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CONTENTS Preface xi 1 Introduction to LC/MS 1 1.1 Why LC/MS?, 1 1.2 Molecular Weights and Structure Studies, 4 1.3 LC/MS Systems, 4 1.4 System Costs, 7 1.5 Competitive Systems, 7 2 The HPLC System 9 2.1 HPLC System Components, 9 2.2 Gradient versus Isocratic Systems, 14 2.3 Micro HPLC Systems, 16 2.4 HPLC Tubing and Fittings, 18 3 The HPLC Column and Separation Modes 21 3.1 Column Construction, 21 3.2 Column Packing Materials, 23 3.3 Normal-Phase Columns, 25 3.4 Other Bonded-Phase Silica Columns, 26 3.5 Optimizing Reverse-Phase Column Use, 28 3.6 Silica Ion-Exchange Columns, 30 3.7 Silica Size-Separation Columns, 31 3.8 Zirconium Bonded-Phase Columns, 31 3.9 Polymer Reverse-Phase Columns, 32 4 HPLC and Column Maintenance 33 4.1 HPLC Maintenance, 33 4.2 Column Maintenance, 37 5 Sample Preparation and Separations Development 41 5.1 Mobile-Phase Preparation, 41 5.2 Mobile-Phase pH Control Using Buffers, 42 5.3 Sample Preparation, 44 5.4 Cartridge Column Cleanup, 44 5.5 On-Column Sample Concentration, 45 5.6 Isocratic and Gradient Methods Development, 46 5.7 Automated Methods Development, 49 6 LC/MS Interfaces 51 6.1 Solvent Removal and Ionization, 51 6.2 Atmospheric-Pressure Interfaces, 52 6.3 Electrospray Interface, 53 6.4 Ion Spray Interface, 54 6.5 Secondary Detectors, 55 7 LC/MS Overview 59 7.1 HPLC and the Ionization Source, 60 7.2 Vacuum Pumps, 61 7.3 Analyzer and Ion Detector Designs, 61 7.4 Data and Control Systems, 66 7.5 Peak Detection, ID, and Quantitation, 69 8 Mass Analyzers 71 8.1 Quadrupole Analyzer, 71 8.2 Ion Trap Analyzer, 74 8.3 Linear Ion Trap Analyzer, 78 8.4 Time-of-Flight Analyzer, 79 8.5 Fourier Transform Analyzer Design, 81 8.6 Magnetic Sector Analyzers, 83 9 Mass Spectrometer Maintenance 85 9.1 High-Vacuum Operation, 85 9.2 MS Hardware Maintenance, 88 9.3 System Electrical Grounding, 92 10 Application Areas for LC/MS 95 10.1 Compound Discovery, 95 10.2 Identification of Complex Biological Compounds, 96 10.3 Analysis of Trace Impurities and Metabolites, 97 10.4 Arson Residue Investigation, 98 10.5 Industrial Water and Pesticide Analysis, 98 10.6 Toxicology and Drugs of Abuse, 98 10.7 Clinical Therapeutic Drug Screening, 99 10.8 Pesticide Manufacturing, 101 11 Trace Analysis and LC/MS/MS 103 11.1 LC/MS/MS Triple-Quadrupole System, 103 11.2 MS/MS Operating Modes, 104 11.3 Ion Trap MS/MS Operation, 106 11.4 Hybrid LC/MS/MS Systems, 108 12 Drug Discovery and Benchtop LC/MS 111 12.1 Activity Screening, 111 12.2 Standardized LC/MS Screening, 113 12.3 Molecular Fragmentation for Structural Determination, 115 12.4 Process Monitoring, 116 13 Proteomics: LC/MALDI/TOF and MS/MS Libraries 119 13.1 Protein Molecular-Weight Determination by LC/MS, 120 13.2 De Novo Protein Purification, 121 13.3 Protein Analysis by Two-Dimensional GEP and LC/TOFMS, 122 13.4 LC/MS/MS Identification of Peptide Structures, 122 13.5 Tracer Labeling for Peptide ID, 124 13.6 Posttranslational Modified Protein, 124 13.7 Transient Peptides and Accumulation Proteins, 124 14 The Future of LC/MS 127 14.1 Instrumentation Improvements, 127 14.2 Affordable Benchtop LC/LITMS, 129 14.3 User-Customized Data Libraries, 129 14.4 Nucleomics and Restriction Fragment Analysis, 130 Appendix A LC/MS Frequently Asked Questions 131 Appendix B Solvents and Volatile Buffers for LC/MS 139 Appendix C Guide to Structure Interpretation 143 Appendix D Glossary of LC/MS Terms 149 Appendix E LC/MS Selective Reading List 155 Index 157
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