Modern HPLC: a guide for practicing scientists

Cover Page......Page 1
Title Page......Page 3
ISBN 047172789X......Page 4
2 Basic Terms and Concepts......Page 5
3 HPLC Columns and Trends......Page 6
4 HPLC Instrumentation and Trends......Page 7
5 HPLC Operation Guide......Page 8
6 Pharmaceutical Analysis......Page 9
8 HPLC Method Development......Page 10
9 Regulatory Aspects of HPLC Analysis: HPLC System and Method Validation......Page 11
11 Modern Trends in HPLC......Page 12
Index......Page 13
Preface......Page 15
1.1.1 Scope......Page 19
1.1.2 What Is HPLC?......Page 20
1.1.3 A Brief History......Page 21
1.1.4 Advantages and Limitations......Page 22
1.2.1 Normal-Phase Chromatography (NPC)......Page 23
1.2.3 Ion-Exchange Chromatography (IEC)......Page 25
1.2.4 Size-Exclusion Chromatography (SEC)......Page 27
1.2.5 Other Separation Modes......Page 28
1.3 Some Common-Sense Corollaries......Page 29
1.4 How to Get More Information......Page 30
1.6 References......Page 31
2 Basic Terms and Concepts......Page 33
2.1 Scope......Page 34
2.2.1 Retention Time (t(R)), Void Time (t(M)), Peak Height (h), and Peak Width (w(b))......Page 35
2.2.2 Retention Volume (V(R)), Void Volume (V(M)), and Peak Volume......Page 36
2.2.3 Retention Factor (k)......Page 37
2.2.4 Separation Factor (a)......Page 38
2.2.5 Column Efficiency and Plate Number (N)......Page 39
2.2.6 Peak Volume......Page 40
2.2.8 Resolution (R(s))......Page 41
2.2.9 Peak Symmetry: Asymetry Factor (A(s)) and Tailing Factor (T(f))......Page 42
2.3.1 General Requirements......Page 45
2.3.2 Solvent Strength and Selectivity......Page 46
2.3.3 Buffers......Page 49
2.3.5 Ion-Pairing Additives......Page 50
2.3.7 Other Operating Parameters: Flow Rate (F) and Column Temperature (T)......Page 51
2.4 The Resolution Equation......Page 52
2.5 The Van Deemter Equation......Page 53
2.6 Isocratic vs. Gradient Analysis......Page 57
2.6.1 Peak Capacity (n)......Page 58
2.6.2 Key Gradient Parameters (Initial and Final Solvent Strength, Gradient Time [t(G)], and Flow Rate)......Page 59
2.7 Concept of Orthogonality......Page 60
2.9 Glossary of HPLC Terms......Page 62
2.10 Summary and Conclusion......Page 63
2.11 References......Page 64
3 HPLC Columns and Trends......Page 65
3.2 General Column Description and Characteristics......Page 66
3.2.1 Column Hardware—Standard vs. Cartridge Format......Page 67
3.3.1 Types Based on Chromatographic Modes......Page 68
3.3.3 Column Length (L)......Page 69
3.4 Column Packing Characteristics......Page 70
3.4.1 Support Type......Page 71
3.4.4 Bonding Chemistries......Page 72
3.4.5 Some General Guidelines for Bonded Phase Selection......Page 74
3.5 Modern HPLC Column Trends......Page 75
3.5.2 Hybrid Particles......Page 76
3.5.3 Novel Bonding Chemistries......Page 77
3.5.4 Fast LC......Page 82
3.5.5 Micro LC......Page 84
3.5.6 Monoliths......Page 86
3.6 Guard Columns......Page 87
3.8 Column Selection Guides......Page 88
3.9 Summary......Page 91
3.10 References......Page 92
3.11 Internet Resources......Page 93
4 HPLC Instrumentation and Trends......Page 95
4.1.1 Scope......Page 96
4.1.2 HPLC Systems and Modules......Page 97
4.2 HPLC Solvent Delivery Systems......Page 99
4.2.1 High-Pressure and Low-Pressure Mixing Designs in Multisolvent Pumps......Page 100
4.2.2 System Dwell Volume......Page 101
4.3 Injectors and Autosamplers......Page 102
4.3.1 Operating Principles of Autosamplers......Page 103
4.3.2 Performance Characteristics and Trends......Page 104
4.5.1 Operating Principles......Page 105
4.5.2 Performance Characteristics......Page 106
4.5.3 Trends in Absorbance Detectors......Page 107
4.6.1 Operating Principles......Page 109
4.7.1 Fluorescence Detector (FLD)......Page 111
4.7.4 Corona-Charged Aerosol Detector (CAD)......Page 112
4.7.8 Radiometric Detector......Page 113
4.8.1 LC/MS, LC/MS/MS......Page 114
4.8.4 Prep LC and Bio-Purification Systems......Page 115
4.8.5 Proteomics Systems: Capillary LC and Multi-Dimensional LC......Page 116
4.8.6 High-Throughput Screening (HTS) and Parallel Analysis Systems......Page 117
4.8.9 Specialized Applications Systems......Page 119
4.9.2 Column Oven......Page 120
4.9.4 Data Handling and HPLC Controllers......Page 121
4.10 Instrumental Bandwidth (IBW)......Page 122
4.11 Trends in HPLC Equipment......Page 125
4.12 Manufacturers and Equipment Selection......Page 126
4.14 References......Page 127
4.15 Internet Resources......Page 128
5 HPLC Operation Guide......Page 129
5.2.1 Safety Concerns......Page 130
5.3.2 Buffers......Page 132
5.3.3 Filtration......Page 133
5.4 Best Practices in HPLC System Operation......Page 134
5.4.2.1 Column Use......Page 135
5.4.2.4 Column Maintenance and Regeneration......Page 136
5.4.4 Detector Operation......Page 138
5.4.5 System Shutdown......Page 139
5.4.6.2 Guidelines for Improving Peak Area Precision......Page 140
5.5 From Chromatograms to Reports......Page 141
5.5.2 Quantitation Analysis Strategies......Page 146
5.7 Guides on Performing Trace Analysis......Page 147
5.9 References......Page 150
6 Pharmaceutical Analysis......Page 153
6.1.2 Overview: From Drug Discovery to Quality Control......Page 154
6.1.4 High-Throughput LC/MS in Drug Discovery Support......Page 155
6.2 Identification......Page 156
6.3 Assays......Page 157
6.3.2 Drug Products......Page 158
6.3.4 Products with Multiple APIs and Natural Products......Page 160
6.4 Impurity Testing......Page 163
6.6 Cleaning Validation......Page 166
6.7 Bioanalytical Testing......Page 168
6.8 Chiral Analysis......Page 169
6.9 Case Study: HPLC Methods in Early Development......Page 170
6.10 Summary......Page 171
6.11 References......Page 173
7 Food, Environmental, Chemical, and Life Sciences Applications......Page 175
7.2 Food Applications......Page 176
7.2.1.1 Sugars......Page 177
7.2.1.2 Fats, Oils, and Triglycerides......Page 178
7.2.1.4 Proteins and Amino Acids......Page 180
7.2.2 Food Additives......Page 182
7.2.2.1 Flavors: A Case Study on HPLC Analysis of Capsaicins......Page 184
7.2.3 Contaminants......Page 185
7.2.3.2 Antimicrobial Additives......Page 186
7.3 Environmental Applications......Page 187
7.3.2.1 Carbamates and Glyphosate......Page 188
7.3.3.1 Case Study: Quick Turnaround Analysis of PAHs by HPLC in Multimedia Samples......Page 190
7.4 Chemical, GPC and Plastics Applications......Page 191
7.4.1 Gel-Permeation Chromatography (GPC) and Analysis of Plastics Additives......Page 193
7.5 Ion Chromatography......Page 196
7.6.1 Proteins, Peptides, and Amino Acids......Page 197
7.6.2 Bases, Nucleosides, Nucleotides, Oligonucleotides, Nucleic Acids, and PCR Products......Page 204
7.7 Summary......Page 206
7.8 References......Page 208
8 HPLC Method Development......Page 211
8.1.2 Considerations Before Method Development......Page 212
8.1.4 HPLC Method Development Trends in Pharmaceutical Analysis......Page 213
8.2.1 Method Goals......Page 214
8.3.1 Defining Sample Preparation Requirements......Page 215
8.4.1 Initial Detector Selection......Page 216
8.4.3 Initial Selection of HPLC Column......Page 217
8.4.4.1 Case Study: Initial Method Development Using a Broad Gradient and Mobile Phase Selection......Page 218
8.5 Method Fine-Tuning......Page 222
8.5.1 Mobile Phase Parameters (%B, Buffer, pH, Solvent Type)......Page 223
8.5.4 Detector Setting and Sample Amount......Page 224
8.5.5 Summary of Method Development Steps......Page 225
8.6 Phase-Appropriate Method Development......Page 226
8.8.1 Composite Assay Method for a Neutral Drug Substance......Page 228
8.8.2 Composite Drug Substance Method for a Basic Drug Substance......Page 232
8.8.3 Impurity Method for a Drug Product with Two APIs......Page 233
8.9 Summary and Conclusions......Page 235
8.10 References......Page 238
9 Regulatory Aspects of HPLC Analysis: HPLC System and Method Validation......Page 239
9.1.2 The Regulatory Environment......Page 240
9.2 HPLC System Qualification......Page 242
9.2.4 Performance Qualification (PQ)......Page 243
9.2.5 Documentation......Page 244
9.3 Method Validation......Page 245
9.3.1 Validation Parameters......Page 248
9.4 System Suitability Testing (SST)......Page 253
9.5 Case Study on Method Validation......Page 255
9.6 Cost-Effective Regulatory Compliance......Page 257
9.8 References......Page 258
9.9 Internet Resources......Page 259
10 HPLC Maintenance and Troubleshooting Guide......Page 261
10.2.1 LC Pump......Page 262
10.2.2 UV/Vis Detectors......Page 265
10.3.1 General Problem Diagnostic and Troubleshooting Guide......Page 266
10.3.2.1 Pressure Problems and Causes......Page 268
10.3.2.2 Baseline Problems (Chromatogram)......Page 269
10.3.2.3 Peak Problems (Chromatogram)......Page 271
10.3.2.4 Data Performance Problems......Page 274
10.4.1 Case Study 1: Reducing Baseline Shift and Noise for Gradient Analysis......Page 275
10.4.2 Case Study 2: Poor Peak Area Precision Encountered During HPLC System Calibration......Page 276
10.4.3 Case Study 3: Poor Assay Accuracy Data, an Out-of-Specification Investigation......Page 278
10.4.4 Case Study 4: Equipment Malfunctioning......Page 279
10.7 Internet Resources......Page 281
11 Modern Trends in HPLC......Page 283
11.3 Pumps......Page 284
11.5 Detectors......Page 285
11.6.3 Multi-Dimensional LC......Page 286
11.9 Regulatory Compliance......Page 287
11.11 Summary and Conclusions......Page 288
11.12 References......Page 289
A......Page 291
C......Page 292
D......Page 294
G......Page 295
H......Page 296
K,L......Page 298
M......Page 299
P......Page 300
S......Page 302
T......Page 303
W,X......Page 304