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HPLC: A Practical User's Guide, 2nd Edition

ISBN: 978-0-471-75401-5
256 pages
December 2006
HPLC: A Practical User


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. Complete with a CD-ROM and appendices, this guide has everything chromatographers need to know to confidently separate, identify, purify, and quantify compounds.

Note: CD-ROM/DVD and other supplementary materials are not included as part of eBook file.

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Table of Contents



1 Advantages and Disadvantages of HPLC.

1.1 How It Works.

1.1.1 A Separation Model of the Column.

1.1.2 Basic Hardware: A Quick, First Look.

1.1.3 Use of Solvent Gradients.

1.1.4 Ranges of Compounds.

1.2 Other Ways to Make My Separation.

1.2.1 FPLC—Fast Protein Liquid Chromatography.

1.2.2 LC—Traditional Liquid Chromatography.

1.2.3 GLC—Gas Liquid Chromatography.

1.2.4 SFC—Supercritical Fluid Chromatography.

1.2.5 TLC—Thin Layer Chromatography.

1.2.6 EP—Electrophoresis.

1.2.7 CZE—Capillary Zone Electrophoresis.

2 Selecting an HPLC System.

2.1 Characteristic Systems.

2.1.1 Finding a Fit: Detectors and Data Processing.

2.1.2 System Models: Gradient Versus Isocratic.

2.1.3 Vendor Selection.

2.1.4 Brand Names and Clones.

2.1.5 Hardware–Service–Support.

2.2 System Cost Estimates.

2.2.1 Type I System—QC Isocratic (Cost: $10–15,000).

2.2.2 Type II System—Research Gradient (Cost: $20–25,000).

2.2.3 Type III System—Automated Clinical (Cost: $25–35,000).

2.2.4 Type IV System—Automated Methods (Cost: $30–50,000).

2.3 Columns.

2.3.1 Sizes: Analytical and Preparative.

2.3.2 Separating Modes: Selecting Only What You Need.

2.3.3 Tips on Column Use.

3 Running Your Chromatograph.

3.1 Set-up and Start-up.

3.1.1 Hardware Plumbing 101: Tubing and Fittings.

3.1.2 Connecting Components.

3.1.3 Solvent Clean-up.

3.1.4 Water Purity Test.

3.1.5 Start-up System Flushing.

3.1.6 Column Preparation and Equilibration.

3.2 Sample Preparation and Column Calibration.

3.2.1 Sample Clean-up.

3.2.2 Plate Counts.

3.3 Your First Chromatogram.

3.3.1 Reproducible Injection Techniques.

3.3.2 Simple Scouting for a Mobile Phase.

3.3.3 Examining the Chromatogram.

3.3.4 Basic Calculations of Results.


4 Separation Models.

4.1 Partition.

4.1.1 Separation Parameters.

4.1.2 Efficiency Factor.

4.1.3 Separation (Chemistry) Factor.

4.2 Ion Exchange Chromatography.

4.3 Size Exclusion Chromatography.

4.4 Affinity Chromatography.

5 Column Preparation.

5.1 Column Variations.

5.2 Packing Materials and Hardware.

5.3 Column Selection.

6 Column Aging, Diagnosis, and Healing.

6.1 Packing Degrading—Bonded-Phase Loss.

6.2 Dissolved Packing Material—End Voids.

6.3 Bound Material.

6.4 Pressure Increases.

6.5 Column Channeling—Center-Voids.

6.6 Normal Phase, Ion Exchange, and Size Columns.

6.7 Zirconium and Polymer Columns.

7 Partition Chromatography Modifications.

7.1 Reverse-Phase and Hybrid Silica.

7.1.1 Ionization Suppression.

7.1.2 Ion Pairing.

7.1.3 Organic Modifiers.

7.1.4 Chelation.

7.2 Acidic Phase Silica.

7.3 Reverse-Phase Zirconium.

7.4 Partition Mode Selection.

8 “Nonpartition” Chromatography.

8.1 Ion Exchange.

8.1.1 Cationic:Weak and Strong.

8.1.2 Anionic:Weak and Strong.

8.2 Size Exclusion.

8.2.1 Organic Soluble Samples.

8.2.2 Hydrophilic Protein Separation.

8.3 Affinity Chromatography.

8.3.1 Column Packing Modification.

8.3.2 Chelation and Optically Active Columns.

9 Hardware Specifics.

9.1 System Protection.

9.1.1 Filters, Guard Columns, and Saturation Columns.

9.1.2 Inert Surfaces and Connections.

9.2 Pumping.

9.2.1 High- and Low-Pressure Mixing Controllers.

9.2.2 Checking Gradient Performance.

9.3 Injectors and Autosamplers.

9.4 Detectors.

9.4.1 Mass Dependent Detectors.

9.4.2 Absorptive Detectors.

9.4.3 Specific Detectors.

9.5 Fraction Collectors.

9.6 Data Collection and Processing.

10 Troubleshooting and Optimization.

10.1 Hardware and Tools—System Pacification.

10.2 Reverse Order Diagnosis.

10.3 Introduction to Data Acquisition.

10.4 Solvent Conservation.


11 Preparative Chromatography.

11.1 Analytical Preparative.

11.2 Semipreparative.

11.3 “True” Preparative.

12 Sample Preparation and Methods Development.

12.1 Sample Preparation.

12.1.1 Deproteination.

12.1.2 Extraction and Concentration.

12.1.3 SFE (Cartridge Column) Preparations.

12.1.4 Extracting Encapsulated Compounds.

12.1.5 SFE Trace Enrichment and Windowing.

12.1.6 Derivatives.

12.2 Methods Development.

12.2.1 Standards Development.

12.2.2 Samples Development.

12.3 Gradient Development.

13 Application Logics: Separations Overview.

13.1 Fat-Soluble Vitamins, Steroid, and Lipids.

13.2 Water-Soluble Vitamins, Carbohydrates, and Acids.

13.3 Nucleomics.

13.4 Proteomics.

13.5 Clinical and Forensic Drug Monitoring.

13.6 Pharmaceutical Drug Development.

13.7 Environmental and Reaction Monitoring.

13.8 Application Trends.

14 Automation.

14.1 Analog-to-Digital Interfacing.

14.2 Digital Information Exchange.

14.3 HPLC System Control and Automation.

14.4 Data Collection and Interpretation.

14.4.1 Preinjection Baseline Setting.

14.4.2 Peak Detection and Integration.

14.4.3 Quantitation: Internal/External Standards.

14.5 Automated Methods Development.

14.5.1 Automated Isocratic Development.

14.5.2 Hinge Point Gradient Development.

14.6 Data Exportation to the Real World.

14.6.1 Word Processors: .ASC, .DOC, .RTF, .WS, .WP Formats.

14.6.2 Spread Sheets: .DIF, .WK, .XLS Formats.

14.6.3 Databases: .DB2 Format.

14.6.4 Graphics: .PCX, .TIFF, .JPG Formats.

14.6.5 Chromatographic Files: Metafiles and NetCDF.

15 Recent Advances in LC/MS Separations.

15.1 A LC/MS Primer.

15.1.1 Quadrupole MS and Mass Selection.

15.1.2 Other Types of MS Analyzers for LC/MS.

15.1.3 LC/MS Interfaces.

15.1.4 LC/MS Computer Control and Data Processing.

15.2 Microflow Chromatography.

15.3 Ultrafast HPLC Systems.

15.4 Chip HPLC Systems.

15.5 Standardized LC/MS in Drug Design.

16 New Directions in HPLC.

16.1 Temperature-Controlled Chromatography.

16.2 Ultrafast Chromatography.

16.3 Monolith Capillary Columns.

16.4 Micro-Parallel HPLC Systems.

16.5 Two-Dimensional HPLC Systems.

16.6 The Portable LC/MS.


APPENDIX A Personal Separations Guide.

APPENDIX B FAQs for HPLC Systems and Columns.

APPENDIX C Tables of Solvents and Volatile Buffers.

APPENDIX D Glossary of HPLC Terms.

APPENDIX E HPLC Troubleshooting Quick Reference.

APPENDIX F HPLC Laboratory Experiments.

Laboratory 1: System Start-up and Column Quality Control.

Laboratory 2: Sample Preparation and Methods Development.

Laboratory 3: Column and Solvent Switching and Pacification.

Appendix G Selected Reference List.


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Author Information

MARVIN C. McMASTER, PhD, is a consultant as well as an Adjunct Professor of Chemistry at the University of Missouri-St. Louis. Dr. McMaster has served as a researcher and product developer for DuPont, Kraft Foods, and Ciba-Geigy. Among his other publications, Dr. McMaster is author of LC/MS: A Practical User's Guide and coauthor of GC/MS: A Practical User's Guide (both published by Wiley).
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