Definition
Retention time is the time between sample introduction and the apex of a compound's peak in a chromatogram. In reversed-phase HPLC, more hydrophobic peptides interact more strongly with the C18 stationary phase and elute later as the acetonitrile gradient rises. Under identical conditions, including column, gradient, flow rate, temperature and mobile-phase additives, a given peptide elutes at a reproducible time, so comparison with a reference standard supports identity. Retention time alone is not proof of identity, because different molecules can co-elute. Small modifications shift it: oxidised methionine usually elutes earlier, and lipidated analogues much later. Values from different laboratories are not directly comparable unless their methods match.