HPLC separates the components of a sample by passing it through a column in a liquid stream. Components that interact more strongly with the column material move more slowly, so substances that entered together emerge at different times.

The output is a chromatogram in which each detected component appears as a peak. Purity is calculated from peak areas: the principal peak's area divided by the total area of all integrated peaks. The result is a relative measure of what the detector registered, under the conditions of that run.

Mass spectrometry converts molecules into ions and separates them by mass-to-charge ratio. Because a peptide's mass follows from its amino acid composition, a measured mass can be compared against the value calculated from the intended sequence.

A match within the instrument's tolerance is meaningful evidence that the material is the intended compound. It does not establish the order of residues, which is why identity work sometimes extends to fragmenting an ion and measuring the pieces.