Mixed-bed ion-exchange columns for protein high-performance liquid chromatography.

Protein retention is investigated on high-performance liquid chromatography columns packed with mixtures of ion exchangers. Retention factors are measured at both low and high salt concentrations in the eluent and their dependence on the bed composition is found to be linear in some cases, but non-linear in others. The physical basis for the observed non-linear retention behavior has not been established and an empirical mixing rule is employed to express the dependence of protein retention on bed composition. Protein separations are carried out on the mixed-bed columns by using gradient elution with increasing salt concentration and the process is modelled mathematically. The retention times predicted by computer calculations correspond closely to the experimental findings. Optimal selection of the mixed-bed composition and the gradient steepness for the separation of four proteins is illustrated by using the window diagram technique. Although the experimental results presented here deal with electrostatic interaction chromatography of proteins only the applicability of mixed sorbents is expected to extend to all branches of liquid chromatography. It is anticipated that mixed-sorbent columns will find extensive use in the large-scale purification of biological compounds and in routine analysis.