Many peptide COAs include a chromatogram: a line graph with one or more peaks. It is the raw material behind the purity percentage, and it is worth a minute of attention, because it shows how the number was reached.
The parts of the picture
- Horizontal axis — time. Retention time in minutes. Each component of the sample leaves the column at its own time.
- Vertical axis — response. For UV detection, how much light was absorbed (often in milli-absorbance units). Taller, wider peaks mean more of that component.
- The main peak. The tall peak at the retention time expected for the peptide.
- Smaller peaks. Other components detected: related peptides, by-products, or the substances the peptide is dissolved in.
- The baseline. The flat line between peaks. A noisy or drifting baseline makes small peaks harder to measure.
- The very start. Early peaks near the beginning often come from the solvent or injection rather than the sample.
How purity comes from it
The software integrates the area under each peak. Purity is usually the main peak’s area divided by the total area of the peaks counted, as a percentage. Two things make that number less absolute than it looks:
- What is counted. Some peaks, such as a preservative in the solution or the solvent front, are legitimately left out. A good report says which, and why. An unexplained exclusion is a question to ask.
- Integration settings. Where a peak starts and ends, and the threshold for what counts as a peak, change the answer slightly.
Things worth a second look
- A chromatogram with no axis labels, no wavelength or no run details.
- One that is identical, down to the noise, to another report’s — see signs a COA may be reused.
- A purity figure that doesn’t match the picture (a visible shoulder or extra peaks, yet “99.9%”).
- A cropped image showing only the main peak.
What it can’t show
A chromatogram shows detected UV-absorbing components in one run. It doesn’t show identity, how much peptide there is, or anything that didn’t absorb at that wavelength. For those you need mass spectrometry and an assay.
Frequently asked questions
- What do the axes of an HPLC chromatogram show?
- The horizontal axis is time, usually retention time in minutes: when each component came off the column. The vertical axis is the detector response, for UV detection the absorbance, so taller and larger peaks mean more of that component was detected.
- How is purity calculated from a chromatogram?
- Typically as the area of the main peak divided by the total area of all integrated peaks, expressed as a percentage. Which peaks are counted, and the integration settings, affect the result, so the report should say what was excluded.
- Is a clean-looking chromatogram proof of a good product?
- No. It shows what the detector saw in one sample at one wavelength. It says nothing about substances that don’t absorb UV, nothing about how much peptide is present, and it can’t on its own show that the main peak is the right molecule.
See it on a real document
PeptideProof reads a COA for you and shows what is only reported, what has no evidence and what looks inconsistent. See an example first, or check your own — new accounts include free analyses.
Educational information about reading laboratory documents. It is not medical, dosing or purchasing advice, and it doesn’t say that any product is safe, legal or suitable for any use. See the medical and research-use disclaimer.
