HPLC and mass spectrometry, explained
What the purity percentage really measures, what mass spectrometry adds, and the limits of both.
HPLC: separating and analyzing
High-performance liquid chromatography (HPLC) separates the components of a sample. A detector, usually UV around 214–220 nm for peptides, draws a chromatogram: each peak is a component. Purity is the area of the main peak divided by the total area of all peaks.
Mass spectrometry: confirming identity
Mass spectrometry (MS) measures the mass of the molecules. If the measured mass matches the theoretical mass of the expected sequence, the identity of the compound is confirmed. HPLC alone cannot tell which molecule the main peak is.
What purity does not tell you
A 99 % HPLC purity does not mean that the powder is 99 % peptide. Lyophilised peptides also contain water and counter-ions (such as acetate or trifluoroacetate). The amount of actual peptide is called the net peptide content and is measured separately.