How HPLC purity analysis works
How reversed-phase HPLC separates a peptide from its impurities and reports a main-peak purity figure — the headline number on a Certificate of Analysis.
High-performance liquid chromatography (HPLC) is the headline purity method on a peptide Certificate of Analysis. It separates a synthetic peptide from the closely related impurities produced during synthesis, and reports how much of the sample is the target molecule.
Principle
In reversed-phase HPLC the sample is pushed through a non-polar stationary phase while a gradient of increasingly organic solvent flows past it. Molecules elute at different times depending on their hydrophobicity. A UV detector — typically at 220 nm, where the peptide backbone absorbs — records absorbance against time, producing a chromatogram of peaks.
Reading the chromatogram
Purity is reported as the main-peak area as a percentage of total integrated peak area. A clean reference standard shows one dominant, symmetrical peak with small, well-resolved impurity peaks. The method (column, gradient, wavelength) should always be stated alongside the figure — a purity number without a method is not interpretable. See our batch traceability overview for how this figure is tied to a specific lot.
Limitations
HPLC purity is an area-percent measure at a single wavelength; it does not by itself confirm identity (that is the role of mass spectrometry) and does not quantify salt content (see counter-ion analysis). A complete picture combines all three. Purity is also lot-specific and can drift once material is reconstituted — see peptide stability.
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