a High Performance Liquid Chromatography Fundamental Principles and Practice Edited by W.J. LOUGH School of Health Sciences University of Sunderland and I.W. WAINER Department of Oncology McGill University Montreal BLACKIE ACADEMIC & PROFESSIONAL ‘An Imprint of Chapman & Hall London - Glasgow - Weinheim - New York - Tokyo - Melbourne - MadrasContents 13 14 24 23 24 25 2.6 Introduction w.J. LOUGH and I.W. WAINER Analysis and chromatography HPLC versus other analytical methods 1.2.1 Volumetric analysis 1.2.2 Ultraviolet spectrophotometry 1.2.3 Using chromatography to improve specificity Historical development of chromatography HPLC today Bibliography Efficiency, retention, selectivity and resolution in chromatography C.M. RILEY Introduction Chromatographic mobility Peak shape 2.3.1. Gaussian distribution 2.3.2 Peak area and peak height 2.3.3 Peak asymmetry Retention relationships 2.4.1 Retention time (¢,) 2.4.2 Retention volume (V;) 2.4.3. Relative-retention parameters Band broadening and column efficiency ‘Number of theoretical plates (N) Height equivalent to one theoretical plate (HETP, H) Reduced plate height (4) Relationships between column efficiency, analysis time and back pressure Relationships between mobile-phase velocity and column efficiency Extra-column contributions to band broadening Separation 2.6.1 Selectivity (a) 2.6.2. Resolution (R,) Effect of peak asymmetry on column efficiency and separation Bibliography Modes of chromatography C.M. RILEY Terminology Chromatographic format Models of retention Choice of mode of chromatography Normal-phase liquid chromatography RBuaavvee 153.6 37 3.8 5.1 5.2 61 62 CONTENTS Retention mechanisms and mobile phase effects in normal-phase liquid chromatography Mobile phase optimisation in normal-phase liquid chromatography 3. Stationary phase effects in normal-phase liquid chromatography 3.5.4 The role of water in normal-phase liquid chromatography Reversed-phased liquid chromatography 3.6.1 Retention mechanisms in reversed-phase liquid chromatography 3.6.2 Mobile phase effects in reversed-phase liquid chromatography 3.6.3. Stationary phase effects in reversed-phase liquid chromatography 3.6.4 Reversed-phase liquid chromatography of ionic compounds 3.6.5 Micellar chromatography Ton-exchange chromatography 3.7.1 Stationary phase effects in ion-exchange chromatography 3.7.2. Retention mechanisms and mobile phase effects in ion-exchange chromatography Size-exclusion chromatography 3.8.1. Separation mechanism in size-exclusion chromatography 38.2 Stationary phase and mobile phase effects in size-exclusion chromatography Bibliography Support materials and solvents P. HAMBLETON Adsorption chromatography Chemically bonded silica Polymer packings Porous graphitic carbon Solvents for HPLC Instrumentation: pumps, injectors and column design T. NOCTOR Introduction $.1.1 The ideal HPLC pump Types of HPLC pump 5.2.1 Constant pressure pumps 52.2 Constant flow pumps Approaches to the reduction of flow pulsation 5.3.1 Mechanical or physical pulse damping 5.3.2. Electronic pulse compensation The modern HPLC pump Valve injectors Column dimensions Column inlet/outlet ‘Connecting tubing and unions Conclusion Instrumentation: detectors and integrators D.K. LLOYD Ideal detectors and real detectors Detector performance criteria 62.1 Noise and drift Sensitivity and limit of detection Linear and dynamic range Band broadening, detector flow cells and time-constant 114 114 116 116 118, 19 19