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The development of a highly sensitive method for the determination of nanomolar concentrations of hydrogen peroxide in the liquid phase is described. This paper demonstrates for the first time a flow injection analysis (FIA) system with immobilized enzyme reactor combined with a total internal reflective cell (a liquid waveguide capillary cell (LWCC)) and spectrophotometric detection, for the development of an improved procedure for the determination of hydrogen peroxide. Moreover, the newly synthesized 4-aminopyrazolone derivative, 4-amino-5-(p-aminophenyl)-1-methyl-2-phenyl-pyrazol-3-one (DAP), is used as a color coupler in its oxidative condensation with the sodium salt of N-ethyl-N-sulphopropylaniline sodium salt (ALPS) which acts as a hydrogen donor. Immobilization of peroxidase is achieved by coupling the periodate-treated enzyme to aminopropyl controlled-pore glass (CPG) beads. The determination of hydrogen peroxide is carried out in a 0.1 M phosphate buffer and the product is monitored at 590 nm with a charge-coupled device (CCD) detector equipped with fiber optics in a fully computerized system. The interference of different species, mainly ionic, was investigated. The method permits detection down to 4 nmol l(-1) hydrogen peroxide (signal-to-noise ratio = 3). A linear calibration graph was obtained over the range 20-700 nmol l(-1). The relative standard deviation (R.S.D.) at 300 nmol l(-1) H,02 is 1.7% (n = 7), The method was successfully applied for the determination of hydrogen peroxide in samples from a vat-cleaning process. (C) 2002 Elsevier Science B.V. All rights reserved. Analytica Chimica Acta

An ascorbate sensor based on a glassy carbon electrode modified with a cellulose acetate polymeric film bearing 2,6-dichlorophenolindophenol (CA/DCPI-CME) was constructed. The overall reaction obeys a catalytic regeneration mechanism (EC mechanism) and the electrochemical rate constant kf for the electrocatalytic oxidation of ascorbic acid was evaluated. The modified electrodes were mounted in a flow injection (FI) manifold, poised at +100 mV versus Ag/AgCl/3 M KCl at pH 6.5 and utilized for the determination of ascorbic acid in beverages and pharmaceuticals. Good correlation with a reference method was attained. Interferents of various molecular sizes were tested. Calibration graphs were linear over the range 0.02-1 and 0.1-6 mM ascorbic acid for CA/DCPI sensors hydrolyzed in KOH and ZnCl2 solution, respectively. The throughput was 25 samples per hour and the CV was for a 0.4 mM ascorbic acid solution 0.75 (n=14) and 1.2% (n=10) for CA/DCPI sensors hydrolyzed in KOH and ZnCl2 solution, respectively. The recovery was 92-110%. The sensors showed very good repeatability and operational stability. (C) 2000 Elsevier Science B.V. All rights reserved. Analytica Chimica Acta

The preparation of a 2,6-dichlorophenolindophenol (DCPI) modified graphite electrode is described. DCPI was successfully immobilized by physical adsorption onto a plain graphite electrode (DCPI-CME) and onto graphite electrodes pretreated with lanthanum nitrate (DCPI-La-CME) or thorium nitrate (DCPI-Th-CME). The electrochemical behavior of DCPI-CME was extensively studied using cyclic voltammetry. The electrochemical redox reaction of DCPI was found to be fairly reversible at low coverage with E degrees' = + 55 mV (vs. Ag/AgCl/3M KCl) at pH 6.5. A pK(a) value of 5.8 +/- 0.1 for immobilized form of DCPI is determined from the intersection of the lines in the plot E degrees' vs. pH. The current I-p has a linear relationship with the scan rate up to 1200 mV s(-1), which is indicative for very fast electron transfer kinetics. The calculated value of the standard rate constant is k degrees = 18 +/- 4 s(-1). No decrease of either the anodic or the cathodic current of the cyclic voltammogram was observed after 500 runs of successive sweeps. The influence of the morphology of the electrode surface on the electrochemical behavior of the DCPI-CME was studied and a mathematical model is proposed, which partly describes the dependence of the geometrical area of the electrode surface on the grid of the emery paper. The modified electrodes were mounted in a flow-injection manifold, poised at +60 mV (vs. Ag/AgCl/3M KCl) and a catalytic current due to the oxidation of NADH was observed reducing thus the oxidation overpotential of NADH for about 400 mV. Interference from various reductive species present in real samples was investigated. The repeatability was 1.2% RSD (n = 10 for 0.1 mM NADH). The sensor showed good operational and storage stability. Electroanalysis

An enzymic method for the determination of citric acid in fruits, juices and sport drinks is proposed, The method is based on the action of the enzymes citrate lyase, oxaloacetate decarboxylase and pyruvate oxidase, which convert citric acid into H2O2 with the latter being monitored amperometrically with a H2O2 probe. The enzymes pyruvate oxidase and oxaloacetate decarboxylase mere immobilized, A multi-membrane system, consisting of a cellulose acetate membrane for the elimination of interferants, an enzymic membrane and a protective polycarbonate membrane were placed on a Pt electrode and used with a fully automated flow injection manifold. Several parameters were optimized, resulting in a readily constructed and reproducible biosensor, Interference from various compounds present in real samples was minimized, Calibration graphs mere linear over the range 0.01-0.9 mM pyruvate, 0.015-0.6 mM oxaloacetate and 0.015-0.5 mM citrate, The throughput was 30 samples h(-1) with an RSD of 1.0% (n = 8); the mean relative error was 2.4% compared with a standard method, The recovery was 96-104%, A 8-10% loss of the initial activity of the sensor was observed after 100-120 injections. Analyst

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-hydroxysuccinimide 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 hexacyanoferrate(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 concentration of hexacyanoferrate(II) at +0.3 V versus an Ag/AgCl reference electrode, The electrochemical behaviour of hexacyanoferrate(III), 2,6-dichlorophenolindophenol and p-benzoquinone was tested with cyclic voltammetry. Parameters such as working and immobilization pH, flow rate, sample size, temperature and NAD(+) and hexacyanoferrate(III) concentrations were investigated, The interference of various ions, amino acids and organic acids present in real samples was also studied, The calibration graph was linear for the range 2 x 10(-5)-4 x 10(-4) mol l(-1) malate, 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% for vegetables, The accuracy of the proposed method was tested by comparison of the results with those obtained by a reference method. Good correlation was attained, Recovery experiments showed results between 95 and 108%. Analyst