HPLC: A Practical User’s Guide

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
Selected Reference List     233
Index     235

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