Selectivity and Detectability Optimizations in HPLC
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1 Selectivity and Detectability Optimizations in HPLC SATINDER AHUJA Development Department Pharmaceuticals Division CIBA-GEIGY Corporation Suflern, New York WILEY A WILEY-INTERSCIENCE PUBLICATION JOHN WILEY & SONS New York / Chichester / Brisbane / Toronto / Singapore
2 CONTENTS CHAPTER 1 THE SCOPE OF SELECTIVITY AND DETECTABILITY OPTIMIZATION IN HPLC Selectivity and Detectability Modesof HPLC Theoretical Considerations Various Routes to High Resolution Optimum Column Selection (Conventional versus Small-Particle versus Microbore Columns) Optimum Analysis Time High-Resolution Evaluations HPLC Optimization Multifactor Optimization 9 References 13 CHAPTER 2 PHYSICOCHEMICAL BASIS OF RETENTION 15 By L. R. Snyder 2.1. General Aspects of Retention Partition Chromatography Retention Involving the Displacement of Mobile-Phase Molecules Adsorption of Mobile Phase by the Stationary Phase Intermolecular Interactions between Solute and Solvent Molecules Ion-Exchange Chromatography Adsorption (Normal-Phase) Chromatography Mobile-Phase Strength 24 vu
3 VW CONTENTS Solute Retention and Localization 2.4. Reversed-Phase Chromatography Mobile-Phase Strength Silanol Effects Solute Retention 2.5. Ion-Pair Chromatography Retention as a Function of Mobile- Phase Composition 2.6. Size-Exclusion Chromatography 2.7. Macromolecular Samples 2.8. Other Chromatographie Procedures Ion Chromatography Hydrophobic-Interaction Chromatography (HIC) Ligand-Exchange Chromatography References CHAPTER 3 PROBING SEPARATION MECHANISM IN HPLC Separations in RPLC versus Octanol Partition Data Impact of Other Physicochemical Parameters on Retention Retention in HPLC versus Solubility Stationary-Phase Effects Adsorption/Normal Phase Reversed Phase Stationary-Phase Dynamics Retention Mechanism Investigations Molecular Probes/Retention Index 65 References 68 CHAPTER 4 CONVENTIONAL APPROACHES TO MOBILE-PHASE SELECTION AND OPTIMIZATION 4.1. General Considerations Properties of Sample or Solute
4 CONTENTS IX Column Selection Column Evaluations Mobile-Phase Selection in HPLC Solvent Classification Mobile-Phase Additives Modes of Chromatography Adsorption Chromatography Normal-Bonded Phases Reversed-Phase Chromatography Ion-Exchange Chromatography 92 References 100 IMPROVING SEPARATIONS IN ADSORPTION FOR NORMAL-PHASE SEPARATIONS Adsorption Chromatography Column Packings Mechanism of Adsorption Chromatography Solvent Selection in LSC Solvent Strength in LSC Role of Modulators Modulators and Selectivity Mobile-Phase Optimization Strategies Effect of Sample Structure on Retention Applications of Adsorption Chromatography Normal-Phase Chromatography Theory Column Packings Mobile-Phase Effects Applications of Normal-Phase Chromatography Miscellaneous Approaches 155 References 156
5 X CONTENTS CHAPTER 6 SELECTIVITY OPTIMIZATION IN REVERSED-PHASE SEPARATIONS 6.1. Stationary Phase Pore Size and Volume Nature of Bonded Phases Column Efficiency Columns Problems with HPLC Columns Column Evaluations 6.2. Retention Mechanism 6.3. Eluotropic Solvent Scale 6.4. Mobile-Phase Selection and Optimization Effect on Selectivity Enthalpy of Binding 6.5. Applications References CHAPTER 7 ION-EXCHANGE CHROMATOGRAPHY OF IONIC AND IONIZABLE COMPOUNDS 7.1. Ion-Exchange Chromatography Column Packings Mobile-Phase Selection Retention Mechanism Applications of IEC 7.2. Ion Chromatography Applications of IC References CHAPTER 8 OPTIMIZING SELECTIVITY OF ION-PAIR SEPARATIONS Nature of Ion Pairs Theory Column Packings Mobile-Phase Selection and Optimization Role of the Counterion Solvent Effects 275
6 CONTENTS XI Ionic Strength and Secondary Ion Effects Association/Dissociation Effects ph Effects Other Separation Variables Retention Mechanism Applications 293 References 310 CHAPTER 9 MACROMOLECULAR SEPARATIONS Types of Macromolecules Proteins Purity Assessment Recombinant-DNA-Derived Proteins Polypeptides Monoclonal Antibodies Nucleic Acids Chromatographie Methods Retention Mechanism Adsorption Reversed Phase Ion Exchange Size-Exclusion Chromatography Mechanistic Studies Proteins Nucleotides Enzymes Miscellaneous Compounds Applications 361 References 387 CHAPTER 10 ISOMERIC SEPARATIONS Chiral Columns Chiral Additives to HPLC Eluents Modes of Separation Chromatography of Diastereomeric Derivatives 408
7 Xll CONTENTS Enantiomeric Resolution Using Chiral Mobile-Phase Additives Enantiomeric Resolution Using Chiral Stationary Phases Applications Reversed- Phase/ Normal- Phase Adsorption Columns Ligand Chromatography Pirkle-Type Columns Cyclodextrin Columns Protein Columns Miscellaneous Applications 451 References 455 CHAPTER11 COMPUTER OPTIMIZATION OF SELECTIVITY Experimental Design Optimization Methods Window Diagram Methods Simplex Methods Gradient Elution Interactive Methods Practical Approaches Computer Simulation and Solvent- Strength Optimization Instrumentation Optimum Method for Optimization 501 References 502 CHAPTER 12 SELECTIVE DETECTORS IN HPLC UV Detectors Applications Fluorescence Detectors Applications Miscellaneous Luminescent Detectors Electrochemical Detectors Applications 525
8 CONTENTS Xlll Refractive Index Detectors Mass Spectrometric Detectors Applications Miscellaneous Detectors 544 References 548 CHAPTER 13 DETECTABILITY OPTIMIZATION Noise and Detection Limits Column Evaluations for Detectability Optimization Limit of Detection Detector Evaluation and Optimizations Ultraviolet Detectors Fluorescence Detectors Novel Detectors and Detection Methods Derivatization Miscellaneous Approaches 595 References 598 INDEX 605
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