How to Read a Peptide Certificate of Analysis

A certificate of analysis is a summary of what an analytical lab observed when it tested a specific batch. Read properly, it tells you whether the vial contains the sequence you ordered, how much of it, in what salt form, and whether anyone actually looked. Read carelessly, it tells you nothing — because a COA that omits the tests you care about still looks like a COA.

Here's how to read one. For background on how the material itself is made, start with our overview of research peptides.

1. Confirm the lot number matches your vial

Start here, because everything downstream is meaningless if it fails.

A COA describes one batch. If the lot on the document doesn't match the lot printed on the vial you received, you have a document about someone else's material. Some suppliers publish a single "representative" COA for a product and reuse it across every batch for years. That is not lot-specific testing, and it defeats the purpose of the document.

Ask for the COA tied to your lot. A supplier who can't produce one is telling you something.

2. Check the analysis date against the manufacture date

These should be close together. A large gap suggests the material sat before testing, or that testing was performed on an earlier batch and applied retroactively.

Also check that the analysis date precedes your ship date. It sounds obvious. It isn't always true.

3. Read the HPLC section properly

The chromatography section is usually the largest block on the page. Look for:

Method — Reversed-phase HPLC, typically C18. The mobile phase should be described (water/acetonitrile with 0.1% TFA is standard).

Detection wavelength — 214 nm detects the peptide bond itself and sees essentially every peptide species present. 280 nm detects aromatic residues (Trp, Tyr, Phe) and will miss sequences that lack them. Purity reported at 280 nm on a peptide with few aromatics is systematically flattering. If the wavelength isn't stated, the number isn't interpretable.

Retention time — Should be reported and should be consistent with prior lots of the same product. A shifted retention time on the same method is worth a question.

Purity (% area) — The proportion of total UV peak area under the main peak. For most research applications, >95% is a working baseline and >98% is common for well-characterized sequences.

The chromatogram itself — This is the part most buyers never request, and it's the most revealing. A clean trace shows one dominant, symmetrical peak. What you're looking for is shoulders on the main peak, which often indicate closely eluting deletion sequences that integration software may have folded into the main peak area. A stated purity of 99% with a visibly shouldered peak is a different product than 99% with a sharp one.

4. Verify identity by mass spectrometry

Purity tells you the sample is homogeneous. It does not tell you the sample is the right molecule. Only mass spec does that.

• Observed mass vs. theoretical mass, agreeing within the tolerance of the instrument (ESI-MS typically ±1 Da for peptides in this size range; higher-resolution methods tighter)
• The actual spectrum, ideally showing the expected charge-state series

A mismatch of ~1 Da can indicate deamidation. A mismatch matching the residue mass of a single amino acid indicates a deletion sequence that co-eluted. Neither is visible on an HPLC trace alone.

A COA without mass spectrometry is not an identity-verified COA. This is the single most common omission in the category.

5. Find the peptide content — and if you can't, ask

This is where most COAs go quiet, and it's the number that most often explains inconsistent results.

HPLC purity is a percentage of the peptide material present. Peptide content is the percentage of the vial's total mass that is peptide at all. The remainder is counterion salt, residual water, and bound solvent — which for a lyophilized TFA-salt peptide commonly runs 15–30% of the mass, and sometimes more.

So a vial can honestly be 99% pure by HPLC, and 70% peptide by mass. If you weigh 10 mg and assume you have 10 mg of peptide, your actual quantity is off by 30%. Every concentration downstream inherits that error. Two lots with different peptide content will produce different results at nominally identical concentrations, and the COA will look fine both times.

Peptide content is determined by amino acid analysis, nitrogen determination, or quantitative NMR. If it isn't on the COA, ask whether it was measured. "Net peptide content not determined" is at least an honest answer.

6. Identify the counterion

Peptides purified with TFA come off the column as TFA salts. Residual TFA is cytotoxic in some cell-based assays and can interfere with certain measurements. If your work is sensitive to it, you need acetate salt exchange, and you need the COA to state which form you received.

If the counterion isn't listed, assume TFA.

7. Check the physical and handling fields

• Appearance — usually "white to off-white lyophilized powder." Discoloration is worth querying.
• Solubility — the recommended solvent, which matters for reconstitution
• Storage conditions — typically -20°C, desiccated, protected from light
• Water content — by Karl Fischer titration, if determined

8. Note who performed the testing

In-house QC is normal and legitimate. Independent third-party testing is stronger, because the party generating the number has no commercial interest in it. If a COA carries a third-party lab's name, letterhead, and analyst signature, that's a meaningfully different document from an unsigned in-house summary.

The quick checklist

Four or more gaps in the "critical/high" rows is a reason to source elsewhere.

All material is supplied for laboratory research use only and is not for human or veterinary use. The framework behind that statement is covered in our research use only compliance guide.