Practical High-Performance Liquid Chromatography

Practical High-Performance Liquid Chromatography......Page 5
Contents......Page 9
Preface to the Fifth Edition......Page 15
Important and Useful Equations for HPLC......Page 17
1.2 A FIRST HPLC EXPERIMENT......Page 21
1.3 LIQUID CHROMATOGRAPHIC SEPARATION MODES......Page 24
1.4 THE HPLC INSTRUMENT......Page 25
1.5 SAFETY IN THE HPLC LABORATORY......Page 26
1.6 COMPARISON BETWEEN HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AND GAS CHROMATOGRAPHY......Page 27
1.7 COMPARISON BETWEEN HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AND CAPILLARY ELECTROPHORESIS......Page 28
1.8 UNITS FOR PRESSURE, LENGTH AND VISCOSITY......Page 29
1.9 SCIENTIFIC JOURNALS......Page 30
1.10 RECOMMENDED BOOKS......Page 31
2.1 THE CHROMATOGRAPHIC PROCESS......Page 33
2.2 BAND BROADENING......Page 35
2.3 THE CHROMATOGRAM AND ITS PURPORT......Page 39
2.4 GRAPHICAL REPRESENTATION OF PEAK PAIRS WITH DIFFERENT DEGREE OF RESOLUTION......Page 46
2.5 FACTORS AFFECTING RESOLUTION......Page 51
2.6 EXTRA-COLUMN VOLUMES (DEAD VOLUMES)......Page 56
2.7 TAILING......Page 57
2.8 PEAK CAPACITY AND STATISTICAL RESOLUTION PROBABILITY......Page 62
2.9 EFFECTS OF TEMPERATURE IN HPLC......Page 65
2.10 THE LIMITS OF HPLC......Page 67
2.11 HOW TO OBTAIN PEAK CAPACITY......Page 71
3.2 THE SHORT-STROKE PISTON PUMP......Page 75
3.3 MAINTENANCE AND REPAIR......Page 78
3.4 OTHER PUMP DESIGNS......Page 79
4.1 SELECTION OF THE MOBILE PHASE......Page 81
4.2 PREPARATION OF THE MOBILE PHASE......Page 83
4.3 GRADIENT SYSTEMS......Page 84
4.4 CAPILLARY TUBING......Page 86
4.5 FITTINGS......Page 88
4.6 SAMPLE INJECTORS......Page 90
4.7 SAMPLE SOLUTION AND SAMPLE VOLUME......Page 94
5.1 TABLE OF ORGANIC SOLVENTS......Page 97
5.3 MISCIBILITY......Page 99
5.4 BUFFERS......Page 100
5.5 SHELF LIFE OF MOBILE PHASES......Page 103
5.6 THE MIXING CROSS......Page 104
6.1 GENERAL......Page 107
6.2 UV DETECTORS......Page 112
6.3 REFRACTIVE INDEX DETECTORS......Page 115
6.4 FLUORESCENCE DETECTORS......Page 117
6.5 ELECTROCHEMICAL (AMPEROMETRIC) DETECTORS......Page 119
6.6 LIGHT-SCATTERING DETECTORS......Page 120
6.7 OTHER DETECTORS......Page 122
6.8 MULTIPLE DETECTION......Page 123
6.9 INDIRECT DETECTION......Page 124
6.10 COUPLING WITH SPECTROSCOPY......Page 125
7.1 COLUMNS FOR HPLC......Page 133
7.2 PRECOLUMNS......Page 135
7.3 GENERAL PROPERTIES OF STATIONARY PHASES......Page 136
7.4 SILICA......Page 141
7.5 CHEMICALLY MODIFIED SILICA......Page 142
7.6 STYRENE-DIVINYLBENZENE......Page 145
7.7 SOME OTHER STATIONARY PHASES......Page 149
7.8 COLUMN CARE AND REGENERATION......Page 152
8.1 SIMPLE TESTS FOR HPLC COLUMNS......Page 157
8.2 DETERMINATION OF PARTICLE SIZE......Page 159
8.3 DETERMINATION OF BREAKTHROUGH TIME......Page 160
8.4 THE TEST MIXTURE......Page 162
8.5 DIMENSIONLESS PARAMETERS FOR HPLC COLUMN CHARACTERIZATION......Page 164
8.6 THE VAN DEEMTER EQUATION FROM REDUCED PARAMETERS AND ITS USE IN COLUMN DIAGNOSIS......Page 168
8.7 VAN DEEMTER CURVES AND OTHER COHERENCES......Page 169
8.8 DIFFUSION COEFFICIENTS......Page 171
9.1 WHAT IS ADSORPTION?......Page 175
9.2 THE ELUOTROPIC SERIES......Page 178
9.3 SELECTIVITY PROPERTIES OF THE MOBILE PHASE......Page 181
9.4 CHOICE AND OPTIMIZATION OF THE MOBILE PHASE......Page 182
9.5 APPLICATIONS......Page 184
10.1 PRINCIPLE......Page 189
10.2 MOBILE PHASES IN REVERSED-PHASE CHROMATOGRAPHY......Page 190
10.3 SOLVENT SELECTIVITY AND STRENGTH......Page 193
10.4 STATIONARY PHASES......Page 197
10.5 METHOD DEVELOPMENT IN REVERSED-PHASE CHROMATOGRAPHY......Page 201
10.6 APPLICATIONS......Page 204
10.7 HYDROPHOBIC INTERACTION CHROMATOGRAPHY......Page 207
11.2 PROPERTIES OF SOME STATIONARY PHASES......Page 211
11.3 HYDROPHILIC INTERACTION CHROMATOGRAPHY......Page 216
12.2 PRINCIPLE......Page 219
12.3 PROPERTIES OF ION EXCHANGERS......Page 220
12.4 INFLUENCE OF THE MOBILE PHASE......Page 223
12.5 SPECIAL POSSIBILITIES OF ION EXCHANGE......Page 224
12.6 PRACTICAL HINTS......Page 226
12.7 APPLICATIONS......Page 229
13.1 INTRODUCTION......Page 233
13.2 ION-PAIR CHROMATOGRAPHY IN PRACTICE......Page 234
13.3 APPLICATIONS......Page 236
13.4 APPENDIX: UV DETECTION USING ION-PAIR REAGENTS......Page 237
14.1 PRINCIPLE......Page 241
14.3 PHASE SYSTEMS......Page 242
14.4 APPLICATIONS......Page 246
15.1 PRINCIPLE......Page 247
15.2 THE CALIBRATION CHROMATOGRAM......Page 250
15.3 MOLECULAR MASS DETERMINATION BY MEANS OF SIZE-EXCLUSION CHROMATOGRAPHY......Page 254
15.4 COUPLED SIZE-EXCLUSION COLUMNS......Page 257
15.5 PHASE SYSTEMS......Page 259
15.6 APPLICATIONS......Page 260
16.1 PRINCIPLE......Page 265
16.2 AFFINITY CHROMATOGRAPHY AS A SPECIAL CASE OF HPLC......Page 267
16.3 APPLICATIONS......Page 268
17.1 THE VARIOUS POSSIBILITIES......Page 271
17.2 METHOD TRANSFER......Page 276
18.1 THE ELUTION PROBLEM......Page 279
18.2 SOLVENT GRADIENTS......Page 280
18.3 COLUMN SWITCHING......Page 286
18.4 COMPREHENSIVE TWO-DIMENSIONAL HPLC......Page 288
18.5 OPTIMIZATION OF AN ISOCRATIC CHROMATOGRAM USING FOUR SOLVENTS......Page 289
18.6 OPTIMIZATION OF THE OTHER PARAMETERS......Page 292
18.7 MIXED STATIONARY PHASES......Page 300
19.1 QUALITATIVE ANALYSIS......Page 301
19.2 TRACE ANALYSIS......Page 303
19.3 QUANTITATIVE ANALYSIS......Page 307
19.4 RECOVERY......Page 312
19.5 PEAK-HEIGHT AND PEAK-AREA DETERMINATION FOR QUANTITATIVE ANALYSIS......Page 315
19.6 INTEGRATION ERRORS......Page 319
19.7 THE DETECTION WAVELENGTH......Page 320
19.8 DERIVATIZATION......Page 322
19.9 UNEXPECTED PEAKS: GHOST AND SYSTEM PEAKS......Page 324
20.1 IS IT WORTH THE EFFORT?......Page 327
20.3 METHOD VALIDATION......Page 328
20.4 STANDARD OPERATING PROCEDURES......Page 330
20.5 MEASUREMENT UNCERTAINTY......Page 331
20.6 QUALIFICATIONS, INSTRUMENT TEST AND SYSTEM SUITABILITY TEST......Page 333
20.7 THE QUEST FOR QUALITY......Page 334
21.1 PROBLEM......Page 337
21.2 PREPARATIVE HPLC IN PRACTICE......Page 338
21.3 OVERLOADING EFFECTS......Page 341
21.4 FRACTION COLLECTION......Page 344
21.5 RECYCLING......Page 346
21.6 DISPLACEMENT CHROMATOGRAPHY......Page 347
22.1 INTRODUCTION......Page 349
22.2 CHIRAL MOBILE PHASES......Page 351
22.3 CHIRAL LIQUID STATIONARY PHASES......Page 352
22.4 CHIRAL SOLID STATIONARY PHASES......Page 353
22.5 INDIRECT SEPARATION OF ENANTIOMERS......Page 361
23.1 MICRO, CAPILLARY AND CHIP HPLC......Page 365
23.2 HIGH-SPEED AND SUPER-SPEED HPLC......Page 368
23.3 FAST SEPARATIONS AT 1000 BAR: UHPLC......Page 369
23.4 HPLC WITH SUPERCRITICAL MOBILE PHASES......Page 371
23.5 HPLC WITH SUPERHEATED WATER......Page 375
23.6 ELECTROCHROMATOGRAPHY......Page 377
24 Appendix 1: Applied HPLC Theory......Page 379
25.2 TEST SEQUENCE......Page 389
25.3 PREPARATIONS......Page 390
25.4 PUMP TEST......Page 393
25.5 UV DETECTOR TEST......Page 395
25.7 COLUMN OVEN TEST......Page 399
25.8 EQUATIONS AND CALCULATIONS......Page 400
25.9 DOCUMENTATION......Page 401
26.1 PRESSURE PROBLEMS......Page 403
26.3 DEVIATING RETENTION TIMES......Page 405
26.5 BASELINE PROBLEMS......Page 406
26.6 PEAK SHAPE PROBLEMS......Page 408
26.7 PROBLEMS WITH LIGHT-SCATTERING DETECTORS......Page 409
26.8 OTHER CAUSES......Page 410
26.9 INSTRUMENT TEST......Page 411
27 Appendix 4: Column Packing......Page 413
Index of Separations......Page 417
Subject Index......Page 419