What a COA Should Contain
A Certificate of Analysis links a specific production lot to the analytical evidence for its identity and quality. At a minimum it should state:
- the product name and batch or lot number, matching the vial label;
- the testing laboratory, test date and the methods used;
- each result alongside its specification (for example, purity ≥99.0%) and a pass/fail status;
- the underlying data — at least the HPLC chromatogram and the mass spectrum.
A summary table without the chromatogram or spectrum asks you to take the numbers on trust.
Reading the HPLC Chromatogram
Peptide purity is usually measured by reversed-phase HPLC: the sample is separated on a C18 column with a water/acetonitrile gradient (commonly with 0.1% trifluoroacetic acid), and a UV detector at 214–220 nm — where peptide bonds absorb — records signal against time.
- The main peak is the target peptide. It should be sharp and roughly symmetrical; shoulders, splitting or strong tailing can indicate closely eluting impurities.
- Minor peaks are synthesis by-products such as deletion or truncated sequences, incompletely deprotected species and oxidation products.
- Retention time — when the main peak elutes — depends on the column and gradient. It should be consistent between batches tested with the same method, but on its own it does not prove identity.
What "≥99% Purity" Really Means
HPLC purity is reported as area percent: the main peak's area divided by the total area of all integrated peaks. A result of 99.4% means that, under that method, 99.4% of the UV-absorbing material eluted as the main peak.
That figure has limits worth understanding. Impurities that co-elute with the main peak are counted as product; species that absorb weakly at the detection wavelength are under-represented; and non-peptide components such as water, salts and counter-ions are invisible to UV detection entirely. Purity is a measure of the peptide fraction's homogeneity, not of how much peptide is in the vial.
Mass Spectrometry: Confirming Identity
Mass spectrometry answers a different question: is this the right molecule? In electrospray ionization (ESI-MS), peptides typically appear as a series of multiply charged ions ([M+2H]²⁺, [M+3H]³⁺ …). Deconvolving these peaks gives the molecular mass, which is compared with the theoretical value calculated from the sequence.
Agreement within about one dalton of the average mass is typical for routine ESI identity testing; high-resolution instruments report errors in parts per million. A large discrepancy — or a missing spectrum — means identity has not been demonstrated.
Metal complexes add a wrinkle. Under ESI conditions GHK-Cu can partially dissociate, so a spectrum may show free GHK (about 340.4 Da) alongside the copper complex. Our GHK-Cu analytical reports list the theoretical masses for exactly this reason.
Purity vs. Net Peptide Content
Synthetic peptides are isolated as salts, typically with trifluoroacetate or acetate counter-ions, and retain some water. Net peptide content — determined by amino acid analysis, elemental nitrogen analysis or quantitative HPLC against a reference standard — expresses how much of the powder's mass is actually peptide. A lot can therefore be 99% pure by HPLC while the peptide itself makes up a smaller fraction of the weighed powder. Fill quantities should be based on net peptide content.
Many COAs also report endotoxin by the LAL test, appearance and solubility — useful additions for cell-based work.
Red Flags to Watch For
- No laboratory name, test date or method description.
- Purity quoted without a chromatogram, or a chromatogram with no axes or integration table.
- Identical chromatograms, down to the baseline noise, across different batches.
- A batch number on the COA that does not match the vial label.
- Identity claimed without a mass spectrum.
Every lot we release is published with its HPLC and mass spectrometry results — you can verify batch numbers against the label on your vial in our COA hub.
References & Further Reading
Mant CT, Hodges RS (eds), HPLC of Biological Macromolecules; Aguilar MI (ed), HPLC of Peptides and Proteins: Methods and Protocols, Humana Press 2004.