Verification Guide
One number describes what the detector saw, the other describes what the powder weighs, and only one of them belongs in a concentration calculation
A certificate reading 99.3% purity invites an easy misreading: that 99.3% of the powder in the vial is peptide. It is not, and the measurement never claimed to be. Purity and peptide content are separate determinations that answer separate questions, and the gap between them carries straight into every concentration a lab works out from the vial.
This piece covers what each number measures, why the second one always sits below 100%, and which one to use at the bench. Every compound mentioned is supplied for laboratory research use only. Nothing below is dosing, medical or legal guidance.
Reversed-phase high-performance liquid chromatography (RP-HPLC) separates the components of a sample by how strongly each one interacts with a hydrophobic column. Peptides are usually detected by UV absorbance at about 210 to 220 nm, the region where the peptide bond absorbs strongly.[2]
The output is a chromatogram: a main peak for the target peptide, and smaller peaks for related impurities such as truncated sequences, deletion sequences, or oxidized and deamidated forms. Purity is reported as main peak area ÷ total peak area × 100, so "99.1% purity" means that 99.1% of the UV-detected, chromatographically separated material is the target peptide.
What HPLC-UV does not see well matters just as much: water, counterions without strong UV absorbance, and inorganic salts. Each of those adds mass to the vial without adding a peak to the chromatogram.
Net peptide content is the fraction of the total powder weight that is peptide. It is measured by amino acid analysis, nitrogen determination or quantitative HPLC against a characterized reference standard. A multi-laboratory USP study compared HPLC assay, amino acid analysis and quantitative NMR for exactly this job; the HPLC assay showed the lowest variability between laboratories.[3]
For synthetic peptides the figure commonly falls well below 100%, and the gap widens for sequences with many basic residues. Three things account for it:
The figures here are illustrative, not taken from a specific lot. A vial is labeled 10 mg, and its certificate reports an HPLC purity of 99.3% and a measured peptide content of 9.6 mg.
The figure to use for concentration is 9.6 mg. Reconstituted in 2.0 mL: 9.6 mg ÷ 2.0 mL = 4.8 mg/mL.
Had you assumed 10 mg, you would have calculated 5.0 mg/mL and overstated every downstream concentration by about 4%. Multiplying the label by purity does not fix it either: 10 mg × 0.993 = 9.93 mg is a statement about composition, not about how much peptide the vial holds.
The peptide calculator takes the measured figure in place of the label mass, so the concentrations it returns reflect what is actually in the vial. The reconstitution math guide carries the same correction through molarity, dilution and aliquots.
Both, for different reasons, and the two failure modes look nothing alike. A high-purity vial that is 20% underfilled gives clean but wrong concentrations. A full vial at 90% purity gives the correct mass but introduces 10% unknown material into the experiment.
Matched to the question being asked:
Every Eppix lot is tested by Janoshik Analytical before sale, and its certificate is published. For a single-compound lot the certificate reports HPLC purity and measured content side by side; a blend has no single purity figure, so its certificate reports measured mass for each component instead.
Most lots measure above 99% purity. An occasional lot comes back around 98%, and it is published as the laboratory reported it rather than left out. A lot can read 98% pure and still hold its full labeled amount, which is the whole argument of this article in one line. Every result is on the certificates of analysis page, the guide to reading a Janoshik COA explains each field, and the supplier-side version of this distinction is in purity vs fill accuracy.
Is TFA salt a problem?
For some cell-based assays residual TFA can affect results, and some labs prefer acetate salts. Check the salt form your protocol calls for.
Why does PubChem list a different molecular weight than my COA?
PubChem typically lists the free peptide. Salt forms add counterion mass, so the molecular weight per salt-form unit is higher.
Can purity be over 100%?
No. Content can exceed the label claim if a vial is overfilled, but purity is a fraction of total peak area and cannot pass 100%.
This article summarizes published preclinical research literature. Compounds referenced are supplied by Eppix Labs strictly as research materials for laboratory investigation within the United States. They are not approved by the FDA for human or veterinary use, and nothing on this page should be interpreted as medical advice or guidance on human or animal administration.
