REVERSED-PHASE HPLC ANALYSIS OF PEPTIDES IN STANDARD AND DAIRY SAMPLES USING ONLINE ABSORBENCY AND POSTCOLUMN OPA-FLUORESCENCE DETECTION

Absorbance and post-column o-phthalaldehyde (OPA)-fluorescent detection were used to analyse standard and dairy peptides following reversed-phase HPLC. Using both detection systems on-line provides additional information on the presence of peptides in dairy products. The detection response depends on the amino acid composition of the peptide involved. Among the peptides used, glutathione, lysine-containing peptides and peptides with glycine as the N-terminal residue give the highest fluorescence after the OPA post-column reaction. Absorbance is more sensitive than fluorescence for peptides with aromatic amino acids. Different parameters, such as the flow rate of OPA, the amount of mercaptoethanol in the OPA reagent and the temperature of reaction, substantially influence the fluorescent response of peptides. The interest of using on-line absorbance and fluorescence detection is highlighted by analysing peptides from skim milk and from a tryptic hydrolysate.