A concise yet comprehensive reference guide on HPLC/UHPLC that focuses on its fundamentals, latest developments, and best practices in the pharmaceutical and biotechnology industries
Written for practitioners by an expert practitioner, this new edition of HPLC and UHPLC for Practicing Scientists adds numerous updates to its coverage of high-performance liquid chromatography, including comprehensive information on UHPLC (ultra-high-pressure liquid chromatography) and the continuing migration of HPLC to UHPLC, the modern standard platform. In addition to introducing readers to HPLC’s fundamentals, applications, and developments, the book describes basic theory and terminology for the novice, and reviews relevant concepts, best practices, and modern trends for the experienced practitioner.
HPLC and UHPLC for Practicing Scientists, Second Edition offers three new chapters. One is a standalone chapter on UHPLC, covering concepts, benefits, practices, and potential issues. Another examines liquid chromatography/mass spectrometry (LC/MS). The third reviews at the analysis of recombinant biologics, particularly monoclonal antibodies (mAbs), used as therapeutics. While all chapters are revised in the new edition, five chapters are essentially rewritten (HPLC columns, instrumentation, pharmaceutical analysis, method development, and regulatory aspects). The book also includes problem and answer sections at the end of each chapter.
- Overviews fundamentals of HPLC to UHPLC, including theories, columns, and instruments with an abundance of tables, figures, and key references
- Features brand new chapters on UHPLC, LC/MS, and analysis of recombinant biologics
- Presents updated information on the best practices in method development, validation, operation, troubleshooting, and maintaining regulatory compliance for both HPLC and UHPLC
- Contains major revisions to all chapters of the first edition and substantial rewrites of chapters on HPLC columns, instrumentation, pharmaceutical analysis, method development, and regulatory aspects
- Includes end-of-chapter quizzes as assessment and learning aids
- Offers a reference guide to graduate students and practicing scientists in pharmaceutical, biotechnology, and other industries
Filled with intuitive explanations, case studies, and clear figures, HPLC and UHPLC for Practicing Scientists, Second Edition is an essential resource for practitioners of all levels who need to understand and utilize this versatile analytical technology. It will be a great benefit to every busy laboratory analyst and researcher.
Table of Contents
Author’s Biography xvii
Biographies of Contributors xix
Preface xxi
Foreword xxiii
Acknowledgments xxv
1 Introduction 1
1.1 Introduction 1
1.1.1 Scope 1
1.1.2 What Is HPLC? 2
1.1.3 A Brief History 3
1.1.4 Advantages and Limitations 4
1.1.5 Ultra-High-Pressure Liquid Chromatography (UHPLC) 4
1.2 Primary Modes of HPLC 4
1.2.1 Normal-Phase Chromatography (NPC) 5
1.2.2 Reversed-Phase Chromatography (RPC) 5
1.2.3 Ion-Exchange Chromatography (IEC) 6
1.2.4 Size-Exclusion Chromatography (SEC) 8
1.2.5 Other Separation Modes 8
1.3 Some Common-Sense Corollaries 10
1.4 How to Get More Information 11
1.5 Summary 11
1.6 Quizzes 11
1.6.1 Bonus Quiz 12
References 12
2 Basic Terms and Concepts 15
2.1 Scope 15
2.2 Basic Terms and Concepts 16
2.2.1 Retention Time (tR), Void Time (tM), Peak Height (h), and Peak Width (wb) 16
2.2.2 Retention Volume (VR), Void Volume (VM), and Peak Volume 16
2.2.3 Retention Factor (k) 18
2.2.4 Separation Factor (𝛼) 19
2.2.5 Column Efficiency and Plate Number (N) 20
2.2.6 Peak Volume 20
2.2.7 Height Equivalent to a Theoretical Plate or Plate Height (HETP or H) 21
2.2.8 Resolution (Rs) 21
2.2.9 Peak Symmetry:Asymmetry Factor (As) and Tailing Factor (Tf) 23
2.3 Mobile Phase 24
2.3.1 General Requirements 24
2.3.2 Solvent Strength and Selectivity 25
2.3.3 pH Modifiers and Buffers 27
2.3.4 Acidic Mobile Phases 28
2.3.5 Ion-Pairing Reagents and Chaotropic Agents 29
2.3.6 High-pH Mobile Phases 29
2.3.7 Other Operating Parameters: Flow Rate (F) and Column Temperature (T) 30
2.4 The Resolution Equation 31
2.5 The Van Deemter Equation 33
2.6 Isocratic vs. Gradient Analysis 35
2.6.1 Peak Capacity (n) 35
2.6.2 Gradient Parameters (Initial and Final Solvent Strength, Gradient Time (tG), and Flow Rate) 36
2.6.3 The 0.25 ΔtG Rule: When Is Isocratic Analysis More Appropriate? 37
2.7 The Concept of Orthogonality and Selectivity Tuning 38
2.8 Sample Capacity 41
2.9 Glossary of HPLC Terms 41
2.10 Summary and Conclusion 42
2.11 Quizzes 42
2.11.1 Bonus Quiz 44
References 44
3 HPLC Columns and Trends 45
3.1 Scope 45
3.1.1 Glossary and Abbreviations 45
3.2 General Column Description and Characteristics 46
3.2.1 Column Hardware - Standard vs. Cartridge Format 47
3.3 Column Type 47
3.3.1 Types Based on Chromatographic Mode 48
3.3.2 Column Types Based on Dimension 48
3.3.3 Column Length (L) 48
3.4 Column Packing Characteristics 50
3.4.1 Support Type 50
3.4.2 Particle Size (dp) 51
3.4.3 Surface Area and Pore Size (dpore) 51
3.4.4 Bonding Chemistries 52
3.5 Modern HPLC Column Trends 54
3.5.1 Silica Support Material 54
3.5.2 Hybrid Particles 55
3.5.3 Novel Bonding Chemistries 58
3.5.4 Shorter and Narrower Columns Packed with Small Particles 61
3.5.5 Micro-LC and Nano-LC 62
3.5.6 Monoliths 64
3.5.7 Superficially Porous Particles (SPP) 65
3.5.8 Micropillar Array Chromatography (μPAC) 67
3.6 Guard Columns 69
3.7 Specialty Columns 69
3.7.1 Bioseparations Columns 69
3.7.2 Chiral Columns 69
3.7.3 Supercritical Fluid Chromatography (SFC) Columns 71
3.7.4 Hydrophilic Interaction Liquid Chromatography (HILIC) Columns 72
3.7.5 Mixed-Mode Chromatography (MMC) Columns 72
3.7.6 Application-Specific Columns 73
3.8 RPC Column Selection Guides 73
3.8.1 Some General Guidelines for Bonded Phase Selection 75
3.9 Summary 76
3.10 Quizzes 76
3.10.1 Bonus Quiz 78
References 78
4 HPLC/UHPLC Instrumentation and Trends 81
4.1 Introduction 81
4.1.1 Scope 81
4.1.2 HPLC Systems and Modules 81
4.1.3 Ultra-High-Pressure Liquid Chromatography (UHPLC) 83
4.2 HPLC and UHPLC Solvent Delivery Systems 83
4.2.1 High-Pressure and Low-Pressure Mixing Designs in Multisolvent Pumps 85
4.2.2 System Dwell Volume 86
4.2.3 Trends 88
4.3 Injectors and Autosamplers 88
4.3.1 Operating Principles of Autosamplers 88
4.3.2 Performance Characteristics and Trends 89
4.4 Detectors 91
4.5 UV/VIS Absorbance Detectors 92
4.5.1 Operating Principles 92
4.5.2 Performance Characteristics 94
4.5.3 Trends in UV/Vis Absorbance Detectors 94
4.6 Photodiode Array Detectors 94
4.6.1 Operating Principles 94
4.6.2 Trends in PDA Detectors 95
4.7 Other Detectors 95
4.7.1 Refractive Index Detector (RID) 96
4.7.2 Evaporative Light Scattering Detector (ELSD) 96
4.7.3 Charged Aerosol Detector (CAD) 97
4.7.4 Conductivity Detector (CD) 97
4.7.5 Fluorescence Detector (FLD) 97
4.7.6 Chemiluminescence Nitrogen Detector (CLND) 98
4.7.7 Electrochemical Detector (ECD) 99
4.7.8 Radiometric Detector 99
4.8 Hyphenated and Specialized Systems 99
4.8.1 LC/MS and LC/MS/MS 99
4.8.2 LC/NMR 100
4.8.3 Other Hyphenated Systems 102
4.8.4 Supercritical Fluid Chromatography (SFC) 102
4.8.5 Preparative LC and SFC 102
4.8.6 Micro- and Nano-LC (Capillary LC) 102
4.8.7 Multidimensional LC 102
4.8.8 Lab-on-a-Chip 104
4.8.9 Specialized Applications Systems 104
4.9 HPLC Accessories 105
4.9.1 Solvent Degasser 105
4.9.2 Column Oven 105
4.9.3 Valves for Column and Mobile Phase Selection 106
4.10 Chromatography Data Systems (CDS) 106
4.10.1 User Interface and CDSWorkflow 107
4.11 Instrumental Bandwidth (IBW) 108
4.11.1 How to Measure IBW 109
4.11.2 IBW of UHPLC Systems 110
4.12 Manufacturers and Equipment Selection 111
4.13 Trends in HPLC and UHPLC Equipment 111
4.14 Summary 112
4.15 Quizzes 112
4.15.1 Bonus Quiz 114
References 114
5 UHPLC: Perspectives, Performance, Practices, and Potential Issues 117
5.1 Introduction 117
5.1.1 Scope 117
5.1.2 Glossary and Abbreviations 117
5.1.3 Historical Perspectives: What Is UHPLC? 118
5.2 Practical Concepts in UHPLC 120
5.2.1 Rationale for Higher System Pressure 120
5.2.2 Rationale for Low-Dispersion Systems 121
5.2.3 Rationale for Low Dwell Volumes 121
5.2.4 Other UHPLC Instrumental Characteristics 122
5.3 Benefits Of UHPLC and Case Studies 122
5.3.1 Benefit #1: Fast Separations with Good Resolution 122
5.3.2 Benefit #2: High-Resolution Analysis of Complex Samples 124
5.3.3 Benefit #3: Rapid HPLC Method Development 124
5.3.4 Flexibility for Customizing Resolution 129
5.3.5 Other Benefits of UHPLC 130
5.4 Potential Issues and How to Mitigate 132
5.4.1 Safety Issues 132
5.4.2 Viscous Heating 133
5.4.3 Instrumental and Operating Nuances 133
5.4.4 Injector Precision 135
5.4.5 UV Detection Noise vs. Mixer Volumes 135
5.4.6 Method Translation (Conversion) 138
5.5 How to Implement UHPLC and Practical Aspects 139
5.5.1 How to Transition from HPLC to UHPLC 139
5.5.2 End-Fittings 140
5.5.3 A Summary of UHPLC System Performance Tradeoffs 140
5.6 Myths in UHPLC 142
5.7 Summary and Conclusions 142
5.8 Quizzes 142
5.8.1 Bonus Quiz 144
References 144
6 LC/MS: Fundamentals, Perspectives, and Applications 147
Christine Gu
6.1 Introduction 147
6.1.1 Scope 147
6.1.2 LC/MS Technology and Instrumentation 147
6.1.3 Basic Terminologies and Concepts for MS 148
6.1.4 Interfacing HPLC and MS 150
6.2 LC/MS Instrumentation 150
6.2.1 Ion Sources 150
6.2.2 Fragmentation 152
6.2.3 Mass Analyzers 153
6.2.4 Detectors 155
6.3 Small-Molecules Drug Research and Development 157
6.3.1 Mass Measurement and Elemental Composition Determination 157
6.3.2 Structural Elucidation 159
6.3.3 Trace Quantitation 162
6.4 Emerging Biopharmaceutical Applications 164
6.4.1 Intact Mass Measurement of Proteins 166
6.4.2 Structural Characterization of Proteins (Bottom-Up and Top-Down Approaches) 166
6.4.3 Peptide Quantitation 170
6.5 Environmental, Food Safety, Clinical, Toxicology, and “Omics” Applications 171
6.6 Future Perspectives 171
6.7 Quizzes 172
6.7.1 Bonus Quiz 174
References 174
7 HPLC/UHPLC Operation Guide 177
7.1 Scope 177
7.2 Safety and Environmental Concerns 177
7.2.1 Safety Concerns 177
7.2.2 Environmental Concerns 179
7.3 Mobile Phase and Sample Preparation 180
7.3.1 Mobile Phase Premixing 180
7.3.2 Mobile Phase Additives and Buffers 180
7.3.3 Filtration 180
7.3.4 Degassing 181
7.3.5 Samples, Diluents, and Sample Preparation 181
7.4 Best Practices in HPLC/UHPLC System Operation 182
7.4.1 Pump Operation 182
7.4.2 HPLC Column Use, Precaution, Connection, and Maintenance 183
7.4.3 Autosampler Operation 184
7.4.4 Column Oven and Switching Valve 186
7.4.5 UV/Vis Detector Operation 186
7.4.6 HPLC System Shutdown 187
7.4.7 Guidelines for Increasing HPLC Precision 187
7.5 From Chromatograms to Reports 189
7.5.1 Qualitative Analysis Strategies 192
7.5.2 Quantitation Analysis Strategies 192
7.6 Summary of HPLC Operation 193
7.7 Guides on Performing Trace Analysis 193
7.8 Summary 195
7.9 Quizzes 195
7.9.1 Bonus Quiz 196
References 196
8 HPLC/UHPLC Maintenance and Troubleshooting 199
8.1 Scope 199
8.2 HPLC System Maintenance 199
8.2.1 HPLC Pump 200
8.2.2 UV/Vis Absorbance or Photodiode Array Detectors (PDA) 202
8.2.3 Injector and Autosampler 204
8.3 HPLC Troubleshooting 204
8.3.1 General Problem Diagnostic and Troubleshooting Guide 205
8.3.2 Common HPLC Problems 206
8.4 Troubleshooting Case Studies 213
8.4.1 Case Study 1: Reducing Baseline Shift and Noise for Gradient Analysis 213
8.4.2 Case Study 2: Poor Peak Area Precision 214
8.4.3 Case Study 3: Poor Assay Accuracy Data 215
8.4.4 Case Study 4: Equipment Malfunctioning and Problems with Blank 216
8.5 Summary and Conclusion 217
8.6 Quizzes 218
8.6.1 Bonus Quiz 219
References 219
9 Pharmaceutical Analysis 221
9.1 Introduction 221
9.1.1 Scope 221
9.1.2 Glossary and Abbreviations 221
9.2 Overview of Drug Development Process 222
9.3 Sample Preparation Perspectives 224
9.4 HPLC, SFC, and HPLC/MS in Drug Discovery 224
9.5 HPLC Testing Methodologies for DS and DP 225
9.5.1 Identification Test (DS, DP) 227
9.5.2 ASSAY (Rough Potency and Performance Testing, DP) 227
9.5.3 Stability-Indicating Assay (Potency and Purity Testing of DS and DP) 230
9.5.4 Assay of Preservatives 238
9.5.5 Assay of Pharmaceutical Counterions 238
9.5.6 Assay of Potential Genotoxic Impurities (PGI) 239
9.6 Cleaning Verification 239
9.7 Bioanalytical Testing 240
9.8 Summary 242
9.9 Quizzes 242
9.9.1 Bonus Quiz 243
References 243
10 HPLC Method Development 245
10.1 Introduction 245
10.1.1 Scope 245
10.1.2 Considerations Before Method Development 245
10.1.3 HPLC Method Development Trends in Pharmaceutical Analysis 246
10.2 A Five-Step Strategy for Traditional HPLC Method Development 246
10.2.1 STEP 1: Defining Method Types and Goals 246
10.2.2 STEP 2: Gathering Sample and Analyte Information 248
10.2.3 STEP 3: Initial HPLC Method Development 248
10.2.4 STEP 4: Method Fine-Tuning and Optimization 253
10.2.5 STEP 5: Method Prequalification 256
10.2.6 Summary of Method Development Steps 257
10.2.7 Phase-Appropriate Method Development and Validation 257
10.2.8 Method Development Software Tools 258
10.3 Case Studies 258
10.3.1 A Phase-0 Drug Substance Method for an NCE 259
10.3.2 Stability-Indicating Method Development for an NCE Using DryLab 260
10.3.3 Stability-Indicating Method for a Combination Drug Product with Two APIs 262
10.3.4 Automated Method Development System Employing Fusion QbD Software 265
10.4 A Three-Pronged Template Approach for Rapid HPLC Method Development 268
10.4.1 Template #1: Fast LC Isocratic Potency or Performance Methods 269
10.4.2 Template #2: Generic Broad Gradient Methods 270
10.4.3 Temple #3 Multisegment Gradient Methods for NCEs 271
10.4.4 Summary of the Three-Pronged Approach 272
10.5 A Universal Generic Method for Pharmaceutical Analysis 272
10.5.1 Rationales for the Generic Method Parameters 272
10.5.2 Adjustment of the Generic Method for Stability-Indicating Assays 273
10.5.3 Summary of the Universal Generic Method Approach 275
10.6 Comments on Other HPLC Modes 276
10.7 Summary and Conclusions 276
10.8 Quizzes 277
10.8.1 Bonus Quiz 278
References 278
11 Regulations, HPLC System Qualification, Method Validation, and Transfer 281
11.1 Introduction 281
11.1.1 Scope 281
11.1.2 Glossary and Abbreviations 281
11.2 Regulatory Environment in the Pharmaceutical Industry 281
11.2.1 Regulations 283
11.2.2 The Role of the United States Food and Drug Administration (U.S. FDA) 284
11.2.3 The United States Pharmacopeia (USP) 284
11.3 HPLC System Qualification 285
11.3.1 Design Qualification (DQ) 285
11.3.2 Installation Qualification (IQ) 285
11.3.3 Operational Qualification (OQ) 287
11.3.4 Performance Qualification (PQ) 287
11.3.5 System Qualification Documentation 287
11.3.6 System Calibration 287
11.3.7 System Suitability Testing (SST) 289
11.4 Method Validation 290
11.4.1 Data Required for Method Validation 291
11.4.2 Case Studies and Summary Data on Method Validation 296
11.5 Method Transfer 298
11.6 Regulatory Filings 298
11.7 Cost-Effective Regulatory Compliance Strategies 298
11.7.1 Regulatory Compliance in Other Industries 301
11.8 Summary and Conclusions 302
11.9 Quizzes 302
11.9.1 Bonus Quiz 303
References 303
12 HPLC and UHPLC for Biopharmaceutical Analysis 305
Jennifer Rea and Taylor Zhang
12.1 Introduction 305
12.2 Size-Exclusion Chromatography (SEC) 308
12.2.1 SEC Introduction 308
12.2.2 SEC Theory and Fundamentals 308
12.2.3 SEC Method Conditions 309
12.2.4 SEC Applications 311
12.3 Ion-Exchange Chromatography (IEC) 312
12.3.1 IEC Introduction 312
12.3.2 IEC Theory and Fundamentals 313
12.3.3 IEC Method Conditions 313
12.3.4 IEC Applications 314
12.4 Affinity Chromatography 314
12.4.1 Affinity Chromatography Introduction 314
12.4.2 Affinity Chromatography Theory and Fundamentals 315
12.4.3 Affinity Chromatography Method Conditions 315
12.4.4 Affinity Chromatography Applications 316
12.5 Hydrophilic Interaction Liquid Chromatography (HILIC) 317
12.5.1 HILIC Introduction 317
12.5.2 HILIC Theory and Fundamentals 317
12.5.3 HILIC Method Conditions 318
12.5.4 HILIC Applications 318
12.6 Reversed-Phase Chromatography (RPC) 320
12.6.1 RPC Introduction 320
12.6.2 RPC Theory and Fundamentals 320
12.6.3 RPC Method Conditions 321
12.6.4 RPC Applications 321
12.7 Hydrophobic Interaction Chromatography (HIC) 322
12.7.1 HIC Introduction 322
12.7.2 HIC Theory and Fundamentals 322
12.7.3 HIC Method Conditions 323
12.7.4 HIC Applications 324
12.8 Mixed-Mode Chromatography (MMC) 324
12.8.1 MMC Introduction 324
12.8.2 MMC Theory and Fundamentals 325
12.8.3 MMC Method Conditions 325
12.8.4 MMC Applications 325
12.9 Multidimensional Liquid Chromatography 326
12.9.1 Multidimensional LC Introduction 326
12.9.2 Multidimensional LC Theory and Fundamentals 326
12.9.3 Multidimensional LC Method Conditions 327
12.9.4 Multidimensional LC Applications 327
12.10 Summary 328
12.11 Quizzes 328
References 329
13 HPLC Applications in Food, Environmental, Chemical, and Life Sciences Analysis 335
13.1 Introduction 335
13.1.1 Scope 335
13.2 Food Applications 335
13.2.1 Natural Food Components 336
13.2.2 Food Additives 341
13.2.3 Contaminants 346
13.3 Environmental Applications 349
13.3.1 Listing of U.S. EPA Test Methods Using HPLC 349
13.3.2 Pesticides Analysis 349
13.3.3 Polynuclear Aromatic Hydrocarbons (PAH) 351
13.3.4 HPLC Analysis of Carbonyl Compounds (Aldehydes and Ketone) 352
13.4 Chemical Industry, GPC, and Plastics Applications 352
13.4.1 Gel-Permeation Chromatography (GPC) and Analysis of Plastics Additives 352
13.5 Ion Chromatography (IC) 356
13.6 Life Sciences Applications 356
13.6.1 Proteins, Peptides, and Amino Acids 357
13.6.2 Bases, Nucleosides, Nucleotides, Oligonucleotides, and Nucleic Acids 363
13.6.3 Bioscience Research in Proteomics, Metabolomics, Glycomics and Clinical Diagnostics 363
13.7 Summary 366
13.8 Quizzes 366
13.8.1 Bonus Questions 368
References 368
Keys to Quizzes 371
Index 373
