A single calibration graph for the direct determination of ascorbic and dehydroascorbic acids by electrogenerated luminescence based on Ru(bpy)3(2+) in aqueous solution

Ascorbic (H(2)A) and dehydroascorbic (DA) acids were for the first time directly determined in a single chromatographic run by means of the tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)(3)(2+)) based electrogenerated chemiluminescence (ECL) detection. For the first time, it was demonstrated that DA, a nonelectroactive compound, is ECL active and is responsible for the ECL behavior of H(2)A. This fact, together with the lack of a DA standard, suggested the use of a calibration graph obtained for H(2)A, for determining both analytes. The proven ECL activity of DA, together with literature data relative to the standard redox potentials of the different species coming from H(2)A, led to a reconsideration of the proposed ECL reaction mechanism for H(2)A. The role of the OH- ion in the reaction mechanism of the two analytes appeared to be crucial, H(2)A and DA could be separated by a suitable C-18-reversed-phase HPLC column using an aqueous 30 mN H3PO4 solution as the mobile phase. The optimal ECL response was achieved by polarizing the working electrode at 1.150 V vs SCE (standard calomel electrode) (oxidation diffusion limiting potential for both H2A and Ru(bpy)(3)(2+)). The Ru(bpy)(3)(2+) solution, at pH 10 for carbonate buffer, was mixed to the eluent solution in a postcolumn system, obtaining, still at pH 10, the final 0.25 mM RU(bpy)(3)(2+) concentration. The detection limit found for the two analytes was I x 10(-7) M. The method was successfully applied to the determination of the analytes in a commercially available orange fruit juice.