Separation of acid whey proteins on the preparative scale by hyperdiffusive anion exchange chromatography

The eluent flow through a fixed bed of a strong onion-exchanger Q Hyper D/F packing has been characterized by mean of the residence time distribution and the separation conditions of acid whey proteins have been established. Myoglobin under non-retaining conditions was used as a test protein because iis molecular weight was close to that of alpha -lactalbumin, the target protein of this study. In the interstitial velocity range of 44 - 350 cm h(-1) a constant reduced height equivalent to a theoretical plate of 13 was observed. Nearly pure fractions of the five main acid whey proteins were obtained on the preparative scale for a gradient slope of NaCl 1 mM ml(-1), in the pH range of 6 - 8 and an interstitial velocity of 127 cm h(-1) (flow rate of 2 mL min(-1)). A separation focused on a pure fraction of alpha -lactalbumin was achieved at pH 7.5 and was effective up to an interstitial velocity of 500 cm h(-1) (flow rate of 8 mt min(-1)). An indepth characterization of alpha -lactalbumin by electrospray ionization - mass spectrometry shaved that 15% of alpha -lactalbumin was lactosylated both in the collected fraction and in the acid whey protein concentrate used as feed.