HPLC and UHPLC for Practicing Scientists

MICHAEL W. DONG, PHD, is the Principal of MWD Consulting, providing expert training and consulting service in HPLC/UHPLC, and pharmaceutical analysis. He is a former senior scientist, for GENENTECH, INC, small molecule analytical chemistry and quality control department. Dr. Dong conducts short courses on HPLC/UHPLC, drug development process, and drug quality at national meetings such as Pittcon, ACS, HPLC, and EAS. He also provides consulting services on CMC, HPLC method development, and solving drug quality issues. He holds a Ph.D. degree in analytical chemistry and has authored 120+ journal articles and three books.

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