Understanding HPLC Testing

HPLC pushes a mobile phase through a packed column. Compounds in the sample interact differently with the stationary phase and so elute at different times. UV-Vis detection (usually 214 nm for peptide bonds) records each compound as a peak.

Reverse-phase C18 with a water/acetonitrile gradient is standard for peptide purity. Method validation parameters — column temperature, flow rate, injection volume — should be documented on the COA.

One dominant peak with minor shoulders is normal. Purity is the main peak area as a percentage of total area. Watch for unresolved shoulders that could indicate a deletion sequence or oxidation product.