Introduction to Modern Liquid Chromatography, Third Edition

INTRODUCTION TO MODERN LIQUID CHROMATOGRAPHY Third Edition......Page 4
CONTENTS......Page 8
PREFACE......Page 34
GLOSSARY OF SYMBOLS AND ABBREVIATIONS......Page 38
1 INTRODUCTION......Page 46
1.1.1 What Is HPLC?......Page 47
1.1.2 What Can HPLC Do?......Page 49
1.2 A Short History of HPLC......Page 51
1.3.1 Gas Chromatography (GC)......Page 53
1.3.2 Thin-Layer Chromatography (TLC)......Page 54
1.3.3 Supercritical Fluid Chromatography (SFC)......Page 55
1.3.5 Countercurrent Chromatography......Page 56
1.4.1 Books......Page 57
1.4.5 The Internet......Page 58
References......Page 60
2 BASIC CONCEPTS AND THE CONTROL OF SEPARATION......Page 64
2.2 The Chromatographic Process......Page 65
2.3 Retention......Page 69
2.3.1 Retention Factor k and Column Dead-Time t(0)......Page 70
2.3.2 Role of Separation Conditions and Sample Composition......Page 73
2.3.2.1 Intermolecular Interactions......Page 75
2.3.2.2 Temperature......Page 79
2.4 Peak Width and the Column Plate Number N......Page 80
2.4.1 Dependence of N on Separation Conditions......Page 82
2.4.1.1 Band-Broadening Processes That Determine Values of N......Page 84
2.4.1.2 Some Guidelines for Selecting Column Conditions......Page 91
2.4.2 Peak Shape......Page 95
2.5 Resolution and Method Development......Page 99
2.5.1 Optimizing the Retention Factor k......Page 102
2.5.2 Optimizing Selectivity α......Page 104
2.5.2.1 \"Regular\" and \"Irregular\" Samples......Page 105
2.5.3.1 Effects of Column Conditions on Separation......Page 106
2.5.3.2 Fast HPLC......Page 108
2.5.4.1 Assessment of Sample Composition and Separation Goals......Page 110
2.5.4.4 Detector Selection......Page 111
2.5.4.6 Anticipation, Identification, and Solution of Potential Problems......Page 112
2.6 Sample Size Effects......Page 114
2.6.1 Volume Overload: Effect of Sample Volume on Separation......Page 115
2.6.2 Mass Overload: Effect of Sample Weight on Separation......Page 116
2.6.3.2 Trace Analysis......Page 118
2.7.1 Column Equilibration......Page 119
2.7.2 Gradient Elution......Page 120
2.7.3 Peak Capacity and Two-dimensional Separation......Page 121
2.7.4 Peak Tracking......Page 122
2.7.5 Secondary Equilibria......Page 123
2.7.6 Column Switching......Page 124
2.7.7 Retention Predictions Based on Solute Structure......Page 125
2.7.7.1 Solvation-Parameter Model......Page 127
References......Page 128
3 EQUIPMENT......Page 132
3.1 Introduction......Page 133
3.2 Reservoirs and Solvent Filtration......Page 134
3.2.1 Reservoir Design and Use......Page 135
3.2.2 Mobile-Phase Filtration......Page 136
3.3.1 Degassing Requirements......Page 137
3.3.2 Helium Sparging......Page 139
3.3.3 Vacuum and In-line Degassing......Page 140
3.4.1.1 Low-Pressure Tubing......Page 141
3.4.1.2 High-Pressure Tubing......Page 142
3.4.2.1 Low-Pressure Fittings......Page 144
3.4.2.2 High-Pressure Fittings......Page 146
3.4.2.3 Specialty Fittings......Page 148
3.5.1 Reciprocating-Piston Pumps......Page 149
3.5.1.2 Accumulator-Piston Pumps......Page 153
3.5.2.1 High-Pressure Mixing......Page 154
3.5.2.3 Hybrid Systems......Page 156
3.5.4.1 Low-Flow (Micro and Nano) Applications......Page 157
3.6 Autosamplers......Page 158
3.6.1.1 Filled-Loop Injection......Page 159
3.6.1.2 Partial-Loop Injection......Page 160
3.6.2 Autosampler Designs......Page 161
3.6.2.1 Pull-to-Fill Autosamplers......Page 162
3.6.2.2 Push-to-Fill Autosamplers......Page 163
3.6.3 Sample-Size Effects......Page 164
3.6.3.1 Injection Volume......Page 165
3.6.3.2 Injection Solvent......Page 166
3.6.4.1 Column Switching......Page 167
3.6.4.2 Fraction Collectors......Page 168
3.6.4.3 Waste Diversion......Page 169
3.7.1 Temperature-Control Requirements......Page 170
3.7.2.3 Peltier Heater......Page 171
3.8.1 Experimental Aids......Page 172
3.8.3 Data Collection......Page 174
3.8.6 Regulatory Functions......Page 175
3.10.1 System-Performance Tests......Page 176
3.10.1.2 Gradient Performance Test......Page 177
3.10.1.3 Additional System Checks......Page 180
3.10.2.1 Periodic Maintenance......Page 183
3.10.2.2 Suggestions for Routine Applications......Page 186
3.10.3.2 Record Keeping......Page 188
References......Page 189
4 DETECTION......Page 192
4.1 Introduction......Page 193
4.2.1 General Layout......Page 194
4.2.2.1 Bulk Property Detectors......Page 196
4.2.3 Signal, Noise, Drift, and Assay Precision......Page 197
4.2.3.1 Noise and Drift......Page 198
4.2.3.2 Signal-to-Noise Ratio (S/N)......Page 200
4.2.4 Detection Limits......Page 202
4.2.5 Linearity......Page 203
4.4 UV-Visible Detectors......Page 205
4.4.1 Fixed-Wavelength Detectors......Page 208
4.4.2 Variable-Wavelength Detectors......Page 209
4.4.3 Diode-Array Detectors......Page 210
4.4.4 General UV-Detector Characteristics......Page 211
4.5 Fluorescence Detectors......Page 212
4.6 Electrochemical (Amperometric) Detectors......Page 215
4.7 Radioactivity Detectors......Page 217
4.9 Chemiluminescent Nitrogen Detector......Page 219
4.10 Chiral Detectors......Page 220
4.11 Refractive Index Detectors......Page 222
4.12 Light-Scattering Detectors......Page 225
4.12.1 Evaporative Light-Scattering Detector (ELSD)......Page 226
4.12.2 Condensation Nucleation Light-Scattering Detector (CNLSD)......Page 227
4.12.3 Laser Light-Scattering Detectors (LLSD)......Page 228
4.13 Corona-Discharge Detector (CAD)......Page 229
4.14 Mass Spectral Detectors (MS)......Page 230
4.14.1.1 Electrospray Interface (ESI)......Page 231
4.14.1.2 Atmospheric-Pressure Chemical-Ionization Interface (APCI)......Page 232
4.14.2 Quadrupoles and Ion Traps......Page 233
4.14.3 Other MS Detectors......Page 235
4.15.1 Infrared (FTIR)......Page 236
4.15.2 Nuclear Magnetic Resonance (NMR)......Page 237
4.16 Sample Derivatization and Reaction Detectors......Page 239
References......Page 241
5 THE COLUMN......Page 244
5.2 Column Supports......Page 245
5.2.1.1 Particle Type......Page 246
5.2.2 Silica Supports......Page 248
5.2.2.1 Column Efficiency......Page 250
5.2.2.2 Nature of the Silica Surface......Page 253
5.2.2.3 Particle Preparation......Page 256
5.2.4 Monoliths......Page 257
5.2.4.1 Silica-Based Monoliths......Page 258
5.2.5 Other Inorganic Particles......Page 259
5.2.5.1 Zirconia......Page 260
5.3 Stationary Phases......Page 262
5.3.1 \"Bonded\" Stationary Phases......Page 263
5.3.2.2 Hybrid Particles......Page 268
5.3.2.3 Columns for Highly Aqueous Mobile Phases......Page 269
5.3.3 Column Functionality (Ligand Type)......Page 270
5.4.1 Basis of RPC Column Selectivity......Page 272
5.4.1.1 Hyperbolic-Subtraction Model......Page 274
5.4.1.2 Shape Selectivity......Page 277
5.4.2 Column Reproducibility and \"Equivalent\" Columns......Page 280
5.4.3 Orthogonal Separation......Page 281
5.4.4.2 Stationary-Phase De-Wetting......Page 282
5.5.1 Column Fittings......Page 283
5.5.2 Column Configurations......Page 284
5.6.2 Slurry-Packing of Rigid Particles......Page 285
5.6.2.1 Selection of Slurry Liquid......Page 286
5.6.2.2 Rigid Polymeric Particles......Page 288
5.7.1 Manufacturing Standards......Page 289
5.7.2 Column Plate Number......Page 290
5.8 Column Handling......Page 291
References......Page 295
6 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES......Page 298
6.1 Introduction......Page 299
6.1.1 Abbreviated History of Reversed-Phase Chromatography......Page 300
6.2 Retention......Page 301
6.2.1 Solvent Strength......Page 302
6.2.2 Reversed-Phase Retention Process......Page 304
6.3.1 Solvent-Strength Selectivity......Page 308
6.3.2 Solvent-Type Selectivity......Page 310
6.3.3 Temperature Selectivity......Page 315
6.3.3.1 Further Observations......Page 317
6.3.4 Column Selectivity......Page 318
6.3.5 Isomer Separations......Page 321
6.3.5.2 Shape Selectivity......Page 322
6.3.6 Other Selectivity Considerations......Page 323
6.3.6.1 Equivalent Separation......Page 324
6.3.6.2 Orthogonal Separation......Page 327
6.4 Method Development and Strategies for Optimizing Selectivity......Page 329
6.4.1 Multiple-Variable Optimization......Page 331
6.4.1.1 Mixtures of Different Organic Solvents......Page 332
6.4.1.2 Simultaneous Variation of Solvent Strength and Type......Page 335
6.4.1.3 Simultaneous Variation of Solvent Strength and Temperature......Page 337
6.4.1.4 Change of the Column with Variation of One or More Other Conditions......Page 338
6.5 Nonaqueous Reversed-Phase Chromatography (NARP)......Page 340
6.6.1 Poor Retention of Very Polar Samples......Page 342
References......Page 343
7 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND IONEXCHANGE CHROMATOGRAPHY......Page 348
7.2 Acid–Base Equilibria and Reversed-Phase Retention......Page 349
7.2.1 Choice of Buffers......Page 354
7.2.1.1 Buffer pK(a) and Capacity......Page 356
7.2.1.2 Other Buffer Properties......Page 359
7.2.1.3 Preferred Buffers......Page 361
7.2.3 Effects of Organic Solvents and Temperature on Mobile-Phase pH and Sample pK(a) Values......Page 362
7.2.3.1 Effect of %B on Values of Effective pK(a) for the Solute......Page 363
7.3 Separation of Ionic Samples by Reversed-Phase Chromatography (RPC)......Page 364
7.3.2.1 Mobile-Phase pH......Page 365
7.3.2.2 Solvent Strength (%B) and Temperature......Page 367
7.3.2.4 Column Type......Page 368
7.3.2.5 Other Conditions That Can Affect Selectivity......Page 371
7.3.3.1 Starting Conditions......Page 372
7.3.3.2 Optimizing Selectivity......Page 373
7.3.4.1 pH Sensitivity......Page 374
7.3.4.2 Silanol Effects......Page 375
7.4 Ion-Pair Chromatography (IPC)......Page 376
7.4.1.1 pH and Ion Pairing......Page 379
7.4.1.2 Ion-Pair Reagent: Concentration and Type......Page 381
7.4.1.3 Simultaneous Changes in pH and Ion Pairing......Page 382
7.4.2 Method Development......Page 384
7.4.2.1 Choice of Initial Conditions......Page 385
7.4.2.2 Control of Selectivity......Page 388
7.4.2.3 Summary......Page 391
7.4.3.2 Slow Column Equilibration......Page 392
7.5 Ion-Exchange Chromatography (IEC)......Page 394
7.5.1 Basis of Retention......Page 396
7.5.2 Role of the Counter-Ion......Page 397
7.5.5 Role of Other Conditions......Page 399
7.5.8 Mixed-Mode Separations......Page 400
References......Page 402
8 NORMAL-PHASE CHROMATOGRAPHY......Page 406
8.1 Introduction......Page 407
8.2 Retention......Page 408
8.2.1 Theory......Page 411
8.2.2 Solvent Strength as a Function of the B-Solvent and %B......Page 415
8.2.3 Use of TLC Data for Predicting NPC Retention......Page 418
8.3.2 Solvent-Type Selectivity......Page 421
8.3.3 Temperature Selectivity......Page 425
8.3.4 Column Selectivity......Page 426
8.3.5 Isomer Separations......Page 427
8.4 Method-Development Summary......Page 430
8.4.1 Starting Conditions for NPC Method Development: Choice of Mobile-Phase Strength and Column Type......Page 433
8.4.2 Strategies for Optimizing Selectivity......Page 434
8.4.3 Example of NPC Method Development......Page 435
8.5.1 Poor Separation Reproducibility......Page 437
8.5.3 Tailing Peaks......Page 439
8.6 Hydrophilic Interaction Chromatography (HILIC)......Page 440
8.6.1 Retention Mechanism......Page 441
8.6.2 Columns......Page 442
8.6.3 HILIC Method Development......Page 443
References......Page 446
9 GRADIENT ELUTION......Page 448
9.1 Introduction......Page 449
9.1.1 Other Reasons for the Use of Gradient Elution......Page 451
9.1.2 Gradient Shape......Page 452
9.1.3.1 The Linear-Solvent-Strength (LSS) Model......Page 454
9.1.3.2 Band Migration in Gradient Elution......Page 456
9.2 Experimental Conditions and Their Effects on Separation......Page 457
9.2.1 Effects of a Change in Column Conditions......Page 460
9.2.2 Effects of Changes in the Gradient......Page 463
9.2.2.1 Initial-%B......Page 464
9.2.2.2 Final-%B......Page 465
9.2.2.3 Gradient Delay......Page 467
9.2.2.4 Dwell-Volume......Page 469
9.2.2.5 Segmented Gradients......Page 470
9.2.3 \"Irregular Samples\"......Page 473
9.2.4 Quantitative Relationships......Page 475
9.2.4.1 Retention Time......Page 476
9.2.4.2 Measurement of Values of S and k(w)......Page 477
9.2.4.3 Peak Width......Page 478
9.3 Method Development......Page 479
9.3.1.1 Choosing between Isocratic and Gradient Elution......Page 482
9.3.1.2 Possible Problems......Page 485
9.3.3 Optimize Gradient Selectivity α*......Page 487
9.3.4 Optimizing Gradient Range......Page 489
9.3.6 Optimizing the Column Plate Number N*......Page 490
9.3.7 Determine Necessary Column-Equilibration Time......Page 491
9.3.8.1 Method Development......Page 494
9.3.8.2 Routine Analysis......Page 495
9.3.9 Peak Capacity and Fast Separation......Page 496
9.3.9.1 Optimized Peak Capacities......Page 498
9.3.9.2 Fast Gradient Separations......Page 501
9.3.10 Comprehensive Two-Dimensional HPLC......Page 502
9.3.10.1 Principles of LC × LC......Page 503
9.3.10.3 Instrumentation for LC × LC......Page 506
9.3.10.4 Method Development for LC × LC......Page 507
9.4 Large-Molecule Separations......Page 509
9.5.1 Theory......Page 510
9.5.2 Normal-Phase Chromatography (NPC)......Page 511
9.5.3.1 Applications......Page 512
9.5.3.2 Separation Conditions......Page 513
9.6.3 Baseline Drift......Page 515
References......Page 516
10.1 Introduction......Page 520
10.1.2 When to Use Computer Simulation......Page 523
10.1.2.1 Advantages......Page 524
10.1.2.2 Disadvantages......Page 525
10.2.1 DryLab Operation......Page 526
10.2.2 Gradient Optimization......Page 528
10.2.3.1 Isocratic Predictions from Gradient Data......Page 530
10.2.3.2 Designated-Peak Selection......Page 531
10.2.3.3 Change in Other Conditions......Page 532
10.2.3.6 Two-Run Procedures for the Improvement of Sample Resolution......Page 533
10.2.5 Sources of Computer-Simulation Software......Page 534
10.3.2 Solute pK(a) Values and Molecular Structure......Page 536
10.4.1 Example 1: Separation of a Pharmaceutical Mixture......Page 537
10.4.2 Example 2: Alternative Method Development Strategy......Page 539
10.4.3 Verifying Method Robustness......Page 541
References......Page 542
11.1 Introduction......Page 544
11.2.1 Integrator Operation......Page 545
11.2.1.1 Data Sampling......Page 546
11.2.1.2 Peak Recognition......Page 548
11.2.1.3 Integration of Non-Ideal Chromatograms......Page 549
11.2.1.4 Common Integration Errors......Page 550
11.2.1.5 Additional Suggestions......Page 551
11.2.2 Retention......Page 552
11.2.4 Sources of Error......Page 553
11.2.4.3 Detection......Page 554
11.2.4.5 Calibration......Page 555
11.2.5 Limits......Page 557
11.2.5.1 Limit of Detection (LOD)......Page 558
11.2.5.2 Lower Limit of Quantification (LLOQ or LOQ)......Page 559
11.2.5.4 Samples Outside Limits......Page 560
11.3.1 Retention Time......Page 561
11.3.2 On-line Qualitative Analysis......Page 562
11.3.2.2 LC-MS......Page 563
11.3.2.8 Off-line Analysis......Page 564
11.4.1.1 External Standardization......Page 565
11.4.1.2 Internal Standardization......Page 568
11.4.1.3 Area Normalization......Page 570
11.4.1.4 Standard Addition......Page 571
11.4.1.5 Evaluating Calibration Curves......Page 572
11.5 Summary......Page 574
References......Page 575
12 METHOD VALIDATION......Page 576
12.1 Introduction......Page 577
12.2 Terms and Definitions......Page 579
12.2.1 Accuracy......Page 580
12.2.2.1 Repeatability......Page 581
12.2.2.3 Reproducibility......Page 582
12.2.2.4 Ruggedness......Page 583
12.2.4 Limit of Detection and Limit of Quantification......Page 584
12.2.6 Robustness......Page 585
12.3 System Suitability......Page 587
12.4 Documentation......Page 588
12.4.2 Test Method......Page 589
12.4.3 Validation Report......Page 590
12.5.1 Category 1 Methods......Page 591
12.5.3 Category 3 Methods......Page 592
12.6 Bioanalytical Methods......Page 593
12.6.2 Bioanalytical Method Development and Validation......Page 594
12.6.2.2 Accuracy, Precision, and Recovery......Page 595
12.6.2.4 Bioanalytical Sample Stability......Page 596
12.6.3 Routine Application of the Bioanalytical Method......Page 597
12.6.4 Bioanalytical Method Documentation......Page 598
12.7 Analytical Method Transfer (AMT)......Page 599
12.7.1.1 Comparative Testing......Page 600
12.7.2 Essentials of AMT......Page 601
12.7.2.4 Acceptance Criteria......Page 602
12.7.3.2 HPLC Columns......Page 603
12.8 Method Adjustment or Method Modification......Page 606
12.8.3 Ratio of Components in the Mobile Phase......Page 608
12.9 Quality Control and Quality Assurance......Page 609
12.10 Summary......Page 610
References......Page 611
13 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS......Page 614
13.1 Biomacromolecules......Page 615
13.2.1.1 Primary Sequence......Page 616
13.2.1.2 Secondary Structure......Page 618
13.2.2.1 Single-Stranded Nucleic Acids......Page 619
13.2.2.2 Double-Stranded Nucleic Acids......Page 620
13.2.3 Carbohydrates......Page 621
13.2.4 Viruses......Page 623
13.3.1.1 Pore Size......Page 624
13.3.1.2 Particle Size......Page 626
13.3.1.3 Support Characteristics and Stability......Page 627
13.3.2 Role of Protein Structure in Chromatographic Behavior......Page 628
13.4.1 Reversed-Phase Chromatography (RPC)......Page 629
13.4.1.2 Mobile-Phase Selection......Page 630
13.4.1.3 Temperature......Page 633
13.4.1.4 Gradient Elution......Page 634
13.4.1.5 Effect of Polypeptide Conformation......Page 638
13.4.1.7 RPC Method Development......Page 640
13.4.2 Ion-Exchange Chromatography (IEC) and Related Techniques......Page 642
13.4.2.1 Column Selection......Page 644
13.4.2.2 Mobile-Phase Selection......Page 646
13.4.2.3 Chromatofocusing......Page 648
13.4.2.4 Hydroxyapatite Chromatography......Page 649
13.4.2.5 Immobilized-Metal Affinity Chromatography (IMAC)......Page 650
13.4.3 Hydrophobic Interaction Chromatography (HIC)......Page 653
13.4.3.1 Supports and Ligands for HIC......Page 654
13.4.3.2 Other Conditions......Page 655
13.4.4.1 Stationary Phases for HILIC......Page 658
13.4.4.4 Electrostatic-Repulsion Hydrophilic-Interaction Chromatography (ERLIC)......Page 659
13.4.5 Multidimensional Liquid Chromatography (MDLC) in Proteomics......Page 661
13.4.5.2 Directly Coupled MDLC......Page 662
13.5 Separation of Nucleic Acids......Page 663
13.5.1 Anion-Exchange Chromatography......Page 664
13.5.2 Reversed-Phase Chromatography......Page 665
13.5.2.3 Denaturing HPLC......Page 666
13.5.2.4 RPC-5 Chromatography......Page 668
13.5.3 Hydrophobic Interaction Chromatography......Page 669
13.6.1 Hydrophilic Interaction Chromatography......Page 670
13.6.2 Ion-Moderated Partition Chromatography......Page 671
13.6.3 High-Performance Anion-Exchange Chromatography......Page 673
13.7 Separation of Viruses......Page 675
13.8 Size-Exclusion Chromatography (SEC)......Page 676
13.8.1 SEC Retention Process......Page 677
13.8.2 Columns for Gel Filtration......Page 678
13.8.2.1 Support Materials......Page 679
13.8.2.2 Pore Size and Porosity......Page 680
13.8.3 Mobile Phases for Gel Filtration......Page 681
13.8.4.2 Use of Denaturing Conditions......Page 682
13.8.5 Advantages and Limitations of SEC......Page 683
13.8.6.1 Analytical Applications......Page 684
13.9.1 Background......Page 686
13.9.2 Production-Scale Purification of rh-Insulin......Page 687
13.9.2.4 Stability of the Product and Column......Page 688
13.9.2.5 Mobile-Phase Composition......Page 689
13.9.2.8 Small-Scale Purification......Page 690
13.9.2.9 Scale-Up......Page 691
13.9.2.10 Production-Scale Purification......Page 692
13.10.1 Background......Page 693
13.10.2 Techniques for Polymer Analysis......Page 696
13.10.3.2 Interactive Liquid Chromatography......Page 698
13.10.3.4 Other Techniques......Page 700
13.10.3.5 Chemical Composition as a Function of Molecular Size......Page 701
13.10.4 Polymer Separations by Two-Dimensional Chromatography......Page 702
References......Page 703
14 ENANTIOMER SEPARATIONS......Page 710
14.2 Background and Definitions......Page 711
14.2.1 Isomerism and Chirality......Page 712
14.2.2 Chiral Recognition and Enantiomer Separation......Page 714
14.3 Indirect Method......Page 715
14.4.1 Chiral Mobile-Phase-Additive Mode (CMPA)......Page 720
14.4.2 Chiral Stationary-Phase Mode (CSP)......Page 722
14.4.3.1 \"Three-Point Interaction Model\"......Page 724
14.4.3.2 Mobile-Phase Effects......Page 725
14.6 Chiral Stationary Phases and Their Characteristics......Page 726
14.6.1 Polysaccharide-Based CSPs......Page 727
14.6.2 Synthetic-Polymer CSPs......Page 734
14.6.3 Protein Phases......Page 736
14.6.4 Cyclodextrin-Based CSPs......Page 742
14.6.5 Macrocyclic Antibiotic CSPs......Page 744
14.6.6 Chiral Crown-Ether CSPs......Page 751
14.6.7 Donor-Acceptor Phases......Page 752
14.6.8 Chiral Ion-Exchangers......Page 756
14.6.9 Chiral Ligand-Exchange CSPs (CLEC)......Page 758
14.7.1 Thermodynamics of Solute-Selector Association......Page 760
14.7.2 Thermodynamics of Direct Chromatographic Enantiomer Separation......Page 761
14.7.3 Site-Selective Thermodynamics......Page 762
References......Page 763
15 PREPARATIVE SEPARATIONS......Page 770
15.1.1 Column Overload and Its Consequences......Page 771
15.1.2 Separation Scale......Page 772
15.1.2.3 Other Considerations......Page 773
15.2.1 Columns......Page 775
15.2.2 Sample Introduction......Page 776
15.2.2.2 Pump Injection......Page 777
15.2.3.2 Other Detectors......Page 778
15.2.5 Product Recovery (Removal of the Mobile Phase)......Page 780
15.3.1.1 Sorption Isotherms......Page 782
15.3.1.2 Peak Width for Small versus Large Samples......Page 783
15.3.2 Touching-Peak Separation......Page 784
15.3.2.1 Column Saturation Capacity......Page 785
15.3.2.3 Sample Solubility......Page 787
15.3.2.4 Method Development......Page 790
15.3.2.5 Fraction Collection......Page 792
15.4.1 Recovery versus Purity......Page 793
15.4.2 Method Development......Page 794
15.4.2.2 \"Crossing Isotherms\"......Page 795
15.5 Gradient Elution......Page 796
15.5.1 Isocratic and Gradient Prep-LC Compared......Page 797
15.5.2 Method Development for Gradient Prep-LC......Page 798
15.6 Production-Scale Separation......Page 799
References......Page 800
16 SAMPLE PREPARATION......Page 802
16.1 Introduction......Page 803
16.2 Types of Samples......Page 804
16.3.1 Sample Particle-Size Reduction......Page 805
16.3.2 Sample Drying......Page 807
16.3.3 Filtration......Page 808
16.5 Liquid–Liquid Extraction......Page 809
16.5.2 Practice......Page 811
16.5.3 Problems......Page 813
16.5.3.4 Mutual Phase-Solubility......Page 814
16.5.4.4 Immobilized Liquid Extraction (ILE)......Page 815
16.6 Solid-Phase Extraction (SPE)......Page 816
16.6.2.1 Interference Removal......Page 817
16.6.2.2 Analyte Enrichment......Page 818
16.6.3.1 Cartridges......Page 819
16.6.3.3 Other SPE Formats......Page 820
16.6.4 SPE Apparatus......Page 822
16.6.5 SPE Method Development......Page 823
16.6.5.1 SPE Steps......Page 824
16.6.5.2 SPE Packings......Page 826
16.6.6 Example of SPE Method Development: Isolation of Albuterol from Human Plasma......Page 829
16.6.7.2 Restricted Access Media (RAM)......Page 830
16.6.7.3 Molecular-Imprinted Polymers (MIPs)......Page 832
16.6.7.4 Immunoaffinity Extraction of Small Molecules......Page 833
16.6.7.6 Class-Specific SPE Cartridges......Page 834
16.7 Membrane Techniques in Sample Preparation......Page 835
16.8 Sample Preparation Methods for Solid Samples......Page 836
16.8.1 Traditional Extraction Methods......Page 837
16.8.2.1 Modern Soxhlet Extraction......Page 838
16.8.2.2 Supercritical Fluid Extraction (SFE)......Page 839
16.8.2.4 Microwave-Assisted Solvent Extraction (MAE)......Page 840
16.9 Column-Switching......Page 841
16.10 Sample Preparation for Biochromatography......Page 842
16.11 Sample Preparation for LC-MS......Page 845
16.12 Derivatization in HPLC......Page 847
References......Page 850
17 TROUBLESHOOTING......Page 854
17.1 Introduction......Page 855
17.2.1 System Performance Tests......Page 856
17.2.2 Periodic Maintenance......Page 857
17.2.4 Historical Records......Page 858
17.2.5.2 Solvent Siphon Test......Page 859
17.2.5.4 Cleaning and Handling Check Valves......Page 860
17.2.5.7 Cleaning Glassware......Page 861
17.2.5.9 Improved Water Purity......Page 862
17.2.5.10 Isolating Carryover Problems......Page 863
17.3.1 Divide and Conquer......Page 864
17.3.5 Module Substitution......Page 865
17.4 Common Symptoms of HPLC Problems......Page 866
17.4.1.1 Pre-pump Leaks......Page 867
17.4.1.2 Pump Leaks......Page 868
17.4.1.4 Autosampler Leaks......Page 870
17.4.1.6 Detector Leaks......Page 873
17.4.2 Abnormal Pressure......Page 875
17.4.2.1 Pressure Too High......Page 876
17.4.2.2 Pressure Too Low......Page 877
17.4.3 Variation in Retention Time......Page 878
17.4.3.3 Mobile-Phase Problems......Page 879
17.4.3.4 Stationary-Phase Problems......Page 880
17.4.3.6 Retention-Problem Symptoms......Page 881
17.4.4 Peak Area......Page 883
17.4.4.1 Peak Area Too Large......Page 884
17.4.4.3 Peak Area Too Variable......Page 885
17.4.5.1 Baseline Drift Problems......Page 886
17.4.5.2 Baseline Noise Problems......Page 889
17.4.5.3 Peak Shape Problems......Page 892
17.4.6 Interpretation of System Performance Tests......Page 901
17.4.6.1 Interpretation of Gradient Performance Tests......Page 902
17.4.6.2 Interpretation of Additional System Tests......Page 909
17.5 Troubleshooting Tables......Page 910
References......Page 921
I.1.1 UV Detection......Page 924
I.1.2 RI Detection......Page 926
I.2 Solvent Polarity and Selectivity......Page 927
I.3 Solvent Safety......Page 930
References......Page 931
II.1 Sequence of Operations......Page 932
II.2 Recipes for Some Commonly Used Buffers......Page 933
Reference......Page 935
Index......Page 936