ساجدة ابراهيم
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water soluble vitamins by HPLC
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mouna.bn
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Description
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-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
Description
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-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