Practical High-Performance Liquid Chromatography

by Meyer, Veronika R.

• ISBN13: 9780470682180
• ISBN10: 0470682183
• Edition: 5th
• Format: Hardcover
• Copyright: 2010-05-17
• Publisher: Wiley

Summary

This textbook aims to present the knowledge which is necessary for everyday HPLC measurements to be made, in a detailed and easy-to-follow manner. It contains a wealth of practical material and covers a wide range of subjects. Topics covered include principles, instrumentation, reverse-phase, ion-exchange, ion-pair, ion, size-exclusion and affinity chromatography, as well as analytical methods, preparative HPLC and enantiomeric separations. Appendices provide information on instrument testing, troubleshooting, column-packing.

Author Biography

Veronika R. Meyer, Department of Biocompatible Materials, Switzerland Swiss Federal Laboratories for Materials Testing and Research, Switzerland.

Table of Contents

Contents

Introduction

HPLC: a powerful separation method

A first HPLC experiment

Liquid chromatographic separation modes

The HPLC instrument

Safety in the HPLC laboratory

Comparison between high-performance liquid chromatography and gas chromatography

Comparison between high-performance liquid chromatography and capillary electrophoresis

Units for pressure, length, and viscosity

Scientific journals

Recommended books

Theoretical Principles

The chromatographic process

Band broadening

The chromatogram and its purport

Graphical representation of peak pairs with different degree of resolution¹¹

Factors affecting resolution

Extra-column volumes (dead volumes)¹⁵

Tailing

Peak capacity and statistical resolution probability¹⁹

Effects of temperature in hplc²³

The limits of hplc

How to obtain peak capacity

Pumps

General requirements

The short-stroke piston pump

Maintenance and repair

Other pump designs

Preparation of equipment up to sample injection

Selection of mobile phase

Preparation of the mobile phase

Gradient systems

Capillary tubing

Fittings¹²

Sample injectors

Sample solution and sample volume

Solvent properties

table of organic solvents

Solvent selectivity

Miscibility

Buffers³

Shelf-life of mobile phases

The mixing cross

Detectors

General

UV detectors²

Refractive index detectors

Fluorescence detectors

Electrochemical (amperometric) detectors³

Light scattering detectors⁶

Other detectors

Multiple detection

Indirect detection

Coupling with spectroscopy

Columns and stationary phases

Columns for HPLC

Precolumns

General properties of stationary phases¹

Silica⁴

Chemically modified silica⁶

Styrene-divinylbenzene¹³

Some other stationary phases

Column care and regeneration

HPLC column tests

Simple tests for HPLC columns

determination of particle size

Determination of breakthrough time

The test mixture

Dimensionless parameters for HPLC column characterization²

The van deemter equation from reduced parameters and its use in column diagnosis

diffusion coefficients³

Adsorption chromatography: normal-phase chromatography

What is adsorption?¹

The eluotropic series

Selectivity properties of the mobile phase

Choice and optimization of the mobile phase

Applications

Reversed-phase chromatography

Principle

Mobile phases in reversed-phase chromatography

Solvent selectivity and strength⁸

Stationary phases

method development in reversed-phase chromatography¹⁵

Applications

Hydrophobic interaction chromatography¹⁵

Chromatography with chemically bonded phases

introduction

properties of some stationary phases

hydrophilic interaction chromatography⁶

Ion-exchange chromatography

Introduction

Principle

Properties of ion exchangers

Influence of the mobile phase

Special possibilities of ion exchange

Practical hints

Applications

Ion-pair chromatography

Introduction

Ion-pair chromatography in practice

Applications

Appendix: UV detection using ion-pair reagents⁵

Ion chromatography¹

Principle

Suppression techniques

Phase systems³

Applications

Size-exclusion chromatography¹

Principle

The calibration chromatogram

Molecular mass determination by means of size-exclusion chromatography⁵

Coupled size-exclusion columns

Phase systems

Applications

Affinity chromatography

Principle¹

Affinity chromatography as a special case of hplc²

Applications

Choice of method

the various possibilities

method transfer⁸

Solving the elution problem

The elution problem

Solvent gradients²

Column switching⁷

Comprehensive two-dimensional hplc¹¹

Optimization of an isocratic chromatogram using four solvents

Optimization of the other parameters

Mixed stationary phases

Analytical HPLC

Qualitative analysis¹

Trace analysis

Quantitative analysis⁷

Recovery¹¹

Peak-height and peak-area determination for quantitative analysis¹⁵

Integration errors

The detection wavelength

Derivatization²⁰

Unexpected peaks: ghost and system peaks

Quality assurance¹

Is it worth the effort?

Verification with a second method

Method validation³

Standard operating procedures (SOP's)

Measurement uncertainty⁶

Qualifications, instrument test, and system suitability test

The quest for quality

Preparative hplc¹

Problem

Preparative hplc in practice²

Overloading effects

Fraction collection

Recycling

Displacement chromatography¹⁰

Separation of enantiomers¹

Introduction

Chiral mobile phases⁴

Chiral liquid stationary phases

Chiral solid stationary phases ⁶

Indirect separation of enantiomers¹⁶

Special possibilities

Micro, capillary and chip hplc¹

High-speed and super-speed hplc⁷

Fast separations at 1000 bar: uplc⁹

HPLC with supercritical mobile phases¹¹

HPLC with superheated water¹⁴

Electrochromatography¹⁵

Appendix 1: applied HPLC theory

Appendix 2: how to perform the instrument test

Introduction

Test sequence

Preparations

Pump test

UV detector test

Autosampler test

Column oven test

Equations and calculations

Documentation

Appendix 3: troubleshooting

Pressure problems

Leak in the pump system

Deviating retention times

Injection problems

Baseline problems

Peak shape problems

Problems with light scattering detectors (elsd)

Other causes

Instrument test

Appendix 4: column packing¹

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