JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 48
Stationary Phases in Gas Chromatography
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JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 48
Stationary Phases in Gas Chromatography
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JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 48
Stationary Phases in Gas Chromatography Harald Rotzsche HULS Aktiengesellschaft, Zentralbereich FE, Marl, F. R. G.
ELSEVIER Amsterdam - Oxford - New York
- Tokyo
1991
This book is distributed in all non-socialist countries by ELSEVIER SCIENCE PUBLISHERS B.V. Sara Burgerhartstraat 25 P. 0. Box 211, loo0 AE Amsterdam, The Netherlands
Distributors for the United States and Canada: ELSEVIER SCIENCE PUBLiSHlNG COMPANY INC. 655 Avenue of the Americas New York, NY 10010, U.S.A.
Library of Congress Cataloging-In-Publlcatlon Data Rotzsche, Harald. Stationary phases in gas chromatography / Harald Rotzsche. 424 p. 16,5 x 24,O cm. chromatography library ; v. 48) Includes bibliographical references and index. ISBN 0-444-98733-9 1. Gas chromatography. 2. Stationary phase (Chromatography) I. Title. II. Series. QD79.C45R673 1990 543’.0896-d~20
- (Journal of
90-3962 CiP
ISBN 0-444-98733-9(Vol. 48) ISBN 0-444-41616-1(Series)
@ Akademische Verlagsgesellschaft Geest Cr Portig K.-G., Leipzig, 1991 Licensed edition for Elsevier Science Publishers B.V., 1991 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, without the prior written permission of the copyright owner. Printed in Germany
Contents
. . . . . . . . . . . . . . . . . . . . . . . .
1
. . . . . . . . . . . . . . . . . . . . . . .
2
1.
Introduction
2.
Basic Concepts
2.1.
Basic Components of a Gas Chromatographic System
2.2. 2.3. 2.3.1. 2.3.2. 2.3.3. 2.3.4. 2.3.5. 2.4. 2.5. 2.6. 2.7. 2.8.
Raw Data Measured from the Chromatogram Derived Basic Chromatographic Parameters . . . . . . . . . . . Retention Volume Terms . . . . . . . . . . . . . . . . . . Distribution Terms . . . . . . . . . . . . . . . . . . . . Temperature Dependence of Distribution and Retention Terms . . . . Parameters Characterizing the Emciency of Columns . . . . . . . Parameters Characterizing the Separation . . . . . . . . . . . Flow of Gases in a Gas Chromatographic Column and Formation of Bands Thermodynamic Bases of Gas Chromatography . . . . . . . . . The Quality of Chromatographic Separation . . . . . . . . . . . The Time of Analysis . . . . . . . . . . . . . . . . . . . Definition of Symbols Used (Table 1) and List of Essential Relationships (Table 2) . . . . . . . . . . . . . . . . . .
3. 3.1. 3.1.1. 3.1.2. 3.1.3. 3.1.4. 3.1.5. 3.1.6. 3.2. 3.3. 3.3.1. 3.3.2. 3.3.3. 3.3.3.1.
. . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . Packed Columns . . . . . . . . . . . . . . . Column Materials . . . . . . . . . . . . . . Column Dimensions . . . . . . . . . . . . . Preparing of the Packing and Packing Procedures . . . .
The Chromatographic Column
Column Conditioning . . . . . . . . . . . . Column Testing . . . . . . . . . . . . . . Pre-columns . . . . . . . . . . . . . . . Micro-Packed Columns . . . . . . . . . . . Open-Tubular Columns . . . . : . . . . . . Tube Material . . . . . . . . . . . . . . Column Dimensions . . . . . . . . . . . . Wall-Coated Open-Tubular (WCOT) Columns . . . . Properties and Pre-treatment of the Inner Tube Surfaces and the Formation of Films . . . . . . . . . . 3.3.3.2. Coating Procedures . . . . . . . . . . . . . 3.3.3.3. Bonding and Cross-Linking of Stationary Phases . . . 3.3.3.4. FilmThicknessoftheStationaryPhase . . . . . . 3.3.3.5. Quality Tests of WCOT Columns . . . . . . . . 3.3.4. Porous-Layer Open-Tubular (PLOT) and Support-Coated Open-Tubular (SCOT) Columns . . . . . . . . 3.3.4.1. PLOT Columns (strictly) . . . . . . . . . . . 3.3.4.2. SCOT Columns . . . . . . . . . . . . . . 3.3.4.3. Thick-Layer Columns . . . . . . . . . . . . Properties and Comparison of the Main Column Types . 3.4. 3.4.1. Packed Columns . . . . . . . . . . . . . . 3.4.2. Micropacked Columns . . . . . . . . . . . . 3.4.3. Support-coatedopen-tubularcolumns . . . . . . 3.4.4. Wall-coated Open-tubular Columns . . . . . . .
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3 4 4 7 9 10 15 17 21 28 33
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35
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2
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43 43 43 45 47 51 52 53 53 54 55 56 57
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57 63 64 71 73
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75 76 76 77 77 78 78 79 79
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VI 4. 4.1. 4.1.1. 4.1.2. 4.1.3. 4.1.4. 4.1.5. 4.2. 4.2.1. 4.2.2. 4.2.3. 4.2.4. 4.2.5. 4.2.6. 4.2.1. 4.2.8. 4.2.9. 5. 5.1. 5.1.1. 5.1.2. 5.5. 5.2.1. 5.2.2. 5.2.3. 5.3. 5.4. 5.4.1. 5.4.2. 5.4.3. 5.5. 5.5.1. 5.5.2. 5.6. 5.6.1. 5.6.2. 5.6.3. 5.6.4. 5.6.5. 5.6.6. 5.6.7. 5.7. 5.7.1. 5.1.2. 6
.
6.1. 6.2. 6.2.1. 6.2.2. 6.2.3.
Contents
. . . . . . Intermolecular Forces . . . . . . . . . . . . London-type Dispersion Forces . . . . . . . . Orientation Forces and Hydrogen Bridge Bonds . . Induction Forces . . . . . . . . . . . . . Donor-Acceptor Interactions . . . . . . . . . Further Intermolecular Forces . . . . . . . . Quantities for the Description of Interactions . . . Characterization of Stationary Phases
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.
80
FurtherQuantitiesfortheDescriptionoftheInteractions . . . . . . . . .
80 80 81 83 83 83 84 84 84 88 89 94 98 99 100 101
Solid Stationary Phases (In part together with W.Engewald and J.P6rschmann)
103
Selectivity Coefficient . . . . . . . . . . . . . . . . . . . . . Retention Index . . . . . . . . . . . . . . . . . . . . . . . Polarity . . . . . . . . . . . . . . . . . . . . . . . . . . Rohrschneider and McReynolds Constants . . . . . . . . . . . . . Thermodynamic Criteria of Polarity . . . . . . . . . . . . . . . The Molecular Retention Index . . . . . . . . . . . . . . . . . The Selectivity Index . . . . . . . . . . . . . . . . . . . . . The Infrared Spectroscopic Frequency Shift . . . . . . . . . . . . .
. . . . . . . .
. Classification of Adsorbents . . . . . . . . . . Classification AccordingtoChemicalStructure . . . Classification According to Geometrical Structure . . Carbon Adsorbents . . . . . . . . . . . . . Graphitized Thermal Carbon Black (GTCB) . . . . . Activated Charcoal . . . . . . . . . . . . . Carbon Molecular Sieve . . . . . . . . . . . BoronNitrideandMolybdenumDisulphide . . . . . . . . Adsorbents with Hydroxylated and Dehydroxylated Surfaces . . . Silica Gels . . . . . . . . . . . . . . . . . . . . Aluminium Oxide . . . . . . . . . . . . . . . . . Porous Glasses . . . . . . . . . . . . . . . . . . Porous Organic Polymers . . . . . . . . . . . . . . . Porous Polyaromatic Beads without Additional Functional Groups . Polar Porous Polymers . . . . . . . . . . . . . . . . Substances Forming Inclusion Compounds . . . . . . . . . Zeolitic Molecular Sieves . . . . . . . . . . . . . . . Bentonites . . . . . . . . . . . . . . . . . . . . Tri-o-thimotide . . . . . . . . . . . . . . . . . . Desoxycholic Acid . . . . . . . . . . . . . . . . . Werner Complexes . . . . . . . . . . . . . . . . . Benzenesulphonates . . . . . . . . . . . . . . . . Urea and Thiourea . . . . . . . . . . . . . . . . . Modified Adsorbents . . . . . . . . . . . . . . . . Modification with High-boiling Liquid and Solid Substances . . . Modification with Non-porous Ionic Adsorbents . . . . . . .
. . . . . . Adsorbents for Bonding Reactions . . . . . . . . Bonding Reactions . . . . . . . . . . . . . . Chemically Bonded Phases with S i - 0 - C Bonds . . . Chemically Bonded Phases with Si-CH2RBonds . . . Chemically Bonded Stationary Phases
Chemically Bonded Phases with Si-0-Si
Bonds
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103 104 106 108 108 113 113 114 114 114 119 120 120 123 128 131 131 134 136 136 136 137 137 137 138 140 142 143 144 144 147 148
VII
Contents 6.3. 6.4.
Properties and Characterization of Chemically Bonded Phases Outlook and Prospects for Chemically Bonded Phases . .
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7.
The Solid Support
7.1. 7.2. 7.3. 1.4. 7.4.1. 1.4.2. 1.4.3. 7.4.4. 1.4.5. 7.5. 1.6. 1.1. 7.8. 1.9.
The Particle Size and Shape . . . . . . . . . The Surface Area . . . . . . . . . . . . Activity of the Original and of the Coated Solid Support Diatomite Supports . . . . . . . . . . . . Acid washing . . . . . . . . . . . . . . Alkali treatment . . . . . . . . . . . . . Silylation . . . . . . . . . . . . . . . Coating of the Support with a Non-extractable Layer . Deactivation of Diatomite by Deposited Pyrocarbon . Synthetic Silica-based Supports (Volaspher and Quartz) Silica Gel . . . . . . . . . . . . . . . Micro Glass Beads and Porous Layer Beads . . . . Fluorocarbon Supports . . . . . . . . . . Other Support Materials . . . . . . . . . .
8.
Liquid Stationary Phases
8.1. 8.1.1. 8.1.2. 8.1.3. 8.1.4. 8.1.5. 8.1.6. 8.1.7. 8.1.8. 8.1.9. 8.2. 8.2.1. 8.2.2. 8.3. 8.3.1. 8.3.2. 8.3.3. 8.3.4. 8.3.5. 8.3.6. 8.3.7. 8.3.8. 8.3.9. 8.3.10. 8.3.11. 8.3.12. 8.3.13. 8.4. 8.4.1. 8.4.2. 8.4.3. 8.4.4. 8.4.4.1. 8.4.4.2. 8.4.4.3.
General Properties of Liquid Stationary Phases . . . . . . . . . . . . Chemical Inertness . . . . . . . . . . . . . . . . . . . . . . Vapour Pressure. Thermal Stability and Maximum Operation Temperature . . Molecular Weight . . . . . . . . . . . . . . . . . . . . . . Viscosity and Minimum Operating Temperatures . . . . . . . . . . . Film Formation . . . . . . . . . . . . . . . . . . . . . . . Solubilizing Power . . . . . . . . . . . . . . . . . . . . . . Purity and Homogeneity . . . . . . . . . . . . . . . . . . . . Selectivity . . . . . . . . . . . . . . . . . . . . . . . . . Specifications of Stationary Phases . . . . . . . . . . . . . . . Hydrocarbons . . . . . . . . . . . . . . . . . . . . . . . . Aliphatic Hydrocarbons . . . . . . . . . . . . . . . . . . . . Aromatic Hydrocarbons . . . . . . . . . . . . . . . . . . . . Silicones . . . . . . . . . . . . . . . . . . . . . . . . . Poly(dimethylsi1oxanes) . . . . . . . . . . . . . . . . . . . . Poly(dimethylsi1oxanes)containing Small Amounts of Vinyl Groups . . . . Poly(dialkylsi1oxanes) . . . . . . . . . . . . . . . . . . . . .
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. . . . Poly(chloroary1siloxanes)and Poly(chloralkylsi1oxanes) . Polyester Silicones . . . . . . . . . . . . . . Chiral Polysiloxanes . . . . . . . . . . . . . Mesogenic Polysiloxanes . . . . . . . . . . . .
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Aminoalkyl-Substituted Polysiloxanes . . . . Other Siloxane Stationary Phases . . . . . . Alcohols. Ethers and Carbohydrates . . . . . Monohydric Alcohols . . . . . . . . . . . Polyhydric Alcohols . . . . . . . . . . . Saccharides . . . . . . . . . . . . . . Polyglycols and Poly(alky1ene oxides) . . . . . Poly(ethy1eneoxides) [Poly(ethyleneglycols)] . . Other Poly(alky1ene oxides) . . . . . . . . Poly(oxyalky1ene) Derivatives . . . . . . .
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160 160 161 164 167 171 172 172 177 177 171 180 181 182 184 186
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Carborane Siloxanes
155 158
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186 186 186 188 189 190 193 194 195 195 196 196 200 201 207 214 215 216 224 227 229 233 236 236 240 242 242 246 246 246 248 249 251 255 256
VIII 8.4.5. 8.5. 8.5.1. 8.5.2. 8.5.3. 8.5.4. 8.5.5. 8.6. 8.7. 8.8. 8.8.1. 8.8.2. 8.9. 8.10. 8.11. 8.12. 8.13. 8.14. 8.14.1. 8.14.2. 8.14.3. 8.14.4. 8.14.5. 8.15. 8.15.1. 8.15.2. 8.15.3. 8.15.4. 8.15.5. 8.16. 8.17.
Contents Aromatic Ethers . . . . . . . . . . . . . . . . . . . Esters . . . . . . . . . . . . . . . . . . . . . . . Esters of Dicarboxylic Acids (Phthalates. Adipates and Sebacates) . . Phosphates . . . . . . . . . . . . . . . . . . . . Stearates. Oleates . . . . . . . . . . . . . . . . . . Acetates. Citrates . . . . . . . . . . . . . . . . . . Polyesters . . . . . . . . . . . . . . . . . . . . . Nitriles and Nitrile Ethers . . . . . . . . . . . . . . . Nitro Compounds . . . . . . . . . . . . . . . . . . Amines . . . . . . . . . . . . . . . . . . . . . . Aliphatic Amines and Imines . . . . . . . . . . . . . . Aromatic Amines . . . . . . . . . . . . . . . . . . Amides . . . . . . . . . . . . . . . . . . . . . . Heterocyclics . . . . . . . . . . . . . . . . . . . . Sulphur Compounds . . . . . . . . . . . . . . . . . Fluorine Compounds . . . . . . . . . . . . . . . . . Fatty Acids and their Salts . . . . . . . . . . . . . . . salts . . . . . . . . . . . . . . . . . . . . . . . Silver Nitrate . . . . . . . . . . . . . . . . . . . . Dissolved Alkali Metal Halides . . . . . . . . . . . . . . Eutectics . . . . . . . . . . . . . . . . . . . . . Molten Organic Salts . . . . . . . . . . . . . . . . . Complexes of Some Transition Metals with N-Duodecylsalicylaldimine and Methyl n-octylglyoxime . . . . . . . . . . . . . . . Chiral Stationary Phases . . . . . . . . . . . . . . . . Peptide Stationary Phases . . . . . . . . . . . . . . . . Diamide Stationary Phases . . . . . . . . . . . . . . . Ureide Stationary Phases . . . . . . . . . . . . . . . . Metal Complexes . . . . . . . . . . . . . . . . . . Recommendable Chiral Stationary Phases and Some Applications . . Liquid Crystals . . . . . . . . . . . . . . . . . . . Mixed Stationary Phases . . . . . . . . . . . . . . . .
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259 261 262 265 266 261 261 210 214 215 215 211 211 218 219 280 282 283 283 283 284 285 286 286 281 288 288 289 289 291 296
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302
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302
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303 308 311
9.
Selection of Stationary Phases
9.1. 9.2.
General Recommendations for Choosing a Suitable Stationary Phase Choosing Stationary Phases for Special Separation Problems with Regard to the Desired Selectivity . . . . . . . . . . . Preferred Stationary Phases . . . . . . . . . . . . . . Approaches to Stationary Phase Selection . . . . . . . . .
9.3. 9.4.
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352
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319
General Subject Index
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393
Stationary Phase Index
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401
Literature Author Index
JOURNAL OF CHROMATOGRAPHY LIBRARY A Series of Books Devoted to Chromatographic and Electrophoretic Techniques and their Applications Although complementary to the Journal of Chromatography, each volume in the Library Series is an 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 Volume 2 Volume 3 Volume 4 Volume 5 Volume 6 Volume 7 Volume 8 Volume 9 Volume 10 Volume 11 Volume 12 Volume 13 Volume 14 Volume 15 Volume 16 Volume 17
Chromatography of Antibiotics (see also Volume 26) by G.H. Wagman and M.J. Weinstein Extraction Chromatography edited by T. Braun and G. Ghersini Liquid Column Chromatography. A Survey of Modern Techniques and Applications edited by Z. Deyl, K. Macek and J. Janhk Detectors in Gas Chromatography by J. SevEik Instrumental Liquid Chromatography. A Practical Manual on High-Performance Liquid Chromatographic Methods (see also Volume 27) by N.A. Paffls Isotachophoresis. Theory, Instrumentation and Applications by F.M. Everaerts, J.L. Beckers and Th.P.E.M. Verheggen Chemical Derivatization in Liquid Chromatography by J.F. Lawrence and R.W. Frei Chromatography of Steroids by E. Heftmann HPTLC- High Performance Thin-Layer Chromatography edited by A. Zlatkis and R.E. Kaiser Gas Chromatography of Polymers by V.G. Berezkin, V.R. Alishoyev and I.B. Nemirovskaya Liquid Chromatography Detectors (see also Volume 33) by R. P. W. Scott Affinity Chromatography by J. Turkovh Instrumentation for High-Performance Liquid Chromatography edited by J.F.K. Huber Radiochromatography. The Chromatography and Electrophoresis of Radiolabelled Compounds by T. R.Roberts Antibiotics. Isolation, Separation and Purification edited by M.J. Weinstein and G.H. Wagman Porous Silica. Its Properties and Use as Support in Column Liquid Chromatography by K. K. Unger 75 Years of Chromatography-A Historical Dialogue edited by L. S. Ettre and A. Zlatkis
X
Journal of Chromatography Library
Volume 18A Electrophoresis. A Survey of Techniques and Applications. Part A: Techniques edited by Z. Deyl Volume 18B Electrophoresis. A Survey of Techniques and Applications. Part B: Applications edited by Z. 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 edited by E.Heftmann Volume 22B Chromatography. Fundamentals and Applications of Chromatographic and Electrophoretic Methods. 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 by V.G. Berezkin Volume 25 Modem Liquid Chromatography of Macromolecules by B.G. Belenkii and L.Z. Vilenchik Volume 26 Chromatography of Antibiotics Second, Completely Revised Edition by G.H. Wagman and M.J. Weinstein Volume 27 Instrumental Liquid Chromatography. A Practical Manual on High-Performance Liquid Chromatographic Methods 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 Chemometric Methods by S.T.Balke 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. ChurlEek
Journal of Chromatography Library
Volume 32 Volume 33
Volume 34 Volume 35
Volume 36 Volume 37
Volume 38
XI
The Science of Chromatography. Lectures Presented at the A. J. P. Martin Honorary Symposium, Urbino, May 27-31,1985 edited by F. Bruner Liquid Chromatography Detectors. Second, Completely Revised Edition by R.P.W. Scott Polymer Characterization by Liquid Chromatography by G. Glockner Optimization of Chromatographic Selectivity. A Guide to Method Development by P. J. Schoenmakers Selective Gas Chromatographic Detectors by M. Dressler Chromatography of Lipids in Biomedical Research and Clinical Diagnosis edited by A. Kuksis Preparative Liquid Chromatography edited by B.A. Bidlingmeyer
Volume 39A Selective Sample Handling and Detection in High-Performance Liquid Chromatography. Part A by R. W. Frei and K. Zech Volume 39B Selective Sample Handling and Detection in High-Performance Liquid Chromatography. Part B by K. Zech and R.W. Frei Volume 40
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Journal of Chromatography Library
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Stationary Phases in Gas Chromatography by H. Rotzsche
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Every chemist is regularly confronted with samples of which the composition, purity, or concentration of by-products or impurities should be determined, and he/she will have to find the most appropriate analytical method. For volatile or vaporizable constituents of the sample, gas chromatography will often be the method of choice. However, like with any other physical method, reliable results can only be obtained when sufficient knowledge of the potential and limitations of the technique is available. The analytical result is influenced primarily by the “heart of the gas chromatograph”, i.e., by the separation column and the stationary phase, and the analyst has to decide carefully which column type and stationary phase to select for the problem at hand. One of the aims of this book is to make the chemist familiar with the numerous stationary phases and column types, with their advantages and disadvantages, to help him/her select the most suitable phase for the type of analytes, and to give him/her detailed information on the chemical structure, physico-chemical behaviour, experimental applicability, physical data of liquid and solid stationary phases and solid supports, which otherwise could only be collected with difficulty from various treatises, handbooks, prospectuses and scientific papers. To understand the processes occurring in the separation column and to offer a manual both to the beginner (for whom a synopsis on practical and theoretical principles could be of help) and to the seasoned chromatographer (for whom it is convenient to have basic equations and physical correlations at hand), one chapter in this book is devoted to basic theoretical aspects. Further, as the effectiveness of the stationary phase can only be considered in relation to the column type, a chapter on different column types and the arrangement of the stationary phase within the column is included. Another aim of this book is to stimulate the development of new and improved standardized stationary phases and columns, in order to improve the reproducibility of separations, as well as the range of applications. This book is also intended for physical chemists, who do not normally deal with chromatography. They can find suggestions for and references to physico-chemical investigations by gas chromatography, particularly in the field of intermolecular interactions and surface studies. It is always risky to write a monograph on a rapidly expanding field-one only needs to think of the development of fused-silica columns and of cross-linked stationary phases and the rapidly growing amount of relevant literature during the last years. Therefore, I hesitated for quite some time to follow the suggestion made some 10 years ago by Professor J. F.K.Huber of the University of Vienna, whom I thank kindly for his proposal and confidence, to write a book on stationary phases in gas chromatography. On the other hand, this task seemed to be attractive, because in our institute and in our chemical plants we have had to deal with problems of stationary phases since our first gas chromatographic separations in 1957, as the samples to be analysed often comprised low- and very-high-boiling, reactive, even offensive, and/or instable compounds. Therefore, we had to develop stationary phases which would withstand such heavy strains and hence we gained quite a lot of experience in synthesizing, evaluating and applying stationary phases. This book is not intended to be a review but rather a working instrument. The literature has been critically evaluated and cited only to a limited extent (which is nevertheless quite comprehensive).
XIV
Preface
I tried to adhere to a systematic representation and nomenclature as far as possible; however, some deviation is intended to serve a better understanding. The order in which the stationary liquid phases are presented may appear to be arbitrary. Except for the hydrocarbons, which are discussed first because of their use as reference liquid phases, the order only indicates to some degree the importance and frequency of application of the phases. Many of the product names, registered designs and patents referred to in this book are in fact registered trademarks or proprietary names even though specific reference to this fact is not made in the text. Therefore, the appearence of a product name, to simplify matters usually without designation as proprietary, is not to be interpreted as a representation by the publisher and the author that it is in the public domain. Any book devoted to a specific scientific area is based on the theoretical and experimental efforts of not only the author, but also his teachers, colleagues and co-workers. I shall always gratefully remember Professor Richard Miiller and the late Professors K. Schwabe and A. Y. Kiseleu for their scientific style which I had the opportunity and pleasure to get acquainted with during the years of collaboration and which had a great influence on so many chemists. It is a pleasure for me to acknowledge the contributions of and helpful discussions with Professor W.Engewald, Dr.J.Porschmann and Dr. D. Glindemann. With gratitude I acknowledge the experimental assistance, in some cases lasting for decades, by my co-workers and colleagues in- and outside of my former department. I only mention here H. Hahnewald. W.Schwenke, J. Grasshog Dr. K. -D. Miiller, S. Langer, Dr. J. Schlapa and H. Steimann. In addition, the service of the technical information staff, especially of the scientific libraries of the Chemical Works Nunchritz and Pharmaceutical Works Dresden, and the diligent typing of the manuscript by the two secretaries, Mrs. K. Weisse and Mrs. K. Kurowski, is gratefully acknowledged. My particular appreciation is due to the publishers for their patience, for the linguistic revision and for the really pleasant cooperation. I wish to thank Huls AG for their support during the final phase of completing this book and Chemical Works Nunchritz for their consent to the publication. Finally, I would like to express particular thanks to my wife, who by her undertaking of many duties and by her patience and understanding made it possible to write this book. Essen, October 1989 Troisdorf, March 1991
HARALD ROTZSCHE
1.
Introduction
Hardly another field in analytical chemistry has devoloped during the past 30 years as rapidly as chromatography. The success of this method is due to its extraordinary performance with regard to separation efficiency and speed. Chromatography enables the analytical chemist to determine the composition of complicated mixtures consisting of individual compounds. At first sight, the principle is relatively simple. In a chromatographic column or bed, repeated transitions of the constituents to be separated take place between two phases, one of which is stationary, has a large surface and retains the compounds more or less, and the other is mobile and carries the compounds along the stationary phase. Provided that the kind or degree of interactions between the individual constituents and an appropriate stationary phase are different, they undergo different retardations in their transport with the mobile phase in a sequence of dynamic solution or/and adsorption equilibria. Although the entire chromatographic process involves sorption equilibration in addition to convective transport and diffusion, the separation is essentially determined by the first of these three terms, resulting in differences in the distribution ratios between both the phases. In gas chromatography, the mobile phase is a gas, thus having a high diffusion coefficient, and the interaction between molecules and the influence of the nature of usually applied gases, if any, on the distribution ratio is very low. This is the reason why selecting the most appropriate stationary phase is especially important in gas chromatography, whereas in liquid chromatography the distribution ratio, i.e., the separation, can be influenced by both the phases. Here the analytical chemist has a further variance: he can select a suitable stationary phase as well as an appropriate mobile phase, which may be varied between non-polar and strongly polar liquids and liquid mixtures. If we return to gas chromatography, the separation occurs in a column, and the sample components must be sufficiently volatile at the column temperature in order to be transported by the carrier gas, i.e., by the mobile phase. Owing to the retardation by the stationary phase, the individual components move with different velocities through the column, and separate bands develop, which are detected, characterized and measured at the column outlet. Depending on the type of column (packed column, packed capillary or microcapillary column, wall-coated open-tubular column) the stationary phase can be an adsorbent, a solid support impregnated with a liquid phase, a thin film of a liquid phase distributed on the inside wall of a capillary tube or a porous layer on the inside wall of the open tube, coated with a liquid phase. As the separability of mixtures is essentially controlled by sorption and desorption processes of its constituents in the interface of the mobile and stationary phases, numerous conditions, in addition to the temperature and the carrier gas flow-rate, have to be considered and optimized, e.g., the particle size and its distribution, the pore size and poresize distribution of the solid support, the type of adsorbent and liquid stationary phase, viscosity, film thickness and interaction forces of the liquid phase. Such practical aspects of stationary phases in gas chromatography will be emphasized in detail in this book.
2.
Basic Concepts
The reader of this book is assumed to be familiar with gas chromatography. Nevertheless, some basic principles, both theoretical and practical, are outlined in t h i s chapter and are intended to provide sufficient help for the beginner and to constitute a useful review for the advanced gas chromatographer. On the other hand, they are not considered to replace a gas chromatographic textbook, and much less are they to enable an excursion into complex gas chromatographic systems or theories. It seems useful, to start with a general definition of the topic. Beginning with chromatography as the general term, one can define it as “the mass transport through a two-phase system being selective for mass transfer. The mass transport is effected by the relative movement of the phases, one of which is compact and the other fluid.” [l]. Gas chromatography, as a special kind of chromatography, can be characterized as follows: “Gas chromatography, abbreviated to GC, comprises all chromatographic methods in which the moving phase is gaseous. The other phase is stationary and may be either a dry, granular solid or a liquid supported by the granules or by the wall of the column, or both. Separation is achieved by differences in the distribution of the components of a sample between the mobile and stationary phases, causing them to move through the column at different rates and from it at different times.”
PI.
The following sections of this chapter go into detail and contain the most important relationships applicable in gas chromatography.
2.1.
Basic Components of a Gas Chromatographic System
Most gas chromatographers have applied the elution method because of its advantages over other gas chromatographic techniques (e.g., frontal analysis, displacement analysis, vacant0 chromatography). Because of their minor importance, these special techniques are not discussed here. The elution technique, introduced into gas chromatography by Cremer [3], is characterized by a continuous flow of the mobile phase through the column, in which the separation of the components of a sample takes place. The individual constituents, together with the mobile phase, leave the column at different times and are detected by a sensitive detection system, which records their concentration in the mobile phase as a function of time, c = f ( t ) , elapsed between sample injection and emergence at the end of the column. The time corresponding to the concentration maximum of a component is its retention time. A diagram of a gas chromatograph is shown in Fig. 1. The essential part of any gas chromatograph is the column (C),which contains the stationary phase. The carrier gas (GIrepresents the mobile phase and is generally delivered from a gas cylinder containing the compressed gas. Constancy of the flow-rate is achieved by a flow controller (F),and the flowrate is measured by a flow meter. An injection system (I)preceding the column permits the sample to be directly introduced and vaporized in the gas stream. The column is followed by a detector ( D ) , in which the separated sample constituents produce electrical analogue signals. After passing an amplifier, in the case of ionization detectors, these signals are displayed on a strip-chart recorder (R),resulting in chromatograms showing individual peaks if the separation was successful. In modem and more expansive apparatus, the detectorlamplifier is connected via an analog-digital converter to a more or less sophisticated data
3
2.2. Raw Data Measured from the Chromatogram
system (9,processing two types of data: the already mentioned retention times, which are used for component identification, and the signal intensities (connected with the amounts of the sample constituents passing the detector), which are utilized, after detailed calibrations, for the determination of the concentrations of the components present in the original sample. Devices for temperature control, frequently with a temperature programmer, and measurement are symbolized by (0). R
r
Fig. 1. Diagram of gas chromatograph. G compressed gas cylinder containing the carrier gas; CG carrier gas cleanup; F flow controller, including flow meter; I injection system; C column; D detector, including supply and amplifrer; R recorder; S data system, including interface, integrator and computer; 0 column oven, including temperature control, measure and programmer
Raw Data Measured from the Chromatogram
2.2.
Although rather simplistic, it may be useful to specify the raw experimental data resulting directly from the gas chromatogram. Fig. 2 shows a one-component chromatogram (apart from the “air peak”) displayed on a common recorder, indicating a compound which was moni-
A
( h 4J L
0 +
Ln
0
Fig. 2. Gas chromatrogram showing the raw gas chromatographic data. t k = tR - tM adjusted retention time Symbol Description 0, start injection point, zero, origin, start y peak width at the inflection points tM mobile time wl peak width at half height tR retention time ?vb peak width at base
4
2. Basic Concepts
tored, after having passed the column, in the mobile phase by the detector. The first parameter usually measured from the chromatogram is the retention time, tR, which represents the time period between the instant of sample injection at (I) (Fig. 1) or Start (Fig. 2) and the emergence of the peak maximum (Fig.2) of a compound retained by the stationary phase. In addition to retention time, sometimes other terms are used for rR, e.g., total retention time of a solute and gross retention time. Unretained compounds, i.e., those not solved or adsorbed by the stationary phase, also need time to pass from (I)to (D). The same transit time through the column and the equipment is required by the mobile phase and it is therefore called the “mobile time“, tM. Other frequently used terms are gas hold-up time and air peak time (as air often represents an unretained substance). Obviously, the part of the retention time due to the residence of the retained compound in the stationary phase is
r;
= t,
- tM
(1) or, in other words, tk represents that portion of time resulting from interactions of the molecules of this compound with the stationary phase, and is characteristic of this compound. Thus, tk is the basis of almost all gas chromatographic calculations (except for the plate number). tk is called the “adjusted retention time”. As other expressions occasionally applied to t k , such as corrected retention time, net retention time and reduced retention time, could be misinterpreted and would confuse the gas chromatographer, only the terms tR = retention time, tM = mobile time, and t k = t R - tM = adjusted retention time
are used in this book. These retention times either can be measured on the recording in units of length and converted into minutes and seconds or the times themselves can be determined directly if a data system is available. The mobile time, being an important value, can be timed in the same way, taking the air peak as the basis, if the stationary phase does not retain air (this generally applies to liquid phases above or near room temperature but not necessarily to solid stationary phases) and if an appropriate detector, e.g., a thermal conductivity detector, is used. With a flame ionization detector methane is often used instead of air. One has to be aware, however, that the measured values are not correct at column temperatures below 50°C.Another possibility is to calculate the mobile time tM [4, 51. Further raw data obtainable from the chromatogram are the peak height, i.e. the distance between the baseline and the maximum peak height, and the peak width at various proportions of the maximum peak height. Three characteristic widths should to be mentioned here (Fig. 2): y = peak width at the inflection points, w, = peak width at half-height, we =peak width at the base (distance between the points of intersection on the baseline by the tangents to the curve through the inflection points).
2.3.
Derived Basic Chromatographic Parameters
2.3.1. Retention Volume Terms Retention Volume, V, The volume of carrier gas flowing through the column during the retention time, tained compound is
zR, of a re-
2.3. Derived Basic Chromatographic Parameters
5
VR = f R F c . (2) F,[mllmin] =volumetric flow rate at the column temperature T, [K] and the column outlet pressure po [MPa]. When measuring the flow rate (e.g. by means of a soap bubble flow meter, determination of the stream of carrier gas volume passing per time unit, stagnation dynamic pressure meter) we do not obtain F, for the column temperature, but Fa at ambient temperature at the column outlet. F. should therefore be corrected for at least the column temperature and, if water is present in the flow meter, for dry carrier gas conditions [5] to obtain F,: Tc Pa-Pw Fc= FaTa Pa
(3)
where column temperature [K], ambient temperature [K], pa = ambient pressure, h = partial pressure of water at ambient temperature (both pa and h are expressed in the same units). T, T,
= =
Mobile Volume, VM The carrier gas volume corresponding to the mobile time, tM, of a non-retained substance is
VM = fMFc.
(4)
Adjusted Retention Volume, VR The carrier gas volume corresponding to the adjusted retention time, t k , is
Vk = tkF,. As eqn. (1) rk = fR - t M , we can write Vk = ( f R - fM) FC= f R F c - tMFc. Substituting the appropriate expressions from eqns. (2) and (4), we obtain
vk= V R -
VM. (6) V i contains, in analogy to t k , only those contributions of retention resulting from interactions with the stationary phase. Hence it is the basis for gas chromatographic separation and identification calculations. Corrected Retention Volume, The carrier gas volume corresponding to the retention volume VR when corrected for gas compressibility is
V::= V R j (7) where j is the James and Martin factor 161: _ _ P2-1 and P=-. Pi j = -3 2 ~ 3 - 1 Po pi = inlet pressure; it shouldbe pointed out here that gauges often do not indicate the true inlet pressure but the difference in pressure from ambient. Therefore, the ambient pressure has to be added to the indication.
6
2. Basic Concepts
= ambient Dressure at the column outlet. (jvalues in dependence on pilpo can be taken from tabular compilations, e.g. in
-~n”
174).
The jfactors which apply to the generally used carrier gases (He, H2, N2, Ar) take into consideration that the carrier gas is compressible and makes it possible to obtain the average linear gas velocity, i,from the velocity measured at the column outlet. Net Retention Volume, VN This is the carrier gas volume corresponding to the adjusted retention volume when corrected for the gas compressibility. From eqns. (l), (5) and (6), it follows that V,
=
V i j = (V, - V d j = (tR - tM)j F,.
(8)
Corrected Mobile Volume, PM The corrected mobile volume or the corrected retention volume of a non-retained compound, according to eqns. (4)and (7), is given by p,=tMFcj=
VMj.
Specific Retention Volume,
(9)
V,
In order to obtain an absolute retention parameter, independent of the amount of the stationary phase, the specific retention volume is calculated from the net retention volume reduced to 273,16K and 0,1013MPa and referred to 1 g of the effective stationary phase:
where
mL = mass of effective stationary phase or in gas-liquid chromatography the mass of effective liquid phase [s], T, = column temperature [K]. Being an absolute term, V, does not depend on the apparatus and operating conditions used. It seems to be an ideal parameter, but its determination is difficult if higher demands on precision are made, since measuring, e.g., the flow-rate and the amount of the effective stationary phase is generally not as accurate as necessary. This is why relative retention parameters (Section 2.7) have proved to be more advantageous. In contrast to gas-liquid chromatography, in gas-solid chromatography the retention volume is proportional to the surface area of the adsorbent in the column. Therefore, a different specific magnitude is commonly used in gas solid chromatography, V,. Just as when calculating V,, the net retention volume VN reduced to 273,16K and 0,1013MPa is divided by the amount of stationary phase, in this instance the adsorbent, but additionally by its specific surface area. Specific Retention Volume in Gas-Solid Chromatography, V, :
V*=-
273,16
VN ~
m,*&
Tc
where
mA = mass of effective stationary phase or in gas-solid chromatography of adsorbent [sl,
7
2.3. Derived Basic Chromatographic Parameters
SA = specific area of adsorbent [m*.g-’].
273’16, V, is connected with V, as follows:
AS mA corresponds to mL, and V, = mL
Tc
and is no longer a volume per gram but a volume per square metre (cm’.m-*).
2.3.2.
Distribution Terms
The retardation of a compound in the gas chromatographic column is a result of the repeated distribution steps between the stationary phase and the mobile phase. In gas-liquid chromatography the retardation is mainly implemented by a sequence of dissolution and volatilization steps to which the volatile compound is subjected in the non-volatile stationary liquid. In gas-solid chromatography retardation is due to repeated adsorption-desorption steps. These steps are characterized by the distribution of the volatile molecules between the two phases and the term describing this distribution is called the distribution coefficient
K=
concentration in the stationary phase concentration in the mobile phase
(13)
‘
The numerical value of this quantity depends on the units in which the concentrations are formulated. In gas-liquid chromatography it is a common practice to define the partition coefficient: KL =
KL=;
solute concentration in the stationary liquid phase solute concentration in the mobile gas phase
7
CL
where cL= mL/ VL, c, = mc/VG.
mL, mo = masses of sample in the liquid and gas phases, respectively, VL,VG = volumes of liquid and gas phases, respectively, in the whole column. The partition coefficient is a fundamental quantity, being constant over wide ranges of concentration provided that they are low. It depends only on the solute-stationary liquid system and on temperature and is connected to the retention terms by VL’KL where VL is the volume of the liquid stationary phase. As V =- VN 273’16 it follows that mL Tc VN=
VN= VB-mL.-
‘C
273,16 ’
and
(15)
8
2. Basic Concepts
From
-_ m L - eLit follows that VL
v, =-K L eL
273,16 Tc
where 4~ is the density of the liquid phase at the column temperature. It can easily be seen that sample components with different partition coefficients leave the gas chromatographic column in order of increasing K, values and are thus separated. It should be remarked in passing that KL, if corrected for the concentrations by the activity coefficients
where
aL = solute activity in the liquid stationary phase, = solute activity in the gas phase, aM k,h =activity coefficients of the sample in the liquid and gas phases, respectively, this partition coefficient has become a thermodynamic quantity and permits e.g., calculations of heat contents from the temperature dependence of retentions [8]. In gas solid chromatography the concentration ratio according to eqn. (13) is formed by the sample concentration on the surface of the solid adsorbent divided by the sample concentration in the mobile gas phase:
xL
and is called the adsorption coefficient. As cs, unlike cL[eqn.(14)], has the dimensions of mass of adsorbed sample per unit adsorbent surface area:
where @
=mass of adsorbed sample,
mA =mass of adsorbent, S, =specific surface area of the adsorbent
and cM= *, K, is not dimensionless in contrast to KL in GLC,but has, with the quantities VO used here for concentration and area, the dimensions cm’ m-2 and is CoMected to the retention (cf. eqn. (12)) by
-
vs=-= vN mA
Ks.
sA
Compared with KL, which according to eqn. (15) is KL = VN/VL, the distribution coefficient Ks is not referred to the volume but to the surface area of the stationary phase present in the column. The retardation of a compound being carried through the column by the mobile phase can be expressed by the ratio
9
2.3. Derived Basic Chromatographic Parameters
where k is the capacity ratio. Applying the retention volume terms, k can be shown to be
This term k expresses how many times longer the compound is retarded in the column by the stationary phase than a completely insoluble compound, e.g., air. As a consequence, in gas-liquid chromatography k is the ratio of the fraction of soluble molecules at equilibrium in the liquid and in the gas phase at a certain time. It is an important quantity, being characteristic of a particular column. The capacity ratio, sometimes denoted retention capacity, depends strongly on the partition coefficient [eqn.(23)] and hence on the temperature, and is directly proportional to mL, the mass of liquid phase, provided that adsorptive interactions can be neglected [eqn.(25)]. Hence it is an expression for the effective amount of stationary phase per unit column length. KL=/?*k,
(23)
where VL = volume of the liquid phase, VG = volume of gas phase in the whole column
=
Vk.
and mL/VL= eL,where & is the density of the liquid phase, we obtain
This means that the capacity ratio is directly proportional to the amount of the liquid stationary phase, as just stated. The proportionality factor /?, termed the phase ratio (i.e., the ratio of mobile to stationary phase, V,/VJ is a column parameter and changes not only from column type to column type but also from column to column even if prepared and operated under the same conditions. As KL is constant for a given stationary phase and is independent, as already mentioned, of the column parameters (but depends on the temperature, see eqn. (26)!), the product k [eqn. (23)] is constant. As a consequence, if the phase ratio is decreased (e.g., by applying a higher degree of loading in packed columns), the capacity ratio must increase, and vice versa. /?a
2.3.3.
Temperature Dependence of Distribution and Retention Terms
The partition coefficient KLdepends strongly on the column temperature:
and so does the capacity ratio k, according to eqn. (23), where k is shown to be directly proportional to KL:
10
2. Basic Concepts C
logk=-++. (27) TC Hence, an increase in k can be achieved by drecreasing the column temperature. As V, is directly proportional to KL [eqn. (16)], the logarithm of the specific retention volume is likewise related to the absolute column temperature. This temperature dependence can be taken advantage of when calculating enthalpies from retention values [8-lo]: log v, = AH' 2,30 RT, +constant
(28)
where enthalpy of vaporization of the solute from an infinitely dilute solution in the investigated liquid stationary phase, R = gas constant. AH: is the difference of two enthalpies: AH:
= molar
AH?= AH,- AH,. (29) AH, = enthalpy of vaporization of 1 mole of pure solute, AH, = excess enthalpy of mixing of 1 mole of solute with the liquid stationary phase at infinite dilution. AH? is dependent on the interaction of the solute with the stationary liquid and on the structure of the liquid. If log V, values are plotted against 1/T, a straight line of slope AHbIR is obtained. It should be mentioned that there is a restriction, because AH? decreases slowly with increasing column temperature over a wide temperature range and the curve is therefore not exactly a straight line. When AHv % AHE (valid in non-polar solutehon-polar liquid systems) the enthalpy of vaporization of the solute can be achieved from the relationship between V, and 1/T [eqn.(28)]. On the other hand, if AH, is known from published data, the excess enthalpy can be calculated by plotting log V, against V T , , measuring AH'/R and applying eqn. (29) [lo] to obtain AH, [lo]. From the sign and magnitude of AHE, conclusions can be drawn about the type and strength of the interactions in the solute-solvent system concerned. By analogy with eqn. (28), which applies to gas liquid chromatography, the temperature dependence of retention that holds for gas-solid chromatography can be derived [ll]:
log v, =
2,30 RT,
+ constant
where AH,, is the partial (differential) molar enthalpy of adsorption (approximating to an infinitesimally small surface coverage of the adsorbent by the adsorbed compound).
2.3.4.
Parameters Characterizing the Ef'ficiency of Columns
Owing to diffusion, convection and delayed mass transfer processes, the residence times of individual molecules of the chromatographed compound diverge more or less from the average. This spreading results in a time-dependent concentration change corresponding to a Gaussian distribution curve (Fig. 2). Assuming that the chromatographic peak (Fig. 2) has an ideal Gaussian profile, its width is characterized by its standard deviation, u, which is a function of band spreading in the column, and the following relationships can be derived:
11
2.3. Derived Basic Chromatographic Parameters
(31)
w,=2u where y is the peak width at the inflection points (Fig. 2).
(32) where wB is the peak width at the base [distance between the intersection points of the tangents (drawn to the inflection points) width the baseline (Fig. 2)]; and wB=4a
where w, is the peak width at half-height (Fig.2). As both u and % can be obtained from the chromatogram only with difficulty it is better to measure the peak width at half-height, %, representing 2.355 u [eqn. (33)]. The quality of a gas chromatographic column is determined by the band spreading in the column and can be expressed as
(+) 2
n=
(34)
where n = number of theoretical plates, u = standard deviation,
rR = retention time. The term “number of theoretical plates” stems from distillation theory, but it should be emphasized that the values of n in chromatography and distillation have been defined completely differently. As a rule of thumb, [12] nDist. dnGC . (35) According to mathematical theory, different equations for determining the number of theoretical plates in practice have been developed, two of which are cited here:
Y:(
n = 16 - , [13] and
y:(
n = 8 ln2 -
y:(
= 5,545 -
[14, 151.
(37)
These equations could be shown to be identical and to be traceable to eqn. (34) [16]. Most commonly eqn. (37) is applied. The number of theoretical plates is dependent on the length of the column. By analogy with distillation (but again note eqn. (35)!) the height equivalent to a theoretical plate has been introduced in order to enable column efficiencies to be compared independent of the column length:
where h = height equivalent to a theoretical plate [cm or mm], L = column length [cm or IIM according to h], n = number of theoretical plates. In eqns. (34), (36) and (37) the retention time tR is used which in eqn. (1)was stated to consist of two terms, the mobile time t M and the adjusted retention time 2;. Hence, eqn. (34) can be written as
12
2. Basic Concepts
Obviously, tM does not contribute to the efficiency of the column. It was therefore eliminated [17], to give
which is called the number of effective plates. Substituting tk for tR in eqns.(36) and (37), we obtain N = 16 2
(5)
and N
= 5,545 (-$)2.
This term N is more appropriate for the evaluation of a gas chromatographic column than n, the number of theoretical plates. According to eqn. (38), where the height equivalent to a theoretical plate is calculated from the column length divided by the number of theoretical plates, a new term is necessary if n is replaced by N :
H = -L N
where H is the height equivalent to an effective plate. The height equivalent to an effective plate, H, and N, the number of effective plates, should be applied instead of h and n especially when comparing different column types. N and n can be correlated by using eqns.(21) (tM = t)R/k), (34a) and (39), and we obtain
n = N(?)’
=N
(1 +
k) 2
(43)
where k = t ) R / t M = capacity ratio. By converting eqn. (43) the relationship between the numbers of effective and theoretical
plates can be clarified:
n * k2 = N ( l + k ) 2 [18]. From eqns. (43) and (42) the following equations can be easily derived:
(43 a)
and inversely H = h l( +kk ) .
(44 a)
The value of h or H is one of the criteria in estimating the efficiency of a column and depends on several variables, one of which is the average carrier gas velocity. This linear velocity (in the direction of the column axis) can be determined by
13
2.3. Derived Basic Chromatographic Parameters
where linear average carrier gas velocity, column length, tM mobile time, and is related to the plate number by a fundamental relationship, the Van Deemter equation, derived in 1956 [19]:
E L
= = =
h=A
B +Y + CP U
(46)
where A, B and C are complex terms. Mathematically, this equation can be described by a hyperbole (Fig. 3) having a minimum which represents the carrier gas velocity at which the height equivalent to a theoretical plate is minimal and conversely the column efficiency is maximal, i.e., the carrier gas velocity is optimal.
'opt.
Average linear gas velocity U in crn.s-'
Fig. 3. Van Deemter curve (hhdependence)
The empirical terms A, B, and C are composite constants consisting of direct physical parameters, e.g., diffusion constants in the liquid and gas phases, capacity ratio and adsorptiondesorption constants. The optimal carrier gas velocity can be deduced from eqn. (46) to be
g.
iopt =
(47)
In practice, it is worth working at a slightly higher velocity than the optimum, i.e., slightly to the right from the minimum of the hyperbole (Fig. 3), as h is then affected only negligibly and the retention time and hence the analytical effort can be decreased, as can be seen from the relationship between retention time and carrier gas velocity in eqn. (48), where tR is inversely proportional to K L L According to eqn.(45), tM = T. From tR = tM + t ; [eqn.(l)] it follows that tR = 1+ r;. As U
tX = k . tM
U
L L [eqn. (21)], we obtain c,=T+ kt,. Replacing rM by T again we obtain U U
14
2. Basic Concepts
The term A represents the effect of the multiplicity of the gas paths, whereas B is an expression of the longitudinal gas diffusion. C, which is composed of C = Cl + C,, is the resistance to mass transfer, related to the diffusion process in the liquid (CJ and gas phases (Cd. In Fig. 4 the Van Deemter curves for packed and open tubular columns are compared. Obviously, the slope of the ascending part of the hyperbole representing the resistance to mass
open-tubular column
uopt.
uopt.
Ip.d
(0.t.c.)
Fig. 4. Van Deemter curves for a packed and an open-tubular column with the contributions of the A-, B- and C-terms and with the optimum practical gas velocity (after Struppe [23]) transfer, C, is smaller for capillary than for packed columns. As a consequence, the dependence of h on the carrier gas velocity is less distinct for capillaries, and thus this column type, according to eqn. (48), enables higher velocities to be used (without a serious loss of efficiency) and hence shorter analysis times to be achieved. Further, there does not exist a multiplicity of the gas path whereby the A term can be omitted, and eqn. (46) varies to become B h=k_+Cii U
(49)
with the restriction that it is not valid for packed columns. This is the so-called Golay equation [20, 211, valid for open tubular columns. Two further points should be mentioned here. First, in the hyperbolic part left from the optimal carrier gas velocity the B term strongly affects the efficiency, and this region has to be avoided. Second, at higher velocities B can be neglected, and eqns. (46) and (49) change to
15
2.3. Derived Basic Chromatographic Parameters ~~
h=A
+ Cii,
(46 a)
(49 a) with the restriction that they are valid only for higher carrier gas velocities. It was proposed [22, 231 to define an optimum practical velocity above which the loss of efficiency is tolerable and eqns. (46a) and (49a) are valid. This value is the average linear velocity, ii, for which the term C is ten times higher than B. In Fig.4 the contributions of the A, B and C terms to the height equivalent to a theoretical plate for packed and open-tubular columns, as a function of carrier gas velocity, are demonstrated. The magnitudes of h and H discussed hitherto are based on the assumption of the chromatographic peak having an ideal Gaussian profile. In practice, however, tailed peaks are often observed as a consequence of internal and extra-column processes leading to peak asymmetry. Apart from recourse to automated data acquisition systems or on-line computers, correct n and h values can be obtained with reasonable accuracy and precision for tailed peaks by a relatively simple graphical method [24], which cannot be described in detail here. It is based on an exponentially modified Gaussian equation [25]. h = Cii
2.3.5.
Parameters Characterizing the Separation
Being an analytical separation technique, gas chromatography is applied to separate individual constituents present in more or less complex mixtures. Hitherto, we have discussed only single peaks. Let us now consider the separation of two compounds, in addition to the unretained air. As already mentioned, absolute retentions, e.g., V, or VN,can be measured with only limited accuracy. Relative data are less inaccurate and less dependent on changes in, e.g., flow-rate or amount of stationary phase. Here corrections, otherwise necessary, can be omitted with the exception of the column temperature (because of the temperature dependence of the partition coefficient). Hence, relative retentions depend only on the pair of compounds taken in relation to each other,.on the special stationary phase and on the temperature. The relative retention, r2,1ris defined as the ratio of the two adjusted retention times of components 1 and 2:
‘X,
r2,1 = t’ Rl
where r2,
‘b ‘
R1
=
1
=
relative retention of compound 2 relative to compound 1, adjusted retention times of compounds 1 and 2.
As a consequence of above relationships, r also corresponds to the ratios of net and specific retention volumes, partition coefficients and capacity ratios:
For the same stationary phase the magnitude of r2, is constant and independent of the kind and dimension of the column, provided that the column temperature is the same when measuring tl; and the conditions do not change during the analysis. Eqn.(51) makes it clear that the relative retention is a ratio of dynamic processes taking place in the column and also a ratio of thermodynamic terms corresponding to an equilibrium state, both describing the separation. In qualitative gas chromatographic analysis, relative retentions are often used for the
16
2. Basic Concepts
identification of unknown peaks because of the mentioned advantages. It is appropriate to relate the unknown peak to a standard: ri* = tki tXS
(51a)
where t ,’
=
adjusted retention time of the compound to be identified,
tL = adjusted retention time of a standard
and to choose a standard, the retention time of which is not too far from that of the compound. Numerous reference substances have been proposed, often of the same type as the investigated compounds. They should be eluted near the centre of the complex chromatogram, with tk values smaller or larger than the tk, and the relative retentions, qS, having values smaller or larger than unity. As the many published standards are not related to one another, the general application of relative data for the identification of unknown compounds offers only limited possibilities, especially as they have not always been determined under comparable or exact conditions. In order to overcome these obstacles satisfactorily it would be advisable to use an only standard. However, this standard would be advantageous only in those cases, where the column temperature permits its elution within a reasonable time. One of the proposed standards is n-nonane [26], the retention time of which could be far from optimum if the compound of interest had a much higher or much lower boiling point. It was therefore suggested to apply homologues of n-nonane, the relative retentions of which can easily be related to that of the standard n-nonane [27]. Relative retentions have not only been used for identification purposes, but also for characterizing the separation efficiency. The term most often used is the separation factor, a, determined from two adjacent peaks, the separation of which is the most problematic:
Obviously, a is identical with r, with the restriction that the numerator of the ratio [eqn.(52)] always contains the adjusted retention time of the later eluted compound, so a is always larger than unity. A value a = 1means that there is no separation at all ( t f l= tR1). The separation of two adjacent peaks depends not only on their adjusted retention times but also on the standard deviations, u, of the peaks, for it can easily be realized that peaks having larger u values._expressed,e.g., in W~ values (peak width at base, eqn. (32)) (Fig. 2), are separated worse than narrow-disperse concentration profiles. The peak resolution, R,,an expression describing the degree of separation, takes account of the column efficiency and is defined as
where tRz - tR1 = tX, - tX1 (since by subtraction tM is eliminated) and is expressed as Ar. For two adjacent peaks eqn. (53) can be reduced by w B 1 - wB2 to give
As can be concluded from Fig. 2, a sufficient separation is achieved if AtR L 4 a, R hence be-
coming 2 1 (provided WE2 s 4 u, Fig. 2). If AtR 2 6 u, R becomes 2 1.5 and the separation is almost complete. For further terms characterizing the separation and resolution, see Section 2.7., and for characterizing the resolution in non-ideal situations, see [27a].
2.4. Flow of Gases in a Gas Chromatographic Column and Formation of Bands
2.4.
17
Flow of Gases in a Gas Chromatographic Column and Formation of Bands
After injection into the gas chromatograph, the sample compounds are carried through the column by the carrier gas, the mobile phase. In a dynamic process each sample component is distributed (according to its interaction possibilities with the stationary phase) between volume elements of the mobile and stationary phase. One can imagine the column being divided into numerous sections (theoretical plates) and the more or less rapid movement through the column being accomplished by a sequence of transport and equilibrium steps. By a large number of such successive steps the components are spatially separated from each other, provided that their interactions with the stationary phase and their properties, especially their vapour pressures, differ sufficiently. If the separation is successful, each compound is eluted purely (with regard to the other components) but mixed with the carrier gas in form of a more or less broadened band, and the elution of the individual bands takes place successively with respect to time. The peak broadening is caused by several parameters, a few of which are discussed later. First, let us examine the carrier gas flow-rate. Its volume flow-rate, Fc [ml/min], is measured according to eqn. (3) considering the column temperature and, if using a soap-bubble flow meter, the partial pressure of water: Fc = Fa T P - P w (for the meaning of the parameters, Ta Pa see eqn. (3)). Another possibility for characterizing the gas flow-rate is its linear velocity, which from the point of view of the gas chromatographic process should be used rather than the flow-rate. Both terms can be related by
where
~0 = velocity at column outlet,
rc
=
column inner radius,
& = interparticle porosity (without regarding the pore volume), &
= interparticle volume, V, = L ma = volume of the column. For empty tubes, which means even for wall-coated open tubular columns, & = Vc because of the lack of particles, and E,, becomes unity. Hence
uo =
FC
(valid only for wall-coated open tubular columns). For packed columns E,, is found to be about 0,4 [28]. Assuming the carrier gas to show ideal behaviour, the outlet velocity for packed columns is ~ 9 1 uo =
x PT-PZO 2 q M goL PO
where x = column permeability (independent of the nature of the carrier gas), pi = inlet pressure, po = outlet pressure,
(57)
18
2. Basic Concepts
tfM = mobile phase viscosity,
L
= column
length,
eo = interparticle porosity.
This is valid, however, only if the velocities are not too high. The permeability of a column determines the value of the inlet pressure necessary to obtain reasonable analysis times and optimum separation conditions. By converting eqn. (57), x is found to be x = 2 uotfM eoL
Po Pi-Po
which is valid for packed columns. With the open tubes, where the outlet velocity has been shown [32] to be
(valid for open tubes), we obtain with the use of eqn. (57a) and substitution of uo by eqn. (58), x = r: eo/8 and for eo = 1, as in open tubes the whole volume [eqn. (56)] is available, % becomes [32]
(valid for wall-coated open-tubular columns). The permeability of a column determines the value of the inlet pressure necessary to obtain reasonable analysis times and optimum separation conditions. Obviously, for packed columns x depends on the particle size and the density of the packing. By using the Kozeny-Carman equation [30, 311, valid for non-compressible media, the column permeability, x,, related to the velocity in an empty tube can be discribed: x, =
dz e: 1808; (1 - eo)2
(59)
where x, = column permeability related to the velocity in an empty tube, dp= average particle diameter of the solid support or adsorbent, 0, = factor describing the deviation of the particle shape from spherical. At least approximately, this equation can be applied to gas chromatographic systems. Hence, the permeability is seen to be proportional to the square of the average particle diameter, but additionally to depend on the packing density (because e0 = &, i.e., the higher is &, the interparticle volume, the less dense is the column packing). In order to obtain efficient columns, the density should be as high as possible, even if it affects the column permeability. The nature of the particles is less dominant than the shape, represented by ap.Spherical particles with Bp = 1 permit twice the permeability of, e.g., Kieselguhr particles of comparable size having 0, values of around 0.7 because of their irregular surfaces. The evaluation of eqns. (57a) and (58a) for the selection of column types and packings will be discussed in a later chapter. Let us return to the carrier gas velocity. From eqns. (57) and (58), it can be concluded that, as p o is almost constant (atmospheric), the gas outlet velocity uo increases considerably faster than the inlet pressure, owing to the gas decompression. The outlet velocity does not reflect, however, the real situation existing in the column. Owing to the compressibility of the mobile phase, at each point of the column the velocity, density and pressure of the gas phase differ, with increasing velocity, e.g., towards the column outlet. The relationship between the
19
2.4. Flow of Gases in a Gas Chromatographic Column and Formation of Bands
outlet velocity, uo, and the already repeatedly used average linear gas velocity (eqns.(45-49)) is given by U = uo.j. where j is the compressibility correction factor (James and Martin factor [6]),where
3 P2-1
i = 2- P3-1
(60)
and P = - Pi
Po
(see eqn. (7)). The flow of the carrier gas is caused by the pressure difference between the column inlet and outlet, pi - p o , and the velocity is not constant either along the column or across the entire cross-section. According to eqn. ( 4 9 , P can be measured by P = UtMand uo by substituting ii in eqn. (60): li 2L P 3 - 1 u --=(61) Oj 3fM P*-1‘ If eqns. (45) and (61) are used, il and uo can be calculated by measuring the column length, the mobile time and the absolute column inlet and outlet (ambient) pressure. This method is simpler and much more accurate [33] than calculating the velocities from the flow rate. At the beginning of this section the band broadening was mentioned. All causes cannot be reviewed in detail, but as peak broadening strongly affects separation and resolution, some causes should be discussed here. In eqn. (46), the meaning of the constants A , B and C is very complex. Numerous investigations, beginning with the basic ones by Van Deemter, Zuiderweg and Klinkenberg [19] for packed colums and by Golay [20] for open-tubular columns, have been carried out [34-371. As a summary, the following equations will be discussed briefly here. Packed columns with liquid stationary phases:
h=2Mp+
2hD,+ 1+6k+llk2 P 96(1+ k)2
#P I ”‘
Dg
2k dili ( 1 + k)2 kd
k d2P 1 3 ( 1 + k)’ Dl .
+ -2
Open-tubular columns with liquid stationary phases:
20 h=-+ u
1+6k+llk2 A r 2 P+2k dlli+ 2k 24(1 + k)’ Dg ( 1 -t k)’ kd 3(1 + k)’
4
Packed columns with solid stationary phases:
h=2Mp+
2 h D g + 1 + 6 k + l l k 2 .x.-e+d21i 2k dali li 96(1 -t k ) 2 D, ( 1 + k)’ kd
where = numerical factor desribing the packing, = mean particle diameter, yN = labyrinth factor, a numerical factor describing path length, obstruction Dg = diffusion coefficient of a solute in the gas phase, Dl = diffusion coefficient of a solute in the liquid phase, dl = mean film thickness of the liquid phase,
A d,
kd, k; = desorption coefficients, r, = column inner radius k = capacity ratio,
factor,
20
2. Basic Concepts = average linear carrier gas velocity, da = mean thickness of the adsorption layer, x = dimensional constant.
Considering the term A = 2Mp (eqns.(62) and (64)), this source of peak broadening can be attributed to the different path lengths and gas flow velocities in the porous packing representing a channel system. The d f i s i o n in the pores which can be described by the coefficient D,,,,, = M, U [37] must be taken into account for the distribution of the solute (or adsorbate). The term B = 2 yN D, (packed columns) (eqns. (62) and (64)) is related to the axial molecular diffusion of the solute (or adsorbate) in the space between the particles and to the sinuosity of the paths the molecules have to cover. Considering the term B = 2 0 , (open-tubular columns), owing to the lack of particles, there is no labyrinth between them, and the correction factor for the axial molecular d f i s i o n is y~ = 1, as there are no extended pathways of the solutes. The term C = C, + C, + C, is a composite term, the composition of which depends on the type of column applied and on the kind of stationary phase. C describes the contribution of the resistances to liquid- and gas-phase mass transfers, related to the difision in the gas and liquid phases, to peak broadening.
c,
=
1 + 6 k + l l k Z ' X .dZ 9 96(1 + k)2 4
c,
=
1+6k+llkZ & 24(1+ k ) z Dg
and
(packed columns) (open tubular columns)
do not represent, in contrast to B, the longitudinal but the lateral diffusion in the mobile phase. Like B, C, is a contribution of the resistance to gas phase mass transfer to the peak broadening. C, is of importance for all types of columns, but especially for open tubular columns, as r, is generally larger than the particle radius, and both r, and d,/2 are squared terms in the equation. Bohemen and Purnell [36] substituted r, by the particle radius. They have considered the packed column as a system of capillaries of radius d,/2.
and
c -!t I-
2k
4 3 ( 1 + k)'
are expressions which, in the case of liquid stationary phases for both packed and open-tubular columns, take into consideration the phenomena diminishing the mass transfer rate of solutes from the stationary phase to the mobile phase and the inverse. The desorption coeflicient, k d , takes into account that the transport of the solute molecules depends on their concentration on the liquid stationary phase/gas (mobile) phase interface, which in turn is determined by an adsorption/desorption process (K = k,/kd where k, = adsorption coefficient). If kd--* 0 3 , which means that sorption equilibrium is established instantaneously, C, becomes zero and we obtain for packed columns (where C, is neglected) the well known Van Deemter equation; kd is related to 4 and dl [37]. The C, term is proportional to the square of the film thickness of the liquid phase, i.e., reducing the film thickness on the particles and on the walls of capillaries will strongly reduce C, and hence reduce that part of peak broadening caused by the resistance to the mass trans-
2.5. Thermodynamic Bases of Gas Chromatography
21
fer in the liquid phase. Unfortunately, the surface properties of the particles or of the inside wall of the tubes limit the decrease in the film thickness. 2k*d, For solid stationary phases (adsorbents), Cl,= -. k)d is applied rather than C,, as (1 + k ) z commonly there is a gas-solid rather than a gas-liquid interface (but several exceptions may occur, which cannot be dealt with here), and C, is not applicable. Solid stationary phases with a uniform surface offer rapid adsorption-desorption steps, and Ci is very small. If the surface is not uniform, a heterogeneity factor (>1) has to be applied, the magnitude of which depends on the distribution of energy centres. To summarize the complicated relationships between column efficiency, expressed as the height equivalent to theoretical plate, h, and the average linear carrier gas velocity can be described in general form as follows: For packed columns with liquid stationary phases: h =A
+ BU + (C, + c, + Cd I7 Y
(624
For packed columns with solid stationary phases: h =A
+ -=BU + (C, + Ci)I7
For open tubular columns:
B h = Y + (C, U
2.5.
+ c, + CJ E
Thermodynamic Bases of Gas Chromatography
The basic relationships between retention values and thermodynamic magnitudes have already been discussed in the Section 2.3.2. The essential term is the partition coefficient, KL (liquid stationary phases) or K, (solid stationary phases). Let us first deal with the thermodynamics in gas-liquid chromatography. In eqn. (21) we have seen that the capacity ratio is k = fk/fM, i.e., the ratio of the adjusted retention time to the mobile time. At equilibrium, k represents the ratio between the fraction (or number) of molecules residing in the liquid and gas phases at that moment: k = nL n0
(65)
If we consider nL, the number of solute molecules present in the liquid stationary phase, it can be neglected in comparison with the number of the molecules of the stationary liquid itself, n,, as the solution is (ideally) infinitely dilute: nL= xn,, where x is the molar fraction of the solute and n, is the number of molecules of the stationary liquid and can be expressed as
where eL and M are the density and the molecular weight, respectively, of the stationary phase, and VL its volume in the column. The number of sample molecules residing, at equilibrium, in the (ideal) gas phase is
22
2. Basic Concepts
where p is the partial pressure of the sample over its solution in the stationary liquid. According to Raoult’s law, the partial pressure over a solution is equal to the product of the saturated vapour pressure of the pure compound, PO, and the molar fraction, x, i.e., p = pox, and is valid for ideal solutions. For real solutions the Lewis activity has to be used instead of x, and a = 9, where y is the activity coefficient and the superscript zero means the limiting value at infinite dilution. Thus, p = poy”x, and no becomes
no
=
POYOX vo RT
Substituting nL in eqn. (65) by eqn. (66) and no by eqn. (67) we obtain
and, owing to eqn. (23a), where KL = k Vo/VL,we obtain for the partition coefficient
The relative retention, r2,1, is an expression describing the separation of two compounds, 2 and 1. Combining eqn.(51), where rz, = KLZ/KLl,with eqn.(69), we obtain, because eL, R , T and M characterize the stationary phase and column temperature and are the same for both compounds,
or, as rz,l = V k / V k l (eqns. (3,(8) and (51))
-
This fundamental relationship, often used in its logarithmic form: log rz, 1 = log
V’
VX1
= log
4 + log L!J P2 YOZ 0
was found by Heringion [38]. It can easily be seen that the separation, expressed by the logarithm of the ratio r2,1 or Vkl VRl,is determined by two terms, one of which is specific for the compounds under investigation (logpylp9 and cannot in practice be changed within the range of column temperatures, whereas the other term (log y!/y$ offers the possibility of strongly influencing the separation by utilizing selective properties of the stationary liquid. The separation can be achieved, with regard to these two compounds 1 and 2, either by taking advantage of differences of their vapour pressures, or by choosing a selective stationary liquid (as f’ is determined by the properties of both the solute and the stationary liquid) or by both. It has been shown [39-411 that the activity coefficients diverge considerably from unity, depending on the type of stationary liquids and solutes, over a range of more than two orders of magnitude. Incidentally, the y” value is only approximate. The accurate limiting activity coefficient can be calculated by adding two complex terms [4]. Despite some restrictions that cannot be discussed here, the determination of the activity coefficient offers the possibility of calculating excess enthalpies and entropies:
23
2.5. Thermodynamic Bases of Gas Chromatography
p E = R T l n yo where pE, the excess chemical potential, is related to the Gibbs-Helmholtz equation:
AGE= AHE- T A P
(71) (72)
where ACE = excess molar free energy, A P = excess (or mixing) enthalpy of 1 mole of the solute with the stationary phase at infinite dilution, excess partial molar enthalpy, A P = excess partial molar entropy.
Using (72a)
pE = ACE
we can combine eqns. (71) and (72) to give AHE- T A S E = R T h y o . (73) If we assume that A P and A P are constant within a small range of temperature and differentiate eqn. (73) with respect to 1/T we obtain
with the consequence that varying the temperature and measuring the change in y" permits the calculation of excess partial molar enthalpies. The excess chemical potential used in eqn. (71) only refers to a dilute real solution (75) p E= p - pideal = R T h y taking only into consideration the deviation of a real dilute solution from ideal behaviour. However, in a gas chromatographic system we meet with a distribution between a gas and a liquid phase, and we therefore have to consider the two phases, as follows. Gas phase (with regard to a real mixed phase): pi' = pi'
+ RT ln ai = p! + RT In xi + RT In y:i
(76)
where pf = chemical potential of component i in a real gaseous mixed phase, pf = chemical potential of the pure gaseous component i in the state of an ideal
gas, ai = activity of component
i in the real gaseous mixed phase,
xi = molar fraction of i in the gaseous mixed phase, yo. = activity coefficient of i in the gaseous mixed phase. 8'.
Liquid (stationary) phase (with regard to a real dilute solution):
R T ~ ~ Y ~
p ) = p: + R T ~ U= )p ; + R T ~ X + ;.
(77)
where pi = chemical potential of component i in a real dilute solution, p ) = chemical potential of the pure component i in an ideal dilute a: = activity of component i in the real dilute solution,
solution,
x ) = molar fraction of component i in the solution, yfi = activity coefficient of component i in the solution. If it is assumed that the partition of i between the liquid and gas phases is at equilibrium, the two chemical potentials are equal, i.e., p f = pf , and using eqns. (76) and (77) we can write p)
+ RT h x ) + RT lnyyi = & + RT h x g + RT h y i i .
24
2. Basic Concepts
Substituting RT In y by the excess chemical potential [eqn. (791,we obtain p)
+R
T ~ X+;pf
= pf
+R
T ~ +Xp ; .~
With Api = pf - p! and A p E= p i - p t , we have
+A
~ =ERT (hX; - h x f )
and
K,, the partition coefficient related to the molar fractions, is defined by K ,
= x,!/x!, and
thus
Analogous to eqn. (72a), we can substitute Ap, and Ape by the corresponding free enthalpies,
AG = AH - TAS, and for 1 mole we obtain AHV+ AHe - T(AS' + AS') K, = exp RT
where AH" and A P are the enthalpy of vaporizing 1 mole of pure solute and the excess (or mixing) enthalpy of 1 mole of solute with the stationary liquid at infinite dilution, respectively, and where AS" and ASe are the corresponding entropies. A P is small compared with AH", and the sum of both AHS= AHv + AHE is the molar vaporization enthalpy of the solute from an infrnitely dilute solution in the stationary liquid. Hence, the vapour pressure of a component is an essential value for the gas chromatographic analysis. AHS is of the order of 42 kJ/mole. Until now, we have only discussed energies of one component. In eqn. (52) the separation of two compounds has been characterized by the separation factor a = f,&/tl;l, which in this investigation can be taken to be identical with rz, in eqn. (70) and hence with the ratio K z / K l in eqn. (51)
i.e., the separation can be described by the ratio of the partition coefficients. Combining eqn. (51b) with eqn. (79) and with eqn. (78), the logarithmic form, respectively, and substituting Api + ApE by AG for both compounds, which are separable when a > 1, we obtain In az, Kz - 1 = In -- -(AGz - AG1) and representing the difference AGz - AG1 by A(AG), we can K1 RT formulate
,
RT ln az,= A(AG),
(81) where A(AG) is the difference between the molar free energies of the vaporization of each of compounds 1 and 2 from their infinitely dilute solution in the stationary liquid. It could be shown that values of the separation enthalpy A(AG) of about 10 Joule/mole are sufficient for separating compounds, in spite of the fact that AG (the molar free energy of vaporization from the infinitely dilute solution in the stationary phase) itself has values of ca. 4 . lo4Joule/ mole. Thus, by gas chromatography small thermodynamic differences can be made visible even though they are based on thermodynamic effects four orders of magnitude higher [42]. Let us reconsider the partition coeficient. In eqn. (79) we used the partition coefficient K,,which is related to the molar fractions, whereas in eqn. (14) K L is the partition coefficient in relation to the concentrations in the gas and liquid phases (in mass per unit volume).
2.5. Thermodynamic Bases of Gas Chromatography
25
They are connected by the equation
where
VmOl,,= molar volume in the mobile (gas) phase, Vmol,I = molar volume in the stationary (liquid) phase. Hitherto we have neglected the influence of the interface between the liquid and gas phases. However, considering the large area of the surface film usually applied in gas chromatography in both packed and open-tubular columns, we have to improve eqn. (15) by a term taking into consideration the adsorption processes on the surface film in a simplified manner [43-45]: VN = VL. KL + ALKA
(83)
where
AL = surface area of the liquid phase, KA = adsorption coefficient of the gas-liquid interface, VL = volume of the liquid stationary phase, KL = bulk liquid partition coefficient. Such processes are mainly caused by polar molecules of the stationary liquid or of the sample components. Additionally, adsorption effects on the liquid-solid interface (stationary liquid/ solid support) may occur, and also between sample molecules and those parts of the liquid phase situated near the liquid-solid interface which, compared with the bulk liquid phase, can be regarded as being modified by the solid support's surface activity. These phenomena contribute to the retention, and eqn. (83) becomes m
n
KLiVLi +
VN = i=1
KAjAAj? j- 1
where KLiis the partition Coefficient between the mobile and gas phases, VLiis the volume of the stationary phase of the i-type (macro- or micro-layer of the liquid on the surface of the solid support, liquid phase in the micropores, etc.), KAj is the partition coefficient between the stationary phase and the interface of the j-type and A , is the area of the j-type in the column, e.g., gas-solid, liquid-solid, gas-liquid. We shall now briefly discuss the thermodynamics of gas-solid chromatography. In eqns. (20) and (30) we have defined the adsorption coefficient and the partial molar adsorption enthalpy, respectively
and log v,
=
2,30 RT,
+ constant*
where AHA approximates to infinitesimally small surface coverage of the adsorbent by the adsorbed compound. In physical adsorption which, in contrast to chemisorption, is considered here, Van der Waals forces between the adsorbent and adsorbate become effective in the boundary layer, and AHA values of <SO kJ mole-' occur (whereas in chemisorption AHA values of >500 kJ mole-' would be observed). Nevertheless, the AHA values in gas-solid chromatography in most instances are higher than comparable AH: values in gas-liquid chromatog-
26
2. Basic Concepts
raphy, GSC thus requiring generally column temperatures more than 100°C higher. The concentration of compound i on the surface of the adsorbent can be expressed by
r-
%a&.
SA
where nil&. = number of adsorbed moles of i , s,, = surface area of the adsorbent, or, in other dimensions, by the adsorption molality:
r - niab. mA
where mA is the mass (weight) of adsorbent. Applying the specific surface area, &[m2 g-'I:
we obtain
With unimolecular localized adsorption, a maximum surface concentration rmm, or rLm,, maximum adsorption molality, can be achieved, and the real coverage can be expressed by 8,the degree of coverage:
r or r' are functions of temperature and pressure or concentration of the sorbate, respectively: or r, r' = g(c, 0. (85) This function is usually determined empirically by plotting r or r' at constant temperature
r, r' = p(p, t )
P
Fig. 5. Typical Langmuir adsorption isotherm
r,-
nirb m.4
[mole.g-'] adsorption molality, p equilibrium pressure of the sorbate
2.5. Thermodynamic Bases of Gas Chromatography
27
against pressure or concentration of the sorbate, i.e., by a system of adsorption isothermes. A typical Langmuir adsorption isotherm is shown in Fig. 5 . The curve, having a linear part only at low pressures, rises continuously with increasing pressure, asymptotically approaching the limiting value rku corresponding to unimolecular coverage of the adsorbent by the sorbate. An equation describing the isotherm can be derived using kinetics: If 8 is that part of the surface covered with sorbate molecules, then 1 - 8 is the free part of the surface. The adsorption rate can be expressed by d l a & .= ap(1 - 8)SA dt where p is the pressure of the sorbate and the desorption rate by dRidcs= b&A dt. At equilibrium dni,& = dnlde8,.and eqn. (86) can easily be developed: @=-
UP b + up a
where a and b are constants. If a’ = - is applied, we obtain b @=-
alp 1+ a l p
and the Langmuir isotherm is [eqn. (84d)l a’P r = rim--1+ a ’ p
‘
From eqn.(87), it can be concluded that at higher pressures r ‘ becomes rk,, and that at very low pressures r‘ is approximately proportional to the pressure, as can be seen in Fig.6. This linear region can also be described by the Henry equation: n i r h = KHP
where
KH= Henry constant [mole. m-2 * Pa-’], = partial pressure of i. KH is related to the partition coefficient used in eqn. (20), K s , by p
K --.Ks
”- RT
Eqns. (86)-(88) are valid only if the adsorption sites have equal or t least similar adsorption potentials. As a consequence of inhomogeneities of the surfaces and of adsorbate-adsorbate interactions that we have not taken into account in the above relationships, numerous investigations have been carried out resulting in modified equations that have been described elsewhere [46-491. Let us finally consider the temperature dependence of the adsorption. From phase equilibrium, where pi = p f , 01; is the chemical potential of i on the surface of the adsorbent and pf is the chemical potential of the pure gaseous compound i in the state of an ideal gas), eqn. (89) can be developed [50]:
28
2. Basic Concepts
where AHA is the partial molar adsorption enthalpy. This relationship is analogous to the Clausius-Clapeyron equation. Again assuming that the gas behaves ideally and applying eqns. (18), (10)-(12) and (20), for constant coverage we obtain
Eqn. (89a) can also be easily derived from eqn. (30): log v, =
2,30 RT,
+ constante.
From the measured adsorption isotherms, the partial molar adsorption enthalpy can be determined by means of eqn. (89), and from the measured V, values adsorption enthalpies are obtained by eqn.(30) or (89a), which represent very low degrees of surface coverage of the investigated adsorbent.
2.6.
The Quality of Chromatographic Separation
In Section 2.3.4 we dealt with parameters characterizing the separation of two compounds. The resolution of two adjacent peaks has been defined by eqn. (53). This relationship for R,, the resolution, only reflects the quality of separation and does not express the causes that result in the measured R value. Other parameters describing the separation are the relative retention rz, (eqn. (50)) and the separation factor a (eqn. (52)). It has been stated that for a given stationary phase, without regard to the influence of temperature, these parameters are constant for the compounds under investigation. Hence, r and a,being characteristic of the stationary phase used, offer the possibility of a correlation between the separation effect and the type of stationary phase if they are correlated with the band spreading (or standard deviation) expressed as column efficiency via the number of theoretical plates necessary for a desirable separation quality. As indicated in Section 2.3, almost complete separation and resolution are attainable when the retention difference of two adjacent peaks is equal to or larger than approximately six times the standard deviation: AfR 2 3 (61+ 62) (90) [this value corresponds to a resolution R,= 1.5 eqn. (53)]. Let us now develop relationships containing only parameters that express the selectivity of the column (AtR or, better!, rz, the separation efficiency (a, peak width, or, better!, n or N) and the solubility (GLC)or adsorbability (GSC) of compounds 1 and 2 (k). Such correlations for the description of the separation quality are necessary as r (or a) alone is not sufficient, because the column quality also depends on the column efficiency. Starting with eqn.(90), giving nearly complete separation (ca.6 a),we can write instead of ArR fu
- rR1=
3 ( q + 62).
This can be rearranged to give
(2, J:
-- 1 = 3 - + - . fR1 a 2
If the fraction -on the right-hand side is reduced to higher terms by tRz
ru/fu,
it follows that
29
2.6. The Quality of Chromatographic Separation
1 = 3 - + Y i h
-t~
I:(
fR1
fR2
tR1
AS f R / U = J;; (eqn. (34)) and, for two adjacent peaks, nl = n2, we can write
or 2
Eqn. (91) provides information on the number of theoretical plates required for a certain ratio of retention times in order to achieve a largely complete separation of two compounds, the separation of which is difficult. If we use N, the number of effective plates, instead of n, and N =
r'Y $
(eqn.(39)), substi-
tuting At = f R Z - fR1 by A f = f l ; - tA1 (which are identical because the mobile time disappears) [51] we obtain
As tl;/tAl
= r2,1,
this can be reduced to
r2, + 1 ~ = 9 r2,1 ( -1) . Assuming that n is used more often than N,we shall continue to deal with eqn. (91). In Chapter 2 the adjusted retention time, &, has been stated repeatedly to be a more characteristic parameter than the retention time, tR. Therefore, the ratios tWlfR1in eqn. (91) have to be altered: --
-
fR1
f&
+ tM
tkl
+ tM
'
Multiplying by fkl/tkl leads to f' + fM R z fR2 --
-
Gl
fk1
fR1
+ tM .
4 1
Substituting f M / f k l= l/kl and I
tR2 -tR1
-
+kl 1 ' 1+kl
r2.1
tk/tkl= r2.
we obtain
30
2. Basic Concepts
Reducing to higher terms by kl/kl it follows that
-_t R z - rz*lkl+ 1 tR1 kl + 1 . This expression substitutes both ratios
tRz/tR1
in eqn. (91) and we obtain
1+ r2,Ikl + 1
kl + 1 If the ratio kl + l/kl
+ 1 is used in place of 1, we can write
Now the fractions may be added or subtracted and simplified and we obtain
We can imagine the capacity ratio kl = f k l / t Mto be of great importance (in addition to relative retention) for the required number of theoretical plates. An example is given below to illustrate this point. If r2, = 1.1, the required plate number is 4761 if kl = 10, but 33 489 if kl is only 0.5. Another expression describing the efficiency of a chromatographic column is the separation number or, in its original German name, Trennzahl (n)[52,53]. As a pair of major (or standard) peaks, two consecutive members of a homologous series are chosen, e.g., CI4 and Cls n-alkanes or slightly lower or higher: t & z + 1)
Tz=
wH(Z+ 1)
- t;(a + wH(Z)
-1.
(94)
where t i ( z + 1) t R ., B. wH(z+ WH(Z)
adjusted retention time of two consecutive n-alkane
} = homologues with 2 and 2 + 1carbon atoms
1
= peak
widths at half-height.
(It does not matter whether t i or t R is used, because the difference in the numerator in eqn. (94) eliminates tM if tR is used). The Trennzahl (which must not be confused with the resolution!) indicates how many peaks can be placed and yet be separated (4,71 cr separation) between two consecutive n-alkane homologues that, by definition (see Chapter 4), have a chromatographic separation of 100 retention index units. The minimum TreMzahl required for the separation of two compounds having retention indices Il and I2 (see Section 4.2.2) can be calculated by the equation [S41
This is valid if the column is not overloaded, if the concentrations of compounds 1 and 2 are similar and if only the k range of 1 and 2 is considered. The separation value 4,71 u corre-
31
2.6. The Quality of Chromatographic Separation
sponds to a resolution Rs = 1.177. It can easily be seen that for an index difference Z2-Zl of 5 the magnitude of TZminhas to be 19. For example, well packed columns have 7Z values up to 30, and open-tubular columns from 20 to more than 80. A further expression for the number of peaks that might be placed between peaks 1 and 2 while guaranteeing a 4 d separation (corresponding to a resolution RS = 1.00) for every peak is the effective peak number (EPN)[ 5 5 ] :
where 1 and 2 are successive n-alkanes. However, eqn. (96) has been derived assuming n to be the same for each compound and that it is strictly valid only for the specially applied pair of n-alkanes. Simplifying eqn. (96) and substituting '(" wB2
- '") by Rs (eq. (53)) we obtain + wB1
EPN= Rs2.1- 1 . (964 Possibly the most important relationship that correlates factors on which the separation is based (selectivity r and capacity ratio k,being an expression for the distribution) with factors of band spreading (a) counteracting the separation will now be derived. Applying eqn.(53) and assuming wB2to be equal to wB1(an approximation not always verified!), we can write
The right-hand side of eqn. (97) substitutes wB2 in eqn. (36):
(k) 2
, we can write
Reducing eqn. (98) to higher terms by
'- , )' (?). 2
n2 = 16 R:(
, tRZ
tRl
Dividing both the numerator and denominator of the first squared term in eqn. (98a) by tkl, we obtain
tL tL-ti1
ti2
AL ti1
$2
k1
+
til
=
*
lR1
IRl
As t&,lt& = r2, we can write tL
-
fL - t i 1
12.1
-1. If in the second squared term in eqn. (98) tRZis expressed by nominator are divided by tM, we obtain 12.1
-_tRZ - & + tM = tL
tL
k+tM & + I tM
tM
& tM
-
tM
tL tM
*
tL + tM and nominator and de-
32
2. Basic Concepts
tRz - kz + 1 However tLltM= k2 and we then have -- -. th kz
These two modified terms are substituted into eqn. (98)
Instead of n2 we can write nrC,,, the number of theoretical plates that are required to achieve a resolution Rs for the two peaks specified by rz, and k2 (56):
Eqn. (99) permits the determination of the number of theoretical plates necessary to attain a certain resolution R between the two peaks under investigation. From eqn. (99) we obtain the resolution
Eqn. (100) states first that the resolution for a given peak pair is proportional to the square root of n and, as n = L / h (eqn.(38)), to the square root of the column length. Second, the capacity ratio affects the resolution more the smaller it is, i.e., the closer to the air peak the compounds are eluted the smaller is the term kzlk2+ 1 and hence the worse is Rs.In other
( kzl )I
words, the reciprocal squared term - in eqn. (99) increases strongly, thus demanding considerably more plates to achieve the same resolution as with higher k values, i.e., low capacity ratios should be avoided in complicated separation problems. Third, we can conclude from eqn. (100) that the selectivity of a stationary phase, expressed here by rz,1, strongly influences the resolution. We shall often return to this important relationship in later chapters. The derivation of this and similar relationships has repeatedly been the subject of thorough investigations [57-601. Because each of the resolution equations depends on special approximations, it has been proposed to re-define the term resolution [61] by
Instead of eqn. (53) it should be emphasized that the magnitude of R, is on no account identical with the above discussed R values. This can easily be realized if one compares eqn. (58) with eqn. (101) and assuming (wB2+ wBl) to amount to about 8 u, hence R, is found to be about the four times Rs.From eqn. (101) another resolution can be derived [61]:
A final relationship describing the separation and revealing the number of peaks that can be placed between two reference peaks, taking into consideration the band spreading (depending on the retention time) and a 4u separation (R = 1.0) can be mentioned here:
log 21.2
=
p
fi+2 log fi-2
2.6. The Quality of Chromatographic Separation
33
where N is the effective plate number:
($) 2
N=
=5.545
(39)
(5) 2
.
(41)
This Z value [62] is an expression characterizing the separation power of a chromatographic column for compounds (of the same class) having boiling points within the boiling point interval of the two reference compounds 1 and 2. The value Z - 1 is the number of peaks that can be placed between peaks 1 and 2. The 2 value is only a relative measure as it is valid for the investigated homologous series and cannot be assigned to other homologous series [63]. If N,the effective number of theoretical plates, has not been determined, Z can be calculated by eqn. (103a) from the adjusted retention times and the peak widths at half-height: 21,2 =0,678
(&+A) log+. wHZ wHl kl
(103a)
It has been shown [63] that if an approximation is applied, Z1,2may be expressed as
and, as In r2,
= A(AG2*1) (eqn. 82), the
RT
2 value is directly proportional to the free enthalpy
of separation.
The Time of Analysis
2.7.
Hitherto we have not dealt with an important factor, especially in industrial chromatographic analysis, namely the time the analysis will require. The numerous influences on the analysis time [amount of stationary phase, column type and length, number of theoretical plates, retention time interval between the first and last eluted compound, column temperature, crux of the separation problem, particle diameter (packed columns) or capillary diameter (opentubular columns, etc.)] cannot be discussed in detail here, and only a few correlations between some chromatographic parameters and the minimum analysis time will be mentioned. In eqn. (21) we have defined k = f k / t M . The adjusted retention time is substituted by the retention time: (105) According to eqn. ( 4 9 , the average linear carrier gas velocity is correlated with the column length and the mobile time, L Z=-
fM
and fR
=
fM
L
and f M = -
L U
in eqn. (105) is substituted by the right hand side of eqn. (106) and we obtain
7(k + 1). With eqn.(38), h = L / n or L = hn, and we can substitute L by hn and obtain
34
2. Basic Concepts
Now the expression for n (eqn.(99)) is substituted into eqn.(l07) and we obtain, using the appropriate subscripts [64], ropt,=--.16R:(+-) h2 ii
rz. 1 -
(k2+ 1)’ k2’
which is the retention time necessary to separate a pair of peaks with relative retention rz, and a desired resolution Rs in a column having a height equivalent to a theoretical plate and a capacity ratio, both referred to the second peak of the pair, at a linear carrier gas velocity ii (on which h2 depends!). This basic relationship is primarily thought to optimize the analysis time and to show the factors to which the gas chromatographer has to pay attention: fmt, using a column having a minimum h value at a maximum possible carrier gas velocity; second, selecting a stationary phase giving walues for the most critical substance pairs that are as far from unity as possible; third, as the term (k + l)’/k2 has minimum at k = 2 [64], the capacity ratio of the most critical pair should be brought close to this value by appropriate selection of the stationary phase, column temperature and column type; and fourth, demanding a resolution R of the critical pair that is only as high as necessary and not as high as possible. Returning to the carrier gas velocity (eqn. (108)), it can be realized that increasing ii means decreasing topt., provided that h is not seriously affected. The column efficiency (or, using a corresponding term, h,& has been shown (see Section 2.3.3) not to be seriously reduced when ii is increased to a magnitude called the optimum practical gas velocity (OPGV) [65]. The value of OPGV is higher than the optimum carrier gas velocity kpt., thus shortening the analysis time without a substantial decrease in efficiency. Depending on the column cross-section, temperature, film thickness and column temperature, it varies widely. As a guide, OPGL is about 1 m/min for packed columns and between 3 and 80 m/min for open-tubular columns [66]. A similar expression for the optimization of the analysis time has been developed [61]:
where topt. is the optimum retention time of compound 2. Increasing the linear gas velocity to values too far from the OPGV has been shown to leave no margin, as the ratio hliiapproximates to a limit for ii+ a. The minimum analysis time, again taking into consideration two compounds the separation of which is difficult, can be calculated by a simple equation [67]:
The time required for a gas chromatographic analysis naturally depends on the retention time of the last eluted compound, rR1, and can be calculated by [68]
where k, is the capacity ratio of the last eluted compound. Further details, especially of the time normalization chromatographic method, which is used for optimizing column length and temperature, are given in Section 3.1.2.
2.8. Definition of Symbols and List of Essential Relationships
2.8.
35
Definitions of Symbols Used (Table 1) and List of Essential Relationships (Table 2)
Table 1. Definitions of Symbols Used Symbol
Eqn. No.
Correlation
A = Idp
B =2bD8 B = 20, CL= mL/V,, C, = molVo
c = c, + c. + c,
2kd. (1 + k)' kh c,=& 1 + 6 k + l l k 2 Dg 9 6 ( l + k)2
c:
Definition
Activity coefficient Activity coefficient in the liquid stationary phase Activity coefficient in the mobile gas phase Term describing the multiplicity of the gas phase in packed columns Surface area of the liquid phase Term describing the longitudinal gas difhsion in packed columns (or in open-tubular columns) Concentration in the liquid stationary phase Concentration in the mobile gas phase Concentration in the solid stationary phase Term describing the resistance to mass transfer in the column Term describing the diminution of the mass transfer rate of solute from the stationary phase to the mobile phase and inversely As above
=
=& 1 + 6 k + l l k 2 D,
c c d2 =l
2 4 ( 1 + k)l
2k D, 3 (1 + k)'
Term representing the lateral diffusion in the mobile phase (packed columns) Term representing the lateral diffusion in the mobile phase (open-tubular columns) Term describing the diminution of the rate of mass transfer Mean thickness of the adsorption layer Mean film thickness of the liquid phase Mean particle diameter Diffusion coefficient of a solute in the gas phase Diffusion coefficient of a solute in the liquid phase
36
2. Basic Concepts
Table 1 (continued) Symbol
Cornlation
Eqn. No.
Dcfmition
Effective peak number Volumetric flow-rate at ambient temperature Volumetric flow-rate at T, [K] and column outlet pressure p o WPa] AGe = AHE- TASe AG2 - AG1
AH: = AHV- AHe AHS = AHV+- AHE
Excess molar free energy Separation enthalpy Height equivalent to a theoretical plate Height equivalent to an effective plate Partial molar adsorption enthalpy Excess partial molar enthalpy (of 1 mole of solute at infinite dilution in the liquid phase) Partial molar vaporization enthalpy Molar vaporization enthalpy of the solute from an infinitely dilute solution in the liquid stationary phase Vaporization enthalpy of the pure solute Retention index James and Martin factor Capacity ratio Adsorption coefficient Desorption coefficient Capacity ratio of the last eluted compound Distribution coefficient Partition coefficient between the stationary phase and the interface Henry constant Partition coefficient (GLC) Thermodynamically based partition coefficient (GLC) Partition coeEcient (GSC) Partition coefficient related to the molar fractions (GLC) Column length
37
2.8. Definition of Symbols and List of Essential Relationships
Table 1 (continued) Symbol
Eqn. No.
Correlation
Defmition
Mass (weight) of the effective adsorbent Mass of effective liquid stationary phase Mass of the solute in the gas phase Mass of the solute in the liquid phase Mass of the sorbate on the surface Molecular weight Number of theoretical plates Number of moles adsorbed Number of moles in the gas phase
P VG n, = -
RT
QLVLX nL = xns = -
M
p = p o x (ideal solution) p = p o y o x (real solution)
Number of moles in the liquid phase Number of moles of the liquid stationary phase Number of effective plates Partial pressure of the sample over its solution in the liquid stationary phase Ambient pressure Inlet pressure Ambient pressure at column outlet Saturated vapour pressure of the pure compound at the column temperature Partial pressure of water at ambient temperature Optimum practical gas velocity Ratio of inlet to outlet pressure in the James and Martin factor Relative retention Column inner radius Relative retention with respect to a standard Particle radius Gas constant Resolution of two peaks (representing two compounds)
38
2. Basic Concepts
Table 1 (continued) Symbol
Cornlation
Eqn. No.
Definition
Resolution related to the standard deviation of the fmt peak of a pair of peaks Surface area of an adsorbent Specific surface area of an adsor. bent Excess partial molar entropy Vaporization entropy of the pure solute Separation number (Trennzahl) Mobile time Minimum retention time Optimum retention time Retention time Adjusted retention time Required analysis time Difference of the retention times of two compounds Column temperature Trennzahl (separation number) Average linear carrier gas velocity Cmier gas velocity at column outlet Optimum carrier gas velocity Volume of the column tube Specific retention volume (GLC)
VN = q j = (tn - th3iF,
v; = v, - v, %= VRj
Volume of the gas phase Interparticle volume Volume of the liquid phase Mobile volume Corrected mobile volume Molar volume in the gas phase Molar volume in the liquid phase Net retention volume Adjusted retention volume Corrected retention volume Specific retention volume (GSC)
39
2.8. Definition of Symbols and List of Essential Relationships Table 1 (continued) Symbol
Correlation
WB
WE
WH
WI
Eqn. No.
= 4u
Peak width at base Peak width at half-height
= 2,355
a
w, = 2 a
Peak width at inflection points Molar fraction
X
2
Z=
Defmition
log r fi+2
log -
Z value characterizing the separation
fi-2
Separation factor (th> tAl)
a
B Y YO
Y: YL
YM
YN
r
r 80 ?lM
BP x
1 P PE P? P?
Pt
Phase ratio of a column k
Activity coefficient Activity coefficient at infinite dilution Activity coefficient in the gaseous mixed phase Activity coefficient in the liquid stationary phase Activity coefficient in the gaseous mobile phase Numerical labyrinth factor Concentration of an adsorbed compound i on the surface Concentration of an adsorbed compound i on the surface (adsorption molality) Interparticle porosity Viscosity of the mobile phase Factor describing the deviation of the particle shape from spherical Column permeability Numerical factor describing the packing Chemical potential' Excess chemical potential Chemical potential of a compound i in a real gaseous mixed phase Chemical potential of a pure gaseous compound i in the state of an ideal gas Chemical potential of a compound i in a real dilute solution
2. Basic Concepts
40 ~
Table 1 (continued) Egn. No.
Detinition
Pt
(77)
eL
(16), ( 2 9 , (66), (691, (68) (34), (34a), (39), (90). (101) (64)
Chemical potential of a pure compound i in an ideal dilute solution Density of the liquid phase
Correlation
Symbol
U X
Standard deviation Dimensional constant
Table 2. List of Essential Relationships and Definitions Eqn. No.
Equation
Defdtion
Adjusted retention time Adjusted retention volume Calculation of the volumetric flow rate Calculation of the net retention volume Specific retention volume (GLC) Specific retention volume (GSC) Correlation of V, with the partition coefficient Correlation of the partition coefficient with the phase ratio and capacity ratio Definition of the capacity ratio Definition of the phase ratio log v, = 2,30RTc constant
Correlation of V, with thermodynamic quantities
log v, = -t constant 2,30RTc
Correlation of V, with adsorption quantities
AH,"
+
n = 16
h = -L n
($)2
'):(
= 5,545
Calculation of the number of theoretical plates Height equivalent to a theoretical plate
41
2.8. Defmition of Symbols and List of Essential Relationships
Table 2 (continued) Eqn. No.
Equation
N = 16
($)’5,545 (6)’
Dchition
Calculation of the number of effective plates
=
Linear average carrier gas velocity Relative retention Separation factor Permeability of packed columns Permeability of wall-coated opentubular columns Van Deemter equation h=214+
2*Dg -+U V
D.
9 6 ( l + k)’
k
Bl a
+--(1 +2kk)’
Dependence of h on the carrier gas velocity for packed columns with liquid stationary phases
Xd’B 1 + 6 k + l l k Z
C, P
dia
kd
1
c.-a
--
d:P + 3 (12k+ k)’ DI c,-a
Dependence of h on the carrier gas velocity for wall-coated open-tubular columns
rta +-- 2k diu h = 20, y + 1+6k+llk2 u 24(1 + k)’ Dg (1 + k)’ kd Bl u
-
+ 3(12k+ k)* d:p 4 c, a
(70)
Separation equation
vx, P? YY log r2,1= log -= log (I+ log -
(70a)
Herington relation, separation equation
RT In rz,l= A(AG)
(81)
Correlation between the relative retention and the separation enthalpy Correlation between V, and the partition coefficient taking adsorption processes on liquid surfaces into account Resolution of two adjacent peaks
vx,
P2
?J!
(83)
R, =
2A t R +
wBI
(53)
42
2. Basic Concepts
Table 2 (continued) Egn. No.
Equation
Defhition
Expression describing the separation quality by nrsguirsd for given rZel and &l values of a column Expression describing the separation quality
(A)
. , = ' a4 ( - )
fq
= (1
+ kJ LU
Y
Fundamental relationship for selecting gas chromatographic columns and parameters Calculation of the required plate number to obtain a certain resolution of two adjacent peaks Similar expression to eqn. (loo), but with the base resolution (other than eqn. (53)):
Expression for optimizing the analysis time by selecting optimum parameters (on the basis of eqn. (100)) Calculation of the analysis time on the basis of the last eluted compound
3.
The Chromatographic Column
The success of a gas chromatographic analysis depends above all on the chromatographic column in which the separation process takes place. This part, often described as the "heart" of the chromatograph, can be designed as the pivot of the gas chromatographic system. The choice of its type, dimensions and stationary phase determines the feasibility, quality and duration of the analysis. In their original work, James and Murtin used columns prepared from tubes of 4 mm I.D. with lengths of 1.2-3.3 m, packed with a stationary phase consisting of a liquid distributed on the surface of relatively inactive porous solid particles. Since then, packed columns have been applied which, as early as 1958, could be optimized to an efficiency of 30000 theoretical plates for a 16 m length and an I.D. of 2.2 mm [70]. By the introduction of more inert supports and very low loadings, the range of application of packed columns could be extended to the analysis of steroids and other biologically important compounds [71]. An extraordinarily significant breakthrough in increasing the column performance is due to Go&, who was the first to report on open-tubular columns [72], a column type revolutionizing high-resolution gas chromatography and offering the facility for achieving column efficiencies of many hundreds of thousands of theoretical plates. Further important steps in column technology were the development of support-coated open-tubular columns [73] with increased sample capacity, of glass [74] and even flexible fused-silica open-tubular columns [75] and of cross-linking the liquid phase on the inside walls of open-tubular columns [76]. A detailed report on the historical development of columns was given by E m [77]. Before dealing in detail with stationary phases themselves, the column, although not being a major topic in this book, should be discussed in more detail than any other part of the chromatograph, as it is the vessel containing the stationary phase. Its function is to expose the stationary phase to the mobile phase such as to guarantee an optimum separation by maximum repeated partition steps between these two phases. There are several possibilities for achieving this goal, each having pros and cons. They will be considered in this section, including the description, where necessary, of pre-treatments and other preparation steps. We shall consider the details of different column types successively.
3.1.
Packed Columns
Originally, these classical columns had lengths of 1-3 m and inner diameters of 4-6 mm and were packed with coated supports having particle diameters of 0.2-0.4 mm. The column materials were copper, aluminium, stainless steel, and glass. As a result of numerous investigations and of the development of detectors of higher sensitivity, column inner diameters and particle diameters could be decreased and column lengths were increased, resulting in highperformance columns. At present the commonly used classical columns have the following characteristics. 3.1.1.
Column Materials
The most commonly used column materials are stainless steel, glass, quartz, nickel and polytetrafluorethylene (PTFE). It is important to note that the gaseous or vaporized sample com-
44
3. The Chromatographic Column
ponents come into contact with the column walls (and the walls of the connecting tubing). Hence, on metallic inner surfaces undesirable effects might occur, especially at higher temperatures, such as decomposition, conversion or adsorption of labile or polar compounds. Such substances should be analysed using glass, especially borosilicate glass, or quartz columns. On the other hand, metallic columns are more advantageous because they are more rigid, have good thermal conductivity and are more easily handled and processed. Nevertheless, the choice of the column material should primarily depend on the type and properties of the sample compounds. Columns made of stainless steel are predominantly applied. They are rigid and commercially available with various lengths and diameters. Before being used they should be washed with ethyl acetate, methanol and distilled water. Subsequently they should be filled with 50% nitric acid and allowed to stand for 10 min, then rinsed with distilled water to neutrality and with methanol and acetone. Finally they are dried in a stream of nitrogen. Lable compounds may be decomposed, e.g., steroids and pesticides, presumably owing to the presence of transition metals in the steel alloys. Glass columns are more inert provided that they do not contain alkali metals or calcium on the inner surface. In the analysis of labile compounds the inner surface must be treated by leaching the metal ions with hydrochloric acid, washing with distilled water to neutrality, then with methanol, drying, filling with a 5% solution of dimethyldichlorosilane in dried toluene, allowing to stand for several hours, draining, rinsing several times with dry toluene and methanol and drying. Another procedure for deactivating glass surfaces [78] involves heating for 4 h at 90°C with concentrated nitric acid, cooling, rinsing with distilled water and allowing to stand for 1h at ambient temperature with concentrated ammonia solution. The column is then rinsed with water and allowed to stand for 1h at ambient temperature with glacial acetic acid, then rinsed again thoroughly with distilled water. Ammonia treatment results in an activated porous surface, fit for the subsequent silylation, and acetic acid removes metal oxides from the surface. The active sites are subsequently blocked with a 20% solution of octadecyltrialkoxysilane in tert.-butanol and diacetone alcohol. Instead of the trifunctional silane, difunctional silanes (dimethyldichlorosilane) and siloxanes and monofunctional silanes (trimethylchlorosilanes), silazanes (hexamethyldisilazane, tetraphenyldimethyldisilazane) can also be used, the success of the procedure depending on the structure of the sample components and the conditions applied, especially temperature [79-871. Soda-lime glass columns, containing more than 20% of sodium oxide + calcium oxide, are less suitable for analysing polar and labile substances than columns made from borosilicate glasses which, however, also contain alkali, at lower levels, but also requiring the above treatment. The treatment with dimethyldichlorosilae is necessary in order to deactivate silanol groups, which are always present on glass surfaces and exhibit harmful adsorption effects. @or a detailed discussion of such surface phenomena and treatments see Section 3.5.1.) With the exception of aggressive fluorine compounds, most organic and inorganic samples, especially those containing strongly polar or sensitive constituents, can be better analysed in glass columns than in columns made of other materials. They also have, moreover, the advantage of being transparent and thus to allow the packing of the column to be inspected and cavities to be seen. A disadvantage is their fragility, especially in the column oven when using hydrogen as the carrier gas, because of the risk of explosion. Glass-lined metal tubing (GLT) columns (Scientific Glass Engineering, Melbourne, Australia) consist of an inner borosilicate glass tube encased in a stainless-steel sleeve, resulting in a column that retains the chemical inertness of conventional glass columns but which can withstand far more careless handling and offer better uniformity of diameter than can be expected from conventional glass tubes. By applying a temperature of about 800°C it can be coiled. The maximum column temperature is otherwise 500°C.
3.1. Packed Columns
45
Nickel columns, if pre-treated in the same way as stainless-steel columns, are almost as inert as glass columns, and steroids, alkaloids, analgesics, barbiturates and catecholamines are readily analysed [88]. PTFE columns are most suitable for investigating aggressive substances, e.g., HCl, Cl,, HF and ClF3, since this material is chemically inert. Nevertheless, caution is required, because owing to their processing, such tubes might be microporous, and traces of ambient air might enter the column and give rise to undesirable effects (oxidation, additional N2and 0,peaks [891). Even organic compounds may be adsorbed and retarded [90]. Owing to its small surface energy and to its relative neutrality [90], quartz offers certain advantages. The most important application of this material in gas chromatography, however, is in open-tubular columns. A detailed discussion is given in Section 3.4.1. Columns made of tantalum, tantalum-tantalum oxide or zirconium-zirconium oxide are appropriate for the analysis of corrosive samples [91]. Finally, it should be mentioned that deactivation even of metallic surfaces has often been carried out with alkylchlorosilanes or dazanes. A temporary improvement may occur, as chemisorption and adsorption sites seem to have been neutralized. However, one should be aware that if a reaction of metallic OH groups with the S i - C l groups of the chlorsilane has taken place, the resulting metal-0-Si bonds are hydrolyzable, and traces of water, which cannot easily be avoided, will soon re-establish the previous state.
3.1.2.
Column Dimensions
For solving not too complicated separation problems, a column length of 1-3 m is sufficient, corresponding, depending on the quality of the packing, to a number of theoretical plates between 500 and 6000, or with high-performance columns even 10000. Simple analyses, e.g., of sample components the boiling points of which are far apart, may be carried out with even shorter columns, having lengths of 0.3-1 m. For the solution of complicated problems gas chromatography offers two possibilities: increasing the efficiency (n) or the selectivity (r), of which the latter, if realizable, is always favoured. A pure or mixed stationary phase giving the best r values for the compounds under investigation is packed into a column of the required length. Originating from eqns. (38) and (93) describing the almost complete separation of the most crucial solute pair, an expression for the required minimum column length Lhcan be derived:
The general column length is 1-6 m. Longer columns are needed for the separation of multicomponent mixtures. Lengths exceeding 12m for packed columns are hardly suitable. It should be emphasized again, and illustrated by an example, importance of the selectivity: let k be m, then when rz,l= 1.05 and h = 0.05 cm a column length of 7.56 m would be necessary, whereas with r2,1= 1.20 only 0.54 m would be required. The resolution can be optimized by varying the column length and temperature by a method called length-temperature time normalization chromatography [92, 931. The basic equation of time normalization is
where L is the column length, r7 is the average linear carrier gas velocity, k is the capacity ra-
46
3. The Chromatographic Column
tio and subscripts A and B identify two sets of chromatographic conditions. As we have seen
L
in eqn. ( l l l ) , the term Y (1 + k ) corresponds to the retention time, and eqn. (112) only exU
presses the fact that tIu = fRB, i.e., the retention time of a certain compound is the same on both columns. If the carrier gas velocity remains constant and if the columns are operated in the vicinity of the Van Deemter equation, we can draw the following conclusions and apply the following relationships. As column B is lengthened with respect to A, the temperature at which B is operated must be higher than that for A in order to decrease the value of kB, and thus keep eqn.'(ll2a) true equality. The required capacity ratio, at any length L,which will keep tR constant, i.e., normalize the system, is calculated from eqn. (112) (subscripts B will henceforth be dropped):
The temperature required to give that k can be obtained from eqn. (114) [94]:
T=
TA 1 - (NAH")T A In (Wkd
(1 14)
where T
R AHV
= absolute temperature, = gas constant, = molar vaporization enthalpy
of the last eluted compound, i.e., the normal-
ized one. For each length change, be it an increase or a decrease, the normalizing temperature can be easily calculated via eqns.(ll3) and (114). AH" can either be approximated from Pictet-Trouton's rule or according to eqns. (28) and (29) from runs on column A at two different temperatures [8]. Gnishka [92] has shown that the optimum capacity ratio is given, using the fundamental relationship of eqn. (lOO), by
where r2.1 is a pair of substances difficult to separate, kptis the optimum capacity ratio giving the maximum resolution, a is a constant that can be determined [95] by running two analyses:
at different teperatures and b is another constant. Once the optimizing k value is known, eqns. (113) and (114) allow the calculation of the column length and temperature that will yield kptand thus maximize the resolution. A convenient procedure is as follows [93]. Install a column of any convenient length in the oven, operate at any sensible temperature, find roughly the velocity for the maximum efficiency and observe the resolution. If it is not sufficient, change the temperature for a second run. This will allow the calculation of the required parameters to solve the left-hand side of eqn. (115). This in return will yield the optimized capacity ratio, kpt, thus allowing the calculation, via eqns.(ll3) and (114). of the maximized lengths and the temperature at which it should be run. Then install in the chromatograph a column of the above length and operate it at the normalized temperature just calculated. The resolution thus obtained should be maximum at the analysis time. The two parameters temperature and length of the column are changed
3.1. Packed Columns
41
concurrently. Although one might be tempted just to increase the column length or decrease the temperature independently, Grushku [931 has shown that frequently a reduction in the column length and temperature might substantially increase the resolution at a constant analysis time. A limitation of the above equations is that they can only be used to give an indication of the parameters to be optimized and not to calculate their actual magnitudes, as complex and not binary mixtures usually have to be separated and as, ideally, the optimization should take into consideration both the column parameters (type, length, diameter, stationary phase, phase ratio) and the operating parameters (temperature, carrier gas nature and velocity). The column inner diameter of classical packed analytical columns ranges from 1.5 mm to 6 mm. Although the limits between the dimensions of classical packed and microbore columns are controversially defined, in this book only columns with diameters d, 5 1 mm are called microbore columns. An important parameter concerning d, is the particle diameter, d , , of the packing material, and the ratio d,ld, should not be lower than 0.03 or higher than 0.3. Each value offers different advantages, depending on the aim of the analytical application. Columns with a ratio of d,/d, near 0.3 are highly permeable, owing to the column permeability, which is proportional to the square of the particle diameter, according to eqn. (59). On the other hand, the efficiency is almost inversely proportional to the mean particle size (which is present explicitly and implicitly in eqn. (62)), and hence smaller particles and columns with narrower bores would be more advantageous. This has been emphasized by Huber et al. [96]. An important restriction in attempting to prefer fine particles is the higher column inlet pressure. Therefore, short columns have been used in such instances. An important factor that should not be overlooked, is the load. At the same impregnation rate, the load is directly proportional to the column cross-section and hence to the square of the inner diameter, i.e., load = constant d : . This is why higher sample weights can be analysed on columns with larger diameters. The sample weight ranges from approximately 3 mg for 5-mmcolumns to 1 mg for 3-mm columns, if other dependences (on temperature, weight of stationary liquid, and type of sample) are disregarded. Hence numerous factors have to be considered when choosing a suitable column diameter. Suitable column inner diameters might be 2-3 mm if the columns are packed with support materials of particle size dp = 0.12-0.15 111111. The shape of the column is most often a helix. Metal columns can easily be coiled by an appropriate device [97]. For the various forms, tubing connectors, etc., see ref. [97].
3.1.3.
Preparing of the Packing and Packing Procedures
In packed columns, the packing may itself be the stationary phase, if it is an adsorbent (GSC),or an appropriate solid support, being as inert as possible, which may be coated with a liquid stationary phase (GLC).Both the adsorbents and the inert solid carriers, and also the liquid stationary phases, will be discussed in later chapters, whereas the coating of the support and the packing of the column with the stationary phase will be dealt with here.
The carrier has to be coated with a liquid film that is as uniform as possible. The wettability of a surface can be expressed in terms of the “critical surface tension” yc [98], which is a value above which liquids with surface tensions yi show a finite contact angle (8) on a smooth surface. The value of y, can be obtained from a plot of cos 8 vs. yi, which is usually a straight line; the intercept with the Cos 8 = 1 line giving yc [99]. On a rough surface, as exists on the usual porous support particles, the (macroscopic) contact angle is decreased (and cos 8 increased) because the liquid phase penetrates into the scratches, holes and pores. The degree of roughness is characterized by rf, the roughening factor [loo]:
48
3. The Chromatographic Column
cos 8’
rr=-=-cos8
s’ s
where 8 = contact angle on the smooth and B on the rough surface, s = macroscopic surface area, s‘ = microscopic surface area. This equation indicates that a porous surface, because s’> s and hence cos 8’ > cos S, can better be coated than a smooth surface. We shall return to this fact when discussing open-tubular columns and column support materials. The weight of the stationary liquid (mJ that is coated on the support material to give a certain impregnation rate depends on several factors. Basically, the weight has to be chosen so as to avoid agglomeration; the material must not be sticky. The area and structure of the support determine the impregnation rate. High impregnation rates have two advantages: the residual activity of the support is less troublesome, and the sample weight can be increased. On the other hand, some disadvantages have to be considered: the efficiency decreases, the h value depends strongly on the gas velocity and the analysis time increases. As a guide for diatomaceous-type supports, 5-20% by weight is advantageous; for other materials, see Chapter 7 and ref.[101]. The optimum value for a certain separation problem can be found [lo21 as follows. The most appropriate stationary liquid is coated on to the support using an impregnation rate of about 5% by weight, and the stationary phase is packed into the column, which serves as a test column. The column temperature that gives the optimum resolution of the investigated sample constituents is determined and applied to the actual column, which will give, independent of the impregnation rate, the most advantageous resolution. The impregnation rate, on the other hand, is selected so as to give the required analysis time. Decreasing the weight of liquid is limited, however, because of possible adsorption effects on the uncovered surface, on the solid-liquid interface and in the pores, which contribute to the retention of polar compounds. However, provided that the support has been well deactivated previously, low impregnation rates are successful even in the analysis of high-boiling compounds. This applies to diatomaceous-type supports, previously deactivated, impregnated in the range ca. 0.1-3% [102]. Nevertheless, one must be aware of the following phenomena. The real stationary phase is not a single-phase but a multiphase system, and the net retention volume is not only determined by solution processes obeying eqn. (15), but additionally by adsorption on the gas-liquid [43] and gas-solid [lo31 interfaces and by adsorption exchanges between liquid and sample molecules interacting with the active sites of the support [104]. Such effects have a relatively greater influence with low impregnation rates because of the lower V, values and hence the relatively higher contributions of the additional effects expressed by the second term of eqn. (83 a) [45]: m
(for definitions, see Section 2.5). The adsorption on the liquid surface is favoured by the adsorption power of the support, which becomes apparent with very thin films. Such a modified layer may develop if a support with a specific surface of a.3 m2/g is less coated than 1% [105]. The minimum possible liquid loading depends not only on the activity of the support, but also on the polarity of the liquid and sample molecules, i.e., when polar compounds are investigated by using non-polar stationary phases, the danger of adsorption processes has to be taken into account. This applies especially to the calculation of retention indices, and differences in experimentally determined retention indices can often be attributed to a non-uni-
3.1. Packed Columns
49
form coating. Retention index differences of 5-10 have been observed with badly deactivated supports [106], whereas relatively inactive supports when coated with 2 2 % of non-polar poly(dimethylsi1oxane)did not indicate a retention index dependence on the liquid loading; merely a decrease from 2 to 1 resulted in a retention index increase of 10 units [107]. Finally, it should be pointed out that if the dimensions of the impregnation rate are as usual weight/ weight, then supports with higher bulk densities (glass beads, Chromosorb G) would contain a larger amount of liquid phase per unit support surface area, and also per unit column packing volume, than supports with lower bulk densities. Hence the degrees of impregnation should rather be related to the support’s surface area in order to be comparable. Supports with a small specific surface area and porosity (glass beads, quartz particles) must be loaded with less than 2% (w/w). There are many coating techniques. Generally the liquid stationary phase is dissolved in an appropriate solvent, from which it is deposited as a film on the support by means of one of the following procedures. The degree of impregnation (the loading) is given either in weight of liquid per 100 g of support (percent by weight) or in weight of liquid per unit packing (i.e. percent liquid stationary phase referred to the weight of impregnated support). Provided that the surface area of the carrier is known, it can also be a basis for the impregnation rate [108], the dimensions of which in this instance would be weight of liquid per square metre of support surface area. Before outlining the various coating procedures, it should be mentioned that detailed discussions have not been included, but only summaries of the principles, with short evaluations and descriptions of techniques to be recommended. For details the reader is referred to a textbook [lo91 and original papers. Filtration technique. The stationary liquid is dissolved in a suitable solvent and the support is added and briefly mixed. After filtration the residue is dried, e.g., by the fluid-bed technique [110-1131. This method is recommended with low loadings (<3%, w/w), the value of which ensues from the difference in added liquid and residual liquid contained in the filtrate. Frontal technique. The support is packed into the column and a solution of the stationary liquid is passed through the packed column until the concentrations of added and draining solution are equal [114]. The solvent is expelled from the column by heating in an inert gas flow. Evaporation technique. A weighed amount of stationary liquid to be coated is dissolved and, after addition of the support, the solvent is evaporated. The simplest way to carry out the evaporation is to use a water-bath and subsequently a steam-bath and a drying cupboard. However, although this method is often applied, it cannot be recommended, for several reasons. The main reason is the danger of oxidation of the liquid stationary phase. If we consider that a small amount of liquid is spread over a large surface area and present as a very thin film, in presence of oxygen partial oxidation of the liquid is likely to occur. For example, a packing with a loading of 5% (w/w) is prepared for a 6 m X 3 mm I.D. column, corresponding to a volume of ca. 42 cm3. Assuming that the support would have a packing density (in the column) of 0.5 g/cm3 and a specific surface area of 4 m2/g,the 1.05 g of stationary liquid would cover a surface of 84 mz. It can be calculated that the average film thickness would be ca. 12 MI, provided that the surface area is covered completely, and we can imagine this thin film to be exposed to atmospheric oxygen at oven temperatures, as often used, above 200°C and hence easily subject to oxidation processes with serious consequences in view of the quality of the packing. Therefore, the evaporation should be performed out of contact with air. A short-cut method con-
50
3. The ChromatographicColumn
sists in evaporating the solvent in a rotary evaporator. At ambient temperature a water-jet vacuum is applied for a few minutes until the support no longer evolves air bubbles. The vacuum is filled up with nitrogen, the bath temperature is elevated to ca. 40°C, then the pressure is reduced cautiously while rotating the flask and the solvent is withdrawn. Once the material is flowing freely, the rotation is stopped and the residual solvent is issued, maintaining the vacuum, at a bath temperature of 80°C [115]. Vacuum-vibration technique The stationary liquid is heated to 20-50K above its maximum operating temperature and by vacuum and vibration impregnated on the support [116]. A maximum loading of 14%is attainable and the occurrence of degradation products is unavoidable. Recommended procedures (a) Prior to coating, the support should be dried in a round-bottomed flask for 8 h at 300°C under vacuum. The cooled flask is vented with dry nitrogen and the solution of the liquid stationary phase is added. Residual oxygen is removed by a partial vacuum, until air bubbles no longer occur. The solvent is mainly evaporated by rotary evaporation and frnally under vacuum at a temperature of 100°C below the maximum operating temperature of the liquid phase overnight [117]. (b) In a special flask (hedgehog flask) the support mixed with the solution of the liquid stationary phase is cautiously evacuated and degassed and after ca. 10min disconnected from the vacuum pump and connected with a rotary evaporator. The temperature of the heating bath should be 20°C above the solvent's boiling temperature. Pure nitrogen is introduced into the flask at ca. 1Ymin. The solvent is vaporized while slowly rotating the flask. A visible reflux should be maintained, thus rinsing the suspension downwards from the walls of the flask [118]. (c) The support is placed in a Pyrex tube (480 X 23 mm) fitted with a sintered glass disc, closed at one end and evacuated at room temperature by connecting the other end to a vacuum pump. After several minutes, the tube is placed in a furnace at 450°C for 4-24 h while maintaining the vacuum, and removed from time to time and shaken to mix the powder. After heating, the tube is cooled to room temperature while still under vacuum. The tap connected to the pump is closed and the dissolved liquid is run into the Pyrex tube via a funnel connected to the tap. This tap is opened and the solution flows over and coats the powder. The tube is allowed to stand overnight. Then the cap, which closed the other end of the tube, is removed and, after the upper surface of the solution has reached the carrier surface, the remaining liquid is forced out under a gentle stream of nitrogen until the solid becomes freeflowing. The purpose of this procedure is to avoid the formation of water, which when using coated support material not been subjected to vacuudheat treatment could be eliminated by condensation of silanol groups to form siloxane bridges by heating in the column. This water could be trapped underneath the liquid phase and thus might account for the gradual deterioration of some types of packed columns [119]. Determination of the degree of impregnation The simplest way to determine the loading is Soxhlet extraction using the same solvent as applied in the coating step [120]. It should be noted that a polar solvent might leach soluble constituents from the support, thus falsifying the impregnation values. The loading with organic stationary liquids can be established by a simple ash determination provided that the liquid is quantitatively oxidizable at 800°C to form only volatile products of combustion such as C02 and H20[121]. Silicone liquid loadings cannot be investigated by this procedure; instead C and H can be determined by elemental analysis.
3.1. Packed Columns
51
It should not be neglected to run a blank with the uncoated carrier, especially ifit has been previously treated with organochlorosilanes or -silazanes. Carboranesiloxanes or organomet a l k phases can be determined by a boron or metal determination, respectively. A method that can be applied to most organic and to poly(dimethylsi1oxane) phases is based on the vaporization and degradation of the stationary liquid phase with exclusion of oxygen. With a temperature programme from ambient to 800°C within 2 h the sample is heated in a flow of suprapure nitrogen. After a further hour the “vaporization” is finished, without the formation of carbon. As water would affect the results, the non-impregnated support has to be analysed in the same way [122]. Finally, it should be pointed out that the determination of the degree of impregnation should be carried out only when the stationary phase was previously conditioned, because during the conditioning residual solvent and vaporizable constituents of the liquid phase could be released, which therefore would not contribute to the retention and must not be taken into account when, e.g., calculating V,. Details concerning the conditioning are given in Section 3.1.4. Packing procedure In a simple way U-tubes are packed by connecting both ends with small funnels through which small portions of the impregnated support are poured alternately while vibrating and slightly tamping until the upper surfaces of the packing can be seen 1 mm underneath the column ends. The ends are loosely closed with deactivated glass-wool in such way that the particles of the packing will be held back but the resistance to flow will not be increased. Packing of coiled columns has to be carried out differently, by tap-filling the precoiled column with a nitrogen flow at the column inlet and a vacuum applied at the column outlet [117, 1231. As the permeability of the packing decreases during its growth, the gas flow-rate produced by the vacuum also decreases, and the packing procedure is prolonged. Therefore, commercially available devices for pressurized packing should be used. To overcome the disadvantage of such appliances, that columns packed in this way are inefficient, a more favourable device has been designed for pouring the stationary phase slowly and continuously under a gas pressure into the column, so ensuring a homogeneous and tight packing [124]. It is especially suitable for laboratories in which many columns have to be packed. Coated PTFE supports should be packed into the column at 0°C to avoid electrostatic charges [125], using vibration [126]. A further recommended procedure applies pressure and ultrasonics. It has been developed especially for the optimum packing of columns with a coated support of the Volaspher type [127]. Finally, it should be emphasized that after finishing the filling of the columns, the pressure and vacuum must be reduced only slowly, as otherwise the tight packing would be loosened and the efficiency diminished. The efficiency of packed columns may also be improved if the packing is completed after having been heated to the maximum column temperature (which depends on the liquid stationary phase) [117].
3.1.4.
Column Conditioning
If the column is used for performing analyses immediately after the packing, then the retention times, selectivity and efficiency will change gradually and the detector will be contaminated. If the packing is not packed densely enough it will become tighter under the influence of the carrier gas flow. Solvent residues, volatile constituents of the liquid stationary phase and its degradation products will evaporate to various extent. At higher temperatures, the stationary phase might be degraded or cross-linked. In order to avoid these and other difficulties, any gas chromatographic column has to be conditioned before operation. Basically, the
52
3. The Chromatographic Column
stationary phase is heated, either in a special tube [128] or in the column itself, whilst (purified, especially oxygen-free) carrier gas is passed through it at a higher temperature than the subsequent maximum operating temperature. When the column itself is used, its effluent end has to be disconnected from the detector. In detail, the following procedures can be recommended: (a) The column containing the packed stationary phase is installed in the oven of the gas chromatograph at ambient temperature; it must not be connected to the detector. A flow of purified carrier gas, which especially has to be oxygen-free, is begun at a flow-rate of 10-20 ml/min. Purging the column for 30-45 min allows air to be removed from the column, which might otherwise cause oxidation of the stationary phase. The column is then heated at 60" for 30 min and subsequently programmed at 2 Umin to the desired upper limit. This temperature, the value of which should be 25OC above the desired operating temperature (but not exceeding the upper temperature limit of the stationary phase), is maintained for at least 12 h. The length of the conditioning time depends on the type of stationary phase, the impregnation rate, the maximum temperature of the analysis relative to the upper temperature limit of the stationary phase and on type and necessary sensitivity of the detector being used, and can require as long as 1 week if extreme parameters (operation at the maximum temperature limit of the stationary phase and at maximum sensitivity of the detector) are inevitable [129]. (b) The packed column is COMeCted with the injector, but not with the detector, [both as in (a)], and a flow of purified carrier gas is begun at the same flow-rate as in (a). After 30 min at ambient temperature the column temperature is raised to 60°C and maintained there for 1 h. After cooling to room temperature the carrier gas flow is stopped, the effluent column end is closed gas-tight and the column is heated to the upper temperature limit of the stationary phase and maintained for 14 h. The column is then cooled, the tube seal is disconnected and the carrier gas is allowed to flow through the column, which is heated to a temperature 20°C below the upper temperature limit of the stationary phase for further 14 h. This method eleminates troublesome surface activity from the support and column walls, especially when using glass columns packed with silicone stationary phases [130]. A shortcoming of both methods (a) and (b) is the lack of control of weight loss of stationary phase, as the relatively high weight of the column generally does not allow exact weighing. 3.1.5.
Column Testing
The column walls and connections must not permit any trace of carrier gas to pass as otherwise losses of carrier gas and sample would occur and, if the carrier gas is hydrogen, an explosion hazard may arise. The column is checked by sealing the effluent end and applying first an Nzpressure which, after interrupting the N2supply, must remain constant for a longer period, and then repeating the procedure with a Hzpressure. By the determination of the permeability, the mobile time, the efficiency, the selectivity, the time of analysis and the degree of impregnation according to the previous chapters and sections, a decision can be made as to whether the column comes up to expectations or preferably should be re-prepared. The peak shape, especially of polar compounds on non-polar stationary phases, indicates residual adsorption sites in the system, and deactivation procedures or a change of support would be required unless the amount of sample is too high for the available loading (in this instance the peak shape, however, would differ from the unsymmetric shape caused by adsorption phenomena). Further details concerning this subject will be dealt with when discussing open tubular columns (Section 3.3) and support materials (Chapter 7).
3.1. Micro-Packed Columns
53
The capacity of the column for the amount of sample can be established by determining the number of theoretical plates as a function of increasing sample volume. If the efficiency is only 90% of the value found for the smallest sample volumes, one has arrived at the maximum capacity. The upper temperature limit and its determination will be described in a subsequent chapter. The packing density (g/ml) can be calculated from the amount of packing and the volume of the column, which was previously determined with any liquid the density of which is known. If water is applied, the remarks in Section 3.1.1. should be considered.
3.1.6.
Pre-columns
A special type of packed columns are pre-columns. These are short columns, having lengths between 2 and 20 cm, and with inside diameters between 2 and 8 mm. They can, generally, be backflushed and are easily interchangeable. Their function consists in saving the column (and detector) from slowly or not eluting sample constituents and from changes in selectivity caused by strongly polar sample constituents having low vapour pressures, in that these are eliminated before arriving at the column by backflushing. Further, the analytical column can be saved from losses of stationary phase in the initial part of the column by using a pre-column with a high degree of impregnation. Moreover, they enable samples that contain nonvaporizable residues, e.g., polymers or salts, to be analysed without changing the quality of the analytical column. It should be borne in mind that the pre-column diameter affects the efficiency of the main column. The higher the pre-column inside diameter, the greater is the loss of efficiency of the column. This loss can partially be diminished by using of smaller diameter solid support particles, but a better method is to increase the pre-column temperature [13Oa]. Pre-columns are packed with an inactive support impregnated with 20-30% of an appropriate silicone phase, and they are connected gastight with the analytical column and operated isothermally at the highest possible temperature, the magnitude of which depends, of course, on the problem to be solved. The principle and installation have been described by Kaiser [131]. Conventional packed columns, if coupled with open-tubular columns, can be considered as pre-columns if they are used to remove the main sample components, thus allowing the trace components to be optimally separated in the capillary column. For trace analysis, this is an important possibility. Both column types are complementary to one another, the packed column with its high sample capacity (2-3 orders of magnitude higher than that of capillary columns) and the open-tubular column with its outstanding performance.
3.2.
Micro-Packed Columns
Owing to their very promising parameters of mass capacity, phase ratio and resolution with respect to the required analysis time, micro-packed columns have increased in importance. Their main features are microbore tubes with inside diameters (4)between 0.3 an 1mm and lengths of ca. 1-15 m, packed with particles of 0.007-0.3 mm diameter (dp).If the ratio of particle diameter to column inside diameter is d,/d, = 0.25, the columns can be packed regularly and densely. For dp/dc> 0.25, the packing becomes increasingly irregular and less dense. Transitions between both classical packed and micro-packed columns and regular and irregular micro-packed columns obviously occur, and an exact classification must therefore be arbitrary. Nevertheless, packed columns ought to be subdivided according to the following
54
3. The ChromatographicColumn
Table 3. Characteristics of Packed Columns Classical packed columns
Regular micro-packed columns
Imgular micro-packed columns
Tube i.d. (d,) [mm] Particle size (d,)
22 0.12-0.30
0.3-1.5 0.04-0.3
0.3-0.5 0.05-0.15
1-1 dP -
0.04-0.10
0.07-0.25
0.25-0.5
> 1000
5-20 0.4-1.2
<15 < 10 OOO*)
1-10 0.15-0.40 5 15 5 50 000
Simple separation problems; trace analysis; coupled techniques with spectrometry
Multi-component analysis; high-resolution coupled technique with MS; trace analysis
Short columns permit high-speedanalyses; coupled techniques with MS
dc
Sample capacity Cg] h [-I L [ml n Preferred applications
>o.s
s6 5 15000
9 n = 30000 for 16 m length obtained by Scott (70) is the exception, not a standard
parameters, as their merits and hence their application fields differ from one another, as shown in Table 3. It should be realized, however, that the values are only guidelines. The term “packed capillary columns”, used repeatedly in the chromatographic literature, has been deliberately avoided in this book in order to prevent misunderstandings. In this we agree with Stnrppe, who also suggested a subdivision into these three types of packed columns [132]. Owing to their high efficiency combined with a sufficient sample capacity, micro-packed columns offer excellent possibilities for the analysis of multi-component mixtures and trace constituents, and, especially, for coupling with mass spectrometry. They further combine high efficiency with short analysis times, thus permitting high-speed analyses. The stationary phases, as in classical packed columns, consist of both adsorbents (GSC)and liquid phases on supports, the particle sizes of which are given in Table 3. With smaller particle diameters (< 160 pm) the above packing procedures have to be modified by using ultrasonic vibration, maintaining an inert gas pressure [133-1351 or at least using electromechanical vibration of 20-100 Hz [136-1381. Apart from the detailed advantages a disadvantage should not be ignored, namely that the pressure drop, depending on particle size and packing density, ranges from 0.03 to 0.3 MPa, thus permitting, in the case of the higher values that occur with smaller particles, only relatively short columns. However, this disadvantage is not serious: owing to the higher efficiency and to the essentially lower C, term of the Van Deemter equation (eqn. (63)) compared with classical packed columns, even short micropacked columns yield high numbers of theoretical plates (e.g., up to 5000-10000 per metre [117, 1351 if operated at high pressures), and higher carrier gas velocities can be applied (lower C, term because of the smaller particle diameters) without seriously increasing the height equivalent to a theoretical plate, both advantageous conditions for high-speed analyses. It should be noted merely that longer columns packed with small particles require modified injection devices applicable to gas inlet pressures above 0.5 MPa [117, 1351.
3.3.
Open-Tubular Columns
Since the first detailed reports by Coluy [20, 211 on open-tubular columns, i.e., tubes that do not contain a packing, and in which the liquid phase is coated on the inside wall and the
3.3. Open-Tubular Columns
55
shape of the free gas cross-section is maintained over the whole length of the column, this gas chromatographic technique has developed considerably and its applicability has widened tremendously. This development is based on the fact that gas chromatography with open-tubular columns represents the most efficient method for separating mixtures of vaporizable constituents regarding information content, resolution and analysis time, and there is no doubt that the future of gas chromatography, disregarding preparative GC, special cases of trace analysis and coupling methods if larger amounts of the sample constituents are required, will lie in the application of open-tubular columns with either thin films or thin layers.
3.3.1.
Tube Material
Essentially, the tube used for the preparation of open-tubular columns consists of stainless steel, glass or fused silica glass, each having advantages and shortcomings. Stainless steel: This is commercially available as tubing with the required internal and external diameters, can easily be bent and connected to the other parts of commercial gas chromatographs, has good thermal conductivity and does not break. On the other hand, the quality of its inner wall is often rough, dirty and active towards polar and labile compounds. This disadvantage is especially important with wall-coated columns, whereas in support-coated columns the inner surface, being covered with porous particles, cannot be reached by the sample molecules. Recently, a rather inert metal column has been developed by Chrompack, HT-SIMDIST, especially for high-temperature application (to 450 "C) [138a]. Glacis: Glass is cheap, easily deformable and has a homogeneous surface. Nevertheless, because of some disadvantages, its application has been restricted to highly skilled chromatographers: glass capillaries are fragile and their dead volume-free connection to the other parts of the gas chromatograph is complicated. The main restriction is the poor wettability, i.e., the difficulty of forming a coherent and stable film of stationary phase on the inner wall of the glass tube, and only after about ten years of intensive investigations by numerous chromatographers did glass open-tubular columns become generally accepted. The production of glass tubes can be implemented by means of a glass capillary drawing machine, invented by Desly et al. [139]. Most often soda-lime glasses are drawn, as capillaries made from this glass type are more elastic than those from borosilicate glass, although the latter are more inert. Fused-silica glacis: Fused-silica glass is a column tube material with outstanding properties. It exhibits flexibility and great inertness, the latter being due to the low metal oxide content compared with soda-lime and borosilicate glasses. Unlike normal glasses with a wall thickness of about 0.3 mm, fused-silica glass capillaries are drawn to give a very thin wall of about 0.05 mm thickness and externally coated, by analogy with the fibre optics process, with an appropriate polymer, e.g., polyimide, to impart flexible tensile strength. The outside coating is thermally stable up to 340°C and has a film thickness of about 0.05 mm. Two types of vitreous silica glasses have been used as starting materials: (a) Conventional fused quartz derived from Brazilian quartz rock crystals by electric or flameCusion [76]. It consists of ca. 99.99%SiO, with a few hundred ppm of N 2 0 3as the main contaminant [76], the content of which can be lowered by further chemical purfication. (b) Fused silica has been produced by gas-phase hydrolysis of a very high purity silicon tetrachloride and fusion of the S O z formed. This synthetic product contains only lppm of impurities. The drawing process is carried out at the melting temperature, which ranges, depending on the composition, between 1700 and 2100°C. An appropriate drawing machine has been described by Lipsky et al. [76].
56
3.3.2.
3. The Chromatographic Column
Column Dimensions
Stainless-steel capillaries for gas chromatography commonly range in length from 5 to 100 m and have outside diameters from 1.0 to 1.6 mm, most often 1.59 mm (1/16 in.), and the inside diameters may vary from 0.05 to 1.55 mm, but commonly only from 0.2 to 0.3 mm (wall coated) or from 0.2 to 0.5 mm (support-coated open-tubular columns). It should be emphasized that the column diameter must be constant all over the column length, thus requiring good column drawing technology. From eqn. (63), it can be derived that the column inside diameter or radius, r,, strongly influences the efficiency (expressed by h), i.e., increasing r, increases h, and hence decreases the efficiency. On the other hand, increasing r, means increasing the amount of stationary phase in the column, because VL= 2 nr, L d,
(118)
(provided r, a dJ, where VL = volume of the stationary phase, L = column length and 4 = average film thickness of the stationary phase, and a larger amount of stationary phase permits the amount of sample to be increased without overloading the column. This is important when coupling techniques are to be applied or trace analyses are to be carried out. A further fact should not be neglected, namely the influence of variations in the column diameter on the phase ratio. This term has been defmed by eqn. (23):
As VG can be expressed by VG = nra L
(118a)
[provided r, % dl because otherwise VQ would be VQ= TI (r, - dd2* L] we obtain, on combining eqns.(23), (118) and (118a),
With jl= K L / k (eqn. (24)) we obtain from eqn. (119)
This means that the smaller the column radius, the higher will be the capacity ratio, k = tlR/tM, or, in other words, increasing the column radius would increase the phase ratio and decrease k (as KLdepends only on the stationary phase and the temperature and does not depend on the column dimensions). Decreasing k would, however, according to eqn. (99), require more theoretical plates to achieve the same resolution of two adjacent peaks. As a consequence, the column diameter has to be suited to the analytical problem. As an average, standard column diameters of 0.25-0.30mm (wall-coated) or 0.5 mm (supportcoated columns) can be recommended, especially for screening purposes. When volatile compounds have to be analysed, the phase ratio must be decreased, which can be achieved by increasing the film thickness or decreasing the column diameter, and vice versa when analysing high boiling samples, where the phase ratio has to be increased. The diameters of glass and fused-silica glass capillary columns are similar to those made of stainless steel. The inside diameter commonly ranges from 0.10 mm to 0.20 mm for high resolution analyses (only split mode) and for mass selective detectors. For split/splitless injec-
3.3. Open-Tubular Columns
57
tion, column diameters of 0.25-0.32 mm can be recommended. In spite of their lower separation efficiency (about one third compared with columns of 0.18mm I.D.), wide bore columns (0.53-0.75 mm I. D.) are favoured for trace analysis (owing to their expanded sample capacity of ca. 2000 ng, i. e., a factor of >20 at comparable film thickness!), for high speed analyses in the case of less complicated separation problems and for packed column conversion. Depending on the inside diameter, the outer diameter is 0.20-0.29 mm (0.10-0.18 mm I. D.), O . ~ ~ I I I I I I (0.25 mm I. D.), 0.40-0.50111111 (0.32I.D.) and 0.66(0.53I. D.). Returning to the column length, which, as it is directly proportional to the number of theoretical plates [eqn.(38)] and to the analysis time [eqn.(lll)], is an essential column parameter, we must carefully consider necessity and possibility. In order to save time when analysing a large number of samples, the column length should be chosen only so as to achieve the necessary resolution of the worst separable pair of sample constituents. This possibility, which saves analysis time contrary to the concept of "open-tubular columns = exceeded columns length = superior efficiency", has often been overlooked. Thus, unless very complex mixtures have to be analysed, relatively short columns can also solve many less complicated problems, in a shorter time. For calculating the required column length and for length-temperature time normalization, see Section 3.1.2.
3.3.3.
Wall-Coated Open-Tubular (WCOT) Columns
Wall-coated open-tubular (WCOT) columns, introduced into gas chromatography by Golay in 1958 [20, 721, contain the stationary phase in form of a thin film deposited on the more or less smooth internal surface of the tube wall. This doubtless most promising column type in addition to the support-coated open-tubular columns, has been developed particularly by Diksfra and De Goey [140], Desty [141], Kaiser and Sfruppe [142], Scoff [143], Condon [144], Zlatkb and Louelock [145], Schomburg [146], Grob [147], Tesaiik and Novofny [148], Eftre [149] and Guiochon [150]. 3.3.3.1.
Properties and Pre-treatment of the Inner Tube Surfaces and the Formation of Films
An essential requirement for high separation efficiency is the formation of a thin and uniform film along the total length of the column, as otherwise the formation of droplets might occur, which would decrease the efficiency of the column to unacceptably low levels, and problems connected with active sites would be even more in evidence. The applicability of a WCOT column stands or falls with the homogeneity and stability of this film on the wall of the column. The wettability of the surface depends, in one respect, on the surface energy of the tube wall, and in the other on both the surface tension of the liquid stationary phase and the (mostly unknown) interfacial tension between liquid and solid. A measure of wettability is the cosine of the contact angle, which is correlated with the mentioned quantities by eqn. (121) [90, 981: Ys = Y L S + YL cos @
(121)
where ys = surface energy of the solid, 12. = surface energy of the liquid stationary phase,
interfacial tension between liquid and solid, 0 = contact angle. In order to wet a surface sufficiently, 0 must be as low as possible and cos 0 as near as possiy~~ =
58
3. The ChromatographicColumn
ble to unity, and ys must be higher than fi. We have seen in Section 3.1.3 that the contact angle can be decreased by roughening the surface [eqn. (117)],and hence the film formation can be improved. Let us now consider the different surfaces. Stainless steel has relatively rough walls, which is advantageous with regard to film formation, as we have just seen. However, this material is active towards polar and labile compounds, and the activity must, prior to or simultaneously with the coating procedure, be reduced, e.g., by the addition of surfaceactive agents [151].Because of their lower activities, siliceous tube materials have been preferred for numerous gas chromatographic applications with the exception, perhaps, of the analysis of hydrocarbons, which is most often carried out with stainless-steel capillaries. Nevertheless, one should be aware of the fact that even glass and to some extent also fusedsilica glass have some activity. Its structure, composition and history of temperature treatment during the manufacture affect its behaviour, and especially its surface, the composition and structure of which influence column performance much more than the bulk glass characteristics [152],and this will be discussed in detail now. If we consider glass surfaces, we can expect metal and boron ions, which can serve as Lewis acid sites, causing adsorptive tailing. Owing to the substantially minor concentrations of these ions on fused-silica glass surfaces, this material is originally more inert [75].To improve the surface of soda-lime and borosilicate glasses, the oxides of metals (Ca, Mg, Al, Fe) and boric oxide have to be removed by leaching or etching procedures. Leaching involves, according to Grob and Grob [154],extensive hydration of the SiOz lattice, which permits soluble ions to be extracted with an appropriate solvent. The procedure consists in, e.g., filling of the tube with 20% HC1(90%of the tube length), sealing of the inlet and outlet and heating for 14 h at 160°C.After cooling the tube it is rinsed with water to neutrality [155].The surface is now a silica gel, which has to be dehydrated, but without removing most of the silanol groups because these are needed for the subsequent deactivation steps (preferably silylation). Therefore, the tube is purged with nitrogen and dried, applying a vacuum at temperatures between 150°C [156]and 300°C [155, 1571. In contrast to leaching, in the etching process the entire surface, including the lattice ions, is attacked and more or less dissolved [154].Etching reagents are gaseous HF or fluorine compounds that liberate HF at elevated temperatures, e.g., 2-chloro-1,1,2-trifluoroethyl methyl ether or NH4HF2[158,1591. The reagent is introduced, the column ends are sealed and the column is placed in an oven, the temperature gradient of which is small, heated (in the case of the ether) to 400°C and maintained at this temperature for 24 h. By the reaction of HF with the glass surface, silicon fluorides are formed, which are decomposed in the closed system to form silicon dioxide, which in turn is deposited in the form of whiskers. This uniform layer of silica whiskers provides, owing to the lack of the metal ions, and to the increased surface area and roughness, a suitable substrate for the liquid stationary phase. Wettability and the achievable (increased) sample capacity favour this procedure and its results. On the other hand, its disadvantage may not be overlooked. In particular, the subsequent, necessary deactivation seems to be difficult [160]. So far we have only considered the conversion of glass surfaces by leaching, rinsing and etching processes to produce a pure silica surface. The physical and chemical state-porosity, ruggedness, SiOH content and entrapped residual reagents-cannot be expected to be the same in each treated column. This is the reason why on the one hand excellent columns can be produced from conventional glasses, but on the other hand some columns are obtained that have to be rejected, as differences in surface density and porosity may affect the chromatographic behaviour [1611.Fused-silica glass columns, in contrast, possess only negligible, if any, Lewis acid sites and exhibit by nature a pure silica surface without any previous treatment and have a high degree of uniformity from column to column. The development of the fused-silica column has thus offered a better understanding of the role of silica surfaces with respect to deactivation and coating procedures, and treatments prior to the coating pro-
59
3.3. Open-Tubular Columns
cess are much more predictable and reproducible and less expansive than those required for conventional glass columns. Nevertheless, owing to the presence of surface SiOH groups, there are active sites on fusedsilica column surfaces and also leachedhinsed or etched conventional glass column surfaces. In an excellent survey, Jennings dealt with such surface phenomena, their origins and methods of deactivation [161]. Hydroxyl groups occur on those lattice silicon atoms, whose interatomic distances preclude the formation of additional siloxane linkages or which are formed after re-reaction of highly strained siloxane bridges with water. Different types of such silanol groups can be present: Lone silanols (a), vicinal silanols (b), geminal silanols or silanediols (c). OH
HO
I
- Si
HO
I -Si-0-5-
I
\ /
I
I (b)
(0)
OH
OH OH
(d
Free silanols are acidic and can react with ion-pair electron donors and hence they exhibit strong adsorption sites. When the interatomic distances of adjacent oxygen atoms are less than 2.8 A (2.8 10-lo m or 0.28 nm), the silanol groups are hydrogen-bonded to one another
(d)
(e)
[161] (d) and exhibit only weak or no adsorption, unless, through interaction with water, they become strong adsorption sites [162, 1631 (e). Heating to ca 165°C results in the desorption of water, which in turn can be readsorbed on cooling. Condensation reactions of spatially appropriate silanol groups to form siloxane bridges,
take place between 150 and >800"C. At first, on increasing the temperature from ambient to ca. 200"C, water from the condensation of geminal hydroxyl groups (c) is degassed, followed by water stemming from types (b), (d) and (e). This dehydration is reversible on heating to 400°C and partially even to 800"C, and increases with increasing temperature. Even above 800°C silanol groups still disappear, and strained, reactive siloxane bridges form. This reaction becomes reversible once again at much lower temperatures and exposure to room air and moisture [164]. We can now conclude that there exist in each glass column (after having removed Lewis acid sites) and fused-quartz and fused-silica glass columns (where Lewis acid sites, if any, would exist in negligible concentrations) a series of gradations from completely free silanols to fully bound silanols and from non-reactive to highly strained, very reactive siloxane linkages and bridges. These surface properties and compositions determine the possibility and degree of reactive and adsorption tendencies and also wettability. A column, just fused, exhibits a very high surface energy and hence can, according to eqn. (121), easily be wetted. On the other hand, a high surface energy means a high concentration of active sites, which has to be avoided in order to produce an inert column. Between these oppositing demands a compromise has to be reached. This is the goal of the following column surface pretreatments, and of the recently developed coating procedures described below and in Section
60
3. The Chromatographic Column
3.5.2., which avoid droplet formation by using gum phases, by bonding phases and by immobilizing the stationary liquid by means of cross-linking reactions. Because of their lesser importance in present, several pre-treatments, even though not long ago frequently applied and investigated, will be mentioned only briefly. In addition to the carbonization of methylene chloride and deposition of the formed carbon black on the inner column walls [165], the controlled deposition of sodium chloride [166, 1671 and barium carbonate [168] have been thoroughly investigated. In lieu of salt deposits, which have been found to decrease the column activity to a certain extent, thin layers of silica can be deposited by the hydrolysis of silicon tetrachloride at high temperatures [169]. These layers increase the surface area and wettability, but simultaneously the column activity is distinctly increased, for obvious reasons, and subsequent deactivation treatments are in any event necessary. Procedures meant to block active sites by surface active groups, by polar polymers or by thermal degradation products of polyethylene glycols sometimes seem to be successful (e.g. ref. [170]). However, the thermal stability is limited, and the column activity is frequently regenerated at elevated temperatures; further, abstraction of reactive sample constituents may occur, and the retention behaviour may be affected. One of the most promising ways of deactivating the inner walls of glass and fused-silica glass columns (which must be free from Lewis acid sites!) is chemical reaction of the surface silanols and highly strained siloxane bridges with thermally stable silylating reagents or other organosilicon compounds when reacted at relatively high temperatures. Let us first consider the true silylation reaction. A reactive silanol reacts with a silylating reagent to form silicon-oxygen-silicon bonds or, in other words, the active hydrogen atom of the SiOH is exchanged by a silyl group:
I I
-SOH
+ A-SiR3
+
I I
-Si-0-SiR3
+ HA
where A-Si is a reactive group (Cl-Si, CH,O-Si, HN-Si, etc.) and R is alkyl (most frequently CH3)or phenyl or, occasionally, if A is C1, a further C1 atom. For details of such reactions, see refs. [171-174, 85 and 1551. Most often hexamethyldisilazane is applied: 2 SiOH + (CH3),SiNHSi(CH3), --* 2 SiOSi(CHJ3 + NH, which, at high temperatures [85], in addition to silylation, might due to attack on the lattice by the liberated ammonia form fresh SiOH groups. However, this seems innocuous, as at such lattice points where the reaction SiOSi + NH, + SiOH + H2NSi might have taken place, in successive reactions, including water from condensation processes, excessive silylation might occur: SiNH2+ H20 + SiOH + NH3 and 2 SiOH would react as above to form 2 SiOSi(CH3),. Hexamethyldisilazane, of course, can also react with water to form hexamethyldisiloxane: (CH3)3SiNHSi(CH3)3 + H20 + (CHJ3SiOSi(CHJ3 + NH3 but this phenomenon is not of great consequence because of the excess of the reagent, unless the amount of ammonia formed is to be measured for elucidating the silylation rate. In lieu of hexamethyldisilazane, its phenyl and fluoroalkyl substitution products, R3SiNHSiR3 (R = CH,, CF3CH2CH2,CsHS),have been applied, which, when using modified conditions, also deactivate well [155, 156, 1751and provide a wettable surface for weakly and medium polar phases, as they increase the surface energy of the column walls [compare eqn.(l21)]. If organic compounds are to be silylated, often a mixture of hexamethyldisilazane and trime-
61
3.3. Open-Tubular Columns
thylchlorosilane is used [173] because of its higher silylation power. This is also true for silylating surfaces under mild conditions. However, such conditions are not sufficient for good deactivation, and high-temperature silylation should rather be used as the presence of additional trimethylchlorosilane is not necessary. Chlorosilanes themselves react with silanols, the reaction rate depending on the number of chlorine atoms in the molecule: CH3SiCI3 > (CH3)2SiC12 % (CH3)3SiC1. Monofunctional chlorosilanes R3SiCI (R = alkyl, phenyl, most often methyl) react according to
I I
I + C1SiR3 + -Si-O-SiR3
-SOH
+ HCl
I
the liberated hydrogen chloride being suspected of affecting the silica lattice. With dimethyldichlorosilane, one reaction course corresponds to the above-mentioned equation:
c1 I I I --SiOH + C12Si(CH3)*4 -Si-OSi(CH3)2 I I
+ HCI
one reactive Sic1 group surviving and being capable of consecutive undesirable reactions. However, as dimethyldichlorosilane is a difunctional chlorosilane, it may react with two properly spaced silanol groups: VH3
OH
OH
I
I
-Si-Si
I
CI
-+
1
-I
CI
\
0-Si-0
/ Si
__c
/ \
CH3
I
CH3
I
I
CH3 -
I1
+ 2 HCI
1
I
This type of reaction is also applicable to a trifunctional chlorosilane, but here unreacted C1 atoms will survive to a greater extent: I
CI
I
-Si-0-Si-CH? I I CI
and
CI I 0-Si-0
I
I
CH3
I
and have to be removed, as with dimethyldichlorosilane, by an additional reaction with moisture or methanol (producing new SiOH groups or hydrolyzable SiOCH3groups in the case of methanol). Regarding these restrictions, the following procedure seems more promising [155, 1611. After the leaching step (for opening of a maximum number of silanol groups) the column is thoroughly dried in order to remove traces of water (but without condensing silanol groups!) and rinsed with hexamethyldisilazane-diphenyltetramethyldisilazane(1:1) in two parts by volume of pentane, subjected to a vacuum, flame sealed and heated at 400°C for ca. 12 h. With fused-silica glass columns, the oven has to be purged with nitrogen in order to protect the polyimide that covers the outer walls of the column. One sealed column end is opened under toluene, which is allowed to fill some of the coils, then toluene is replaced with methanol and finally with diethyl ether. The amount of reacted reagent, recognizable by the ammonia liberated, is determined by measuring that part of the column which remains un-
62
3. The Chromatographic Column
filled when the pressure has equilibrated (as mentioned earlier, this only applies if water, which also could have reacted, can be excluded). According to Grob et al. [155], the proper degree of silylation has been reached when a third to a half of the column is filled with NH3, which prevents it filling with solvent. Further phenyl-substituted silylating reagents were used by Grob and Grob [176]. Deactivation can also be achieved using organosiloxanes. Schomburg et al. [177, 1781 coated dynamically the leached glass column with a poly(dimethylsiloxane),filled it with inert gas, sealed it and heated it at 450°C for 2-20 h. The degradation products formed under these severe conditions were considered to react with surface silanol groups or be adsorbed. The column was then opened and subjected to solvent extraction to remove that part of the decomposed polysiloxane which was not bound. It is well known in silicone chemistry that by thermal degradation of poly(dimethylsiloxanes), in addition to linear fragments of varying chain length, oligomeric cyclosiloxanes (hexamethylcyclotrisiloxane,octamethylcyclotetrasiloxane, and in decreasing amounts the higher homologues) are the main products that are formed. It can be assumed, and this would be analogous to ref. [179], where cyclosiloxanes were applied for deactivation of silanols on capillary walls from the start, that at elevated temperatures the cyclosiloxanes react with the silanols via ring opening of the cyclosiloxane and substitution of the reactive SiOH proton:
,
IjI.." P-EjiR2
- si -0
I
.... SiRZ I
0
I
0 -SiR2
--
I -Si-O-SiRZ
-0SiRZOSiR20H
I
on the surface. These groups exhibit less active sites and, on the other hand, create a better wettable surface for non- or weakly polar stationary phases, and are thermally stable with regard to chromatographic requirements. Similar ways of deactivating the inside wall and simultaneously improving the wettability have been the subject of recently published and current investigations [180- 1851. Murkides et al. [180] treated fused silica columns (0.20 mm i.d.) as follows. A plug of halfconcentrated hydrochloric acid ( ~ 1 8 % HC1 w/w) (10% of the column length) was forced through the tube by nitrogen until it had left the end of the tube. Both ends were carefully sealed and the capillary was heated at 140°C. The water may substantially increase the number of silanol groups on the surface and hence increase the reaction between these groups and functional groups of the consecutively applied organosiloxanes, being silanol-terminated or having become silanol-terminated by ring-opening reactions. The acid was displaced by one capillary length of dilute HCl (PH3), followed by one capillary length of methanol. Dehydration was carried out at 260°C for 5 h under a slow stream of nitrogen. The column was then dynamically coated (for deactivation!) (for true coating procedures see below) with 20% (w/v) solution of bis(cyanopropy1)cyclotetrasiloxane in CH2C12at a constant rate of 20 mm/s in order to cover the silica walls with an even film. After evaporation of the solvent (6 h), the tube ends were carefully sealed under vacuum, preventing any trace of air from entering the column. Chemical modification was achieved at 395°C (the outer surface of the fused-silica tube has to be taken into account, however, when choosing the temperature, as with a polyimide outside coating immediately after the drawing process its thermal stability is limited to ca. 340°C unless a protective gas (N,) is purged through the oven) for 2 h, the oven being heated and cooled slowly (5 Wmin). The column end was opened under methylene chloride, which was used to rinse the column free from excess of cyclosiloxane. The column was dried and was then ready for the true coating procedure with the stationary phase. In lieu of cyclosiloxanes, which have to undergo ring opening during the treatment prior to the reaction with surface silanols, silanol-terminated organosiloxanes can be directly applied for covalent bonding to the inner walls of the column.
3.3. Open-Tubular Columns
63
Verzele et al. [1811 synthesized an a,w-oligo(methylpheny1siloxane)diol CH3
I
HO-(Si-O)nH
I C6H5
and deactivated the tube by dynamically coating it with a 0.3% solution of the diol in CHzC12 at a speed of 2 cm/s. The capillary was evacuated, sealed and heated to 300°C at 3 K/min and held at this temperature for 15 h. Excess of reagent was removed as above [180]. Even with untreated fused-silica glass tubing exhibiting only relatively few silanol moieties on the surface, specially prepared non-polar, high-molecular-weight silanol-terminated polysiloxane stationary phases can be bonded covalently to the walls [182, 1831. Untreated fusedsilica glass columns were purged with nitrogen at room temperature before use to remove residual HC1 (unreacted Sic& present in the various procedures used in the drawing of narrow-bore tubing above 2000°C is converted by moisture into HCl, which clings to the inner surfaces of the tube unless preferentially removed [183]. For a 25-m column, all traces of hydrogen chloride disappeared, as indicated by moist pH paper, in ca. 5 min. Non-polar silanol-terminated poly(dimethylsi1oxanes) or weakly polar poly(methylphenylsi1oxane) mixtures with poly(dimethylsi1oxane) (5% phenyl), partially cross-linked by heat before use, were dissolved (30% in CHzC12for dynamic coating or 0.3-1.2% in pentane or CHzC12for statical coating). Traces of solvent were subsequently removed by gently purging the column with nitrogen for 2-3 h at 60°C and, with N2still flowing, the column was allowed to cool to ambient temperature. The ends were placed under vacuum for 30 min and then carefully sealed. The temperature of the column was slowly increased at 2-4"C/min to 370°C and maintained at this temperature for 5-15 h. After cooling, the seals were broken and the column was again purged with nitrogen for 1 h and rinsed with methylene chloride and pentane. The polymers, contacted directly to the untreated surface of the fused-silica capillary tubing at these high temperatures, readily deactivate and wet the column inner surface by distributing themselves as a homogeneous film throughout the whole length. Following the extraction with methylene chloride and or pentane, 85-95 % of the film appeared to be cross-linked in the case of partially gummified polymers prior to coating; if not previously cross-linked, only 50-60% of the condensed film was immobilized. From numerous results (e.g., refs.[177-186]), it can be concluded that no single pre-treatment can be used for the preparation of the surface even of as a unique glass as fused-silica which would enable one to coat it effectively with stationary phases of any polarity. Thus, surface pre-treatment has to be selected carefully, depending on the stationary phase to be employed. A step forward in this direction is the surface deactivation of untreated fused-silica glass with non-polar and polar prepolymers having various chemical compositions and subsequent cross-linking of the prepolymer to obtain the proper stationary phase film. 3.3.3.2.
Coating Procedures
As already emphasized in Section 3.1.3, a detailed discussion of coating procedures is not given in this book; only summaries of the principles and brief mentions of their pros and cons are included. For more details the reader is referred once again to handbooks (e.g., ref. [187]) and to the original papers cited. A practicable procedure is the dynamic technique introduced by Dijkstra and Goey [40]. The stationary phase is dissolved in a suitable, carefully purified solvent to give a concentration of 5-30% w/w. The solution is poured into a coating reservoir according to Schomburg and Husmann [188] together with mercury in order to produce reproducibly homogeneous films.
64
3. The Chromatographic Column
The coating device is connected with the tube, and by means of nitrogen, the flow-rate of which is controlled by a precision pressure regulator, a plug (10-30 cm long) of the impregnation solution, followed immediately by a mercury plug (3-15 cm long which corresponds to 0.02-0.11 g of mercury at a tube i.d. of 0.25 mm), is forced through the tubing at a velocity of 1-2 cm * s-*. The nitrogen flow is continued, even if both plugs have left the column, in order to remove the solvent; if the boiling point of the solvent is not too high, purging with nitrogen for 3 h at 60°C will be sufficient. For details of this method, see refs. [187-1911. The advantage of this method is that it is easily and quickly practicable. The disadvantages are that: The amount of the stationary phase in the column cannot be determined exactly; deviations of the inside diameter of the tube result in variations of the film thickness; and the efficiencies are lower than with static procedures. The static vacuum method first applied by Bouchd and Venele [191] consists in slowly vaporizing the solvent from the stationary phase solution in the column. The dilute solution (5l%),prepared with highly purified solvent, is degassed and sucked into the column. After complete filling, one column end has to be sealed, avoiding any air bubbles, e.g., by means of a mechanical device [192], and the column is placed in a suitable thermostat the temperature of which depends on the solvent. This is volatilized slowly, cautiously maintaining a vacuum. When the volatilization has finished, the sealed end is opened and traces of solvent and residues are removed by an inert gas stream. For details, see refs. [192-1981. The advantages of this method are that it is especially applicable to high-viscosity stationary phases, high-efficiency columns are produced; and the amount of stationary phase and the film thickness can be calculated. The disadvantage is that it is time consuming. The static evaporation procedure was introduced by Golay [20]. As above, the impregnation solution is sucked into the column, carefully avoiding evaporation of the solvent. After having sealed one end, the column, starting with the open end, is moved through a relatively hot zone into a thermostat, thus avoiding re-condensation of the already vaporized solvent. In the hot region the stationary phase film develops. Several parameters (velocity of the column movement through the heated zone, the applied temperature etc.) have to be optimized. For details, see refs. [20, 187 and 199-2031. The advantages of this method are that the amount of stationary phase in the column can be measured and the film thickness easily be calculated; viscous liquids can be coated; and it is not time consuming. The disadvantage is that occasionally inhomogeneous films are formed with low viscosity stationary phases (owing to non-uniform evaporation of the solvent).
3.3.3.3.
Bonding and Cross-Linking of Stationary Phases
Recent advances in bonding phases on to the inside walls of fused-silica glass columns and in immobilizing stationary phases in fused-silica glass columns have led to enhanced stationary phase stability. Owing to its outstanding advantages compared with the usual coated open-tubular columns, in all likelihood this method will, in conjunction with fused-silica glass column material, substantially extend the application of WCOT columns to increasing numbers of laboratories and fields. This is why this procedure, although still in mid-development, will be discussed here in detail. Let us first define the suitable chemical reactions taking place (a) on the wall surface between the surface silanol groups or strained siloxane bridges and appropriate stationary phase reactants, and (b) within the stationary phase itself prior to, during or after coating. Type (a), which we have already dealt with in the previous section, is the actual bonding reaction, which can be assumed to proceed according to one of the following simplified schemes:
65
3.3. Open-Tubular Columns
-SiOH
+
Si - R
HO-
I
I
-
I
I
R
+
CI
I
- Si-R
I
I
--
18
-
I I
R
R
I
-R
SiOH + H - 3
I
I
-
1 +(R2SiO)nt)
I
R
I
I
+ H2
-SiOSiR
R
-SiOH
+ HCI
-SiOSiR
R
I
H20
R
R -SiOH
+
-5iOSiR
I I
R
-
followed by ++I
R = C 3 z n + 1 , C ~ H> SClzHg, CnH7 CF3CHzCH2 CN(CHz)n, OH 9
9
*) cyclic Siloxanes **) thus linking two surface silanol groups, which otherwise would for steric reasons not react with them-
selves, via a short diorganosiloxane chain [small number n of D-units (R2SiO)].
\SI "'Si
/
\o/
/'
+
HOSiR,
\
-
OH
OSlR3
\ ISI - 0 - 5 - '
/ \
/
(highly strained) OH
\s/o\si/ H20 \o/ \ +
/
OH
\ I si -0 - si I/ /
\
/I
(being reactive os above shown) Si R2
66
3. The Chromatographic Column
PH
(being reactive as above shown)
*) Cyclic siloxane
Type (b) is a reaction taking place in the liquid stationary phase. Several terms have been used, e.g., curing, vulcanization, immobilization, cross-linking, in situ cross-linking, polymerization and condensation of prepolymers, free radical cross-linking, auto cross-linking, gummification, in situ vulcanization and production of non-extractable (or insoluble) films. Some terms stem from polymer chemistry, others from elastomer and rubber technology and two can be traced back to gas chromatography. In this book, we prefer in general to use those terms that describe the chemical reaction (cross-linking, polymerization, condensation) if the reaction itself is dicussed. The main reasons for the extensive investigations in this field are that on glass and fused-silica glass walls in open-tubular columns, column deterioration was often observed as a consequence of a physical rearrangement of the stationary phase (formation of droplets) at elevated temperatures and that the wettability of the surface with polar stationary phases, if any, was considerably obstructed. The first attempts to bond the phase to the surface in combination with cross-linking were carried out by Grob [165], Bossurf [203] and Mudani [204] and soon afterwards by Blornberg and co-workers [205-2071. So far, bonding and cross-linking has chiefly been restricted to silicone-type phases. This is based on the fact that these phases have been most widely used in gas chromatography because of their numerous merits, which will be discussed later in this book, and not least by the possibilities that they offer for tailormaking stationary phases. Bonding reactions have already been discussed and will only be described here when experimental details prior to the cross-linking reaction have to be given. Increasing the viscosity (i.e., increasing the chain length in the case of linear polymers) has been shown to improve film formation and film stability of liquid stationary phases [208], and polysiloxane gum phases with long chains have been synthesized, thus increasing the column efficiencies of coated fused-silica open-tubular columns [209-2 111. In spite of these improvements, the bulk phases are not yet immobilized, even when partially bonded to the surface. By introducing a slight degree of cross-linking, however, in the stationary phase itself, better immobilization can be achieved [205-207, 1211. Occasionally, a,o-(polysiloxane)diols have been partially bonded to the surface or/and partially coated and subsequently heat-cured after addition of a catalyst, e.g., tetramethylammonium hydroxide. Such coatings are less extractable, but the reaction having taken place is a polycondensation, leading to high-molecular-weight materials, and is not a desired cross-linking reaction. Cross-linking is a decisive step for immobilization. With the mentioned siloxanediols, tri- or tetrafunctional silanes, e.g., RSi(OR)3 or Si(OR)4, have to achieve a true cross-linking reaction (184, 185, 204-207) by condensation of hydroxy and, e.g., alkoxy groups with elimination of water, alcohol or ether and formation of Si-0-Si cross-links.
67
3.3. Open-Tubular Columns
Even if the thermal stability, due to the stability of the Si-0-Si bond, is outstanding, this type of cross-linked stationary phase has crucial disadvantages. High cross-linking levels, which give rise to diminished solubilities of sample molecules, and an undesirable activity of the column, are caused by residual SiOH and SiOCzHScontent [213].Further, residual alkoxy groups are susceptible to hydrolysis. Hence it is much more promising at present to use free radical generators to form carbon- carbon cross-links, as very little cross-linking (0.1-1%) is necessary to change long polymeric chains into insoluble rubbers, hence requiring only low levels of cross-linking agents [213].Free radicals can be generated by decomposition of organic peroxides, of azo compounds or by the use of high-energy radiation. The linking occurs according to the following scheme in the case of pure poly(dimethylsi1oxanes) (R' = radical):
)
CH3- Si - CH3
s I
R' R' I T
)
2R'
CH3 -5- CH3 -CH3 I
P
)
-Si -CH2
I
>
- CH2-Si
+2 R H
-CH3
I
Groups other than methyl present in the poly(diorganosi1oxane)molecule have a decisive effect on the cross-linking rate and the nature of the cross-linked product. If vinyl groups are present (in addition to the bulk of methyl groups), the methyl-to-methyl cross-linking is no longer dominant, but rather methyl-to-vinyl cross-linking [213,2141:
1
CH3-Si-CH I
R'
2
R'
2
CH3-Si -CH~-+CH3-Si-CH2-CH~-C~-Si-CH3
= CH2 T
}
I
I
I
P
P
P
+ R'
Because of the greater tendency of vinyl groups to cross-link, as is well known in silicone chemistry [215],lower levels of free radical generators are required, in order to achieve the same or even a higher degree of cross-linking. Numerous free radical generators have been used for the initiation of cross-linking. First, peroxides are most effective in forming insoluble stationary phases. Benzoyl peroxide, bis (2,4-dichlorobenzoyl) peroxide, dicumyl peroxide and di-tert.-butyl peroxide have, for example, been applied, of which dicumyl peroxide can be considered to give decomposition products that affect the column stability less than the other peroxides, and hence is the most often used. It would be beyond the scope of this book to review the numerous investigations in this area and therefore only a few bibliographic details are cited [210,212, 213, 216-2311. Second, in situ generation of free radicals can alternatively be accomplished by using azo compounds, e.g., azo-tert.-butane, azoisobutyronitrile, or azo-tert.-dodecane. Owing to its main decomposition products, nitrogen, isobutane and isobutene, which are non-polar and do not react with the polysiloxane chain, the azo-tert.-butane, in contrast to peroxides, cannot form acidic products which would attack the column. Unfortunately, it is less reactive than the peroxides and requires higher temperatures to obtain acceptable free radical generation rates, and hence comparable cross-linking [213].Nevertheless, apart from dicumyl peroxide, azo compounds can be used; for details see refs. [182,211, 213, 227 and 232-2371. A third procedure for cross-linking silicone, especially polar silicone, stationary phases consists in applying radiation, either gamma radiation from a 6oCosource [227,238, 2391 or accelerated electrons from a Van de Graaff generator [240].It offers the advantages that no chemicals with harmful effects have to be added, that the reaction takes place at ambient tempera-
68
3. The Chromatographic Column
ture and that the columns may be tested before cross-linking. Owing to the expansive equipment required, its application will be limited, however. More readily available is the equipment for a fourth method, in situ cross-linking with ozone [241, 2421. Immobilization can be achieved at low temperatures (ambient for dimethylsiloxanes) or medium temperatures (150°C for phenyl- or cyanopropyl-substituted siloxanes). Nevertheless, we feel that this reaction type should be treated with caution. Even if silicones have proved to be relatively resistant towards oxidation, the presence of ozone must have some effect. The absence of an infrared carbonyl band, or at least only a very weak absorption, observed after curing, [242], may not be admissible evidence, nor are the marginal differences in Kovhts retention indices [242]. Naturally, this also applies to peroxide-initiated cross-linking. More thorough investigations need to be carried out if small alterations, which could affect the analysis of labile or trace compounds, are to be recognized. A few cross-linking procedures will now be given in detail.
Procedure (a), according to Wright, Peaden and Lee [213]. Deactivation consists in rinsing the fused-silica capillary tubing with 5-10 ml of methanol at room temperature, then purging with Nz for several hours to remove any traces of methanol. Next, octamethylcyclotetrasiloxane (D4 in organosilicon chemistry nomenclature) is dynamically coated by filling ca. 20% of the column and then rapidly pushing the D4 plug through the column with Nz until it has been expelled. After sealing both ends, the column is heated for 2 h at 420°C (whilst protecting the polyimide outer coating with N2!),and subsequently the column is purged with Nz for about 30 min at 350°C to remove any residual D4. The column is statically coated. The long-chain poly(dimethylsi1oxane) containing vinyl groups is dissolved in purified pentane and the concentration is selected to give a film thickness of 0.1-0.5 pm. Solid dicumyl peroxide, dissolved in methylene chloride (l%),is added to the coating solution 30 min prior to coating to give a concentration of 0.28%.After the usual coating the column is purged with nitrogen, sealed and temperature-programed from 40 to 175°C at 4 W rnin and held at 175°C for 15 min. After cross-linking, the column is washed for 30-60 rnin with 5-10 ml of methylene chloride. Subsequently it is conditioned with a slow carrier gas flow for 1h at 40°C to desorb any residual solvent from the stationary phase left from the washing procedure and then temperature programmed to 260°C at O.S"C/min and held there for 8 h. When using azo-tert.-butane, which is a liquid at room temperature, the column is coated in the usual way, without the free radical generator prior to cross-linking. The coating is followed by saturation of tke stationary phase with the vapour of azo-tert.-butane by bubbling nitrogen through the azo compound and purging the coated column at 40°C for 2 h. Subsequently, for dynamic curing the column is attached to an argon manifold in an oven and heated at 5-1O"Umin to 220°C and maintained there for 15 min; the argon linear velocity is 10 c d s . After curing, the column is rinsed with 10-25 column volumes of methylene chloride-acetone (5050,v/v) and then conditioned with a rapid carrier flow for 30-60min at room temperature. After being reconnected to the argon manifold, the column is heated at 5"C/min to 350°C and held there for 4 h with an argon velocity of 25-30 cm/s. Procedure (b), according to Lee and co-workers [210, 2361. First, a mixture of dimethyldichlorosilane, methylphenyldichlorosilane, diphenyldichlorosilane, methylvinyldichlorosilane and 1,4-dimethyl-1,1,4,4-tetrachlorodisilethyleneis prepared for synthesizing a polymer with the desired phenyl and vinyl contents. Hydrolysis of the mixture is accomplished by dissolution in an equal volume of acetonitrile and adding an equal volume of water, followed by an equal volume of methylene chloride. The methylene chloride layer is then extracted with distilled water to neutrality. The methylene chloride is finally removed by gentle warming under a nitrogen purge. Polymerization
3.3. Open-Tubular Columns
69
is accomplished by adding 0.05 wt-% of the catalyst tetramethylammonium hydroxide and heating at 110-130°C until the viscosity of the polymer ceased to change. The catalyst is then rendered inactive and the polymer is end-capped by the addition of trimethylchlorosilane. As the polymerized product contains both low- and high-molecular-weight moieties, fractionations have to be performed in order to remove the lower-molecular-weight materials. This is achieved by dissolving the polymer mixture in four times of its volume of CHzClzand adding an equal volume of methanol, which causes the higher-molecular-weight moiety to precipitate. After repeating this precipitation four times, the final precipitate is collected. In case of a 50% phenyl-, 49% methyl-, 1%vinyl-polysiloxane, the solvent used to prepare the coating solution is ratio n-pentane:methylene chloride 2:1, v/v. An appropriate amount of the polymer is dissolved to give the desired film thickness. The column is coated statically. To give a film thickness of 0.25 pm in a 15-m column of 0.1 mm I.D. in a reasonable time, the coating temperature can be chosen to be as low as 28°C. After coating, the column is purged with nitrogen for 30 min at room temperature. For cross-linking, azo-tert.-butane is purged through the column for 2.2 h using a special purging device [236]. Cross-linking twice, each time after purging azo-tert.-butane from opposite ends of the column, increases the successful cross-linking rate. After azo-tert.-butane purging, the column is sealed and temperature programmed from 40 to 220°C at 4"C/min and held at 220°C for 1 h for cross-linking. After cross-linking twice, the column is normally temperature programmed from 40 to 250°C at 0.5 Wmin under a gas stream (N2or He) and held at 250°C overnight. The finished column is then evaluated as usual and subsequently washed with 50-100ml of methylene chloride by means of an HPLC pump. Washout data can be obtained from the difference in k values before and after washing. Using this procedure, nearly 100%non-extractability can be achieved. Procedure (c), according to Venele et a1 [181]. Although not a cross-linking reaction, the following procedure is described here for two reasons. First, because free radical generators are avoided, decomposition products cannot occur and hence cannot give rise to column activity. Second, the polarity and selectivity are well defined and equal to those of, e.g., OV-17, because the prepolymer does not contain any vinyl or tolyl groups, which otherwise are necessary to achieve cross-linking of phenylsiloxanes initiated by free radical generators. Further, it is easily practicable. A prepolymer 1, synthesized by alkaline hydrolysis of methylphenyldichlorosilane, is used to deactivate the previously leached fused silica column. Deactivation is achieved by dynamically coating the column with a 0.3% solution of prepolymer 1 in CH2C12at a velocity of 2 cm/s. The column is then evacuated, sealed and heated to 300°C at 3 K/min and held isothermally at 300°C for 15 h. Excess of reagent is removed by rinsing the column with 10 ml of CHzClz.Prepolymer 1 is further polymerized to a semi-gum, called prepolymer 2, by heat-curing under nitrogen whilst continuously controlling the viscosity to avoid complete gummification and loss of solubility. The deactivated column is then statically coated with a 0.1-0.3% (w/v) solution of prepolymer 2 in CHzClz.The silanol-terminated prepolymer 2, coated on the column wall, is immobilized by in situ gummification by heat-curing. This is achieved by heating the column repeatedly from 150 to 250°C at 3 K/min for 15 h under a low flow of carrier gas (0.1 ml/ min). The column is then rinsed with 5 ml of CHzClzand conditioned. The immobilization yield was 100%. Residual terminal silanol groups can be capped by hexamethyldisilazane at elevated temperatures. Procedure (d), according to Markides, Elomberg, Buijten and Wannman [223, 223al. Bis(cyanopropy1)dichlorosilane is hydrolyzed with acid catalysis to form octakis(cyanopr0py1)cyclotetrasiloxane [212], which is coated dynamically in a 20% (w/v) or 2-5% (w/v) [223a] solution in methylene chloride at a constant rate of 20 mm/s on the inner walls of a fused-silica column. Prior to the coating, the column has to be leached with 20% hydrochloric acid at
70
3. The Chromatographic Column
100°C for 12 h, rinsed and dried [224, 1551 as described in Section 3.3.3.1, as a fused-silica surface seems to contain too few silanol groups to be sufficiently modified by this cyclosiloxane. To avoid any contamination, leached glass vessels, high-purity chemicals and freshly prepared solutions should be used for the leaching procedure. The dynamic coating has to be performed carefully in order to form an even film along the length of the capillary, and no air or solvent residues should be present. During the dynamic coating, a buffer capillary is connected to the column end. Immediately after the coating plug has left the column, the flow of dry nitrogen passing through the column is drastically increased, and the evaporation of the solvent is allowed to proceed for 6 h. The capillary is then evacuated and both ends are sealed. The sealed capillary is heated in an oven to 395°C at 5 Wmin and held at the fmal temperature for 1.5 h, after which the oven is allowed to cool slowly to room temperature. One end of the column is opened under the surface of CHzClz, thus forming a plug with which the column is rinsed using nitrogen. Finally, the column is dried by flushing with dry nitrogen. Now, the column is modified for good wettability with polar cyanopropylsiloxane phases. Prepolymers with a high degree of cyan0 substitution are prepared from bis(cyanopr0pyl)dichlorosilane, methyl(toly1)dichlorosilaneand dimethyldichlorosilane by basic reversed hydrolysis (in methylene chloride solution, according to Patnode and Wilcock [223b]). A linear methyl(viny1)pentasiloxane is synthesized from methyl(viny1)cyclopentasiloxane by ring-opening with boiling butanol [243]; 1.7-3 mol-% of this vinyl siloxane is included in the reaction mixture to incorporate vinyl groups into the gums as methyl(viny1)siloxanesegments rather than single methyl(viny1)-siloxane units in order to facilitate cross-linking of gums containing bulky groups. Further, 0.5 mob% of 1,4-dimethyl-1,1,4,4,-tetrachlorodisilethylene is added [analogous to procedure (b)] in order to introduce a slight degree of crosslinking in the prepolymer. The synthesis is carried out in acetonitrile. Polymerization is carried out at 110°C for 10 min using ammonia solution as catalyst [215, 2441. The reaction is performed with stirring under nitrogen. The gum is dissolved and purified by washing with dilute hydrochloric acid, followed by water, to remove catalyst residues. Finally, after drying the gum with calcium sulphate, residual silanol groups are capped by reaction with 1,3-divinyltetramethyldisiloxanein refluxing acetonitrile for 6 h under an atmosphere of nitrogen. This polymer is dissolved in acetonitrile-diethyl ether (3:2), and 5% of dicumyl peroxide, calculated from the amount of stationary phase, is added to the coating solution. Before filling the column, the coating solution is filtered and centrifuged; only freshly prepared solutions are used. Coating is performed by the static method and with the column immersed in a horizontal position in a water-bath. The coated column is opened under an atmosphere of dry nitrogen, 1min before disconnecting the vacuum, and is then directly purged with dry nitrogen for 30 min. The gum phase is then cross-linked in situ by dynamic curing in a GC oven, programmed from 40 to 170°C at 5 Wmin, the final temperature being maintained for 40 min. During curing the column is rinsed with a slow stream of dry hydrogen (0.1 ml/min). After cross-linking, the column is rinsed with 5 ml of methylene chloride and conditioned in a gas chromatograph programmed to 250°C at 1Wmin. Cyanopropylsilicone rubber, cross-linked via Si-0-Si, can be obtained according to ref. [212], and trifluoropropylsilicone rubber with cross-links of the type Si-C-C-Si according to ref. [220]. Conclusions concerning bonding and cross-linking of stationary phases With the development of immobilized phases by bonding and in situ cross-linking reactions during the past 5 years, the overall quality of WCOT columns has been vastly increased. The following advantages have become apparent: enhanced film stability involving long column lifetimes and high efficiencies of both non-polar and polar stationary phases on the col-
3.3. Open-Tubular Columns
71
umn wall; minimal phase stripping from sample injection solvents, thus allowing the injection of large amounts of sample, including samples dissolved in both non-polar and polar solvents, even in water; the possibility of washing the columns to remove non-volatile compound deposits, provided these are soluble; higher film thicknesses while maintaining stable films can be chosen; in spite of cross-linking, no significant differences in diffusion coefficients compared with conventional stationary phases [245] have been observed; cross-linking suppresses the glass transition mechanism of poly(dimethylsiloxanes), thus allowing efficient separations of, e.g., CI-C5 hydrocarbons at sub-ambient temperatures, even at - 70°C; and reduction of column bleeding can generally be observed as a consequence of cross-linking (including conditioning) and end-capping [which converts terminal silanols (which would give rise to degradation reactions) into innocuous SiOSiR,-groups, in the case of silicone stationary phases]. There are also a few disadvantages: temporarily, the immobilization is, with the exception of poly(ethy1ene oxides), restricted to silicone phases at present, but as different functional groups can easily be attached to the siloxane skeleton (C6H5,CN(CH2),, CF3(CH&, C6HS, C6H4,etc.), a wide range of selectivity can be achieved; cross-linking of polar silicone phases is more difficult than cross-linking of poly(dimethylsi1oxanes) and generally requires additional vinyl or tolyl groups and higher amounts of the free radical generator, e.g., peroxides; and decomposition of the cross-linking initiators, especially of peroxides, may cause undesirable column activity. In spite of these disadvantages, it can once again be concluded that fused-silica glass capillaries of different diameters, containing bonded and/or cross-linked tailor-made stationary phases, of different film thicknesses will increase the applicability of WCOT columns tremendously, at the expense of packed columns.
3.3.3.4.
Film Thickness of the Stationary Phase
We have seen in eqns. (62) and (63) that it is preferable to choose a stationary phase film that is as thin as possible, as both the C, and C, terms, i.e., terms describing the resistance to mass transfer from and in the stationary phase, respectively, depend on the stationary film thickness, d , . The first term is proportional to d : , and the second term is proportional to d , . Hence a reduction in d, will result in a decrease in C, and, owing to the square dependence, a greater decrease in C,, hence reducing hmin,i.e., increasing the column efficiency. However, this is only one side of the picture. On the other side we have to take into account, that a reduction in d, leads to an increase in the phase ratio, B, as
p , 2Ldl
(119)
and a higher B value means, according to eqn. (24) ( B = K L / k ) ,a smaller k value (because KL is a constant). A smaller k, in turn, would require, corresponding to eqn. (99), more theoretical plates to achieve the same resolution. This would be especially serious when analysing low-boiling compounds, which exhibit very small capacity ratio values. Therefore, we have to reach a compromise depending on the tube material (which might be active!), on the column temperature (which can be reduced when the film thickness is reduced) and on the physical and chemical properties of the sample components. If we return to the phase ratio, B = VG/VL [eqn. (23)], it is clear, that it can easily be adjusted by changing either the film thickness (which is contained in VJ or the column diameter (expressed as V,). Volatile compounds, for example, can well be separated in columns with an increased film thickness of up to 5 pm, compensating for the lower separation efficiency by an increase in
72
3. The ChromatographicColumn
column length, whereas the separation of high-boiling compenents can be accomplished on shorter columns with relatively thin stationary phase films of dl = 0.1 pm [2471. Although hitherto the exception, open-tubular columns of inside diameter ~ 0 . mm 5 and with film thicknesses of d, = 2 to 7 pm, rendered possible by cross-linking, can be prepared, thus permitting the injection of larger sample amounts but nevertheless maintaining better performances than with packed columns, especially shorter analysis times, lower adsorption of polar compounds and higher thermal stability [248, 2491. The lower limit of the film thickness may be 0.1 pm; the usual range on glass or fused-silica glass walls is between 0.2 and 0.3 pm and on stainless steel walls owing to their activity, between 0.5 and 0.6 pm. Using the static coating method, the film thickness can easily be calculated from dl = 5r,c
(122)
where dl = film thickness [pm], r, = column radius [mm] and c = concentration of the stationary liquid phase in the coating solution [%, w/w], as all of the stationary phase being contained in the coating solution will remain in the colUmn. If the column is to be coated dynamically, the film thickness may be predicted by one of the following equations (Fairbrother and Stubbs [250]):
dl = rcc
4 4 + tl/n
where d, = film thickness [pm], r, = column radius [mm], u, = coating velocity [mm/s], 9 = viscosity of the coating solution
Pa. s],
[
yc = surface tension of the coating solution 10-
c
= concentration
4
of the stationary liquid phase in the coating solution [%, w/w] (Nouotny et al. [251])
or (Guiochon [252])
4=
134r c
u,q 3 ( 7 T .)
The best accord with experimental results is given by eqn. (124), except with very thin films or very low viscosities of the coating solution, where eqn. (125) is to be preferred (2521. The film thickness can, however, also be determined from gas chromatographic data (Crumers et al. [245]): d-I-
kr, 273 2YBeL T
where
k = capacity ratio, V, = specific retention volume of a solute at the column temperature T [K], eL = density of the liquid stationary phase at the column temperature.
13
3.3. Open-Tubular Columns
The values of r, and eL are known, T and k can be measured and V, can be obtained advantageously from, e.g., corresponding data from packed columns.
3.3.3.5.
Quality Tests of WCOT Columns
In addition to the usual determination of the values of k [eqn. (21)], B [eqns.(23) and (24)], n or N [eqns.(36), (37), (40) and (41)], the following characterizations of the column ought to be carried out: (a) Determination of hdn (smallest experimental h value by plotting h =f(@ at Copt.(see Fig. 4). (b) Calculation of the coating efficiency, i.e., the ratio of the theoretical and experimental minimum height equivalent to a theoretical plate [192]: CE =
hmh (theor.)
(exp.) using the k value of a standard compound, the selection of which depends on the operating temperature. hmin(theor.) can be calculated from hmin
hmin(theor.) = r,
JW,
r, = internal column radius, k = capacity ratio.
Hence it follows that
or, if CE is to be given as a percentage, (128a) Instead of CE, the term UTE (utilization of the theoretical best efficiency) has also been used, see, e.g., ref. [223]. At this point it should be noted, that eqn. (127) is a simplified expression, neglecting both the pressure drop in the column and C, [eqns. (62) and (63)]. Provided that the dif€usion coefficients are known, an expression developed by Cramers et al. [253] can be utilized, which has been proved to correspond better with the experimental data [252]. (c) Determination of the loading, i.e., of the amount of sample that can be injected without a serious loss of efficiency. An approximation is [254] SL [g] = 0.05 M .$(1
+ k)
*
(129)
where SL = sample loading = molecular weight of the standard compound, d, = column inside diameter, k = capacity ratio of the standard compound, which should be chosen such as to give k > 10.
M
A simple experimental means of obtaining the maximum solute load consists in injecting increasing amounts of sample and measuring the peak width at half-height until the value of
74
3. The ChromatographicColumn
wH [eqn.(33)] has increased to 5% above the average wH value. This value is the maximum solute load (MSL). (d) Determination of undesirable column activity. A serious problem, especially in the trace analysis of polar compounds, has been the possible residual column activity. The column therefore has to be checked by an appropriate test method. One must be aware of the fact, however, that deleterious effects might be caused by the injector, injector sleeves, the tube connectors, etc., unless these parts of the instrument have been properly deactivated. The inertness of a column can be tested by two approaches. Reversible adsorption can be observed by peak tailing of selected compounds having special functional groups. This tailing can be quantitied by an asymmetry factor [255]: A, =
a+b (a + b ) - ( b - a )
where A,
= asymmetry
(I
= front
factor, half of the peak, measured from the perpendicular drawn through the peak maximum, b = back half of the peak. A totally symmetrical peak would have, according to this equation, an A, value of 1.00, and tailing on the rear edge, being an indication of reversible adsorption would give asymmetry factors greater than 1.00. Irreversible adsorption and partial decomposition are even more detrimental when carrying out trace analyses. This effect, which would falsify the analytical results, can be detected by comparing the ratios of the peak areas of selected compounds with that of a hydrocarbon contained in the test mixture, as compounds being subject to such effects would exhibit reduced peak areas. This method holds true, however, only for the investigated concentration range. Hence it is recommended that as small amounts of the test mixture as possible are injected as at higher concentrations the effects might become less evident. The composition of the test mixture can be chosen according to Table 4.
Table 4. Standard Test Mixtures Schombug et al. 1178.2581
Rohrschneider I2561
MeReynolds
benzene ethanol methyl ethyl ketone nitromethane pyridine
benzene n-Clo, n-Cll, 1-butan01 n-C12alkanes 2-pentanone G o - , c11-, nitropropane C12-acid methyl pyridine esters 2-methyl-2-pen- 1-octanol tan01 n-octylamine 1-iodobutane 2,6-dimethyl2-octyne aniline 1,4-dioxane dicyclohexylcis-hydrindane amine 2,6-dimethylphenol
12571
Gmb
[W ~~~~
n-Clo, n-CI1-al- n-Clo, nkanes C12-alkanes 1-0ctanol c10-9c11-, Clz-acids octanediol methyl esters octanoic acid 1-octanol trichlorophenol nonanal nitrophenol 2.3-butane-diol n-Cs-, n-Clo-, 2,6-dimethylani- n-Clz-amines line naphthalene 2,6-dimethylphe- biphenyl no1 dicyclohexylamine 2-ethylhexaneacid
n-Cs-C12-alkanes dibutyl ketone nonanal nonylamine nitrohexane 4-tert-butylpyridine nonanol naphthalene 4-propylphenol
3.3. Open-Tubular Columns
75
Owing to different causes of adsorption, it is necessary, as can be seen from Table 4, to test compounds with different structures. In any event, an m i n e ought to be present in the test mixture, because amines are very sensitive indicators of adsorption effects. Pyridine, used in the test mixtures of Rohrschneider and McReynolds (Table 4), seems to be less suitable. This is not surprising, as both of these test mixtures were developed for other reasons (see Chapter 4) and not to evaluate adsorption. Nevertheless, both of these mixtures have also been applied for this purpose occasionally, because they are necessary for determining the column selectivity, and dominant adsorption phenomena may occur. (e) Long-Term Stability Test Depending on numerous parameters the column can be subjected to changes during the operation. Therefore, several checks ought to be carried out at suitable intervals, e.g., once per week. An easily measured value is the capacity ratio, k, of a standard, which would give information on eventual losses of the stationary phase. Occasionally the Kovhts retention indices (see Chapter 4) of the Rohrschneider or McReynolds test mixture ought to be measured. Changes in the retention index of any of the components would indicate changes in column selectivity. Finally, any increase in adsorption effects can be realized by injecting a standard mixture according to (d) (Table 4).
3.3.4.
Porous-Layer Open Tubular (PLOT) and Support-Coated Open-Tubular (SCOT) Columns
In order to increase the sample capacity of open-tubular columns while decreasing the stationary phase film thickness, porous layers were first prepared on the inside walls of the column tubing by Halasr and Horuath [260, 2611 and thoroughly investigated and made commercially available by Ettre et al. [262, 2631. These porous layers increase the surface area of the original geometric area of the inner walls of the tubing, thus increasing the amount of stationary phase and with it the load of the column, in spite of the simultaneous reduction of the film thickness, in order to increase the efficiency according to eqn. (63) (decreasing both the C, and C, terms). Compared with WCOT columns, the phase ratio is much smaller, because the amount of stationary phase will be 10- to 50-fold higher (maintaining the high permeability and the efficiency of WCOT columns!). An additional advantage of this type of column is that the layer surface can be better coated than the normal wall surface, as the contact angle on the rough surface to be wetted by the liquid is smaller than that on smooth surfaces [compare eqn. (117)l. Porous layers can be applied to the inner tube wall by static coating with a suspension of particles of, e.g., graphitized carbon black [264, 264a], activated charcoal [265], modified silica [266], silanized silicic acid [268, 268a], molecular sieves or Chromosorb-based support materials [263, 2671, porous polyaromatic polymers [267a].Another possibility consists in the formation of porous layers direct from the column wall material by etching procedures (especially in glass capillaries), which will lead to silica gel-type layers [269, 2701, or to silica whiskers [158] (discussed in Section 3.3.3.1). The layer may be very thin, or thick, may be more or less active, and may be coated with a liquid stationary phase or may be used unmodified as an adsorbent, e.g., for the separation of non-or weakly polar low-boiling compounds. All these feasibilites exist when using the general term porous-layer open-tubular (PLOT) columns. In order to take into account the type and the geometric dimensions of the layer, we shall subdivide this type of column into subgroups: PLOT columns (strictly), SCOT columns and thick-layer columns; although this cannot be claimed to be scientifically exact, it is expedient for practical purposes.
76 3.3.4.1.
3. The Chromatographic Column
PLOT Columns (strictly)
Principle of preparation. Open tubes, most often having an inside diameter of 0.5 mm, are coated with adsorbent particles finely dispersed in a vaporizable liquid, which is subsequently evaporated so as to leave the particles as a thin porous layer on the column walls. Occasionally, the porous adsorbent layer can be modified by previously dissolving, e.g., a polar stationary liquid, in a dispersion, leaving, after evaporation of the solvent/dispersion liquid, a wet porous layer. The tube has first to be rinsed with purified solvents, e.g., in the order methylene chloride, methanol, acetone and R113 (CFZC1-CFClz),and subsequently filled with the solvent used for the dispersion (e.g., R113, carbon tetrachloride, chloroform or another vaporizable liquid of relatively high density). The particles generally have sizes of about 1 pm or less but larger particles have also yielded good efficiencies provided that the particle size distribution is narrow. Usually, adsorbents are applied, e.g., graphitized carbon black, activated charcoal, silica or porous polymers. They are added to the vaporizable dispersing liquid and dispersed by rapid stirring or sonication. The dispersion is forced through the tube by means of pressurized nitrogen, similar to the static coating procedure described in Section 3.3.3.2,avoiding the formation of gas bubbles. After being filled, one end of the tube is sealed, and the tube is slowly, beginning with the unsealed end, drawn or pushed through an oven, the temperature of which depends on both the tube length and the boiling point of the liquid to be vaporized. When the evaporation is completed, the column is purged with nitrogen to remove residual liquid. The particles adhere to the column walls by Van der Waals forces. The thickness of the layer depends on the particle size and structure, and generally ranges from 1 to 30 pm. If a wet, modified porous layer is to be prepared, the stationary liquid is added to the dispersion prior to stirring. This procedure for preparing porous layers allows a fairly unrestricted selection of particle size, layer thickness and composition, whereas etching of the inner walls to form the layer, produces porous layers, the structure of which depends on the tube material [269,270].These chemically produced layers (e.g., by the action of ammonia water or sodium hydroxide solution) may also act as an active porous layer per se or, if coated with a stationary liquid, as a modified adsorbent. 3.3.4.2.
SCOT Columns
This type of column differs from the previous type in the porous material used and by the fact that the layer, formed on the inside column walls, will always be wetted with a stationary liquid. The particles consist of relatively inert column support material (e.g., Chromosorb) having d, 5 1 pm. Silanized silicic acid has also been used [267](e.g., Silanox 101). They are coated on to the inner walls of the tube, together with the stationary liquid, both dispersedl dissolved in an appropriate solvent having a density L 1.5 g/cm3 and a boiling point between 50 and lOO"C,e.g., R113 (1,1,2-trifluoro-1,2,2,-trichloroethane), analogously to the procedure described in Section 3.3.4.1. Two further coating procedures are described in detail below. (a) Two-step procedure (according to Van Hour et al. [267].A plug consisting of Silanox 101 (silanized silicic acid) dispersed in a dilute solution of a polar phase in chloroform is forced through the column at a rate of 5-8mWs. The dispersion is prepared, immediately prior to use, by dissolving 0.1g of polar phase in 100 ml of chloroform, then adding 1.0g of Silanox and sonicating the mixture at 35°C for 15 min. A plug of about 1 ml is propelled through the capillary, and the solvent is removed by a flow of nitrogen through the column for 2 h. A thin layer of the polar phase containing Silanox remains on the walls. In the second step, a solution of the polar phase in acetone is prepared free from dissolved gases by dissolving 72 mg of the polar phase in 40 ml of acetone and removing 10 ml of the
3.4. Properties and Comparison of the Main Column Types
77
solvent by vacuum. The capillary is immediately filled with the solution using reduced pressure. One end of the column is warmed to expel a drop of the coating solution and immediately sealed, avoiding trapping of air. The opposite end of the tube is connected to a vacuum pump, and the solvent is slowly evaporated at room temperature. A 20-m column requires 36-48 h for solvent evacuation. (b) One-step procedure (according to Chauhan and Darbre [271]) A glass capillary previously treated with hydrogen fluoride is deactivated with benzyl triphosphonium chloride and pre-coated with poly(dimethy1oiloxane). A solution/dispersion is then prepared by dissolving 5% stationary liquid phase and intensively dispersing 5% of Silanox 101 or Chromosorb R-6470-1. A plug of this dispersion having a length of one quarter of the column length is propelled through the column at a rate of 4 c d s . After this dynamic coating the solvent is removed by a flow of nitrogen at 15 ml/min for 4 h. These SCOT columns, which belong, besides WCOT columns, to the most efficient gas chromatographic columns, exhibit lengths generally in the range 10-40 m, most often 15-20 m. The inside diameter is usually 0.5 mm, occasionally lower (down to 0.25 mm), and the layer consisting of support stationary liquid has a thickness from less than 1to a few pm. Their advantages and disadvantages in comparison with WCOT columns are discussed in Section 3.4. Due to the development of cross-linked surface-bonded liquid stationary phases, the application of SCOT columns has considerably decreased. 3.3.4.3.
Thick-Layer Columns
A special case of porous layer open-tubular columns, when considering only the presence of a layer, is the thick-layer column. One could argue, however, over the group of columns to which this type ought to be assigned. Regarding the layer type, either the PLOT or the SCOT type would be possible; regarding large layer thicknesses, with respect to the column inside diameter, the term open-tubular would no longer hold good, and these columns ought to be considered as a kind of micro-packed column. Nevertheless, as they are often thought to fill a gap between open-tubular and packed columns and as their properties, disregarding the thickness of the layer, are essentially the same as those of PLOT and SCOT columns, they are listed here. Their preparation [272-2741 differs from that of the other open-tubular columns. A glass tube with an inside diameter of, e.g., 3 mm is loosely filled with relatively coarse particles (d, = 60-90 pm) and drawn over a steel core of 0.3 mm to give a capillary tube with an inside diameter of, e.g., 0.5 m. The particles adhere tightly to the walls, and form a layer the thickness of which is about 100 pm. The particle material may be an adsorbent, and thus the column can be used in the unimpregnated form, or it may be an appropriate support material, which has to be coated with a stationary phase. Owing to the essentially smaller phase ratio, the amount of sample injected can be considerably larger than that with either of the other open-tubular column types, hence facilitating the method of sample introduction. Conventional gas chromatographs are applicable, on-column injection can easily be arranged, and routine analyses are simple. Nevertheless, the decrease in efficiency caused by the thick layer must not be overloocked.
3.4.
Properties and Comparison of the Main Column Types
At the beginning it must be emphasized that each column type has advantages and disadvantages. An absolute answer to the question of which column type is to be preferred cannot be given, as the decision is dependent on the equipment available, on the expenditure that can be devoted to research and development and, especially, on the analytical problem to be
78
3. The ChromatographicColumn
solved. In order to facilitate such a decision, several characteristic values of the different column types are listed in Table 5 and the pros and cons and the field of application of the most important column types are discussed below. The magnitudes in Table 5 are only average values, and should not be thought to represent all experimental results that have been obtained so far: nor was it possible to take all dependences into account. Table 5. Characteristic Properties of Different Column Types
Phase ratio, B Characteristic /J values k i n
1-1
[cm/sI Optimum practical gas velocity [ c d s ] L [ml Column inside diameter d , &p,
1-1
Film thickness, dl, or layer thickness, dl, , Cm] Sample capacity Cg] Column permeability, x [lo-’ cm2] n (maximum) AZmh, Minimum retention index difference necessary for two compounds to be separated at optimum column length (R,= 1.5)
3.4.1.
Conventional packed Columns
Micropacked Columns
SCOT
PLOT
WCOT
Columns
Columns
Columns
10-30 15 0.5-2 2-10 8-12
10-100 50 0.2-1 10-20 20-50
50-150 80 0.5-1 10-20 20-80
20-150 80 0.5-2 10-20 20-80
80-500 250 0.2-1 8-15 15-30
2-12 2-4
1-15 0.3-1
10-30
10-30
0.5
0.5
10-100 0.05-0.7
d10.01-0.3
d10.01-0.1
d1,O.S-S
dl,0.5-30
d10.1-1
1000 1-10
1-50 5-40
0.3-10 750- 1000
0.3-10 500-800
10 000 10
50 000 3
60 000 2
60 000 2
0.02-2 10- 1500, mostly 200 500 000 0.5
Packed Columns
Advantages: Inexpensive, can be laboratory-made, easy to use, require only simple apparatus, high sample capacity, no injection problems. Shortcomings: High pneumatic resistance (low permeability), restricted column length, low efficiency, low resolution, long analysis time. Application fields: Routine analysis provided that the mixtures to be separated are not too complex; simple separation problems; trace analysis; coupled techniques; process gas chromatography.
3.4.2.
Micro-packed Columns
Advantages: High efficiency enables short columns to be operated at high gas velocities and hence high-speed analysis to be implemented. Inexpensive, can be laboratory-made, sufficient sample capacity. Shortcomings: Pressure drop generally between 0.03 and 0.3 MPa may cause instrumental problems; e.g., at 0.5 MPa a special injector is necessary. Application fields: Multi-component analysis, high-resolution coupled techniques, trace analysis, high-speed analysis using short but efficient columns, high performance gas solid chromatography.
3.4. Prouerties and ComDarison of the Main Column Types
3.4.3.
79
Support-coated Open-tubular Columns
Advantages: High efficiency at high gas velocity permits high-speed analysis; higher sample capacity, lower column bleed, simpler wettability, smaller phase ratios than WCOT columns; splitless injection possible; low detection limits. Shortcomings: Less inactive and shorter column lengths than WCOT columns. Application fields: High performance gas chromatography, high-speed analysis, trace analysis of complicated mixtures, multi-component analysis, high-resolution coupled techniques, analysis of low-boiling compounds.
3.4.4.
Wall-coated Open-tubular Columns
Advantages: Highest efficiencies; possibility of optimization of analysis by changing 4 , 4 and L;owing to the low flow resistance long columns are applicable; analysis time 1-2 orders of magnitude shorter than with conventional packed columns; high-resolution at short analysis times, separability of compound pairs having small retention index differences; lowest adsorption of polar compounds; wall-coated fused-silica open-tubular columns, when combined with the most advanced GC hardware and when the stationary phase is immobilized, offer an almost ideal gas chromatographic system permitting highly reproducible retention values. Shortcomings: Complicated injection, lower sample capacity and more unfavourable phase ratios than other GC columns; only limited trace analysis practicable ( 210 ppm for 4 5 0.5 pm). Application fields: General purpose GC separation system for screening complex mixtures of different structure compounds; high-speed, high-resolution analysis; analysis of very complex mixtures the separation of which would otherwise be impossible; highly reproducible retention index measurements that will offer the possibility of identifying unknown components only by means of a comprehensive retention index library.
4.
Characterization of Stationary Phases
4.1.
Intermolecular Forces
The high selectivity of gas chromatography is based on molecular interactions between the molecules to be separated and the molecules of the stationary phase, be it an adsorbent or a stationary liquid. The intermolecular forces occur between molecules the valencies of which are saturated. Hence the appertaining interaction energies are mostly two orders of magnitude lower than the energies of chemical bonds and are generally < 60 kJ mole-' for both stationary liquids and adsorbents. The molecular interaction can be subdivided into four general types: London-type disperson forces, orientation forces including hydrogen bridge bonds, induction forces and donor-acceptor forces.
4.1.1.
London-type Dispersion Forces
As a result of the investigations of London [275], these forces represent the main interactions of non-polar or weakly polar components. They result from induced dipole/induced dipole and multi-polar attractions, as each atom and each molecule, even if it does not exhibit a measurable dipole moment, is unsymmetric at each point in time because of the permanent movement of electrons and exhibits dipole moments that cancel out only in the temporal average. Such an electron system acts as a fluctuating dipole polarizing the electron systems of its neighbouring atoms and molecules, achieving an attraction force. Hence the interaction depends on the polarization and on the ionization potentials of the interacting partners (London [275]):
where a;,a; = polarizability of the coherent partners 1 and 2, Zpl, Zpz = ionization potentials of the coherent partners 1 and 2, fD = distance between the coherent partners' centres, EL = interaction energy resulting from London-type dispersion forces. This type of interaction is independent of temperature. Based on this London relationship, Korol [276] developed the dispersion index for non-polar stationary liquids containing methylene groups in a non-ordered concentration, interacting with the molecules to be separated:
(132)
where
Ri, Rj= atomic refraction of the atomic groups i and j, = sum of the Van der Waals radii of the atomic groups i and j. rG The dispersion index, 4, represents the energy of the interaction between two atomic groups i and j. The intermolecular interaction energy, in the case of predominant dispersion forces, e.g., beween sample components consisting of saturated hydrocarbons, with carbon atoms ex-
4.1. Intermolecular Forces
81
hibiting only a-bonds and molecules of the stationary liquid phase having the same structure, or between such hydrocarbons and graphitized thermal carbon black, ranges from 0.1 to 40 kJ/mole, depending on the molecular distances [r-6 in eqn.(131)] between the sample and stationary phase molecules.
4.1.2.
Orientation Forces and Hydrogen Bridge Bonds
Forces between dipolar molecules result from the spatial orientation of the interacting dipoles. That is, if this spatial orientiation of the stationary dipoles and the sample dipoles were the same, attraction and repulsion would counterbalance. However, as the dipole orientation with the lower potential energy is favoured statistically, an attraction results. Because of the thermal motion which has a contrary effect, it depends strongly on the temperature. From this interaction, energy is liberated [277]; (Keesom 1921):
where permanent dipoles of the dissolved molecules and of the molecules of the liquid stationary phase, respectively, = distance between these dipoles, r, T = absolute temperature. With increasing temperature, accordingly, the orientation forces decrease, and liquid stationary phases that are selective at low temperatures because of a high proportion of orientation interactions, become less or even non-selective at high temperatures. A particular case of this electrostatic orientation effect is the hydrogen bridge bond that occurs between proton donor groups X-H and proton acceptor groups Y: p,,
=
X-H...Y where X is a strongly electronegative atom, especially 0, N and F, the donor group X-H thus having a partially ionic character. In addition to these three types of atoms, X may also be their higher homologues, e.g., S, P, C1, Br and I, and also C; the strength of the resulting hydrogen bond in these instances, however, is only of the order of magnitude of normal orientation forces. The higher the electronegativity of X, the more positive H becomes in the X-H bond. The proton acceptor group, Y,may be either an electronegative atom having polarizable lone pair orbitals or an unsaturated or aromatic system with sr-electrons. In Table 6 common proton donor and acceptor groups are listed [278]. Depending on the presence of donor and/or acceptor groups, four types of molecules with different hydrogen bonding tendencies can be classified [278] (Table 7 a). Systems consisting of two groups, one of which representing the dissolved/adsorbed compound and the other the stationary liquid phase/adsorbent, may or may not form more or less strong hydrogen bonds as can be seen in Table 7 b. When combining two partners of Type I or 11, for example, the retention is increased by hydrogen bridge bonds. Hydrogen bonds are also formed between molecules of type I (or 11) and I11 or IV. If molecules of types 1-111 and IV are interacting, donor-acceptor complexes emerge, the strength of which depends on the acidity of the proton donor I, I1 or I11 and the basicity of the acceptor IV (which increases approximately in the order sr-electron systems < nitriles < ketones < ethers < amines) [278]. When donor and acceptor groups are present in type I and I1 molecules, donor-acceptor complexes are also formed within such systems. The nature of the hydrogen bond equilibria, however, is more complicated than that between type I11 and IV molecules [280].
82
4. Characterization of Stationary Phases
Table 6. Proton Donor and Proton Acceptor Groups (2781 Proton Donor Oroups-X-H
Proton Acceptor Oroups Y
Examples
Examples
a) Lone pair orbitals OH
Water, alcohols, phenols, carboxylic acids, silanols primary and secondary amines, pyrroles, amides thiols hydrohalic acids haloforms, ethinyl hydrocarbons, nitro or cyan0
NH SH Hal-H CH
\ \
/ O!
\
\
o/\
oximes, nitroso compounds, pyridine N-oxides nitro compounds
o/\
sulphoxides
-N=0/
-N= II
compounds with H-atoms in aposition PH
\ /
/C = 0
\o/ /s= \
water, alcohols, phenols, carboxylic acids, silanols, ethers ketones, carboxylic acids, esters
sulphones
phosphines fP=O \
/
phosphineoxides primary, secondary and tertiary amines, pyrroles, pyridines
-j \ NI
b) rr-electrons
-c=c-
alkynes
\
/
/
\
c=c
alkenes aromatics
-CENI
nitriles
Table 7a. Classification of Molecules According to the Presence or Absence of Proton Donor or Acceptor Groups [278, 2791 Type
H+-donor groups present
H+-acceptor groups present
Examples
I
Yes, several
Yes, several
I1
Yes
Yes
I11 IV
Yes No
No Yes
V
No
No
water, polyols, aminoalcohols, hydroxy acids, polyphenols,polybasic acids alcohols, phenols, monobasic carboxylic acids, oximes, primary and secondary amines, nitriles and nitro compounds with a-H atoms haloforms ketones, esters, ethers, alkenes, aromatics, tertiary amines, aldehydes, nitriles and nitro compounds without a-H atoms alkanes, perhalogenated hydrocarbons
83
4.1. Intermolecular Forces ~~~
Table 7 b. Strength of Hydrogen Bonds of Groups I-V
I
I1 111
IV
V
I
I1
111
Iv
V
Verystrong strong
strong strong medium-weak
medium medium-weak none weak none
medium medium weak none none
none none none none none
medium medium none
medium
none
In systems that contain only groups I11 or IV or V, hydrogen bridge bonds cannot be developed. This also holds for all the combinations of group V with molecules of the other groups (disregarding the self-association of type I and I1 molecules). Therefore, systems of this kind behave nearly ideally (activity coefficients =l),and the elution order of the dissolved compounds occurs in the order of decreasing vapour pressures at the column temperature. In all other systems in Table 7b, marked contributions to the intermolecular interaction are made by the formation of hydrogen bridges, and the hydrogen-bridge bond enthalpies range between 1.6 kJ.mole-' (S-H...n) and 38 kJ. mole-' (0-H ...N) [281]. Hence they are relatively high compared with other polar interaction enthalpies.
4.1.3.
Induction Forces
Induction forces represent only a relatively small portion of the Van der Waals forces. They were investigated by Delye [282] and are based on the fact that an electric field of a dipolar molecule induces an electrical moment in another, polarizable molecule. The resulting attraction is generally independent of temperature, as can be seen in the Debye equation
where 01
=
polarizability,
p = dipole moment, rj
4.1.4.
=
distance between the coherent partners' centres.
Donor-Acceptor Interactions
If one of two partners has a high electron affinity (acceptor) and the other a n-electron system with a low ionization energy (donor), a partial transfer from the loaded orbital of the donor to the vacant orbital of the acceptor is possible. Typical acceptors applied in gas chromatography include nitro aromatics, nitrile ethers and tetrachlorophthalate. They retain appropriate donors, e.g., aromatics and olefins, selectively.
4.1.5.
Further Intermolecular Forces
Coordinatively unsaturated stationary phases can retain selectively these sample components which are suitable ligands and form complexes the stability of which is low enough at column temperature to transport the volatile pre-ligands through the column. The underlying forces are ion-dipole interactions [283].
84
4. Characterization of Stationary Phases
4.2.
Quantities for the Description of Interactions
4.2.1.
Selectivity Coefficient
From eqn. (70) it can be derived that the separation even of two equally boiling compounds can be accomplished by utilizing the selective properties of stationary phases expressed by the selectivity term:
Provided that the vapour pressures of the two compounds are identical at the column temperature, the vapour pressure or volatility term becomes unity and r2,1becomes q,l, a quantity that has been called the (special) selectivity coefficient [284, 2851: r2,1= V L
= u2,1=
(+) y2
R1
(135) PY-P:'
This coefficient can be elucidated either by calculation from the adjusted or relative retentions of two equally boiling compounds or by interpolation from two homologous series in a log Vk/Tbdiagram (Tb = boiling points of the members of the two homologous series), as it is well known in isothermal gas chromatography that graphs of the logarithm of adjusted retentions against the boiling points of the members of a homologous series are linear. The hypothetical compounds 1and 2 would have, in the case of identical (hypothetical) boiling points, more or less widely differing retentions, the retention ratio being an expression of the differences in their interactions with the stationary phase. The more u deviates from unity, the more selective is the stationary phase for the separation of compounds 1 and 2 and, moreover, for the separation of the two homologous series, as equally boiling compounds do not belong to the same homologous series but have different constitutions. For details and tables, see ref. [286]. It should be mentioned that u owing to the temperature dependence of the orientation forces, frequently changes over a wide temperature range.
4.2.2.
Retention Index
From Equation 51b: and Equation 8 1:
K2 r2,1= az,l= -
K1 RTln a2,1 = A (AG)
it can be seen that differences between the molar free energies of vaporization of two compounds 1 and 2 from their infinitely dilute solution in the stationary liquid phase (i.e., the separation enthalpy), render possible the separation expressed as r2,1or E ~ , On ~ . the other hand, if the vapour pressures are not taken into consideration in eqn. (70), r2,1is an appropriate quantity for the description of the interaction differences between the two samples and the stationary phase. Therefore, one could be tempted to choose a standard compound (s) according to eqn. (51a) r\, = t)Ri/t)Rsthe interactions of which with the stationary phase are well known, in order to obtain the interaction differences of i and s with the stationary phase. This relative retention related to a standard has been widely applied for identification purposes. There is, however, a serious disadvantage when using only one standard: if the retentions of the sample compound and the standard differ greatly (and this will often be the case because
4.2. Quantities for the Description of Interactions
85
of the great temperature range of gas chromatography, the numerous stationary phases and the wide range of samples), the accuracy will be far from acceptable. Thus, a multitude of standards have been proposed, most often diversely interacting with the stationary phase. These problems could be overcome by the introduction of the retention index system proposed as early as 1958 by Koucits [287] and revised by himself [288] and by Etfre [289]. The retention index is defined as the carbon number (multiplied by 100) of a hypothetical n-alkane that would have an adjusted retention time identical with that of the sample compound of interest, and which is bracketed by two n-alkane standards:
where
4
=
Z t'
= =
tzz tX(Z+
1)
}
=
retention index of substance i, carbon number of an n-alkane, adjusted retention time of the substance, adjusted retention times of alkanes with carbon numbers z and (z + l), where
As
and
we can write the retention index eqn. (136) as (136a)
In addition to n-alkanes (general formula C,H2,+ Z)r which exhibit only dispersive interactions, other compounds (general formula C,H2,+ X ) can additionally interact with polar groups of the stationary phase, and the resulting retention is an indicator of the group X and also of the type of stationary phase. We shall return to this fact in the following section. Because of the great importance of the retention index in gas chromatography, we shall in eqn. (136) means that the discuss this quantity in detail. The restriction t X , 5 t ; I&(,+ compound of interest, i, is eluted just after the n-alkane of carbon number z and just prior to that of carbon number z + 1. As the selection of an n-alkane pair suitable for the compound in question and for the applied column temperature is not difficult, standards from other types of compounds can be abandoned. According to definition, the retention indices of n-alkanes are I = 100 (CH,), 200 (C2H6), 300 (n-C3H8), ... z 100 (n-C,H2,+ A compound with an adjusted retention time between that of n-octane and n-nonane would therefore have a retention index, I, between 800 and 900. In a homologous series the retention indices increase by 100 units for each CH2-groupon both polar and non-polar stationary phases. The retention indices are completely independent of the experimental equipment and operating conditions, with the following exceptions. First, according to definition, the retention indices of n-alkanes are the same on all stationary phases and independent of temperature. However, this does not hold for all other compounds; their retention indices depend on both the stationary phase used (because of the relationship of ?Xi [eqn.(Sla) and (136a)l with H [eqn.(70)] and fi, strongly depending on the
86
4. Characterization of Stationary Phases
stationary phase) and on the applied column temperature (because the orientation forces decrease with increasing temperature according to eqn. (133), through which y iand r4 are again effected. Second, the retention index depends to some extent on both the residual activity of the liquid-solid interface (solid support or column wall, respectively) used and the stationary phase loading or film thickness, respectively. Precautions can be taken against these restrictions by choosing a less active solid support or column wall and deactivating it prior to use and by avoiding very low loadings or film thicknesses, respectively [290]. The temperature dependence of Z can be calculated by the Antoine equation (291): Z=A+-
B
(137) T+C where A, B and C are constants, depending on the solute and the liquid stationary phase. Within the temperature range usually applied in gas chromatographic practice, the function Z = f(2') exhibits, to a first approximation, a straight-line portion that is longer for non-polar than for polar stationary liquids. It was shown that one unit per degree frequently applies [292] and that similarly boiling isomers with a boiling point difference of x K differ in their retention indices by 5 x units (2871. The dependence of Z on boiling point, 4 , for homologous series of hydrocarbons, ethers, aldehydes, ketones, esters and alcohols on numerous stationary phases has been investigated by many workers [293-2981 and was found to be described by the linear correlation [298] Z=arb+ b
where ti,
= boiling
temperatures of the homologues,
a, b = constants. The constant a, valid for the homologous series concerned, is related only to the functional
group X and is therefore independent of the stationary phase, whereas 6 is related to both the stationary phase and the functional group of the sample homologues. As 6 includes y, it will vary with temperature (temperature dependence of the orientation forces!), whereas the constant a will remain constant [298]. At first sight, the simple linearity of eqn.(138) seems to be strange, yet it is easily explicable. It is well known in isothermal gas chromatography that the relationship between the logarithmus of the adjusted retention times and the boiling temperatures is linear for homologous series, this behaviour being a consequence of the relationship between the specific retention volume (V& and the vapour pressure (Po) of a solute:
v, = 273,16 R
(139) Y0P0MP where Mp = relative molecular mass of the stationary phase and yo = activity coefficient of the solute, which can consisdered to be approximately constant for a given homologous series. Combining the Clausius-Clapeyron equation and Trouton's rule, it was derived [298] that hl vg= Arb + B (138a) i.e., a logarithmic dependence. Because, according to definition, the retention index is logarithmically correlated with the retention r& [eqn. (136)], the combination with (138a) and (8) results in direct linearity. The retention index, Z, of a compound can be calculated from basic factors (corresponding to the carbon skeleton) and increments [resulting from functional groups and their position relative to each other (methylene increment, homomorphic increment, phase increment, substitutions increment)]; for details, see ref. [299]. The exact calculation of the essential data and various mathematical methods for the calculation of the Kovhts retention index have
4.2.
Quantities for the Description of Interactions
87
been thoroughly investigated by Smith, Haken et al. [300, 3011, Ettre [302], Guardino et al. [303], Budahegy et al. [304] and others [305-3111. The reproducibility of I values is not only dependent on the mathematical method used for processing the experimental data, but also on these data themselves. The combination of a well designed experimental technique, an accurate timing mechanism (nearest tenth of a second) and an appropriate mathematical method will give an inter-laboratory reproducibility of one unit for larger values of Kovhts indices, and two units for indices below ca. 400 [300, 3011. Owing to the development of fusedsilica wall-coated open-tubular columns (described in detail in Chapter 3), when combined with advances in gas chromatography hardware, it is now practical to compile a retention index library that is reproducible enough for the qualitative analysis fo unknown compounds. Sadtler Research Laboratories introduced such a Gas Chromatography Retention Index Library of chemical compounds measured on fused-silica WCOT capillary columns (stationary phases: cross-linked methylsilicone, cross-linked 5% phenylmethylsilicone, polythylene glycol Carbowax 20M bonded phase and cross-linked polyethylene glycol Carbowax 20M) [312]. The reproducibility is one retention index unit for non-polar stationary phases and two units for polar phases. Hence the measurement of the retention index of an unknown on, e.g., two different columns, varying in stationary phase polarity and under the same column temperature conditions, is usually sufficient for the positive identification of an unknown that is in the retention index library [312]. Finally, it should be mentioned that we have only dealt with the isothermal (standard) Kovats retention index. For the analogous quantity in temperature programmed gas chromatography, see refs. [312-3141. The retention index in gas-solid chromatography can be defined, in the case of wide-pore or non-porous adsorbents, by the equation
SA = specific surface area of the adsorbent, m, = mass of the adsorbent.
The retention volumes apply to an infinitesimally low surface coverage of the adsorbent by the adsorbed compounds, i, and the n-alkanes. The differences in the logarithms in eqn.(l40) correspond to the relative retentions (for zero coverage): (140a) The dependence of the retention indices ZAi on molecular structure and on temperature is more complicated than the dependence of Z in gas liquid chromatography. These dependences of ZA, are even more complex than those of the Henry constant [315]:
(88a and 20) The retention indices ZA of numerous hydrocarbons with different structures and of their oxygen and chlorine derivatives have been determined on graphitized thermal carbon black and on hydrogenated thermal carbon black [316].
88 4.2.3.
4. Characterization of Stationary Phases
Polarity
At the beginning it must be emphasized that the terms “selectivity” and “polarity”, often used synonymously in gas chromatographic papers, are not synonyms in practice. As described in the previous sections, the selectivity is an expression for the different interactions between the solutes adsorbates and the stationary phase. In contrast, the polarity is a term for substances (both stationary phases and sample molecules) having a significant concentration of polar substituent groups and hence having a measurable dipole moment. Thus, for example, a non-polar stationary phase is not automatically non-selective. The situation is additionally complicated by the polarizability of one of the two or of both the partners, if one is considering the term polarity. Therefore, it is not surprising that correlations between adjusted retentions or retention indices and electrical quantities such as dipole moments or dielectric constants of the stationary phases and solutes/adsorbates have only rarely been observed. In fact, correlations occured only between those phases and samples the structures of which were relatively simple, and the portions of polar groups of which were not too high. Especially compounds with hydrogen bonds showed no regularity. On the other hand, when taking into account all types of interactions (which contribute to the retention) as a characteristic of polarity, we had to consider even n-alkanes to be polar, which is absurd. In Section 4.2.2 we stated that the retention index of a compound other than an n-alkane would be an indicator of its group X (in C,H2,+J) and also the type of stationary phase. On this basis, and considering that the polarity in gas chromatography has most often been used in connection with elution order, a gas chromatographic polarity, Pp,can be defined by the retention ratio of a polar or polarizable solute (adsorbate) R-X, compared with a non-polar solute (adsorbate) R-Hon the stationary phase in question related to a standard phase: (141) Pp= l o g ( V y / y ) - l o g ( V y v y ) , = polarity of the polar phase p, PP Vy,Vy = retention volume of RX and RH, respectively, on the polar phase, Vp = retention volume of RX and RH, respectively, on the non-polar standard phase, RX = polar solute, RH = non-polar solute. The retention ratio for another compound, RX’, on the stationary phase p is connected with the polarity Ppby
F,
l o g ( V ~ / V =~ A“’Pp )
+ log(Vy/Vy),
(141a)
Am’ = characteristic constant of the solute RX’. On the basis of eqn.(141), a polarity scale of stationary phases can be advanced if both an appropriate polar or polarizable compound RX and a non-polar compound RH are selected and their retentions are determined on all suitable stationary phases [317]. Rohrschneider selected butadiene as the polar compound RX and n-butane as the non-polar compound RH and chose squalane as the non-polar stationary phase, the polarity of which was assigned by definition to be P = 0. For the pair of compounds butadieneh-butane, mainly the olefinic interactions are reflected. Hence we cannot expect to obtain a general polarity, but rather a special olefinic polarity, Paler. A polarity scale on this basis was established in 1966 by Rohrschneider [317, 3181 for 29 stationary phases, with squalane Polcf= 0 and o,@’-oxydipropionitrile Polef= 100 bracketing the whole polarity range. The retention ratio of two successive (non-polar!) n-alkanes also depends on the polarity of the stationary phase; it is the smaller the more polar is the stationary phase [319, 3201: g z , z + 1 = VB(Z+1)/
VBW *
(142)
4.2. Quantities for the Description of Interactions
89
From this, the polarity can be expressed on an empirical scale by the logarithm of the relative retention (0 for squalane, 1 for BIB'-oxydipropionitrile).As Vg(z+l)/Vg(,) is not independent of the number of carbon atoms (the reasons for this fact are dealt with in Section 4.2.5), the polarity expressed by this term is not the same for any pair of n-alkanes. Koucits established a system of retention indices that offered the facility of characterizing liquid stationary phases by retention index differences of varied homologous series [287,292, 3211. For example consider a compound RX (where the group X is polar or polarizable) that is chromatographed on two stationary phases a and b, where the retention indices I' and Ib can be calculated from the chromatographic data. If the retention index on phase b, Ib,is higher than that on phase a, In, then phase b can be regarded as more polar than phase a, and the difference in the gas chromatographic polarity is I=Ib-I'. (143) This relationship is the essential basis for the following generalizations. If I of a given solute/adsorbate is higher on another stationary phase, then the polarity of that phase would be higher or, in the case of the same phase, the dissolved/adsorbed compound would be more polar. It follows that
I = ax,
(143 a)
a = polarity factor of the dissolved/adsorbed compound, x = polarity factor of the stationary phase.
On this basis, as absolute values of polarity cannot be obtained. Rohrschneider was the first to develop a system utilizing relative values [317, 322, 3231. Based on the non-polar series of nalkanes used to measure and calculate retention indices and based on the non-polar liquid stationary phase squalane, he applied certain probes differing in the type of polarity and POlarizability. This system was extended by McRtynolds [324]. Because of the importance of this system and its great applicability and widespread use, it will be discussed in detail in the following section.
4.2.4.
Rohrschneider and McReynolds Constants
Eqn. (143 a), using one probe and one stationary phase only, is not sufficient to describe the behaviour of any solute on each stationary phase. Rohrschneider [317, 322, 3231 found that the retention index differences consist of several contributions, which are always determined by two types of factor: those which are specific for the sample and do not change when the stationary phase is varied, and those which are specific for the stationary phase and do not depend on the solute concerned, but are related to a limited number of selected standard compounds. The first are symbolized by a, b, c, d and e, and the latter by x, y , z, u and s (the reason for the alphabetically irregular order of the stationary phase factors is that Rohrschneider originally used only three test samples (symbols x-z), and later realized that it is necessary to take two additional compounds into consideration, and that he wanted to maintain the symbols x, y and z of the phase specific constants as they had already been published). For a given system stationary phase - solute, we obtain from eqn.(143a), applying five factors in each instance:
I = ax + by + cz + du + es,
(143b)
where x, y, z, u and s are polarity factors characterizing the stationary phase and a. b, c, d and e are polarity factors characterizing the solute. If m probe solutes are chromatographed on each of n stationary phases, we obtain mn results, which describe 5m polarity factors of the
90
4. Characterization of Stationary Phases
sample solute and 5n polarity factors of the stationary phases in a system of mn equations with 5 (rn + n ) unknowns: Z i = ulxl + bty, + clzl + dlul + elsl, I: = u1x2+ btyz + clzz + dluz + els2, Zf, = ulxn+ bty, + clzn + dlu, + elsn, Z: = u2x1+ bzYl + c2zl + d2u1+ e2s1, Z;=U,X,,+ b~n+Cmzn+dmUn+emsn.
In order to solve this indeterminate system, Rohrschneider selected arbitrary, yet typical, values for the stationary phase polarity factors: (143 c) (143 d)
- zqualane
zphaae
= AZnitrometimnc/100
= nltromctbPDc
-
s = AZpMdi,,e/lOO
nl-
3
100
100
Hence it follows for a stationary phase j and for a dissolved compound i that AI{
AI&H6
= II] -
100
+
Az&,oH
bi
100
A&H,COC,H, +
ci
100
AI&H3NOl +
dl
100
+ ei-
Azis~,~ 100
. (143 h)
The calculation of all factors is very time consuming. If, however, the factors a, b, ..., e of a compound in question are determined on at least five different stationary phases, the polarity factors of which, x, y, z, u and s, have been established by means of the five sample solutes benzene, ethanol, methyl ethyl ketone, nitromethane and pyridine, then the behaviour of the compound in question on each stationary phase can be predicted, provided that the factors of which x, y, z, u and s are known and the equations for the constants a-e can be solved. As the stationary phase specific factors x...s represent Z / 1 0 0 values for the specific test substances on this stationary phase, the factors u, ., e of the five test substances used for the determination of the phase specific factors are constants by definition (Table 8).
..
Table 8. Solute Specific Constants of the Five Test Substances used by Rohmchneider
benzene ethanol methyl ethyl ketone nitromethane pyridine
a
b
C
100 0 0 0 0
0 100
0 0 100 0 0
0 0 0
d
c
0
0 0 100
0
0 0 0 0 100
Although Rohrschneider proposed both stationary phase-specific constants and solute-specific constants, the expression “Rohrschneider constants” is used almost exclusively for the
4.2.
91
Quantities for the Description of Interactions
stationary phase-specific factors x, y, z, u and s. The five test substances have been selected for their specific interactions: induction forces (acceptor), donor-acceptor forces hydrogen bridge bond (H+ donor) hydrogen bridge bond (H+ acceptor) induction forces (donor), orientation forces, donor-acceptor forces orientation forces
benzene: ethanol: methyl ethyl ketone: nitromethane : pyridine:
Let us repeat and summarize the meaning, origin and object of Rohrschneider constants. The I;::; values (Kovfits retention indices) are measured for five deliberately chosen solutes on the stationary phase in question and on squalane as a non-polar reference phase. The differential Kovats retention indices, AI, are calculated from eqn. (143 c-g), and the resulting five A I values are divided by 100 to give x, y, z, u and s. These Rohrschneider constants are intended to characterize the selectivity and/or polarity of the stationary phase in question. Although not absolutely correct, the term "constant" should be retained because it has become common practice. Determination of Rohrschneider constants [325-3281 (Example) Stationary phases: (1) 20% Ucon LB 550X polypropylene glycol on deactivated Kieselguhr (2) 20% Squalane on deactivated Kieselguhr Less than 1pl of the test substance (benzene for x, ethanol for y, methyl ethyl ketone for z, nitromethane for u and pyridine for s) is chromatographed at a column temperature of 100°C on polypropylene glycol and squalane. From their retention times and from the retention times of the appropriate n-alkanes, the retention indices are calculated (see Section 4.2.2). In our example, the following values were found: qolypropylcneglycol = 753 Isqualane = 649 enzcne benzene , x = (763 - 649). 0.01 = 1.14; Ipolypropylene glycol = 660 Isqualane = 384 ethanol ethMOl v = (660 - 384) * 0.01 = 2.76; pO&OpylCnCglycol'= 699 ISqULhlle methyl ethyl ketone = 53 zmethyl ethyl ketone z = (699 - 531) eO.01 = 1.68; Ipolypropylene glycol = 769 Imgualane nitromethane = nitromethane u = (769 - 457). 0.01 = 3.12; polmropylenealycol = 903, zwy@ = 695 Ipyridine pyndme s = (903 - 695) * 0.01 = 2.08. 9
9
9
3
9
4573
9
Hence the liquid stationary phase Ucon LB 550X polypropylene glycol can be characterized at 100°C by the Rohrschneider constants x = 1.14, y = 2.76, z = 1.68, u = 3.12 and s = 2.08. As the retention indices are temperature dependent (see Section 4.2.2), the Rohrschneider constants also depend on temperature. However, this effect can be neglected in practice because it affects at best only the second decimal place. Numerous Rohrschneider constants have been determined. They are listed in Section 9.4, using the publications of Supinu and Rose [329] and Huken [330]. As the retention times of the bracketing low homologous n-alkanes, necessary for the calculation of the Kovits retention indices, raise objections if (and this is an absolut requirement) they have to be determined accurately [300-3111, McReynolds proposed the application of higher homologous of three probe compounds: he recommended n-butanol instead of ethanol, 1-nitropropane instead of nitromethane and 2-pentanone instead of methyl ethyl ke-
92
4. Characterization of Stationary Phases
tone. Additionally, McReynolds investigated further typical compounds in order to characterize special interactions and found that the supplementary application of 2-methyl-2-pentanol and 1-iodobutane increases the accuracy of the prediction of the retention indices of branched-chain and halogen compounds, respectively. On the other hand, 2-octyne, dioxane and cis-hydrindane only negligibly improved the accuracy of prediction and can generally be neglected when characterizing stationary phases [331, 3321. McReynolds did not correlate the stationary phase specific constants with the sample-specific constants, as he intended only to characterize liquid stationary phases. Therefore, he did not need to divide AZ by 100 as Rohrschneider proposed because the sample-specific constants a-e were defrned as 100. This is why McReynolds could use the AZ value itself. Therefore, the Rohrschneider constants range between ca 0.10 and 13.00, whereas the McReynolds constants range from 10 to 1300 (see Chapter 4). In Table 9 the sample compounds proposed by McReynolds and the types of substances they represent are listed. Table 9. McReynolds Test Substances Symbol
Test Substances
Retention index on Squalane
Compound types Characterize
(I'puhe)
X Y 2
U S
H I
K L
M
benzene butand-1 2-pentanone (methyl n-propyl ketone) 1-nitropropane pyridine 2-methyl-2-pentanone 1-iodobutane 2-octyne 1,4-dioxane cis-hydrindane
653 590 62 I
aromatics,olefins alcohols, nitriles, acids ketones, ethers, aldehydes, esters, epoxides, dimethyl amino derivatives nitro and nitrile compounds N-heterocycles branched-chaincompounds, especially alcohols halogen compounds acetylenic compounds ethers, polyols polycyclic compounds, steroids
652 699 690 818 841 654 1006
The values of X and S correspond to the original 100-fold Rohrschneider constants, whereas for X Z and V the retention differences AZ = ZPolar - Inon-Pohr h ave been determined for the higher homologues of the Rohrschneider sample compounds. McReynoZh showed, after having evaluated 226 stationary phases, that the selectivity and gas chromatographic polarity of liquid stationary phases can be characterized by the differential Kovhts retention indices, AZ = zpolar - Inon-polar, measured for the solutes in Table 9 on the given stationary phase and on squalane as a non-polar reference stationary phase. By analogy with the previous examples, the meaning of the symbols X...M, again, is
x= Azbcnzcnc = Y = AZbutanol etc.
cnzcnc
= @a*e utanol - 1
- zbenzene qualane - qualane zbutanol
9
- 19
These so-called McReynolds constants, symbolized by the capital letters X...M, were determined by McReynolds [324] at 120°C instead of 100°C as used by Rohrschneider. By means of the stationary phase-specific Rohrschneider and McReynolds constants, polarity scales of stationary phases can be formulated. For example, they are graduated according to increasing X values and hence a gas chromatographic polarity scale (elution order on the stationary phases concerned) for aromatics and olefins (relative to alkanes) can be obtained. Similarly, a gas chromatographic polarity order for different stationary phases can be given for alcohols, acids by comparing the Y values, for ketones, ethers, aldehydes, esters (Z values), nitro and cyan0 compounds ( V values) and N-heterocycles (Svalues).
93
4.2. Quantities for the Description of Interactions
Hence the constants can be used to forecast whether different stationary phases will behave similarly or differently and hence to facilitate the choice of a stationary phase. This aspect will be discussed in detail later (Chapter 9). Hitherto, we have only considered the selective interactions, expressed by the different constants, and not the general polarity. However, attempts have been made starting from A1 values to find the polarity order of stationary phases towards an “average” functional group. For example, “general polarity” can be defined by
P,,,
=
x+
Y + z+ u+ s.
(144) In Table 10 several selected liquid stationary phases are listed with their McReynolds constants and arranged according to their general polarities. Table 10. Order of Gas Chromatographic Polarity Pkenof Some Selected Liquid Stationary Phases (General Polarity = Sum of the McReynolds Constants, X + Y + 2 + II + S) Stationary Phase
X
Y
Z
U
S
General polarity
methylsilicone oil OV-101 methylphenylsilicone oil OV-17 poly(oxyethyleneoxypropy1ene) Ucon LBSSOX methylphenylsilicone oil, OV-25 polypropylene glycol, Ucon 50 HB-2000 cyanoethylmethylsiliconeoil, XE-60 cyanopropylmethylphenylsiliconeoil, OV-225 Carbowax 600 diethylene glycol succinate cyanopropylsiliconeoil Silar 1OC tris(cyanoethoxy)propane cyanopropylsiliconeoil, OV-275 bis(cyanoethy1)fomamide
17 119 118 178 202 204 228 350 470 523 593 629 690
57 158 271 204 394 381 369 631 705 757 857 872 991
45 162 158 208 253 340 338 428 558 659 752 763 853
67 243 243 305 392 493 492 632 788 942 1028 1106 1110
43 207 206 280 341 361 396 605 779 801 915 849 1000
229 884 996 1175 1582 1785 1823 2646 3300 3682 4145 4219 4644
By P,,, , the predominant interactions altogether are considered. However, special interactions cannot be recognized by this term. For example, the polyethers with their hydrogen bridge bonding tendency exhibit relatively high Y values; hence the order of the Y values in Table 10 is not the same as the order based on P,,,. It can be stated that the general polarity is much less clear than the special Rohrschneider/McReynoldsconstants. Instead of the sum of the McReynolds constants, the polarity has also arbitrarily been defmed as the arithmetic mean of the constants:
1 P=r 1A l ; i
(145)
for i= benzene, 1-butanol, 2-pentanone, 1-nitropropane and pyridine [324, 3331. This “average McReynolds constant” consists of the average interactions of the stationary phase with an “average” functional group of a solute. On the basis of AZ values for the solutes ethanol, 1,4-dioxane and nitromethane, which are intended to characterize hydrogen-bonding interactions (ethanol as a proton donor and dioxane as a proton acceptor) and dipole interactions (nitromethane), stationary phases have been characterized by selectivity parameters [334-3371. Multiplying these AZ values by the logarithm of the retention increments of an additional methylene group in the solute, Snyder [335] obtained corrected logarithms of the partition coefficients the sum of which he defined as polarity P’.
94
4. Characterization of Stationary Phases
A series of solvents including some gas chromatographic stationary phases has been investigated, and polarities P' ranging from 0.1 for n-hexane to 10.2 for water have been calculated for the solvents and from 1.2 (squalane) to 6.8 (B,fl-oxydipropionitrile) for the stationary phases. The restrictions already mentioned when using the term polarity also apply to P,as this quantity only reflects the sum of special interactions and does not indicate the selection of a stationary phase for the numerous special separation problems. It has been shown [338] that in some instances the use of n-alkanes as the basis of Kovhts retention indices, I, and the Rohrschneider-McReynoldsconstants, AZ, fails owing to adsorption effects from highly polar stationary phases or from the column supports at low stationary phase loadings. The n-alkane standard series may exaggerate the relative polarity of the more polar stationary phases and n-alkanes are badly resolved on some highly polar columns. Therefore, another series of polar compounds such as alcohols or aldehydes should also be used together with n-alkanes [338, 3391.
4.2.5.
Thermodynamic Criteria of Polarity
In Section 4.2.2 we considered a more or less polar monofunctional solute with the general formula C,Hzz+]X, where X represents the polar group. We can, instead, write (CH,),(CH,),X, where m + n = z, and n is the only variable in a given homologous series. According to Martin [340] and Rorh and Nouak [333], the standard molar Gibbs function of sorption of a solute i (the free standard enthalpy in the partition equilibrium), AGo(i), represents the sum of the standard Gibbs function contributions corresponding to the individual groups of the solute i: AGo(i) = mAGo(CH3)+ nAGo(CHz) + AGo(x). Approximately, AGo(CH3)= AGo(CH2), and the'Kovbts retention index of the solute i on a given stationary phase, I,(eqn. (136), Section 4.2.2), can be expressed by [341]
4 = 100
[
z, + AQ(CH2)
I
where z, is the number of carbon atoms in the solute molecule. The retention index difference AZi for a solute i on a stationary phase y and on squalane (sq) is then given by
The ratio AGo(X)/AGo(CHz) will increase on the stationary phase y with increasing interactions of i with y, and hence the value of AZ,, the retention index difference of the solute i on stationary phase y with respect to squalane, is a measure of the extent to which non-dispersive intermolecular forces contribute to the retention of i. In Section 4.2.4 we defined the average McReynolds constant as the arithmetic mean of representative interactions between a stationary phase and five selected solutes, P = 1/5 AZ, (145). This expression, according to I
eqn. (146) with a thermodynamic basis, reflects the relative affinity of the stationary phase towards an "
