Modification of reversed-phase columns with dyed surfactants: Preparation of mechanically resistant efficient immobilized dyes for protein purification

• Published in: Journal of Chromatography A 1991-09, Vol.558 (1), p.43-54
• Main Authors: Kong Sing, Youne Lie, Algiman, Etienne, Kroviarski, Yolande, Massot, Christian, Dhermy, Didier, Bertrand, Olivier
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Proteins
• ISSN: 0021-9673
• DOI: 10.1016/0021-9673(91)80110-3

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.