Practical High-Performance Liquid Chromatography

Jump into the HPLC adventure! Three decades on from publication of the 1st German edition of Veronika Meyer's book on HPLC, this classic text remains one of the few titles available on general HPLC aimed at practitioners. New sections on the following topics have been included in this fifth edition: Comparison of HPLC with capillary electrophoresis How to obtain peak capacity van Deemter curves and other coherences Hydrophilic interaction chromatography Method transfer Comprehensive two-dimensional HPLC Fast separations at 1000 bar HPLC with superheated water In addition, two chapters on the instrument test and troubleshooting in the appendix have been updated and expanded by Bruno E. Lendi, and many details have been improved and numerous references added. A completely new chapter is presented on quality assurance covering: Is it worth the effort? Verification with a second method Method validation Standard operating procedures Measurement uncertainty Qualifications, instrument test, and system suitability test The quest for quality Reviews of earlier editions "That this text is written by an expert in both the practice and teaching of HPLC is evident from the first paragraph....not only an enjoyable, fascinating and easy read, but a truly excellent text that has and will serve many teachers, students and practitioners very well." —The Analyst “…provides essential information on HPLC for LC practitioners in academia, industry, government, and research laboratories…a valuable introduction." - American Journal of Therapeutics

Preface to the Fifth Edition xiii Important and Useful Equations for HPLC 1 vii 1 Introduction 5 1.1 HPLC: A powerful separation method 5 1.2 A first HPLC experiment 5 1.3 Liquid chromatographic separation modes 8 1.4 The HPLC instrument 9 1.5 Safety in the HPLC laboratory 10 1.6 Comparison between high-performance liquid chromatography and gas chromatography 11 1.7 Comparison between high-performance liquid chromatography and capillary electrophoresis 12 1.8 Units for pressure, length and viscosity 13 1.9 Scientific journals 14 1.10 Recommended books 15 2 Theoretical Principles 17 2.1 The chromatographic process 17 2.2 Band broadening 19 2.3 The chromatogram and its purport 23 2.4 Graphical representation of peak pairs with different degree of resolution 30 2.5 Factors affecting resolution 35 2.6 Extra-column volumes (dead volumes) 40 2.7 Tailing 41 2.8 Peak capacity and statistical resolution probability 46 2.9 Effects of temperature in HPLC 49 2.10 The limits of HPLC 51 2.11 How to obtain peak capacity 55 3 Pumps 59 3.1 General requirements 59 3.2 The short-stroke piston pump 59 3.3 Maintenance and repair 62 3.4 Other pump designs 63 4 Preparation of Equipment up to Sample Injection 65 4.1 Selection of the mobile phase 65 4.2 Preparation of the mobile phase 67 4.3 Gradient systems 68 4.4 Capillary tubing 70 4.5 Fittings 72 4.6 Sample injectors 74 4.7 Sample solution and sample volume 78 5 Solvent Properties 81 5.1 Table of organic solvents 81 5.2 Solvent selectivity 83 5.3 Miscibility 83 5.4 Buffers 84 5.5 Shelf life of mobile phases. 87 5.6 The mixing cross 88 6 Detectors 91 6.1 General 91 6.2 UV detectors 96 6.3 Refractive index detectors 99 6.4 Fluorescence detectors 101 6.5 Electrochemical (amperometric) detectors 103 6.6 Light-scattering detectors 104 6.7 Other detectors 106 6.8 Multiple detection 107 6.9 Indirect detection 108 6.10 Coupling with spectroscopy 109 7 Columns and Stationary Phases 117 7.1 Columns for HPLC 117 7.2 Precolumns 119 7.3 General properties of stationary phases 120 7.4 Silica 125 7.5 Chemically modified silica 126 7.6 Styrene-divinylbenzene 129 7.7 Some other stationary phases 133 7.8 Column care and regeneration 136 8 HPLC Column Tests 141 8.1 Simple tests for HPLC columns 141 8.2 Determination of particle size 143 8.3 Determination of breakthrough time 144 8.4 The test mixture 146 8.5 Dimensionless parameters for HPLC column characterization 148 8.6 The van Deemter equation from reduced parameters and its use in column diagnosis 152 8.7 van Deemter curves and other coherences 153 8.8 Diffusion coefficients 155 9 Adsorption Chromatography: Normal-Phase Chromatography 159 9.1 What is adsorption? 159 9.2 The eluotropic series 162 9.3 Selectivity properties of the mobile phase 165 9.4 Choice and optimization of the mobile phase 166 9.5 Applications 168 10 Reversed-Phase Chromatography 173 10.1 Principle 173 10.2 Mobile phases in reversed-phase chromatography 174 10.3 Solvent selectivity and strength 177 10.4 Stationary phases 181 10.5 Method development in reversed-phase chromatography 185 10.6 Applications 188 10.7 Hydrophobic interaction chromatography 191 11 Chromatography with Chemically Bonded Phases 195 11.1 Introduction 195 11.2 Properties of some stationary phases 195 11.3 Hydrophilic interaction chromatography 200 12 Ion-Exchange Chromatography 203 12.1 Introduction 203 12.2 Principle 203 12.3 Properties of ion exchangers 204 12.4 Influence of the mobile phase 207 12.5 Special possibilities of ion exchange 208 12.6 Practical hints 210 12.7 Applications 213 13 Ion-Pair Chromatography 217 13.1 Introduction 217 13.2 Ion-pair chromatography in practice 218 13.3 Applications 220 13.4 Appendix: UV detection using ion-pair reagents 221 14 Ion Chromatography 225 14.1 Principle 225 14.2 Suppression techniques 226 14.3 Phase systems 226 14.4 Applications 230 15 Size-Exclusion Chromatography 231 15.1 Principle 231 15.2 The calibration chromatogram 234 15.3 Molecular mass determination by means of size-exclusion chromatography 238 15.4 Coupled size-exclusion columns 241 15.5 Phase systems 243 15.6 Applications 244 16 Affinity Chromatography. 249 16.1 Principle 249 16.2 Affinity chromatography as a special case of HPLC 251 16.3 Applications 252 17 Choice of Method 255 17.1 The various possibilities 255 17.2 Method transfer 260 18 Solving the Elution Problem 263 18.1 The elution problem 263 18.2 Solvent gradients 264 18.3 Column switching 270 18.4 Comprehensive two-dimensional HPLC 272 18.5 Optimization of an isocratic chromatogram using four solvents 273 18.6 Optimization of the other parameters 276 18.7 Mixed stationary phases 284 19 Analytical HPLC 285 19.1 Qualitative analysis 285 19.2 Trace analysis 287 19.3 Quantitative analysis 291 19.4 Recovery 296 19.5 Peak-height and peak-area determination for quantitative analysis 299 19.6 Integration errors 303 19.7 The detection wavelength 304 19.8 Derivatization 306 19.9 Unexpected peaks: Ghost and system peaks 308 20 Quality Assurance 311 20.1 Is it worth the effort? 311 20.2 Verification with a second method 312 20.3 Method validation 312 20.4 Standard operating procedures 314 20.5 Measurement uncertainty 315 20.6 Qualifications, instrument test and system suitability test 317 20.7 The quest for quality 318 21 Preparative HPLC 321 21.1 Problem 321 21.2 Preparative HPLC in practice 322 21.3 Overloading effects 325 21.4 Fraction collection 328 21.5 Recycling 330 21.6 Displacement chromatography 331 22 Separation of Enantiomers 333 22.1 Introduction 333 22.2 Chiral mobile phases 335 22.3 Chiral liquid stationary phases 336 22.4 Chiral solid stationary phases 337 22.5 Indirect separation of enantiomers 345 23 Special Possibilities 349 23.1 Micro, capillary and chip HPLC 349 23.2 High-speed and super-speed HPLC 352 23.3 Fast separations at 1000 bar: UHPLC 353 23.4 HPLC with supercritical mobile phases 355 23.5 HPLC with superheated water 359 23.6 Electrochromatography 361 24 Appendix 1: Applied HPLC Theory 363 25 Appendix 2: How to Perform the Instrument Test 373 25.1 Introduction 373 25.2 Test sequence 373 25.3 Preparations 374 25.4 Pump test. 377 25.5 UV detector test 379 25.6 Autosampler test 383 25.7 Column oven test 383 25.8 Equations and calculations. 384 25.9 Documentation 385 26 Appendix 3: Troubleshooting 387 26.1 Pressure problems 387 26.2 Leak in the pump system 389 26.3 Deviating retention times 389 26.4 Injection problems 390 26.5 Baseline problems 390 26.6 Peak shape problems 392 26.7 Problems with light-scattering detectors 393 26.8 Other causes 394 26.9 Instrument test 395 27 Appendix 4: Column Packing 397 Index of Separations 401 Subject Index 403