HPLC: A Practical User's Guide
Here is the perfect handbook for both novice and experienced chromatographers. For novices, HPLC, Second Edition cuts through the technical detail, theory, and confusing variety of column types, enabling them to easily set up and maintain an HPLC system. For more experienced practitioners, the book explains how to fully exploit the most recent innovations and improvements in column technology. The latest information and techniques in high-temperature, micro-flow, and ultra-fast...
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Everything you need to set up an HPLC systemthat takes advantage of the latest innovations Here is the perfect handbook for both novice and experienced chromatographers. For novices, HPLC, Second Edition cuts through the technical detail, theory, and confusing variety of column types, enabling them to easily set up and maintain an HPLC system. For more experienced practitioners, the book explains how to fully exploit the most recent innovations and improvements in column technology. The latest information and techniques in high-temperature, micro-flow, and ultra-fast chromatography, as well as the linking of an HPLC to a mass spectrometer, represent just some of the new material featured in this Second Edition. Readers benefit from the author's twenty-five years of experience in HPLC, first as a user, then as a field sales and application support representative, and finally as a teacher and consultant. The book's wealth of tips and troubleshooting advice originate from the author's own laboratory and field work. HPLC's structure is carefully designed to give readers a solid foundationin the basics and then methodically build and refine their skills:Part I, HPLC Primer, presents the essentials needed to get an HPLC system up and runningPart II, HPLC Optimization, explains how to make the best use of the common columns and troubleshoot problemsPart III, HPLC Utilization, focuses on real-world applications, including interfacing HPLC systems with computers and robotic workstations A CD-ROM with the author's PowerPoint® presentation, Practical Course in HPLC, is included with the book. Plus, new appendices offer answers to frequently asked questions, tables of solvents and volatile buffers, an updated glossary of terms, and laboratory experiments. In short, everything that you need to know to confidently separate, identify, purify, and quantify compounds is here.
Preface xiHPLC Primer 1Advantages and Disadvantages of HPLC 3How It Works 4A Separation Model of the Column 5Basic Hardware: A Quick, First Look 7Use of Solvent Gradients 8Ranges of Compounds 9Other Ways to Make My Separation 9FPLC-Fast Protein Liquid Chromatography 10LC-Traditional Liquid Chromatography 10GLC-Gas Liquid Chromatography 11SFC-Supercritical Fluid Chromatography 11TLC-Thin Layer Chromatography 12EP-Electrophoresis 12CZE-Capillary Zone Electrophoresis 13Selecting an HPLC System 15Characteristic Systems 16Finding a Fit: Detectors and Data Processing 16System Models: Gradient Versus Isocratic 16Vendor Selection 17Brand Names and Clones 17Hardware-Service-Support 18System Cost Estimates 19Type 1 System-QC Isocratic (Cost: {dollar}10-15,000) 19Type II System-Research Gradient (Cost: {dollar}20-25,000) 19Type III System-Automated Clinical(Cost: {dollar}25-35,000) 20Type IV System-Automated Methods (Cost: {dollar}30-50,000) 21Columns 21Sizes: Analytical and Preparative 21Separating Modes: Selecting Only What You Need 22Tips on Column Use 23Running Your Chromatograph 25Set-up and Start-up 25Hardware Plumbing 101: Tubing and Fittings 26Connecting Components 28Solvent Clean-up 30Water Purity Test 33Start-up System Flushing 34Column Preparation and Equilibration 35Sample Preparation and Column Calibration 36Sample Clean-up 36Plate Counts 37Your First Chromatogram 37Reproducible Injection Techniques 38Simple Scouting for a Mobile Phase 39Examining the Chromatogram 40Basic Calculations of Results 41HPLC Optimization 43Separation Models 45Partition 45Separation Parameters 48Efficiency Factor 49Separation (Chemistry) Factor 53Ion Exchange Chromatography 56Size Exclusion Chromatography 57Affinity Chromatography 59Column Preparation 61Column Variations 61Packing Materials and Hardware 64Column Selection 66Column Aging, Diagnosis, and Healing 73Packing Degrading-Bonded-Phase Loss 74Dissolved Packing Material-End Voids 77Bound Material 78Pressure Increases 81Column Channeling-Center-Voids 83Normal Phase, Ion Exchange, and Size Columns 84Zirconium and Polymer Columns 86Partition Chromatography Modifications 89Reverse-Phase and Hybrid Silica 89Ionization Suppression 90Ion Pairing 91Organic Modifiers 92Chelation 92Acidic Phase Silica 93Reverse-Phase Zirconium 93Partition Mode Selection 94"Nonpartition" Chromatography 95Ion Exchange 96Cationic: Weak and Strong 96Anionic: Weak and Strong 97Size Exclusion 98Organic Soluble Samples 98Hydrophilic Protein Separation 99Affinity Chromatography 101Column Packing Modification 102Chelation and Optically Active Columns 103Hardware Specifics 105System Protection 105Filters, Guard Columns, and Saturation Columns 106Inert Surfaces and Connections 107Pumping 108High-and Low-Pressure Mixing Controllers 109Checking Gradient Performance 112Injectors and Autosamplers 113Detectors 116Mass Dependent Detectors 116Absorptive Detectors 119Specific Detectors 122Fraction Collectors 123Data Collection and Processing 123Troubleshooting and Optimization 125Hardware and Tools-System Pacification 125Reverse Order Diagnosis 129Introduction to Data Acquisition 132Solvent Conservation 133HPLC Utilization 135Preparative Chromatography 137Analytical Preparative 138Semipreparative 139"True" Preparative 139Sample Preparation and Methods Development 143Sample Preparation 143Deproteination 144Extraction and Concentration 145SFE (Cartridge Column) Preparations 145Extracting Encapsulated Compounds 147SFE Trace Enrichment and Windowing 148Derivatives 151Methods Development 151Standards Development 152Samples Development 154Gradient Development 156Application Logics: Separations Overview 159Fat-Soluble Vitamins, Steroid, and Lipids 159Water-Soluble Vitamins, Carbohydrates, and Acids 160Nucleomics 161Proteomics 162Clinical and Forensic Drug Monitoring 163Pharmaceutical Drug Development 164Environmental and Reaction Monitoring 164Application Trends 165Automation 167Analog-to-Digital Interfacing 167Digital Information Exchange 169HPLC System Control and Automation 169Data Collection and Interpretation 170Preinjection Baseline Setting 171Peak Detection and Integration 171Quantitation: Internal/External Standards 172Automated Methods Development 172Automated Isocratic Development 173Hinge Point Gradient Development 176Data Exportation to the Real World 177Word Processors; .ASC, .DOC, .RTF, .WS, .WP Formats 177Spread Sheets: .DIF, .WK, .XLS Formats 178Databases: .DB2 Format 178Graphics: .PCX, .TIFF, .JPG Formats 178Chromatographic Files: Metafiles and NetCDF 178Recent Advances in LC/MS Separations 181A LC/MS Primer 181Quadrupole MS and Mass Selection 183Other Types of MS Analyzers for LC/MS 185LC/MS Interfaces 187LC/MS Computer Control and Data Processing 189Microflow Chromatography 191Ultrafast HPLC Systems 192Chip HPLC Systems 192Standardized LC/MS in Drug Design 193New Directions in HPLC 195Temperature-Controlled Chromatography 195Ultrafast Chromatography 196Monolith Capillary Columns 196Micro-Parallel HPLC Systems 197Two-Dimensional HPLC Systems 197The Portable LC/MS 198Appendices 201Personal Separations Guide 203FAQs for HPLC Systems and Columns 205Tables of Solvents and Volatile Buffers 211Glossary of HPLC Terms 213HPLC Troubleshooting Quick Reference 221HPLC Laboratory Experiments 227System Start-up and Column Quality Control 227Sample Preparation and Methods Development 229Column and Solvent Switching and Pacification 231Selected Reference List 233Index 235
