High-performance liquid chromatography

High-performance liquid chromatography (HPLC) separates the components of a sample on a packed column and is the standard method for measuring a peptide's chromatographic purity.

In reversed-phase HPLC, the usual method for peptides, the sample is pumped through a column under high pressure while the solvent mixture changes gradually. Different molecules leave the column at different times, and a UV detector, typically reading near 214–220 nm where the peptide bond absorbs, records each one as a peak. Purity is the main peak's area as a percentage of all the peak area detected.

That makes HPLC purity a precise but narrow figure. It counts only what elutes and absorbs at the detection wavelength, so water, salts and counter-ions do not appear at all. Closely related impurities, such as a sequence missing one amino acid or with one residue flipped to its mirror form, can elute inside the main peak and be counted as the compound.

Above all, HPLC purity does not confirm identity. A chromatogram shows that one substance dominates the sample, not that it is the substance on the label. Identity needs a separate test, usually mass spectrometry or a comparison against a reference standard.

Related terms

• Mass spectrometry
• Reference standard
• Net peptide content

UPDATED OCTOBER 26, 2026 · REVIEWED BY DEL POTTER, PHD