The dissolved sample is pushed at high pressure through a column packed with very fine particles. Each component interacts with that packing with a different strength and leaves the column at a different moment, its retention time. A detector (for peptides, almost always ultraviolet at about 214-220 nm, where the peptide bond absorbs) records each component as a peak.
The result is the chromatogram. Purity is calculated by dividing the area of the main peak by the total area of all peaks. “≥99% by HPLC” means that, under those conditions, 99% of what the detector sees is the main peptide.
For peptides, reversed-phase HPLC is used most (a C18 column and a gradient of water and acetonitrile with an acidic modifier). Method quality matters: an impurity that elutes at the same time as the main peak is invisible, which is why methods are optimised and complemented with mass spectrometry.
HPLC purity says how much of the material is the right peptide compared with other similar peptides, but it does not say how much peptide is in the vial (that is peptide content) nor does it confirm identity on its own (mass does that).
Related terms
Learn it in depth at the Peptide University
- HPLC: the measure of purity · Module 7
- Related impurities and counterions · Module 7
- Peptide content, water, counterion and reading the COA · Module 7
- Mass spectrometry and identity · Module 7
- Classification and solid-phase synthesis · Module 1
Definition for educational and scientific purposes. It is not medical advice or a recommendation for use. NeoPeptidos products are sold labelled for research use only (RUO).