A pulsed potential waveform displaying enhanced detection capabilities towards sulfur-containing compounds at a gold working electrode

Pulsed electrochemical detection of sulfur-containing compounds was successfully investigated by applying a four-step potential waveform at a gold working electrode. This potential waveform called APAD, which stands for activated pulsed amperometric detection, is composed of an activation potential step added to a conventional three-step potential waveform. A key advantage of the APAD at the Au electrode is the ability to enhance sensitivity through the use of a short potential pulse (E(ACT) = +750 mV versus Ag/AgCl and tACT approximately 90 ms) during which the formation of redox active species (presumably OH*) are able to efficiently oxidize organosulfur compounds. The APAD waveform parameters were optimized to maximize the signal-to-noise ratio (S/N) and successfully applied for the sensitive detection of lipoic acid, biotin, iminobiotin, methionine, cystine, cysteine, homocysteine, homocystine, N-acetylcysteine and glutathione, following their separations by high-performance anion-exchange chromatography (HPAEC) using alkaline mobile phases. The detection limits (S/N = 3, 10 microL injected) ranged from 0.3 for cysteine (400 pg) to 0.02 micromol/L for biotin (50 pg) and methionine (30 pg). The response of sulfur-, amine- and alcohol-based compounds was compared by using four selected pulsed potential waveforms. It was found that the APAD exhibits excellent sensitivity for thiocompounds outperforming all other pulsed potential waveforms. Ratios of the peak areas for APAD and the six-step potential integrated waveform increased from 3.2 +/- 0.4 to 13.5 +/- 0.6 for lipoic acid and biotin, respectively.     

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.     

Integrated pulsed amperometry for the analysis of organic compounds

Integrated pulsed amperometric detection (IPAD) was applied for the detection of organic compounds for flow injection analysis. The pulse waveform used in the integrated pulsed amperometry consisted of three steps: detection potential, oxidation potential, and adsorption potential. The pulse waveform was applied to the working electrode as the analyte flowed through the electrochemical cell. Unlike ordinary pulsed amperometry, a faradaic current was integrated over the duration period of the detection potential in the IPAD. Therefore, the total charge was measured by integrating the current after the detection potential was applied. The current for the initial 10 ms, after applying the detection potential, was excluded from the integration due to a large charging current at the initial period. Compared with pulsed amperometry, integrated pulsed amperometry provides a better signal-to-noise ratio and a lower detection limit. This method was applied to the quantitative analysis of thiourea as a representative analyte of organosulfur compounds in a flow injection analysis.     

离子色谱中的安培检测方法及其应用

介绍了离子色谱中的安培检测方法(包括恒电位安培检测法、脉冲安培检测法和积分脉冲安培检测法)的原理和应用。脉冲安培检测法与高效阴离子交换色谱结合(HPAEC-PAD)是一种新的分析糖类化合物的方法;积分脉冲安培检测法与高效阴离子交换色谱结合(HPAEC-IPAD)是一种新的氨基酸分析方法。     

高效液相色谱-脉冲安培检测法测定脑微透析溶液中单胺类神经递质

详细对比了脉冲安培与恒电位安培检测单胺类神经递质的响应值,结果表明冲安培检测上述物质的响应值是恒电位安培检测的1.2倍。建立了以3.4 -二羟基苄胺（DHBA）为内标的定量方法。在微透析溶液中的微量神经递质的回收率在74％－101％之间。     

Pulsed coulometric detection of carbohydrates at a constant detection potential at gold electrodes in alkaline media

A significant increase in the signal-to-noise ratio for the pulsed amperometric detection (PAD) of carbohydrates at gold electrodes is obtained by increasing the length of the current integration period (t_i) from the traditional value of 16.7 ms (i.e., $\text{1}\text{60}$ Hz). For t_i > 16.7 ms, the integrated response (q, coulombs) is plotted as the signal. This pulsed coulometric detection (PCD) is applied in a flow-injection system. For t_i = 500 ms, the detection limit with the instrumentation used is 1 μM (S/N = 2) for glucose which is a significant improvement on the value 35 μM found with PAD. The absolute detection limits for glucose and sucrose are ca. 50 pmol and 125 pmol, respectively, in 50-μl samples. Calibration plots (q_p vs. C^b) for PCD are linear over significantly larger dynamic ranges than those observed for PAD because of the lower detection limits.     

Coupling Capillary Electrophoresis and Pulsed Electrochemical Detection

Pulsed electrochemical detection (PED) is an excellent method for detection of analytes that normally foul electrodes. In PED, the detection electrode is first cleaned at a high positive potential, then reactivated at a negative potential dissolving the surface oxide, and finally used to oxidize the analyte at a moderate positive potential. Due to the advantages and versatility of PED, many different variations of the detection waveform can be found in literature. This review focuses on application of PED to CE and in particular, the most commonly used modes: pulsed amperometric detection (PAD) and integrated pulsed amperometric detection (iPAD).     

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.     

Pulsed voltammetric/amperometric detection of polycyclic aromatic sulfur heterocycles (PASHs) at the gold disc electrode for studies in petroleum asphalts

This work describes the voltammetric and amperometric behavior of a high number of PASHs (sulfides, thiophenes, benzothiophenes, dibenzothiophenes, indenothiophenes, naphtothiophenes, thienothiophenes, phenanthrothiophenes, and acenaphtothiophenes) at gold disc electrodes aiming at their identification and determination in petroleum asphalts. The adsorption/redox processes expected for sulfur compounds at gold electrodes could be observed in all the studied PASHs in DMSO and hydromethanolic medium. Differential pulse (DP) voltammetry in non-aqueous solutions (0.1 mol L⁻¹ NaClO₄ in DMSO) was approached for determining non-volatile PASHs in asphalts submitted to different aging processes. It was found herein that the DP voltammetric monitoring of PASH oxidation at + 0.7 V (vs. Ag/AgCl/LiCl 3 mol L⁻¹) for virgin/aged asphalts can be used for the comparative study of asphalts based on the consumption of PASHs. Additionally, pulsed amperometric detection (PAD) in hydroalcoholic solution (10 mmol L⁻¹ acetate buffer in 65% methanol) coupled with a chromatographic separation was approached for determining volatile PASHs in asphalts submitted to thermal decomposition processes. A detection cycle of 2 s involving oxidative (0.4 s at + 0.4 V) and reductive (1.2 s at − 1.0 V) cleaning pulses after a detection pulse of − 0.8 V (0.4 s) applied successively to the gold electrode (vs. Pd/PdO) was found to be optimal for regenerating the gold surface during successive chromatographic runs of PASHs. Thus, reversed-phase liquid chromatography (LC)–coupled PAD was found useful to separate a complex mixture of PASHs. The optimized PAD and LC separation was further applied to investigate the presence of electroactive PASHs as volatile compounds in asphalt fumes generated at 260 °C.

     

Simplified current decoupler for microchip capillary electrophoresis with electrochemical and pulsed amperometric detection

There is a need to develop broadly applicable, highly sensitive detection methods for microchip CE that do not require analyte derivatization. LIF is highly sensitive but typically requires analyte derivatization. Electrochemistry provides an alternative method for direct analyte detection; however, in its most common form, direct current (DC) amperometry, it is limited to a small number of easily oxidizable or reducible analytes. Pulsed amperometric detection (PAD) is an alternative waveform that can increase the number of electrochemically detectable analytes. Increasing sensitivity for electrochemical detection (EC) and PAD requires the isolation of detection current (nA) from the separation current (muA) in a process generally referred to as current decoupling. Here, we present the development of a simple integrated decoupler to improve sensitivity and its coupling with PAD. A Pd microwire is used as the cathode for decoupling and a second Au or Pt wire is used as the working electrode for either EC or PAD. The electrode system is easy to make, requiring no clean-room facilities or specialized metallization systems. Sensitive detection of a wide range of analytes is shown to be possible using this system. Using this system we were able to achieve detection limits as low as 5 nM for dopamine, 74 nM for glutathione, and 100 nM for glucose.     

Platinum particles-modified electrode for HPLC with pulsed amperometric detection of thiols in rat striatum

A new chemically modified electrode based on the immobilization of Pt particles is fabricated and exhibits electrocatalytic oxidation for L-cysteine (L-Cys), glutathione (GSH) and penicillamine (PEN) with relatively high sensitivity. It is also adaptable to HPLC for pulsed amperometric detection (PAD) of these thiols. PAD largely improves the detection sensitivity because the alternated polarizations can effectively clean and reactivate the electrode surface. It is shown that the peak currents of L-Cys, GSH and PEN are linear to their concentrations, with the calculated detection limit of 1.1 x 10(-7), 1.8 x 10(-7) and 3.8 x 10(-7) mol L(-1), respectively (S/N = 3). The method has been successfully applied to assess the contents of L-Cys and GSH in rat striatal microdialysates. The average contents of the two analytes in rat striatum are 2.6 x 10(-6) and 2.8 x 10(-6) mol L(-1), respectively.     

Direct detection of renal function markers using microchip CE with pulsed electrochemical detection

Creatinine, creatine, and uric acid are three important compounds that are measured in a variety of clinical assays, most notably for renal function. Traditional clinical assays for these compounds have focused on the use of enzymes or chemical reactions. Electrophoretic microchips have the potential to integrate separation power of capillary electrophoresis with devices that are small, portable, and have the speed of conventional sensors. The development of a microchip CE system for the direct detection of creatinine, creatine, and uric acid is presented. The device uses pulsed amperometric detection (PAD) to detect the nitrogen-containing compounds as well as the easily oxidizable uric acid. Baseline separation of creatinine, creatine and uric acid was achieved using 30 mM borate buffer (pH = 9.4) in less than 200 s. Linear calibration curves were obtained with limits of detection of 80 microM, 250 microM and 270 microM for creatinine, creatine and uric acid respectively. An optimization of the separation conditions and a comparison of PAD with other amperometric detection modes is also shown. Finally, analysis of a real urine sample is presented with validation of creatinine concentrations using a clinical assay kit based on the Jaffé reaction.     

Simultaneous determination of amino acids and carbohydrates by anion-exchange chromatography with integrated pulsed amperometric detection

A direct, sensitive, simple and practical method for simultaneous determination of amino acids and carbohydrates by anion-exchange chromatography with integrated pulsed amperometric detection was developed. The retention behavior of amino acids and carbohydrates on the anion-exchange column and the detection of amino acids and carbohydrates at different integrated pulsed amperometric detection waveforms were investigated. The optimized gradient eluent conditions for analysis of 17 amino acids and nine carbohydrates were obtained. Separation time was 100 min. Detection limits for amino acids and carbohydrates were 5.2-207.1 nM under injection volume of 25 microl. The RSDs of peak area were 1.2-3.3%. The calibration graphs of peak area for the analytes were linear over about three orders of magnitude with a correlation coefficient of 0.9950-0.9999. The method was applied to determine amino acids and carbohydrates in a liquid condiment with satisfactory results.     

Determination of phytosiderophores by anion-exchange chromatography with pulsed amperometric detection

Phytosiderophores of the mugineic acid family are separated by anion-exchange HPLC using NaOH gradient elution. Separation of mugineic acid (MA), 2'-deoxymugineic acid (DMA), 3-hydroxymugineic acid (HMA) and 3-epi-hydroxymugineic acid (epi-HMA) is obtained within 15 min. Detection of the underivatised phytosiderophores is performed using pulsed amperometric detection (PAD) at pH 13. The sensitivity of the detection increases in the order DMA < MA < HMA < epi-HMA and respective detection limits of 5 microM (DMA), 1 microM (MA) and < 0.5 microM (HMA, epi-HMA) are achieved. PAD is discussed in comparison with the well-established fluorimetric detection method after post-column derivatisation with ortho-phthaldialdehyde. The main advantage of PAD is the simplicity of the method (no derivatisation) and the high sensitivity for hydroxylated mugineic acids. The method is used for the determination of phytosiderophores in root washings of iron-deficient and non-deficient wheat and barley plants.     

A consideration of electrode polishing prior to application of pulsed amperometric detection

An examination was made of the effect of electrode polishing on the direct pulsed amperometric detection (PAD) and indirect PAD response to penicillin G. The polishing procedure produced a temporary loss of sensitivity for both types of PAD, which over time relaxed back to higher levels. A temporary increase in the electrode kinetics can be observed as well, particularly for a PAD waveform in the crossover region between direct and indirect PAD. In extreme cases, the penicillin G response can undergo a complete polarity reversal. By observing post-polish equilibration times, it was recommended that the direct and indirect PAD user observe 3 and 5 h delay times, respectively, to insure stable electrode response following an electrode polish.     

Use of disposable gold working electrodes for cation chromatography-integrated pulsed amperometric detection of sulfur-containing amino acids

We have prepared disposable thin-film gold working electrodes on polymeric substrates. Our microfabrication process allows for inexpensive and reproducible mass production of such electrodes. We utilize this new type of electrode in flow-through electrochemical cells to replace the conventional non-disposable gold working electrodes for integrated pulsed amperometric detection (IPAD) of compounds separated by high-performance cation-exchange chromatography. Using two S-containing amino acids (homocysteine and cysteine) as test compounds, we have modified a previously reported waveform for optimum performance with disposable gold electrodes. With the help of the same two test substances we have characterized the analytical performance of disposable gold electrodes under the new conditions. Compared to non-disposable working electrodes, the disposable working electrodes generated equal or better results in the limit of detection, linearity of calibration and reproducibility. When used with a new IPAD waveform, the disposable electrodes functioned reproducibly for 3 days. At the end of the specified usage period of 3 days, the disposable electrodes are simply replaced. Reconditioning by polishing is thus no longer required.     

铜/金修饰电极-阴离子交换色谱-直流安培法测定阿米卡星注射液组分

建立了一种用阴离子交换色谱分离、以自制铜/金修饰电极为工作电极的直流安培电化学法(DC)直接检测硫酸阿米卡星注射液中主要组分及杂质含量的分析方法。考察了流动相浓度、测定电位等参数对色谱分离和测定的影响。在固定相为CarboPacPA10阴离子交换柱、流动相为26 mmol/L NaOH的色谱条件下,检测电位为0.64 V时,阿米卡星在0.0005~0.02 g/L(r=0.9989)和0.02~0.2 g/L(r=0.9991)两个浓度范围内呈线性。与裸Au电极在采用脉冲安培检测模式(PAD)时相比,电化学检测所需要的碱性强度低(pH<13),而且测定灵敏度高,线性范围宽。本方法不需要柱前和柱后衍生化,能同时测定硫酸阿米卡星注射液中的主要组分和杂质组分。修饰电极制作方法简单,催化稳定性好,可望被应用到流动注射、毛细管电泳等其它流动体系中,对硫酸阿米卡星原料药、注射液等实际样品中的各组分进行测定。     

Pulsed amperometric detection of carbohydrates on an electrophoretic microchip

The first report of pulsed amperometric detection (PAD) on an electrophoretic microchip is presented. A hybrid poly-(dimethylsiloxane)/glass device was coupled with a platinum working electrode for the electrochemical detection of glucose, maltose, and xylose. Under optimized detection conditions, glucose was found to respond linearly from 20 to 500 microM with a measured detection limit of 20 microM. The coupling of PAD with a microchip provides a straightforward approach to the analysis of a wide range of carbohydrates using microfluidics.     

Sub-millimolar determination of formalin by pulsed amperometric detection

Formalin, formaldehyde in the presence of methanol, was determined by pulsed amperometric detection (PAD). A triple waveform using E_det=−0.3 V (t_det=30 ms), E_oxd=+0.8 V (t_oxd=200 ms), and E_red=−0.8 V (t_red=350 ms) versus Ag/AgCl was applied at a Au electrode for detection in a flow injection (FI) system. The approach was rapid and yielded a sub-millimolar detection limit (0.0129 mM) with a dynamic range up to 100 mM. A precision of 8.8% R.S.D. at 1.0 mM for two hundred repetitive injections by the FI-PAD was obtained, whereas holding at a constant potential (−0.3 V versus Ag/AgCl) for anodic oxidation of formaldehyde caused the response to decrease dramatically after a few measurements. The method developed was used to analyze the formalin contents of water from rinsed samples of vegetables and fruit and ice-melt from seafood, and the method showed good agreement with the liquid chromatography (LC) method.     

高效阴离子交换色谱-脉冲安培检测法分析啤酒和麦汁中的糖

建立了高效阴离子交换色谱-脉冲安培检测(HPAEC-PAD)同时测定单糖、二糖和多种低聚糖的方法。采用水、0.25 mmol/L NaOH溶液和1 mol/L NaAc溶液三元梯度淋洗,在CarboPac PA-100色谱柱上,11种糖在40 min内达到良好分离;采用积分脉冲安培检测方法,无需对样品进行复杂的前处理或衍生反应便可直接检测。11种糖的检出限(S/N=3)在13～88 pg范围内。将该方法用于啤酒和麦汁样品中单糖、二糖及低聚糖的分析取得了很好的结果,样品中的加标回收率为81%～107%。