Quantitative evaluations of surface-concentrated amino groups on monolithic-type solid supports prepared by copolymerization method

Abstract

We have prepared novel monolithic-type highly cross-linked solid supports having surface amino group using a copolymerization method of functional monomers and cross-linking monomer. From the view point of controlled surface functionalities, we have firstly certified and proved quantitative determination methods of the surface amino groups to utilize and evaluate those solid supports prepared as affinity resins. We utilized a typical titration method and ninhydrin method that have been effectively utilized for quantitative determination of primary amine and amino acid. The ninhydrin method was calibrated using octylamine as a standard compound (R2 = 0.998) and proved applicability for the quantitative determination of other primary amines. A commercially available solid support, Affigel, was able to be quantitatively evaluated only by the ninhydrin method, while Toyopearl afforded just compatible amino group density to the value certified by the manufacturer only through titration method. The obtained incompatible results using both determination methods have been unclear at this moment. On the other hand, both determination methods afforded compatible amino group density of the prepared solid support. The obtained value was up to 126 μmol/ml, which was 94% of the calculated theoretical value based on the feed composition. The results suggested surface-concentrated introduction of amino groups. Based on the determination methods, especially ninhydrin method, we prepared the monolithic-type solid supports having various densities of surface amino groups and proved quantitative and surface-concentrated introduction of amino groups. Those amino groups were able to react with a relatively large ligand molecule, methotrexate, quantitatively and also with controlled density. The obtained results strongly suggest that the novel monolithic-type solid supports have possible advantages when we utilize those as affinity resins for target peptide analyses.