Lloyd R. Snyder, PhD, is a Principal at LC Resources in Walnut Creek, California. He is the author or coauthor of several books including An Introduction to Separation Science, Introduction to Modern Liquid Chromatography, Second Edition, the bestselling Practical HPLC Method Development, Second Edition, and the comprehensive High-Performance Gradient Elution, all published by Wiley.

Joseph J. Kirkland, PhD, is Vice President of Research and Development for Advanced Materials Technology, Inc., and coauthor of Introduction to Modern Liquid Chromatography, Second Edition, Practical HPLC Method Development, Second Edition, and Modern Size-Exclusion Liquid Chromatography, Second Edition, all published by Wiley.

John W. Dolan, PhD, is a Principal at LC Resources. He is author of the popular "LC Troubleshooting" column in LCGC magazine and coauthor with Lloyd Snyder of Troubleshooting LC Systems and High-Performance Gradient Elution.

Preface xxxi Glossary of Symbols and Abbreviations xxxv 1 Introduction 1 1.1 Background Information 2 1.2 A Short History of HPLC 6 1.3 Some Alternatives to HPLC 8 1.4 Other Sources of HPLC Information 12 References 15 2 Basic Concepts and the Control of Separation 19 2.1 Introduction 20 2.2 The Chromatographic Process 20 2.3 Retention 24 2.4 Peak Width and the Column Plate Number N 35 2.5 Resolution and Method Development 54 2.6 Sample Size Effects 69 2.7 Related Topics 74 References 83 3 Equipment 87 3.1 Introduction 88 3.2 Reservoirs and Solvent Filtration 89 3.3 Mobile-Phase Degassing 92 3.4 Tubing and Fittings 96 3.5 Pumping Systems 104 3.6 Autosamplers 113 3.7 Column Ovens 125 3.8 Data Systems 127 3.9 Extra-Column Effects 131 3.10 Maintenance 131 References 144 4 Detection 147 4.1 Introduction 148 4.2 Detector Characteristics 149 4.3 Introduction to Individual Detectors 160 4.4 UV-Visible Detectors 160 4.5 Fluorescence Detectors 167 4.6 Electrochemical (Amperometric) Detectors 170 4.7 Radioactivity Detectors 172 4.8 Conductivity Detectors 174 4.9 Chemiluminescent Nitrogen Detector 174 4.10 Chiral Detectors 175 4.11 Refractive Index Detectors 177 4.12 Light-Scattering Detectors 180 4.13 Corona-Discharge Detector (CAD) 184 4.14 Mass Spectral Detectors (MS) 185 4.15 Other Hyphenated Detectors 191 4.16 Sample Derivatization and Reaction Detectors 194 References 196 5 The Column 199 5.1 Introduction 200 5.2 Column Supports 200 5.3 Stationary Phases 217 5.4 Column Selectivity 227 5.5 Column Hardware 238 5.6 Column-Packing Methods 240 5.7 Column Specifications 244 5.8 Column Handling 246 References 250 6 Reversed-phase Chromatography for Neutral Sam- Ples 253 6.1 Introduction 254 6.2 Retention 256 6.3 Selectivity 263 6.4 Method Development and Strategies for Optimizing Selectivity 284 6.5 Nonaqueous Reversed-Phase Chromatography (narp) 295 6.6 Special Problems 297 References 298 7 Ionic Samples: Reversed-phase Ion-pair and Ion- Exchange Chromatography 303 7.1 Introduction 304 7.2 Acid-Base Equilibria and Reversed-Phase Retention 304 7.3 Separation of Ionic Samples by Reversed-Phase Chromatography (RPC) 319 7.4 Ion-Pair Chromatography (IPC) 331 7.5 Ion-Exchange Chromatography (IEC) 349 References 357 8 Normal-phase Chromatography 361 8.1 Introduction 362 8.2 Retention 363 8.3 Selectivity 376 8.4 Method-Development Summary 385 8.5 Problems in the Use of NPC 392 8.6 Hydrophilic Interaction Chromatography (HILIC) 395 References 401 9 Gradient Elution 403 9.1 Introduction 404 9.2 Experimental Conditions and Their Effects on Separation 412 9.3 Method Development 434 9.4 Large-Molecule Separations 464 9.5 Other Separation Modes 465 9.6 Problems 470 References 471 10 Computer-assisted Method Development 475 10.1 Introduction 475 10.2 Computer-Simulation Software 481 10.3 Other Method-Development Software 491 10.4 Computer Simulation and Method Development 492 References 497 11 Qualitative and Quantitative Analysis 499 11.1 Introduction 499 11.2 Signal Measurement 500 11.3 Qualitative Analysis 516 11.4 Quantitative Analysis 520 11.5 Summary 529 References 529 12 Method Validation 531with Michael Swartz 12.1 Introduction 532 12.2 Terms and Definitions 534 12.3 System Suitability 542 12.4 Documentation 543 12.5 Validation for Different Pharmaceutical-Method Types 546 12.6 Bioanalytical Methods 548 12.7 Analytical Method Transfer (AMT) 554 12.8 Method Adjustment or Method Modification 561 12.9 Quality Control and Quality Assurance 564 12.10 Summary 565 References 566 13 Biochemical and Synthetic Polymer Separations 569with Timothy Wehr, Carl Scandella, and Peter Schoenmakers 13.1 Biomacromolecules 570 13.2 Molecular Structure and Conformation 571 13.3 Special Considerations for Biomolecule HPLC 579 13.4 Separation of Peptides and Proteins 584 13.5 Separation of Nucleic Acids 618 13.6 Separation of Carbohydrates 625 13.7 Separation of Viruses 630 13.8 Size-Exclusion Chromatography (SEC) 631 13.9 Large-Scale Purification of Large Biomolecules 641 13.10 Synthetic Polymers 648 References 658 14 Enantiomer Separations 665 with Michael Lämmerhofer, Norbert M. Maier and Wolfgang Lindner 14.1 Introduction 666 14.2 Background and Definitions 666 14.3 Indirect Method 670 14.4 Direct Method 675 14.5 Peak Dispersion and Tailing 681 14.6 Chiral Stationary Phases and Their Characteristics 681 14.7 Thermodynamic Considerations 715 References 718 15 Preparative Separations 725 with Geoff Cox 15.1 Introduction 726 15.2 Equipment for Prep-LC Separation 730 15.3 Isocratic Elution 736 15.4 Severely Overloaded Separation 748 15.5 Gradient Elution 751 15.6 Production-Scale Separation 754 References 755 16 Sample Preparation 757 with Ronald Majors 757 16.1 Introduction 758 16.2 Types of Samples 759 16.3 Preliminary Processing of Solid and Semi-Solid Samples 760 16.4 Sample Preparation for Liquid Samples 764 16.5 Liquid-Liquid Extraction 764 16.6 Solid-Phase Extraction (SPE) 771 16.7 Membrane Techniques in Sample Preparation 790 16.8 Sample Preparation Methods for Solid Samples 791 16.9 Column-Switching 796 16.10 Sample Preparation for Biochromatography 797 16.11 Sample Preparation for LC-MS 800 16.12 Derivatization in HPLC 802 References 805 17 Troubleshooting 809 Quick Fix 809 17.1 Introduction 810 17.2 Prevention of Problems 811 17.3 Problem-Isolation Strategies 819 17.4 Common Symptoms of HPLC Problems 821 17.5 Troubleshooting Tables 865 References 876 Appendix I. Properties of HPLC Solvents 879 I.1 Solvent-Detector Compatibility 879 I. 2 Solvent Polarity and Selectivity 882 I. 3 Solvent Safety 885 References 886 Appendix II. Preparing Buffered Mobile Phases 887 II.1 Sequence of Operations 887 II.2 Recipes for Some Commonly Used Buffers 888 Reference 890 Index 891