Oxidative Amperometric Detection Research Articles

Detection of Hydrogen Peroxide Produced during Electrochemical Oxygen Reduction Using Scanning Electrochemical Microscopy

The substrate-generation/tip-collection mode of scanning electrochemical microscopy was used to detect hydrogen peroxide formed as an intermediate during oxygen reduction at various electrodes. The experiment is conceptually similar to rotating ring-disk experiments but does not require the production of a ring-disk assembly for the specific electrode material in question. In order to limit the extension of the diffusion layer above the sample, the sample electrode potential is pulsed while the Pt ultramicroelectrode probe (UME) is held at a constant potential for oxidative amperometric detection of hydrogen peroxide. The signal at UME is influenced by the sample region within the diffusion length of hydrogen peroxide during the pulse of 2.5 s. The method is tested with three model electrodes showing different behavior with respect to the oxygen reduction reaction (ORR) in acidic solution. Simple analytical models were used to extract effective rate constants for the most important reaction paths of ORR at gold and palladium-cobalt samples from the chronoamperometric response of the UME to a reduction pulse at the sample electrode.

Determination of diflubenzuron and its main metabolites in forestry matrices by liquid chromatography with on-line diode-array and electrochemical detection

A liquid chromatography (LC) method with on-line diode-array (DAD) and electrochemical detection (ED) has been developed for the determination of the pesticide diflubenzuron (DFB) and its main metabolites: 2,6-diflurobenzamide (2,6-DFBA), 4-chlorophenylurea (4-CPU), 4-chloroacetanilide (4-CAA), 4-chloroaniline (4-CA) and N-methyl-4-chloroaniline (NM-4-CA) in forestry matrices. Liquid chromatography was performed on a Spherisorb C 18 column 5 μm (4.1×150 mm), using acetonitrile/methanol/citrate-disodium hydrogenphosphate buffer (50:2:48) at pH 7 as mobile phase, pumped at a flow rate of 1 ml min −1. The electrochemical study of the compounds proves the oxidation processes of 4-CPU, 4-CA, NM-4-CA and DFB on a glassy carbon electrode, which allows to determine them by oxidative amperometric detection. The amperometric detector equipped with a glassy carbon working electrode was operated at +1350 mV vs Ag/AgCl. The DAD detector was set at 245 and 260 nm, where the compounds show secondary maxima of absorbance which do not interfere with the matrix. The detection limits were 2.0–25.2 μg l −1 for the DAD detection and 0.07–1.8 μg l −1 for the amperometric detection. The method was applied to the determination of the pesticide and these metabolites in pine-needles. Samples were extracted into acetonitrile and further cleaned-up by solid-phase extraction through aminopropyl cartridges. Recoveries of the compounds from supplied pine-needle samples were higher than 90% in most of the cases, with relative standard deviations lower than 3.7%.

On-line deoxygenation in reductive (and oxidative) amperometric detection: environmental applications in the liquid chromatography of organic peroxides

Cyclic voltammetry was used qualitatively to characterize and determine the feasibility of the oxidation and reduction of selected organic peroxides and hydroperoxides at a glassy carbon electrode. Organic peroxides were determined using reversed-phase high-performance liquid chromatography with simultaneous reductive and oxidative amperometric detection in a thin-layer dual parallel–adjacent electrode configuration. An on-line deoxygenator allowed the removal of molecular oxygen from the mobile phase and this resulted in an extension of the negative potential range of the glassy carbon electrode by approximately 750 ± 50 mV vs. the Ag/AgCl reference electrode. Chromatographically assisted hydrodynamic voltammetric measurements, in the dual parallel–adjacent electrode configuration, provided confirmation of the feasibility of simultaneously monitoring two independent redox reactions, for a single analyte, and allows for both the qualitative and quantitative analysis of organic peroxides (and hydroperoxides). The reductive amperometric responses were, on average, 2–5 times greater, depending on the particular organic peroxide, than the corresponding oxidative amperometric responses. An empirically determined parameter was evaluated and developed, {[iOxiRed–1] or [iaic–1]}, from two independent electrode reactions, that allows the qualitative identification of organic peroxide analytes by comparison of samples with standard injections. Estimated method detection limits (MDLs) for butan-2-one (butan-2-one) peroxide (2-BP), tert-butyl hydroperoxide (t-BHP) and cumene hydroperoxide (CHP) in reductive amperometry are approximately 0.8, 0.4 and 5 ppb, respectively and the oxidative amperometric MDLs are about 3, 2.5 and 12 ppb, respectively. The reductive amperometric responses for 2-BP, t-BHP and CHP are linear over 4–5 orders of magnitude of concentration, extending from ca. 1 µg l–1 to ca. 100 mg l–1, and the corresponding correlation coefficients are of the order of 0.9997–0.99994.

High-performance liquid chromatography of selected organic peroxides with oxidative amperometric detection.

Reversed-phase high-performance liquid chromatography with oxidative amperometric detection was optimized for the determination of several organic peroxides in drinking water under ideal conditions. The determinations were performed under isocratic conditions using acetonitrile and methanol as the organic modifiers with 0.05 M potassium phosphate buffer solution. The oxidative amperometric response of the organic peroxides was dependent on the concentration of organic modifier and the electrode potential. The optimum electrode potential (EOxAPP), for the simultaneous determination of the organic peroxides was approximately +1.150 +/- 0.05 V versus the Ag/AgCl reference electrode. The maximum analytical signal for butan-2-one peroxide and tert-butyl hydroperoxide, when using acetonitrile, was obtained with 20% v/v organic modifier. For cumene hydroperoxide, the maximum analytical signal was achieved with approximately 35% v/v acetonitrile. The retention time of cumene hydroperoxide, on an octyldecylsilane column (250 x 4 mm id), decreased sharply from > 100 to < 10 min when the organic modifier concentration was varied from 5 to 50% v/v. The retention time of butan-2-one and tert-butyl hydroperoxide, under the same conditions, varied by < 10 min. The calibration curves for the aliphatic peroxides and aromatic peroxide were linear from 2 to 200 ng and from 0.2 to 200 ng injected, respectively.

Electrochemical study of the flavour enhancer maltol. Determination in foods by liquid chromatography with amperometric detection

The electrochemical behaviour of maltol on a glassy carbon electrode was studied. The results were applied to electrochemical detection in liquid chromatography (LC-ED). Liquid chromatography (LC) and oxidative amperometric detection with a glassy carbon electrode were used for the determination of maltol in food samples. A mobile phase of methanol-acetonitrile-citrate/phosphate buffer, pH = 2.4 (50:48:2) was used with the detector operated at E = 1000mV vs. Ag AgCl reference electrode. Under optimal conditions detection and determination limits of 0.1 and 3.7 ng respectively were obtained. The method was used for the determination of maltol in cakes. Conditions for clean-up of the sample were established using solid phase extraction cartridges.

Studies on electrochemical reactions at metal‐oxide electrodes for combination with high‐performance liquid chromatography

AbstractVarious electrodes, such as nickel, copper, and cobalt metal electrodes and nickel‐, copper‐, and cobalt‐modified glassy‐carbon electrodes, are investigated for the oxidative amperometric detection of alcohols, polyols, and carbohydrates after separation by high‐performance liquid chromatography (HPLC). These electrode materials are compared with respect to stability, detection limits, and an eventual necessity of electrode surface pretreatment. Detection limits of 4 ng and 10 ng glucose injected can be achieved using a metallic copper electrode and a metallic nickel electrode, respectively. Investigations at the surface of a nickel electrode reveal the existence of a reversible layer of nickel oxide with higher valence which is formed in an alkaline medium at an applied potential of at least 400 mV versus Ag/AgCl/KCl(sat). Its thickness strongly depends on electrochemical pretreatment procedures. This layer can be converted into a layer of lower valence nickel oxide either electrochemically or chemically. Investigations of the electrode surface are carried out by in situ ultraviolet‐visible (UV‐vis) reflectance spectroscopy and by Fourier‐transform infrared spectroscopy (FTIR). Controlled‐potential experiments at various metal‐oxide electrodes and separation of the oxidation products on ion‐exchange columns reveal some of the reaction mechanisms for the oxidation of alcohols, polyols, carbohydrates, and similar organic compounds.

Determination of the catecholamines and serotonin, their precursors tyrosine and tryptophan, and their main metabolites in rat brain using reversed-phase high-performance liquid chromatography with fluorimetric and oxidative amperometric detection in series

A high-performance liquid chromatographic method for the determination of catecholamines and serotonin, their precursors and their main metabolites was developed applied to rat cerebellum, hypothalamus, striatum and cortex. A fluorimetric and an oxidative amperometric detector were used in series. For both detectors, detection limits (25–520 pg) were useful for this application, linearity of standards was excellent (average r > 0.9997), between-run precision for sample analytes was generally acceptable (coefficient of variation < 10% with appreciable concentrations present) and average recoveries of standard additions to sample analytes were better than 90%. Particular attention was paid to peak identification, including both a thorough treatment of retention time agreement of peaks in standards and sample analytes, and a comparison of results for the seven compounds amenable to quantitation by both detectors. Considerable attention was also given to determining the stability of standards and sample analytes under a wide variety of conditions, and practical recommendations were made.

Determination of Deoxynivalenol (DON, Vomitoxin) in Wheat by High-Performance Liquid Chromatography with Photolysis and Electrochemical Detection (HPLC-hv-EC)

Deoxynivalenol (DON, vomitoxin) is a naturally occurring toxic fungal metabolite found in grains such as wheat, corn, rye, and barley. Current methods of analysis for DON and related trichothecene mycotoxins involve gas chromatography with electron capture detection, thin-layer chromatography, and high-performance liquid chromatography with ultraviolet detection. Improved sensitivity and selectivity for DON, as well as for the related compounds nivalenol and fusarenon-X, are made possible by the use of high-performance liquid chromatography with online, postcolumn photolysis and oxidative amperometric detection (HPLC-hv-EC). Conditions are optimized with respect to residence time in the photolytic reactor, applied potentials, mobile phase pH, and chromatographic parameters for the separation of DON, nivalenol, and fusarenon-X, and the technique is found to be linear over a range of 10 ppb-2 ppm with minimum detection limits on the order of 1-2 ng on-column (10-20 ppb). Since these compounds are not electroactive in the absence of photolysis, an additional mode of specificity is realized in addition to the chromatographic retention time and the dual-electrode response ratio. Some market samples of wheat naturally contaminated with DON are analyzed by this method, and the results are in agreement with those obtained by HPLC-UV and TLC analyses of the same samples.

Phase-selective alternating current polarographic assay of methotrexate in human serum

The a.c. polarographic behaviour of methotrexate (MTX) was studied and the nature of the process taking place at the dropping mercury electrode was elucidated. Both the molecule and its reduced product appeared to be absorbed at the surface of the electrode. The observed electrochemical behaviour was in close agreement with theoretical predictions for an absorbed species which is reversibly reduced. This permitted a very sensitive and selective determination of MTX in serum samples by phase-selective a.c. polarography. The method, which involves a very simple and rapid previous clean-up procedure, has a limit of detection of 3.0 × 10 −7 M in serum and the results compare well with those obtained by liquid chromatography with oxidative amperometric detection (LC-ED) at +1.1 V. Both methods form an interesting alternative to others previously reported for monitoring MTX in cancer patients under treatment with high doses of the drug.

Electrochemical Detectors in HPLC and Ion Chromatography.

Back in 1952, the renowned Polish electrochemist Wiktor Kemula introduced chromato-polarography,1 i.e., polaro-graphic detection for liquid chromatography. This technique continued to develop slowly until the early 1970s (for a review see Reference 2) when modem high-performance liquid chromatography (HPLC) emerged. This new, highly efficient chromatographc method could only be. used with detectors ensuring low dispersion. It was not easy to modify the dropping mercury electrode cells to satisfy this requirement. However, at the same time, electroanalytical chemists, who already had much experience in using carbon-based electrodes for oxidative detection in flow analysis, put forward the idea of oxidative amperometric detection in liquid chromatography.3,4 In this technique, solid or quasi-solid (paste) electrodes were used and this made possible the construction of miniaturized cells with just a few microliter volume.

Determination of Fluazifop-Butyl and Fluazifop Acid in Soybeans and Soybean Oil Using Liquid Chromatography with Oxidative Amperometric Detection

A new method is described for the determination of the herbicide fluazifop-butyl, and its metabolite fluazifop acid, in soybeans and soybean oil as fluazifop acid. Liquid chromatography with amperometric detection (LC/AD) is used to determine fluazifop acid produced from the metabolism or base hydrolysis of fluazifop-butyl in soybeans and soybean oil. These foods were spiked with fluazifopbutyl at 0.05, 0.10, and 0.50 ppm and hydrolyzed with 0.2N NaOH in methanol. The hydrolysate (adjusted to pH less than or equal to 1) is extracted with dichloromethane and the extract is washed with 1.0% NaHCO3. The NaHCO3 is acidified to pH less than or equal to 1 and extracted with dichloromethane; the partitioning is repeated 2 more times. The dichloromethane is removed, mobile phase solvent is added, and aliquots are injected onto a PRP-1 liquid chromatographic column; fluazifop acid is separated from coextracted compounds and detected at an applied potential of + 1.25 V, using an amperometric electrochemical detector in the oxidation mode. Recoveries ranged from 69 +/- 6.5 to 101 +/- 18% and from 72 +/- 7.5 to 88 +/- 11% for soybeans and soybean oil, respectively. Accuracy of these recoveries was confirmed by use of 14C-radiolabeled fluazifop-butyl and by liquid scintillation spectrometry of the 14C-fluazifop acid released.

Determination of nitrite and nitrate by reversed-phase high-performance liquid chromatography using on-line post-column photolysis with ultraviolet absorbance and electrochemical detection

Ion-pair (paired-ion) reversed-phase HPLC has proved to be an effective technique for the analysis of inorganic anions. Also referred to as ion-interaction chromatography, it has bee used with conductimetric, ultraviolet (UV) absorbance, refractive index, and electrochemical detection. Nitrite and nitrate are amenable to UV detection at wavelengths of approximately 240 nm and below. In addition, nitrite is readily oxidized at potentials of +0.9 V or higher at a glassy carbon electrode. While nitrate is electrochemically unreactive under these conditions, it will undergo photolysis, with the conversion product (in all likelihood the nitrite anion) generating an oxidative signal. This paper describes the use of a Teflon TM knitted open tubular (KOT) reactor, which when wrapped around a UV source, provides a means of continuous, on-line photolysis. This derivatization step, combined with high-performance liquid chromatography, permits the determination of both nitrite and nitrate using oxidative amperometric detection. We have applied this technique to a number of samples (cured meats, smoked and fresh salmon, smoked cod, spiked water solutions) and have also obtained comparative data for nitrite by UV and direct ( i.e. non-photolytic) oxidative electrochemical detection, as well as by a standard spectrophotometric procedure.

Separation of sulphite adducts by ion chromatography with oxidative amperometric detection

Separation of sulphite adducts by ion chromatography with oxidative amperometric detection

An electroanalytical study of the anticancer drug dacarbazine

The electrochemical behaviour of dacarbazine [5-(3,3-dimethyl-1-triazenyl) imidazole-4-carboxamide; DTIC] was investigated by Tast and differential pulse polarography (d.p.p.) at the dropping mercury electrode, by cyclic and differential pulse voltammetry at the hanging mercury drop electrode and by anodic voltammetry at the glassy carbon electrode. Calibration graphs were obtained for 2×10 −8−2×10 −5 M DTIC by d.p.p., for 5×10 −9−1×10 −5 M by adsorptive stripping voltammetry ar a hanging mercury drop electrode, and for 1−10×10 −5 M by high-performance liquid chromatography with oxidative amperometric detection at a glassy carbon electrode. The methods are compared and applied to determine DTIC added to blood serum after a simple clean-up procedure.

Photolytic derivatization for improved LCEC determinations of pharmaceuticals in biological fluids.

Although electrochemical (EC) methods have been demonstrated to be sensitive and selective, wide use of EC detection in high performance liquid chromatographic (HPLC) assays in forensic and clinical toxicology laboratories has not been forthcoming. This fact is due to the general difficulty involved with the use of reductive EC detection methods, as well as to the lack of EC response in either the oxidative or reductive mode, for a number of classes of drugs having substantial clinical and forensic importance. The use of an on-line, post-column, continuous photolytic derivatization step, followed by conventional oxidative amperometric detection, alleviates many of these problems. In this report, the use of HPLC-photolysis-EC (HPLC-h nu-EC) for the trace determination of a number of controlled substances in biological fluids is presented. Following system optimization, the determination of phenobarbital, cocaine, methylphenidate, and several 1,4-benzodiazepines (and metabolites) is linear over three orders of magnitude. In addition, HPLC-h nu-EC offers a sensitive approach for these compounds, in that limits of detection (LODs) are all below 1 microgram/ml, ranging from 1 ng/ml to 750 ng/ml. The validity of this newer method is demonstrated in collaborative studies involving the trace determinations of phenobarbital in human serum, and chlordiazepoxide and its major metabolite, norchlordiazepoxide, in human urine. Finally, the authors' view of the role of HPLC-h nu-EC in the clinical and forensic toxicology laboratory is presented.

A Simple Electrochemical Method for the Quantitative Determination of Acetaminophen in Serum

Abstract A simple electrochemical method for the determination of acetaminophen in serum is described. The eleotrode and associated electronics are simple, reliable, and inexpensive to build. The apparatus can be operated at a rate of 2–3 determinations per minute using only 10 μl serum per determination. The procedure includes extraction of acetaminophen in ethyl acetate and subsequent oxidative amperometric detection of the drug by a form of flow-injection analysis. The system parameters of buffer, pH, and redox potential have been optimized to permit measurement of less than 10 μg/ml of acetaminophen. The determination is linear over the range of 10–300 μg/ml with a C.V. of less than 3% for replicate analysis of the same sample.