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Analytical Methods

HPLC Testing Methods for Peptide Purity

Overview of reverse-phase HPLC methodology for determining peptide reference material purity.

Reverse-phase high-performance liquid chromatography (RP-HPLC) is the standard analytical method for evaluating peptide reference material purity. C18 stationary phases with acetonitrile/water + 0.1% TFA gradients are typical. Detection at 214 nm captures the peptide backbone amide absorption. Quantitative purity is reported as the percent area of the principal peak at the specified wavelength. Mass spectrometric coupling (LC-MS) provides confirmatory mass identity. This article does not constitute guidance for any non-laboratory use.