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

Summary The eluent flow through a fixed bed of a strong anion-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 its molecular weight was close to that of α-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 α-lactalbumin was achieved at pH 7.5 and was effective up to an interstitial velocity of 500 cm h⁻¹ (flow rate of 8 mL min⁻¹). An indepth characterization of α-lactalbumin by electrospray ionization—mass spectrometry showed that 15% of α-lactalbumin was lactosylated both in the collected fraction and in the acid whey protein concentrate used as feed.