HPLC

MARVIN C. MCMASTER, PHD, is a consultant as well as an Adjunct Professor of Chemistry at the University of Missouri— St. Louis. Dr. McMaster has served as a researcher and product developer for DuPont, Kraft Foods, and Ciba-Geigy. Among his other publications, Dr. McMaster is author of LC/MS: A Practical User's Guide and coauthor of GC/MS: A Practical User's Guide (both published by Wiley).

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