Understanding HPLC Chromatograms on a COA

Many peptide COAs include an HPLC chromatogram — a plot of detector response over time. Vendors treat it as proof of purity, but the image is only useful if you know what you are looking at. This guide walks through the basics without requiring an analytical chemistry degree.

Axes and peaks

The x-axis is retention time: how long each component takes to travel through the column. The y-axis is detector response — usually UV absorbance at a wavelength chosen for the peptide. Each bump is a peak representing a compound (or group of co-eluting compounds) passing the detector.

The main peak should correspond to your target peptide. Smaller peaks before or after it are impurities — deletion sequences, incomplete couplings, or oxidized forms depending on the preparation.

What "clean" looks like

A strong chromatogram shows:

• One dominant peak with a clear, symmetric shape.
• Impurity peaks that are small relative to the main peak.
• A stable baseline without excessive noise or drift.
• Labeled retention time and peak-area integration.

A quoted "99% purity" number without the chromatogram — or with an unlabeled screenshot — deserves skepticism. The integration table matters as much as the picture.

Red flags on chromatograms

• No peak labels or integration table — you cannot verify the purity claim.
• Multiple large peaks of similar size — the sample may be a mixture, not a single species.
• Flat line or obvious editing artifacts — the image may not reflect the actual run.
• Missing method details — column type, gradient, and detection wavelength should appear in the COA methods section.

Pair with identity data

HPLC tells you how pure the dominant peak is, not what molecule it is. Always read chromatograms alongside mass spectrometry identity confirmation. For a full COA walkthrough, start with COA literacy.