A certificate of analysis is the only objective evidence you have about what is in a vial. It is also the document most researchers skim for a single number before filing it. That number — the purity percentage — is the least informative figure on a well-constructed certificate, and the easiest to produce without doing much work.
This is what each section actually establishes, in the order it usually appears, and what its absence should tell you.
Identity: does the molecule match the label?
Identity is established by mass spectrometry. The instrument measures the molecular weight of what is in the vial, and that measurement is compared against the theoretical weight calculated from the sequence.
A certificate should report both numbers and let you check the arithmetic yourself. Agreement within about 1 Da is normal. A larger discrepancy is not a rounding issue — it means the molecule is not the one on the label, or it has been modified, most commonly by oxidation of a methionine or cysteine residue.
Identity is the section people skip and the one that matters most. Purity without identity tells you the vial contains mostly one thing. It does not tell you that thing is what you ordered. A 99.5% pure preparation of the wrong compound is a perfectly achievable result.
Purity: what fraction is the target compound?
Purity comes from reverse-phase HPLC, and it is reported as area percent — the area under the main peak divided by the total area of all peaks, usually with UV detection at 214 nm.
That definition carries three limitations worth internalising:
- It only counts what the detector sees. Detection at 214 nm responds to the peptide bond, so it sees peptide-like impurities well. Non-peptide contaminants — residual salts, solvent, scavengers — may be invisible, and therefore excluded from the denominator entirely.
- It only counts what elutes. Anything that stays on the column or comes off in the void volume is not in the calculation.
- Area percent is not mass percent. Different species have different molar absorptivity. Two peaks of equal area do not represent equal masses.
A purity figure is meaningful, but it is a statement about the chromatogram, not a complete inventory of the vial.
What to look for in the chromatogram itself
Ask for the trace, not just the number. A certificate that reports 99.2% without showing you the chromatogram is asking you to trust an unverifiable claim.
On the trace, check three things. The main peak should be symmetrical — significant tailing suggests either column problems or a co-eluting species. The baseline should be flat and return cleanly between peaks. And the gradient should be long enough that impurities are actually resolved: a steep, fast gradient compresses everything into one peak and will report a flattering number for a mediocre preparation.
Net peptide content: how much peptide is in the milligrams?
This is the section most commonly missing, and its absence is usually deliberate.
A lyophilized peptide is not pure peptide by mass. It contains counter-ions from purification — typically trifluoroacetate — plus residual water. Together these routinely account for 10–20% of the powder, and for highly charged sequences they can exceed 30%.
Net peptide content, measured by nitrogen analysis or amino-acid analysis, tells you what fraction of the powder is actually peptide. It is what you should use when calculating concentrations.
Consider a vial labelled 10 mg with 80% net peptide content. It contains 8 mg of peptide, not 10. Any calculation that starts from the label figure rather than the certificate figure carries that 20% error forward silently.
A supplier who quotes gross weight without disclosing net content is selling you salt at peptide prices, and any concentration you derive from that label will be systematically wrong.
Water content
Determined by Karl Fischer titration. It matters for two reasons: it is part of the mass you are not getting as peptide, and high residual moisture shortens shelf life considerably. Below about 8% is unremarkable. Substantially above that suggests incomplete lyophilization.
The contaminant panel
Purity describes the ratio of target to non-target peptide species. It says nothing about the four contaminant classes that will ruin an experiment independently:
| Contaminant | Method | Why it matters |
|---|---|---|
| Endotoxin | LAL, kinetic chromogenic | Bacterial pyrogens survive sterile filtration and confound any inflammatory or immune readout |
| Residual TFA | Headspace GC | Cytotoxic in culture at concentrations far below what a purity assay would register |
| Heavy metals | ICP-MS | Catalyst and reagent carryover from synthesis |
| Bioburden | Plate count | Microbial contamination in a preparation stored above freezing |
Residual trifluoroacetic acid deserves particular attention because it is the one that quietly invalidates cell work. TFA is the standard ion-pairing agent in peptide purification, and it persists as a counter-ion. At concentrations that would barely register as an impurity by area percent, it is measurably cytotoxic to sensitive lines. A preparation can be 99% pure and still unusable in culture for exactly this reason.
Chain of custody
The last section, and the one that determines whether any of the preceding sections mean anything.
A certificate should identify the specific lot, when it was synthesised, when it was assayed, who performed the analysis, and who authorised release. Without a lot number tying the document to physical material, the certificate describes an abstraction.
This is the mechanism behind the most common documentation failure in this market: the representative certificate. A supplier tests one good lot, publishes that certificate, and continues serving it for every subsequent production run. The document is genuine. It simply has nothing to do with the vial you received.
You can detect this. Ask for the certificate matching your lot number. If what arrives has a different lot number, or no lot number, or the same certificate every customer receives, you have learned something important.
A practical checklist
Before accepting a certificate as evidence:
- Does it name a lot number that matches your vial?
- Does it report measured mass against theoretical mass?
- Is the HPLC chromatogram shown, not just the percentage?
- Is net peptide content reported, and is it salt-corrected?
- Is there a contaminant panel, or only a purity figure?
- Is there an independent laboratory result alongside the in-house one?
- Is it signed and dated by a named analyst?
A certificate missing items 1, 3, or 4 is not necessarily fraudulent. But it is not doing the job a certificate exists to do, and you should price the uncertainty accordingly.
Why we publish ours before purchase
Every certificate we hold is searchable in the COA library without an account, an email address, or a purchase. That is not generosity — it is the only arrangement that makes the documentation worth anything.
A certificate you can only see after paying cannot inform the decision to pay. If a supplier will show you their analytical data only once your money is committed, the document is functioning as reassurance rather than evidence, and that tells you what the document is for.
For laboratory research use only
This article is technical background for laboratory professionals. Nothing in it is medical advice, and nothing sold on this site may be administered to humans or animals.