Definition
An analytical separation technique used to measure peptide purity by resolving the target from synthesis impurities.
High-performance liquid chromatography pumps a sample through a packed column under pressure, separating components by their interaction with the stationary phase. For peptides, reversed-phase HPLC (RP-HPLC) on a C18 column with a water–acetonitrile gradient is standard, and detection is usually UV absorbance at 214–220 nm, where the peptide bond absorbs. Purity is calculated as the main peak area divided by the total integrated area. HPLC resolves truncated, deletion and oxidised by-products from the target sequence. It does not confirm identity: a wrong sequence can still give a single sharp peak. That is why a complete certificate of analysis pairs HPLC with mass spectrometry.