Simultaneous Quantification of Free Amino Acids and Peptide Fractions from Keratin Hydrolysates by SE-HPLC/UV

An original SE-HPLC/UV methodology was developed to quantify the main fractions of keratin protein hydrolysates – peptides and free amino acids (FAA) – from absorbance signals. The method processes SEC chromatograms assuming a boundary retention time between the two fractions before signals conversion to concentrations using key parameters: mean molecular weight (MW) by amino acid (\(\:{{-}{MW}}ₐₐ\)) and mean molar extinction coefficient by amino acid (\(\:{{-}{ε\:}}ₐₐ\)). 28 keratin protein hydrolysates produced by the acid hydrolysis of poultry feathers under various conditions of temperature and HCl/feather ratio, and sampled at multiple times were analyzed by SE-HPLC to identify the boundary retention times from the chromatograms. The hydrolysates were further assayed by a classic amino acid analyzer to obtain their free and total amino acids aminograms, capturing the concentrations of the Peptide and FAA fractions. Based on this, key parameters \(\:{{-}{MW}}ₐₐ\) and \(\:{{-}{ε\:}}ₐₐ\) were deduced from hydrolysate aminograms, and by nonlinear regression considering the mathematical expression of \(\:{{-}{ε\:}}ₐₐ\) as a function of concentration, respectively. Across 14 hydrolysates used for parameters estimation, key parameters calculated for each fraction remained largely constant with RSDs below 10%, leading to averaged values being used for the final implementation of the SE-HPLC method in MATLAB. Method validation against the reference method showed high accuracy (errors inferior to 15%) for 81 out of 84 quantifications of Peptide, FAA and soluble total amino acids. The validated method was ultimately applied for monitoring the peptide MW distribution and concentration kinetics of peptide classes of custom MW range, as additional capabilities.