This Second Edition of the classic handbook details how to set up an HPLC system that capitalizes on the latest innovations. It covers new techniques in high-temperature, micro-flow, and ultra-fast chromatography, the linking of an HPLC to a mass spectrometer, and more. Complete appendices and supplementary material online, this guide has everything chromatographers need to know to confidently separate, identify, purify, and quantify compounds.

Table of Contents:
Preface xi I HPLC Primer 1 1 Advantages and Disadvantages of HPLC 3 1.1 How It Works 4 1.1.1 A Separation Model of the Column 5 1.1.2 Basic Hardware: A Quick, First Look 7 1.1.3 Use of Solvent Gradients 8 1.1.4 Ranges of Compounds 9 1.2 Other Ways to Make My Separation 9 1.2.1 FPLC—Fast Protein Liquid Chromatography 10 1.2.2 LC—Traditional Liquid Chromatography 10 1.2.3 GLC—Gas Liquid Chromatography 11 1.2.4 SFC—Supercritical Fluid Chromatography 11 1.2.5 TLC—Thin Layer Chromatography 12 1.2.6 EP—Electrophoresis 12 1.2.7 CZE—Capillary Zone Electrophoresis 13 2 Selecting an HPLC System 15 2.1 Characteristic Systems 16 2.1.1 Finding a Fit: Detectors and Data Processing 16 2.1.2 System Models: Gradient Versus Isocratic 16 2.1.3 Vendor Selection 17 2.1.4 Brand Names and Clones 17 2.1.5 Hardware–Service–Support 18 2.2 System Cost Estimates 19 2.2.1 Type I System—QC Isocratic (Cost: $10–15,000) 19 2.2.2 Type II System—Research Gradient (Cost: $20–25,000) 19 2.2.3 Type III System—Automated Clinical (Cost: $25–35,000) 20 2.2.4 Type IV System—Automated Methods (Cost: $30–50,000) 21 2.3 Columns 21 2.3.1 Sizes: Analytical and Preparative 21 2.3.2 Separating Modes: Selecting Only What You Need 22 2.3.3 Tips on Column Use 23 3 Running Your Chromatograph 25 3.1 Set-up and Start-up 25 3.1.1 Hardware Plumbing 101: Tubing and Fittings 26 3.1.2 Connecting Components 28 3.1.3 Solvent Clean-up 30 3.1.4 Water Purity Test 33 3.1.5 Start-up System Flushing 34 3.1.6 Column Preparation and Equilibration 35 3.2 Sample Preparation and Column Calibration 36 3.2.1 Sample Clean-up 36 3.2.2 Plate Counts 37 3.3 Your First Chromatogram 37 3.3.1 Reproducible Injection Techniques 38 3.3.2 Simple Scouting for a Mobile Phase 39 3.3.3 Examining the Chromatogram 40 3.3.4 Basic Calculations of Results 41 II HPLC Optimization 43 4 Separation Models 45 4.1 Partition 45 4.1.1 Separation Parameters 48 4.1.2 Efficiency Factor 49 4.1.3 Separation (Chemistry) Factor 53 4.2 Ion Exchange Chromatography 56 4.3 Size Exclusion Chromatography 57 4.4 Affinity Chromatography 59 5 Column Preparation 61 5.1 Column Variations 61 5.2 Packing Materials and Hardware 64 5.3 Column Selection 66 6 Column Aging, Diagnosis, and Healing 73 6.1 Packing Degrading—Bonded-Phase Loss 74 6.2 Dissolved Packing Material—End Voids 77 6.3 Bound Material 78 6.4 Pressure Increases 81 6.5 Column Channeling—Center-Voids 83 6.6 Normal Phase, Ion Exchange, and Size Columns 84 6.7 Zirconium and Polymer Columns 86 7 Partition Chromatography Modifications 89 7.1 Reverse-Phase and Hybrid Silica 89 7.1.1 Ionization Suppression 90 7.1.2 Ion Pairing 91 7.1.3 Organic Modifiers 92 7.1.4 Chelation 92 7.2 Acidic Phase Silica 93 7.3 Reverse-Phase Zirconium 93 7.4 Partition Mode Selection 94 8 “Nonpartition” Chromatography 95 8.1 Ion Exchange 96 8.1.1 Cationic: Weak and Strong 96 8.1.2 Anionic: Weak and Strong 97 8.2 Size Exclusion 98 8.2.1 Organic Soluble Samples 98 8.2.2 Hydrophilic Protein Separation 99 8.3 Affinity Chromatography 101 8.3.1 Column Packing Modification 102 8.3.2 Chelation and Optically Active Columns 103 9 Hardware Specifics 105 9.1 System Protection 105 9.1.1 Filters, Guard Columns, and Saturation Columns 106 9.1.2 Inert Surfaces and Connections 107 9.2 Pumping 108 9.2.1 High- and Low-Pressure Mixing Controllers 109 9.2.2 Checking Gradient Performance 112 9.3 Injectors and Autosamplers 113 9.4 Detectors 116 9.4.1 Mass Dependent Detectors 116 9.4.2 Absorptive Detectors 119 9.4.3 Specific Detectors 122 9.5 Fraction Collectors 123 9.6 Data Collection and Processing 123 10 Troubleshooting and Optimization 125 10.1 Hardware and Tools—System Pacification 125 10.2 Reverse Order Diagnosis 129 10.3 Introduction to Data Acquisition 132 10.4 Solvent Conservation 133 III HPLC Utilization 135 11 Preparative Chromatography 137 11.1 Analytical Preparative 138 11.2 Semipreparative 139 11.3 “True” Preparative 139 12 Sample Preparation and Methods Development 143 12.1 Sample Preparation 143 12.1.1 Deproteination 144 12.1.2 Extraction and Concentration 145 12.1.3 SFE (Cartridge Column) Preparations 145 12.1.4 Extracting Encapsulated Compounds 147 12.1.5 SFE Trace Enrichment and Windowing 148 12.1.6 Derivatives 151 12.2 Methods Development 151 12.2.1 Standards Development 152 12.2.2 Samples Development 154 12.3 Gradient Development 156 13 Application Logics: Separations Overview 159 13.1 Fat-Soluble Vitamins, Steroid, and Lipids 159 13.2 Water-Soluble Vitamins, Carbohydrates, and Acids 160 13.3 Nucleomics 161 13.4 Proteomics 162 13.5 Clinical and Forensic Drug Monitoring 163 13.6 Pharmaceutical Drug Development 164 13.7 Environmental and Reaction Monitoring 164 13.8 Application Trends 165 14 Automation 167 14.1 Analog-to-Digital Interfacing 167 14.2 Digital Information Exchange 169 14.3 HPLC System Control and Automation 169 14.4 Data Collection and Interpretation 170 14.4.1 Preinjection Baseline Setting 171 14.4.2 Peak Detection and Integration 171 14.4.3 Quantitation: Internal/External Standards 172 14.5 Automated Methods Development 172 14.5.1 Automated Isocratic Development 173 14.5.2 Hinge Point Gradient Development 176 14.6 Data Exportation to the Real World 177 14.6.1 Word Processors: .ASC, .DOC, .RTF, .WS, .WP Formats 177 14.6.2 Spread Sheets: .DIF, .WK, .XLS Formats 178 14.6.3 Databases: .DB2 Format 178 14.6.4 Graphics: .PCX, .TIFF, .JPG Formats 178 14.6.5 Chromatographic Files: Metafiles and NetCDF 178 15 Recent Advances in LC/MS Separations 181 15.1 A LC/MS Primer 181 15.1.1 Quadrupole MS and Mass Selection 183 15.1.2 Other Types of MS Analyzers for LC/MS 185 15.1.3 LC/MS Interfaces 187 15.1.4 LC/MS Computer Control and Data Processing 189 15.2 Microflow Chromatography 191 15.3 Ultrafast HPLC Systems 192 15.4 Chip HPLC Systems 192 15.5 Standardized LC/MS in Drug Design 193 16 New Directions in HPLC 195 16.1 Temperature-Controlled Chromatography 195 16.2 Ultrafast Chromatography 196 16.3 Monolith Capillary Columns 196 16.4 Micro-Parallel HPLC Systems 197 16.5 Two-Dimensional HPLC Systems 197 16.6 The Portable LC/MS 198 Appendices 201 Appendix A Personal Separations Guide 203 Appendix B FAQs for HPLC Systems and Columns 205 Appendix C Tables of Solvents and Volatile Buffers 211 Appendix D Glossary of HPLC Terms 213 Appendix E HPLC Troubleshooting Quick Reference 221 Appendix F HPLC Laboratory Experiments 227 Laboratory 1—System Start-up and Column Quality Control 227 Laboratory 2—Sample Preparation and Methods Development 229 Laboratory 3—Column and Solvent Switching and Pacification 231 Appendix G Selected Reference List 233 Index 235