Liquid Chromatography - Method Development and Validation

Types of Chromatography Paper Chromatography (Ascending, Descending) Column Chromatography Thin Layer Chromatography Gas Chromatography Liquid Chromatography

Basic Types of Liquid Chromatography Ion Exchange Chromatography Size Exclusion Chromatography Supercritical Fluid Chromatography/ SFE Liquid-Solid Chromatography (Adsorption) Liquid-Liquid Chromatography (Partitioning)

Size Exclusion Gel Particle

Modes of Liquid-Liquid Chromatography Reverse Phase Normal Phase

Liquid-Liquid Chromatography (HPLC) (Partitioning) (most common LC) Polarity Like dissolves like Distribution Coefficient /Capacity factor/ Selectivity factor pH & pKa Henderson Hasselbalch Equation

Schematic Diagram of HPLC

Components of HPLC Pump (Isocratic/HP/LP Gradient) Pump heads Autosampler Injector Column Detector Data Recorder

Schematic Diagram of Injector

HPLC/UPLC Column

Separation Process on Column

Silanol deactivation Base Deactivation

Silanol deactivation Contd. End Capping

Types of Detectors UV/PDA 200-800nm, most common 254 nm Refractive Index ELSD (Evaporative light scattering) Fluorescence Conductivity ECD

HPLC in 1990’s

HPLC – Recent Model Waters Alliance

Ultra Performance Liquid Chromatography (UPLC)- Waters Aquity

Comparison HPLC UPLC Dead volume 0.65 mL 0.25 mL Pressure 6000 psi Run time Abt 10 fold Abt 1 fold Flow cell volume 8 µL 0.5 µL

System Suitability Theorotical Plates (N) Tailing Factor (T) Resolution (R) Relative Retention Time (RRT) Kapacity Factor/Selectivity Factor (K) Relative Standard Deviation (RSD)

Adjustments to chromatographic systems pH of mobile phase Conc. of Buffer Organic Ratio of mobile phase Column Length Particle Size Flow rate Injection Volume Column Temperature

Method Development Literature survey Solubility pKa - Henderson Hasselbalch Equation Selection of wavelength Selection of column-based on compound Chemistry (Size exclusion, ion exchange, MW, chromophore) Selection of buffer Selection of org. modifier based on UV cut offs

Method Development Contd. Selection of pH Optimization of chromatographic Conditions(flow,inj. vol, pressure, RT, R, N, T, solution stab, filter compatibility) Forced Degradation – Stability Indicating Method System suitability finalization

Method Validation (ICH & USP) Specificity Forced Degradation (Stability Indicating) Filter paper study Solution stability Linearity Range Accuracy (Bias) Repeatability (method precision) Intermediate Precision Reproducibility (Interlaboratory Precision) Limit of Detection Limit of Quantification Robustness Statistical analysis

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