Assay for determining the number of reactive groups on gels used in affinity chromatography and its application to the optimisation of the epichlorohydrin and divinylsulfone activation reactions

An assay has been developed for measuring the number of reactive groups on gels activated for use in affinity chromatography with epichlorohydrin or divinylsulfone. The activated gel is reacted with mercaptoacetic acid and the number of acidic groups introduced is determined by titration. The results obtained are in good agreement with those obtained by elemental analysis and are significantly higher than those obtained by reaction with sodium thiosulfate. Using this assay, the epichlorohydrin and divinylsulfone activation reactions were optimised so that, by varying the amount of activating reagent and time of the reaction, Sepharose CL-4B can be activated in a controlled manner, to between 2 and 34 μmol active groups per ml of gel with epichlorohydrin, and to between 2 and 55 μmol active groups per ml of gel with divinylsulfone. Activation at pH 12 was found to be optimal for divinylsulfone activation. The stability of these activated gels to incubation for 40 h over the pH range 2–14 was examined, as was the stability to long-term storage at 4°C.