TestingGuide
How to Verify a Peptide
Reading an HPLC trace and a mass spectrum.
On this page6 sections
Quick answer
Mass spectrometry tells you what it is. HPLC tells you how much of it is that thing. The lot number tells you whether either document has anything to do with the vial on your bench. All three, or you are taking it on trust.
You read the certificate of analysis — and you check that it belongs to the vial in your hand. This page explains what each section of a COA is actually measuring, and what it is not.
Everything below is about material characterisation. All products on this site are supplied for research use only.
A COA answers two separate questions
People talk about a certificate of analysis as if it reports one number. It reports two different things, measured by two different instruments, and confusing them is the single most common mistake when reading one.
Identity — is this the right molecule? Answered by mass spectrometry. The instrument measures the molecular weight of what is in the sample and compares it to the theoretical weight calculated from the sequence (which is not simply the sum of the amino acid weights). If the sequence is right, the observed mass matches. If the supplier sent you a different compound, the mass will not match, no matter how pure it is.
Purity — how much of the material is that molecule? Answered by high-performance liquid chromatography (HPLC). The instrument separates everything in the sample and reports each component as a share of the total. This is where the percentage comes from.
A sample can be 99% pure and still be the wrong compound. It can also be the right compound at 70% purity. You need both numbers, and they come from different pages of the document.
Reading the HPLC trace
The chromatogram is a line with peaks on it. Time runs along the bottom; detector response runs up the side. Each peak is something that came off the column at a particular moment.
The large peak is your peptide. The small peaks are everything else — most often closely related by-products of the synthesis itself. The purity figure is that main peak’s area divided by the area of all the peaks together.
What to look at, in order:
| Check | What good looks like | What should give you pause |
|---|---|---|
| Main peak shape | Narrow, symmetrical, returns to baseline | Broad, tailing, or merged with a neighbour |
| Baseline | Flat and quiet between peaks | Drifting or noisy, which hides small impurities |
| Integration | Peaks marked and totalled on the trace | A percentage with no visible trace behind it |
| Wavelength | Stated, commonly 214 nm for the peptide bond | Not stated at all |
| Gradient and column | Stated, so the run could be repeated | Omitted |
A purity percentage printed on its own, with no chromatogram behind it, is a claim rather than a measurement. The trace is the evidence; the number is the summary.
Reading the mass spectrum
The mass spectrometry page reports an observed mass against a theoretical mass. For a peptide these should agree closely — to within a fraction of a mass unit on a high-resolution instrument.
Two details catch people out. First, peptides often appear as multiply charged ions, so the raw spectrum may show a series of peaks rather than one, and the stated mass is calculated from them. Second, the theoretical mass is for the free peptide, while the material in the vial is usually a salt. That difference is accounted for separately, and it is the subject of net peptide content, not of identity.
Make sure the document matches the vial
This is the part that gets skipped. A certificate is specific to a batch, not to a product. A supplier can publish a genuine COA for a batch they no longer ship.
Check that:
The batch or lot number on the document matches the one printed on your vial. If there is no lot number on the vial, the document cannot be tied to it at all.
The compound name and sequence on the document match what you ordered. Closely related compounds have confusingly similar names.
The date is present, so you can tell how old the analysis is relative to the material.
The testing laboratory is named, so the analysis is attributable to someone.
What a COA cannot tell you
A certificate characterises a sample of material. It is silent on everything else, and it is worth being explicit about that.
It does not tell you how the material was handled after the sample was taken, whether the cold chain held in transit, or whether the vial was stored correctly once it arrived. It does not tell you anything about biological activity — purity and identity are chemistry, not function. And it does not transfer to a different batch, however similar the two may look.
Those gaps are why handling matters as much as documentation. If you want the storage and transit side, see storage and shipping in cold weather.
In short
Mass spectrometry tells you what it is. HPLC tells you how much of it is that thing. The lot number tells you whether either document has anything to do with the vial on your bench. All three, or you are taking it on trust.