Pulsed amperometric detection of carbohydrates, amines and sulfur species in ion chromatography —the current state of research

A review is given of so-called pulsed amperometric detection at Au and Pt electrodes. Of greatest interest is the application of pulsed amperometric detection for polar aliphatic compounds not easily detected by conventional photometric or fluorometric techniques. The anodic detection mechanisms are electrocatalytic in nature under the control of potential-dependent surface states. Oxidations of carbohydrates at Au electrodes in alkaline media occur in a potential region where a submonolayer of adsorbed hydroxyl radicals (·OH_ads) is formed and speculation is offered on the role of this species in the anodic mechanisms. Very little anodic signal is obtained at Au electrodes for low-molecular-mass n-alcohols; however, a large response is obtained from oxidation of the alcohol moiety of n-alkanolamines. This is attributed to the beneficial effect of adsorption via the amine moiety with the result that the residence time of the molecules at the electrode surface is increased to give a high probability for ultimate oxidation. Amines and sulfur compounds with non-bonded electrons on the N and S atom, respectively, are adsorbed at Au electrodes and are oxidatively desorbed concomitantly with formation of inert surface oxide (AuO). The simultaneous formation of surface oxide produces a large background signal in pulsed amperometric detection. Hence, a modification of the pulsed waveform is described whose application is called integrated pulsed amperometric detection. Applications are shown for pulsed amperometric detection and integrated pulsed amperometric detection in ion chromatography to illustrate strengths of these combined technologies.     

Pulsed amperometric detection of sulfur compounds: Part I. Initial studies of platinum electrodes in alkaline solutions

Conventional voltammetric and amperometric techniques at Pt or Au electrodes have not been considered applicable for quantitative detection of most sulfur compounds. This is the result of the observed loss of electrode activity caused by accumulation of sulfurous adsorbates and surface oxides. Pulsed Amperometric Detection (PAD) at Pt and Au is highly sensitive for the anodic detection of sulfur compounds, organic and inorganic, which adsorb on the electrode surface. The detection of thiourea by PAD at Pt is described here, and is concluded to correspond to the surface-oxide catalyzed oxidation of the sulfur moiety of adsorbed thiourea to sulfate. Results reported here for detection in a flow-injection system (FI/PAD) produce linear calibration plots of I_p vs. c^b at low concentration and linear plots of 1/I_p vs 1/c^b at high concentrations. The detection limit for thiourea is 0.38 ppm (i.e., ca 17 ng/45 μl sample) for the FI system used.     

Amperometric detection of simple alcohols in aqueous solutions by application of a triple-pulse potential waveform at platinum electrodes

Application of a triple-pulse waveform is described for the anodic detection of methanol, ethanol, and ethylene glycol. The execution of the waveform incorporates the cleaning and reactivation of the platinum electrode, by alternate anodic and cathodic polarization, with measurement of the faradaic signal for the analyte at the reduced electrode surface. Some results for formic acid are also presented. The waveform is completed within approximately 2 s and the faradaic signal exhibits no decay with time as would be the case for amperometric detection at a constant applied potential. Calibration curves made by plotting —1/I vs. 1/C are linear. This is consistent with a reaction mechanism in which the analyte is adsorbed prior to anodic detection. The technique is applicable for detection in chromatographic and flow-injection systems.     

Pulsed amperometric detection of underivatized amino acids using polypyrrole modified copper electrode in acidic solution

The successful pulsed amperometric detection of underivatized amino acids have been carried out in an acidic media on a polypyrrole (PPy) modified Cu electrode. The formation of PPy film doped with glutamate (glu) on a Cu electrode surface changes the mechanism of Cu dissolution. After application of multistep potential waveform, the PPy film was glu free due to the electro-reduction and overoxidation. High anodic potential polarization treatment yielded partially overoxidized PPy film as long as the Cu surface dissolution and amino acid permeation through the film was well controlled. This overoxidized PPy film acted as a charge and size exclusion barrier in order to improve the selectivity and stability of a Cu electrode. Various process parameters such as film modification time, detection and cleaning potential and pH of solution have been optimized to maximize the beneficial electrocatalytic properties of the electrode surface. At an optimized condition, detection limits for positively charged histidine and arginine are 19 and 22 pg respectively, whereas the neutral amino acids detected in amounts of 0.9–2.3 ng. Furthermore, the PPy coated Cu electrode response was long lived, stable and reproducible.     

Pulsed amperometric detection of sulfur-containing pesticides in reversed-phase liquid chromatography

Pulsed amperometric detection at a gold electrode is demonstrated for the separation of eight sulphur-containing pesticides on a C-18 reversed-phase column with 50% (v/v) acetonitrile in acetate buffer (pH 5.0) as the mobile phase. Detection was based on a two-step potential waveform with adsorption of the analyte during cathodic polarization and subsequent amperometric detection catalyzed by oxide formation following anodic polarization. The mechanism of the anodic detection involves prior adsorption of the sulfur compounds. Hence, the shape of the calibration curve is strongly influenced by the adsorption isotherm of the analyte and, therefore, deviates from linearity at high concentrations. Detection limits below 100 ng ml^?1 in 20-μl samples (e.g., 0.8 ng of dimethoate) were obtained by using preconcentration from a larger sample onto a C-18 fore column prior to injection into the separation column.     

Nickel-titanium alloy electrodes for stable amperometric detection of underivatized amino acids in anion-exchange chromatography

Nickel-titanium (Ni-Ti) alloy electrode was used as an electrochemical detector for the analysis of underivatized amino acids in flow systems. In strong alkaline solution, an oxide film on the Ni-Ti alloy electrode surface exhibited a high catalytic activity toward the oxidation of amino acids. Cyclic voltammetry experiments confirmed that electrogenerated Ni(III)O(OH) functioned as the key redox mediator associated with the oxidation of the amine group in amino acids. The electrochemical behavior of the Ni-Ti electrode in alkaline medium was very similar to the Ni electrode. However, the oxide film was found to be much stable on Ni-Ti than on Ni. Consequently, the Ni-Ti alloy electrode exhibited an excellent stability for constant-potential amperometric detection of amino acids in flow systems. For example, the relative standard deviation (R.S.D.) for the repetitive 100 injections of 50 muM (1.2 nmol) glycine over 10 h was less than 1%. It was postulated that the presence of Ti in the alloy stabilizes the microstructure of oxide layer on the electrode surface. The sensitivities of amino acids at the electrode were different, depending on their chemical structures. The detection limits obtained in a range from 0.9 pmol for arginine to 90.2 pmol for leucine and isoleucine. The Ni-Ti alloy electrodes have been demonstrated to be very suitable for the amperometric detection of underivatized amino acids in anion-exchange chromatography.     

Determination of Amino Acids at a Silver Oxide/Silver Phosphate Electrode and the Analysis of Structure‐Response Relationships

When a silver electrode is conditioned in a solution of 0.5 M sodium hydroxide with added sodium phosphate and using a dual pulse (500 mV/750 mV vs. Ag/AgCl), a stable silver(I)/silver(II) oxide surface is formed. It has been previously shown that various moieties react with the silver(II) oxide in a chemical oxidation at the outer surface of the oxide layer. This is then followed by re‐oxidation of the silver with the generation of current at approximately 500 mV relative to the silver/silver chloride electrode. Previously we found the need to remove carbon dioxide from the base and condition the electrode in a solution containing phosphate ion in order to provide mechanical stability to the oxide layer. We have previously shown this electrode to be applicable to the detection of a variety of carbohydrates. The applicability of the silver oxide/silver phosphate electrode to the post‐chromatographic amperometric detection of amino acids was investigated. Calibration studies of amino acids representative of the various classes demonstrated good sensitivity and linearity in the 1–100 μM range. Responses of amino acids were measured using glucose as an external standard, in order to correct for variability of the oxide layer. Relative responses of the amino acids ranged from 3 down to 0.1. Correlation with structure suggested the importance of absorption in determining the rate of oxidation. Comparison of arginine with n‐benzoyl‐L ‐arginine ethyl ester indicated that side chains as well as the backbone amino group can be oxidized. A Levitch plot of alanine was shown to be linear from approximately 30 to 300 radians per second spin rate at a scan rate of 50 mV per second. Application to post‐chromatographic detection was demonstrated.     

Determination of the anticancer drug 5-fluorouracil added to blood serum by liquid chromatography with anodic amperometric detection

Liquid chromatography with on-line anodic amperometric detection is used after a liquid/solid extraction step for the determination of the anticancer drug 5-fluorouracil added to blood serum. A detection limit of 15 ng ml^?1 can be achieved. The anodic electrochemical behaviour of the drug at a mercury electrode, which is the basis of the detection principle, is also described. Anodic amperometric detection at a mercury electrode is compared with detection at a glassy carbon electrode in terms of sensitivity, linearity and selectivity.     

Operational parameters of voltammetric high-performance liquid chromatographic detectors with copper electrodes and application to a determination of some fodder biofactors

Two versions of an amperometric detector with a copper working electrode have been constructed and tested for high-performance liquid chromatography (HPLC). The performance of the detectors was studied using selected amino acids. The dependence of the detector response on the mobile phase flow-rate was studied in the range common in both macro- and microcolumn HPLC (5 microliter/min to 1.0 ml/min). It has been found that the detection sensitivity generally increases with decreasing flow-rate, i.e., the detector response is governed by the rate of the complexation reaction between the cupric ions and the solutes. This fact makes amperometric detection with a copper electrode especially useful for microcolumn separations. For all 20 amino acids studied, calibration curve parameters and detection limits have been determined; the latter vary from 0.4 to 18 ng in the injected volume. The amino acids can also be sensitively detected in a medium of 0.1-1.0 M ammonia, which is promising for the use of strong anion exchangers in amino acid separations. Choline can also be detected at a copper electrode, with a detection limit of 40 ng. An HPLC method with amperometric detection at a copper electrode has been developed for the determination of lysine, methionine and choline in fodder biofactors.     

检测CO_2的脉冲电势调制库仑技术

应用脉冲电势调制技术,通过控制电极电势可将CO2还原为以吸附COads为主的中间物;之后再将电极电势阶跃到该物种能发生电化学氧化的电势,测量它的暂态氧化电流,得到积分电量Q随被测CO2浓度C的变化关系.对给定的还原时间,在CO2浓度为0.015%～20%的范围内,氧化电量Q与CO2浓度C有确定的对应关系.其中,当还原时间较短及CO2浓度较低情况下,两者有近似线性关系.这是一种可在较宽浓度范围内测定CO2的简便有效方法.     

Comparison of metallic electrodes for constant-potential amperometric detection of carbohydrates,amino acids and related compounds in flow systems

Constant-potential amperometric detection of carbohydrates, amino acids, and other aliphatic organic compounds is possible by means of their oxidation in alkaline solution at a variety of metal/metal oxide electrodes including Pt, Au, Cu, Ni, Ag and Co. The experimental conditions required for optimum detection and the analytical performance obtainable vary widely for different electrode materials and analytes. In this work, the cyclic voltammetric behavior exhibited by selected analytes (glucose, glycine, lactic acid, ethylamine and ethanol) at each of these electrodes was used to determine the optimum potentials suitable for flow detection so that the capabilities of the different metal electrodes could be evaluated and systematically compared. In general, the Cu electrode was found to provide superior detection capabilities in terms of its range of response, detection limits and especially stability. Despite the fact that Pt and Au are typically used only with a pulsed applied potential, both can provide long-lived constant-potential detection of carbohydrates and other analytes at low concentrations if the potentials ere carefully chosen and the electrodes are allowed to undergo an initial stabilization period.     

Chemical Adsorption onto an ITO Substrate of Single‐Wall Carbon Nanotube Functionalized by Carboxylic Groups as an Efficient Support for Electrocatalyst

A single‐wall carbon nanotube functionalized by carboxylic groups (SWNT‐CA) was found to be adsorbed on an indium tin oxide (ITO) electrode by chemical interaction between carboxylic groups and the ITO surface. The adsorption experiments indicated that the narrow pH conditions (around pH 3.0) exist for its adsorption which is restricted by preparation of stable fluid dispersion (favorable at higher pH) and by the chemical interaction (favorable at lower pH). Atomic force microscopic (AFM) measurements suggest that fragmented SWNT‐CA are adsorbed, primarily lying on the surface. Electrochemical impedance analysis indicated that an electrochemical double layer capacitance of the SWNT‐CA/ITO electrode is considerably higher than that for the ITO electrode, suggesting that the interfacial area between the electrode surface and the electrolyte solution is enlarged by the SWNT‐CA layer. Pt particles were deposited as a catalyst on the bare ITO and SWNT‐CA‐coated ITO (SWNT‐CA/ITO) electrodes to give respective Pt‐modified electrodes (denoted as a Pt/ITO electrode and a Pt/SWNT‐CA/ITO electrode, respectively). The cathodic current for the Pt/SWNT‐CA/ITO electrode was 1.7 times higher than that for the Pt/ITO electrode at 0.0 V, showing that the Pt/SWNT‐CA/ITO electrode works more efficiently for O₂ reduction at 0.0 V due to the SWNT‐CA layer. The enhancement by the SWNT‐CA layer is also effective for electrocatalytic proton reduction. It could be ascribable to the enlarged interfacial area between the electrode surface and the electrolyte solution.     

Amperometric determination of underivatized amino acids at a nickel-modified gold electrode by anion-exchange chromatography

A nickel-based composite electrode obtained by anodic electrodeposition of nickel (III) oxyhydroxide film on the gold electrode substrate was characterized as an amperometric sensor and successfully applied to the determination of underivatised amino acids in flow-through systems. The electrodeposition of nickel oxyhydroxide films was obtained by cycling a gold electrode between 0.0 V and +1.0 V vs. a saturated calomel electrode in a 80 microM Ni2+ solution buffered at pH 10 with NaHCO3/Na2CO3. The resulting Au-Ni composite electrode exhibits good stability in alkaline medium and can be used as an amperometric sensor of underivatised amino acids at a fixed applied potential (+0.55 V vs. Ag/AgCl). The detection limits (S/N=3) for all investigated compounds ranged between 5 and 30 pmol injected, while the linear ranges spanned over two or three orders of magnitude. The contents of several free amino acids in two sample cheeses from different brands were evaluated by calibration graphs.     

Modification of a Pt surface by spontaneous Sn deposition for electrocatalytic applications. 2. Oxidation of CO,formaldehyde, formic acid,and methanol

The electrocatalytic activity of a spontaneously tin-modified Pt catalyst, fabricated through a simple dip-coating method under open-circuit conditions and characterized using surface analysis methods, was studied in electrooxidation reactions of a preadsorbed CO monolayer and continuous oxidation of methanol, formic acid, and formaldehyde in the potentiodynamic and potentiostatic modes. The catalytic activity of the tin-modified Pt surface is compared with that of a polycrystalline Pt electrode. Spontaneously Sn-modified Pt catalyst shows a superior activity toward adsorbed CO oxidation and thus can be promising for PEFC applications. The methanol oxidation rate is not enhanced on the Sn-modified Pt surface, compared to the Pt electrode. Formic acid oxidation is enhanced in the low potential region on the Sn-modified surface, compared to the Pt electrode. The formaldehyde oxidation rate is dramatically increased by modifying tin species at the most negative potentials, where anodic formaldehyde oxidation is completely suppressed on the pure Pt electrode. The results are discussed in terms of poisoning CO intermediate formation resulting from dehydrogenation of organic molecules on Pt sites, and oxidation of poisoning adsorbed CO species via the surface reaction with OH adsorbed on neighboring Sn sites.     

基于纳米金与碳纳米管-纳米铂-壳聚糖纳米复合物固定癌胚抗原免疫传感器的研究

采用纳米金(nano-Au)、多壁碳纳米管-纳米铂-壳聚糖的纳米复合物(MWNT-Pt-CS)及电子媒介体硫堇(Th)固载抗体制得高灵敏癌胚抗原免疫传感器．首先, 于壳聚糖溶液中用NaBH4还原H2PtCl6, 并将多壁碳纳米管分散于其中制得碳纳米管-纳米铂-壳聚糖纳米复合物, 并将其滴涂在玻碳电极上成膜; 然后, 吸附电子媒介体硫堇制得硫堇/碳纳米管-纳米铂-壳聚糖(Th/MWNT-Pt-CS)修饰电极．利用壳聚糖和硫堇分子中大量的氨基固定纳米金并吸附癌胚抗体(anti-CEA); 最后, 用辣根过氧化物酶(HRP)封闭活性位点从而制得高灵敏电流型免疫传感器．在优化的实验条件下, 该传感器响应的峰电流值与癌胚抗原(carcinoembryonic antigen)浓度在0.5～10和10～120 ng/mL的范围内保持良好的线性关系, 检测限为0.2 ng/mL．     

The effect of iodide on the electrochemical reduction of thick oxide films formed on platinum electrodes in sulfuric acid solution

The reduction of thick oxide films formed on Pt under severe anodic conditions was studied in the presence of adsorbed I^?. The Pt electrode covered with a thick oxide film does not adsorb I^?. However, when a superficial monolayer oxide on the thick oxide has been reduced, I^? is irreversibly adsorbed. Iodide adsorbed on its surface blocks the adsorption of hydrogen and retards markedly the cathodic reduction of the inner thick oxide remaining. It was found that the reduction rate of the inner oxide depends only on the coverage by hydrogen, which coexists with adsorbed I^?. These results support the proton-electron theory which has previously been proposed for the explanation of the characteristic reduction of the thick oxide films.     

Simultaneous Detection of Peracetic Acid and Hydrogen Peroxide by Amperometry at Pt and Au Electrodes

Based on preliminary voltammetric investigations at both Pt and Au electrodes in aqueous solutions buffered at different pH values in the range 0–10, two possible profitable triple‐pulse amperometric approaches were developed for determining simultaneously peroxyacetic acid (PAA) and hydrogen peroxide present in the same samples. At both surfaces a pulsed waveform applied at rotating‐disc electrodes was adopted to take advantage on one hand of the optimized signal reproducibility achieved by this potential multistep antifouling approach and on the other hand of the constant thickness of the diffusion layer, which is necessary when the recording of time‐independent currents is desired. At a rotating‐disc Pt electrode an anodic selective signal was indeed recorded for H₂O₂ alone, while PAA contents could be inferred only from the difference of convenient signals, since at all pHs explored its sole cathodic reaction could be observed at potentials coincident with those proper for the reduction of H₂O₂ too. The same pulse approach at Au electrodes instead provided totally independent signals for the two analytes considered, thus proving to be suitable for their independent detection. In fact, H₂O₂ alone undergoes anodic oxidation also at this surface, while the reduction of PAA occurs at potentials less cathodic than those required for H₂O₂. At both electrodes, the best results turned out to be achieved at pH 0 in terms of both precision (±2–4%) and detection limits (0.2–0.3 mM), as well as of linear range which extended for about three orders of magnitude. The kinetics of the equilibrium involving the generation of H₂O₂ from the reaction of PAA with water was also evaluated, since it was suspected of making unreliable the proposed amperometric approaches.     

Carbon-nanotube/copper composite electrodes for capillary electrophoresis microchip detection of carbohydrates

The preparation of carbon nanotube (CNT)/copper composite electrodes, based on co-mixing CNT and Cu powders within mineral oil, is described. The new composite electrode is used for improved amperometric detection of carbohydrates following their capillary electrophoresis (CE) microchip separations. The CNT/Cu composite electrode detector displays enhanced sensitivity compared to detectors based on copper or CNT alone. The marked catalytic action of the CNT/Cu composite material permits effective low potential (+0.5 V vs. Ag/AgCl) amperometric detection, and is coupled to the renewability, bulk modification and versatility advantages of composite electrodes. The CNT/Cu composite surface also leads to a greater resistance to surface fouling compared to that observed at the copper electrode. Factors affecting the electrocatalytic activity and the CE microchip detection are examined and optimized. The CNT/Cu composite electrode is also shown to be useful for the detection of amino acids as indicated from preliminary results. While the present work has focused on the enhanced CE microchip detection of carbohydrates and amino acids, the CNT/metal-composite electrode route should benefit the detection of other important groups of analytes.     

Pulsed electrochemical detection of orotic acid by an activated potential waveform at a gold working electrode following anion-exchange chromatography

The application of activated pulsed amperometric detection (APAD) for the determination of orotic acid (OrA) in real samples at a gold working electrode in alkaline solutions, in combination with anion-exchange chromatography, is reported. Such an activated potential waveform was designed with an initial step that involves the formation of redox active species (e.g., adsorbed AuOH/AuO), which in turn is halted upon lowering the applied potential at the detection value while the adsorbed gold hydroxide/oxide species are still catalytically active. A direct comparison between the activated potential waveform and the more commonly used pulsed amperometric detection showed roughly a 20-fold increase in sensitivity. The chromatographic separation of OrA was accomplished by using a microbore anion-exchange column eluted with an isocratic mobile phase composed of 100 mM NaOH+40 mM NaNO(3). Orotic acid was determined at the concentration ranges of 0.2-30 microM (r=0.9997) with an absolute detection limit of 80 pg (10 microL injected). The levels of OrA in cows' milk samples evaluated by standard additions, using 5-aminoorotic acid as an internal standard, ranged from 56 to 126 mg/L. Lower levels were found in raw sheep's milk (<20 mg/L). The assay is shown to be very useful in clinical investigations where relatively high levels of OrA in human urine are correlated to metabolic diseases.     

Determination of polar organic compounds by potentiometry at an anodically pretreated nickel oxide electrode

The open-circuit potential drop of an oxidatively pretreated nickel electrode in 0.1 M NaOH was used to develop a technique for the determination of alcohols, amino acids, carbohydrates, etc., in aqueous solution. The electrode pretreatment consisted of the formation of nickel hydrated oxides on the electrode surface with an oxidation state > 2. Both electrochemical and chemical pre-oxidation of the electrode surface were examined. The analytical signal was the enhancement of the potential drop corresponding to analyte concentration. The analytical signal was linearly related to the logarithm of the analyte concentration. The limits of determination ranged from 1 mM for low-molecular-weight to 0.02 mM for alkyl polyether alcohols. The flow-injection technique allows convenient pretreatment and direct observation of the analytical signal. Interferences from chloride and calcium can be readily eliminated by excluding them during electrode pretreatment. Potentiometric measurements were correlated with amperometric anodic responses at the nickel oxide electrode, allowing an unusual direct comparison of the two methods.