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 detectio n were used to analyse standard and dairy peptides following reversed- phase HPLC. Using both detection systems on-line provides additional i nformation on the presence of peptides in dairy products. The detectio n response depends on the amino acid composition of the peptide involv ed. Among the peptides used, glutathione, lysine-containing peptides a nd peptides with glycine as the N-terminal residue give the highest fl uorescence after the OPA post-column reaction. Absorbance is more sens itive than fluorescence for peptides with aromatic amino acids. Differ ent parameters, such as the flow rate of OPA, the amount of mercaptoet hanol in the OPA reagent and the temperature of reaction, substantiall y influence the fluorescent response of peptides. The interest of usin g on-line absorbance and fluorescence detection is highlighted by anal ysing peptides from skim milk and from a tryptic hydrolysate.