DEVELOPMENT OF DIHYDRAZIDE-ACTIVATED SILICA SUPPORTS FOR HIGHPERFORMANCE AFFINITY-CHROMATOGRAPHY

A method of preparing dihydrazide-activated silica was developed for u se in high-performance affinity chromatography (HPAC). This support wa s made by oxidizing diol-bonded silica and reacting it with oxalic or adipic dihydrazide. The steps involved in this synthesis were studied and confirmed by FTIR. Items considered in optimizing the preparation of the support included the amount of dihydrazide added and the reacti on time or pH used. Control of dihydrazide bifunctional attachment was obtained by varying the extent of diol-bonded silica oxidation. This support was successfully used in the immobilization of oxidized antibo dies, horse radish peroxidase (a glycoenzyme) and transfer RNA. In eac h case, data indicated that immobilization was through site specific c oupling rather than non-specific adsorption. Dihydrazide-activated sil ica was found to be stable for 2-6 weeks after preparation when stored at 5 to 25 degrees C. The linkage between oxidized biomolecules and t his support was stable for at least one month in the presence of vario us solvents commonly used in HPAC.