High-Performance Liquid Chromatography (HPLC)
The standard analytical technique for assessing peptide purity, separating components based on their interaction with a column material under high pressure. Reversed-phase HPLC is most common for peptides. Purity is reported as a percentage — pharmaceutical-grade peptides typically require above 95-99%.
Technical Context
HPLC system components: solvent delivery pump (generating pressures up to 400-600 bar), injector (introducing sample onto column), column (typically C18 reversed-phase, 4.6mm × 150-250mm with 3-5μm particles for analytical; 21-50mm × 150-250mm for preparative), detector (UV at 214nm for peptide bond absorption, 280nm for aromatic amino acids; or mass spectrometer for identification), and data system. Method parameters for peptide analysis: mobile phase A (water + 0.1% TFA or formic acid), mobile phase B (acetonitrile + 0.1% TFA or formic acid), gradient elution (typically 1%B/min linear gradient), flow rate (1mL/min analytical, 10-50mL/min preparative), and column temperature (typically 25-60°C). Ultra-HPLC (UHPLC) systems use sub-2μm particles and higher pressures for faster, higher-resolution separations. Method validation follows ICH Q2(R1): specificity, linearity, accuracy, precision, range, detection/quantification limits, and robustness.