e d and c r y o f o c u s e d a t -25OC. A f t e r e x t r a c t i o n a n d c o l l e c t i o n , t h e t r a p was b a c k f l u s h e d a n d t h e e x t r a c t e d component w a s f l u s h e d i n t o t h e a n a l y t i c a l SFC p o l y e t h y l e n e g l y c o l column. Each a d d i t i v e was i d e n t i f i e d a n d q u a n t i t a t e d a t 200-300 ppm levels. F i g u r e 18 shows t h e e x t r a c t i o n a n d s e p a r a t i o n o f t h r e e d i f f e r e n t a d d i t i v e s from a n o t h e r p o l y e t h y l e n e concentrate. A a i n , a f t e r e x t r a c t i o n (450 atm, 100°C) a n d collect i o n (-25 C ) , t h e e x t r a c t e d components were b a c k f l u s h e d i n t o t h e SFC o c t a d e c y l column, w i t h e a c h a d d i t i v e b e i n g d e t e r m i n e d a t 1 0 0 - 2 0 0 ppm l e v e l s .

8

3 A

1. 2 3. 4. 5.

BKT BHEB tsonoxl29 lrganox 1070 lrganox 1010

2

100 I 0

10

20

450 485 30 40

50

PRESSURE, atm TIME, mlnuter

Figure 17: SFE/SFC o f d i f f e r e n t a d d i t i v e s from low d e n s i t y polyethylene.

220

2

100 0

I . lrgafos 168 2. Cyasorb 3346 3. Cyanox1790

450 485

10

Figure 18: concentrate.

20 SFE/SFC

30 of

40 additives

je

PRESSURE, atm TIME, mlnutes from

polyethylene

CONCLUSIONS The multidimensional use of supercritical fluid technology specifically implies the use of on-line SFE/GC and SFE/SFC methodologies. Both of these directly coupled methodologies fall into the realm of problem-solving tools that car, be effectively utilized by analytical chemists for

qualitative or quantitative characterizations or deterrinations. For GC, S F E presents itself as a selective sa-qple introduction means for liquid or solid sample matrices with volatile and non-volatile analytes. The nature of SFE instrumentation provides an added feature with the potential capability of doing sample preparation in the field with analytical determinations using an on-line GC/MS. On-line SFE/SFC complements SFE/GC when target analytes are therr&ally labile or beyond the volatility range of GC. An added feature with SFE/SFC is the depth of method development capability since SFC can be interfaced to a full array of GClike and LC-like detectors (e.g. flame ionization, ultraviolet absorbance, mass spectrometer, infrared, nitrogen-phosphorus and sulfur chemilluminescence to narne a few). As S F E technology further evolves certainly additional capabilities will be added and refined in the areas of selectivity enhancement (i.e. alternate fluids, modifiers, absorbents, in-situ derivatizaton), operational parameters optimization (what conditions to use for specific analytes and sample matrices), automation (sequential and parallel) and new on-line interfaces (LC and G P C ) . Acknowledgments The following people need to be identified and acknowledged for their experimental work which was described in this chapter: Athos Rosselli, David Boyer, Eugene Storozynsky, Kathryn Cross, Robert Ravey and Carol Hamilton (the ever-loyal secretary)

.

References S.B. Hawthorne and D . J . 258-264

Miller.

J. Chromatogr. Sci. 2 4 :

(1986).

S.B. Hawthorne, M.S. Krieger, and D . J . Chem. 61: 7 3 6 - 7 4 0 ( 1 9 8 9 ) .

Miller.

S.B. Hawthorne, M.S. Krieger, and D . J . Chem. 60: 472-477 (1988).

Miller.

Anal. Anal.

J . M . Levy and A.C. Rosselli. Chromatographia. 2 8 : 1 1 / 1 2 , 613-616 ( 1 9 8 9 )

B.W. Wright, S.R. Frye, D.G. McMinn, and R . D . Anal. Chem. 5 9 : 640-644 ( 1 9 8 7 )

issue

Smith.

Levy, J . P . Guzowski and W.E. Huhak. J . High Resolut. Chromatogr. Chromatogr. Commun. 10: 3 3 7 - 3 4 7

J.M.

(1987).

J.M. Levy, J . P . 207-210 (1988)

Guzowski.

Fresenius Z. Anal. Chem. 3 3 0 :

222

8 J.M. Levy, R.A. Cavalier, T.N. Bosch, A.F. F.ynaski, and W.E. Huhak. J. Chromatogr. Sci. 27: 341-346 (1989). 9 S.B. Hawthorne and D.J. Miller. (1987).

J. Chromatogr. 403: 63-76

10 S.B. Hawthorne, D.J. Miller, and M.S. Krieger. Chromatogr. Sci. 27: 347-354 (1989).

J.

11 M.W.F. Nielen, J.T. Sanderson, R.W. Frei, and U.A.T. Brinkman. J. Chromatogr. 474: 388-395 (1989). 12 S.B. Hawthorne, D.J. Miller, and J.J. Langenfeld. Chromatogr. Sci. 28: 2-8 (1990) 13 K. Sugiyama, M. Saito, T. Hondo, and M. Senda. Chromatogr. 332: 107-116 (1985).

J.

J. of

14 S.B. Hawthorne. Anal. Chem., 62 (No. 11): 633A-624A (1990) 15 R.J. Houben, H.G.M. Janssen, P.A. Leclercq, J.A. Rijks, and C.A. Cramers, J. High Resolution Chromatogr. 13: 669673 (1990) 16J.M. Levy, A.C. Rosselli, D.S. Boyer and K. Cross, J. High Resolution Chromatogr. 13: 416-421 (1990). 17 J.M. Levy, D.S. Boyer, E. Storozynsky, R. Ravey, M. Ashraf-Khorassani. LC/GC in press (1991)

.

18 S.B. Hawthorne, unpublished results. 19 J.M. Levy, "Use of Modifiers in SFE", ACS Symposium Series, M.E. McNally, in press (1991). 20 J.M. Levy, unpublished results. 21 Jerry King, personal communication. 22 J.W. Wheeler, M.E. McNally, J. Chromatogr. Sci., 27, 534 (1989). 23 M. Ashraf-Khorassani, L.T. Taylor, LC-GC, 8, 314 (1990). 24 R.J. Skelton Jr., C.C. Johnson, L.T. Taylor, Chromatographia, 21, 3-12 (1986)

.

25 G. Mapelli, C. Borra, F. Munari, S. Trestiance, Tenth International Symposium on Capillary Chromatography, 1, 430 (1989). 26 W.P. Jackson, K.E. Markides, M.L. Lee, J. High Resolu. Chromatogr. & Chromatogr. Commun., 9, 213-218 (1986).

2 7 M. Ashraf-Khorassani, L.T. Taylor, Anal. Chem., 148 ( 1 9 8 9 ) .

61, 1 4 5 -

2 8 M.R. Anderson, J.T. Swanson, N.L. Porter, B.E. Richtey, J. Chromatogr. Sci., 27, 3 7 1 - 3 7 7 ( 1 9 8 9 ) .

29 Q.L. X i e , K.E. Markides, M.L. L e e , 27,

365-370

J. Chromatogr. Sci.,

(1989).

30 M. Ashraf-Khorassani, M.L. Kumar, D.J. Koebler, G.P. Williams, J. Chromatogr. Sci., 28, 5 9 9 - 6 0 4 ( 1 9 9 0 ) .

31 M. Ashraf-Khorassani, J.M. Levy, J. High Resolution Chromatogr., 13, 742-747, ( 1 9 9 0 ) .

This Page Intentionally Left Blank

K. Jinno (Ed.), Hy henated Techniques in Supercritical FluifChrornatography and Extraction Journal of Chromatography Library Series, Vol. 53 0 1992 Elsevier Science Publishers B.V. All rights reserved.

225

Chapter 12 ADVANCES IN ANALYTICAL SUPERCRITICAL FLUID EXTRACTION (SFE)

Steven B. Hawthorne, David J. Miller, and John J. Langenfeld Energy and Environmental Research Center University of North Dakota Grand Forks, North Dakota, USA 5 8 2 0 2 INTRODUCTION

The ability of supercritical fluids to dissolve low vapor pressure solids was first reported by Hannay and Hogarth in 1879 (l), and numerous process-scale applications of SFE have been developed since the mid 1900s ( 2 ) . In contrast, the use of SFE for the extraction of trace and minor components on the analytical scale has only received attention during the last few years. For example, at the first International Workshop on Supercritical Fluid Chromatography (Park City, Utah, USA) held in January, 1988, only one paper focused on SFE. Since then, the number of investigators developing analytical-scale S F E methods has increased rapidly, as evidenced by the fact that 25 SFE papers were presented at the next meeting held in June, 1989. Compared to the incredible development of chromatographic techniques and detectors which has occurred since Tswett's first report of chromatography in 1906, relatively few advances in sample extraction/preparation methods have been developed. Indeed, one of the most commonly-used extraction methods, liquid solvent extraction with a Soxhlet apparatus, is still performed in essentially the same manner as it was before Tswett's 1906 report. Since the extraction of organic compounds from sample matrices is often the most error-prone and slowest of an entire analytical scheme, the replacement of liquid solvent extractions with SFE has several potential advantages. These advantages can be summarized as: Speed: Mass transfer is faster in a supercritical fluid than in liquid solvents because supercritical fluids have lower viscosities (10" vs. N-sec/m2) and higher cm2/sec). Since mass solute diffusivities (10" vs. transfer limitations often control extraction rates, quantitative SFE can usually be completed in 10 to 60 minutes, compared to several hours for liquid solvent extractions.

Variable solvent strength: Since the solvent strength of

a supercritical fluid depends primarily on its density (2,3,4), the solvent strength can easily be manipulated by changing the pressure and temperature of the extraction. This allows SFE parameters to be optimized for a target analyte, and provides a method to achieve class-selective extractions ( e . g . alkanes versus PAHs) from a single sample by simply extracting the sample at two different pressures with the same supercritical fluid. The large volumes of liquid solvents used for conventional extractions have caused recent concern because of their potentially toxic nature and rapidly-increasing disposal costs. Since most commonly-used supercritical fluids are gases at ambient conditions and SFE effluents are typically collected in only a few mL of liquid solvent (or no liquid solvent for on-line SFE methods), the need for liquid solvents is dramatically reduced. In a similar manner, the need to concentrate extracts prior to analysis of trace analytes is also greatly reduced.

Reduction of liquid solvent usage:

Simplified on-line coupling with chromatographic techniques: The gaseous nature (at ambient conditions) of most supercritical fluids also facilitates the direct

coupling of SFE with GC and SFC (as discussed in Chapter 9)

-

It must be emphasized that SFE is a sample extraction/ preparation technique (analogous to liquid solvent extraction), and has no inherent need to be coupled with a chromatographic technique. While on-line techniques such as coupled SFE-GC and SFE-SFC (discussed in Chapter 9) are viable approaches to many analytical problems, the majority of SFE studies have been performed using off-line collection of the extracted analytes followed by analysis using a variety of measurement techniques including (but not limited to) chromatographic, spectroscopic, gravimetric, and radiochemical. Factors that contribute to the choice of offline or on-line SFE are outlined in Table I. The major advantage of on-line approaches is the potential to transfer every extracted analyte molecule to the chromatographic system. Thus, on-line techniques are preferred when maximum sensitivity is needed from small samples. However, on-line SFE techniques generally require that the chromatographic system be used for sample collection during the SFE step, thus reducing the number of chromatographic runs that can be performed during a set period of time. Off-line SFE is inherently simpler to perform, since only the extraction parameters need to be understood, and several analyses can be performed on a single extract. The list in Table I should only be viewed as general guideline, since the continued development and refinement of on-line and off-line techniques

227

will likely change the factors that control the choice between these two approaches. Table I Comparison of Off-Line and On-Line SFE Off-Line GC or SFC needed for extraction? 100% transfer of analytes? multiple injections per extract? polarity modifiers useful? sample handling between SFE and analysis? maximum convenient sample size?

no no Yes Yes yes 10-15g

On-Line Yes Yes no ??

no 1-3g

Because the development of analytical SFE has occurred almost exclusively since 1986, the related literature has tended to be applications oriented, and the development of analytical SFE methods has been largely empirical. Since pure supercritical fluids that are convenient to use (e.g., CO,) are relatively non-polar, the majority of SFE studies reported to date have focused on the extraction of relatively non-polar and low molecular weight analytes (e.g., those amenable to separation using GC) although there are several notable reports of the extraction of more polar analytes. A survey of the literature also makes it apparent that the processes that control the extraction rates and ultimate recoveries achieved using SFE are poorly understood. Numerous qualitative and quantitative applications of analytical SFE have been reported during the last three years for a wide variety of sample matrices including (but not limited to) environmental solids (5-27), sorbent resins (9,18,28-33), food products and biological tissues (18,22,23,34-55), polymers (30,50,56-59), and petroleum-related samples (17,25,60-64). A review of analytical SFE applications is beyond the scope of this chapter, and the interested reader is referred to the above references and recent review articles (3,65). Instead, this chapter includes a brief description of off-line SFE techniques and instrumentation, and focuses more on the mechanical and chemical principles that need to be considered to successfully develop an analytical SFE method for both nonpolar and polar analytes from a variety of matrices. Approaches to extending SFE to more polar and higher molecular weight species including the use of polarity modifiers and insitu chemical derivatization, and the recent extension of SFE to water samples will also be discussed. Examples will be given of recent results from the literature and our laboratory.

GENERALIZED METHODS FOR SFE A brief description of SFE techniques and instrumentation is given below. However, since instrumentation and methods are rapidly evolving, the reader should consult individual publications and instrument suppliers for details. A recent review ( 3 ) and the references therein describe various approaches to SFE in detail.

In contrast to a popular misconception, analytical-scale SFE is inherently simple to perform, and need not be unreasonably expensive. (For purposes of this chapter, analytical SFE will be restricted to sample sizes
229

Figure 1: Components of a simple SFE instrument. The fluid reservoir (A) is connected using 1/16 inch stainless steel tubing to a shut-off valve (B) mounted on a pressurecontrolled syringe pump (C). During extraction, valve (B) is closed, and shut-off valve (D) is opened to supply the pressurized extraction fluid to the extraction cell (E) which is placed in a tube heater (F) to maintain the extraction temperature. The extracted analytes then flow out of the restrictor (G) into the collection vessel (H). For static extractions, an additional shut-off valve (I) can be placed between the extraction cell and the restrictor as indicated on the figure. Two common modes are used for SFE; dynamic and static. For dynamic SFE the sample is constantly swept with fresh supercritical fluid at a flow rate determined by the extraction pressure and the dimensions of the outlet restrictor. For static SFE the sample cell is pressurized with the fluid and the sample is extracted with no outflow of the supercritical fluid. After the extraction is thought to be completed, a valve is opened at the outlet of the cell (Figure 1) to allow the analytes to be swept from the cell into the collection device. Typically, a static extraction is followed by several minutes of dynamic extraction to recover the analytes. Dynamic SFE continually provides new fluid to the sample, and is more effective when the supercritical fluid is likely to be saturated with the target analytes. Static extraction has the advantages that less fluid is used and that liquid polarity modifiers can be used by simply adding them to the cell prior to pressurization. Absolute criteria for selecting dynamic or static modes are not yet clear, and both have been widely used to achieve quantitative extractions. Additional considerations will be discussed later in the text. While it is not the purpose of this work to rank SFE instrumentation (a process that would not be useful given the rapid changes in commercial instruments), some general guidelines to consider before selecting SFE pumps and extraction cells are listed in Table 11. In general,

230

characteristics in the llminimalll column will allow essentially the same SFE experiments to be performed as those in the lldesirablell column (with the exception of pressure limits); however, the number of extractions per day and the overall convenience will be lower. We have found the m o s t important characteristics of the SFE pump to be fast recovery after pressurizing the cell, fast refill and pressurization to working pressure, and a continuous display of the fluid flow rate. For extractions requiring polarity modifiers (discussed below), dual pumps that can provide known fluid mixtures are convenient, however, a single pump can also be used by mixing the modified supercritical fluid in the pump. Untilrecently, many SFE cells (including commercial cells and those made in the lab) were subject to leaks and had very limited lifetimes. Fortunately, commercial SFE cells are now available at a cost of ca. $100 to $1000 (US) that are extremely reliable, and some cells in our lab have been used for more than 1000 extractions without leaking. Table I1 Characteristics of SFE Pumps and Cells minimal

PUP maximum flow rate (at full P) recovery time (after pressurizing cell)' fill rate total fill cycle timeb upper pressure limit flow readout (real time) modifier mixing (variable and controlled by pumps)

1 mL/min 5

min

desirable

mL/min

>50

< l o sec

10 mL/min 30 min 300 atm

<5

100 mL/min

should be no

Yes Yes

min

600 atm

extraction cells

pressure rating no wrenches needed to assemble' finger tight connections to pumpC replaceable frits and seals' dead volume lifetime (number of extractions)

300 atm

>600

no no no

Yes Yes Yes


atm

<0.1 mL >loo0

'Time required for the pump to regain the working pressure after pressurizing the sample cell. bTotal time required for an empty pump to be filled and returned to the working pressure. 'These characteristics add convenience, but are not necessary.

231 SELECTING SFE FLUIDS AND CONDITIONS

Criteria for selecting supercritical fluids and SFE conditions have been previously discussed (3), and will only be briefly addressed here. Table I11 shows a list of several fluids that have been used for SFE, along with their critical temperatures and pressures, Hildebrand solubility parameters, and dipole moments. (Note that the Hildebrand parameters given in Table I11 are maximum values that are approached at very high pressures and are based on a correlation suggested by Giddings, ref. 4. Because of pressure limitations, typical extractions are performed at conditions which yield lower solvent strengths.) Carbon dioxide has been the most widely used fluid for SFE, primarily because it is relatively non-reactive, nontoxic, inexpensive, and has a relatively low critical temperature and pressure. Unfortunately, CO, is not sufficiently polar to extract many analytes of interest. As a very general rule, organic compounds that can be separated using conventional GC techniques can be extracted at high pressure (e.g. , 400 atm) with CO,, providing that there are no strong interactions between the analytes and the sample matrix (discussed below) Since the solvent strength of a supercritical fluid increases with its density, and since CO, is rarely too polar, SFE with CO, is generally performed at the highest pressure compatible with the extraction system.

.

Table I11 Characteristics of Representative Supercritical Fluids Used for SFE

co2 N2O NH3 F6

CHF, CHClF, ethane n-butane xenon

72.8 71.5 112.0 37.1 48.0 49.1 48.2 37.5 57.6

31.0 36.4 132.2 45.6 26.1 96.1 32.2 152.0 16.5

10.7 10.6 13.2 7.6 8.7 8.8 8.7 7.7 9.5

0.0 0.2

1.5 0.0

1.6 1.4 0.0 0.0 0.0

'Hildebrand solubility parameter in (cal/cm3) 'Dipole moment.

232

The lack of polar supercritical fluids that also have attractive practical characteristics has been a major frustration for developing SFE methods for polar and higher molecular weight analytes. For example, ammonia would be an excellent SFE fluid from a polarity standpoint, except that it is highly reactive and toxic. Nitrous oxide has some advantages over CO, for some applications (discussed below), and recent investigations have shown some polar freons such as chlorodifluoromethane (freon-22) to have potential for yielding more efficient extractions (discussed below). However, successful applications of SFE to polar analytes have so far relied on the addition of organic solvents to increase the polarity of the supercritical fluid. While methanol has been the most widely used, a variety of organic solvents have been used including (but not limited to) lower alcohols, organic acids, propylene carbonate, acetone, 2-methoxyethanol, and methylene chloride. An example of the use of organic modifiers in CO, for the extraction of the ionic surfactant, linear alkylbenzenesulfonate ( U S ) from municipal wastewater Note that treatment digester sludge is shown in Figure 2 . neither pure CO, nor N,O gave any detectable recovery of the LAS, which is not surprising since LAS is an ionic compound with an average molecular weight of ca. 340 amu. However, when modifiers were added to the CO,, a 15-minute extraction at 380 atm (125 "C) yielded much higher recoveries, and SFE with methanol-modified CO, yielded quantitative recovery of the LAS. 100

so

5'

60

8

5

2

- P a,

m C

u

40

0

f

u n

a,

20

-a a C x

2

a

sam, x x 0

f

2 .A

0

sI

Y

J? 3

0

Fisure 2: Recoveries (2f LAS from municiDal wastewater treatment sludge using a 15-minute SFE extraction at 380 atm with pure CO, and N,O, and different polarity modifiers in CO,.

233

As shown in Figure 2, the use of different modifiers can yield quite different recoveries, and no clear criteria exist for the selection of the best modifier for a particular extraction. A modifier may act by increasing the solubility of the analyte in the extraction fluid, or by competing with the target analytes for the active sites on the sample matrix. Until a better understanding of the action of modifiers and the factors that control S F E efficiencies is gained, selection of modifiers for a particular extraction will largely be empirical, although a reasonable starting point would be to select a modifier that is a good solvent for the target analyte in its liquid state. FACTORS THAT CONTROL SFE RATES AND RECOVERIES

While SFE is experimentally simple to perform, the development of quantitative extraction conditions has been severely hampered by the lack of overall understanding of the factors that control SFE extraction rates and ultimate extraction efficiencies. Several of the parameters (grouped loosely into IIExperimental parameters" and I'Sample/analyterelated parameters") that can control S F E recoveries are listed in Table IV.

Table IV Parameters That Can Control S F E Rates and Recoveries Experimental parameters: 1 Choice of supercritical fluid and modifier (if used). 2 Extraction T and P (which control fluid density). 3 The flow rate and total volume of the supercritical fluid used. 4 Extraction time. 5 Volume and dimensions of the extraction cell. 6 Dead volume of the extraction cell. 7 Sample size (related to cell size). 8 Efficiency of the post-extraction analyte collection method. Sample/analyte-related parameters: 1 Analyte solubility in the supercritical fluid. 2 Sorption strength of analytes onto matrix active sites. 3 Ability of the supercritical fluid to compete with analytes for active sites on the matrix. 4 Physical location of the analyte in or on the sample matrix.

234

Initial SFE conditions have most often been based on the solubility of the analytes in the supercritical fluid, but it has become increasingly clear that the interactions among the matrix, analytes, and the supercritical fluid frequently control the extraction rates to a much larger extent than can be explained by solubility considerations. Additionally, sample size, extraction flow rate and time, and the dimensions of the extraction cell all contribute to the final recoveries achieved. When developing an SFE method it is useful to divide the SFE process into steps that are summarized by the following

three questions: 1.

Are the analytes partitioned from the sample matrix into the supercritical fluid ?

2.

Once in the supercritical fluid, are the analytes swept efficiently out of the cell ?

3.

Are the analytes depressurization ?

collected

efficiently

after

These three factors interrelate to ultimately determine the optimal way to conduct an SFE sample preparation, and to some extent they can be evaluated independently. The last two questions are simplest to address and can provide a good starting point for developing an SFE method for specific target analytes. Once analytes are partitioned into the supercritical fluid, they are swept from the cell at a rate related to the sample size (and total void volume of the cell) and the flow rate. Thus, if the analytes are rapidly partitioned into the supercritical fluid (and saturation is not approached), the extraction rate is limited primarily by the need to sweep the void volume of the sample cell. For such extractions, extracting the sample with a few cell volumes is often sufficient to achieve quantitative recovery. (However, for most samples the partitioning of the analytes from the matrix into the supercritical fluid is not instantaneous, as discussed below.) Based on these considerations, relatively fast flow rates (e.g., >2 mL/min of the supercritical fluid) could be used to extract relatively large samples (e.g., > 2 0 gram) in a reasonable time (e.g., < 3 0 minutes). Unfortunately, the useful flow rate is limited by the need to quantitatively trap the extracted analytes upon depressurization (question 3 ) . As previously mentioned, SFE extracts are most commonly collected off-line into a few mL of liquid solvent. This approach is useful for supercritical fluid flows of up to ca. 1-2 mL/min which corresponds to gas flows of ca. 5 0 0 to 1000 mL/min. Thus, assuming a 3 0 % void volume for a packed solid sample and a supercritical fluid flow of ca. 1 mL/min, sweeping 10 void volumes through a cell that has been packed

235

full with 10 grams of sample would require ca. 30 minutes. Such flow considerations combined with the difficulty in making large extraction cells indicate that sample sizes are best limited to ca. 10 grams for analytical SFE, unless larger samples are needed to insure homogeneity. The efficiency of the trapping step (question 3 ) is important to distinguish from the efficiency of the extraction Poor SFE recoveries have often step (questions 1 and 2). mistakenly been blamed on the extraction step, when the actual reason for poor recoveries was inefficient trapping of the A s shown in Figure 3 by the SFE extracted analytes. extraction of n-alkanes from Tenax with collection in 2 mL methylene chloride, the more volatile alkanes were not quantitatively trapped in the methylene chloride during a 30minute extraction with a flow rate of ca. 1 mL/min supercritical CO, (500 mL/min of gas) at 400 atm. However, switching the solvent to hexane or increasing the volume of methylene chloride allowed the n-alkanes to be efficiently trapped.

lo(1

so e

?

a

2, 2 40

20

0

1

j

Q

m

B

U d

c

4

Collection efficiency of n-alkanes in 2 mL Figure 3 . methylene chloride during a 3 0-minute SFE extraction. Extraction conditions are given in the text. Whether the extracted analytes are collected off-line into a liquid solvent or sorbent trap, or on-line into a chromatographic system, testing the efficiency of the trapping system for each analyte of interest is imperative. Regardless of the trapping system, quantitative recovery of spikes placed into empty cells (or cells packed with a non-absorbing clean matrix) indicates that the trapping system is efficient, and such spike recovery studies are a necessary preliminary to performing the extraction of real samples (note however, as discussed below, that good spike recoveries do not necessarily mean that the same analytes are quantitatively extracted from

real-world matrices). When liquid solvents are used for analyte collection, an additional simple test of the trapping system is to prepare a standard solution of the target analytes in the proposed trapping solvent, then use a few mL of the standard solution as the trapping solvent for a blank No significant loss of the (empty cell) SFE experiment. standard compounds from the standard solution used as a collection solvent indicates that purging of the target analytes will not occur during the extraction of real-world samples. If quantitative collection can not be achieved with a particular solvent based on this purging test, the collection efficiencies can generally be increased by one of 1. Select a liquid with better the following methods: solvating properties for the target analytes 2. Increase the collection solvent volume and/or use a taller capped collection vial (making sure that a vent hole is provided for venting the depressurized fluid) . 3 . Reduce the SFE flow rate and/or extraction time (if possible). While the flow and analyte trapping considerations discussed above are relatively straight-forward to understand, the factors that control partitioning of the analytes from the sample matrix into the bulk supercritical fluid (question 3 , above) are far more complex and less understood. At least three general factors can be identified, any or all of which can control the rate of SFE recoveries from different samples. These factors, analyte solubility, analyteimatrix interactions (including the ability of the supercritical fluid to displace the analytes from active sites on the matrix), and diffusion of the analyte in the matrix particles (not in the supercritical fluid) are each discussed below along with examples of real-world extraction data. It must be emphasized that all three of these factors likely contribute to some extent to controlling SFE rates and efficiencies for every sample. The following discussions are presented only as examples where it appears that one of the factors appears to predominately control the extraction, and interpretations of the experimental results may change as the SFE mechanisms are better understood. Analyte solubility:

Early SFE studies often determined the extraction conditions (pressure and temperature) based on maximizing the solubility of the target analytes. (A recent review contains extensive lists of the solubilities of organic compounds in CO,, ref. 66). While this approach is quite useful when the target analytes represent a large percentage of the bulk matrix (e.g, the extraction of fats from meat products), maximum solubility considerations are less useful when the target analytes are present in minor and trace amounts. In these cases, the analyte need only be soluble enough to be

237

transported out of the extraction vessel, and the concept of threshold solubility (the pressure where an analyte becomes significantly soluble) suggested by King (34) becomes a useful guide. SFE rates appear to be controlled by solubility limitations for two general cases. First, when the analytes are present in very high concentrations (e.g., fats from meats), saturation of the supercritical fluid can occur. In this case, optimizing the SFE conditions for maximum solubility and increasing the amount of supercritical fluid used will yield higher extraction efficiencies. The second general case where analyte solubility controls the extraction rate occurs when the analytes do not have sufficient solubility to be transported from the extraction vessel (i.e., the threshold pressure has not been reached). This is frequently the case when the target analytes are polar and/or have high molecular weights. A s previously discussed, pure fluids such as CO, and N,O do not have sufficient polarity to dissolve such analytes, and the addition of polarity modifiers is needed. The extraction of the ionic surfactant, linear alkylbenzenesulfonate ( U S ) , from municipal wastewater treatment sludge and the extraction of pure abietic acid provide good examples (67). A s previously shown in Figure 2, LAS could not be extracted with pure CO, or N,O. However, when methanol modifier was added to CO,, quantitative recoveries were achieved. The extraction of a 200-mg sample of abietic acid (Figure 4 ) was also very slow with pure CO,, and only 4 % of the material was extracted after 30 minutes at 400 atm (50 "C) . However, when 1 mL of methanol was added to the cell 100% of the abietic acid was prior to extraction with CO, recovered in 20 minutes.

Figure 4.

Structure of abietic acid.

AnalyteIMatrix Interactions: A s mentioned above, solubility considerations frequently do not predict the results of SFE extractions, primarily because they fail to consider interactions between the analytes and active sites on (or in) the sample matrix. For example, both CO, and N,O have similar solubility parameters (Table 111), which indicate that their SFE extraction efficiencies should be similar. However, several comparisons of SFE recoveries have been reported (7,16,27) Which show that, at least for some matrices, N,O yields considerably better extraction efficiencies than CO, when the same sample is extracted under identical conditions. A dramatic example has been reported by Alexandrou (7) for the extraction of chlorinated dibenzo-p-dioxins from incinerator fly ash. While N,O yielded essentially quantitative recoveries of the dioxins, extraction with CO, yielded virtually zero recoveries. Interestingly, if the fly ash was first treated with acid, CO, then yielded good recoveries, demonstrating that analytelmatrix interactions were more important than solubility considerations for controlling the extraction efficiencies.

For many samples, the superiority of N,O over CO, is not dramatic, but is still significant. For example, the extraction of polycyclic aromatic hydrocarbons (PAHs) from marine sediment (16) shows similar recoveries of the lower molecular weight PAHs from a 15-minute extraction using N,O and CO,, but the N,O yields much higher efficiencies for the higher molecular weight PAHs (Figure 5). The reason for the higher recoveries with N,O is unclear, but is likely related to the fact that N,O has a permanent dipole while CO, does not (Table 111), and therefore N,O can better compete with the active sites on the fly ash and marine sediment matrices. so

This explanation is further supported by comparing the extraction rates achieved using CO, (no dipole moment) , N,O (small dipole moment), and CHClF, (relatively strong dipole moment, Table 111) for the extraction of polychlorinated biphenyls (PCBs) from a river sediment and PAHs from a petroleum waste sludge. Each extraction was performed at 4 0 0 atm and a few degrees above the critical temperatures of the individual fluids (extraction temperatures of 4 5 'C for co, and N,O, and 100 "C for CHClF,). Supercritical fluid flow rates N,O yielded somewhat were maintained at ca. 1 mL/minute. faster recoveries of the PCBs from the sediment and PAHs from the waste sludge than CO,, but CHClF, yielded much faster This extraction rates of all of the PAHs and PCBs than N,O. is demonstrated in Figure 6 by the extraction kinetic curves for the PAH chrysene and in Figure 7 for the trichlorobiphenyl PCB isomers. Since all of the PCBS and PAHS studied should have more than sufficient solubility in all three fluids, these results indicate that the factor controlling the

239 1.6

1.4 1.2

,i

1.0

g

0.8

2

0.6 0.4

0.2 0.0

Figure 5. SFE recoveries of PAHs from marine sediment with CO, and N,O at 400 atm (45 "C) with a 15-minute extraction. Results are adapted from reference 16.

-0

10

20

$0

40

SO

60

70

SO

90

Extraction Time (min) ,CO2

+NZO

,Freon-ZZ

Figure 6. SFE extraction kinetics of chrysene from a petroleum waste sludge using CO,, N,O, and CHClF,. SFE conditions are given in the text.

10

20 Extraction Time (min)

30

40

Figure 7. SFE extraction kinetics of trichlorobiphenyl PCBs from river sediment using CO,, N,O, and CHClF,. SFE conditions are given in the text. Values are normalized to CO,. extraction rates was more related to the ability of the fluid to displace the analytes from the sorptive sites on the sample matrices. Similar increases in extraction efficiencies of steroids using CHClF, were recently reported by Li et al. (52). For example, recoveries of estrone spikes were quantitative in 15 minutes using CHClF,, but were only 16% using CO, for 30 minutes (both extractions at 18 MPa). Diffusion Limitations: As discussed above, mass transfer in supercritical fluids is much better than in liquids, which is the major reason why quantitative SFE can often be completed in much less time than liquid solvent extractions. However, a recent report has shown that SFE extraction rates can be explained by a model which describes diffusion of the analytes in the sample matrix itself (as opposed to the diffusion of the analytes in the supercritical fluid, ref. 58). The model assumes that the analytes are evenly distributed throughout spherical sample particles, and that the concentration of the analytes in the supercritical fluid is always low. While these assumptions

24 1

are clearly not true for most real-world samples, all of the samples tested (including the extraction of PAHs from contaminated soil, flavor and fragrance compounds from rosemary spice, the ionic surfactant LAS from wastewater treatment sludge, and Tinuvin-326 from polypropylene beads) conformed to the diffusion model after an initial extraction time. To test the diffusion model, fractions were collected during SFE of each sample and analyzed. The results were then plotted as the In (m/m,) versus extraction time, where m, is the total mass of extractable analyte and m is the mass of the analyte remaining in the sample after extraction time t. While the model assumes an even distribution of the analytes throughout the matrix particle, distribution of analytes in the sample matrix is generally not known for real-world samples. However, an example that almost ideally fits this theoretical model is the extraction of the polymer additive Tinuvin-326 [2-(3-tertiarybutyl-2-hydroxy-5-methylphenyl)-2-H5-chlorobenzotriazole] from polypropylene, since it can be assumed that the additive is evenly distributed in the polymer particles. Figure 8 shows the extraction efficiency curves that resulted from the CO, extraction of two particle sizes of the ground polypropylene beads. The top of the figure shows the cumulative % recovery of the Tinuvin-326, while the bottom part of the figure shows the diffusion model plots. The extractions show nearly ideal conformity with the diffusion model, and clearly demonstrate that recovery is much faster from the smaller particles (95% recovery at ca. 45 minutes for the c0.6 mm particles compared to ca. 250 minutes for the 0.6 mm to 1.2 mm particles). The original sample was supplied as ca. 5 mm beads, which required more than 80 hours to achieve These results clearly near quantitative extraction ( 5 8 ) . demonstrate the great advantage in extraction rates achieved by grinding samples when analytes are distributed throughout the matrix. The majority of samples tested, however, showed a faster initial extraction (corresponding to a steeper initial drop in the diffusion model curve) than predicted by the model, indicating that analytes have higher concentrations at or near the surface of the sample particles. For example, after an initial rapid extraction period which removed ca. 90% of the LAS, the extraction rate then conformed to the diffusion model. This is demonstrated in Figure 9 for the extraction of LAS from municipal wastewater treatment sludge. The initial sharp drop corresponds to faster extraction of the LAS during the first 15 minutes than the model predicts, indicating that the LAS was present at higher concentrations at the surface of the sludge particles. However, the plot becomes linear after ca. 15 minutes, and the remaining extraction conforms to the diffusion model.

242

rn particles

< 0.6 m m I

4

0

50

0.6 mm

< particles <

150

100

t

1.2 m m

250

200

(llli11s)

0

-1

a E \

--cE

-2

-3

I

\

\ 1

1

I

I

I

Figure 8. SFE extraction kinetic curves (top) and diffusion model plots (bottom) for the extraction of Tinuvin-326 from two particle sizes of polypropylene. Extractions were performed with CO, at 400 atm (45 "C) at a flow rate of ca. 0.5 mL/min of supercritical fluid. Bottom figure was adapted with permission from reference 58.

243

0

10

20

30

40

50

60

Time, minutes

Figure 9. Diffusion model SFE plots for the extraction of LAS from municipal wastewater treatment sludge. Even though the multitude of parameters that can control SFE rates are not well understood, a consideration of the

factors just discussed, analyte solubility, matrix/analyte interactions, and diffusion limitations in the sample matrix can help to design quantitative SFE conditions. For example, if an extraction is primarily limited by diffusion in the matrix (e.g., the extraction of Tinuvin-326 from polypropylene discussed above), the extraction rate will be greatly increased by grinding the sample, but will be little affected by changing the extraction flow rate. However, if the extraction is limited by solubility, increasing the flow rate may yield faster extractions, or the analyst should change extraction conditions to increase the fluid's solvent strength (e.g., by using higher pressure or adding a modifier) as discussed above for the LAS and abietic acid extractions. Increasing the solvent strength should also increase extraction rates that are limited by analytelmatrix interactions, and the use of fluids with high dipole moments such as CHClF, also appears to be effective.

244

CLASS-SELECTIVE SFE

As discussed above, one of the potential advantages of over liquid solvent extraction is that the solvent strength of a supercritical fluid can be changed by simply changing its density. Thus, the potential to achieve classselective extractions exists by simply extracting the same sample at two different pressures with the same fluid. For example, diesel exhaust particulates contain pgfg levels of PAHs with mutagenic activity, and much higher concentrations of branched and normal alkanes which can interfere with the determination of the PAHs when conventional methylene chloride extraction is used to recover the PAHs. However, sequential extractions at a relatively low pressure (to extract the nonpolar alkanes) followed by a high pressure extraction (to extract the PAHs) can be used to yield a cleaner PAH fraction. Figure 10 shows the relative concentrations of representative PAHs and alkanes in extracts using a 5-minute SFE at 75 atrn with CO, (fraction 1) followed by a 15-minute extraction of the same sample at 300 atm (9). Note that ca. 85% of the alkanes were removed in the first extract, while (with the exception of phenanthrene) > 9 0 % of the PAHs were found in the 300 atm extract. Unfortunately, similar approaches to the selective extraction of the same alkanes and PAHs from petroleum industry waste sludges were not so successful, and selectivities of only ca. 60% were achieved compared to the 85-90% selectivities achieved for the diesel exhaust particulate sample. SFE

I00

-5

Alkanes

PAHs

22

178 202 228 228 276

19

26

75

u

t 5

50

Y

25

0

175 atm CO,,

0-5 rnin

300 atm CO,, 5-90 min

Figure 10. Class-selective S F E of alkanes and PAHs from diesel exhaust particulate matter using sequential CO, extractions at 75 atm (fraction 1) followed by 300 atm (fraction 2). Adapted from reference 9.

245

Class-selective SFE has also been applied to the extraction of target analytes from a bulk matrix (e.g., fat) which is itself highly soluble in supercritical CO, under most conditions. Even though fat components are highly soluble above pressures of ca. 120 atm, selective extracts of nonpolar analytes have been achieved by extracting the samples at lower pressures. With this approach, King has achieved quantitative recovery of pesticides from fat samples, yet the extracts were sufficiently fat-free to allow direct GC analysis (34). Selective extraction of lactones from milk fat triglycerides has also been recently reported (55). A single extraction concentrated the lactones by 2 0 to 50 times, while a two-step extraction yielded a concentration factor of ca. 500 times. When sufficient selectivity can not be achieved by simply extracting the sample under different SFE conditions, classselective extractions have also been achieved by depositing the analytes onto a sorbent column, then eluting them with the supercritical fluid in an SFC mode. Such approaches have been used to achieve rapid fractionation of alkanes, alkenes, and aromatics from gasoline (17, 61) , and fractionation of saturates, aromatics, and asphaltenes from crude oil ( 6 4 ) . France et al. have recently described an elegant technique using silica columns to selectively recover pesticides from fat samples (68). For example, the recovery of several pesticides from lard at concentrations of ca. 0.5 to 2 ppm were excellent (Table V) using CO, with 2% (v/v) methanol modifier, yet the extracts were sufficiently fat-free to be analyzed directly by capillary GC. Table V Class-selective Recoveries of Pesticides from Lard" pesticide lindane heptachlor heptachlor epoxide dieldrin endrin 2 ,4 -DDT

%

recoverv 103 107 102 98

95 103

'Adapted from reference 68. Despite the attractive potential of class-selective SFE, relatively few quantitative applications have been described. Experience in our lab has shown that one sample may yield encouraging results, while the next sample will not, even though the same analytes are being studied (as described above In addition to for the extraction of alkanes and PAHs).

246

solubility considerations, the nature of analyte/matrix interactions clearly has an influence on the success of classselective extractions, and an increased understanding of these interactions would greatly facilitate the development of class-selective SFE. SFE OF WATER SAMPLES

Nearly all of the SFE investigations to date have focused on the recovery of organic analytes from a solid matrix. However a few recent studies have demonstrated the potential to use SFE to recover analytes from water and water-based Special extraction cells have been fluids (44,45,69-71). developed to ensure that the supercritical fluid (usually CO,) percolates through the water sample as shown by the cell reported by Hedrick et al. in Figure 11. The supercritical CO, enters the extraction cell through the top tube and exits into the bottom of the water sample. Since the supercritical CO, is less dense than water, the CO, percolates through the water sample to the top of the cell, where it exits for analyte collection. The most common application has been the extraction of phenols from water, and quite acceptable recoveries of 80 to 85% with RSDs
CO? in 1

co.

out

Figure 11. Extraction cell for performing SFE of water samples. Adapted from reference 69.

247

SFE WITH IN-SITU CHEMICAL DERIVATIZATION

As discussed above, popular supercritical fluids generally do not have sufficient solvent strength to achieve the extraction of polar analytes, and for such extractions the addition of polarity modifiers has been needed to obtain sufficient solubility (as previously described for the extraction of LAS). An alternate approach to extracting highly polar analytes is to reduce their polarity by chemical derivatization, thus making the analytes easier to extract under conventional SFE conditions. This approach would seem to be particularly elegant, since many polar species need to be derivatized prior to performing chromatographic analysis of the extracts. For example, conventional methods for the analysis of acid herbicides (e.g., 2,4-dichlorophenoxyacetic acid) require liquid solvent extraction followed by diazomethane derivatization of the herbicides to their methyl derivatives prior to capillary GC analysis. With in-situ derivatization during the SFE step, the herbicides can be converted to their methyl derivatives (which also makes them easier to extract) and extracted in a single step. Two examples of SFE derivatizationjextraction are shown in Figures 12 and 13 for bacterial phospholipids from a 10-mg sample of Bacillus subtilis and several polar pesticides from a 2-gram sample of river sediment. To perform the SFE derivatizationjextraction, 0.5 to 1 mL of methanol which contained 0.1 M of the transesterification reagent, TMPA (trimethylphenylammoniumhydroxide),was added to the sample in the extraction cell, and the cell was pressurized to 400 atm with CO,. The derivatization step was carried out for 10 minutes at 80"C in the static mode, then the SFE was continued in the dynamic mode for 15 minutes with pure CO, to recover the derivatized analytes in a few mL of methanol. The extracts were then analyzed without any additional preparation using capillary GC. The extraction of the underivatized bacterial phospholipids was difficult even when methanol was added as a modifier. However, with the derivatization under SFE conditions, the fatty acid methyl esters from the derivatized phospholipids were very easy to extract, even with pure CO,. Figure 12 shows the chromatogram of the fatty acid methyl esters resulting from the 25-minute SFE derivatizationextraction procedure. Performing the procedure a second time on the same sample showed no additional peaks, indicating that the first extraction/derivatization was quantitative. Additionally, comparisons of the chromatograms of the extracts from the SFE procedure with the conventional procedure which requires liquid solvent extraction followed by a separate derivatization step (a process requiring several hours), show good agreement.

248

i15:C

\

16:o

17:O

1

I

a17:O

I 19:o

st

SFE

111 2nd SFE

L

i

:0

L

L

Figure 12. GC/FID analysis of the phospholipid-derived fatty acid methyl esters from a 20-mg sample of Bacillus subtilis. SFE derivatizationjextraction procedures are given in the text. Figure 13 shows the capillary GC analysis using an electron capture detector (ECD) of the extract from the SFE derivatizationjextraction of polar pesticides from the river sediment. (Names shown on the figure are the parent compounds prior to derivatization. Identities of the methyl derivatives were confirmed by GC/MS analysis). Each of the spiked pesticides showed the appropriate methyl derivatives in the SFE extract. Although quantitative work has only been done with the acid form of 2,4-dichlorophenoxyacetic acid (2,4-D), these results are encouraging, since recoveries of the 2,4-D acid as the methyl ester are >90$ with an SFE extraction1 derivatization procedure which requires only 3 0 minutes. These initial studies demonstrate that performing derivatizations under SFE conditions can achieve two goals at once: making the target analytes easier to extract, and making the analytes easier to measure. Although SFE derivatizationextraction studies are very limited, a wealth of chemical derivatization techniques are available for liquid phase reactions which should be applicable to SFE conditions. In addition to decreasing the polarity of target analytes, SFE derivatizations could be used to enhance their detection, e.g., dansylation for fluorescence detection, or trifluoroacetylation to enhance ECD sensitivities. The potential for

249 performing class-selective derivatizations could also enhance the ability to achieve class-selective extractions under SFE conditions.

/

\

Pentachlorophenol

Dicamba

:Ioram

I

0

c

\Dinoseb

Q)

c a 0

Dalapon /

1

&

Y

5

ib

Retention Time (min) Figure 13. GC/ECD analysis of polar pesticides from river sediment that were derivatized and extracted under SFE conditions as described in the text. CONCLUSIONS

The rapid increase in publications describing analyticalscale SFE over the last four years attests to both the interest in, and the need for new sample extraction methods. A wide range of quantitative SFE applications has been reported, and several investigators have demonstrated the ability of SFE to drastically reduce both the time required, and the quantities of waste solvents produced for sample extractions. Further developments in the use of modifiers, the extraction of water samples, class-selective extractions, and chemical derivatizations under SFE conditions will doubtless increase the range of quantitative SFE applications in the near future. However, analytical SFE is still a very new field, and a much deeper understanding of the physical and chemical factors that control extraction rates and efficiencies is needed to support the development of routine and widely-applicable SFE methods, particularly for polar and high molecular weight analytes.

2'

250 ACKNOWLEDGEMENTS

The authors would like to thank the U.S. Environmental Protection Agency (Cincinnati) and British Petroleum (USA) for financial support. Instrument support from ISCO (Lincoln, NB, USA) and Suprex (Pittsburgh, PA, USA) is also acknowledged. REFERENCES 1

J.B. Hannay and J. Hogarth, Proc. R.

SOC. London, 29

(1879) 324. 2

M. McHugh and V. Krukonis, Supercritical Fluid Extraction; Principles and Practice, Butterworths, Boston, 1986.

3

S.B. Hawthorne, Anal.Chem., 62 (1990) 633A.

4

J.C. Giddings, M.N. Myers, L. McLaren and R.A. Keller, Science, 162 (1968) 67.

5

J.R. Wheeler and M.E. McNally, J. Chromatogr. sci., 27 (1989) 534.

6

.

F.I. Onuska and K. A. Terry , J. High Resolut Chromatogr

.,

12 (1989) 357. 7

N. Alexandrou and J . Pawliszyn, Anal. Chem., 6 1 (1989) 2770.

8

B.W. Wright, C.W. Wright and J . S . Fuels, 3 (1989) 474.

9

S.B. Hawthorne and D . J .

Fruchter, Energy and

Miller, J. Chromatogr. Sci., 24

(1986) 258.

10

S.B. Hawthorne and D.J. Miller, Anal. Chem., 59 (1987) 1705.

11

P. Capriel, A. Haisch ana S . U . Chem., 34 (1986) 70.

12

V. Janda, G. Steenbeke and P. Sandra, J . Chromatogr. , 479 (1989)

13

Khan, J. Agric. Food

200.

M.M. Schantz and S.N. Chesler, J. Chromatogr. , 363 (1986) 397.

14

B.O. Brady, C.P.C. Kao, K.M. Dooley, F.C. Knopf and R . P . Cambrell, Ind. Eng. Chem. Res., 26 (1987) 261.

15

M.A. Schneiderman, A.K. Sharma and Chromatogr., 409 (1987) 343.

D.C.

Eocke, J.

251 Langenfeld , J.

16

S.B. Hawthorne, D.J. Miller and J.J Chromatogr. Sci., 28 (1990) 2.

17

J.M. Levy, R.A. Cavalier, T.N. Bosch, A.M. Rynaski and W.E. Huhak, J. Chromatogr. Sci., 27 (1989) 341.

18

S.B. Hawthorne, D.J. Miller and Chromatogr. Sci. , 27 (1989) 347.

19

B.W. Wright, S.R. Frye, D.G. McMinn and R.D. Smith, Anal. Chem. , 59 (1987) 640.

20

S.B. Hawthorne and D.J. Miller, J. Chromatogr., 403

M.S.

Krieger,

J.

(1987) 63. 21

.

F.I. Onuska and K.A. Terry , J. High Resolut Chromatogr

.,

12 (1989) 527.

B.E.

22

M.R. Andersen, J.T. Swanson, N . L . Porter and Richter, J. Chromatogr. Sci., 27 (1989) 371.

23

R.J. Skelton, C.C. Johnson Chromatographia, 2 1 (1986) 3.

24

M.W. Raynor, I . L . Davies, K.D. Bartle, A.A. Clifford, A. Williams, J.M. Chalmers and B.W. Cook, J. High Resolut. Chromatogr. Chromatogr. Commun., 11 (1988) 766.

25

K.R. Jahn and B. Wenclawiak, Chromatographia, 26 (1988)

and

L.T.

Taylor,

345. 26

M. Ashraf-Khorassani, L.T. Taylor and P. Zimmerman, Anal. Chem. , 62 (1990) 1177.

27

X. Yu, X. Wang, R. BarYha and J.D. Rosen, Environ. Sci. Technol., 24 (1990) 1732.

28

S.B. Hawthorne, M.S. Chem., 6 1 (1989) 736.

29

M.W.F. Nielen, J.T. Sanderson, R.W. Frei and U.A.T. Brinkman, J. Chromatogr., 474 (1989) 388.

30

S.A. Liebman, E.J. Levy, S . Lurcott, S . O'Neil, J. Guthrie, T. Ryan and S. Yocklovich, J. Chromatogr. Sci.,

Krieger and D.J.

Miller, Anal.

27 (1989) 118. 31

B.W. Wright, C.W. Wright, R.W. Gale and R.D. Smith, Anal. Chem. , 59 (1987) 38.

32

J.H. Raymer and E.D. Pellizzari, Anal. Chem., 59 (1987) 1043.

252 33

J.H. Raymer, E.D. Pellizzari and S.D. Chem., 59 (1987) 2069.

Cooper, Anal.

34

J.W. King, J. Chromatogr. Sci., 27 (1989) 355.

35

K.S. Nam, S. Kapila, G. Pieczonka, T.E. Clevenger, A.F. Yanders, D . S . Viswanath and B. Mallu, Proceedings of the International Symposium on Supercritical Fluids, (1988) 743.

36

J.W. King, J.H. Johnson and J.P. Friedrich, J. Agric. Food Chem., 37 (1989) 951.

37

M.A. Schneiderman, A.K. Sharma, K.R.R. Mahanama and D.C. Locke, J. Assoc. Off. Anal. Chem., 7 1 (1988) 815.

38

M.A. Schneiderman, A.K. Sharma and Chromatogr. Sci., 26 (1988) 458.

39

K. Sugiyama and M. Saito, J. Chromatogr. , 442 (1988) 121.

40

E.D. Ramsey, J.R. Perkins, D.E. Games and J.R. Startin, J. Chromatogr., 464 (1989) 353.

41

Q.L. Xie, K.E. Markides and M.L. Lee, J. Chromatogr. sci., 27 (1989) 365.

42

M. Saito, Y. Yamauchi, K. Inomata and W. Kottkamp, J. Chromatogr. Sci., 27 (1989) 79.

43

H. Engelhardt and A. Gross, J. High Resolut. Chromatogr. Chromatogr. Commun. , 11 (1988) 38.

44

D. Thiebaut, J.P. Chervet, R.W. Vannoort, G.J. DeJong, U.A.T. Brinkman and R.W. Frei, J. Chromatogr. , 477 (1989)

D.C.

Locke, J.

151. 45

J. Hedrick and L.T. Taylor, Anal. Chem., 6 1 (1989) 1986.

46

S . B . Hawthorne, M . S . Krieger and D. J. Miller, Anal. Chem., 6 0 (1988) 472.

47

W. Gmuer, J.O. Bosset and E. Plattner, J. Chromatogr., 388 (1987) 335.

48

K. Anton, R. Menes and H.M. Widmer, Chromatographia, 26 (1988) 221.

49

K. Sugiyama, M. Saito, T. Chromatogr., 332 (1985) 107.

Hondo and M.

Senda, J.

50

R.M. Campbell, D.M. Meunier and H.J. Cortes, J. Microcol. Sep., 1 (1989) 302.

253 51

P. Sandra, F. David and E. Stottmeister, J. High Resolut. Chromatogr. , 13 (1990) 284.

52

S.F.Y. Li, C.P. Ong, M.L. Chromatogr. , 515 (1990) 515.

53

C.P. Ong, H.K. Lee and S.F.Y. Lit J. Chromatogr., 515

Lee

and

H.K.

Lee,

J.

(1990) 509. 54

K. Sugiyama, T. Shiokawa and T. Moriya, J. Chromatogr., 515 (1990) 555.

55

A.B. de Haan, J. de Graauw, J.E. Schaap and H.T. Badings, J. Supercritical Fluids, 3 (1990) 15.

56

J. Hirata, F. Nakata and M. Horihata, J. High Resolut. Chromatogr. Chromatogr. Commun., 1 1 (1988) 81.

57

Y. Hirata and Y. Okamoto, J. Microcol. Sep., 1 (1989) 46.

58

K.D. Bartle, A . A . Clifford, S.B. Hawthorne, J.J. Langenfeld, D.J. Miller and R. Robinson, J. Supercritical Fluids, 3 (1990) 143.

59

R.C. Wieboldt, K.D. Kempfert and D.L. Dalrymple, Appl. Spectrosc., 44 (1990) 1028.

60

J.C. Monin, D. Barth, M. Perrut, M. Espitalie and B. Durand, Adv. Org. Geochem., 13 (1988) 1079.

61

J.M. Levy and J.P. Guzowski, Fresenius Z. Anal. Chem., 330 (1989) 207.

62

J.M. Levy, J.P. Guzowski and W.E. Huhak, J. High Resolut. Chromatogr. Chromatogr. Commun., 10 (1987) 337.

63

G. Hopfgartner, J.L. Veuthey, F.O. Gulacar and A. Buchs, Org. Geochem., 15 (1990) 397.

64

H. Skaar, H.R. Norli, E. Lundanes and T. Greibrokk, J. Microcol. Sep., 2 (1990) 222.

65

R.W. Vanoort, J.-P. Chervet, H. Lingeman, G.J. Dejong and U.A. Th. Brinkman, J. Chromatogr., 505 (1990) 45.

66

K.D. Bartle, A.A. Clifford, S . A . Jafar and Shilstone, J. Phys. Chem. Ref. Data (in press).

67

S.B. Hawthorne, D.J. Miller, D.D. Walker, D.E. Whittington and B.L. Moore, J. Chromatogr. (in press).

68

J.E. France and J.W. King, J. Ag. review).

Food Chem. ,

G.F.

(in

254 69

J.L. Hedrick and L.T. Taylor, Chromatogr. , 13 (1990) 312.

J.

High

Resolut.

70

D. Thiebaut, J . P . Chervet, R.W. Vannoort, G.J. Dejong, U.A. Th. Brinkman and R.W. Frei, J. Chromatogr., 477 (1989) 151.

71

R.K.

Roop, A. Akgerman, B. J. Dexter and T.R. Irvin, J. Supercritical Fluids, 2 (1989) 51.

K. Jinno (Ed.), Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction Journal of Chromatography Library Series, Vol. 53 0 1992 Elsevier Science Publishers B.V. All rights reserved.

255

Chapter 13

Introduction of Directly Coupled SFE / GC Analysis Tsuneaki Maeda Research Center, DKK CORPORATION,4-13-14, Kichijoji Kitamachi, Musashinoshi, Tokyo, Japan

Toshiyuk~Hoboa ‘Department of Chemistry, Tokyo Metropolitan University, 1-1, Minami Ohsawa, Hachioji-shi, Tokyo, Japan

INTRODUCTION Recently, supercritical fluid is widely used for industrial processing such as decaffenation of coffee beans, extraction of bitter essence from hop and so on. It is because it has unique phase properties for separation as well as extraction. Since supercritical fluid is much easily produced than before using the advanced technologies, it became much popular and created new fields like supercritical fluid chromatography (SFC) and supercritical fluid extraction (SFE). Application of these techniques is increasing in these days. The supercritical region is defined as the one above the critical temperature and critical pressure. This region gives a single phase and the substance in this region is called supercritical fluid. It is the forth state of the substance. One of the unique properties of the fluid, solubility, depends on the density which can be controlled by changing the pressure and temperature. The diffusion coefficient of the fluid lays in the midst of gas and liquid, which means that the mass transfer rate is faster in the fluid than liquid. Then if it is used for extraction in place of liquid, the extraction time could be made shorter. The low viscosity allows it to be used as the mobile phase for capillary and micropacked column SFC. These fundamental part of supercritical fluid techniques and applications of both SFE and SFC have been described in many literatures [l61. The analytical SFE is often compared with solvent extraction methods and between them there exist large differences. Actually they have many advantages and disadvantages. For analytical purposes, particularly for solid sample analysis, SFE is superior t o liquid extraction method, namely Soxhlet method, in speed, solvent waste problem, and so on. As the sample preparation method for chromatography, SFE is a relatively new method of choice. In this chapter, directly coupled SFE / GC analysis method which is one of so called hyphenated methods being used in the instrumental analysis is described. It has such a convenience that the sample preparation, separation and detection can be done in a straight line. Advantageously the technique can provide the followings; a) simplify the sample preparation procedure. b) make the analysis time shorter.

256

c) minimize the contamination. d) maximize the use of capabilities of both methods. At the same time, however, the method requires the understanding of the both methods coupled. This chapter starts with the overview of SFE for GC,then describes on the chracteristics of GC and SFE, and finally on the on-line SFE / GC interface and its application. SFE FOR GC SFE has been used intensively and shown that it has a great capability in the sample preparation for GC as well as for other chromatographies, i.e, SFC, LC, IR, MS and so on. Many reviews pointed out the prospect of SFE and described opinions on the analytical scale SFE [7-121. Basically, there is almost no difference between off-line and on-line SFE. Limitation of On-line SFE I GC It is reasonable to think that the sample range applicable t o the on-line SFE / GC is limited by the fundamental circumstances of GC. Although supercritical fluid can dissolve liquid samples of high boiling point, GC can not analyze those samples which has low volatility and thermally unstable substances. In Figure 1, molecular ranges of substances which can be handled by the ordinary chromatographies and SFE are presented. Another aspect is that the solubility of a compound in the supercritical fluid can not exceed that in liquid of same material. It means that the compound that is hard to dissolve i n t o the liquid is harder to dissolve into the solvent in the supercritical fluid state. In addition, the higher molecular weight or polar compounds are difficult t o be dissolved i n t o the supercritical fluid as far as non-polar C02 or slightly polar NzO are used as the fluid material. Probably the limitation also comes from SFE side. One of the limitation from GC side can be overcome by using a high temperature capillary column such as aluminum claded fused silica capillary column with stable stationary phase. Nowadays, up to CIOO can be analyzed using these technically advanced systems [13-181. It is, however, still not enough to cover the whole range of SFE. It can be pointed out that unexpected or undesirable components extracted by a SFE will be carried into the GC system and disturb analysis. In case of an off-line SFE, further clean-up or pretreatment step can be employed t o eliminate such interferences [8]. Although online SFE / GC is a simple technique, aplicable sample is limited compared t o the offline SFE. As the result, on-line SFE / GC method requires suitable sample selection and appropriate setting of extraction conditions.

SFE as a sample preparation technique From the sample preparation point of view, SFE has two types of technique for controlling the solubility strength. One is to use a single solvent and control its strength by changing the pressure and temperature. The solubility,power of dissolution, in supercritical fluid is related to the density of the fluid which can be controlled by the temperature and pressure. Thereafter, using single solvent, selective extraction could be expected. Several off-line SFE studies reported the possibility of fractionation or class-selectiveextraction [8,12,19-221. These techniques are also useful for the on-line SFE. Various kinds of fluid materials have been investigated and compared each other. From the gas chromatographicpoint of view, SFE using

257

Molecular weight range applicable to the on-line SFE / GC

LC(SEC)

SFC

GC

SFE 100

lo*

lo2

lo3

lo4

lo5

10 lo7 Molecular weight (daltons)

Figure 1. Molecular weight range applicable to the chromatography and SFE.

liquefied gas is desirable. Further, intentional solvent-solute separation is not required because of its property; liquefied gas is evaporatively separated from the solute in the collection device by depressurization. Usually, carbon dioxide is the primary choice for the experiments, but it has no polarity. Therefore, many other fluid materials including NzO, CHF3, CHClF2, SF6, C2H6, i-CdH10are used as they show characteristic properties [7,23-261. The other technique is solvent polarity control method either by adding a polar solvent, which is called modifier or entrainer, or by changing solvent itself. An example for the latter case is from non polar C02 to slightly polar N20. As the modifier, methanol is commonly used. These techniques are applicable to the on-line SFE / GC, too. Basically, the role of SFE is just to dissolve the component in the sample matrix and transfer to the collection place. It is advisable to do the preliminary test and select the optimal solvent and extraction conditions using an off-line SFE before applying sample to the on-line SFE. Important thing to think about when the technique is chosen is what is the goal of analysis, whether it requires trace analysis or profile analysis. It is also important to get the information about sample matrix, properties, interferences, co-existing components and the concentration of analytes. Putting these information into consideration the best sample preparation procedure should be constructed. Basically, the process of construction is same as in other sample

258

preparation technique.

Optimization of SFE conditions There are many factors to be considered depending on the goal of analysis, so as to achieve optimum extraction efficiency . If the goal is to get the profile or matrix composition of a sample, it is required to use the fluid at the maximum solubility. On the contrary, if the trace components in the sample are the analytes, it does not need to think about maximum solubility. In this case, as King [8] described, it is better t o choose the condition that can separate the analytes from the matrix without interference. The density of fluid increases with increasing pressure and the diffusivity increases with the temperature, which means that time saving extraction can be achieved at high temperature. These points are described by the reports showing the change of k at different temperatures [27-291. Supercritical fluid behaves like gas at high temperature and low density, and like liquid a t low temperature and high density. Next point is the elimination of the matrix effect. If a test gave a sufficient solubility and good recovery from standard solution,but poor recovery from actual sample, the interaction between targeted component and matrix must be considered. In this case, first the extraction time should be extended. Then, either the use of polar modifier o r change of the fluid to a more polar one such as N2O and Freon should be tried. Another way t o eliminate matrix effect is the addition of some other modifier which can break the interaction between solute and matrix. The selection of such a modifier should be done depending not on the solubility, but on the interaction. These are just rough guidelines to pursue the optimum SFE condition. Another approach to obtain good recovery of polar components is a derivatization of analytes in the fluid [30,311. Lastly, it is important t o remember that SFE is the time saving technique, but still requires time to dissolve the analytes. It can not dissolve the objectives in a moment. CONSTRUCTION of ON-LINE SFE / GC SYSTEM On-line SFE / GC construction is shown in Figure 2. There are three important parts. They are a solvent delivery pump, a SFE oven and a GC. The system is very simple to build up. The high pressure pump deliver the fluid at a higher pressure than the critical one and the extraction vessel is put in a oven to keep the temperature higher than the critical one. The examples of extraction vessel design are shown in Figure 3. An empty column or a guard column are commonly used for the vessel. The restrictor is connected after the SFE vessel t o maintain the extraction pressure high, and passing in it the pressure is reduced to meet the GC analysis. As a restrictor, fused silica capillary tubing or crimped tubing are usually used. The former is a linear restrictor and the latter is a tapered one. Extraction solvent is introduced into the GC as a gas and the solutes in the gas are concentrated in the inlet of the column during extraction. The most important part of on-line SFE / GC is the interface between SFE and GC.

SFE / GC INTERFACE There are many types of sample introduction system for capillary gas chro-

259

matography developed aiming to get a good separation. These systems allow liquid or gaseous samples introduced into the GC in a narrow band. Selection of a system is dependent on the sample to be analyzed. But in case of SFE, special consideration different from the direct sample introduction technique in the GC will be required. The solvent commonly used in SFE is liquefied gas (CO2, N20, SF6 etc.), which changes to the gaseous state after depressurization. Therefore, the solvent is a gas when it is introduced into the interface of a on-line SFE / GC. The volume of extraction solvent will expand about a thousand times ( I d of liquid C02 expands to about 560ml CO2 gas). Besides, for SFE it requires a few minutes to finish in the case of a micro extractor is used. It must be considered that a large amount of gaseous solvent must be separated and removed from the solutes in the interface. It takes long time for the completion of a SFE when a micro scale extraction vessel is used. Therefore a cryogenic focusing technique should be applied t o prevent the band spreading. In this section, the interfaces so far reported are classified and overviewed. Direct injection system Figure 4. shows the schematic diagram of direct injection system [32-351. This type of system uses a heated tee piece joint to prevent the plugging. The end of the restrictor is put inside of the retention gap and the carrier gas enters coaxi-

Extraction oven Extraction vessel

Flow restrictor

Interface

I I

1

I

Caniergas

L

-Detector

.Column Liq. CO2

Solvent delivery system for SFE (High pressure pump)

Gas chromatograph

Figure 2. Schematic diagram of a typical on-line SFE / GC.

260

ally along the restrictor. Whole solvent will flow through the column and whole extracted compounds are introduced into the column. The extracted compounds are focused in the retention gap. The retention gap also prevents the deposition of the non-volatile components in the column. During extraction, the carrier gas flow is stopped in order to get rid of the high back pressure effects. In this system, the flow rate of a SFE solvent is restricted by the capillary column and utilization of a micro extraction system is just recommended. Thus, when a vessel of large volume is used, intermittent introduction of the extractant, Like a heart cutting technique in multidimensional chromatography,should be employed [33,34]. Wright et al. [321

1/16"Tube 4to

restrictor

Ferrule 1/16"o.d.tube 1.Micro extraction vessel(
Il16"Tube

__+

q , , &---/

Joint body

Nut

J o i n t body

wlqy

Extraction cell Ferrule lI4"o.d.tube 2. Semi micro extraction vesselb0.lml)

Filter

&/16"Tube --+to restrictor Ferrule

-b

Filter Extraction cell Ferrule 1/4"o.d. or 318"o.d. tube 3. Conventional extraction vessel bO.5m.l) using empty column for liquid chromatography

t o restrictor

Ferrule

Figure 3. Examples of extraction vessels designed for on-line SFE /GC.

26 1

Extraction solvent inlet

Extraction solvent inlet

joint

+

Carrier gas inlet

-

ii

Retention gap

, I, I

o : C 0 2 , x : Light,

Figure 4. Direct injection system.

0

: Heavy

Figure 5. Direct cool on column injection system.

and Lohleit et al. [34] reported selective extraction method with some examples. Lohleit et al. [34] also investigated the performance of this system by comparing with the thermal desorption analysis. They showed that the former can analyze the higher molecules than the latter. This system might be applicable to the small volume extraction and wide range of boiling point samples.

Cool on column injection system This system is similar to the direct injection system but the depressurised extraction solvent is led out from the interface. A conventional cool on column injector was used after only taking out the septum [36-411. Figure 5. shows the schematic of this system. As the restrictor is inserted into the analytical column, the solutes are deposited in the column while it is cooled for a cryogenic focusing. The extracted components are retained in the liquid stationary phase and volatile components are vented with solvent. Since the restrictor is not heated in this system, plugging will be happened easily during extraction. Some modifications from on-line SFE / SFC t o on-line SFE / GC and another system similar t o the direct

262

Extraction solvent inlet

v

Extraction solvent inlet

Extraction vessel

P-

Restrictor Septum

/

o O* O01

Column

Figure 6. Split I' splitless injection system.

/---I

Column i

Figure 7. Programmed temperature vaporizer injection system.

injection system using a tee piece with a vent line were reported [12,42-441. These systems might be applicable to the trace analysis where large amount of matrix is not coextracted.

Split I' splitless injection system This system is the simplest one that does not need any modification of a conventional split I' splitless injector [45-491. Figure 6. shows the schematic diagram of this system. The restrictor is just inserted through the septum during the extraction and withdrawn after it. The injector is heated t o protect a plugging. The column is initially cooled to focus the extracted components. The solvent is finally

263

led to the split vent or septum purge vent. In this system, sample is splitted regardless of split or splitless mode, and the split ratio depends on the flow rate of extraction solvent. It means that the higher the extraction pressure is, the higher the split ratio is. This system is very easy to use but sample injection takes long time, and be resulted in a discrimination. This system is also similar t o the hot needle injection method where sample is injected very slowly. Though there is no maximum volume of extraction vessel, relatively high molecular weight compounds can not be introduced uniformly.

Programmed temperature vaporizer injection system For this system a conventional programmed temperature vaporizer injection system P"v)can be used without any modification [501. Figure 7. shows the schematic diagram of this system. The restrictor is just inserted through the septum during the extraction and withdrawn after it. The extraction solvent and the components with high vapor pressure exit from the solvent vent line. The injector is kept at sub-ambient temperature for trapping the extracted components inside the injector. At the end of the extraction the restrictor is withdrawn from the septum, the solvent vent line is closed and the programmed temperature operation of the injector is started. Then the whole trapped components kom the sample is introduced into the column and analysis is started. Houben et al. [501 described that since the extracted components are trapped by reduced vapor pressure, in this system, the complete trapping of the components is accomplished if they have boiling points at least 250°C higher than the trapping temperature. It seems that it has a chance t o have the plugging problem, because the restrictor is not heated. Similar range of samples to the cool on column injection system could be applicable. As for the volume of the extraction vessel, there is no restriction. Thermodesorption I cold-trap iltjection system This type of system is the modification of a thermodesorption I cold-trap injector [511. Figure 8. shows this system. In order to prevent plugging the restrictor is inserted into the heated inlet region, where usually an adsorbent-packed trap is placed. During the extraction the extracting solvent is wasted through the purge vent. The extracted components are trapped on the surface of the cold trap placed under the heated region. The property of this trapping system is the same as one in the PTV injection system. When the extraction is completed, the purge vent line is closed and the carrier gas line is opened with a rapid heating of the cold trap. The whole trapped compounds are led into the column. The original system [52] is developed for the volatile components. The maximum operating temperature of the cold trap region is 350°C. Since it is placed on the GC oven, relatively cool part exists between the cold trap and the analytical column. The semi-volatile components may deposit on this cool part. This system might be applicable to the volatile components. Miscellaneous irqiection system Some other systems were reported [53,541. The extracted sample is trapped in a loop which is connected to a 6 way port valve. After trapping, the loop is heated to introduce the extracted sample into the column. Figure 9 shows the schematic of this system. This system might be applcable to the volatile components.

264

Extraction solvent inlet

Extraction solvent inlet

Extraction vessel

Restrictor

Carrier gas

d?

/

Valve

i

Column

Figure 8. Thermodesorption-cold trap injection system.

Figure 9. Loop injection system.

Conclusion Above mentioned studies suggest that a restrictor heating and a cryogenic focusing should be used during extraction. There are a number of variations of the focusing technique for the volatile sample introduction [55-581. Since the focusing temperature should be higher than the freezing point of SFE solvent, the cryogenic focusing using liquid nitrogen can't be applied. It's also important to understand the properties of a conventional GC injection system when a conventional injection system is used as the interface. There are many literatures on the capillary column GC sample injection system [59-691. It is recommended to refer those information before selecting the interface. The sample injection system is very important part of the capillary column GC. Though many systems are developed and applied, those words of which Sandra described in his book [591 are still alive; "Sample introduction in capillary gas chromatography has always been, and in fact still is, a problem. There is still no such thing as a universal injection system and there probably never will be." The situation about on-line SFE / GC interface is absolutely the same, as far as it is using capillary column.

265

PRACTICE OF ON-LINE SFE / GC Most of the papers reported on the instrumentation and application of online SFE / GC have been referred in the previous section. Here, evaluation of practical on-line SFE / GC system using a conventional split / splitless injector will be presented on the basis of authors' experiments. The flow diagram of the system developed is shown in Figure 10 [701. A micro extraction vessel, approximately 110 pl, was used. An inner diameter of 20 pm and 10 cm long fused silica capillary

High pressure C02

\L

----------

6 way port

Extraction vessel

Septum purge vent Split / splitless injector Backpressure valve Column head pressure

Column oven i _____---__________--_____--

to FID S.V.; 3 way port solenoid valve Splitless injection; Carrier gas flow through N.C. to C. Figure 10. Flow diagram of on-line SFE / GC system.

266

Table 1 Anakytical GC conditions Column Purge vent flow Carrier gas Column head pressure Injector temperature Detector Detector temperature

Methyl silicone, 0.25mm i.d., 5m long, film thickness 0. lum (Quadrex) 28dmin He 0.7kg/cm2 300°C FID 300°C

(A)

yo 1

c[

-

.o

I 15

I 20

I 10

I

I

15

20

I

2.5 Time(mm)

I

10

I

15

I

20

I

25

I

30 Time(min

I

25 Time(mn)

Figure 11.Effect of cryogenic focusing on separation. (A) Without focusing, column oven temp. 100°C, (B) with focusing, column oven temp. lOO"C, (C) without focusing, column oven temp. 50°C, (D) with focusing, column oven temp. 50°C.

267

(B) C 14

C 14

c20

C30

c20

C30

-

.5

20

25

30 Time(min)

1

I

I

15

20

25

I

30 Timebin)

Figure 12. Chromatograms obtained using different injection modes. Conventional split / splitless injector for GC was used; (A) Split injection mode, (B) splitless injection mode. tubing was used as a restrictor. The cryogenic focusing system is adapted inside the column oven t o focus the extracts in the front part of the column. Analytical conditions are presented in Table 1. Alkanes were used for the standard sample. A narrow-bore capillary column with thin film stationary liquid phase was used t o separate high molecular weight alkanes.

Cryogenic focusing The cryogenic focusing effect is shown in Figure 11. In this system, since the extracted components are trapped in the stationary liquid phase of the column, the cryogenic focusing depends on the oven temperature, film thickness of the liquid phase and focusing time. As the boiling points of target components are high enough, focussing can be effected at the top of the column, when the column oven is kept at near ambient temperature. The effect of focusing was reported elsewhere [38,41,711. Differences of split I splitless injection This system has a conventional split / splitless injector for capillary column GC, but its function is different from conventional use described in previous discussion. Figure 12 shows the chromatograms obtained using both split and splitless injections. Figure 12 (A) was obtained at the split ratio of 1/ 25 and (B) was obtained using the splitless injection. The chromatogram(B) shows the extracted components were escaped with extraction solvent even in the case of splitless mode. Extraction time and extraction pressure When the supercritical fluid has enough solubility for the sample components, two important factors should be considered, in order to complete the extraction successfully. The first is the sample transfer time and the second is the time to dissolve in the solvent. Figure 13 shows the recovery changes under various pressure and extraction time. Figure 13 (A) shows that at 200 atm., the fluid has

-

c20 c30 DI c40

g

3

5

3

5

10

15

10

15

Extraction time (min)

1.0

% W b

?

3

2

0.5

2 0.0

Extraction time(min) figure 13.Effect of extraction time on relative recovery (peak height of each component after 15 min extraction = 1.00). Sample; C20, C30, C40 n-alkanes (50ng/ each component), Volume of extracion vessel 110~1,Extraction temperature 50°C, Extraction pressure; (A) 2OOatm, (B) 150atm.

enough solubility but 3 minute extraction time is not enough because of the inunifonnity of the recoveries of each component. The reason is that the transfer time depends on the void volume of the extraction vessel. Figure 13 (B) shows the differences of dissolving time among the compounds tested. It shows the higher molecular weight hydrocarbons dissolved slowly. The extraction time required is related to the total volume of extraction solvent and the void volume of extraction system.

269

Selective extraction In SFE, simple separation can be achieved by selecting the solubility. Figure 14 shows an example of selective extraction. It shows the C40 has low solubility at the extraction conditions of 100 atm., 80 "C and 15 minute. Figure 15 also shows the selective extraction. Figure 15 (B) shows the selective discrimination of molecules whose molecular weights are higher than C50. The large molecules required a longer time to dissolve in the extraction solvent than a smaller one. It was also slowly injected into the column, which caused discrimination. The class-selective analysis using different extraction pressure was demonstrated [32,34,39].

APPLICATION OF ON-LINE SFE / GC Typical applications of SFE / GC were listed in the reviews [6,7,711.They are roughly divided into followings; the pollutant from environmental solids [32,3537,47,48],the adsorbed compounds from sorbent resins [33,34,40,511, polymer In additives [441,and the flavor o r fragrance from food products [38,39,41,43,54]. these applications, the most emphasized is the merit of analysis time saving. Compared to the thermal desorption method, on-line SFE / GC technique can recover the higher molecules without using high temperature [35,36,40,42,1. Figure

g 1.00 El

0

P Q)

k

4 Q)

4

2 c20 c30 c40

0 200/15/50

150/15/50 100/15/50 100/15/80

Extraction condition (at& time/ temp.) Figure 14. Effect of extraction conditions (C20=1.00). Peak heights of alkanes extracted under various SFE conditions. Volume of extraction vessel 110~1,Sample; C20, C30,C40 n-alkanes (50ng / each component).

270

I

I

I

I

20

25

30

35

40 I Timebin)

!6 C30 c22 c20

j

C32 C36 C40 C44

*C50 \

I

'/ F"" ~

Figure 15. Selective &actionation of C12-C60 mixture. Column oven temperature profile; 100°C 2min up t o 380°C a t the rate of 10°C / min hold 15 min. Injector and detector temperature 380°C. SFE conditions; (A) 100atm, 8O"C, 15min, (B) 300atm, 50"C, 15min. (GC analysis start at 15 minutes). * Apparently discriminated components

271

16 shows the chromatograms obtained using carbon dioxide as a carrier gas [72]. The separation is carried out under a low linear flow velocity of carrier gas on a narrow bore column. It shows that a high resolution is obtained using a dense gas. In this case, the extraction solvent (carbon dioxide) does not need to be replaced by the other carrier gas such as helium. Another example of on-line SFE / GC is shown in Figure 17. On-line SFE / GC is powerful system for the GC analysis of various kind of samples. It has a short history, but it will become an important technique for the sample introduction method in GC analysis. Separation techniques such as on-line SFC / GC [73] and on-line SFE / SFC / GC will also make GC widely applicable.

E h I

I

I

I

0

10

20

30

I

40 Time(min)

(B)

I

I

0

10

II I

I

20

30

I

40

Timebin)

Figure 16. Effect of mobile phase on on-line SFE / GC; (A) carbon dioxide, (B) helium. Sample; low density polyethylene, GC column inlet pressure 0.8atm, Column oven temperature profile; 50°C 2min up to 300°C a t the rate of 8°C / min. hold 15 min. Capillary column; 15m x 250pm i.d., methylsilicone O . l p m , Detector FTD, SFE conditions; 200atm, 50"C, lmin, cell volume 1lOpl.

272

I/ 10

20

I

30 40 Time(min)

10

20

40 Time(min)

30

Figure 17. Application of on-line SFE / GC on tea samples; (A) tea 7.9mg, (B) green tea 15mg. Analytical conditions; Mobile phase : C02, Column oven temperature profile; 50°C 2min up to 300°C at the rate of 8"C/min hold 15 min. Capillary column; 15m x 250pm i.d. methylsilicone O.lpm, Detector; FID range lO"1, SFE conditions; 250atm 60°C lmin , cell volume 110~1,solvent CO2.

ACKNOWLEDGMENT We thanks Hiroko Tatematsu for the assistance of the study.

REFERENCES 1 J. M. L. Penninger, M. Random, M. A. McHugh, V. J. Krukonis (eds.),

Supercritical Fluid Techonlogy, Elsevier, Amsterdam, 1985. 2 K. P. Johnston, J. M. L. Penninger (eds.), Supercritical Fluid Science and Technology, American Chemical Society, Washington, DC, 1989. 3 B. A. Charpentier, M. R. Sevenants (eds.), Supercritical Fluid Extraction and Chromatography. Techniques and Applications,American Chmical Society, Washington, DC, 1988. 4 R. M. Smith (ed.), Supercritical Fluid Chromatography, The Royal Society of Chemistry, London, 1988. 5 C. M. White (ed.), Modern Supercritical Fluid Chromatography,Huthig, Heidelberg, 1988. 6 M. L. Lee, K. E. Markides (eds.),Analytical Supercritical Fluid Chromatography and Extraction, Chromatography Conferences, Inc., Utah, 1990. 7 S. B. Hawthrne, Anal. Chem., 62 (1990) 633A. 8 J. W. King, J. Chromatogr. Sci., 27 (1989)355.

273

9 K. G. Furton, J. Rein, Anal. Chim. Acta, 236 (1990) 99. 10 S. K. Poole, T. A. Dean, J . W. Oudsema, C. F. Poole, Anal. Chim. Acta, 236 (1990) 3. 11 W. Pipkin, LC. GC., 10 (1992) 14. 12 M. R. Andersen, J. T. Swanson, N. L. Porter, B. E. Richter, J. Chromatogr. Sci., 27 (1989) 371. 13 S. R. Lipsky, M.L.Duffy, LC. GC., 4 (1986) 898. 14 S. R. Lipsky, M.L.Duffy, HRC & CC, 9 (1986) 376. 15 S. R. Lipsky, M.L.Duffy, HRC & CC, 9 (1986) 725. 16 Y. Takayama, T. Takeichi, S. Kawai, HRC, 11(1988) 732. 17 T. Welsch, U. Teichmann, HRC, 14 (1991) 153. 18 I. Hagglund, K. Janhk, L. Blomberg, A. BemgrAd, S.G. Claude, M. Lymann, R. Tabacchi, J. Chromatogr. Sci., 29 (1991) 396. 19 R. M. Campbell, M. L. Lee, Anal. Chem., 58 (1986) 2247. 20 M. Nishioka, D. G. Whiting, R. M. Campbell, M. L. Lee, Anal. Chem., 58 (1986) 2251. 2 1 K. S. Nam,S. Kapila, A. F. Vanders, P. K. Pun, Chemosphere, 20 (1990) 873. 22 D. M. Kassim, M. S. Hameed, Sep. Sci. and Tech., 24 (1990) 1427. 23 S. B. Hawthorne, D. J. Miller, Anal. Chem., 59 (1987) 1705. 24 B. W. Wright, C. W. Wright, R. W. Gale, R. D. Smith, Anal. Chem. 59 (1987) 38. 25 K. Sakaki, T. Yokochi, 0. Suzuki, T. Hakuta, JAOCS, 67 (1990) 553. 26 S. F. Y. Li, C. P. Ong, M. L. Lee, H. K. Lee, J. Chromatogr., 515 (1990) 515. 27 M. G. Randon, T. A. Nonis, Am. Lab. (1984) 17. 28 T. L. Chester, D. P. Innis, HRC & CC, 8 (1985) 561. 29 A. Wilsch, G. M. Schneider, J. Chromatogr. 357 (1986) 239. 30 J. W. Hills, H. H. Hill, Jr., T. Maeda, Anal. Chem. 63 (1991) 2152. 3 1 S. B. Hawthorne, C.J.Miller, D. E. Nivens. D. C. White, 64 (1992) 405. 32 B. W. Wright, S. R. Frye, D. G. McMinn, R. D. Smith, Anal. Chem., 59 (1987) 640. 33 M. Lohleit, K. Bachmann, J. Chromatogr. 505 (1990) 227. 34 M. Lohleit, R. Hillmann, K. Bachmann, Z. Anal. Chem., 339 (1991) 470. 35 R. F. Mauldin, J. M. Vienneau, E. L. Wehry, G. Manmantov, Talanta, 37 (1990) 1031. 36 S. B. Hawthorne, D. J. Miller, J. Chromatogr. Sci., 24 (1986) 258. 37 S. B. Hawthorne, D. J. Miller, J. Chromatogr., 403 (1987) 63. 38 S. B. Hawthorne, M. S. Krieger, D. J. Miller, Anal. Chem., 60 (1988) 472. 39 S. B. Hawthorne, D. J. Miller, M. S. Krieger, Z. Anal .Chem., 330 (1988) 211. 40 S. B. Hawthorne, D. J. Miller, Anal. Chem., 6 1 (1989) 736. 4 1 S. B. Hawthorne, D. J. Miller, M. S. Krieger, J . Chromatogr. Sci., 27 (1989) 347. 42 J. H. Raymer, G. R. Velez, J. Chromatogr. Sci., 29 (1991) 467. 43 B. J. Murphy, B. E. Richter, J. Microcol. Sep., 3 (1991) 59. 44 S. Schmidt, L. Blomberg, T. Wannnman, Chromatgraphia,28 (1989) 400. 45 J. M. Levy, J. P. Guzowski, W. E. Huhak, HRC&CC, 10 (1987) 337. 46 S. A. Liebman, E. J. Levy, S. Lurcott, S. O'Neil, J. Guthrie, T. Ryan, S. Yocklovich, J. Chromatogr. Sci., 27 (1989) 118. 47 J. M. Levy, R. A. Cavalier, T. N. Bosch, A. F. Rynaski, W. E. Huhak, J. Chromatogr. Sci., 27 (1989) 341. 48 S. B. Hawthorne, D. J. Miller, J. J . Langenfeld, J. Chromatogr. Sci., 28(1990)

274

2.

49 J . M. Levy, E. Storozynsky, R. M. Ravey, HRC, 14 (1991) 661. 50 R. J. Houben, Hans-G. M. Janssen, P. A. Leclercq, J. A. Rijks, C. A. Cramers, HRC,13 (1990) 669. 51 M. W. F. Nielen, J. T. Sanderson, R. W. Frei, U. A. Th. Brinkman, J. Chromatogr., 474 (1989) 388. 52 H. T. Badings, C. de Jong, R. P. M. Dooper, HRC & CC, 8 (1985) 755. 53 K. Hartonen, M. Jussila, P. Manninen, Maja-L. Riekkola,J. Microcol. Sep., 4 (1992) 3. 54 F. I. Onuska, K. A. Terry, HRC, 12 (1989) 527. 55 K. Grob, A. Habich, J. Chromatogr., 321 (1985) 45. 56 J. F. Pankow, HRC & CC, 9 (1986) 18. 57 B. Kolb, B. Liebhardt, L. S. Ettre, Chromatographia, 21 (1986) 305. 58 Y. Yokouchi, Y. Ambe, T. Maeda, Anal. Sci., 2 (1986) 571. 59 P. Sandra (ed), Sample Introduction in Capillary Gas Chromatography Volume 1,Huethig, Heidelberg, 1985. 60 K. Grob (ed), Classical Split and Splitless Injection in Capillary GC, Huethig, Heidelberg, 1988. 61 K. J. Hyver (ed), High Resolution Gas Chromatography, Hewlett-Packard, Avondale, 1989. 62 R. L. Grob (ed), Modern Practice of Gas Chromatography, J o h n Wiley & Sons, New York, 1985. 63 J. V. Hinshaw, Jr., J. Chromatogr. Sci., 25 (1987) 49. 64 J . V. Hinshaw, J. Chromatogr. Sci., 26 (1988) 142. 65 M. Herraiz, G. Reglero, E. Loyola, T. Herraiz, HRC&CC, 10 (1987) 598. 66 K. Grob, Z. Li, HRC, 11(1988) 626. 67 A. Tipler, G. Johnson, HRC, 13 (1990) 365. 68 B. W. Hermann, L. M. Freed, M. Q. Thompson, R. J. Phillips, K. J. Klein, W. D. Snyder, HRC, 13 (1990) 361. 69 P .L. Wylie, R. J. Phillips, K. J . Klein, M. Q. Thompson, B. W. Hermann, HRC, 14 (1991) 649. 70 T. Maeda, H. Tatematsu, F. Morishita, Proceedings of the 14th International Symposium of Capillary Chromatography, Baltimore, U.S.A.,(1992). 71 I. L. Davies, M. W. Raynor, J. P. Kithinji, K. D. Bartle, P. T. Willams, G. E. Andrews, Anal. Chem., 60 (1988) 683A. 72 T. Maeda, H. Tatematsu, F. Morishita, Anal. Sci. 7 (1991) 219. 73 J . M. Levy, J. P .Guzowski, Z. Anal. Chem., 330 (1989) 207.

K. Jinno (Ed.), Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction Journal of Chromatograph Library Series, Vol. 53 0 1992 Elsevier Science Jublishers B.V. All rights reserved.

275

Chapter 14 SFE, SFE/GC and SFE/SFC: Instrumentation and Applications Marja-Liisa Riekkola, Pekka Manninen and Kari Hartonen

Division of Analytical Chemistry, Department of Chemistry, University of Helsinki, Vuorikatu 20, SF-00100 Helsinki, Finland

INTRODUCTION

In contrast to the rapid developments that have taken place in instrumental identification, separation and analytical techniques, most sample preparation methods (Soxhlet, sonication) are still carried out according to the procedures described many decades ago. Very often these methods are time-consuming, and the opportunities for error diminish the reliability of the total analysis. The use of large solvent volumes not only means high costs but also disposal problems. The development of multidimensional techniques, such as LC/GC, SFE/GC and SFE/SFC, have started from this need for efficient, simple and economical sample preparation methods. Zosel [l]was one of the first to use supercritical fluid as an extraction medium for organic material. Since his work in the seventies, supercritical methods have developed into industrial-scale extraction techniques [2, 31 and, slowly in the 1980s, into analyticalscale techniques [4-61. During the last five years tremendous effort has gone into the development of the method, as can be seen in the increased number of papers published on the subject [7]. In addition to enhanced sensitivity and easy automation, the direct coupling of SFE with high resolution chromatographic techniques such as GC and SFC provides the extra benefits of analysis without exposure of the extract to oxygen, light or high temperatures. Further, in SFE/SFC, the SFE eliminates the injection problems that plague quantitative SFC analysis. Off-line SFE nevertheless is the simpler system from the experimental point of view, as has been described in chapter 12.

276

In this chapter we mainly describe the experiences of our laboratory with off-line SFE, SJ?E/GC and SFE/SFC in terms of instrumentation, practical aspects that need to be taken into consideration and applications.

SOLUTE SOLUBILITY' IN SUPERCRITICAL FLUIDS

Many studies on phase equilibrium and attempts to create models for supercritical mixtures have been described in the literature [3]. These theoretical methods require a large number of physical and chemical parameters, and since the phase behaviour of components is affected by their molecular size and chemical similarities, even the modelling of binary mixtures is complicated. Considering that the time available for obtaining the analytical results is often limited, extraction conditions in practice tend to be chosen empirically, particularly if the sample matrix is unknown. Solubility theory provides the most convenient theoretical approach to obtaining information about the solubility of the analyte in supercritical fluid [8]. The solubility of a compound in supercritical fluid can be approximated with the solubility parameter (S),

where AH is the heat of vaporization and V is the molar volume. Giddings has applied the theory to describe quantitatively the solvent power of dense gases PI,

where 6, is the solubility parameter of the dense gas, P, is the critical pressure, er,gis the reduced density of the gas and e,,, is the reduced density of the liquid of the given mobile phase. King has further widened this approach to calculate the conditions needed to achieve maximum solubility of the analyte in supercritical fluid [lo]. This approach is based on the solute/solvent interaction parameter (q),

277

where $H is the enthalphic interaction parameter, 6, is the solubility parameter of the is the molar volume of the supercritical fluid. solute and

vl

The solubility parameters and the molar volume of the supercritical fluid are a function of pressure and density, and therefore the interaction parameter is a function of these thermodynamic variables. The presentation of the solute/solvent interaction parameter as a function of pressure gives the maximum solubility of the solute at the minimum of the curve. Equation 3 shows that if the solubility parameters of the supercritical fluid and the solute are about the same, sufficient solubility of the solute in the supercritical fluid can be expected. Table 1 lists the solubility parameters of some supercritical fluids [111.

Table 1. Solubility parameters of selected pure compounds [ 111. Compound Helium Hydrogen Neon Nitrogen Argon Krypton Ethene Xenon Carbon dioxide Ethane Nitrous oxide Sulfur hexafluoride

ti( cal-"cm") 1.0 2.6 4.2 4.7 5.5 5.9 5.8 6.1 7.5 5.8 7.2 5.5

Compound

6( cal"cmH)

9.3 Ammonia 5.2 n-Butene 5.3 n-Butane 5.4 Diethyl ether 5.1 n-Pentane 4.9 n-Hexane 7.4 2-Propanol 8.9 Methanol 5.7 Ethyl acetate Tetrahydrofuran 6.2 Acetonitrile 6.3 13.5 Water

(p/p, = 2, T/T, = 1.02)

Solute solubility in CO, has been experimentally studied by Stahl [ 2 ] ,who summarized that typically lipophilic organic compounds of relatively low polarity and benzene derivatives with up to three phenolic hydroxyls or one carboxyl and two hydroxyl groups are

278 extractable. More strongly polar substances cannot be extracted below 400 atm. Hyatt [12] and Dandge [13] correlated the solute solubilities in supercritical fluid using a reduced solubility parameter, suggesting that solubility decreases with the increase in the molecular mass of a homologous or oligomeric series. However, Schultz and Randall [14] observed an exception to this trend for the partitioning of homologues between water and liquefied CO,. They found that solute partitioning from the aqueous medium into the nonpolar phase continued to occur when the number of methylene groups was increased. The same was found for the solute partitioning between polar solvents and supercritical CO,.

EFFECT OF TEMPERATURE

Slightly above the critical point even a small rise of temperature at constant pressure increases the solubility of the solute in supercritical fluid. However, if the pressure is raised, the increase of temperature will decrease the solubility. This result is explained by the decrease in the density of the fluid: the effect of density on the solute concentration in the supercritical fluid is greater than the effect of the temperature in increasing the vapour pressure of the solute. The increase in temperature has a smaller effect on density at high than at low pressures, so the solute concentrations will be larger in supercritical fluid.

SUPERCRITICAL FLUID EXTRACTION

Supercritical fluid extraction can roughly be divided into the following three steps: 1) The release of the analytes from the sample matrix into the supercritical fluid (solubility, diffusion, analyte/matrix interactions)

2) The sweep of the analytes out from the extraction vessel and their transfer to the collection system 3) The efficient collection of the analytes after SFE by depressurizing the supercritical fluid by passage through a restrictor There are two types of SFE, dynamic and static, and both can be carried out as separate or as combined techniques.

279

DYNAMIC SFE

Dynamic SFE is carried out by flushing the sample continuously with supercritical fluid (ca. 0.1 - 4 ml/min). It is widely used in off-line and especially in on-line methods, where the goal is to extract all analytes from the matrix. The rate of fluid flow through the extraction cell must be carefully optimized. Fractionation of the sample can sometimes be achieved by changing the temperature and pressure/density during the extraction. Use of a short static period at the beginning of the extraction is common. Modifier or derivatization reagent can be added directly to the extraction cell and allowed to interact with analytes and/or the matrix before flushing begins.

STATIC SFE

Static SFE is less frequently used than dynamic SFE. The reason for this is the interest in residue analysis, or at least in extracting analytes totally from the matrix, which is not possible with static SFE. The static mode is useful for solute solubility measurements and can be used as a preoptimizing method for dynamic SFE. It also allows easy study of the effect of modifiers and derivatization since a known amount of modifier or derivatization reagent can be directly added to the extraction vessel. We do not know enough about the solubility of analytes in supercritical fluid and, as has often been pointed out, the nature of the matrix (particle size, shape, porosity, surface area, activity, moisture etc.) plays an important role in real sample extraction. This means that the use of spiked samples for SFE optimization does not provide enough information. McHugh et al. [15] have described a static method for measuring solubilities in supercritical fluid. The instrumentation described later in this chapter can be used for the same kind of purposes.

INSTRUMENTATION FOR SFE

SFE equipment is now commercially available from several suppliers. In our laboratory we prefer a self-made instrument, for several reasons. SFE equipment is relatively easy to construct, cost savings are considerable and modifications are easily incorporated. Commercial equipment often restricts one to the use of specific extraction vessels, restrictors and analyte collection procedures.

280

The main components of our SFE system (see Figure 1) are a high pressure pump to pressurize the supercritical fluid, a heating block or oven to maintain SFE temperatures and an extraction vessel. In addition, some metal tubings, connectors and a few on/off valves are required. All fittings and sealings must be carefully tested to ensure against hazardous leakages. Analytical-scale SFE systems differ from each other mainly in the use of extraction vessels, restrictors and extract collection methods.

Heater and thermo-

Heated o njoffvalve

I

I I -

I

restrictor inserted into the solvent

I I.

PUMP

/

000 *** .....................

+ Extraction vessel LGC oven for SFE

-

Oven control

Figure 1. An example of off-line SFE instrumentation.

EXTRACTION VESSELS An extraction vessel for solid samples can be made from LC column tubing by connecting, for example, 1/4" standard Swagelog female fittings (with 1/4" 0.d. tube) and stainless steel frits to both ends of the rube. Alternatively one can use an empty LC column, an empty LC guard column cartridge or a commercially available extraction vessel (e.g. Keystone Scientific, Inc., USA). The extraction vessel should be selected so that the sample to be extracted fills almost the whole volume; that is, dead volumes should be avoided. Typical extraction cell volumes are 0.1 - 10 ml; the small volumes are mainly used in on-line applications. The extraction cell must be able to withstanc pressures up to 500 atm and temperatures up to 250 "C depending on the instrument anc

28 1

the fluid used. Fittings and especially sealing materials must be chemically inert against the supercritical fluid, and maintain their size, shape and hardness under a wide range of working pressures and temperatures. For liquid samples the fluid flow through the vessel must be arranged in such a way that the incoming fluid is led by a capillary into the bottom of the extraction vessel and the outlet is in the upper part of the vessel, or arranged with another capillary as in Figure 2. With this kind of arrangement, analytes can be extracted from liquid samples while the liquid matrix remains in the extraction vessel. However, the solubility of the matrix in the supercritical fluid must also be taken into consideration; with aqueous samples, for example, one must remember that water is slightly soluble (0.3 %) in supercritical CO, and could freeze at the restrictor exit.

0.5 ml Vessel

co, in

Extraction Cartridge

om I I

Internal Washer Seal Snap Ring

External Snap Ring

1 ml to 12 mi Vessels

Ill1

Frit and Seal Unit

Extender

Extraction Cartridge

Retaining Sleeve

Aqueous extraction VeSel Distributor

Figure 2. Some typical extraction vessels [16, 171.

RESTRICTORS The restrictors used in SFE are usually straight silica capillaries or metal capillaries with a crimped end (Figure 3). Restrictors that are narrowed at the end keep the analyte solubility (fluid density) unchanged all the way to the restrictor exit. A frit restrictor

282

generates small gas bubbles, but for concentrated samples it has too low a flow rate and quickly tends to break if the solvent trap is used to collect the extract. The breakage is probably due to the joint effect of freezing of the frit and the trapping solvent. In our laboratory we have also tested the integral restrictors (White Associates Inc., USA) designed for packed SFC columns. Serious plugging problems arose with plant matrices; restrictors were plugged after less than one minute of extraction.

A: Linear capillary restrictor

3

B: Polished “integral” restrictor

B

C

n =rzL n 5 D

E

C: Fast drawn capillary

restrictor

D: Porous frit restrictor E: “Pinched restrictor

Figure 3. Restrictors suitable for SFE.

EXTRACT COLLECTION METHODS

There are three methads for the extract collection: 1) Solvent trapping by bubbling the expanded fluid through a few millilitres of solvent, 2) Sorbent trapping by adsorbing extracted compounds to suitable sorbent material or a mixture of sorbents and 3) Cold trapping where the temperature of the container at which the fluid is depressurized is decreased so that the analytes are deposited on the walls of the cold container. An advantage of the cold trapping is that the cooling of the expanding fluid can be exploited. The cooling effect may also be of assistance in the first two trapping methods. Solvent trapping is perhaps the most convenient of the trapping methods because it involves simply immersing the end of the restrictor through the septum seal of a

283

collection vial containing a few millilitres of the trapping solvent. Factors affecting the trapping efficiency are the analyte solubility in the trapping solvent (solvent polarity), the volume of the solvent, the time that bubbles spend in the solvent, the size of the bubbles and the temperature of the solvent. A small id. trapping vial provides a longer path for the bubbles and the volume of the solvent can be held to a minimum, that is, to avoid an extra concentration step. In practice, flow rates in off-line SFE are high - with a 25 pm i d . silica capillary about 200 ml/min (gaseous) - and with such high flow rates the size of the bubbles may be very large. The collection efficiency can be increased by using a solvent filter or screen with small bore size to reduce the bubble size. Another approach is to reduce the SFE flow rate by using a smaller i.d. capillary as restrictor. However, this increases the extraction time. Fast and violent gas purging enhances the rate of solvent evaporation from the trapping vial, which can then be decreased by the methods mentioned above. However, the cooling of the expanding gas will lower the temperature of the solvent, decreasing the evaporation of the solvent. The addition of the trapping solvent during the extraction, especially with long extraction times, is necessary to keep the trapping efficiency constant. The trapping capacity of a solvent trap, as well as other kinds of traps, may be exceeded if samples are highly concentrated. The efficiency of the traps can be monitored with spiked samples and by using traps in series. The use of a solvent trap allows immediate analysis of extracted compounds by chromatographic or other analytical methods, in contrast to sorbent traps where an extra procedure is needed to desorb adsorbed analytes. In the case of the sorbent trap, analytes are washed out from the sorbent with a small volume of solvent, or in on-line methods with thermodesorption and direct flushing with eluent into the chromatographic column. Analytes must not only be retained quantitatively by selected sorbent material but they must be easily and quantitatively washed out. For a larger group of analytes with different polarities it is worthwhile to use a mixture of different sorbent materials to obtain high collection efficiency for all analytes. Extra restrictor heating and additional heated gas flow are applied to avoid restrictor plugging due to freezing [ 181. Sorbent trap systems can also be constructed by filling solid phase extraction tubes with suitable sorbent material (Figure 4). Cold trapping usually involves cooling the container to the point where the fluid expands, and the analytes are deposited on the walls. The trapping temperature depends on whether the analytes are to be separated from fluid or whether the fluid used is liquefied and the collection container is sealed to prevent the loss of analytes. An example of the latter case is presented in Figure 5. The container is cooled with liquid nitrogen.

284

Solid phase extraction tube Pulled restriction

Silicon rubber seal

Heated stainless steel block gas line

Figure 4. Sorbent trap for SFE using a solid phase extraction tube [17].

In cold trapping some of the analytes, even though their volatility is low, may be lost due to crystallization and aerosol formation [19]. This problem can be eliminated by use of a sealed liquefying cold trap system. Recoveries are much higher with a system like this than with an open collection to narrow-necked flask at 0 "C [20]. With such extreme cooling as with liquid nitrogen it is necessary to ensure a sufficient restrictor heating to avoid two-phase formation [19] and restrictor plugging. The cold trapping methods also require an extra sample preparation step before analysis, in which some of the analytes may be lost. The same is true in the sealed liquefying cold trap, where the liquefied fluid has to be evaporated and the analytes dissolved in suitable solvent. The sampling from liquefied fluid phase will be difficult if the low temperature causes nucleation and crystallization of the analytes.

285 718 in. S.S. Tubing

1/16 in. S.S. Tubing Controller

Heated Transfer Line Sealeff Collection

Heated Restrictor ( 5 0 prn S.S.) Gbs~-tind S.S. Extraction Vesd

Uquid Nitrogen Dewar

Figure 5. SFE with sealed liquefymg fluid collection system [20].

PRACTICAL ASPECTS

All tubings, fittings, pump cylinders and valves must be carefully cleaned before construction of the SFE system, as these are the main source of fluid contamination. Success in experimental SFE largely depends on how the SFE conditions to the restrictor exit are maintained, to ensure against restrictor plugging and allow effective extraction of the solutes of interest (see chapter 12 for extraction kinetics). Sample sizes are usually much larger in off-line SFE than in on-line applications, leading with some types of sample (e.g. samples containing fats or waxes) to restrictor plugging. Plants that contain flavour and fragrance compounds, usually inside cells, must be finely ground to loosen analytes from the matrix, i.e. to increase the mass transfer rate and to allow extraction of the analytes in reasonable time. However, grinding will also cause partial loss of volatiles.

If the density of supercritical fluid and solubilities of analytes decrease inside the restrictor, concentrated samples containing high molecular weight and polar compounds could deposit on the restrictor walls, causing plugging. To avoid these density and

286

solubility changes the restrictor should be heated at least to the temperature used in SFE; a higher temperature should be used to avoid freezing at the restrictor tip. A somewhat higher restrictor temperature than extraction temperature will not necessarily lead to density changes because heat transfer through the capillary into the fluid is not so fast. It has been shown that the easiest way to avoid restrictor freezing is to keep the analyte collection vessel inside the heated block or water bath to ensure that the temperature of the trapping solvent is above zero (for example, 5 "C) 1211. Additionally, if samples are large and finely ground, small nonsoluble particles may pass through the extraction vessel exit frit, or they may get stuck in the frit and plug it. We placed extra frits after the extraction vessel to prevent restrictor plugging due to small particles. Even then, sudden plugging sometimes occurred, though probably for other reasons. In connecting the restrictor capillary with a polyimide ferrule, one should be careful not to tighten the nut too much, as this could break the ferrule allowing polyimide particles to enter the restrictor. The restrictors used in off-line SFE are usually linear fused silica capillaries (i.d. 15 - 30 pm) or metal capillaries that are crimped at the end or made similar to the integral restrictors. Flow rates are normally higher in off-line SFE than in on-line techniques owing to the larger sample size. Although the frit restrictors used in SFC do not plug so easily as others, they are not suitable for off-line SFE because of the low flow rate and short life-time. Figure 1 shows our off-line SFE system where the on/off valve allows the use of either static or dynamic periods. Straight deactivated fused silica capillaries are used as restrictors because they are easy to use, readily available and inexpensive. They also work relatively well, although for some complex samples and with longer restrictors, plugging due to analyte deposition on the capillary walls may be a problem and change of the restrictor between extractions may be necessary. The use of solvent trapping is convenient and easy, but quantitative trapping of high volatiles may be a problem.

OFF-LINE SFE APPLICATIONS

Interest in SFE is rapidly increasing, as the area of applications has widened and people involved in routine analyses have become aware of the benefits. In laboratories carrying out routine analyses, it is typical that many time-consuming and error prone sample preparation steps are carried out before the application of highly sensitive analytical methods. There is thus broad scope for the replacement of conventional extraction methods by SFE. The main field at present is in solid sample matrices, which tend to be the most difficult to handle and require long sample preparation times 122. 231. SFE has also proved to be suitable for liquid samples [17, 241.

287

Many environmental and biological applications require the extraction of spiked samples. Although it must always be borne in mind that the environment of spiked analytes is different from that of real samples, valuable information about solute solubilities in a certain fluid, extraction efficiencies, method capabilities and system validation can be obtained through study of spiked samples. Schafer and Baumann [25] have shown the effect of moisture on the extraction of selected pesticides from glass wool, Nam et al. [26] the extractability of polychlorinated organics from biological tissue samples and the dependence of this on lipid content and lipid extractability, and Wright ef al. [20] the benefits of SFE over Soxhlet extraction for semivolatile and higher molecular weight compounds from different adsorbent and particulate matrices. Extraction recoveries of steroids from glass wool obtained with supercritical Freon-22, supercritical CO, and Soxhlet with methanol were compared by Li et af. [27] using SFC for the analysis. They found Freon-22 to be superior to CO,, although the extraction time with CO, (30 min) was twice that with Freon-22. The time needed to obtain 100 % recoveries with Soxhlet extraction was more than seven hours. With UV detection at 254 nm, higher signal-to-noise ratios were obtained with Freon-22 than with CO,. Wright et af. [28] have described an interesting application of ultrasound during SFE to increase overall extraction efficiency. Ultrasound creates intense sinusoidal variations in density and pressure which improve solute mass transfer. The density waves were presumed to induce convection of solutes from the inner pores of material because the density waves are much smaller in the matrix than in the fluid. Acoustic streaming [29] in supercritical fluid was expected to decrease the resistance to external mass transfer. Ultrasound may enhance the desorption rates through the localized heating that it creates. Furthermore, in the case of porous materials, some improvement in extraction can be gained through changes to the internal structure of the material. Increased extraction rates were found for crysene extractions from various adsorbents and for extraction of caffeine from coffee beans when ultrasound was applied. Ultrasound seems to be especially useful with small extraction cells where conventional stirring methods cannot be used. Various environmental applications where compounds of different polarity have been extracted from soil samples by SFE are reported in the literature [30, 311. One application of interest to us is the extraction of chemical warfare (CW) agents from soil samples [32, 331.

288

7-

L

:R

Sam Soman

Tabun H CR

Recovery 1%) 22

53 20 98 43

Figure 6. Two-channel GC chromatogram of CW agents extracted from soil by SFE at 200 atm and 45 OC. Temperature program: 40 "C (1 min), increasing 10 "C mid to 280 "C (10 min) [33].

Spiked CW agents and their degradation products were extracted from soil using SFCgrade CO, (Scotts Specialty Gases); agents were trapped in 2 ml ethyl acetate and degradation products in 2 ml ethanol. Even though most of the agents and their degradation products are polar, many of them can be extracted with SFE, and with better recoveries than achieved by solvent extraction with sonication. Although our extractions were made at rather low pressure (100 atm and 200 atm), the results showed SFE to be a more efficient extraction method for chemical warfare agents than the conventionally used sonication. Figures 6 and 7 show two-channel GC chromatograms for CW agents and their degradation products.

289

NPO HP-5 EMTPA

MPA EMPA IPMPA EMTPA PMPA

Recovery (961

3 8 10 6 12

Mf

Figure 7. Two-channel GC analysis of an SFE extract of soil containing degradation products of CW agents. Temperature program: 40 "C (1 min), then 10 "C m i d to 280 "C (10 min) [33].

The wide variations in recoveries (Table 2) can be reduced by using restrictor and collection system temperature control. Degradation products were poorly recovered (ca. 10 %) and VX was not recovered at all, which indicates that these methylphosphonic acids are strongly bound to soil even in spiking. The addition of polar modifiers and the use of higher sample moisture content would probably improve the recoveries. It would be of interest to create laboratory-scale conditions better corresponding to real ones. Spiking would then be done in an airtight system and soil samples collected after certain defined periods. Owing to the rapid degradation of many warfare agents, however, one might find only degradation products.

290

Table 2. SFE recovery (%) of selected CW agents by SFE at 45 "C and 200 atm [33]. Recovery (%) CW agent

Trial 1

Trial2

TriaI3

soman

22 53

Tabu

20

66 91 66 nd

H CR

nda 98 43

45 12 46 nd

90 39

42

Sarin

vx

Trial4

Trial 5

Trial 6

20

44

52 15 nd 86 44

84 29 nd 102 37

21 44 15 nd

70

X

k S.D.

31 61

** 2118

32 f 20 89 k 12 41 f 3

~

aNot detected (recovery SO.01 W).

INSTRUMENTATION FOR SFE/GC

Deciding upon the interface for an SFE/GC instrument is not easy. Several solutions are reported in the literature and they all have disadvantages as well as advantages. In most cases, the SFE system has been connected directly to the GC column or through an on-column or split/splitless injector (Figure 8) [34, 351. Recently increasing interest has been shown in the use of a programmed temperature vaporizing (PTV)injector as an interface between the SFE pressure restrictor and the GC column [36]. Extra switching valves can be connected to the system to allow the use of both static and dynamic extraction and to increase system flexibility (e.g. multiple extractions, multiple trapping systems, operation with different supercritical fluids and a switch between different columns). Cold trapping is the most common extract collection method in SFE/GC, but sorbent trapping can be used as well. The development of our SFE/GC system began in 1990. The system (Figure 9) includes an external cold trap from which the analytes are thermodesorbed to the column. A heat insulated transfer line between the SFE and GC ovens ensures effective transport of the extracted analytes from the extraction vessel to the cold trap. A linear deactivated fused silica capillary restrictor is connected to a 6-port valve, which is used to switch between SFE and GC operation modes. In SFE mode the fluid is circulated through the stainless steel trapping capillary and led out of the system, while the carrier gas from the GC injector flows through the column. When the valve is turned to the GC position and the trap heating starts, the analytes are therrnodesorbed from the trapping

291

capillary and flushed into the column by carrier gas. The fluid flows through the valve and directly out of the system.

A

Pump

ilcr 15

"c

Exlractlon

-30 to 5 OC CaplllJry

,GC

Column

Figure 8. SFE/GC interface using (A) on-column injector [34] and (B) split/splitless injector [35].

In a first approach, the extraction vessel was placed inside a heated aluminium block to maintain the extraction temperature. The temperature was different in the middle of the block and at the side where the temperature was measured. The actual fluid temperature was probably different as well, since the heat transfer was almost certainly insufficient, so that preheating of the fluid would have been required. We subsequently abandonned this approach and instead placed the extraction vessel in a gas chromatograph oven, where preheating was easily arranged by coiling some of the tubing in front of the extraction vessel. The temperature can be controlled more precisely with this system. Using the oven to control the extraction temperature also makes it more convenient to change the extraction vessel. And since the extraction vessel and most of the tubings are inside the oven, the user is protected from any hazardous explosions that might occur at high pressures, especially with the large supercritical fluid volumes associated with large extraction vessels.

292

GC

oven for SFE

co,

GC oven

Figure 9. SFE/GC instrumentation used in our laboratory [37].

In our trapping system we used a mixture of ice and salt and solid CO, and ethanol baths to cool the trapping capillary. Thermodesorption was first attempted by heating the part of the trapping capillary that was external to the GC oven with a hot air blower. However, this proved inefficient for the desorption of analytes that were deposited in the first few centimetres of the trapping capillary inside the oven and were eluted only during an additional GC run. Because extracted analytes began to deposit right after the restrictor there was a need for more efficient heating, and this was arranged through electrical heating of the trapping capillary. It was necessary to heat the trapping capillary from end to end in order to get all the analytes desorbed to column. Even then, however, some analytes might be deposited inside the valve to which the restrictor was connected and from which the trapping capillary exited to the cold bath. The system employing electrical trap heating (Figure 9) gave good reproducibility and symmetrical peaks in GC chromatograms. Focusing of the sample to the first part of the column was enhanced by using as low a trapping temperature as possible and a low initial GC temperature, and by leading the CO, (SFE) and carrier gas flows inside the 6-port valve and trapping capillary in different directions. Samples could be small because of the high sensitivity, and heating of the restrictor (150 "C) ensured that restrictor plugging seldom occurred. With quick thermodesorption all analytes were transferred to the column. Electrically actuated valves and a trapping system cooled with liquid nitrogen cooled gas would allow the system to be automated and controlled by computer.

293 SFE/GC APPLICATIONS

Direct coupling of SFE to the GC has several advantages. First, sample handling and preparation steps can be minimized, decreasing the time required for analysis and the possibility of loss and degradation of analytes. Because the SFE/GC method is highly sensitive, sample size can be reduced. On-line SFE/GC has mainly been used for trace analysis such as the analysis of PAHs in environmental solids [38] and flavour and fragrance compounds in various natural products [39]. Hawthorne et af. [38] used on-line SFE/GC to extract, separate, identify and quantify PAHs in sediment, urban dust and lampblack, PCBs in sediment samples and heterocyclic organics in cigarette ash. N,O provided the extraction fluid and the SFE unit was coupled to the GC by inserting the restrictor through the on-column injector. Good chromatographic peak shapes were obtained after all extractions and analytes were mostly recovered during the first 15 min of extraction; subsequent extractions from the same samples generated few or no peaks. Hawthorne and co-workers also evaluated the ability of the SFE/GC technique to provide quantitative extraction and on-column trapping of PAHs in river sediment. The results showed SFE/GC (FID) analysis to give quantitative results except for more volatile compounds (2-methylnaphthalene, 1methylnaphthalene and acenaphthene) where recoveries were as low as 70 %. With SFE/GC/MS quantitative data was obtained for urban dust samples in less than one hour. Hawthorne et. af [39] analyzed various samples including spices, chewing gum, orange peel, spruce needles and cedar wood by SFE/GC with mass spectrometric and flame ionization detection. Extraction times as low as ten minutes yielded quantitative recoveries of extractable analytes. Ten minutes SFE extraction of rosemary gave recovery efficiencies similar to four hours of sonication with methylene chloride. Replicate SFE/GC (FID) analyses showed good reproducibilities with relative standard deviations for the individual species ranging from 6 % to 14 %. The results demonstrate the usefulness of SFE/GC for rapid and reproducible determinations of flavour compounds. We have used SFE/GC for the analysis of volatiles in thyme [37]. All extractions were carried out with SFC-grade CO, (Scotts Specialty Gases, USA) at 200 atm and 54 "C for 30 minutes. Thyme (0.5 - 0.9 rng) was also used to test the reproducibility of our SFE/GC system (see Figure 9, and for further details the instrumentation section below). Reproducibility was excellent for the manual system, especially with the 6C6W-Vespel6port valve (Table 3).

294

Table 3. Reproducibility of our SFE/GC system with 6C6W-Vespel valve (results calculated on the basis of six replicate analyses of thyme) [37]. Thyme

Trial 1

Trial2

Trial 3

Trial4

Trial5

Trial6

Ave.

SD

a-Terpinene

Area %

1.1 9.174

1.7 9.177

1.8 9.180

1.2 9.173

2.1 9.174

1.1 9.183

1.5 9.177

0.4 0.004

y-Terpinene

Area % tR

0.4 10.065

0.2 10.065

0.3 10.063

0.2 10.065

0.4 10.064

0.4 10.075

0.3 10.066

0.1 0.004

Thymol

Area % t~ (mid

72.0 16.900

71.6 16.867

71.6 16.915

73.3 16.925

72.7 16.852

73.0 16.927

72.4 16.908

0.7 0.04

Carvacroi

Area %

4.3 17.037

4.3 17.005

4.3 17.101

4.3 17.056

4.2 16.992

4.2 17.058

4.3 17.042

0.1 0.04

tR b i n )

1

Figure 10. SFE/GC chromatogram of thyme. Conditions: 6C6W-Vespel valve, 0.65 mg sample extracted at 200 atm and 54 "C for 30 min; during the extraction the GC oven and trap cooling bath temperatures were 130 "C and -63 "C, respectively; the trap (90 cm) was heated to 130 "C. Peak identifications: (1) a-terpinene, (2) y-terpinene, (3) thymol, (4) carvacrol. Temperature program: from 50 OC (2 min) to 100 "C at 10 "C m i d , from 100 "C to 180 "C at 5 "C m i d , and from 180 "C to 240 "C (5 min) at 10 "C mid. Detector temperature was 280 "C [37].

295 Excellent peak shapes were achieved for thyme components and chromatograms were comparable to those obtained by GC after hydrodistillation. There was much greater divergence in the recoveries of thymol and earlier eluting components (a-terpinene and y-terpinene) with SFE/GC than with hydrodistillation followed by GC. The recoveries of hydrodistilled oil obtained by GC with silylation and by SFC without silylation were similar [40]. An SFE/GC chromatogram of thyme is shown in Figure 10 and a GC (splitless) chromatogram of thyme oil in Figure 11.

1

Figure 11. GC (splitless) chromatogram of thyme oil. Conditions: temperature program from 50 "C ( 2 min) to 80 "C at 10 "C min-', then 80 "C to 170 OC at 5 "C min-', and 170 "C to 200 "C ( 2 min) at 10 "C mid'. Detector temperature was 280 "C and injector temperature 200 "C. Peak identifications (area %): (1) a-terpinene (16.4), (2) y-terpinene (9.1), (3) thymol (50.2), (4) carvacrol (2.3) [37j.

INSTRUMENTATION FOR STATIC SFE/SFC

The static SFE/SFC equipment used in our laboratory comprises a commercid SFC instrument (Lee Scientific Series 600 supercritical fluid chromatograph, Lee Scientific, Salt Lake City), equipped with a flame ionization detector, Valco valves and 1/16 0.d.

296

stainless steel capillary tubings. A scheme of the instrument is shown in Figure 12. The two 10-port valves are used to change the direction of fluid flow during the pressurization, sample introduction and cleaning steps, and the 3-port valve functions as an onJoff valve. The system is constructed so that in the equilibration stage the tubings and the extraction vessel form a closed circle. The extraction vessel is a guard column cartridge (Brownlee Labs) with an internal volume of 150 pl. Samples are introduced simply by turning one of the 10-port valves to the other position. No trap is needed because the sample is introduced with a sample loop that takes a cut from the equilibrium phase. The sample is introduced to the column by fluid flow. The column can be coupled with or without a split interface to the 10-port valve. SFC-grade CO, is used as supercritical fluid.

SCF INLET

3 9 0 R T VALVE TO SELECT SFEEFC OR SFC MODE

SFC INJECTOR

-

SCF OUTLET

SPLIT RESTRICTOR

SFC COLUMN

Figure 12. Static SFE/SFC equipment.

STATIC SFE/SFC APPLICATIONS

Our interest in static SFE was excited by a proposal made by Prof. R. Hiltunen, who is experienced in headspace methods. He suggested to us a comparison of the

297

supercritical and headspace methods in the analysis of essential oils and herbs. In a first step we compared the recoveries of terpenes by capillary SFC and capillary GC [40]. Recoveries, especially for the oxygenated terpenes, were more quantitative with SFC, and there was no need for derivatization as in the case of GC. We then analysed herbs by static SFE/SFC using CO, as supercritical fluid. The choice of static mode was based on the analogy between static SFE and the static headspace method. The plant materials studied were thyme (Thymus vufgaris L.), basil (Ocimum busificum L.) and chamomile (Matticuriarecutitu L.). The plant materials were dried and stored at room temperature, so that their humidity was about 10 %. Although moisture can cause problems, especially in the extraction of nonpolar compounds, it was not entirely removed because drying of the sample by heat would have resulted in loss of analytes. Moreover, homogeneous mixing of the sample with sorbent is difficult with small samples (15 - 25 mg). The static SFE/SFC began with the extraction of authentic components spiked into starch. The strach, which gave little or no background in the chromatogram, was intended to simulate the plant matrix. However, as mentioned earlier in this chapter, spiked samples do not necessarily behave in exactly the same way as real samples. The spiked components of the analytes were easily extracted, whereas recoveries from real samples under the same conditions were much smaller. The nature of the matrix was clearly responsible for the poorer recoveries. The plant material cannot be ground to very fine particles because the essential oils are present as small droplets between the cells and the most volatile components would easily be lost during the grinding. Therefore, the matrix must be handled in the form of relatively large particles. The matrix also contains many active sites, e.g. free hydroxyl groups, which hinder the analyte partition into supercritical CO,. The effect of active sites can be partially eliminated by derivatization. This was demonstrated with chamomile herb, where only the active sites of the matrix, not the main components of the volatile oil, are silylated by BSTFA “,Obis(trimethylsilyltrifluoroacetamide)]. The silylation reagent was added directly to the extraction vessel so that it was flushed through the sample during the pressurizing step. The better recovery of terpenes with the silylation is explained by the silylation of the active sites of the matrix (Figure 13). The effect of modifiers could not be tested with our instrument because of the large response they excited from the flame ionization detector.

298

A

., L

k -

TERPENES

TERPENES

Figure 13. The effect of silylation in chamomile extraction: (A) unsilylated, (B) silylated sample [41].

Figures 14 and 15 show static SFE/SFC chromatograms for thyme extraction at 65 and 75 "C, and at densities of 0.4, 0.5 and 0.6 g/ml. The proportion of thymol was highest at a density of 0.5 g/ml at both temperatures (see Table 4) and much lower at 75 "C than at 65 "C, particularly at lowest and highest density. This is related to the effect of increasing temperature at moderate pressures on the decrease of solute solubility in supercritical fluid. In addition, wax components and higher hydrocarbons are seen at the end of the chromatograms only at lower temperature and at higher densities. The average recovery of thymol at 75 "C corresponded to that obtained from hydrodistilled oil by GC with silylation, and by SFC without silylation [40].

299

p = 0.4 g mL"

p = 0.5 g mL"

Figure 14. SFE/SFC analysis of thyme. Extraction: 65 "C, sample amount 25 mg. from , 0.450 Chromatography: from 0.145 g ml-' with 0.012 g ml" min-' to 0.450 g d-' g d1 with 0.018 g d'min" to 0.790 g r d ' . Column SB-Octyl-30 (10 m x 50 pm, 0.25 pm) [42].

Thymol p = 0.4 g mC'

Thymol

p = 0.5 g mu'

Thymol

p = 0.6 g mL"

L Figure 15. SFE/SFC analysis of thyme. Extraction: 75 Zhromatography: same as in Figure 14 [42].

OC,

sample amount 25 mg.

300 Table 4. The proportion of thymol calculated as percentage of the total recovery of terpene compounds [42].

0.4

0.5

0.6

65

78

85

83

75

52

72

68

Temperature ("C)

A typical static SFE/SFC chromatogram of basil extraction at 75 "C and at 0.5 g/ml is shown in Figure 16. The peaks in the terpene area (the first half of the chromatogram) are relatively small compared with those of thyme under the same conditions. Evidently, the main volatiles of basil are more poorly soluble in CO,, as would be expected from their higher functionality and more polar nature.

Figure 16. SFE/SFC analysis of basil. Extraction: 75 O C , sample amount 35 mg Chromatography: from 0.145 g d1 with 0.018 g d'mid' to 0.470 g ml-', from 0.470 g d1 with 0.010 g ml-' min-' to 0.730 g ml-'. Column SB-Octyl-30 (10 m x 50 pm, 0.25 pm) [42].

301 Matricine in chamomile flower heads has anti-inflammatory properties, and it is therefore of great interest to know the content in the herb. Matricine is usually determined indirectly by GC as chamazulene, to which it is converted during isolation of the essential oil by hydrodistillation or in headspace vial after addition of water and by heating. The isolation of pure matricine from matrix is troublesome because it requires several liquid extractions. We are currently working on the development of a direct method for matricine determination by off-line SFE/SFC and the isolation of matricine by SFE. To this end, the solubility of matricine in supercritical CO, was examined by static SFE/SFC. When the stability of matricine in SFC was tested at temperatures up to 100 "C with pure component, no hydrolysation was observed. Extractions of chamomile were made at temperatures between 60 and 85 "C and densities between 0.4 and 0.6 g/ml. Tables 5 and 6 show the general trends in terpene and matricine recoveries. The small recoveries of terpenes at high temperatures indicate that the increase of temperature more strongly affects density, and thence solute solubility in the supercritical fluid phase, than it does the vapour pressures of the analytes. The results suggest that the effect of increasing temperature could be exploited to isolate matricine from chamomile by dynamic SFE.

Table 5. The relative percentage (%) of terpene compounds from chamomile by static SFE/SFC [41].

Density (g/ml) Temperature ("C)

65 75 80 85

0.4

0.5

0.6

74.1 49.2 44.5 56.6

76.6 76.6 28.1

68.5 23.3

302

Table 6. The proportion of matricine calculated as percentage (%) of the recovery of terpenes [41].

Density (g/ml) Temperature (“C)

0.4

0.5

0.6

+

65 75 80

t

t

6.4

9.4

15.6

85

3.8

23.0

23.3

CONCLUSIONS

SFE and on-line SFE methods such as SFE/GC and SFE/SFC are highly sophisticated techniques for sample preparation from a variety of matrices. They offer speed, minimum sample contamination and sample losses, sensitivity, low cost and the possibility to automate the total sample preparation and analytical procedure. In our laboratory the extraction of herbs by SFE is more than one hour faster than the normal sample preparation by hydrodistillation. In addition, the drastic effects of heating (degradation and formation of artifacts) involved in hydrodistillation can be avoided. However, SFE methods are not always as easy to carry out as they are supposed to be, owing to instrumental shortcomings such as restrictor plugging and the lack of efficient and practical extract collection systems. Moreover, the knowledge of analyte/matrix interactions in the supercritical phase is incomplete.

Although SFE methods continue to be plagued by a few problems, for many applications they unquestionably offer an excellent alternative to other sample preparation techniques. There is also every reason to believe that many of the problems will be eliminated in the near future.

303 REFERENCES

1. K. Zosel, Angew Chem., 90 (1978) 748. 2. G. M. Schneider, E. Stahl, G. Wilke (eds.), Extraction with Supercritical Gases, Verlag Chemie, Weinheim, 1980. 3. M. A. McHugh and V. J. Krukonis (eds.), Supercritical Fluid Extraction; Principles and Practice, Butterword, US, 1986. 4. E. Stahl, W. Schilz and E. Willing, Angew. Chem., 90 (1978) 778. 5. K. Sugiyama, M. Saito, T. Hondo and M. Senda, J. Chromatogr., 332 (1985) 107. 6. S . B. Hawthorne and D. J. Miller, J. Chromatogr. Sci., 24 (1986) 258. 7. S . B. Hawthorne, Anal. Chem., 62 (1990) 883. 8. J. H. Hildebrand and R. L. Scott (eds.), The Solubility of Nonelectrolytes, Reinhold, New York 1950. 9. J. C. Giddings, M. N. Myers, L. McLaren and R. A. Keller, Science 162 (1968) 67. 10. J. W. King, J. Chromatogr. Sci., 27 (1989) 355. 11. P. J. Scoenmakers and L. G. M. Uunk, Eur. Chromatogr. News, 1 (1987) 14. 12. J. A. Hyatt, J. Org. Chem., 49 (1984) 5097. 13. D. K. Dange, J. P. Hellr and K. V. Wilson, Ind. Eng. Chem. Prod. Res. Dev., 24 (1985) 162. 14. W. G. Schultz and J. M. Randall, Food Tech., 24 (1970) 94. 15. M. A. McHugh, A. J. Seckner and T. J. Yogan, Ind. Eng. Chem. Fundam., 23 (1984) 493. 16. M. L. Lee and K. E. Markides (eds.), Analytical Supercritical Fluid Chromatography and Extraction, Chromatography Conferences, Inc., Provo, Utah, 1990, p. 349. 17. J. L. Hedrick and L. T. Taylor, J. High Resolut. Chromatogr. 13 (1990) 312. 18. L. J. Mulcahey, J. L. Hedrick and L. T. Taylor, Anal. Chem. 63 (1991) 2225. 19. R. D. Smith, J. L. Fulton, R. C. Petersen, A. J. Kopriva and B. W. Wright, Anal. Chem. 58 (1986) 2057. 20. B. W. Wright, C. W. Wright, R. W. Gale and R. D. Smith, Anal. Chem. 59 (1987) 38. 21. J. J. Langenfeld, M. D. Burford, S. B. Hawthorne and D. J. Miller, J. Chromatogr., submitted for review. 22. F. I. Onuska and K. A. Terry, J. High Resolut. Chromatogr., 12 (1989) 357. 23. M. Richards and R. M. Campbell, LC-GC International, 4 (1991):7, 33. 24. C. P. Ong, H. M. Ong, S . F. Y. Li and H. K. Lee, J. Microcol. Sep. 2 (1990) 69. 25. K. Schiifer and W. Baumann, Fresenius’ Z. Anal. Chem. 332 (1989) 884. 26. K. S . Nam, S . Kapila, D. S . Viswanath, T. E. Clevenger, J. Johansson and A. F. Yanders, Chemosphere 19 (1989) 33. 27. S . F. Y. Li, C. P. Ong, M. L. Lee and H. K. Lee, J. Chromatogr. 515 (1990) 515. 28. B. W. Wright, J. L. Fulton, A. J. Kopriva and R. D. Smith, Supercritical Fluid Extraction and Chromatography, Techniques and Applications, ACS Symposium

304

Series 366, B. A. Charpentier and M. R. Sevenants (eds.), Am. Chem. SOC.,Washington, DC, 1988, p. 44. 29. H. S. Fogler, Sonochemical Engineering, H. S. Fogler (ed.), AIChE, New York, 1971, Vol. 67, p. 1. 30. P. Capriel, A. Haisch and S . U. Khan, J. Agric. Food Chem., 34 (1986) 70. 31. V. Lopez-Avila, N. S. Dodhiwala and W. F. Beckert, J. Chromatogr. Sci., 28 (1990) 468. 32. M.-L. Kuitunen, P. Manninen and K. Hartonen, Presented in Int. Symp. on Supercritical Fluid Chromatogr. and Extraction, Park City, Utah, January 14-17, 1991. 33. M.-L. Kuitunen, K. Hartonen and M.-L. Riekkola, J. Microcol. Sep., 3 (1991) 505. 34. S . B. Hawthorne, D. J. Miller and M. S . Krieger, Proc. 10th Int. Symp. on Capillary Chromatography, P. Sandra and G. Redant (eds.), Huethig, Heidelberg, Germany, 1989, Vol. I, p. 305. 35. J. M. Levy and A. C. Rosselli, Chromatographia, 28 (1989) 613. 36. R. J. Houben, H.-G. M. Janssen, P. A. Leclercq, J. A. Rijks and C. A. Cramers, J. High Resolut. Chromatogr., 13 (1990) 669. 37. K. Hartonen, M. Jussila, P. Manninen and M.-L. Riekkola, J. Microcol. Sep., 4 (1992) 3. 38. S . B. Hawthorne and D. J. Miller, J. Chromatogr., 403 (1987) 63. 39. S. B. Hawthorne, M. S. Krieger and D. J. Miller, Anal. Chem., 60 (1988) 472. 40. P. Manninen, M.-L. Riekkola, Y. Holm and R. Hiltunen, J. High Resolut. Chromatogr., 13 (1990) 167. 41. P. Manninen, K. Saarinen and M.-L. Riekkola, Presented in 13th Int. Symp. on Capillary Chromatogr., Riva del Garda, Italy, May 13-16, 1991 42. P. Manninen and M.-L. Riekkola, Presented in Int. Symp. on Supercritical Fluid Chromatogr. and Extraction, Park City, Utah, January 14-17, 1991.

K. Jinno (Ed.), Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction Journal of Chromatography Library Series, Vol. 53 0 1992 Elsevier Science Publishers B.V. All rights reserved.

305

Chapter 15 COMPUTER ENHANCED HYPHENATION IN CHROMATOGRAPHY PRESENT AND FUTURE Eldon R. Baumeister and Charles L. Wilkins Department of Chemistry University of California, Riverside Riverside, CA 92521

Chromatographers have always been interested in obtaining the maximum amount of information from their separations. From the outset, practitioners of chromatography have sought detectors of greater specificity, sensitivity, and generality. Thus, it is not surprising that combinations of separation systems with information-rich forms of spectroscopy have been a recurring theme in analytical methods development. With the advent of fast inexpensive laboratory computers, more ambitious combinations involving multiple detectors have become common. This development is primarily the result of the availability of computers with sufficient computational speed and data storage capacity to deal with the large amount of information inherently available from such integrated analytical systems. Collectively, chromatographycomputer-spectroscopy systems have come to be known as “hyphenated analysis systems”. Thus, most of the chapters in this book, although sharing the specific emphasis on supercritical fluid chromatography, also discuss hyphenated systems. Within this context, it is the objective of this chapter to review the development of this concept and conclude with some discussion of recent work in SFC-mass spectrometry. GC-MS - The combination of gas chromatography with mass spectrometry represents one of the earliest success stories in development of hyphenated techniques. The first report of the now-ubiquitous gas chromatography-mass spectrometry (GC-MS) combination appeared in a 1957 paper by Holmes and Morrell [l]. Although the scan rate of the magnetic mass spectrometer they used was very slow, a mass spectrum of n-butane was observed with an oscilloscope. Because of the need to rapidly acquire enough spectra to properly define chromatographic peaks, many early GC-MS instruments utilized time of fight mass spectrometers [2-41. Quadrupole mass analyzers were introduced for GC-MS in the late 1960’s. The convenience and improved mass spectrometric performance quadrupole mass spectrometers for many applications, including GC, contributed to the rapiddevelopment of the GC-MS technique. Early systems had high sensitivity but limited mass ranges. By 1970, the method had developed to the point where it was appropriate for Merritt to write an extensive review on GC-MS with special emphasis on the GC-MS interface [5]. As a result of the high data acquisition rates of GC-MS measurements, it soon became clear that computers would be needed to manage the information. When the costs andcapabilities of laboratory computers finally became compatible with the requirements of GC-MS, the method was even more widely accepted. One of the earliest reviews of this aspect of GC-MS development was contributed by Karasek [6]. A real-time

computer-interfaced GC-MS system utilizing aquadrupole mass analyzer was developedin 1967 at Stanford University [7]. Biemann, in 1968, introduced the first real-time data system for a sector instrument [8]. By the 1970’s,GC-MS was an established analytical technique. Two other early summarized the historical development of GC-MS from the perspective of the early to mid1970s [9,10]. The introduction during the 1980s low cost quadrupole mass selective detectors and ion trap mass analyzers has made GC-MS even more widespread and, at the time of this writing, there are thousands of such systems in use all over the world. GC-IR Paralleling, to some extent, the development of GC-MS, the first combinations of gas chromatography with infrared spectroscopy [IR] usually involved off-line separations followed by spectral measurements. Early attempts at GC-IR employed packed column GC and dispersive instruments. On-line systems using stopped flow gas cells were employed, when faster scan rates became available. These flow cells were rectangular in shape and referred to as “lightpipes”. It was the development of infrared spectrometers based upon interferometry that really opened the door for practical GC-IR. Thus, development of Fourier transform infrared spectrometry (FTIR) was the key step in achieving analytically practical GC-IR. One of the earliest such spectrometers was the one described by Low and Coleman [111for measurement of weak emission spectra. That same year, Low published the first report of gas chromatography combined with infrared interferometry [ 121. The heated flow cell used consisted of an 8 cm long cylindrical tube with KBr end fittings. Several other publications by Low describing applications of GC-FTIR soon followed [ 13-15]. As computer technology advanced, digital data processing made new advances in GC-FTIR possible. The development of the fast Fourier transform algorithm (FFT)by Cooley and Tukey [ 161 coupled with its application by Forman [ 171 laid the groundwork for the later explosive development of not only FTIR,but all of the Fourier spectroscopies. During the 1970’s, commercial development of FTIR instrumentation allowed its utility to be demonstrated in a variety of applications. As a result of developments in gas chromatography, flow cell design, digital electronics and computer technology, GC-FTIR has become an well-accepted analytical technique. Many excellent reviews of GC-FTIR describing the instrumental development and related applications have been published [ 18-25]and the reader is referred to those for more in-depth discussion of it development. One area of progress in GCFTIR involved the development of lightpipes with reduced volumes. Following early descriptions of gold coated lightpipes for GC-IR [26], the manufacture of lightpipes made form thick walled glass tubing internally coated with gold, having length to inner diameter ratios of 100 or more, was described [27-301. Such lightpipes are now utilized in the great majority of commercial GC-IR systems. The exceptions are those instruments relying on cryogenic trapping methods (vide infru). As capillary column technology progressed, so did its utilization for complex mixture analysis by GC-FTIR. Azzaraga was one of the leaders in early capillary GCFTIR applications in environmental analysis and reported some of the first results establishing its potential [27,31,32]. Wallcoatedopen tubular(WC0Tl columns wereeventually incorporated with GC-FTIR to take full advantage of their superior separation efficiency [28,33,34]. The more recent introduction of fused silica columns has made possible routine high resolution GC-FTIR as a common analytical tool. In fact, it is sufficiently widely accepted that Gurka and co-workers have suggested GC-FTIR data collection guidelines for U.S.Environmental Protection Agency applications [35,36]. Other applications of GC-FTIR have been reviewed [20]. GC-IR-MS - In one of the very first papers on the topic of GC-IR (in 1968) Low and Freeman suggested the potential utility of a GC-IR-MS system [37]. Thus, the simultaneous linkage of both a mass and an infrared spectrometer with a gas chromatograph has been of interest

-

307 to analytical chemists for more than 20 years. One of us has authored several reviews on combined GC-IR-MS over the past few years [38-401. A plan for construction of an integrated GC-FTIRMS system was published in 1977 [41]. Prior to the first demonstration of an operational GCFTIR-MS combination [42], several investigators reported joint use of various instruments for generation of complementary information in organic analysis [43-471. The f i s t actual demonstrationofadirectlinkedGC-IR-MS systemoccurredin 1980 andwaspublishedinearly 1981, thirteen years after Low and Freeman suggested the idea [42]. In the initial report of GC-IR-MS, a packed GC column was used with a jet separator which removed much of the carrier gas prior to introduction into the mass spectrometer [42]. This approach was abandoned in later work because of the reIatively high gas load and inefficiency of packed columns. In the second report of GC-IR-MS, Hirschfeld and co-workers used a SCOT column linked in parallel to a quadrupole mass spectrometer (via a splitter) and an FTIR spectrometer [48]. Because of the nondestructive nature of FTIR, it is clear that both serial and parallel interfacing with mass spectrometry is possible. In the serial mode, the lightpipe dead volume is interposed between the GC and the mass spectrometer. In an early report of direct linked GCFTIR with a Fourier transform mass spectrometer, the chromatographic resolution of a SCOT column was reduced by 50% for the last-eluting substances in two complex mixture separations [49]. For GC-FTIR-MS systems using lightpipes, a parallel split interface between the GC and the two spectrometers is most desirable. This arrangement allows for most of the sample (> 95%) to flow to the IR spectrometer while the balance flows to the more sensitive mass spectrometer. The full potential of GC-IR-MS is realized only when each detector is able to obtain data for all separated mixture components. Early work in GC-IR-MS generally required the use of very expensive IR and MS spectrometers [38,42,48,49]. As a result, the general utility of GC-IR-MS was in doubt. Fortunately, at about the same time as GC-IR-MS was maturing, low cost mass spectrometers suchas theHewlettPackardmass selectivedetector (MSD) and the 1aterFinniganion trapdetector (ITD) became available. Simultaneously, unprecedented competition in the FTIR field lead to the introduction of many affordable, yet relatively high performance FTIR instruments, including the Hewlett Packard infrared detector (IRD), which is a special-purpose GC accessory, designed specifically for GC-IR. There are currently two commercially available GC-IR-MS systems. The first introduced to the market was the Hewlett Packard GC-IRD-MSD system, employing the optimized lightpipe-based infrared detector (IRD) mentioned above in combination with the MSD mass spectrometer [50].The second system is the Mattson Instruments Cryolect GC-matrix isolationFTIR-MSD. This instrument, based on a matrix isolation GC-IR approach [51] utilizes a matrix isolation FTIR spectrometer in conjunction with the Hewlett Packard MSD. Another cryogenic trapping system which is potentially adaptable to GC-IR-MS is the Bio-RadTracer FTIR system. With this system chromatographic eluents are trapped on a 77K sample stage for spectral measurement, using methods based on those developed by Griffiths, Bourne, and co-workers [52,53] and the direct liquid nitrogen temperature trapping as demonstrated earlier by Brown and Wilkins [54]. The important advance in Griffiths and Bourne’s latest work is the use of a microscope accessory, together with the direct trapping method, to obtain real-time GC-IR spectra from as little as 160picograms of sample injected, and post-run spectra from 50 picogram samples [53]. This approach appears to make real-time GC-IR on 1 nanogram quantities of analytes aroutine operation. The relative merits of matrix isolation versus lightpipe detection has been the subject of a recentreview [ 5 5 ] . In anotherreview, Wilson and Childers discuss the more

308 general topic of matrix isolation FTIR [56]. HPLC-MS The combination of chromatography with mass spectrometry currently provides the broadest analytical approach for the characterization of complex mixtures. Over the past decade or so, the GC-MS technique has become the method of choice for characterization of volatile thermally stable compounds. Much recent research has focused on providing similar analytical technology for the vast majority of compounds either too non-volatile or too thermally unstable to be separated by GC. Recent advances in liquid chromatography have helped to focus commercial attention on the development and application of liquid chromatography-MS. Many of these developments are covered in a recent comprehensive review of HPLC-MS [57]. There are a number of commercially available interfaces for mating liquid chromatography with mass spectrometry. Three of the most popular are the moving belt interface, the direct liquid interface and thermospray interface. The concept of the moving belt interface (MBI) was first demonstrated by Scott, et.QZ. [58] who designeda system using amoving wire to carry the solventholute into the MS source. Unfortunately, the efficiency of this early system was only 1%. The development of Kapton belts soon allowed design of an interface to introduce samples directly into the source region, permitting higher sensitivity [59,60]. The utility of the MBI has been exploited by Games for analysis of a wide range of compounds and of various polarities and demonstrates the versatility of such a system [61]. Arpino recently reviewed the moving belt interfacein depth [62]. By far the simplest approach toHPLC-MS is thedirect liquidintroduction (DLI) technique pioneered by Tal’Rose, et. al. and Baldwin and McLafferty [63,64]. These early DLI interfaces were generally designed to be compatible with the direct probe inlet systems of conventional mass spectrometers. A common feature of the many such interfaces reported in the literature is use of a 3-5kmopening in the probe tip leading into the MS source. These dimensions permit a fine liquid stream of sample into the source region. A recent detailed review discusses the direct liquid interface, in addition to several others. [65]. The historical limitations of the MBI and DLI interfaces are their inability to handle l-2mL/min flows of high polarity solvents and the difficulties encountered with nonvolatile and thermally unstable compounds. The development of thermospray (TSP) mass spectrometry by Vestal [66] provided solutions to both problems. The TSP interface can handle both normal andreverse-phase solvent flow rates between 0.1 and 2mL/ min. In early versions, it was required that a buffer be added to permit efficient ionization [67]. The use of micro column HPLC has solved several of the problems stemming from introduction of too much solvent vapor into the MS source region [68]. Thermospray is currently the most widely used HPLC interface and its use has been recently reviewed [57,65]. In addition to the commercially available interfaces, several other methods that show much promise for extending HPLC-MS technology, are being developed . The performance of an improved monodisperse aerosol generation interface for HPLC (MAGIC) was recently described by Browner and coworkers [69]. Levi recentlyreported aparticle beaminterface forHPLC andcomparedit toTSP using several organic compounds [70]. For polar analytes, development of continuous flow FAB (CF-FAB) interfacing for HPLC-MS shows some promise [57]. Ion spray [71] and atmospheric pressure ionization [72,73] techniques for HPLC-MS have also recently been reported. It also appears that glow discharge [74] or electrospray ionization methods [75] may have some promise for use in HPLC-MS applications. SFC-MS Because of its much-reduced mobile phase flow rate, capillary supercritical fluidchromatography has madeit feasible to directly interface SFC with mass spectrometers. The wide acceptance of supercritical fluid chromatography-mass spectrometry (SFC-MS) as a potentially important tool for analysis of nonvolatile and thermally unstable compounds is

-

-

309 evidenced by numerous recent publications and review articles [76-811. The surge of interest in SFC and SFC-MS can be explained by two factors. First, the diffusion rates and viscosities of supercritical fluids allow for fast and efficient separations of nonvolatile or thermally unstable compounds. Such separations are generally not possible by GC-MS. The physical properties of supercritical fluids allow the use of liquid phase detectors and, after decompression through an appropriate restrictor, use of many gas phase detectors. Second, SFC-MS has several potential advantages over HPLC-MS. In particular, direct interfacing is simpler and shorter analysis times can be achieved. Furthermore, SFC-MS chromatographic resolution and sensitivity compares favorably with HPLC-MS. Most important, mobile phase removal is much easier. Although supercritical fluids work well with most GC detectors if compatible mobile phases are used, the greater information provided by mass spectrometers makes MS attractive as an SFC detector. Following the pioneering work by Smith and coworkers [82, 831, a great many SFC-MS interfaces have been constructed. Most are describedin thereview articles cited, but recent papers of particular pertinence are those by Kalinoski and Hargiss [84], Sheeley and Reinhold [78] and Huang and coworkers [@I. SFC-FTMS Fourier transform mass spectrometers have the necessary scanning speed, high mass range and high resolution to fully exploit SFC. However, FTMS does require that relatively low analyzer pressures (lower than Torr) be maintained for optimal results. The feasibility of interfacing SFC with Fourier transform mass spectrometers was first demonstrated by Lee, et. af. in 1987 [86]. Their results showed that on-line SFC-FTMS could be accomplished with continuous introduction of supercriticai C 0 2 via a 50 pm i.d. 5 m bonded fused silica

-

Figure 1. ( a ) Schematic diagram of differentially-pumped dual cell FTMS system used as a detector for SFC. (b)Expanded view of SFCIFTMS transfer line and interface. Reprinted from reference 87 with permission of the American Chemical Society.

310

capillary column maintained at 1000. These workers reported separation and identification of the components of a simple synthetic mixture of methyl stearate and caffeine. A 25 ng on-column injection of caffeine was analyzed under high pressure EI conditions and found to give excellent mass measurement accuracy (within 0.36 ppm), with mass resolution of 8,300. The caffeine mass spectrum was similar to that obtained under conventional EI GC-MS conditions, thus demonstrating the feasibility of determining exact masses by EI SFC-FTMS. In addition, the self-chemical ionization (CI) mass spectrum of a PFTBA fragment ( d z 131) was measured with mass resolution of 12,100. These values were used to calibrate the self-CI ions from the SFC-FTMS spectrum of caffeine. Rapid mass spectral scans were obtained by SFC-FTMS under EI, selfCI and isopentane CI conditions. Because EI mass spectra are not usually available from compounds injected directly as liquids, these experiments demonstrated a new approach to obtaining structurally important information under such conditions. However, as these workers observed, their interface had a number of limitations. In particular, the transfer line and restrictor could not be heated, making analysis of high mass analytes problematic. Additionally, the interface was constructed by modifying a fixed standard GC-FTMS interface. This arrangement prevented other easily interchangeable modes of chromatographic sample introduction from being used concurrently. A second demonstration of SFC-FTMS, employing an improved heated interface was reported by Laude and co-workers later that year [87]. That design also involved a fixed transfex line-restrictor configuration (Figure 1), with a probe-mounted electron filament. Several SFC separations of relatively simple mixtures including substituted aromatics, polyaromatics, barbiturates and pesticides were described. Figure 2 is a typical reconstructed chromatogram obtained in the study. Chromatographic conditions that emphasized resolution at the expense of

Figure 2 . Segment of a reconstructed SFC-FTMS chromatogram (1.50-230 dafton integration window)for a 56 nanogram per component injection of a six-component mixture of substitutea aromatic compounds. Reprinted from reference 87 with permission of the American Chemical Society.

31 1

speed were chosen. Thus, although sensitivity was not outstanding, mass resolution was exceptionally good for on-line SFC-MS measurements. High source pressures of C 0 2 hindered sample ionization. With 70 eV elecn-on ionization and continuous ejection of C02 ions, a few micrograms of sample were required. Trapping was inefficient due to space-charge effects from the excess of un-ejected carbon dioxide ions. However, by reducing the ionizing potential to 13 eV, also with continuous ejection of C 0 2 ions, analyte detection limits were at low nanogram levels. When conditions optimized to obtain pressure-limited resolution were used, high resolution mass spectra of naphthalene and bromonaphthalene with mass resolutions of 10,700

Figure 3. FTMS spectra of (a)naphthalene and (b)1-bromonaphthalene molecular ion regions with data acquistion parameters chosen to allow pressure-limited mass spectral resolution. Reprintedfrom reference 87 with permission of the American Chemical Society. and 6,900 (Figure 3), respectively, were obtained. Under conditions optimized for sensitivity, separations of barbiturates and pesticides mixtures with detection limits of 1- 10 nanograms were obtained. As in the earlier work, the interface was useful for SFC introduction, but other

31 2 concurrent modes of sample introduction were made inconvenient. Because neither of the previously-discussed approaches to the SFC-FTMS interface utilized a completely removable probe-mounted arrangement, use, modification and servicing of the interfaces was inconvenient. Furthermore, without a completely removable probe-mounted interface, it was inconvenient to change from SFC-FTMS to other modes of sample introduction. The initial work in SFC-FTMS prompted the design of a self-containedprobe-mounted interface which is convenient to use, has good temperature control, and is contained entirely within a standard FTMS probe [88, 891. Good temperature control is a prerequisite if thermally labile materials are to be analyzed. As noted by the authors, a convenient and practical interface should incorporate an easily exchanged integral restrictor and be operable for extended periods with the tip temperature above 600OC. For maximum sensitivity, it should also have a tip position which is easily adjustable, relative to the FTMS cell, during operation. Ideally, the interface should be useable for GC-MS without any significant alterations. The supercritical fluid chromatography-Fourier transform mass spectrometry system employed in this most recent work is diagrammed in Figure 4. To evaluate the new design, pure

F

Figure 4 . Block diagram of SFC-FTMS system. ( A ) carbon dioxide cylinder; ( R ) high pressure oven; ( F ) dual syringe pump; (C) 0.45 mm filter; (D)injection valve; (E) gas chromat~~raph transfer line; (G)probe interface; ( H ) Fourier transform mass spectrometer. Reprinted from reference 88 with permission of the American Chemical Society.

313

CO:! was used as mobile phase and SFC-FTMS runs were pressure programmed with initial linear flow velocities of 4 cm/sec at 1800 psi. The transfer line containing the last meter of the capillary SFC column was heated and delivered eluant directly to the interface. In this research, instrumental parameters were chosen for maximum sensitivity, rather than optimal mass resolution. The parameters used maximize the number of ions in the cell, up to the space charge limit. Under these ionization conditions a combination of both C02 chargeexchange and direct electron ionization probably occurs. Chromatography was conducted to minimize analysis time, rather than to achieve high chromatographic resolution. Thus, a very sharp programmed pressure ramp was utilized. An initial isobaric period of 10 minutes at 1800 psi was followed programming the pressure linearly to 5000 psi over the next 5 minutes, followed by a secondisobaricperiodof lominutes at 5OOOpsi. Initial testingof the probeinterfaceinvolved evaluation of the temperature control of the various heated zones and pressure/flow characteristics of the integral restrictor and heater tip. For this design, the source/analyzer pressure ratio always remains over 100, (averaging 128 over the inlet pressure range of 1500-6000psi). Thus, acceptable analyzer cell pressures (on the order of 10-7 Tom) are maintained. In fact, this may be the lowest analyzer pressure for an SFC-MS system yet reported.

Figure 5 . SFC-FTMS total ion reconstructed chromatogram of 100 nanograms of cholesterol injected. Reprinted from reference 88 with permission of the American Chemical Society. Historically, cholesterol has been used as a sensitive test for thermal activity of GC-MS interfaces andjet separators [79]. Because cholesterol dehydrates readily, for typical cholesterol mass spectra obtained by GC-MS or with heated probes, the base peak is the dehydration product ion (m/z 368), and only a low abundance molecular ion peak (m/z 386) is observed. Thus, the appearance of the cholesterol mass spectrum can serve a diagnostic indicator of the ability of an interface to handle thermally labile compounds. Figure 5 is the SFC-FTMS reconstructed total ion chromatogram obtained when 100 ng of cholesterol is injected, and its SFC-FTMS spectrum is shown in Figure 6. Judging by the signal to noise ratio of about 20:1, a lower detection limit of 5-10 ng would have been possible under the not fully optimized, SFC-FTMS conditions used.

0

100

150

200

250

MASS I N A . M . U

300

350

4

Figure 6 . SFC-FTMS spectrum of 100 nanograms of cholesterol injected. Reprinted from reference 88 with permission of the American Chemical Society.

Figure 7. SFC-FTMS total ion reconstructed chromatogram of 500 picograms of pyrene injected. Reprintedfrom reference 88 with permission of the American Chemical Society.

315 The base peak in the mass spectrum is the molecular ion (m/z 386) with the dehydration product ion (m/z 368) having a relative abundance of only 26% . Aside from the high relative abundance of the molecular ion, the spectrum has much i n common with conventional EI spectra. A four component mixture of polyaromatic hydrocarbons was analyzed to demonstrate the capability for on-line detection under actual separation conditions and sub-nanogram detection limits demonstrated. To further characterize system sensitivity, 500 picograms of pyrene was injected. The reconstructed chromatogram is shown in Figure 7 and the corresponding mass spectrum in Figure 8. From this spectrum a detection limit of about 100 picograms was estimated. It seems clear, even on the basis of only three literature reports of SFC-FTMS, that detection limits of less than 100picograms should be achievable using optimized conditions. However, presently obtainable sensitivity is already similar to thatobtainedfor PAHanalysis with other EI SFC-MS systems (e.g. full-scan spectra obtained at the “low nanogram level” with a Hewlett Packard 5988 SFC-MS system with single ion monitoring detection limits of ca. 25 pg estimated) [go] and should approach the sensitivity obtained with GC-MPI-FTMS, as reported by Sack and co-workers [91]. Equally clear is the potential of SFC-FTMS for providing high resolution SFC-mass spectra, when conditions are chosen to optimize that aspect of the analysis.

0

100

‘’O

MASS

200

IN A.M.U.

250

Figure 8 SFC-FTMS spectrum of 500 picograms of pyrene injected. Reprinted from reference 88 with permission of the American Chemical Society

316

References J.C. Holmes and F.A. Morrell, Appl. Spectrosc., 2 (1957) 86. R.S. Golhke, Anal. Chem., 31 (1959) 535. R.S. Golhke, Anal. Chem., 34 (1962) 1332. W.H. McFadden and R. Teranishi, Nature, 200 (1963) 329 C. Memtt, Jr., Appl. Spectrosc. Rev., 4 (1970) 263. F.W. Karasek, Anal. Chem., 44 (1972) 32A. W.E. Reynolds, J.C. Bridges, T.B. Cobum and R.B. Tucker, Tech. Rep. No. IRL-1062, Dept. of Genetics, Stanford Univ. School Medicine, Palo Alto, (1967). R.A. Hites and K. Biemann, Anal. Chem., 40 (1968) 1217. G.A. Junk, Int. J. Mass Spectrom. Ion Phys., 8 (1972) 1. B.J. Gudzinowicz, M.J. Gudzinowicz and H.F. Martin (eds.), Fundamentals of In tegrated GC/MS, Vol. 7 (1-111), Marcel Dekker, NY, 1976. M.J.D. Low and I. Coleman, Spectrochim. Actu, 22 (1966) 369. M.J.D. Low, Chem. Commun., 12 (1966) 371. M.J.D. Low and S.K. Freeman, Anal. Chem., 39 (1967) 194. M.J.D. Low, H. Mark and A.J. Goodsel, J. Paint Technol., 43 (1971) 49. M.J.D. Low, J. Agric. FoodChem., 19 (1971) 1124. J.W. Cooley and J.W. Tukey, Math. Comput., 19 (1965) 297. M.L. Forman, J. Opt. SOC.Am., 56 (1966) 978. M.J.D. Low, Anal. Chem., 41 (1969) 97A. P.R. Griffiths (ed.); Transform Techniques in Chemistry, Plenum Press, IVY, 1978. P.R. Griffiths and D.E. Henry, Prog. Anal. Spectrosc., 9 (1986) 455. P.R. Griffiths, J.A. de Haseth and L.V. Azarraga, Anal. Chem., 55 (1983) 1361A. P.R. Griffiths; Advances in Infrared and Raman Spectroscopy, (1983) vol. 10, Chapter5, 277. L. R.A. Nyquist, M.A. Leugers, M.L. McKelvy, R.R. Papenfuss, C.L. Putzig and Yurga, Anal. Chem.., 62 (1990) 223R. P.R. Griffiths and J.A. de Haseth, Fourier Transform Infrared Spectrometry, John Wiley & Sons, NY, 1986. R.L. White, Appl. Spectrosc. Rev., 23 (1987) 165. B. Krakow, Anal. Chem., 41 (1969) 815. L.V. Azarraga, “GC/IR With Submicrogram Sensitivity”, Presented at the 5th Annual Symposium on Recent Advances in the Analytical Chemistry of Pollutants, Jekyll Island, GA, (May 1975). L.V. Azarraga, “Improved Sensitivity of On-The-Fly GCIR Spectroscopy’’, Presented at the 27th Annual Pittsburgh Conference & Expos., Cleveland, OH, (March 1-5,1976) Paper #334. L.V. Azarraga, Appl. Spectrosc., 34 (1980) 224. P.W.J Yang, E. L. Ethridge, J. L. Lane and P. R. Criffiths, Appl. Spectrosc., 38 (1984) 813 L.V. Azarraga and A.C. McCall, “Infrared Fourier Transform Spectrometry of Gas Chromatography Effluents”, Presented at the 25th Annual Pittsburgh Conference & Expos., Cleveland, OH, (March 4-8, 1974), Paper #328.

317

(32) (33) (34) (35)

(45)

(47) (48) (49)

(53) (54) (55)

L.V. Azarraga and A.C. McCall “Infrared Fourier Transform Spectrometry of Gas Chromatography Effluents”, EPA-600/2-73-034, (1974), 61 pp. K.H. Shafer, A. Bjorseth, J. Tabor and R.J. Jakobsen, J . High Resolut. Chromatogr. & Chromatogr. Commun., 3 (1980) 87. L.V. Azarraga, and C.A. Potter, J . High Resolut. Chromatogr. & Chromtogr. Commun., 4 (1981) 60. D.F. Gurka, M. Umana, E.D. Pellizzari, A. Moseley and J.A. de Haseth, Appl. Spectrosc., 39 (1985) 297. D.F. Gurka, Appl. Spectrosc., 39 (1985) 827. M.J.D. Low and S.K. Freeman, J. Agric. Food Chem., 16 (1968) 525. C.L. Wilkins, Science, 222 (1983) 291. C.L. Wilkins,Anal. Chem., 59 (1987) 571A. C.L. Wilkins, Science, 243 (1989) G14. C.L. Wilkins, J . Chem. Inf. Comput. Sci. ,17 (1977) 242. C.L.Wilkins, G.N. Giss, G.M. Brissey and S . Steiner,Anal. Chem., 53 (1981)113. P.C. Uden, D.E. Henderson and R.J. Lloyd, J . Chromatogr., 126 (1976) 225. P.C. Uden, A.P. Carpenter, Jr., H.M. Hackett, D.E. Henderson and S . Siggia, Anal. Chem., 51 (1979) 38. P.C. Jurs, B.R. Kowalski, T.L. Isenhour, and C.N. Reilly, Anal. Chem., 4 1 (1969) 1949. S. Sasaki, H. Abe, T. Ouki, M. Sakamoto and S . Ochiai, Anal. Chem., 40 (1968) 2220. T. Hirschfeld, Anal. Chem., 52 (1980) 297A. R.W. Crawford, T. Hirschfeld, R.H. Sanbom and C.M. Wong, Anal. Chem., 54 (1982) 817. C.L. Wilkins, G.N. Giss, R.L. White, G.M. Brissey and E.C. Onyiriuka, Anal. Chem., 54 (1982) 2260. E.J. Darland, L.D. Mealer, J.A. Michnowicz, C. Tang, T. Hirschfeld, “An Infrared Detector (IRD) for Gas Chromatography”,Presented at 27th Annual Pittsburgh Con ference & Expos., Atlantic City, NJ, (March, 1986), Paper #494. S. Bourne, “Mamx Isolation GC/FTIR”, Presented at 13th Annual FACSS Meeting, St. Louis, MO, September-October, 1986, Paper #632. A.M. Haefner, K.L.Norton, P.R. Griffiths, S. Bourne and R. Curbelo, Anal. Chem., 60 (1988) 2441. S. Bourne, A.M. Haefner, K.L. Norton and P.R. Griffiths, Anal. Chem., 62 (1990) 2448. R.S. Brown and C.L.’Wilkins, Anal. Chem., 60 (1988) 1483. J.F. Schneider, J.C. Demirgian and J.C. Stickler, J . Chromatographic Science, 24 (1986) 330. N.W. Wilson and J.W. Childers, Appl. Spectr. Rev., 25 (1989) 1. R.E. Synovec, E.L. Johnson, L.K. Moore and C.N. Renn, Anal. Chem., 62 (1990) 357R. R. Scott, C. Scott, M. Munroe and J. Hess, J . Chromatogr., 99 (1974) 395. R.M. Caprioli, T. Fan and J.S. Cottrell, Anal. Chem., 58 (1986) 2949. D.E. Games, S. Pleasance, E.D. Ramsey and M.A. McDowell, Biomed. Environ Mass Spectrom, 15 (1988) 179.

D.E. Games, Adv. Mass Specrrom., 10B (1986) 323. P. Arpino, Mass Spectrom Rev., 8 (1989) 35. V. Tal’Rose, G. Karpov, I. Gordoetshii and V. Skurat, Russ. J. Phys. Chem., 42 (1968) 1658 M. Baldwin and F. McLafferty, Org. Mass Spectrom., 7 (1973) 111. P. Arpino, Mass Spectrom Rev., 9 (1990) 63 1. C. Blakely and M. Vestal, Mass Spectrorn. Rev., 2 (1983) 447. R.D. Voyksner, J.T. Bursey and E.D. Pellizzari, Anal. Chem., 56 (1984) 1507. A.C. Barefoot and R.W. Reiser, J. Chromatogr., 398 (1987) 217. P.C. Winkler, D.D. Perkins, W. K. Williams and R.F. Browner, Anal. Chem., 60 (1988) 489. P. Levi, Analusis, 16 (1988) XLVII-L. T.R. Covey, A.P. Bruins and J.D. Henion, Org. Mass Spectrom., 23 (1988) 178. M. Sakairi and H. Kambara, Anal. Chem., 60 (1988) 744. M. Sakairi and H. Kambara, Anal. Chem., 61 (1989) 1159. F.L. King and W.W. Harrison, Mass Spectrom Rev., 9 (1990) 285. J.B. Fenn, M. Mann, C.K.Meng and S.F. Wong, Mass Spectrom Rev., 9 (1990) 37. T.L. Chester and J.D. Pinkston, Anal. Chem., 62 (1990) 394R. R.D. Smith and B.W. Wright, In Microbore Column Chromatography ;F.J. Yang (ed.) Marcel Dekker, NY, 1989, pp 307-368. D.M. Sheeley and V.N. Reinhold, J. Chrornatogr., 474 (1989) 83. G.D. Owens, L.J. Burkes, J.D. Pinkston, T. Keough, J.R. Simms and M.P. Lacey, In Supercritical Fluid Extraction and Chromatography; B.A. Charpentier and M.R. Sevenants (eds.) American Chemical Society; Washington, DC, 1988, 191. R.D. Smith, H.T. Kalinoski and H.R. Udseth, Mass Spec. Rev., 6 (1987) 445. B.W. Wright, H.T. Kalinoski, H.R. Udseth and R.D. Smith, J. High Resolur. Chromatogr. & Chromatogr. Commun., 9 (1986) 145. R.D. Smith, J.C. Fjeldsted and M.L. Lee, J. Chromatogr., 247 (1982) 231. R.D. Smith, H.R. Udseth and H.T. Kalinoski, Anal. Chern.,56 (1984) 2971. H.T. Kalinoski and L.O. Hargiss, J. Chromatogr., 474 (1989) 69. E.C. Huang, B.J. Jackson, K.E. Markides and M.L. Lee, Anal. Chem., 60 (1988) 2715. E.D. Lee, J.D. Henion, R.B. Cody and J.A. Kinsinger, Anal. Chern., 59 (1987) 1309. D.A. Laude, S.L. Pentoney, P.R. Griffiths and C.L. Wilkins, Anal. Chem., 59 (1987) 2283. E. R. Baumeister, C. D. West, C. F. Ijames andC. L. Wilkins, Anal. Chem., 63 (1991) 261. E.R. Baumeister, C.D. West and C.L. Wilkins. “An Improved Probe Interface For SFC-FTMS.” Presented at the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tucson, AZ, (June 6, 1990), Paper # WP-45. S.B. Hawthorne and D.J. Miller, J. Chromatogr., 468 (1989) 115. T.M. Sack, D.A. McCrery and M.L. Gross, Anal. Chem., 57 (1985) 1290. Acknowledgments Support by the National Science Foundation (CHE-89-11685) and the US-EPA (CR81-4755-02-0) is gratefully acknowledged.

319

Subject index Absorbents in SFE 207,208 Absorption 138, 141, 143, 144, 145 Acetophenone 123 Adiabatic expansion 129 After-run 129, 130, 148 Aldehydes 123 Aldicarb 121 Alkane 244 Alkyl 143 Alkyl-ethoxy carboxylate 28 Allyldiisopropylamineoxide 123 Alternate -fluids 206 - supercritical fluids 207 Amines 13 Analytical figures of merit 22 Analytical performance 182 Anisole 123 Annularplasma 175 Anti-dandruff shampoos 38 Antioxidants 107, 110 Arachlor 19 Aroma 143 Aromatic isocyanates 113 Arrival time 9 Aryl 143 Atmospheric pressure electrophoretic separation 9 Atomic emission line 181 Background 143 Band broadening 106 Barbiturates 116,310 Beam dimension 106 Beenakker cavity 174 Benz[a]anthracene 119 Benzene modifier 206 Benzonitrile 123 Binary fluid of C02 and methanol 49 Binary fluid of carbon dioxide and an organic solvent 47 Bio-Rad Tracer FTIR system 307 Biphenyl polysiloxane column 121 Boron-selective detection 189 Broadening peak 133, 134 Bromonaphthalene 3 1 1 Butter 113 Butylphthalates 130, 132 Caffeine 106, 310 Capillary SFC-MEJ 179 Captan 121 Carbamate 121 Carbonyl 145, 146

320 Cell volume 106, 121 Charge 13 Chemical warfare (CW) agent 287 Chester restrictor 3 , 4 Chlorinated pesticides 121 Chloroform 135 Cholesteral 19 Cholesterol 3 13 Chrysanthemum cinerarioefolium 121 Chrysene 119 C-H stretching absorption 121 Cinerin I 121 CinerinII 121 Circulating 131, 136 Class-selectiveextraction 244,245 "closed" design 2 Cluster reactions 13 C02 charge exchange 31 Coconut oil 113 Coefficients, absorption 141 Co-eluted component 138, 141 Cold trapping 282-284 Collection 131, 135, 136 Collision-induced dissociation 32 Column pressure 160 Comparison with other selective detectors 190 Composition programming of binary fluid 49 Condensation alcohol (C3o-C38) 34 Condensed-phase library 104, 106 Contour plot 129, 136-138 Cool on column injector 261 Cooled trapping device 53 Corona ionization 13 Cortiocosterone 116 Cry0 focusing 218 Cryogenic collection 216-218 Cryogenic cooling trap 2 15,2 16 Cryogenic focusing 264 Cryogenic trapping 203 Cryolect GC-mamx isolation 307 Cryopumping 27 CW agents 288,289 Cyanopropyl-phenyl-methylpolysiloxane116 Deactivation of sites 91 Decafluorotriphenylphosphate 30 Decomposition products 123 Density 129, 130, 141, 148 Density profile of the binary fluid 52 Detected volume 5 Detection 129, 130, 141, 148 Detection Limit 8,66, 106, 188 Detector-cell broadening 5 Detector "spiking" 177

321 Dibutylphthalate 130, 132 Diglycerides 147, 148 Diffuse 130, 135, 143 Diffuse restrictor 85 Diffusion 15 Diffusion during SFE 240 Diffusivity 133 Dimethylpolysiloxane 135 Dimethylsiloxaneoligomers 107 Di-n-propyl adipate 119 Diphenyl 135 Direct fluid introduction interface 27 Direct injection system 259 Direct liquid interface 308 Directly coupled SFE/GC 255 Direct on-line coupling of SFC to FAB/MS 47 Discrimination 269 Dispersive 308 Dodecane 123 Double-base propellant extracts 119 Drift time windows 16 Dual supply pump system 214 Dynamic range 8 Dynamic SFE 229,279,301 ECD-like response 18 Ecdysone 116 EI - GC-MS 310 -inSFC-MS 29 - SFC-FTMS 310 -SFC-MS 315 -spectra 315 Electronegative compounds 18 Electronic broadening 5 Element selective detectors 151 ELSD, Varex Marx 2A 53 Erucamide 110 Essential oils 113 Eucalyptus leaves 199 Extra-column 133 Extraction 136 Extraction vessel 199,201,280,281 Extracts 136 Fast Atom Bombardment Mass Spectrometer 49 Fat 245 Fat-soluble 148 Fatty acids 113 Fatty alcohol acid esters 36 Fermi resonance 18 FID 135, 146, 147 FID-like response 18 Figures of Merit 6

322 Fjeldsted restrictor 3 Flavours 113 Flow cell 65, 83,84, 103, 106, 123 - SFC-FTIR 116 Flow restrictor 106 Fluid 129, 131, 133, 135, 149 Fluid materials 257 Foods 116 Fossil fuel 116 Fourier transform infrared 65 Fourier transform ion mobility spectrometry 9 Fractionation 129, 130, 135, 137, 138, 141, 148 Free fatty acids 79 Freon-22 287 Freons 17 Frequency 11 Frit restrictor 113 FTIR microscope 107 Fuel contaminated sediment 199 Gas phase diffusion coefficient 9 Gas phase mobility 9 Gate functions 12 Gating correlation function 12 Gaussian 133, 141 GC --FTIR 306 - -1CP 169 --IR 306 - -1R-MS 306,307 - -like detectors 171 --MPI-FTMS 315 --MS 305 - sample introduction means 199 Gel 132,135 Gennacrene 113 Germ, wheat 134,136, 137, 146,148 GLC 146,147 Glyme column 113 Glyme stationary phase 110 Gold coated light pipes 306 Gram-Schmidt reconstruction 67,94-96, 103 Grape juice 113 Guthrie resmctor 3 , 4 Halogenated compounds 14 Heated coil 177 Heated flow cell 306 Heated interface 176 He-CO2 plasma 176 Helium MIP 174 Herbicides 121,247 Hit quality index 93

323 HF'LC 129, 132,134,135 --FTIR 103 --ICP 151 - - M S 308 Hydraulic 131, 135, 136 Hydrocarbons 116,218 12-Hydroxystearicacid 110 Hyphenated 129 Hyphenated analysis systems 305 Hyphenated systems 305 Hyphenated use of ELSD 47 Hyphenation 53, 129, 130 ICP nebulizer 153 Identification 143,146 Impurity 141, 145,147, 148 Indoles 123 Inductively coupled plasma (ICP) 151 Infrared interferometry 306 Infrared spectrum 83,84 In situ derivatization 247 Integral restrictor 181 Integrated absorbance 95-97 Interface mechanics 212 Interferogram 11 Ion detectors 2 Ion gates 11 Ionization 135 Ionization from 63Ni 13 Ion mobility spectrum 12 Ion-molecule reactions 13, 14 Ion trap detector 307 Ion trap mass analyzers 306 IR-compatible mobile phase 104 Irgacure 907 110 Irganox 1010 110 IR transparent support 104 Isolation FTIR 307 Isomer differentiation 119 Isomers 130, 132, 148 Jasmolin I 121 Jasmolin I1 121 .Jet separators 313 Joule-Thompson effect 153, 154 KBr 135,144 Ketones 123 Kinetic energy 153, 154

Lamp black 119 LC 129 LC-FAB/MS frit interface 59 LC/GC 275 Lightpipe 307

324 Lightpipe dead volume 307 Light scatteringdetector 53 Light stabilizers 107 Linear alkyl benzene sulphonate 232,248 Linear dynamic range 160 Linearity and precision 190 Lipids 113, 137 Longitudinal diffusion 5 Loop 131, 135 Low density 129 Low pressure 129 Lowest detectable concentration 160 MAGIC 308 Make-up fluid 110 Mass resolutions 3 11 Mass selective detectors 306 Mass sensitive 160 - detector 156 Matrices 148 Matrix effects 258 - on SFE 238 Matrix isolation 307 Mercury cadmium telluride detectors 87 Metal selective monitoring 161 Methanol modifier 206 Methomyl 121 Methyl phenyl siloxane 107 Microcolumn SFC-ICP 169 Microscope 85, 88 Microwave induced plasma (MIP) 151, 174 Migration 135 Mist 148 Modifier 16,130-137,142,148,204,206 Modifiers on SFE 232 Molar selectivity 175 Molecular sieve absorbent 208 Mono disperse aerosol generation interface 308 Monosaccharideby-product 116 Moving belt interface 308 MS chromatogram 60 Multidimensional techniques 275 Multi-element detection capability 173 Multi vessel SFE 208-211 Munitions 119 Naphthalene 31 1 Natural products 113 Near-infrared spectral region 181 Nebulizer 153, 154, 160 - Ar gas flow rate 155 - gas flow rare 156, 158, 160 Negative ammonia chemical ionization 55 Negative NH4Cl 27

325 Nitroaniline 119 Nitrobenzene 123 2-Nitrodiphenylamine 119 Nitroglycerine 119 Nitrosoanilines 119 Nitrous oxide 232 Non-destructive detection 179 Non-selective detection 18 No spray-chamber 153 octadecanol 34 - Reaction-Mixture Residue 34 Octylphthalate 132, 133 Off-line - and on-line SFE comparison 227 - measurements 88,95 -SEE 226 -SFE/SFC 301 Oligomers 110 On-column SFE/GC 201 On-line - FTIR detection 121 - interfacing 197 - measurements 88,93,98 - SFC-FTIR 113 - SFC/GC vs syringe GC 199 -SFE 302 -SFE/GC 258 - SFE/GC interface 256 -SFE/SFC 212 Open cell detectors 3 Open tubular column 95 Optical detectors 2 Optimization of the system 180 Optimum nebulization temperature 55 Orange juice 208 Oxygenated and nitro-containingcompounds 14 PAC 119 Packed column SFC 66,91-93, 113, 116, 121, 160, 179 PAH 238,244,315 Parallel split 307 PCBs 238 PDA 129,148 Pesticides 19, 71, 121,245, 310 Phase changes 2 Phenmedipham 121 Phenolic acids 1 13 Phenols 123 Phosphates 247 Phospholipids 247 Photodiode array 129,131, 135,136, 141 Photoinitiators 110 Photoionization 13

326 Phthalates 132 Pictorial SFC/GC 202 Plasma detectors 173 Plasticisers 107 Plugging 286 - problems 282 Polyammatics 3 10 Polycyclic aromatic compounds 116 Polycyclic aromatic hydrocarbons 119 Polyethylene 219,220 Poly(ethy1ene glycol) 29 Polymer additives 90,9 1, 107, 110, 2 19,220, 243 Polymeric compounds 19 Polynuclear aromatic hydrocarbons 204,205,209,210 Polyphenyls 119 Polypropylene 107 Poor IR absorber 106 Positive EI 27 Positive electron ionization 39 Positive Methane CI 27 Post-run signal averaging 88 Practical use of modifiers 204 Pre-heat 131, 136 Preparative 129 Prep-LC 129, 130 Prep-SFC 129-136,148 Pressure programming 161 Production 13 - region 16 Progesterone 116 Propellants 119 Propylene carbonate modifier 206 Prospects for the future 191 Proton affinities 13 - transfer 13 Pyrene 315 Pyrethrin 121 - I 121 - I1 121 Quadruple mass spectrometers 305 Quartznozzle 178 Quinones 94-98, 123, 149 Radio-frequencyplasma detector (WD)173 Reactive diluent 110 Reactive oligomers 110 Recirculating 131 Recoveries 148 Recycling 131-133,135,137, 138 Reduced mobility constant 10 Re-mixing 135 Resolution 132, 148 Response 147

327 Restrictor 104,228, 309 Richter restrictor 3, 4 Root-mean square noise 8 Sample gas flow rate 158 Sample introduction 176 Sample introduction system 258 Sample preparation 256 Selective extraction 256 Selective response 14 Selectivity enhancements 204 Selenium Sulfides 38 Semi-micro packed column 47 Sensitivity 8,85,86,88, 89,98, 188 - Of SFC/FTIR 106 Sequential 208,209,211 Sesquiterpene hydrocarbons 113 SF6 206,207 SFC --FAB/MS 62 - -FID 26.107 - -FTIR 103,104 -/FI’IR 123 -/FT-IR 83-99 - -FTMS 309,310,312,313,315 --GC 130 - -1CP 152, 153, 155, 160-162, 169 - -MS 26,308,313 --LJV 167 --UV-ICP 167 SFE - analyte trapping 235 - applications 227 -cells 230 - collection methods 228 - collection solvents 236 - fluids 231 - -GC 275,290-295,302 - /GC schematic 200 - instrumentation 228 - introduction 197 -kinetics 239 - mechanisms 234 - of water samples 246 -pumps 230 - rate controlling factors 234 - recoveries 233 -scheme 198 - /SFC 213-218,275,295,299,302 - -SFC-FTIR 119 Signal-to-noise ratio 85, 86 Silica discharge tube 174 Silicons 94,95 Single pump system 212, 213

328 Slip agents 107 Smith restrictor 3, 4 Soap oil 113 Solubility effects on SFE 236 Solubility parameter 276,277 Solute focusing 202 Solute solubility 276, 277 Solvent elimination 107, 116, 123 - interface 107 - methods 104 -SFC/FTIR 106 Solvent trapping 282 Sorbent trapping 282 Soybean oil 113 Space-chargeeffects 3 11 Spectral searching 84,92 Spiking 154 Split SFE/GC 201 Split/splitless injector 262 Stagnant-volumebroadening 5 Static SFE 229,279,296 Stationary phases 116 Step-wise pressure programming 161 Steroids 71, 116 Styrene 107 Subnanogram detection level 107 Substituted aromatics 310 Substrate translation speed 89 Sugar derivatives 116 Sulfanilamides 91,93 Sulfer 182 Sulfer-containingpolyaromatics 182 Sulfide 182 SupercriticalC02 119 - fluid extraction 119,225 SupercriticaIfluid extraction (SFE) 255 Surfatron 134 Temperature gradient elution 54 Temperature programming 49 Testosterone 116 Thermal decomposition 39 Thermally degradable compounds I2 I Thermodesorptiodcold-trapinjectror 263 Three domensional 129, 130, 148 Time domain 12 Time of flight mass spectrometers 215 Total ion current 12 Transfer 13 Transfer-line broadening 5 Triacetin 119 2,4,5-Trichlorophenylaceticacid 121 Triglycerides 113, 16, 137, 138, 146

329 Undirectional flow design 11 Universal detection 186 Ultrasound 287 UV absorbers 107 UV-curing coating 110 UV-ELSD 54,57 UV-FAB/MS hyphenation system 60 UV FAB/MS system 57 UV labeled compounds 56 UV multichannel detector 163 Vanillic acid 113 Variation of modifier identity 206 Void volume 200 Waxextract 121 Wheat germ 134,136,137,146,148 Whisky 113 Xenon 77,110,119 Yiang-yiang oil 113

This Page Intentionally Left Blank

331

JOURNAL OF CHROMATOGRAPHY LIBRARY A Series of Books Devoted to Chromatographic and Electrophoretic Techniques and their Applications Although complementary to the Journal ofChromatography, each volume i n the Library Series is a n important and independent contribution in the field of chromatography and electrophoresis. The Library contains no material reprinted from the journal itself.

Other volumes in this series Volume 1

Chromatography of Antibiotics (see alsoVolume 26) by G.H. Wagman andM.J. Weinstein

Volume 2

Extraction Chromatography edited by T. Braun and G. Ghersini

Volume 3

Liquid Column Chromatography.A Survey of ModernTechniques and Applications edited by Z. Deyl, K. Macek and J . Janhk

Volume 4

Detectors in Gas Chromatography by J. SevEik

Volume 5

Instrumental Liquid Chromatography.A Practical Manual on HighPerformance Liquid Chromatohraphic Methods fsee alsoVolume 27) by N.A. Parris

Volume 6

Isotachophoresis. Theory, Instrumentation and Applications by F.M. Everaerts, J.L. Beckers andTh.P.E.M. Verheggen

Volume 7

Chemical Derivatization in Liquid Chromatography by J.F. Lawrence and R.W. Frei

Volume 8

Chromatography of Steroids by E. Heftmann

Volume 9

HPTLC -High Performance Thin-LayerChromatography edited by A. Zlatkis andR.E. Kaiser

Volume 10

Gas Chromatographyof Polymers by V.G. Berezkin, V.R. Alishoyev and I.B. Nemirovskaya

Volume 11

Liquid ChromatographyDetectors fsee also Volume 33) by R.P.W. Scott

Volume 12

Affinity Chromatography by J.Turkova

Volume 13

Instrumentation for High-Performance Liquid Chromatography edited by J.F.K. Huber

Volume 14

Radiochromatography.The Chromatography and Electrophoresis of Radiolabelled Compounds byT.R. Roberts

Volume 15

Antibiotics. Isolation, Separation and Purification edited by M.J. Weinstein and G.H. Wagman

332 Volume 16

Porous Silica. Its Properties and Use as Support in Column Liquid Chromatography by K.K. Unger

Volume 17

75 Years of Chromatography A Historical Dialogue

-

edited by L.S. Ettre and A. Zlatkis Volume 18A

Electrophoresis. A Survey of Techniques and Applications. Part A Techniques edited by 2. Deyl

Volume 18B

Electrophoresis. A Survey of Techniques and Applications. Part B: Applications edited by 2. Deyl

Volume 19

Chemical Derivatization in Gas Chromatography by J. Drozd

Volume 20

Electron Capture.Theory and Practice in Chromatography edited by A. Zlatkis and C.F. Poole

Volume 21

Environmental Problem Solving using Gas and Liquid Chromatography by R.L. Grob and M.A. Kaiser

Volume 22A

Chromatography. Fundamentals and Applications of Chromatographic and Electrophoretic Methods. Part A Fundamentals andTechniques edited by E. Heftmann

Volume 22B

Chromatography. Fundamentals and Applications of Chromatographic and ElectrophoreticMethods.Part B: Applications edited by E. Heftmann

Volume 23A

Chromatography of Alkaloids. Part A: Thin-Layer Chromatography by A. Baerheim Svendsen and R. Verpoorte

Volume 23B

Chromatography of Alkaloids. Part B: Gas-Liquid Chromatography and High-Performance Liquid Chromatography by R. Verpoorte and A. Baerheim Svendsen

Volume 24

Chemical Methods in Gas Chromatography byV.G. Berezkin

Volume 25

Modern Liquid Chromatography of Macromolecules byB.G. Belenkii and L.Z. Vilenchik

Volume 26

Chromatography of Antibiotics. Second, Completely Revised Edition by G.H. Wagman andM.J. Weinstein

Volume 27

Instrumental Liquid Chromatography. A Practical Manual on HighPerformane Liquid ChromatographicMethods. Second, Completely Revised Edition by N.A. Parris

Volume 28

Microcolumn High-Performance Liquid Chromatography by P. Kucera

Volume 29

Quantitative Column Liquid Chromatography. A Survey of ChemometricMethods

333 Volume 30

Microcolumn Separations. Columns, Instrumentation and Ancillary Techniques edited by M.V. Novotny and D. Ishii

Volume 31

Gradient Elution in Column Liquid Chromatography.Theory and Practice by P. Jandera and J. ChuraEek

Volume 32

The Scienceof Chromatography. Lectures Presented at t h e A.J.P. Martin Honorary Symposium, Urbino, May 27-31,1985 edited by F. Bruner

Volume 33

Liquid Chromatography Detectors. Second,Completely Revised Edition by R.P.W. Scott

Volume 34

Polymer Characterization by Liquid Chromatography by G. Glockner

Volume 35

Optimizationof Chromatographic Selectivity.A Guide t o Method Development by P. J. Schoenmakers

Volume 36

Selective Gas Chromatographic Detectors by M. Dressler

Volume 37

Chromatography of Lipids in Biomedical Research and Clinical Diagnosis edited by A. Kuksis

Volume 38

Preparative Liquid Chromatography edited by B.A. Bidlingmeyer

Volume 39A

Selective Sample Handling and Detection in High-Performance Liquid Chromatography. Part A edited by R.W. Frei and K. Zech

Volume 39B

Selective Sample Handling and Detection in High-Performance Liquid Chromatography. Part B edited by K. Zech and R.W. Frei

Volume 40

Aqueous Size-ExclusionChromatography edited by P.L. Dubin

Volume 41A

High-Performance Liquid Chromatography of Biopolymers and Bio-oligomers.Part A: Principles, Materials andTechniques by 0.MikeS

Volume 41B

High-Performance Liquid Chromatography of Biopolymers and Bio-oligomers.Part B: Separation of Individual Compound Classes by 0.Mike6

Volume 42

Quantitative Gas Chromatography for Laboratory Analyses and On-LineProcess Control by G. Guiochon and C.L. Guillemin

Volume 43

Natural Products Isolation. Separation Methods for Antimicrobials, Antivirals and Enzyme Inhibitors edited by G.H. Wagman and R. Cooper

Volume 44

Analytical Artifacts. GC, MS, HPLC,TLC and PC byB.S. Middleditch

334 Volume 45A

Chromatography and Modification of Nucleosides. Part A: Analytical Methods for Major and Modified Nucleosides - HPLC, GC, MS, NMR, UV and FT-IR edited by C.W. Gehrke and K.C.T. Kuo

Volume 45B

Chromatography and Modification of Nucleosides. Part B: Biological Roles and Function of Modification edited by C.W. Gehrke and K.C.T. Kuo

Volume 45C

Chromatography and Modification of Nucleosides. Part C: Modified Nucleosides in Cancer and Normal Metabolism -Methods and Applications edited by C.W. Gehrke and K.C.T. Kuo

Volume 46

Ion Chromatography. Principles and Applications by P.R. Haddad and P.E. Jackson

Volume 47

Trace Metal Analysis and Speciation edited by1.S. Krull

Volume 48

Stationary Phases in Gas Chromatography by H. Rotzsche

Volume 49

Gas Chromatography in Air Pollution Analysis by V.G. Berezkin and Yu. S. Drugov

Volume 50

Liquid Chromatography in Biomedical Analysis edited by T. Hanai

Volume 51A

Chromatography, 5th edition. Fundamentals and Applications of Chromatography and Related Differential Migration Methods. Part A Fundamentals andTechniques edited by E. Heftmann

Volume 51B

Chromatography, 5th edition. Fundamentals and Applications of Chromatography and Related Differential Migration Methods. Part B: Applications edited by E. Heftmann

Volume 52

Capillary Electrophoresis. Principles, Practice and Applications by S.F.Y. Li

Volume 53

Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction edited by K. Jinno