Chromatographie 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 BLACKIE ACADEMIC & PROFESSIONAL An tmprint of Chapman & Hall London Glasgow Weinheim New York Tokyo Melbourne Madras
Contents 1 Introduction 1 1.1 Introduction to chromatography 1 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 Chromatographie 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 Peakwidth 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
Vlll 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 Typesofpaper 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 chromatography 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 111 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 IX 4.4 Adsorption chromatography 120 4.4.1 Solvents 120 4.4.2 Adsorbents 121 4.5 Partition column chromatography 124 4.5.1 Solid Supports 124 4.5.2 Solvents 125 4.6 Ion exchange chromatography 127 4.6.1 Properties desirable in resins 129 4.6.2 Ion exchange capacity 130 4.6.3 Selectivity of resins 130 4.6.4 Nature of the resin 131 4.6.5 Separation methods 132 4.7 Inorganic ion exchangers 135 4.8 Gel ion exchangers 137 4.8.1 Dextrans 138 4.8.2 Agarose gels 139 4.8.3 Cellulose 140 4.9 Gel chromatography 141 4.9.1 Column parameters and separations 143 4.9.2 Nature of the gel 146 4.9.3 Methodology 150 4.9.4 Applications 153 4.10 Affinity chromatography 156 4.10.1 Principles 156 4.10.2 Column materials 157 4.10.3 Applications 159 4.11 Covalent chromatography 159 4.12 Chromatofocusing 160 4.12.1 Ionic properties of amino acids and proteins 161 4.13 Flash chromatography 162 References 163 5 Gas chromatography 165 5.1 Introduction 165 5.2 Principles of gas chromatography 165 5.3 Resolution 167 5.3.1 10% valley resolution 172 5.3.2 Separation number 172 5.3.3 Asymmetry 172 5.4 Columns and stationary phase 173 5.4.1 Gas chromatography columns and column oven 173 5.4.2 Column oven and column temperature 174 5.4.3 Packed columns 175 5.4.4 Stationary phases for packed columns 177 5.4.5 Specialised stationary phases 178 5.4.6 Micropacked columns 180 5.5 Choice of stationary phase 181 5.6 Capillary column gas chromatography 183 5.6.1 Band broadening and analysis time 185 5.6.2 Optimum practical gas velocity 186 5.6.3 Column efficiency 187 5.6.4 Coating efficiency 189 5.6.5 Wall coated open tubulär columns 189 5.6.6 Stationary phases for wall coated open tubulär columns 191 5.6.7 Phase ratio, film thickness and column internal diameter 193
X CONTENTS 5.6.8 Performance of wall coated open tubulär columns 198 5.6.9 Porous layer open tubulär columns 199 5.7 Gas-solid chromatography 200 5.7.1 5.7.2 5.7.3 5.7.4 5.7.5 Alumina adsorbents Silica gel Molecular sieves Carbon molecular sieves and graphitised carbon Microporous polymers 5.8 Gas chromatography Instrumentation 5.8.1 5.8.2 5.8.3 5.8.4 5.8.5 5.8.6 5.8.7 5.9 Sampling techniques 5.9.1 5.9.2 5.9.3 5.9.4 5.9.5 5.9.6 5.9.7 5.9.8 Carrier gas supply and control Column switching Sample inlet Systems Sample preparation Gaseous samples Liquid sample inlet Systems Capillary column gas chromatography injectors Derivatisation of samples Silylation Acylation Alkylation (esterification) Headspace sampling Sample introduction by pyrolysis Automated sample introduction Sampling by adsorption tubes 5.10 Detectors for gas chromatography 5.10.1 Detector Performance 5.10.2 Signal noise and detection limits 5.10.3 Detector response factors 5.10.4 Flame ionisation detector 5.10.5 Electron capture detector 5.10.6 Thermal conductivity detector 5.10.7 Nitrogen-phosphorus detectors 5.10.8 Flame Photometrie detector 5.10.9 Photoionisation detector 5.10.10 Miscellaneous detectors 5.11 Supercritical gas chromatography 5.12 Applications of gas chromatography 5.12.1 5.12.2 5.12.3 5.12.4 5.12.5 5.12.6 5.12.7 5.12.8 References Headspace gas chromatography analysis Food analysis Drugs Pyrolysis gas chromatography Metal chelates and inorganic materials Dual detector applications Dual column applications Environmental analysis 202 203 203 204 204 209 209 211 213 215 215 215 216 217 217 218 219 220 221 221 223 224 228 229 230 231 232 234 236 239 241 242 244 247 248 249 250 251 251 252 252 253 253 254 6 High Performance liquid chromatography 258 6.1 6.2 Introduction Modes of chromatography 6.2.1 Adsorption 6.2.2 Bonded phase chromatography 6.2.3 Ion exchange 6.2.4 Ion pair partition 6.2.5 Gel permeation-gel exclusion 258 260 260 262 263 264 265
CONTENTS XI 6.2.6 Affinity 266 6.2.7 Chiral chromatography 266 6.3 Overview of high Performance liquid chromatography Instrumentation 267 6.3.1 Packings 268 6.3.2 Pumps 268 6.3.3 Detectors 269 6.3.4 Solvents 269 6.3.5 Columns 269 6.3.6 Injection 270 6.4 Theory 270 6.4.1 Retention 270 6.4.2 Column efficiency 271 6.4.3 Rate theory 272 6.4.4 Extra column band broadening 276 6.4.5 Temperature effects and difmsion 277 6.5 Detailed discussion of high Performance liquid chromatography Instrumentation 278 6.5.1 Solvent delivery system 278 6.5.2 Equipment for gradient elution 282 6.5.3 Sample introduction 286 6.5.4 Detectors 288 6.5.5 Specific property detectors 290 6.6 Column packings and stationary phases for liquid chromatography 311 6.6.1 Totally porous beads 311 6.6.2 Pellicular packings 312 6.6.3 Microporous particles 313 6.7 Adsorption chromatography 313 6.7.1 Solvent Systems 315 6.7.2 Solvent optimisation 316 6.8 Liquid-liquid partition chromatography 317 6.9 Chemically bonded stationary phases for high Performance liquid chromatography 318 6.9.1 Synthesis of bonded phase materials 318 6.10 Chiral chromatography.. 327 6.10.1 Chiral stationary phases 328 6.11 Ion exchange chromatography 332 6.11.1 Latex-based ion exchange materials 333 6.11.2 Ion exchange with conductivity suppression 334 6.12 Ion pairing 336 6.13 Size exclusion chromatography 338 6.13.1 Column packings 340 6.14 Liquid chromatography method development 341 6.14.1 Solvent optimisation techniques 342 6.15 Quantitative analysis 352 6.16 Preparative liquid chromatography 353 6.16.1 Practical aspects 354 6.17 Microcolumns in liquid chromatography 355 6.17.1 Microbore packed columns 357 6.17.2 Narrow bore packed columns 357 6.17.3 Open tubulär columns 357 6.17.4 Microcapillary packed columns 358 6.17.5 Instrumentation 359 6.17.6 Solvent delivery 359 6.17.7 Injection Systems 359 6.17.8 Microcolumn applications 359 6.18 Applications 361 References 362
Xll CONTENTS 7 Chromatography and spectroscopic techniques 366 7.1 Introduction 366 7.2 Chromatographie requirements 367 7.3 Chromatographie and mass spectrometry techniques 369 7.3.1 Ion source and inlet System 369 7.3.2 Mass analysers 373 7.3.3 The quadrupole mass analyser 373 7.3.4 Magnetic analysers 374 7.4 Gas chromatography interfacing techniques 375 7.4.1 Direct interfacing 376 7.4.2 Indirect interfaces 377 7.5 High Performance liquid chromatography interfacing 379 7.5.1 Moving belt interface 379 7.5.2 Thermospray interfaces 380 7.5.3 Atmospheric pressure ionisation interfaces 382 7.5.4 Electrospray interface 382 7.5.5 Particle beam interface 383 7.6 Chromatography and mass spectrometry data Systems 386 7.7 Infrared spectrophotometry 388 7.7.1 Data collection and processing 390 7.8 Ultraviolet-visible spectrophotometry 391 7.9 Atomic spectroscopy 393 References 397 8 Processing Chromatographie data 399 8.1 Introduction 399 8.2 The chromatogram 400 8.2.1 Chart recorders 401 8.2.2 Integrators 404 8.3 Data collection and processing 405 8.3.1 Signal digitisation 406 8.3.2 Data processing 407 8.3.3 Computer hardware 409 8.4 Chromatography Software 411 8.4.1 Calculation of peak area 411 8.4.2 Area percentage and area calculations 412 8.4.3 Peak detection 413 8.4.4 Baseline correction and overlapping peaks 415 8.5 Method development and optimisation 417 8.5.1 High Performance liquid chromatography optimisation 419 8.5.2 Gas chromatography method development 421 8.5.3 Expert Systems 421 8.6 Laboratory Information management Systems 423 References 425 9 Model or practical experiments in Chromatographie techniques 426 9.1 Introduction 426 9.2 List of experiments 427 9.3 Section A. Paper chromatography 428 9.3.1 Experiment 1. 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 electrophoresis 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 ofproteins 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 by GC 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 ofpaint 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 cyclohexyl 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 9.9.1 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 510 10.1 Introduction 510 10.2 Plate theory and resolution 510 10.3 Van Deemter 514 10.4 Volatility and temperature in gas chromatography 516 10.5 Qualitative analysis 516 10.6 Quantitative analysis 518 10.7 Composite questions 523 Appendix 1 Glossary of Chromatographie terms 525 Appendix 2 Table of chromatography Symbols 546 Index 548