MODIFICATION OF REVERSED-PHASE COLUMNS WITH DYED SURFACTANTS - PREPARATION OF MECHANICALLY RESISTANT EFFICIENT IMMOBILIZED DYES FOR PROTEIN-PURIFICATION

The properties of reversed-phase chromatographic supports can be substantially altered by saturating the hydrophobic sites at their surface with easily prepared dyed non-ionic surfactants. The dissociation constants governing the interaction between a reversed-phase support and a model dyed surfactant were found to be in the micromolar range under several mobile phase conditions. The total amount of modified surfactant immobilized on the reversed-phase support was also measured and found to be as great as that usually immobilized on agarose supports (expressed as micromoles per millilitre of support). A reversed-phase column saturated with dyed surfactant can be used with the same aqueous mobile phases as used with immobilized dye columns prepared with agarose as a supporting matrix, but the mechanical sturdiness of the silica matrix allows the use of higher flow-rates. This methodology was used to screen several dyes to find the one best suited for a given purification. A convenient procedure (with affinity elution) was devised for the purification of pancreatic ribonuclease and chymotrypsinogen.