Indirect amperometric detection of underivatized amino acids in microcolumn liquid chromatography with carbon film based ring-disk electrodes

An indirect amperometric detection of underivatized amino acids has been developed using a carbon film based ring-disk electrode (CFBRDE) in microcolumn liquid chromatography (LC). Bromide present in the mobile phase could be efficiently oxidized to bromine at the upstream (disk) electrode, and was subsequently detected at the downstream (ring) electrode. Most of the underivatized amino acids that are electroinactive under conventional amperometric conditions react rapidly with the electrogenerated bromine, the concentration of amino acids can therefore be indirectly determined by continuously monitoring the reduction current of bromine. The signal monitored at the downstream electrode was largely dependent on the bromide concentration in the mobile phase. Under optimized conditions, the response linearly increased with the concentration for most of the amino acids over a concentration range of 1-100 microM, with a correlation coefficient of 0.990-0.993. The detection limits for most of the amino acids were below 1 microM (0.2 pmol). It was demonstrated that detection with a ring-disk electrode offers the advantages of achieving a much higher collection efficiency caused by a decrease in flow rate in the microcolumn LC.     

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.     

Electrochemical detectors – tailormade techniques for liquid chromatography and capillary electrophoresis?

Electrochemical detectors for liquid chromatography and capillary electrophoresis are reviewed with special emphasis on electrode materials that allow the amperometric detection of otherwise non-electroactive compounds such as aliphatic alcohols, carbohydrates or amino acids. Noble metal electrodes can catalyze the oxidation of aliphatic compounds in alkaline media if multistep potential-time waveforms are employed. Various metal and metal oxide electrodes such as Ni, Cu or Co allow the detection of carbohydrates and similar compounds under constant potential conditions. Metallic copper electrodes operating in an amperometric mode or in a potentiometric mode can also serve as selective detectors for complexing species. A range of applications in combination with chromatography and electrophoresis is summarized. The current state of electrochemical detectors indicates that both amperometric and potentiometric detectors are on the verge of becoming tailormade detectors for micro-separation techniques.     

Isocratic ion chromatographic determination of underivatized amino acids by electrochemical detection

An isocratic chromatographic separation with amperometric detection of underivatized amino acids at a copper oxyhydroxide modified glassy carbon electrode is described. A simple and sensitive quantitation procedure of amino acids without the need of tedious and time-consuming derivatization step was achieved by coupling anion-exchange chromatography with electrochemical detection. The effects of hydroxide, nitrate and acetonitrile concentration in the mobile phase on the capacity factors and peak resolution was evaluated. Under the optimized isocratic chromatographic conditions (i.e. 60 mM NaOH) and under constant applied potentials (i.e. 0.55 V versus Ag/AgCl) the detection limit ranged between 4 and 24 pmol injected and the linear dynamic range spanned generally over three or four order of magnitude for all investigated amino acid compounds. Direct determination of several common free amino acids in beer and milk samples were performed.     

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.     

Amperometric detection of ultra trace amounts of Hg(I) at the surface boron doped diamond electrode modified with iridium oxide

Iridium oxide (IrOx) films formed electrochemically on the surface boron doped diamond electrode by potential cycling in the range −0.2 to 1.2 V from a saturated solution of alkaline iridium(III) solution. A strongly adherent deposit of iridium oxide is formed after 5–10 potential scans. The properties, stability and electrochemical behavior of iridium oxide layers were investigated by atomic force microscopy and cyclic voltammetry. The boron doped diamond (BDD) electrode modified with electrodeposition of a thin film, exhibited an excellent catalytic activity for oxidation of Hg(I) over a wide pH range. The modified electrode shows excellent analytical performance for Hg(I) amperometric detection. The detection limit, sensitivity, response time and dynamic concentration ranges are 3.2 nM, 77 nA μM⁻¹, 100 ms and 5 nM–5 μM. These analytical parameters compare favorably with those obtained with modern analytical techniques and recently published electrochemical methods.     

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.     

Flow injection-pulse amperometric detection of ephedrine at a cobalt phthalocyanine modified carbon paste electrode

Direct detection of ephedrines and other underivatized amino compounds (amines, alicyclic amines, alkanolamines, and amino acids) can be carried out via electrocatalytic oxidation at a carbon paste electrode (CPE) modified with cobalt phthalocyanine (CoPC) in alkaline solution (0.10 mol L-1 NaOH). Most of the amino compounds tested could be determined using the CoPC/CPE in an amperometric flow detector. The analytical signal of ephedrine was stabilised by alternating the potential between an anodic detection potential of +0.30 V (+0.45 V for other amino compounds) applied for 220 ms and a cathodic reactivation potential of -0.30 V applied for 100 ms (potentials versus SCE). The linear response range for ephedrine was within 1-100 mumol L-1 and the detection limit was 0.8 mumol L-1 with a 100 microL sample loop and a typical sampling ra 60 h-1. The signal (oxidation peak current) reproducibility was 2-3%. The method was applied to the determination of ephedrine in pharmaceutical formulations with results comparable to those obtained with a standard spectrophotometric method.     

The electrochemical oxidation of homocysteine at boron-doped diamond electrodes with application to HPLC amperometric detection

The electrochemical oxidation of homocysteine was studied at as-deposited and anodized (oxidized) boron-doped diamond (BDD) thin film electrodes with cyclic voltammetry, flow injection analysis and high-pressure liquid chromatography with amperometric detection. At anodized boron-doped diamond electrodes, highly reproducible, well-defined cyclic voltammograms for homocysteine oxidation were obtained in acidic media, while as-deposited diamond did not provide a detectable signal. In alkaline media, however, the oxidation response was obtained both at as-deposited and anodized diamond electrodes. The potential sweep rate dependence of homocysteine oxidation (peak currents for 1 mM homocysteine linearly proportional to v(1/2), within the range of 0.01 to 0.3 V s(-1)) indicates that the oxidation involves a diffusing species, with negligible adsorption on the BDD surface at this concentration. In the flow system, BDD exhibited a highly reproducible amperometric response, with a peak variation less than 2%. An extremely low detection limit (1 nM) was obtained at 1.6 V vs. Ag/AgCl. In addition, the determination of homocysteine in a standard mixture with aminothiols and disulfide compounds by means of isocratic reverse-phase HPLC with amperometric detection at diamond electrodes has been investigated. The results showed excellent separation, with a detection limit of 1 pmol and a linear range of three orders of magnitude.     

The Direct Electrochemical Detection of Amino Acids at a Platinum Electrode in an Alkaline Chromatographic Effluent

Abstract

It is the general experience that most organic compounds including amino acids do not produce reversible or even quasi-reversible anodic waves at a Pt electrode under conditions of conventional cyclic voltammetry. Furthermore, amperometric detection of these compounds at a constant electrode potential is not successful because of the accumulation of adsorbed reaction products and/or an oxide film at the electrode surface. However, it is observed that a Pt electrode surface is cleaned quite effectively of adsorbed organic molecles and radicals simultanaeously with the anodic formation of the oxide layer. This oxidation of adsorbed organic species is concluded to be electrocatalyzed by PtOH formed as the first step in the production of the oxide layer (PtO). A pulsed-potential waveform applied at a frequency of ca. 1 Hz is demonstrated to provide direct amperometric detection of adsorbed amino acids at a Pt electrode. Satisfactory analytical precision (i.e., < 3% rel. std. dev.) results because the waveform reproducibly generates the catalytically active surface state at the Pt electrode. Both primary and secondary amino acids are determined with satisfactory detection limits: e.g., ca. 13 ng for glycine, 7 ng for phenylamine and 23 ng for hydroxyproline in 50-μL samples. Analytical response is concluded to depend on the adsorption isotherm of the amino acid being detected. Hence, the calibration plot of I/I_peak vs. 1/C^D is linear for low surface coverages. Results are shown for amperometric detection of a synthetic mixture of amino acids by anion-exchange chromatography using NaOH as the eluent and supporting electrolyte.     

Direct RP‐HPLC determination of underivatized amino acids with online dual UV absorbance,fluorescence, and multiple electrochemical detection

The combined use of a dual‐UV detector, a fluorimetric one and of a multiple electrochemical (EC) detector equipped with a dual electrode, consisting of a conventional size 3 mm diameter glassy carbon electrode (GCE) and of a pair of 30 μm thick carbon microfibers, is proposed for the determination of 15 amino acids, two dipeptides and creatinine. This online coupling of the above detection modes could partially replace amino acid analysis by derivatization methods, since it solves problems concerning the direct detection of selected underivatized amino acids. Additionally, it was proved that the use of multiple‐detection allows positive peak identification in a single chromatographic run, yields more information for free amino acids and solves in some cases the problem of chromatographic resolution. In order to optimize the detection conditions of the underivatized amino acids and related compounds by different detectors, their detection characteristics were determined by adequate preliminary experiments. The electro‐oxidation characteristics of the underivatized compounds of interest were determined by hydrodynamic voltammetry using a flow cell with a macrodisc GCE and by ex‐situ voltammetry using both a GCE of conventional size and a carbon fiber disk microelectrode. Important practical advantages of microfiber and microdisk electrodes with respect to macroelectrodes were demonstrated.     

Integrated pulsed amperometric detection of glufosinate, bialaphos and glyphosate at gold electrodes in anion-exchange chromatography.

A rapid and practical method for direct detection of the herbicides (glufosinate, bialaphos and glyphosate) in anion-exchange chromatography has been developed with integrated pulsed amperometric detection (IPAD). The electrochemical behavior of these herbicides showed catalytic currents based on the oxidation of amines in their structures. Waveform in IPAD was similar to that for amino acids, which exhibited adsorption/desorption catalytic features at gold electrode surface in alkaline solution. Under optimized conditions, detection limits (signal-to-noise ratio of 3) for glufosinate, bialaphos and glyphosate were 20, 65 and 50 ng ml(-1), respectively, with correlation coefficients of 0.995, 0.997 and 0.996 over concentration ranges of 0.1-45, 0.3-32 and 0.1-50 microg ml(-1), respectively. The relative standard deviations (n=5) were 1.7-3.0%. The present method was successfully applied to the determination of glyphosate in urine and serum.     

Determination of underivatized amino acids by high-performance liquid chromatography and electrochemical detection at an amino acid oxidase immobilized CuPtCI6 modified electrode

An amperometric sensor for amino acids based on the immobilization of amino acid oxidase on the surface of a CuPtCl(6)/GC is described. The amperometric current is due to the oxidation of H2O2 liberated during the enzyme reaction on the surface of the enzyme electrode. The electrode response characteristics as well as kinetic parameters have been evaluated. The enzyme electrode was characterized as an electrochemical biosensor, which was used as detector in high performance liquid chromatography (HPLC) for the determination of a mixture of amino acids with satisfactory results.     

Electrocatalytic oxidation and flow-injection determination of sulfur amino acids on a glassy carbon electrode modified by a nickel(II) polytetrasulfophthalocyanine film

The electrochemical oxidation of sulfur amino acids, i.e., cysteine, cystine, and methionine, is studied on a glassy carbon electrode modified by a film of nickel(II) polytetrasulfophthalocyanine (poly-NiTsPc). Poly-NiTsPc demonstrates a selective mediator activity in the oxidation of sulfur amino acids, depending on the pH of solution. The proper conditions for fabricating a polymer film on the surface of glassy carbon are found and the conditions of registering the maximal electrocatalytic effect on the modified electrode are determined. A procedure is proposed for the voltammetric determination and amperometric detection of cysteine, cystine, and methionine on an electrode coated by a poly-NiTsPc film under the conditions of flow-injection analysis (FIA). The linear relation of the electrocatalytic response of a composite electrode to amino acid concentration is observed to the level n × 10^?6 M in the static mode and n × 10^?9 M under FIA conditions.     

Electrooxidation of Aliphatic and Aromatic Amines at a Ni,Al Based Hydrotalcite Modified Electrode

The electrocatalytic oxidation of a wide series of aliphatic and aromatic amines was investigated in alkaline solutions at a gold electrode modified with a Ni,Al based hydrotalcite. All the amino compounds, with the exception of pyridine, were detected by the amperometric sensor. Chronoamperometric measurements at a rotating disk allowed us to use the same modified electrode for sequential analysis of several analytes in order to investigate the factors affecting the electrochemical response. By varying the characteristics of the substrate, parameters like steric hindrance, kind of amino group, and number of oxidizable sites have been studied. The concentration ranges, where the amperometric response was linear, have been determined for the amines investigated.     

Preparation of multiwall carbon nanotubes film modified electrode and its application to simultaneous determination of oxidizable amino acids in ion chromatography

A multiwall carbon nanotubes (MWNTs) film modified electrode was prepared and used as an amperometric sensor for the simultaneous determination of oxidizable amino acids including cysteine, tryptophane and tyrosine. The electrochemical behaviors of these amino acids at this modified electrode were studied by cyclic voltammetry (CV). The results indicated that the MWNTs chemically modified electrode (CME) exhibited efficient electrocatalytic activity towards the oxidation of these amino acids with relatively high sensitivity, stability and long-life. Following separation by ion chromatography (IC) with 2.0×10⁻³ mol l⁻¹ citric acid buffer solution (pH 6.5) as eluent, cysteine, tryptophane and tyrosine could be determined by the MWNTs CME successfully. Under the optimal conditions, the detection limits were 7.0×10⁻⁷ mol l⁻¹ for cysteine, 2.0×10⁻⁷ mol l⁻¹ for tryptophane and 3.5×10⁻⁷ mol l⁻¹ for tyrosine at the signal-to-noise of 3, respectively. The method was applied successfully to the determination of these substances in plasma.     

Determination of electroactive organic acids by anion-exchange chromatography using a copper modified electrode

An ion-chromatographic method combined with electrochemical detection at a copper-based chemically modified glassy carbon electrode (Cu-GC) has been shown to provide a simple analytical approach for the determination of some common organic acids in alkaline medium. Under the optimized isocratic chromatographic conditions (i.e. 0.1 M NaOH plus 80 mM CH3COONa), organic acids such as gallic, ascorbic, gluconic, lactobionic, galacturonic and glucuronic acid could be separated in less than 20 min. Under constant potential amperometric detection (i.e. 0.55 V vs. Ag-AgCl) the Cu-GC modified electrode allowed detection limits between 2 and 5 pmol for all investigated organic acids while the linear dynamic range spanned generally over three orders of magnitude. Examples of applications included the separation and quantitation of some common organic acids in vinegar, honey and tea samples, are given.     

Electrocatalysis and determination of hydrazine compounds in liquid chromatography at a mixed-valent cobalt oxide/cyanocobaltate film electrode

A glassy carbon electrode coated with an electrodeposited film of mixed-valent cobalt oxide/cyanocobaltate (CoO/CNCo) enabled hydrazine compounds to be catalytically oxidized at the greatly reduced overpotential and in a wide operational pH range (pH 2.0-7.0). Electrocatalytic activity at the CoO/CNCo modified electrode was evaluated with respect to solution pH, film thickness, supporting electrolyte ions, potential scan rate, operating potential, concentration dependence and other variables. The CoO/CNCo film electrode was completely compatible with a conventional reversed-phase liquid chromatographic (RP-LC) system. Practical RP-LC amperometric detection (RP-LCEC) of hydrazines was performed. A dynamic linear response range over three orders of magnitude and a detection limit at the pmol level were readily obtained. The CoO/CNCO film electrode exhibited excellent electrocatalytic stability in the flowing streams.     

基于表面活性剂单分子层修饰碳糊电极的一氧化氮电化学传感器及其应用

报道了一种表面活性剂单分子层修饰碳糊电极,并用于NO的高灵敏电化学检测。研究表明,表面活性剂通过烷基链在电极表面形成的疏水性单分子层微环境对NO的电化学响应具有较好的促进作用。其中,阳离子表面活性剂十六烷基三甲基溴化铵(CTAB)对NO的电化学氧化表现出最强的催化活性和增敏作用。在Nafion膜覆盖的CTAB修饰碳糊电极上,NO的安培响应与其浓度在3.6×10-8～1.8×10-5mol/L范围内呈良好的线性关系,检出限为1.8×10-8mol/L。该电极作为低成本、高灵敏的NO电化学传感器,被成功应用于大鼠肺组织细胞中NO释放的实时监测。