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Hplc. A Practical User's Guide. 2nd Edition

  • ID: 449868
  • Book
  • 256 Pages
  • John Wiley and Sons Ltd
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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 running
  • Part II, HPLC Optimization, explains how to make the best use of the common columns and troubleshoot problems
  • Part 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.

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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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Marvin C. McMaster
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