184 J, A. Asenjo and J B Chaudhuri PEG/Dextran systems with addition of small concentration of salts whose charged ions will partition between the phases (e. g. 0. 1 M NaCl or 0.05 M Na2SO4) and also by manipulating the pH of the system. In PEG/salt systems an increase in the pH usually increases the value of K. Figure 7.3 shows the increase in K with pH for a-amylase in a PEG/phosphate system. As the bottom phase has a high concentra tion of salt, it is the charges in the top phase that affect partitioning; thus the top (4) Biospecific affinity. The affinity between sites on the proteins and ligands attached to one of the phase polymers is used for separation. Dyes, inhibitors, fatty acids glutathione, Protein A and several other ligands have been used. Particularly impressive results for selective partitioning have been obtained with metal ions (e.g. Cu**)both in PEG/Dextran but also in PEG/salt systems(Wuenschell et al (5) Solubility dependent. In addition it is important to know the actual concentration of the protein in the extractant phase. In typical PEG/salt systems, protein solubility tends to be higher in the PEG and lower in the salt phase. It is clear that, in the region near saturation of the protein in one of the phases, a constant partition Fig. 7.3. Partition of pure a-amylase()in PEG 4000(10% w/w)/phosphate(11.5% w/w) According to this, it is possible to split the partition coefficient into different terms K=KhfobKe kmw ksol where hfob, el, mw, aff and sol stand for hydrophobicity, electrostatic, size(molecular weight), affinity and solubility contributions to the partition coefficient In practical terms partitioning in aqueous two-phase systems is influenced by many ystem variables. Generally, the higher the molecular weight of the polymers the lower the concentration needed for the formation of two phases. Also, the larger the molecular weight of the PEG, the lower the value of K. Work is presently being carried out on elucidating how the different physicochemical properties of individual proteins determine1.0 Y g 0.5 - 0 - - I I