HPLC and UHPLC for Practicing Scientists

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

About the Author :
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.