Anion-exchange chromatographic properties of alpha-lactalbumin eluted from quaternized polyvinylimidazole. Study of the role of the polymer coating.

The anion-exchange elution behaviour of alpha-lactalbumin was studied on cross-linked and quaternized polyvinylimidazole, deposited on various high-performance liquid chromatographic supports (porous silica and diol silica). The influence of the nature and thickness of the coating layer on the retention and band-width properties of the protein elution peak was examined by isocratic elution. The retention properties of alpha-lactalbumin were studied from the plot of log k' vs. log(NaCl]), where k' is the capacity factor and NaCl] the displacer salt concentration in the aqueous phase. The retention depends on the amount of stationary phase deposited on the support, but an increased hydrophobic effect is found when the polymer films do not coat the chromatographic support uniformly. Band broadening of the elution peaks was studied in terms of plots of plate height vs. mobile phase velocity. An important mass-transfer contribution is found, which decreases with increasing k' and increases with the thickness of the coating layer. These effects reveal that the diffusion into the polymer layer is the controlling step of the ion-exchange process with non-uniform polymer layers of large mean thickness.