Certificates of analysis: what purity testing actually shows

The two tests that matter

Mass spectrometry answers “is this the right molecule?”. It measures molecular mass, which for a peptide of known sequence is a fixed, predictable number. A measured mass matching the theoretical mass to within a fraction of a unit is strong evidence of identity; a mass that is off by tens of units means something else is in the vial.

HPLC answers “how much of it is the right molecule?”. The sample is pushed through a column that separates compounds by how strongly they stick to it, and the output is a chart of peaks. Purity is reported as the area of the main peak as a percentage of all the peak area — so 98% purity means 98% of what the detector saw was the target, and 2% was something else, usually closely related fragments left over from synthesis.

What a purity figure does not include

HPLC purity is a percentage of what the detector could see, and the detector cannot see everything. Water, residual salts, counter-ions such as acetate or trifluoroacetate, and endotoxins do not show up as peaks. That is why a vial can be 99% pure by HPLC and still contain noticeably less peptide by weight than the label says — the missing mass is water and salt, not impurity. Better certificates report water content separately, by Karl Fischer titration or loss on drying.

Reading the document itself

1. 1Check it names a batch or lot number, and that the number matches the vial in your hand — a COA for a different lot is not evidence about yours.
2. 2Check the date. Testing happens once, at manufacture; an old COA describes the powder as it was then.
3. 3Look for the chromatogram and the mass spectrum as images, not just a typed summary table. A table with no underlying trace is a claim, not a result.
4. 4Check who ran it. Third-party testing by a named lab is a different kind of evidence from a seller's own in-house sheet.
5. 5Check the sequence printed on the sheet against the compound you ordered, one letter at a time.

Third-party testing, and why it exists

Because a COA can be produced by whoever is selling the vial, an independent test is the usual answer to the obvious conflict of interest. Several labs will test a vial sent in by a buyer and publish the result, and communities pool those results per batch. It costs money and consumes a vial, which is why it tends to be done per batch rather than per order.