Gel permeation chromatography of proteins in partly aqueous eluents

Gel permeation chromatography of a number of proteins has been performed with Sephadex G-100 and G-200, using mixtures of water and organic solvents as eluents. It appears that the chromatographic distribution coefficients of the proteins are enhanced by the addition of organic co-solvents to the eluent. Possible causes of this phenomenon are (1) adsorption of the proteins to the gel matrix in the partly aqueous eluents and (2) partition of the proteins between two phases of different composition, viz., the eluent and the liquid in the gel beads. This phenomenon has been applied to resolve a mixture of ribonuclease and cytochrome c, using 5% (w/v) polyethylene glycol 20,000 as the eluent. These proteins cannot be separated with an aqueous eluent, due to the small difference between their molecular weights