HPLC (high-performance liquid chromatography) pushes a dissolved sample through a packed column under high pressure so that components separate by how strongly they interact with the stationary phase. For peptides the standard method is reverse-phase HPLC on a C18 column with a water/acetonitrile gradient and 0.1% trifluoroacetic acid, detected by UV absorbance at 214 nm, where the peptide bond itself absorbs.
What the purity number means
Reported purity is normally area percent: the main peak's area divided by the total area of all integrated peaks. A 98.6% result means 98.6% of the detected UV signal eluted as one peak. It is a relative measure, not an assay of how much peptide is in the vial — water, counter-ion and non-absorbing excipients are invisible to it.
What it does not tell you
HPLC separates; it does not identify. A closely eluting deletion sequence can hide under the main peak, and an impurity with no chromophore may not register at 214 nm. That is why a credible certificate pairs the chromatogram with a mass spectrometry identity result for the same lot.
Related terms
certificate of analysis · third-party testing. Read what ≥98% actually means and how to read a COA.