AMPEROMETRIC DETERMINATION OF L-MALIC ACID IN A FLOW-INJECTION ANALYSIS MANIFOLD USING PACKED-BED ENZYME REACTORS

An enzymic method for the determination of L-malic acid in fruits and vegetables is described, Malate dehydrogenase (MDH) and diaphorase (DI ) were co-immobilized on aminopropyl-, isothiocyanate- and N-hydroxysu ccinimide ester (N-HSE)-modified controlled-pore glass, which was then used for the construction of a packed-bed reactor, MDH catalyses the reaction between malate and NAD(+) and produces NADH, which converts h exacyanoferrate(III) into hexacyanoferrate(II) in the presence of the DI, Amperometric measurements with pure graphite electrodes in a fully automated flow injection manifold were used to monitor the concentrat ion of hexacyanoferrate(II) at +0.3 V versus an Ag/AgCl reference elec trode, The electrochemical behaviour of hexacyanoferrate(III), 2,6-dic hlorophenolindophenol and p-benzoquinone was tested with cyclic voltam metry. Parameters such as working and immobilization pH, flow rate, sa mple size, temperature and NAD(+) and hexacyanoferrate(III) concentrat ions were investigated, The interference of various ions, amino acids and organic acids present in real samples was also studied, The calibr ation graph was linear for the range 2 x 10(-5)-4 x 10(-4) mol l(-1) m alate, The reactors remained active for a period of more than 3 months under specified storage conditions, The maximum sample throughput was 30 h(-1) and the relative standard deviation of the method was better than 1.5% (n = 5), The mean relative error was 4% for fruits-and 2% f or vegetables, The accuracy of the proposed method was tested by compa rison of the results with those obtained by a reference method. Good c orrelation was attained, Recovery experiments showed results between 9 5 and 108%.