MODERN HPLC FOR PRACTICING SCIENTISTS
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1 MODERN HPLC FOR PRACTICING SCIENTISTS Michael W. Dong Synomics Pharmaceutical Services, LLC Wareham, Massachusetts WILEY- INTERSCIENCE A JOHN WILEY & SONS, INC., PUBLICATION
2 Preface xv 1 Introduction Introduction / Scope / What Is HPLC? / A Brief History / Advantages and Limitations / Modes of HPLC / Normal-Phase Chromatography (NPC) / Reversed-Phase Chromatography (RPC) / Ion-Exchange Chromatography (IEC) / Size-Exclusion Chromatography (SEC) / Other Separation Modes / Some Common-Sense Corollaries / How to Get More Information / Summary / References / 13 2 Basic Terms and Concepts Scope / Basic Terms and Concepts / Retention Time (t R ). Void Time (t M ). Peak Height (h). and Peak Width (w b ) / Retention Volume (V R ). Void Volume (V M ). and Peak Volume / Retention Factor (k) / Separation Factor (a) / Column Efficiency and Plate Number (N) / Peak Volume / Height Equivalent to a Theoretical Plate or Plate Height (HETP or H) / 23
3 Vi CONTENTS Resolution (R s ) / Peak Symmetry: Asymetry Factor (A s ) and Tailing Factor (T f ) / Mobile Phase / General Requirements / Solvent Strength and Selectivity / Buffers / Acidic Mobile Phases / Ion-Pairing Additives / High ph Mobile Phase / Other Operating Parameters: Flow Rate (F) and Column Temperature (T) / The Resolution Equation / The Van Deemter Equation / Isocratic vs. Gradient Analysis / Peak Capacity (n) / Key Gradient Parameters (Initial and Final Solvent Strength, Gradient Time [t G ], and Flow Rate) / The 0.25 At G Rule: When Is Isocratic Analysis More Appropriate? / Concept of Orthogonality / Sample Capacity / Glossary of HPLC Terms / Summary and Conclusion / References / 46 3 HPLC Columns and Trends 3.1 Scope / General Column Description and Characteristics / Column Hardware Standard vs. Cartridge Format / Column Types / Types Based on Chromatographic Modes / Types Based on Dimensions / Column Length (L) / Column Packing Characteristics / Support Type / Particle Size (d p ) / Surface Area and Pore Size (d pore ) / Bonding Chemistries / Some General Guidelines for Bonded Phase Selection / Modern HPLC Column Trends / High-Purity Silica / 58 47
4 Vii Hybrid Particles / Novel Bonding Chemistries / Fast LC / Micro LC / Monoliths / Guard Columns / Specialty Columns / Bioseparation Columns / Chiral Columns / Application-Specific Columns / Column Selection Guides / Summary / References / Internet Resources / 75 4 HPLC Instrumentation and Trends Introduction / Scope / HPLC Systems and Modules / HPLC Solvent Delivery Systems / High-Pressure and Low-Pressure Mixing Designs in Multisolvent Pumps / System Dwell Volume / Trends / Injectors and Autosamplers / Operating Principles of Autosamplers / Performance Characteristics and Trends / Detectors / UV/VIS Absorbance Detectors / Operating Principles / Performance Characteristics / Trends in Absorbance Detectors / Photodiode Array Detectors / Operating Principles / Trends in PDA Detectors / Other Detectors / Fluorescence Detector (FLD) / Refractive Index Detector (RID) / Evaporative Light Scattering Detector (ELSD) / Corona-Charged Aerosol Detector (CAD) / Chemiluminescence Nitrogen Detector (CLND) / Electrochemical Detector (ECD) / 95
5 4.7.7 Conductivity Detector / Radiometric Detector / Hyphenated and Specialized Systems / LC/MS, LC/MS/MS / LC/NMR / Other Hyphenated Systems / Prep LC and Bio-Purification Systems / Proteomics Systems: Capillary LC and Multi-Dimensional LC / High-Throughput Screening (HTS) and Parallel Analysis Systems / Ultra-High-Pressure Liquid Chromatography / Lab-on-a-Chip / Specialized Applications Systems / HPLC Accessories and Data Handling Systems / Solvent Degasser / Column Oven / Column Selector Valve / Data Handling and HPLC Controllers / Instrumental Bandwidth (IBW) / Trends in HPLC Equipment / Manufacturers and Equipment Selection / Summary / References / Internet Resources / HPLC Operation Guide Scope / Safety and Environmental Concerns / Safety Concerns / Environmental Concerns / Mobile Phase Preparation / Mobile Phase Premixing / Buffers / Filtration / Degassing / Best Practices in HPLC System Operation / Pump Operation / HPLC Column Use, Connection, and Maintenance / Column Use / Column Precautions / 118
6 ix Column Connection / Column Maintenance and Regeneration / Autosampler Operation / Detector Operation / System Shutdown / Guidelines for Increasing HPLC Precision / Guidelines for Improving Retention Time Precision / Guidelines for Improving Peak Area Precision / From Chromatograms to Reports / Qualitative Analysis Strategies / Quantitation Analysis Strategies / Summary of HPLC Operation / Guides on Performing Trace Analysis / Summary / References / Pharmaceutical Analysis Introduction / Scope / Overview: From Drug Discovery to Quality Control / Sample Preparation Perspectives in Drug Product Analysis / High-Throughput LC/MS in Drug Discovery Support / Identification / Assays / Drug Substances / Drug Products / Content Uniformity / Products with Multiple APIs and Natural Products / Assay of Preservatives / Impurity Testing / Trends in Impurity Testing / Dissolution Testing / Cleaning Validation / Bioanalytical Testing / Chiral Analysis / Case Study: HPLC Methods in Early Development / Summary / References / 155
7 Food, Environmental, Chemical, and Life Sciences Applications 7.1 Introduction / Scope / Food Applications / Natural Food Components / Sugars / Fats, Oils, and Triglycerides / Free Fatty Acids and Organic Acids / Proteins and Amino Acids / Food Additives / Flavors: A Case Study on HPLC Analysis of Capsaicins / Contaminants / Mycotoxins / Antimicrobial Additives / Pesticide Residues / Environmental Applications / Listing of Important U.S. EPA HPLC Methods / Pesticides Analysis / Carbamates and Glyphosate / Polynuclear Aromatic Hydrocarbons (PAH) / Case Study: Quick Turnaround Analysis of PAHs by HPLC in Multimedia Samples / Chemical, GPC and Plastics Applications / Gel-Permeation Chromatography (GPC) and Analysis of Plastics Additives / Ion Chromatography / Life Sciences Applications / Proteins, Peptides, and Amino Acids / Bases, Nucleosides, Nucleotides, Oligonucleotides, Nucleic Acids, and PCR Products / Summary / References / t HPLC Method Development Introduction / Scope / Considerations Before Method Development / Strategy for Method Development / 195
8 Xi HPLC Method Development Trends in Pharmaceutical Analysis / Defining Method Types and Goals / Method Goals / Gathering Sample and Analyte Information / Defining Sample Preparation Requirements / Initial HPLC Method Development / Initial Detector Selection / Selection of Chromatographic Mode / Initial Selection of HPLC Column / Generating a First Chromatogram / Case Study: Initial Method Development Using a Broad Gradient and Mobile Phase Selection / Method Fine-Tuning / Mobile Phase Parameters (%B, Buffer, ph. Solvent Type) / Operating Parameters (F,T, A< >. t G ) / Column Parameters (Bonded Phase Type. L. d p, d c ) / Detector Setting and Sample Amount / Summary of Method Development Steps / Phase-Appropriate Method Development / Method Development Software Tools / Case Studies / Composite Assay Method for a Neutral Drug Substance / Composite Drug Substance Method for a Basic Drug Substance / Impurity Method for a Drug Product with Two APIs / Summary and Conclusions / References / 220 Regulatory Aspects of HPLC Analysis: HPLC System and Method Validation Introduction / Scope / The Regulatory Environment / HPLC System Qualification / Design Qualification (DQ) / Installation Qualification (IQ) / Operational Qualification (OQ) / 225
9 Xii CONTENTS Performance Qualification (PQ) / Documentation / System Calibration / Method Validation / Validation Parameters / System Suitability Testing (SST) / Case Study on Method Validation / Cost-Effective Regulatory Compliance / Summary and Conclusions / References / Internet Resources / HPLC Maintenance and Troubleshooting Guide Scope / HPLC System Maintenance / LC Pump / UV/Vis Detectors / Injector and Autosampler / HPLC Troubleshooting / General Problem Diagnostic and Troubleshooting Guide / Common HPLC Problems / Pressure Problems and Causes / Baseline Problems (Chromatogram) / Peak Problems (Chromatogram) / Data Performance Problems / Case Studies / Case Study 1: Reducing Baseline Shift and Noise for Gradient Analysis / Case Study 2: Poor Peak Area Precision Encountered During HPLC System Calibration / Case Study 3: Poor Assay Accuracy Data, an Out-of- Specification Investigation / Case Study 4: Equipment Malfunctioning / Summary and Conclusion / References / Internet Resources / Modern Trends in HPLC Columns: Shorter and Narrower Packed with Small Particles / 266
10 Xiii 11.2 Column Packing: Novel Bonded Phases / Pumps / Autosamplers / Detectors / HPLC Systems / Low-Dispersion Instruments / Ultra-High-Pressure LC / Multi-Dimensional LC / Parallel Analysis / Lab-on-a-Chip / Data Handling / Regulatory Compliance / Greener HPLC Methods / Summary and Conclusions / References / 271 Index 273
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