Peptide COA: HPLC, mass spec, water, endotoxin
A Certificate of Analysis is the document that separates a verifiable research peptide from a label claim. Done properly, a COA contains four independent tests, each addressing a different failure mode in peptide synthesis. Done improperly, a COA is a marketing artefact. Here is how to tell the two apart in under two minutes.
The Four Tests You Should See
1. HPLC Chromatogram
High-performance liquid chromatography separates the peptide from impurities by polarity. The chromatogram is a plot of detector signal against elution time. You are looking for one tall, narrow peak that integrates to ≥99% of the total peak area. The HPLC method should specify the column, mobile phase gradient, flow rate, and detection wavelength (typically 214 nm or 220 nm). A bare "purity 99%" statement without the chromatogram itself is a red flag.
2. Mass Spectrometry
HPLC tells you the sample is pure; mass spec tells you the pure thing is what you ordered. The mass spec spectrum should show a peak at the theoretical molecular weight of the target peptide, plus the expected adduct ions (typically [M+H]+, [M+2H]2+, [M+Na]+). The reported mass should match the theoretical mass to within 1 Da for most peptides, tighter for ESI-TOF instruments.
3. Water Content (Karl Fischer or TGA)
Lyophilised peptides retain residual water that affects mass-based dosing calculations and stability. A typical specification is ≤8% water by mass. If a peptide is dispensed by mass and water content is unreported, downstream concentration calculations carry a hidden error.
4. Endotoxin (LAL Assay)
For peptides destined for cell-based or in-vivo research work, endotoxin contamination from manufacturing creates artefacts that can be misattributed to the peptide itself. The LAL (Limulus amebocyte lysate) assay quantifies endotoxin in EU/mg. Research-grade peptides should test ≤1 EU/mg.
Reading the HPLC Chromatogram
| What you see | What it means |
|---|---|
| One dominant peak, baseline elsewhere | Clean synthesis, well-optimised purification |
| Dominant peak plus small early-eluting peaks | Truncated or deletion sequences; acceptable if main peak ≥99% |
| Dominant peak plus late-eluting peaks | Hydrophobic impurities; check if main peak still ≥99% |
| Two peaks of similar size | Likely a stereoisomer or oxidation product; suspect |
| No chromatogram, only a number | Discard; not a real COA |
Common Red Flags
- COA dated months or years before the labelled batch date
- COA copy-pasted between batches with only the batch ID changed
- HPLC method information missing (no column specification, no gradient)
- Mass spectrum showing a peak at the wrong mass with vague hand-waving
- No water content reported for lyophilised material
- Endotoxin reported as "passes" without a numerical EU/mg value
- Boilerplate "purity ≥X%" statement with no underlying instrument data
What to Demand from a Supplier
For a research-grade supplier, a real COA package per batch includes the chromatogram image, the mass spectrum image, the Karl Fischer or TGA water-content report, and the LAL endotoxin number. Bonus marks for: third-party retest reports, batch traceability to the synthesis run, and a documented chain-of-custody from synthesis to vial.
We do not currently issue a third-party certificate of analysis, and would rather say so than imply one.
Why This Matters
Research on a 95% pure peptide is research on the peptide plus a 5% cocktail of side products. For most experimental designs the side products are uncontrolled variables that can produce confounding results. The cost of demanding a real COA is a few extra euros per vial; the cost of skipping it is research conclusions you cannot reproduce.
For in-vitro research and laboratory use only. Not for human or animal consumption.
