HPLC purity testing, explained

The sample is pushed under high pressure through a column packed with a stationary phase. Components travel at different speeds depending on how strongly they interact with that phase, so they leave the column at different times and reach the detector separately.

Reverse-phase HPLC, the usual choice for peptides, separates primarily by hydrophobicity. Each separated component produces a peak, and the area under that peak is proportional to how much of it is present.

A purity figure is the target peak area divided by the total area of all peaks. It is a relative measure, not an absolute one: it describes the proportion of detected material, not the mass of peptide in the vial.

Residual solvents, water content and counter-ions are not captured by this figure, which is why peptide content is sometimes reported separately.

Chromatography tells you how much of one thing is present; mass spectrometry tells you what that thing is. Run together, they confirm both identity and purity, which is the combination we require before a lot is released.