What HPLC purity does and does not tell you
Area percent is the most quoted and least understood number on a peptide certificate. A short methods note on detection thresholds, co-elution, and why two labs can honestly report different purity for the same vial.
Two accredited laboratories can analyze the same vial and report 98.6% and 99.3% purity without either being wrong. The difference is method, not material, and understanding why is the fastest way to read peptide certificates critically.
Area percent is a ratio of what the detector sees
HPLC-UV purity is computed from integrated peak areas at a fixed wavelength. Anything that does not absorb at that wavelength — salts, water, some counterions, some deletion products — contributes nothing to the denominator. The reported figure is therefore a purity-with-respect-to-detectable-related-substances, and the honest framing on a certificate says so.
Gradient and column choice move the number
A shallow gradient over a long column resolves closely-related impurities that a fast screening method merges into the main peak. More resolution usually means more detected impurities, which means a lower area percent for identical material. A higher number from a coarser method is not a better peptide.
What to compare instead
- Method parameters: column, gradient, wavelength, injection mass.
- The chromatogram itself, including baseline noise and the smallest labeled peak.
- Whether the certificate reports the detection limit for related substances.
- Identity confirmation (MS) on the same batch, not a different lot.