Chromatography

Biography

Biography: Romain Kapel

Abstract

Nowadays, protein hydrolysates and fractions are of great interest because of their nutritional or bioactive properties. The quantification of total peptide concentration is of a crucial importance in order to establish mass balance of fractionation processes. This is commonly done either by Kjeldhal analysis, or by colorimetric assays, whose are laborious and time-consuming. This work describes an original methodology to quantify complex peptide mixtures by size-exclusion high-performance liquid chromatography (SE-HPLC), already used to characterize the molecular weight distribution of hydrolysate. In the proposed methodology, each point of the complex mixture chromatogram is regarded as a mixture of peptides sharing same molar extinction coefficient and molar weight, estimated from its retention time and the hydrolysate aminogram. This allows a conversion of absorbance into concentration (using Beer-Lambert law) and the integration of the overall signal gives the peptide concentration of the analysed fraction. The methodology was first tested on simulated elutions of peptide mixtures and a good estimation of the total peptide concentration was observed (error less than 10%). Then 30 fractions obtained by ultrafiltration of hydrolysates from two different sources were titrated by Kjeldahl or BCA analysis and analysed by SE-HPLC for an experimental validation of the methodology. Very good matchs between methods were obtained (error less than 15%). Moreover, the presence of organic solvents or salts in samples does not impact the accuracy of the methodology contrary to common quantification methods.