Chromatographic Methods

Chromatographic Methods Fifth Edition

A. BRAITHWAITE Department of Physical Sciences

Nottingham Trent University

and

F. J. SMITH Department of Chemistry and Chemical Engineering

University of Paisley

SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

FIN editlOn 1963 Second edltlOn 1967 Thlrd edltlOn 1974 Repnnted 1977 Fourth edltlon 1985 Flfth edltlon 1996 Repnnted 1999 Repnnted 2001

© 1999 Springer Science+Business Media Dordrecht Originally published by Kluwer Academic Publishers in 1999 ISBN 978-0-7514-0158-5 ISBN 978-94-011-0599-6 (eBook) DOI 10.1007/978-94-011-0599-6

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Library of Congress Catalog Card Number: 95-80922

Printed an acid-free paper

Preface

Since the inception of chromatography in 1903, the principal landmarks in its progress have been the virtual rediscovery of the technique in the 1930s, invention of synthetic resins in 1935, introduction of paper chromatography in the 1940s followed by that of thin layer, gas-solid and gas-liquid chroma­tography in the early 1950s. Whilst the theoretical aspects of HPLC were developed in the 1960s, it was the late 1970s before commercial instruments appeared. Developments through the 1980s in microelectronics and micro­processor technology afforded enhanced control, data acquisition and processing capabilities, and improved technologies for the manufacture of instrumentation. Developments in chromatographic media and packings and rapid scanning spectroscopic instruments have enabled combination techniques such as GC-MS, GC-IR, HPLC-MS and HPLC-IR to reach maturity and become standard routine techniques for the analyst. Further considerable research activity in the 1980s and early 1990s has led to the development of supercritical fluid chromatography (SFC), and high perfor­mance capillary electrophoresis is a technique that has proved invaluable in the genome project and the separation and typing of DNA fragments. Applications in environmental, health and safety, foods analysis and medical studies have contributed significantly to the advancement of these tech­niques. All of the instrumental chromatographic techniques are now used routinely by academic and industrial analysts. An understanding and experi­ence of such techniques is fundamental to the training of today's science undergraduates studying a range of disciplines reflecting the application areas mentioned above.

A number of specialist books have been published over the years though many are concerned only with particular aspects of the subject whilst others are essentially literature surveys which, though comprehensive, are somewhat uncritical, lack background information and are rather formid­able to someone seeking an introduction to chromatography. Thus the aim of the first edition published in 1963 'to present a unified account of the techniques in current use' is in many ways just as relevant today as it was then, although clearly a more extensive text is required to reflect the changes and advances described above.

The 5th edition of Chromatographic Methods reflects these many changes across all fields of chromatography. The principles of chromatography, how­ever, remain the same although an understanding of the theoretical aspects and principles has become more important in order to appreciate the influence various parameters have on the separation process, particularly

vi PREFACE

in high performance high resolution techniques such as capillary column GC, HPLC, HPIC and HPTLC. The 5th edition therefore includes an extended introduction to chromatographic techniques and a more in-depth discussion of the theory. Each subsequent chapter is updated to reflect the current status of the technique, particularly the instrumentation, detectors and column technology in for example, GC and HPLC. Hybrid techniques for GC and HPLC are now commonplace with bench-top instruments available. The chapter on spectroscopic techniques and chromatography looks at the principles and practice of these trends and discusses typical applications. Data systems have been a feature of the chromatography laboratory for many years; however, the power of modern PCs and workstations have been programmed for use as sophisticated integrators and to provide data processing and reporting and a gateway onto computer networks. Integrated laboratory data stations and information management systems are a familiar feature of QA and GLP laboratories. These and the many other aspects of the modern GCjHPLC laboratory are included in a much expanded chapter on data processing.

The 5th edition also includes an updated and expanded set of model experiments which reflect the current practice of chromatography. These are supported by a new chapter of a comprehensive set of problems with answers. A further new feature is a glossary of chromatographic terms and a list of symbols as an aide-memoire and to support the preceding chapters.

The individual chapters have been written to be self contained so that readers may peruse particular topics but can pursue the background in more depth if they so wish. The comprehensive contents of Chromatographic Methods (5th edition) thus reflects the increased range of knowledge and expertise required of today's students and chromatographers.

Finally we would like to acknowledge the patience and support from our families and in particular our wives during the preparation of this text.

A. Braithwaite F.J. Smith

November 1995

Contents

1 Introduction 1

1.1 Introduction to chromatography I 1.1.1 Definition of chromatography 2 1.1.2 IUPAC definition of chromatography (1993) 2

1.2 History of chromatography 2 1.2.1 Paper chromatography 4 1.2.2 Thin layer chromatography 4 1.2.3 Ion exchange chromatography 5 1.2.4 Gel permeation chromatography (size exclusion) 5 1.2.5 Affinity chromatography 6 1.2.6 Gas chromatography 6 1.2.7 Supercritical fluid chromatography 7 1.2.8 High performance liquid chromatography 7 1.2.9 Capillary (zone) electrophoresis 7

1.3 Classification of chromatographic methods 8 1.3.1 According to separation procedure 8 1.3.2 According to development procedure 9

References 14

2 Theoretical considerations 17

2.1 Introduction 17 2.1.1 Distribution ratio and separations 18

2.2 Factors influencing retention 20 2.2.1 Coulomb's Law (like attracts like) 21 2.2.2 Polar retention forces 21 2.2.3 Non-polar retention forces 22

2.3 Retention and equilibrium in chromatography 23 2.3.1 Retention time, retention volume and retardation 23 2.3.2 Retention factor 25 2.3.3 Separation factor 26

2.4 Separating efficiency of a column 26 2.4.1 Peak width 27 2.4.2 Column efficiency 28 2.4.3 Peak asymmetry 29

2.5 Band broadening processes 30 2.5.1 Van Deemter model of band broadening 31 2.5.2 A term ('eddy' diffusion and unequal pathways) 32 2.5.3 B term (longitudinal diffusion) 33 2.5.4 C terms (resistance to mass transfer) 34 2.5.5 Optimum mobile phase velocity 34 2.5.6 Alternative methods for band broadening 36

2.6 Resolution 37 2.6.1 10% valley resolution 38 2.6.2 Separation number 39 2.6.3 Resolution and selectivity 39

2.7 Quantification in chromatography 40 2.7.1 Normalising peak areas 40

VIll CONTENTS

2.7.2 Internal standard 41 2.7.3 External standards 41 2.7.4 Standard addition 42

References 43

3 Planar chromatography 44

3.1 Thin layer chromatography 44 3.1.1 Theory and principles 45 3.1.2 Outline of the method 49 3.1.3 Surface adsorption processes and spot shape 49 3.1.4 Comparison of thin layer with other forms of chromatography 50 3.1.5 Adsorbents 51 3.1.6 Preparation of plates 59 3.1.7 Application of samples 64 3.1.8 Documentation 65 3.1.9 Development 65 3.1.10 Overpressured thin layer chromatography 71 3.1.11 Solvents 72 3.1.12 Location of separated substances 73 3.1.13 Preparative thin layer chromatography 80 3.1.14 High performance thin layer chromatography 80

3.2 Paper chromatography 81 3.2.1 Origin 82 3.2.2 Overview of the technique 82 3.2.3 Sample preparation 83 3.2.4 Types of paper 83 3.2.5 Solvents 86 3.2.6 Equilibrium 87 3.2.7 Development 88 3.2.8 Sample application and detection 90 3.2.9 Identification 91 3.2.10 Quantitative methods 91 3.2.11 Applications of paper chrOlpatography 91

3.3 Electrophoresis 92 3.3.1 Procedure of zone electrophoresis 93 3.3.2 Factors affecting migration rates 94 3.3.3 Supporting media 97 3.3.4 Techniques of electrophoresis 100

3.4 Capillary electrophoresis 104 3.4.1 Overview of instrument operation 105 3.4.2 Theory and principles 106 3.4.3 Electroendosmotic flow 107 3.4.4 Separation modes 108 3.4.5 Instrumentation III 3.4.6 Applications 114

References 114

4 Liquid phase chromatography on open columns 117

4.1 Introduction 117 4.2 Practical aspects and considerations 117

4.2.1 Columns and packing procedures 117 4.2.2 Sample application 118 4.2.3 Elution procedures 118

4.3 Modes of chromatography 119

CONTENTS

4.4 Adsorption chromatography 4.4.1 Solvents 4.4.2 Adsorbents

4.5 Partition column chromatography 4.5.1 Solid supports 4.5.2 Solvents

4.6 Ion exchange chromatography 4.6.1 Properties desirable in resins 4.6.2 Ion exchange capacity 4.6.3 Selectivity of resins 4.6.4 Nature of the resin 4.6.5 Separation methods

4.7 Inorganic ion exchangers 4.8 Gel ion exchangers

4.8.1 Dextrans 4.8.2 Agarose gels 4.8.3 Cellulose

4.9 Gel chromatography 4.9.1 Column parameters and separations 4.9.2 Nature of the gel 4.9.3 Methodology 4.9.4 Applications

4.10 Affinity chromatography 4.10.1 Principles 4.10.2 Column materials 4.10.3 Applications

4.11 Covalent chromatography 4.12 Chromatofocusing

4.12.1 Ionic properties of amino acids and proteins 4.13 Flash chromatography References

5 Gas chromatography

5.1 Introduction 5.2 Principles of gas chromatography 5.3 Resolution

5.3.1 10% valley resolution 5.3.2 Separation number 5.3.3 Asymmetry

5.4 Columns and stationary phase 5.4.1 Gas chromatography columns and column oven 5.4.2 Column oven and column temperature 5.4.3 Packed columns 5.4.4 Stationary phases for packed columns 5.4.5 Specialised stationary phases 5.4.6 Micropacked columns

5.5 Choice of stationary phase 5.6 Capillary column gas chromatography

5.6.1 Band broadening and analysis time 5.6.2 Optimum practical gas velocity 5.6.3 Column efficiency 5.6.4 Coating efficiency 5.6.5 Wall coated open tubular columns 5.6.6 Stationary phases for wall coated open tubular columns 5.6.7 Phase ratio, film thickness and column internal diameter

ix

120 120 121 124 124 125 127 129 130 130 131 132 135 137 138 139 140 141 143 146 150 153 156 156 157 159 159 160 161 162 163

165

165 165 167 172 172 172 173 173 174 175 177 178 180 181 183 185 186 187 189 189 191 193

X CONTENTS

5.6.8 Perfonnance of wall coated open tubular columns 198 5.6.9 Porous layer open tubular columns 199

5.7 Gas-solid chromatography 200 5.7.1 Alumina adsorbents 202 5.7.2 Silica gel 203 5.7.3 Molecular sieves 203 5.7.4 Carbon molecular sieves and graphitised carbon 204 5.7.5 Microporous polymers 204

5.8 Gas chromatography instrumentation 209 5.8.1 Carrier gas supply and control 209 5.8.2 Column switching 211 5.8.3 Sample inlet systems 213 5.8.4 Sample preparation 215 5.8.5 Gaseous samples 215 5.8.6 Liquid sample inlet systems 215 5.8.7 Capillary column gas chromatography injectors 216

5.9 Sampling techniques 217 5.9.1 Derivatisation of samples 217 5.9.2 Silylation 218 5.9.3 Acylation 219 5.9.4 Alkylation (esterification) 220 5.9.5 Headspace sampling 221 5.9.6 Sample introduction by pyrolysis 221 5.9.7 Automated sample introduction 223 5.9.8 Sampling by adsorption tubes 224

5.10 Detectors for gas chromatography 228 5.10.1 Detector perfonnance 229 5.10.2 Signal noise and detection limits 230 5.10.3 Detector response factors 231 5.10.4 Flame ionisation detector 232 5.10.5 Electron capture detector 234 5.10.6 Thennal conductivity detector 236 5.10.7 Nitrogen-phosphorus detectors 239 5.10.8 Flame photometric detector 241 5.10.9 Photoionisation detector 242 5.10.10 Miscellaneous detectors 244

5.11 Supercritical gas chromatography 247 5.12 Applications of gas chromatography 248

5.12.1 Headspace gas chromatography analysis 249 5.12.2 Food analysis 250 5.12.3 Drugs 251 5.12.4 Pyrolysis gas chromatography 251 5.12.5 Metal chelates and inorganic materials 252 5.12.6 Dual detector applications 252 5.12.7 Dual column applications 253 5.12.8 Environmental analysis 253

References 254

6 High performance liquid chromatography 258

6.1 Introduction 258 6.2 Modes of chromatography 260

6.2.1 Adsorption 260 6.2.2 Bonded phase chromatography 262 6.2.3 Ion exchange 263 6.2.4 Ion pair partition 264 6.2.5 Gel penneation-Gel exclusion 265

CONTENTS

6.2.6 Affinity 6.2.7 Chiral chromatography

6.3 Overview of high performance liquid chromatography instrumentation 6.3.1 Packings 6.3.2 Pumps 6.3.3 Detectors 6.3.4 Solvents 6.3.5 Columns 6.3.6 Injection

6.4 Theory 6.4.1 Retention 6.4.2 Column efficiency 6.4.3 Rate theory 6.4.4 Extra column band broadening 6.4.5 Temperature effects and diffusion

6.5 Detailed discussion of high performance liquid chromatography instrumentation 6.5.1 Solvent delivery system 6.5.2 Equipment for gradient elution 6.5.3 Sample introduction 6.5.4 Detectors 6.5.5 Specific property detectors

6.6 Column packings and stationary phases for liquid chromatography 6.6.1 Totally porous beads 6.6.2 Pellicular packings 6.6.3 Microporous particles

6.7 Adsorption chromatography 6.7.1 Solvent systems 6.7.2 Solvent optimisation

6.8 Liquid-liquid partition chromatography 6.9 Chemically bonded stationary phases for high performance liquid

chromatography 6.9.1 Synthesis of bonded phase materials

6.10 Chiral chromatography 6.10.1 Chiral stationary phases

6.11 Ion exchange chromatography 6.11.1 Latex-based ion exchange materials 6.11.2 Ion exchange with conductivity suppression

6.12 Ion pairing 6.13 Size exclusion chromatography

6.13.1 Column packings 6.14 Liquid chromatography method development

6.14.1 Solvent optimisation techniques 6.15 Quantitative analysis 6.16 Preparative liquid chromatography

6.16.1 Practical aspects 6.17 Microcolumns in liquid chromatography

6.17.1 Microbore packed columns 6.17.2 Narrow bore packed columns 6.17.3 Open tubular columns 6.17.4 Microcapillary packed columns 6.17.5 Instrumentation 6.17.6 Solvent delivery 6.17.7 Injection systems 6.17.8 Microcolumn applications

6.18 Applications References

Xl

266 266

267 268 268 269 269 269 270 270 270 271 272 276 277

278 278 282 286 288 290 311 311 312 313 313 315 316 317

318 318 327 328 332 333 334 336 338 340 341 342 352 353 354 355 357 357 357 358 359 359 359 359 361 362

xii CONTENTS

7 Chromatography and spectroscopic techniques 366

7.1 Introduction 7.2 Chromatographic requirements 7.3 Chromatographic and mass spectrometry techniques

7.3.1 Ion source and inlet system 7.3.2 Mass analysers 7.3.3 The quadrupole mass analyser 7.3.4 Magnetic analysers

7.4 Gas chromatography interfacing techniques 7.4.1 Direct interfacing 7.4.2 Indirect interfaces

7.5 High performance liquid chromatography interfacing 7.5.1 Moving belt interface 7.5.2 Thermospray interfaces 7.5.3 Atmospheric pressure ionisation interfaces 7.5.4 Electrospray interface 7.5.5 Particle beam interface

7.6 Chromatography and mass spectrometry data systems 7.7 Infrared spectrophotometry

7.7.1 Data collection and processing 7.8 Ultraviolet-visible spectrophotometry 7.9 Atomic spectroscopy References

8 Processing chromatographic data

8.1 Introduction 8.2 The chromatogram

8.2.1 Chart recorders 8.2.2 Integrators

8.3 Data collection and processing 8.3.1 Signal digitisation 8.3.2 Data processing 8.3.3 Computer hardware

8.4 Chromatography software 8.4.1 Calculation of peak area 8.4.2 Area percentage and area calculations 8.4.3 Peak detection 8.4.4 Baseline correction and overlapping peaks

8.5 Method development and optimisation 8.5.1 High performance liquid chromatography optimisation 8.5.2 Gas chromatography method development 8.5.3 Expert systems

8.6 Laboratory information management systems References

366 367 369 369 373 373 374 375 376 377 379 379 380 382 382 383 386 388 390 391 393 397

399

399 400 401 404 405 406 407 409 411 411 412 413 415 417 419 421 421 423 425

9 Model or practical experiments in chromatographic techniques 426

9.1 Introduction 426 9.2 List of experiments 427 9.3 Section A. Paper chromatography 428

9.3.1 Experiment I. Separation of cobalt, manganese, nickel and zinc by ascending and horizontal development 429

9.3.2 Experiment 2. Quantitative separation of copper, cobalt and nickel by ascending development 431

9.3.3 Experiment 3. Ascending chromatography on ion exchange paper 432

CONTENTS Xlll

9.4 Section B. Electrophoresis and related techniques 434 9.4.1 Experiment 4. Horizontal low voltage electrophoresis of amino

acids 434 9.4.2 Experiment 5. Cellulose acetate and polyacrylamide gel

electropho~esis of proteins 435 9.4.3 Experiment 6. Horizontal agarose gel electrophoresis of DNA 438

9.5 Section C. Thin layer chromatography 440 9.5.1 Experiment 7. Preparation of microplates and separation of

aromatic amines 440 9.5.2 Experiment 8. Separation of simple organic compounds on

fluorescent silica plates 442 9.5.3 Experiment 9. Separation of sugars on bisulphite and acetate

modified silica 443 9.5.4 Experiment 10. Analysis of analgesics using normal and reverse

phase TLC 445 9.6 Section D. Column chromatography 449

9.6.1 Experiment 11. Separation of dichromate ion and permanganate ion using an alumina column 449

9.6.2 Experiment 12. Determination of the exchange capacity and exchange efficiency of a cation exchange resin 450

9.6.3 Experiment 13. Complex elution of iron and copper using a cation exchange resin 452

9.6.4 Experiment 14. Purification of proteins on DEAE-cellulose 453 9.6.5 Experiment 15. Separation of a mixture of analgesics by flash

column chromatography 456 9.7 Section E. Gas chromatography 457

9.7.1 Experiment 16. GC of alcohols 458 9.7.2 Experiment 17. Determination of ethanol in an aqueous solution

byGC 460 9.7.3 Experiment 18. Determination of barbiturates using an internal

standard 462 9.7.4 Experiment 19. Determination of whisky congeners by capillary GC 465 9.7.5 Experiment 20. Qualitative analysis by GC using retention data

from two columns (polar and non-polar) 468 9.7.6 Experiment 21. Study of some important parameters in GC 471 9.7.7 Experiment 22. The determination of some chlorinated insecticides

by capillary GC using an EC detector 477 9.7.8 Experiment 23. Analysis of paint shop vapours 478 9.7.9 Experiment 24. The determination of water content in solvents by

capillary and packed GC using thermal conductivity detection 481 9.7.10 Experiment 25. Analysis of mineral acids as their cyc\ohexyl

derivatives by capillary GC and GC-MS 484 9.8 Section F. High performance liquid chromatography 488

9.8.1 Experiment 26. Analysis of barbiturates by reverse phase isocratic chromatography 488

9.8.2 Experiment 27. Ion pair chromatography of vitamins 489 9.8.3 Experiment 28. Techniques in HPLC analysis of analgesics 493 9.8.4 Experiment 29. Analysis of amino acids as their DNP derivatives 495 9.8.5 Experiment 30. Analysis of paraben preservatives by HPLC with

photodiode array detection 497 9.8.6 Experiment 31. Determination of the amino acid composition of a

peptide using pre-column derivatisation with o-phthalaldehyde and reverse phase HPLC and fluorescence detection 499

9.8.7 Experiment 32. Analysis of inorganic anions in aqueous samples 501 9.9 Section G. Capillary electrophoresis 504

9.9.) Experiment 33. Determination of inorganic cations by CE 504 9.9.2 Experiment 34. Analysis of analgesics by CZE 507

References 509

xiv CONTENTS

10 Illustrative problems in chromatography

10.1 Introduction 10.2 Plate theory and resolution 10.3 Van Deemter 10.4 Volatility and temperature in gas chromatography 10.5 Qualitative analysis 10.6 Quantitative analysis 10.7 Composite questions

Appendix 1 Glossary of chromatographic terms

Appendix 2 Table of chromatography symbols

Index

510

510 510 514 516 516 518 523

525

546

548