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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. Completewith a CD-ROM and appendices, this guide has everything chromatographers need to know to

confidently separate, identify, purify, and quantify compounds .

Table of Contents .

Preface xi

HPLC Primer 1

Advantages and Disadvantages of HPLC 3

How It Works 4

A Separation Model of the Column 5

Basic Hardware: A Quick, First Look 7

Use of Solvent Gradients 8

Ranges of Compounds 9

Other Ways to Make My Separation 9

FPLC-Fast Protein Liquid Chromatography 10

LC-Traditional Liquid Chromatography 10

GLC-Gas Liquid Chromatography 11

SFC-Supercritical Fluid Chromatography 11

TLC-Thin Layer Chromatography 12

EP-Electrophoresis 12

CZE-Capillary Zone Electrophoresis 13

Selecting an HPLC System 15

Characteristic Systems 16

Finding a Fit: Detectors and Data Processing 16

System Models: Gradient Versus Isocratic 16

Vendor Selection 17

Brand Names and Clones 17

Hardware-Service-Support 18

System Cost Estimates 19

Type 1 System-QC Isocratic (Cost: {dollar}10-15,000) 19

Type II System-Research Gradient (Cost: {dollar}20-25,000) 19

Type III System-Automated Clinical(Cost: {dollar}25-35,000) 20

Type IV System-Automated Methods (Cost: {dollar}30-50,000) 21

Columns 21

Sizes: Analytical and Preparative 21

Separating Modes: Selecting Only What You Need 22

Tips on Column Use 23

Running Your Chromatograph 25

Set-up and Start-up 25

Hardware Plumbing 101: Tubing and Fittings 26

Connecting Components 28

Solvent Clean-up 30

Water Purity Test 33

Start-up System Flushing 34

Column Preparation and Equilibration 35

Sample Preparation and Column Calibration 36

Sample Clean-up 36

Plate Counts 37

Your First Chromatogram 37

Reproducible Injection Techniques 38

Simple Scouting for a Mobile Phase 39

Examining the Chromatogram 40

Basic Calculations of Results 41

HPLC Optimization 43

Separation Models 45

Partition 45

Separation Parameters 48

Efficiency Factor 49

Separation (Chemistry) Factor 53

Ion Exchange Chromatography 56

Size Exclusion Chromatography 57

Affinity Chromatography 59

Column Preparation 61

Column Variations 61

Packing Materials and Hardware 64

Column Selection 66

Column Aging, Diagnosis, and Healing 73

Packing Degrading-Bonded-Phase Loss 74

Dissolved Packing Material-End Voids 77

Bound Material 78

Pressure Increases 81

Column Channeling-left-Voids 83

Normal Phase, Ion Exchange, and Size Columns 84

Zirconium and Polymer Columns 86

Partition Chromatography Modifications 89

Reverse-Phase and Hybrid Silica 89

Ionization Suppression 90

Ion Pairing 91

Organic Modifiers 92

Chelation 92

Acidic Phase Silica 93

Reverse-Phase Zirconium 93

Partition Mode Selection 94

& 8220;Nonpartition& 8221; Chromatography 95

Ion Exchange 96

Cationic: Weak and Strong 96

Anionic: Weak and Strong 97

Size Exclusion 98

Organic Soluble Samples 98

Hydrophilic Protein Separation 99

Affinity Chromatography 101

Column Packing Modification 102

Chelation and Optically Active Columns 103

Hardware Specifics 105

System Protection 105

Filters, Guard Columns, and Saturation Columns 106

Inert Surfaces and Connections 107

Pumping 108

High-and Low-Pressure Mixing Controllers 109

Checking Gradient Performance 112

Injectors and Autosamplers 113

Detectors 116

Mass Dependent Detectors 116

Absorptive Detectors 119

Specific Detectors 122

Fraction Collectors 123

Data Collection and Processing 123

Troubleshooting and Optimization 125

Hardware and Tools-System Pacification 125

Reverse Order Diagnosis 129

Introduction to Data Acquisition 132

Solvent Conservation 133

HPLC Utilization 135

Preparative Chromatography 137

Analytical Preparative 138

Semipreparative 139

& 8220;True& 8221; Preparative 139

Sample Preparation and Methods Development 143

Sample Preparation 143

Deproteination 144

Extraction and Concentration 145

SFE (Cartridge Column) Preparations 145

Extracting Encapsulated Compounds 147

SFE Trace Enrichment and Windowing 148

Derivatives 151

Methods Development 151

Standards Development 152

Samples Development 154

Gradient Development 156

Application Logics: Separations Overview 159

Fat-Soluble Vitamins, Steroid, and Lipids 159

Water-Soluble Vitamins, Carbohydrates, and Acids 160

Nucleomics 161

Proteomics 162

Clinical and Forensic Drug Monitoring 163

Pharmaceutical Drug Development 164

Environmental and Reaction Monitoring 164

Application Trends 165

Automation 167

Analog-to-Digital Interfacing 167

Digital Information Exchange 169

HPLC System Control and Automation 169

Data Collection and Interpretation 170

Preinjection Baseline Setting 171

Peak Detection and Integration 171

Quantitation: Internal/External Standards 172

Automated Methods Development 172

Automated Isocratic Development 173

Hinge Point Gradient Development 176

Data Exportation to the Real World 177

Word Processors; .ASC, .DOC, .RTF, .WS, .WP Formats 177

Spread Sheets: .DIF, .WK, .XLS Formats 178

Databases: .DB2 Format 178

Graphics: .PCX, .TIFF, .JPG Formats 178

Chromatographic Files: Metafiles and NetCDF 178

Recent Advances in LC/MS Separations 181

A LC/MS Primer 181

Quadrupole MS and Mass Selection 183

Other Types of MS Analyzers for LC/MS 185

LC/MS Interfaces 187

LC/MS Computer Control and Data Processing 189

Microflow Chromatography 191

Ultrafast HPLC Systems 192

Chip HPLC Systems 192

Standardized LC/MS in Drug Design 193

New Directions in HPLC 195

Temperature-Controlled Chromatography 195

Ultrafast Chromatography 196

Monolith Capillary Columns 196

Micro-Parallel HPLC Systems 197

Two-Dimensional HPLC Systems 197

The Portable LC/MS 198

Appendices 201

Personal Separations Guide 203

FAQs for HPLC Systems and Columns 205

Tables of Solvents and Volatile Buffers 211

Glossary of HPLC Terms 213

HPLC Troubleshooting Quick Reference 221

HPLC Laboratory Experiments 227

System Start-up and Column Quality Control 227

Sample Preparation and Methods Development 229

Column and Solvent Switching and Pacification 231

Selected Reference List 233

Index 235 .

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