PERFUSION LIQUID-CHROMATOGRAPHY OF WHEY PROTEINS

A perfusion reversed-phase (RP) HPLC method was developed for the rapi d separation of the main bovine whey proteins: alpha-lactalbumin (alph a-LA), serum albumin (BSA) and the genetic variants of beta-lactoglobu lin (A and B) (beta-LG A and beta-LG B). For the method development, t he influence of factors favouring structural changes of proteins (temp erature and organic acid concentration in the mobile phase), gradient and other chromatographic conditions and the mass of protein injected was examined. The optimized method allowed the separation of proteins in about 1.5 min (cycle time 3.5 min) with resolution around 1.0 for t he beta-lactoglobulins, The method was applied to the determination of proteins in a whey from raw bovine milk. The precision of the determi nations was less than or equal to 3.75 mg per 100 ml (S.D.). With resp ect to the accuracy, errors less than or equal to 7.0% in the determin ation on alpha-LA, beta-LG A and beta-LG B were obtained, compared wit h an RP-HPLC reference method. However, higher errors in the quantific ation of BSA were found owing to the lack of purity of the peak assign ed. In addition, the proposed method has proved to be very useful in t he detection of homologous whey proteins from different species (cow, sheep and goat) in milk mixtures.