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How to read an HPLC chromatogram

The number on a certificate is a summary. The trace is the evidence. Here is what each part of the plot actually means.

The axes

Time runs left to right in minutes; absorbance runs bottom to top in milli-absorbance units at a fixed wavelength — for peptides, almost always 214 nm, where the peptide backbone absorbs.

The main peak

The tall peak is your compound eluting off the column. Its retention time is a weak identity check: it tells you the compound behaves consistently, not that it is the right molecule.

Area percent

Purity is the area under the main peak divided by the total area of all peaks. That is why a purity figure is only as honest as the integration — how the software decided where one peak ends and the next begins.

Small peaks are the story

A cluster of small peaks before the main one usually means incomplete synthesis or degradation. Peaks after it are often aggregates or oxidation products. A 99% purity figure with a messy shoulder is not a clean batch.

What it cannot tell you

HPLC says how much of the UV-absorbing material is the main species. It cannot tell you what that species is. Only mass spectrometry can.

Put it into practice. Every Verdica Labs certificate ships with the chromatogram and mass spectrum attached, and a report ID you can resolve on the verify page. See the tests →