Perfusion liquid chromatography of whey proteins

A perfusion reversed-phase (RP) HPLC method was developed for the rapid separation of the main bovine whey proteins: alpha-lactalbumin (alpha-LA), serum albumin (BSA) and the genetic variants of beta-lactoglobulin (A and B) (beta-LG A and beta-LG B). For the method development, the influence of factors favouring structural changes of proteins (temperature 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 the beta-lactoglobulins. The method was applied to the determination of proteins in a whey from raw bovine milk. The precision of the determinations was < or = 3.75 mg per 100 ml (S.D.). With respect to the accuracy, errors < or = 7.0% in the determination of alpha-LA, beta-LG A and beta-LG B were obtained, compared with an RP-HPLC reference method. However, higher errors in the quantification of BSA were found owing to the lack of purity of the peak assigned. In addition, the proposed method has proved to be very useful in the detection of homologous whey proteins from different species (cow, sheep and goat) in milk mixtures.