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.

"In summary, I would strongly recommend this book for anyone working with LC and LC/MS. It is a finely crafted introduction that adequately covers nearly every aspect of the science. The emphasis on basic principles and practical aspects ensures that it will be a useful reference for many years to come." (J Am Soc Mass Spectrom, 2011) "This third edition is highly cross-referenced, so as to allow the reader to follow up on topics of special interest, or to clarify questions that may arise during reading. The third edition of Introduction to Modern Liquid Chromatography will continue to be the HPLC reference book for thousands of readers, either experienced workers who may wish to explore topics of his/her choice, or find an answer to specific problems, or beginners who would like to understand and know the possibilities offered by thetechnique." (Chemistry Journals, 11 April 2011)"This classic text on liquid chromatography has been thoroughly updated through the addition of information on modern instrumentation, columns, and troubleshooting. It is a valuable resource for practicing chromatographers at all levels.." (Anal Bioanal Chem, 2011)"In summary, I would strongly recommend this book for anyone working with LC and LC/MS. It is a finely crafted introduction that adequately covers nearly every aspect of the science. The emphasis on basic principles and practical aspects ensures that it will be a useful reference for any years to come." (American Society for Mass Spectrometry, 21 Januay 2011)"The text is illustrated with many figures and tables originating from authors' own work or taken from the literature . . . both groups of readers will find in this book plenty of information and inspiration." (Journal of Separation Science, 1 June 2010)"Following their highly successful second edition (1979), which appeared more than 30 years ago, Snyder (LC Resources) and Kirkland (Advanced Materials Technology) have teamed with Dolan (LC Resources) and additional collaborators to provide an excellent update of their earlier work." (CHOICE, July 2010)"It is difficult in these times to ever call a scientific book a "bargain", but this truly is. The wealth of information contained in these almost 1000 pages is invaluable. The book is comprehensive, eminently readable and approachable, and highly useful for both the skilled chromatographer and those new to the technique. Everyone using a liquid chromatographic instrument would benefit from owning a copy." (JACS, 2010)"It's current, clearly-indexed, well-referenced, and comprehensive. The inclusion of John Dolan as author has significantly expanded the sections on troubleshooting, and additional expert contributors have enhanced the coverage of the specialist chapters." - Bruce Hamilton posted on the Chromatography Forum

• Preface xxxiGlossary of Symbols and Abbreviations xxxv1 Introduction 11.1 Background Information 21.2 A Short History of HPLC 61.3 Some Alternatives to HPLC 81.4 Other Sources of HPLC Information 12References 152 Basic Concepts and the Control of Separation 192.1 Introduction 202.2 The Chromatographic Process 202.3 Retention 242.4 Peak Width and the Column Plate Number N 352.5 Resolution and Method Development 542.6 Sample Size Effects 692.7 Related Topics 74References 833 Equipment 873.1 Introduction 883.2 Reservoirs and Solvent Filtration 893.3 Mobile-Phase Degassing 923.4 Tubing and Fittings 963.5 Pumping Systems 1043.6 Autosamplers 1133.7 Column Ovens 1253.8 Data Systems 1273.9 Extra-Column Effects 1313.10 Maintenance 131References 1444 Detection 1474.1 Introduction 1484.2 Detector Characteristics 1494.3 Introduction to Individual Detectors 1604.4 UV-Visible Detectors 1604.5 Fluorescence Detectors 1674.6 Electrochemical (Amperometric) Detectors 1704.7 Radioactivity Detectors 1724.8 Conductivity Detectors 1744.9 Chemiluminescent Nitrogen Detector 1744.10 Chiral Detectors 1754.11 Refractive Index Detectors 1774.12 Light-Scattering Detectors 1804.13 Corona-Discharge Detector (CAD) 1844.14 Mass Spectral Detectors (MS) 1854.15 Other Hyphenated Detectors 1914.16 Sample Derivatization and Reaction Detectors 194References 1965 The Column 1995.1 Introduction 2005.2 Column Supports 2005.3 Stationary Phases 2175.4 Column Selectivity 227  5.5 Column Hardware 2385.6 Column-Packing Methods 2405.7 Column Specifications 2445.8 Column Handling 246References 2506 Reversed-phase Chromatography for Neutral Sam- Ples 2536.1 Introduction 2546.2 Retention 2566.3 Selectivity 2636.4 Method Development and Strategies for Optimizing Selectivity 2846.5 Nonaqueous Reversed-Phase Chromatography (narp) 2956.6 Special Problems 297References 2987 Ionic Samples: Reversed-phase Ion-pair and Ion- Exchange Chromatography 3037.1 Introduction 3047.2 Acid–Base Equilibria and Reversed-Phase Retention 3047.3 Separation of Ionic Samples by Reversed-Phase Chromatography (RPC) 3197.4 Ion-Pair Chromatography (IPC) 3317.5 Ion-Exchange Chromatography (IEC) 349References 3578 Normal-phase Chromatography 3618.1 Introduction 3628.2 Retention 3638.3 Selectivity 3768.4 Method-Development Summary 3858.5 Problems in the Use of NPC 3928.6 Hydrophilic Interaction Chromatography (HILIC) 395References 4019 Gradient Elution 4039.1 Introduction 4049.2 Experimental Conditions and Their Effects on Separation 4129.3 Method Development 4349.4 Large-Molecule Separations 4649.5 Other Separation Modes 4659.6 Problems 470References 47110 Computer-assisted Method Development 47510.1 Introduction 47510.2 Computer-Simulation Software 48110.3 Other Method-Development Software 49110.4 Computer Simulation and Method Development 492References 49711 Qualitative and Quantitative Analysis 49911.1 Introduction 49911.2 Signal Measurement 50011.3 Qualitative Analysis 51611.4 Quantitative Analysis 52011.5 Summary 529References 52912 Method Validation 531with Michael Swartz12.1 Introduction 53212.2 Terms and Definitions 53412.3 System Suitability 54212.4 Documentation 54312.5 Validation for Different Pharmaceutical-Method Types 54612.6 Bioanalytical Methods 54812.7 Analytical Method Transfer (AMT) 55412.8 Method Adjustment or Method Modification 56112.9 Quality Control and Quality Assurance 56412.10 Summary 565References 56613 Biochemical and Synthetic Polymer Separations 569with Timothy Wehr, Carl Scandella, and Peter Schoenmakers13.1 Biomacromolecules 57013.2 Molecular Structure and Conformation 57113.3 Special Considerations for Biomolecule HPLC 57913.4 Separation of Peptides and Proteins 58413.5 Separation of Nucleic Acids 61813.6 Separation of Carbohydrates 62513.7 Separation of Viruses 63013.8 Size-Exclusion Chromatography (SEC) 63113.9 Large-Scale Purification of Large Biomolecules 64113.10 Synthetic Polymers 648References 65814 Enantiomer Separations 665 with Michael Lämmerhofer, Norbert M. Maier and Wolfgang Lindner14.1 Introduction 66614.2 Background and Definitions 66614.3 Indirect Method 67014.4 Direct Method 67514.5 Peak Dispersion and Tailing 68114.6 Chiral Stationary Phases and Their Characteristics 68114.7 Thermodynamic Considerations 715References 71815 Preparative Separations 725 with Geoff Cox15.1 Introduction 72615.2 Equipment for Prep-LC Separation 73015.3 Isocratic Elution 73615.4 Severely Overloaded Separation 74815.5 Gradient Elution 75115.6 Production-Scale Separation 754References 75516 Sample Preparation 757 with Ronald Majors 75716.1 Introduction 75816.2 Types of Samples 75916.3 Preliminary Processing of Solid and Semi-Solid Samples 76016.4 Sample Preparation for Liquid Samples 76416.5 Liquid–Liquid Extraction 76416.6 Solid-Phase Extraction (SPE) 77116.7 Membrane Techniques in Sample Preparation 79016.8 Sample Preparation Methods for Solid Samples 79116.9 Column-Switching 79616.10 Sample Preparation for Biochromatography 79716.11 Sample Preparation for LC-MS 80016.12 Derivatization in HPLC 802References 80517 Troubleshooting 809Quick Fix 80917.1 Introduction 81017.2 Prevention of Problems 81117.3 Problem-Isolation Strategies 81917.4 Common Symptoms of HPLC Problems 82117.5 Troubleshooting Tables 865References 876Appendix I. Properties of HPLC Solvents 879I.1 Solvent-Detector Compatibility 879I. 2 Solvent Polarity and Selectivity 882I. 3 Solvent Safety 885References 886Appendix II. Preparing Buffered Mobile Phases 887II.1 Sequence of Operations 887II.2 Recipes for Some Commonly Used Buffers 888Reference 890Index 891

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.

"In summary, I would strongly recommend this book for anyone working with LC and LC/MS. It is a finely crafted introduction that adequately covers nearly every aspect of the science. The emphasis on basic principles and practical aspects ensures that it will be a useful reference for many years to come." (J Am Soc Mass Spectrom, 2011) "This third edition is highly cross-referenced, so as to allow the reader to follow up on topics of special interest, or to clarify questions that may arise during reading. The third edition of Introduction to Modern Liquid Chromatography will continue to be the HPLC reference book for thousands of readers, either experienced workers who may wish to explore topics of his/her choice, or find an answer to specific problems, or beginners who would like to understand and know the possibilities offered by thetechnique." (Chemistry Journals, 11 April 2011)"This classic text on liquid chromatography has been thoroughly updated through the addition of information on modern instrumentation, columns, and troubleshooting. It is a valuable resource for practicing chromatographers at all levels.." (Anal Bioanal Chem, 2011)"In summary, I would strongly recommend this book for anyone working with LC and LC/MS. It is a finely crafted introduction that adequately covers nearly every aspect of the science. The emphasis on basic principles and practical aspects ensures that it will be a useful reference for any years to come." (American Society for Mass Spectrometry, 21 Januay 2011)"The text is illustrated with many figures and tables originating from authors' own work or taken from the literature . . . both groups of readers will find in this book plenty of information and inspiration." (Journal of Separation Science, 1 June 2010)"Following their highly successful second edition (1979), which appeared more than 30 years ago, Snyder (LC Resources) and Kirkland (Advanced Materials Technology) have teamed with Dolan (LC Resources) and additional collaborators to provide an excellent update of their earlier work." (CHOICE, July 2010)"It is difficult in these times to ever call a scientific book a "bargain", but this truly is. The wealth of information contained in these almost 1000 pages is invaluable. The book is comprehensive, eminently readable and approachable, and highly useful for both the skilled chromatographer and those new to the technique. Everyone using a liquid chromatographic instrument would benefit from owning a copy." (JACS, 2010)"It's current, clearly-indexed, well-referenced, and comprehensive. The inclusion of John Dolan as author has significantly expanded the sections on troubleshooting, and additional expert contributors have enhanced the coverage of the specialist chapters." - Bruce Hamilton posted on the Chromatography Forum

• Preface xxxiGlossary of Symbols and Abbreviations xxxv1 Introduction 11.1 Background Information 21.2 A Short History of HPLC 61.3 Some Alternatives to HPLC 81.4 Other Sources of HPLC Information 12References 152 Basic Concepts and the Control of Separation 192.1 Introduction 202.2 The Chromatographic Process 202.3 Retention 242.4 Peak Width and the Column Plate Number N 352.5 Resolution and Method Development 542.6 Sample Size Effects 692.7 Related Topics 74References 833 Equipment 873.1 Introduction 883.2 Reservoirs and Solvent Filtration 893.3 Mobile-Phase Degassing 923.4 Tubing and Fittings 963.5 Pumping Systems 1043.6 Autosamplers 1133.7 Column Ovens 1253.8 Data Systems 1273.9 Extra-Column Effects 1313.10 Maintenance 131References 1444 Detection 1474.1 Introduction 1484.2 Detector Characteristics 1494.3 Introduction to Individual Detectors 1604.4 UV-Visible Detectors 1604.5 Fluorescence Detectors 1674.6 Electrochemical (Amperometric) Detectors 1704.7 Radioactivity Detectors 1724.8 Conductivity Detectors 1744.9 Chemiluminescent Nitrogen Detector 1744.10 Chiral Detectors 1754.11 Refractive Index Detectors 1774.12 Light-Scattering Detectors 1804.13 Corona-Discharge Detector (CAD) 1844.14 Mass Spectral Detectors (MS) 1854.15 Other Hyphenated Detectors 1914.16 Sample Derivatization and Reaction Detectors 194References 1965 The Column 1995.1 Introduction 2005.2 Column Supports 2005.3 Stationary Phases 2175.4 Column Selectivity 227  5.5 Column Hardware 2385.6 Column-Packing Methods 2405.7 Column Specifications 2445.8 Column Handling 246References 2506 Reversed-phase Chromatography for Neutral Sam- Ples 2536.1 Introduction 2546.2 Retention 2566.3 Selectivity 2636.4 Method Development and Strategies for Optimizing Selectivity 2846.5 Nonaqueous Reversed-Phase Chromatography (narp) 2956.6 Special Problems 297References 2987 Ionic Samples: Reversed-phase Ion-pair and Ion- Exchange Chromatography 3037.1 Introduction 3047.2 Acid–Base Equilibria and Reversed-Phase Retention 3047.3 Separation of Ionic Samples by Reversed-Phase Chromatography (RPC) 3197.4 Ion-Pair Chromatography (IPC) 3317.5 Ion-Exchange Chromatography (IEC) 349References 3578 Normal-phase Chromatography 3618.1 Introduction 3628.2 Retention 3638.3 Selectivity 3768.4 Method-Development Summary 3858.5 Problems in the Use of NPC 3928.6 Hydrophilic Interaction Chromatography (HILIC) 395References 4019 Gradient Elution 4039.1 Introduction 4049.2 Experimental Conditions and Their Effects on Separation 4129.3 Method Development 4349.4 Large-Molecule Separations 4649.5 Other Separation Modes 4659.6 Problems 470References 47110 Computer-assisted Method Development 47510.1 Introduction 47510.2 Computer-Simulation Software 48110.3 Other Method-Development Software 49110.4 Computer Simulation and Method Development 492References 49711 Qualitative and Quantitative Analysis 49911.1 Introduction 49911.2 Signal Measurement 50011.3 Qualitative Analysis 51611.4 Quantitative Analysis 52011.5 Summary 529References 52912 Method Validation 531with Michael Swartz12.1 Introduction 53212.2 Terms and Definitions 53412.3 System Suitability 54212.4 Documentation 54312.5 Validation for Different Pharmaceutical-Method Types 54612.6 Bioanalytical Methods 54812.7 Analytical Method Transfer (AMT) 55412.8 Method Adjustment or Method Modification 56112.9 Quality Control and Quality Assurance 56412.10 Summary 565References 56613 Biochemical and Synthetic Polymer Separations 569with Timothy Wehr, Carl Scandella, and Peter Schoenmakers13.1 Biomacromolecules 57013.2 Molecular Structure and Conformation 57113.3 Special Considerations for Biomolecule HPLC 57913.4 Separation of Peptides and Proteins 58413.5 Separation of Nucleic Acids 61813.6 Separation of Carbohydrates 62513.7 Separation of Viruses 63013.8 Size-Exclusion Chromatography (SEC) 63113.9 Large-Scale Purification of Large Biomolecules 64113.10 Synthetic Polymers 648References 65814 Enantiomer Separations 665 with Michael Lämmerhofer, Norbert M. Maier and Wolfgang Lindner14.1 Introduction 66614.2 Background and Definitions 66614.3 Indirect Method 67014.4 Direct Method 67514.5 Peak Dispersion and Tailing 68114.6 Chiral Stationary Phases and Their Characteristics 68114.7 Thermodynamic Considerations 715References 71815 Preparative Separations 725 with Geoff Cox15.1 Introduction 72615.2 Equipment for Prep-LC Separation 73015.3 Isocratic Elution 73615.4 Severely Overloaded Separation 74815.5 Gradient Elution 75115.6 Production-Scale Separation 754References 75516 Sample Preparation 757 with Ronald Majors 75716.1 Introduction 75816.2 Types of Samples 75916.3 Preliminary Processing of Solid and Semi-Solid Samples 76016.4 Sample Preparation for Liquid Samples 76416.5 Liquid–Liquid Extraction 76416.6 Solid-Phase Extraction (SPE) 77116.7 Membrane Techniques in Sample Preparation 79016.8 Sample Preparation Methods for Solid Samples 79116.9 Column-Switching 79616.10 Sample Preparation for Biochromatography 79716.11 Sample Preparation for LC-MS 80016.12 Derivatization in HPLC 802References 80517 Troubleshooting 809Quick Fix 80917.1 Introduction 81017.2 Prevention of Problems 81117.3 Problem-Isolation Strategies 81917.4 Common Symptoms of HPLC Problems 82117.5 Troubleshooting Tables 865References 876Appendix I. Properties of HPLC Solvents 879I.1 Solvent-Detector Compatibility 879I. 2 Solvent Polarity and Selectivity 882I. 3 Solvent Safety 885References 886Appendix II. Preparing Buffered Mobile Phases 887II.1 Sequence of Operations 887II.2 Recipes for Some Commonly Used Buffers 888Reference 890Index 891

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.

"In summary, I would strongly recommend this book for anyone working with LC and LC/MS. It is a finely crafted introduction that adequately covers nearly every aspect of the science. The emphasis on basic principles and practical aspects ensures that it will be a useful reference for many years to come." (J Am Soc Mass Spectrom, 2011) "This third edition is highly cross-referenced, so as to allow the reader to follow up on topics of special interest, or to clarify questions that may arise during reading. The third edition of Introduction to Modern Liquid Chromatography will continue to be the HPLC reference book for thousands of readers, either experienced workers who may wish to explore topics of his/her choice, or find an answer to specific problems, or beginners who would like to understand and know the possibilities offered by thetechnique." (Chemistry Journals, 11 April 2011)"This classic text on liquid chromatography has been thoroughly updated through the addition of information on modern instrumentation, columns, and troubleshooting. It is a valuable resource for practicing chromatographers at all levels.." (Anal Bioanal Chem, 2011)"In summary, I would strongly recommend this book for anyone working with LC and LC/MS. It is a finely crafted introduction that adequately covers nearly every aspect of the science. The emphasis on basic principles and practical aspects ensures that it will be a useful reference for any years to come." (American Society for Mass Spectrometry, 21 Januay 2011)"The text is illustrated with many figures and tables originating from authors' own work or taken from the literature . . . both groups of readers will find in this book plenty of information and inspiration." (Journal of Separation Science, 1 June 2010)"Following their highly successful second edition (1979), which appeared more than 30 years ago, Snyder (LC Resources) and Kirkland (Advanced Materials Technology) have teamed with Dolan (LC Resources) and additional collaborators to provide an excellent update of their earlier work." (CHOICE, July 2010)"It is difficult in these times to ever call a scientific book a "bargain", but this truly is. The wealth of information contained in these almost 1000 pages is invaluable. The book is comprehensive, eminently readable and approachable, and highly useful for both the skilled chromatographer and those new to the technique. Everyone using a liquid chromatographic instrument would benefit from owning a copy." (JACS, 2010)"It's current, clearly-indexed, well-referenced, and comprehensive. The inclusion of John Dolan as author has significantly expanded the sections on troubleshooting, and additional expert contributors have enhanced the coverage of the specialist chapters." - Bruce Hamilton posted on the Chromatography Forum

• Preface xxxiGlossary of Symbols and Abbreviations xxxv1 Introduction 11.1 Background Information 21.2 A Short History of HPLC 61.3 Some Alternatives to HPLC 81.4 Other Sources of HPLC Information 12References 152 Basic Concepts and the Control of Separation 192.1 Introduction 202.2 The Chromatographic Process 202.3 Retention 242.4 Peak Width and the Column Plate Number N 352.5 Resolution and Method Development 542.6 Sample Size Effects 692.7 Related Topics 74References 833 Equipment 873.1 Introduction 883.2 Reservoirs and Solvent Filtration 893.3 Mobile-Phase Degassing 923.4 Tubing and Fittings 963.5 Pumping Systems 1043.6 Autosamplers 1133.7 Column Ovens 1253.8 Data Systems 1273.9 Extra-Column Effects 1313.10 Maintenance 131References 1444 Detection 1474.1 Introduction 1484.2 Detector Characteristics 1494.3 Introduction to Individual Detectors 1604.4 UV-Visible Detectors 1604.5 Fluorescence Detectors 1674.6 Electrochemical (Amperometric) Detectors 1704.7 Radioactivity Detectors 1724.8 Conductivity Detectors 1744.9 Chemiluminescent Nitrogen Detector 1744.10 Chiral Detectors 1754.11 Refractive Index Detectors 1774.12 Light-Scattering Detectors 1804.13 Corona-Discharge Detector (CAD) 1844.14 Mass Spectral Detectors (MS) 1854.15 Other Hyphenated Detectors 1914.16 Sample Derivatization and Reaction Detectors 194References 1965 The Column 1995.1 Introduction 2005.2 Column Supports 2005.3 Stationary Phases 2175.4 Column Selectivity 227  5.5 Column Hardware 2385.6 Column-Packing Methods 2405.7 Column Specifications 2445.8 Column Handling 246References 2506 Reversed-phase Chromatography for Neutral Sam- Ples 2536.1 Introduction 2546.2 Retention 2566.3 Selectivity 2636.4 Method Development and Strategies for Optimizing Selectivity 2846.5 Nonaqueous Reversed-Phase Chromatography (narp) 2956.6 Special Problems 297References 2987 Ionic Samples: Reversed-phase Ion-pair and Ion- Exchange Chromatography 3037.1 Introduction 3047.2 Acid–Base Equilibria and Reversed-Phase Retention 3047.3 Separation of Ionic Samples by Reversed-Phase Chromatography (RPC) 3197.4 Ion-Pair Chromatography (IPC) 3317.5 Ion-Exchange Chromatography (IEC) 349References 3578 Normal-phase Chromatography 3618.1 Introduction 3628.2 Retention 3638.3 Selectivity 3768.4 Method-Development Summary 3858.5 Problems in the Use of NPC 3928.6 Hydrophilic Interaction Chromatography (HILIC) 395References 4019 Gradient Elution 4039.1 Introduction 4049.2 Experimental Conditions and Their Effects on Separation 4129.3 Method Development 4349.4 Large-Molecule Separations 4649.5 Other Separation Modes 4659.6 Problems 470References 47110 Computer-assisted Method Development 47510.1 Introduction 47510.2 Computer-Simulation Software 48110.3 Other Method-Development Software 49110.4 Computer Simulation and Method Development 492References 49711 Qualitative and Quantitative Analysis 49911.1 Introduction 49911.2 Signal Measurement 50011.3 Qualitative Analysis 51611.4 Quantitative Analysis 52011.5 Summary 529References 52912 Method Validation 531with Michael Swartz12.1 Introduction 53212.2 Terms and Definitions 53412.3 System Suitability 54212.4 Documentation 54312.5 Validation for Different Pharmaceutical-Method Types 54612.6 Bioanalytical Methods 54812.7 Analytical Method Transfer (AMT) 55412.8 Method Adjustment or Method Modification 56112.9 Quality Control and Quality Assurance 56412.10 Summary 565References 56613 Biochemical and Synthetic Polymer Separations 569with Timothy Wehr, Carl Scandella, and Peter Schoenmakers13.1 Biomacromolecules 57013.2 Molecular Structure and Conformation 57113.3 Special Considerations for Biomolecule HPLC 57913.4 Separation of Peptides and Proteins 58413.5 Separation of Nucleic Acids 61813.6 Separation of Carbohydrates 62513.7 Separation of Viruses 63013.8 Size-Exclusion Chromatography (SEC) 63113.9 Large-Scale Purification of Large Biomolecules 64113.10 Synthetic Polymers 648References 65814 Enantiomer Separations 665 with Michael Lämmerhofer, Norbert M. Maier and Wolfgang Lindner14.1 Introduction 66614.2 Background and Definitions 66614.3 Indirect Method 67014.4 Direct Method 67514.5 Peak Dispersion and Tailing 68114.6 Chiral Stationary Phases and Their Characteristics 68114.7 Thermodynamic Considerations 715References 71815 Preparative Separations 725 with Geoff Cox15.1 Introduction 72615.2 Equipment for Prep-LC Separation 73015.3 Isocratic Elution 73615.4 Severely Overloaded Separation 74815.5 Gradient Elution 75115.6 Production-Scale Separation 754References 75516 Sample Preparation 757 with Ronald Majors 75716.1 Introduction 75816.2 Types of Samples 75916.3 Preliminary Processing of Solid and Semi-Solid Samples 76016.4 Sample Preparation for Liquid Samples 76416.5 Liquid–Liquid Extraction 76416.6 Solid-Phase Extraction (SPE) 77116.7 Membrane Techniques in Sample Preparation 79016.8 Sample Preparation Methods for Solid Samples 79116.9 Column-Switching 79616.10 Sample Preparation for Biochromatography 79716.11 Sample Preparation for LC-MS 80016.12 Derivatization in HPLC 802References 80517 Troubleshooting 809Quick Fix 80917.1 Introduction 81017.2 Prevention of Problems 81117.3 Problem-Isolation Strategies 81917.4 Common Symptoms of HPLC Problems 82117.5 Troubleshooting Tables 865References 876Appendix I. Properties of HPLC Solvents 879I.1 Solvent-Detector Compatibility 879I. 2 Solvent Polarity and Selectivity 882I. 3 Solvent Safety 885References 886Appendix II. Preparing Buffered Mobile Phases 887II.1 Sequence of Operations 887II.2 Recipes for Some Commonly Used Buffers 888Reference 890Index 891