HPLC COLUMNS WILEY-VCH. Theory, Technology, and Practice. Uwe D. Neue with a contribution from M. Zoubair El Fallah

Transcription

1 HPLC COLUMNS Theory, Technology, and Practice Uwe D. Neue with a contribution from M. Zoubair El Fallah WILEY-VCH New York Chichester Weinheim Brisbane Singapore Toronto

2 CONTENTS Preface ix 1 Introduction History Scope of This Book What Is HPLC? Separation Mechanisms Used in HPLC 4 2 Theory of Chromatography Definitions Isocratic and Gradient Chromatography The Height Equivalent to a Theoretical Plate Resolution Efnciency, Plate Count Peak Asymmetry Linear Velocity Reduced Parameters Hydrodynamics of Chromatography Van Deemter Equation Giddings Equation Knox Equation Comparison of the Different Equations Permeability Estimation of the Diffusion Coefficient in the Mobile Phase Performance Index and Separation Impedance Thermodynamics of Chromatography Definition of the Retention Factor Retention Factor and Chemical Equilibrium Relative Retention a Relationships to the Free Energy 38 References 39 Additional Reading 40

3 viii CONTENTS Column Design Choice of Column Dimensions and Particle Size Influence of Particle Size and Column Length Influence of Column Diameter Design Limits Heat of Friction Extracolumn Effects Column Hardware End-Fitting Design Tubing Columns or Cartridges? Alternative Column Designs Radial Compression Axial Compression Alternatives to Packed Beds Membranes Monolithic Structures Fibers Evaluating Alternative Designs 73 References 79 4 Physical Properties of HPLC Packings Particle Size, Particle Shape, and Particle Size Distribution Porosity, Pore Size, and Specific Surface Area Porous versus Nonporous Particles Particle Strength 90 References 91 5 Column Packing and Testing Column Packing Principles Apparatus Slurry Preparation Packing Techniques Column Testing Summary 103 References 104 Additional Reading 104 Other Literature Related to Column Packing 104

4 CONTENTS ix 6 Column Chemistry Inorganic versus Polymerie Packings Silica-Based Bonded Phases Bonding Technology Monomeric versus Polymerie Bonding Types of Bonded Phases Reproducibility of Bonded Phases Alternative Bonding Technologies The Chemistry of Polymerie Packings Divinylbenzene-Based Packings Methacrylate-Based Packings Vinylalcohol-Based Packings Other Surface Modification Techniques for Inorganic Packings Properties of Other Inorganic Packings Other Surface Modification Techniques 127 References 128 Additional Reading 129 Column Selection 7.1 Introduction 7.2 Polymer Analysis 7.3 Protein Analysis 7.4 Peptide Analysis 7.5 Nucleic Acid Analysis 7.6 Carbohydrate Analysis 7.7 Analysis of Inorganic Ions 7.8 Analysis of Low-Molecular-Weight Organic Compounds 7.9 Summary Size-Exclusion Chromatography Separation Mechanism Selection of Mobile Phase and Column Type Selection of the Pore Size Selection of the Particle Size Polymerie Packings for Organic SEC Polymerie Packings for Aqueous SEC Silica-Based Packings Practical Aspects of SEC 161 References 162

5 x CONTENTS 9 Normal-Phase Chromatography Retention Mechanism Silica Silica-Based Bonded Phases Alumina Special Techniques Practical Aspects of Normal-Phase Chromatography 180 References 181 Additional Reading Reversed-Phase Chromatography Retention Mechanism Silica-Based Bonded Phases Silanol-Group Activity Characterization of Packings Reproducibüity of Reversed-Phase Packings Polymerie Reversed-Phase Packings Styrene-Divinylbenzene-Based Packings Methacrylate-Based Packings Polyvinylalcohol-Based Packings Other Types Porous Graphitized Carbon Polymer-Coated Alumina Internal-Surface Reversed-Phase Packings Immobilized Artificial Membranes Special Techniques Paired-Ion Chromatography RPLC of Peptides Practical Aspects of Reversed-Phase Chromatography Column Selection Mobile-Phase Considerations Column Care 214 References 215 Additional Reading Hydrophilic Interaction Chromatography Retention Mechanism Packings for HILIC Applications Practical Aspects 221 References 223

6 CONTENTS xi 12 Ion-Exchange Chromatography Retention Mechanism General Ion-Exchange Mechanism Behavior of Coions Elution Strength Scales Influence of ph Ion-Exchange Capacity Nonideal Interactions Ion-Exchange Packings Silica-Based Ion Exchangers Ion Exchangers Based on Organic Polymers Ion Exchangers for Ion Chromatography Ion Chromatography Ion-Exchange Separations of Biopolymers Proteins Nucleic Acids Practical Aspects 247 References 249 Additional Reading Hydrophobie Interaction Chromatography Retention Mechanism Operating Conditions Packings for HIC Applications Practical Aspects References Additional Reading Special Techniques References Column Coupling Principles of Column Coupling Guard Columns Column Switching Recycling and Walking Column Simulated Moving Bed Continuous Chromatography Preparative Chromatography Principles 276

7 xii CONTENTS Influence of the Injection Volume Influence of Mass Overload Influence of Column Dispersion Combined Effect of Injection Volume, Mass Overload, and Dispersion Technologies for Laboratory-Scale Preparative Chromatography Practical Aspects Loading Studies Scaling Laws Particle Size 294 References 295 Additional Reading HPLC Methods Development Introduction Theoretical Considerations Linear-Solvent-Strength Theory Characteristic Parameters in Linear-Solvent-Strength Gradients Retention Time Peak Width Resolution Relationship between Solvent Strength and Mobile-Phase Composition Measuring the Quality of Separation between Adjacent Peaks Resolution Other Separation Criteria The Discrimination Factor Basis of Mobile-Phase Selectivity in HPLC Method Development Strategy Considerations in the Choice of Isocratic versus Gradient Elution Developing Isocratic Separations Binary Mobile Phases Solvent Selectivity and Ternary Mobile Phases Separation of Totally or Partially Unknown Mixtures Developing Gradient Separations Computer-Assisted Method Development 332

8 CONTENTS xüi 16.4 Enhancing Sensitivity Temperature Effects 337 References Column Maintenance Care and Use of Chromatographie Columns Installation and Equilibration Column Use Performance Monitoring During Use Column Protection Storage Troubleshooting of Column Problems Peak-Shape Problems Extra-Column Effects Extra-Column Band Spreading Detector Time Constant Column Overload Volume Overload Mass Overload Detector Overload Mobile Phase Overload ph-control Incorrect Sample Solvent Solubility Tailing Due to Secondary Interactions Sample Distribution Column Collapse Secondary Equilibria Retention-Time Problems Retention Time Reproducibility Temperature Mobile-Phase Composition Retention Time Drift Column Equilibration Stationary-Phase Stability Column Contamination Mobile-Phase Composition Other Column Aging Mechanisms Hydrophobie Collapse Column-to-Column Reproducibility 374

9 xiv CONTENTS Miscellaneous Problems Extra Peaks Excessive Backpressure Baseline Drift 379 References 379 Appendix: Teaching Column Chromatography 380 List of Symbols 383 Index 389