A certificate of analysis is the only document that allows objective checking of what a supplier claims about a batch. This article describes which fields it must contain, how a chromatogram is interpreted, and which elements indicate that a certificate is not verifiable.
What is a peptide certificate of analysis?
A certificate of analysis, abbreviated COA, is the report an analytical laboratory issues on a specific batch of material. It declares measured purity, molecular identity, batch number, analysis date and the responsible laboratory.
The relevant distinction is between a figure and a claim. A supplier publishing a COA issued by an identifiable laboratory provides checkable information. A supplier declaring "purity above 99%" without naming a laboratory or batch provides a claim nobody can verify.
Which fields must a verifiable COA contain?
Six fields are indispensable. The absence of any one makes the document non-verifiable, regardless of the purity figure it declares.
| Field | Expected content | Red flag |
|---|---|---|
| Issuing laboratory | Full name, country and contact details of an independent laboratory. | Generic logo or the phrase "certified laboratory" with no name. |
| Batch number | Code identical to the one printed on the vial label. | Batch absent or different from the physical product. |
| Analysis date | Later than and consistent with the batch production date. | No date, or a date preceding production. |
| Analytical method | Technique explicitly declared for each measured parameter. | The word "analysed" without specifying technique or conditions. |
| Raw data | Chromatogram and spectrum reproduced in the document. | Only a final percentage, with no supporting plots. |
| Molecular identity | Measured mass and calculated theoretical mass, which must match. | Absence of mass spectrometry. |
The six fields are cumulative: a certificate with five of them still does not allow complete verification.
How is the HPLC chromatogram interpreted?
The chromatogram plots detector signal against time. Each compound in the sample crosses the column at a different speed and produces a peak at a characteristic retention time. Purity is calculated by dividing the main peak area by the total area of all peaks.
A chromatogram with one dominant peak and a clean baseline corresponds to homogeneous material. Appreciable secondary peaks indicate impurities, which in peptide synthesis are typically truncated sequences, deletion products or incompletely deprotected material.
Three specific elements deserve attention. First, the relative height of secondary peaks against the main one. Second, the shape of the main peak, since a peak with shoulders can conceal coelution of two species. Third, the vertical axis scale, because a compressed scale can make non-trivial impurities appear negligible.
What does mass spectrometry add?
Mass spectrometry measures the mass-to-charge ratio of ionised molecules in the sample. The result is compared with the theoretical molecular mass, calculated by summing the masses of the declared sequence's amino acids minus the water released at each peptide bond.
If measured mass matches theoretical mass within instrument error, identity is confirmed. If it does not match, the material is not the declared peptide, regardless of how pure the sample is.
This is why a COA with HPLC but no mass spectrometry is insufficient. It establishes that 99% of the content is a single compound, but not which one.
Why is the batch number the most important field?
Because it is the only element linking the document to the physical object. A certificate describes a specific batch analysed on a specific date. If the certificate code does not match the one printed on the vial, the document describes material other than the one at hand.
This point also delimits the scope of a manufacturer COA. It certifies what was produced at origin. It does not by itself demonstrate that the vial received after packing, export and transit belongs to that same batch. Closing that distance requires an additional analysis of the material that actually arrives, covered in the article on double purity verification.
What purity threshold is reasonable to require?
In synthetic peptides for research use, the usual standard sits above 98% by HPLC, and many suppliers declare values above 99%. The figure alone carries less informational value than it appears to.
A verifiable 99%, with published chromatogram and spectrum and a traceable batch, provides more information than a 99.9% declared without documentary support. The relevant question is not what the figure is, but what supports it.
References
- Mant CT, Chen Y, Yan Z, et al. HPLC analysis and purification of peptides. Methods Mol Biol. 2007;386:3-55. PMID: 18604941.
- Domon B, Aebersold R. Mass spectrometry and protein analysis. Science. 2006;312(5771):212-217. DOI: 10.1126/science.1124619.
- Aebersold R, Mann M. Mass-spectrometric exploration of proteome structure and function. Nature. 2016;537(7620):347-355. DOI: 10.1038/nature19949.
