Reading a Certificate of Analysis
What HPLC purity actually measures, why identity confirmation is a separate question, and how to spot a certificate that proves nothing.
Purity and identity are two different claims
All material below describes laboratory handling and analytical practice for reference compounds supplied strictly for in-vitro research. Nothing here is guidance for human or animal use.
A certificate answers two independent questions, and conflating them is the most common error in the field. HPLC purity asks: of the material detected, what fraction is the dominant species? Mass spectrometry asks: is that dominant species the molecule it is supposed to be? A sample can be 99% pure and 100% the wrong compound. Both tests are required; neither substitutes for the other.
Reading the chromatogram
In reverse-phase HPLC the sample is driven through a hydrophobic column and its components separate by retention time. The detector plots absorbance — usually at 214 nm, where the peptide bond absorbs — against time. The target elutes as a dominant peak, and purity is that peak's integrated area divided by total integrated area.
Look past the headline percentage. A clean trace shows a symmetrical main peak and a flat baseline. Peak tailing suggests column interaction or a degraded sample. A cluster of small peaks immediately adjacent to the main one often indicates closely related synthesis by-products — deletion sequences — which a summary number alone will not reveal.
Identity by mass
Mass spectrometry compares the measured mass-to-charge ratio against the theoretical mass computed from the intended sequence. A match within instrument tolerance confirms identity. A consistent offset is diagnostic rather than random: a shortfall matching the mass of a single amino acid points to a deletion during synthesis; an excess of 16 Da suggests oxidation.
Quantitative content
Purity is a ratio, not an amount. Quantitative assay answers how many milligrams of target the vial actually contains — which determines every concentration you calculate downstream. A vial can pass purity and still be under-filled. Where content testing is reported, it is the number that governs your arithmetic.
Red flags
A certificate is a claim about a specific batch of material on a specific date. Anything less is marketing.
- No lot or batch number tying the document to the vial in your hand.
- No issuing laboratory, no analyst, no test date.
- A summary table with no chromatogram or spectrum attached.
- An identical document reused across visibly different production runs.
- Purity quoted without stating the detection wavelength or method.
Independent verification
Every batch we supply carries a third-party report from an accredited analytical laboratory, published against the product and searchable by batch on our verification page. Independence matters: a supplier grading its own homework is not a control.