ABSTRACT
This chapter reviews aspects of instrument optimization for the separation of biomolecules in analytical, micropreparative as well as laboratory preparative scale using packed columns for ‘gel’ filtration’, ‘ion-exchange’, ‘hydrophobic interaction’ chromatography including ‘reversed-phase’ chromatography, and ‘affinity’ chromatography. Chromatography is a technique for separating compounds by exploiting differences in their interaction with a stationary phase when transported by a second, mobile phase and simultaneously allowed to interact. In column liquid chromatography, the mobile phase is, thus, a liquid and the stationary phase is packed into a column and retained by the inlet/outlet frits; alternatively, the stationary phase can be chemically bonded to the wall of the column. Knowledge of the titration curve for a protein mixture can greatly simplify establishing the conditions for purification of the sample by high-performance ‘ion-exchange’ chromatography. Considering chromatography, it is worth remembering that biochemists have for decades achieved a reasonable quality of separation by using the available soft gel media.
