Modern HPLC for Practicing Scientists

Abstract

Preface. 1 Introduction. 1.1 Introduction. 1.1.1 Scope. 1.1.2 What Is HPLC? 1.1.3 A Brief History. 1.1.4 Advantages and Limitations. 1.2 Modes of HPLC. 1.2.1 Normal-Phase Chromatography (NPC). 1.2.2 Reversed-Phase Chromatography (RPC). 1.2.3 Ion-Exchange Chromatography (IEC). 1.2.4 Size-Exclusion Chromatography (SEC). 1.2.5 Other Separation Modes. 1.3 Some Common-Sense Corollaries. 1.4 How to Get More Information. 1.5 Summary. 1.6 References. 2 Basic Terms and Concepts. 2.1 Scope. 2.2 Basic Terms and Concepts. 2.2.1 Retention Time (tR),Void Time (tM), Peak Height (h), and Peak Width (wb). 2.2.2 Retention Volume (VR),Void Volume (VM), and Peak Volume. 2.2.3 Retention Factor (k). 2.2.4 Separation Factor (alpha). 2.2.5 Column Efficiency and Plate Number (N). 2.2.6 Peak Volume. 2.2.7 Height Equivalent to a Theoretical Plate or Plate Height (HETP or H). 2.2.8 Resolution (Rs). 2.2.9 Peak Symmetry: Asymetry Factor (As) and Tailing Factor (Tf). 2.3 Mobile Phase. 2.3.1 General Requirements. 2.3.2 Solvent Strength and Selectivity. 2.3.3 Buffers. 2.3.4 Acidic Mobile Phases. 2.3.5 Ion-Pairing Additives. 2.3.6 High pH Mobile Phase. 2.3.7 Other Operating Parameters: Flow Rate (F) and Column Temperature (T). 2.4 The Resolution Equation. 2.5 The Van Deemter Equation. 2.6 Isocratic vs. Gradient Analysis. 2.6.1 Peak Capacity (n). 2.6.2 Key Gradient Parameters (Initial and Final Solvent Strength, Gradient Time [tG], and Flow Rate). 2.6.3 The 0.25tG Rule:When Is Isocratic Analysis More Appropriate? 2.7 Concept of Orthogonality. 2.8 Sample Capacity. 2.9 Glossary of HPLC Terms. 2.10 Summary and Conclusion. 2.11 References. 3 HPLC Columns and Trends. 3.1 Scope. 3.2 General Column Description and Characteristics. 3.2.1 Column Hardware-Standard vs. Cartridge Format. 3.3 Column Types. 3.3.1 Types Based on Chromatographic Modes. 3.3.2 Types Based on Dimensions. 3.3.3 Column Length (L). 3.4 Column Packing Characteristics. 3.4.1 Support Type. 3.4.2 Particle Size (dp). 3.4.3 Surface Area and Pore Size (dpore). 3.4.4 Bonding Chemistries. 3.4.5 Some General Guidelines for Bonded Phase Selection. 3.5 Modern HPLC Column Trends. 3.5.1 High-Purity Silica. 3.5.2 Hybrid Particles. 3.5.3 Novel Bonding Chemistries. 3.5.4 Fast LC. 3.5.5 Micro LC. 3.5.6 Monoliths. 3.6 Guard Columns. 3.7 Specialty Columns. 3.7.1 Bioseparation Columns. 3.7.2 Chiral Columns. 3.7.3 Application-Specific Columns. 3.8 Column Selection Guides. 3.9 Summary. 3.10 References. 3.11 Internet Resources. 4 HPLC Instrumentation and Trends. 4.1 Introduction. 4.1.1 Scope. 4.1.2 HPLC Systems and Modules. 4.2 HPLC Solvent Delivery Systems. 4.2.1 High-Pressure and Low-Pressure Mixing Designs in Multisolvent Pumps. 4.2.2 System Dwell Volume. 4.2.3 Trends. 4.3 Injectors and Autosamplers. 4.3.1 Operating Principles of Autosamplers. 4.3.2 Performance Characteristics and Trends. 4.4 Detectors. 4.5 UV/VIS Absorbance Detectors. 4.5.1 Operating Principles. 4.5.2 Performance Characteristics. 4.5.3 Trends in Absorbance Detectors. 4.6 Photodiode Array Detectors. 4.6.1 Operating Principles. 4.6.2 Trends in PDA Detectors. 4.7 Other Detectors. 4.7.1 Fluorescence Detector (FLD). 4.7.2 Refractive Index Detector (RID). 4.7.3 Evaporative Light Scattering Detector (ELSD). 4.7.4 Corona-Charged Aerosol Detector (CAD). 4.7.5 Chemiluminescence Nitrogen Detector (CLND). 4.7.6 Electrochemical Detector (ECD). 4.7.7 Conductivity Detector. 4.7.8 Radiometric Detector. 4.8 Hyphenated and Specialized Systems. 4.8.1 LC/MS, LC/MS/MS. 4.8.2 LC/NMR. 4.8.3 Other Hyphenated Systems. 4.8.4 Prep LC and Bio-Purification Systems. 4.8.5 Proteomics Systems: Capillary LC and Multi-Dimensional LC. 4.8.6 High-Throughput Screening (HTS) and Parallel Analysis Systems. 4.8.7 Ultra-High-Pressure Liquid Chromatography. 4.8.8 Lab-on-a-Chip. 4.8.9 Specialized Applications Systems. 4.9 HPLC Accessories and Data Handling Systems. 4.9.1 Solvent Degasser. 4.9.2 Column Oven. 4.9.3 Column Selector Valve. 4.9.4 Data Handling and HPLC Controllers. 4.10 Instrumental Bandwidth (IBW). 4.11 Trends in HPLC Equipment. 4.12 Manufacturers and Equipment Selection. 4.13 Summary. 4.14 References. 4.15 Internet Resources. 5 HPLC Operation Guide. 5.1 Scope. 5.2 Safety and Environmental Concerns. 5.2.1 Safety Concerns. 5.2.2 Environmental Concerns. 5.3 Mobile Phase Preparation. 5.3.1 Mobile Phase Premixing. 5.3.2 Buffers. 5.3.3 Filtration. 5.3.4 Degassing. 5.4 Best Practices in HPLC System Operation. 5.4.1 Pump Operation. 5.4.2 HPLC Column Use, Connection, and Maintenance. 5.4.2.1 Column Use. 5.4.2.2 Column Precautions. 5.4.2.3 Column Connection. 5.4.2.4 Column Maintenance and Regeneration. 5.4.3 Autosampler Operation. 5.4.4 Detector Operation. 5.4.5 System Shutdown. 5.4.6 Guidelines for Increasing HPLC Precision. 5.4.6.1 Guidelines for Improving Retention Time Precision. 5.4.6.2 Guidelines for Improving Peak Area Precision. 5.5 From Chromatograms to Reports. 5.5.1 Qualitative Analysis Strategies. 5.5.2 Quantitation Analysis Strategies. 5.6 Summary of HPLC Op