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HPLC：A Practical User's Guide[2007]
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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-Center-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 "Nonpartition" 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 "True" 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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