HPLC (high-performance liquid chromatography)

High-performance liquid chromatography pushes a dissolved sample through a column packed with fine particles under high pressure. Different molecules travel through the column at different speeds depending on how strongly they interact with the packing, so they emerge at different times and are detected as peaks. The area of the peak for the intended peptide, divided by the total area of all peaks, is the purity figure a certificate of analysis reports.

A purity of 98% or higher means that at least 98% of the peptide material in the vial is the intended sequence. The remaining fraction is made up of related impurities: sequences missing a residue, sequences with an oxidised side chain, and fragments left from synthesis. HPLC does not by itself prove which peak is the right peptide; that is the job of mass spectrometry, which confirms the molecular weight of the main peak.

HPLC purity also says nothing about how much peptide is in the vial or what else is present that is not a peptide, such as residual trifluoroacetate or water. The guide to reading a certificate of analysis explains how the numbers fit together and the lab-testing page describes the testing behind each lot sold here.