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).     

Characterization of etched electrochemical detection for electrophoresis in micron inner diameter capillaries

An enhanced etched electrochemical (EC) detection technique has been developed for CE in micron inner diameter capillaries. The design improvements allow for better alignment between the capillary bore and the electrode. This new method involves utilizing a carbon fiber microelectrode and etching both the carbon fiber and the detection end of a micrometer-sized inner diameter capillary to limit dead volume and analyte diffusion at the amperometric EC detector. To understand the factors affecting enhanced detector efficiency, a detailed examination of the relationship between detector design and performance has been completed by exploring the effects of varying electrode diameter, tip shape, and size, in addition to the etch length of the capillary outlet. The enhanced detection provides peak efficiencies as high as 75000 theoretical plates and estimated detection limits as low as 40 nM for dopamine. This etched detection method should further facilitate volume-limited sample analysis by CE.     

A glass capillary ultramicroelectrode with an electrokinetic sampling ability.

A glass capillary ultramicroelectrode (tip diameter approximately 1.2 microm) having an electrokinetic sampling ability is described. It is composed of a pulled glass capillary filled with an inner solution and three internal electrodes (Pt working and counter electrodes and an Ag/AgCl reference electrode). The voltammetric response of the capillary electrode is based on electrokinetic transport of analyte ions from the sample solution into the inner solution across the conical tip. It was found that the electrophoretic migration of analytes at the conical tip is faster than electroosmotic flow, enabling electrokinetic transport of analyte ions into the inner solution of the electrode. By using [Fe(CN)6]4- and (ferrocenylmethyl)trimethylammonium (FcTMA+) ions as model analytes, differential pulse voltammetric responses of the capillary electrode were investigated in terms of tip diameter of the capillary, sampling voltage, sampling time, detection limit and selectivity. The magnitude of the response depends on the size and charge of analyte ions. With a capillary electrode having a approximately 1.2-microm tip diameter, which minimizes non-selective diffusional entry of analytes, the response after 1 h sampling at +1.7 V is linearly related to [Fe(CN)6]4- concentration in the range of 0.50-5.0 mM with the detection limit of 30 microM. Application of a potential of the same sign as that of the analyte ion forces the analyte to move out from the electrode to the solution, enabling reuse of the same capillary electrode. The charge-selective detection of analytes with the capillary electrode is demonstrated for [Fe(CN)6]4- in the presence of FcTMA+.     

毛细管电泳安培检测技术进展

对毛细管电泳离柱和柱端安培检测方式、不同形式电极在安培检测中的应用、安培检测在芯片毛细管电泳中的应用、安培检测池等内容进行了总结和讨论 ,并预测了安培检测技术未来发展方向     

Use of multiple colorimetric indicators for paper-based microfluidic devices

We report here the use of multiple indicators for a single analyte for paper-based microfluidic devices (μPAD) in an effort to improve the ability to visually discriminate between analyte concentrations. In existing μPADs, a single dye system is used for the measurement of a single analyte. In our approach, devices are designed to simultaneously quantify analytes using multiple indicators for each analyte improving the accuracy of the assay. The use of multiple indicators for a single analyte allows for different indicator colors to be generated at different analyte concentration ranges as well as increasing the ability to better visually discriminate colors. The principle of our devices is based on the oxidation of indicators by hydrogen peroxide produced by oxidase enzymes specific for each analyte. Each indicator reacts at different peroxide concentrations and therefore analyte concentrations, giving an extended range of operation. To demonstrate the utility of our approach, the mixture of 4-aminoantipyrine and 3,5-dichloro-2-hydroxy-benzenesulfonic acid, o-dianisidine dihydrochloride, potassium iodide, acid black, and acid yellow were chosen as the indicators for simultaneous semi-quantitative measurement of glucose, lactate, and uric acid on a μPAD. Our approach was successfully applied to quantify glucose (0.5-20 mM), lactate (1-25 mM), and uric acid (0.1-7 mM) in clinically relevant ranges. The determination of glucose, lactate, and uric acid in control serum and urine samples was also performed to demonstrate the applicability of this device for biological sample analysis. Finally results for the multi-indicator and single indicator system were compared using untrained readers to demonstrate the improvements in accuracy achieved with the new system.     

Determination of active constituents in Lonicera confusa DC. by capillary electrophoresis with amperometric detection

A method based on capillary electrophoresis with amperometric detection has been developed for the determination of luteolin, chlorogenic acid, 3,5-dicaffeoylquinic acid and caffeic acid in the dried flower buds, leaves and stems (three medicinal parts) of Lonicera confusa DC., respectively. The effects of several important factors such as detection potential, the concentration of the running buffer, separation voltage and injection time were investigated to acquire the optimum conditions. The detection electrode was a 300 microm diameter carbon disc electrode at a working potential of + 0.90 V (vs saturated calomel electrode). The four analytes can be well separated within 10 min in a 40 cm-long fused silica capillary at a separation voltage of 12 kV in a 50 mM borate-25 mM phosphate buffer (pH 8.0). The relationship between peak current and analyte concentration was linear over about 3 orders of magnitude with detection limits (S/N = 3) ranging from 0.35 to 0.52 microM for all analytes. The proposed method has been successfully applied to the monitoring of bioactive constituents in the real plant samples with satisfactory assay results.     

Sensitive chemically amplified electrochemical detection of ruthenium tris-(2,2′-bipyridine) on tin-doped indium oxide electrode

Optimized combination of chemical agents was selected for sensitive electrochemical detection of dissolved ruthenium tris-(2,2′-bipyridine) (Ru-bipy). The detection was based on the chemical amplification mechanism, in which the anodic current of a redox-active analyte was amplified by a sacrificial electron donor in solution. On indium-doped tin oxide (ITO) electrodes, electrochemical reaction of the analyte was reversible, but that of the electron donor was greatly suppressed. Several transition metal complexes, such as ferrocene and tris-(2,2′-bipyridine) complexes of osmium, iron and ruthenium, were evaluated as model analyte. A correlation between the amplified current and the standard potential of the complex was observed, and Ru-bipy generated the largest current. A variety of organic bases, acids and zwitterions were assessed as potential electron donor. Sodium oxalate was found to produce the largest amplification factor. With Ru-bipy as the model analyte and oxalate as the electron donor, the analyte concentration curve was linear up to 50 μM, with a lower detection limit of approximately 50 nM. Preliminary work was presented in which a Ru-bipy derivative was attached to bovine serum albumin and detected electrochemically. Although the combination of Ru-bipy, oxalate and ITO electrode has been used before for electrochemiluminescent detection of Ru-bipy and oxalate, as well as electrochemical detection of oxalate, its utility in amplified voltammetric detection of Ru-bipy as a potential electrochemical label has not been reported previously.     

Improvement of Pulse Amperometric Detection Integrated Automated Flow Injection Analysis of Ethylenethiourea Determination

Improvement of pulse amperometric detection (PAD) method is demonstrated in determination of ethylenethiourea (imidazolidine‐2‐thione, ETU). The anodic detection of ETU will produce polymeric film on an electrode leading to an inactive electrode surface. Here, the PAD method was used to remove the polymeric film formed on the electrode surface between ETU detection. Further, the scheme was integrated with automated flow injection analysis (AFIA) for determining ETU. The operational parameters of PAD in the AFIA system were discussed thoroughly. The analytical characteristics of the system were evaluated at optimum conditions. The linear range of calibration plot was between 20 to 300 μM (the correlative coefficient, r = 0.999) and the detection limit was 0.9 μM (S/N = 3). The relative standard deviations of detection of 50 μM ETU were 0.82% with and 9.07% without PAD scheme. The results indicate the system is a very promising tool for ETU determination. Finally, the matrix effects of two water samples that were collected from a campus and a farm show good recoveries of 92% and 96%.     

Indirect detection method for flow systems based on perturbation of oxyen reduction at gold and gold chloride electrodes

A simple scheme for potential indirect electrochemical detection in flow analyses (liquid chromatography and ion chromatography) with experimental examples is presented. The proposed scheme exploits the influence of the analyte on the reduction current of an intrinsic mobile phase additive-oxygen-at the detector electrode, and utilizes the resulting changes in background current as an analytical signal. Several analytes: chloropromazine, ascorbic acid, nitrate and sulfate, cause a shift in the oxygen reduction peak, particularly at the AuCl electrode, that results in a cathodic or anodic shift in the current at a fixed potential.     

Simultaneous electrochemical and electrochemiluminescence detection for microchip and conventional capillary electrophoresis

A simultaneous electrochemical (EC) and electrochemiluminescence (ECL) detection scheme was introduced to both microchip and conventional capillary electrophoresis (CE). In this dual detection scheme, tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)3(2+)) was used as an ECL reagent as well as a catalyst (in the formation of Ru(bpy)3(3+)) for the EC detection. In the Ru(bpy)3(2+)-ECL process, Ru(bpy)3(3+) was generated and then reacted with analytes resulting in an ECL emission and a great current enhancement in EC detection due to the catalysis of Ru(bpy)3(3+). The current response and ECL signals were monitored simultaneously. In the experiments, dopamine and three kinds of pharmaceuticals, anisodamine, ofloxacin, and lidocaine, were selected to validate this dual detection strategy. Typically, for the EC detection of dopamine with the presence of Ru(bpy)3(2+), a approximately 5 times higher signal-to-noise ratio (S/N) can be achieved than that without Ru(bpy)3(2+), during the simultaneous EC and ECL detection of a mixture of dopamine and lidocaine using CE separation. The results indicated that this dual EC and ECL detection strategy could provide a simple and convenient detection method for analysis of more kinds of analytes in CE separation than the single EC or ECL detection alone, and more information of analytes could be achieved in analytical applications simultaneously.     

Simultaneous determination of aminothiols, ascorbic acid and uric acid in biological samples by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection has been employed for the separation and determination of homocysteine, cysteine, reduced glutathione, ascorbic acid and uric acid. Effects of several important factors such as the acidity and concentration of the running buffer, separation voltage, injection time and detection potential were investigated to acquire the optimum conditions. The detection electrode was a 500 microm diameter platinum disk electrode at a working potential of +1.05 V (vs saturated calomel electrode). The five analytes were well separated within 10 min in a 50 cm long fused silica capillary at a separation voltage of 18 kV in a 100 mm phosphate buffer (pH 7.8). The relation between peak current and analyte concentration was linear over about 3 orders of magnitude with the detection limits (S/N = 3) ranging from 0.83 to 2.58 microm. The proposed method was successfully applied to determine cysteine, reduced glutathione, ascorbic acid and uric acid in human whole blood and rat brain tissues with satisfactory assay results and should find a wide range of bioanalytical applications.     

Microextraction combined with derivatization

Microextraction is still finding its niche in the world of analytical sample-preparation techniques and several alternatives have been explored for their ability to cover a wide range of analytes. Derivatization has been another important tool for analysis, especially using chromatography, and great strides have been made in developing key reactions for several classes of compounds. Combining microextraction with derivatization not only enhances analyte recovery, but also improves separation, detectability and compound identification. This article highlights operational principles, advances, directions and key strategies of microextraction combined with derivatization in analytical chemistry. The subjects covered also include design of microextraction–derivatization systems, integration of detection systems, operational requirements, derivatization reactions, typical applications and future prospects.     

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.     

Derivatizations for Improved Detection of Alcohols by Gas Chrohatography and Photoionization Detection (GC-PID)

Abstract

Pentafluorophenyldimethylsilyl chloride (flophemesyl chloride, F1) is a well known derivatization reagent for improved electron capture detection (ECD) in gas chromatography (GC)(GC-ECD), but it has never been utilized for improved detectability and sensitivity in GC-photoionization detection (GC-PID). We have now utilized a wide variety of flophemesyl alcohol derivatives in order to show a new approach for realizing greatly reduced minimum detection limits (MDL) of virtually all alcohol derivatives in GC-PID analysis. This particular derivatization approach is inexpensive and easy to apply, leading to quantitative or near 100% conversion of the starting alcohols to the expected flophemesyl ethers (silyl ethers). Detection limits can be lowered by 2–3 orders of magnitude for such derivatives when compared with the starting alcohols, along with calibration plots that are linear over 5–7 orders of magnitude. Specific GC conditions have been developed for many flophemesyl derivatives, in all cases using packed columns. Both ECD and PID relative response factors (RRFs) and normalized RRFs have been determined, and such ratios can now be used for improved analyte identification from complex sample matrices, where appropriate.

We have attempted to describe in this preliminary report some interesting results and approaches for improved GC-PID detection of a large number of alcohols and alcohol analogs. The method of derivatization is extremely simple to perform, and appears to lead to a single, well-defined product of known structure in 100% yield or thereabouts. Chromatography for typical flophemesylalcohol analytes can be excellent, as in Figure 1, with symmetrical peak shape, little or no tailing, and overall excellent MDLs. With GC-detector-computer interfacing, we are able to obtain both chromatograms and preliminary as well as calculated data within a single 5–10 minute time span²⁷ The total amount of time per analysis will obviously depend on the particular analyte derivative and the chromatography obtained. RRFs and normalized RRFs are quite easy to determine, they are fully reproducible, and can serve as good markers for a particular alcohol and its flophemesyl derivative. In view of the calibration plots possible and MDLs, these overall analytical methods for GC-ECD-PID using flophemesyl derivatization should, we hope, find widespread and ready acceptance and utility by the analytical community.     

Extending the dynamic range of electrochemical sensors using multiple modified electrodes

Multiple electrodes, combined with a chemometric strategy to calibrate the measurement response, have been used for the determination of an analyte across a broader dynamic range than is possible with a single electrode. The model system used for the detection of copper comprised electrodes modified with a self-assembled monolayer. The electrodes were modified with the copper-complexing species (3-mercaptopropionic acid, thioctic acid, and the peptides cysteine and Gly-Gly-His) and copper was determined over concentrations ranging from nanomolar to millimolar using voltammetric analysis. We have demonstrated that by combining the calibration functions from the four electrodes a better estimate (i.e. with smaller variance) of the concentration of the analyte is obtained. Measurement uncertainty is expressed for independently prepared electrodes, which allows the possibility of commercial production and factory calibration. The principles of using multiple electrodes modified with recognition elements with different affinities for the target analyte to extend the dynamic range of sensors is a general one that could be applied to other analytes.     

Microfabricated chemical preconcentrators for gas-phase microanalytical detection systems

The gas or vapor preconcentrator is an analytical device that significantly improves the detection limit of a microanalytical system by preconcentrating the analyte. The preconcentrator performs front-end sampling and preconcentration of analyte by collecting and concentrating analyte over a period of time. After the analyte-collection phase is complete, a heat pulse releases the analyte as a concentrated wave into the detector. Desirable features of the preconcentrator device include the capability of operating at high flow rates, thermal heating with short-time constants, and selective collection of the analyte(s) of interest. The preconcentrators presented in this review are used as a generic front-end modification to gas-phase microanalytical detection systems, such as gas chromatographs, mass spectrometers, ion-mobility spectrometers, and microelectromechanical system (MEMS)-based chemical sensors. The advantages of the detector in incorporating a preconcentrator device are enhanced sensitivity and improved selectivity. Target analytes concentrated by the preconcentrators described in this review include various organic compounds in gas or vapor phase, such as explosives 2,4,6-trinitrotouluene (TNT) and 1,3,5 trinitro-1,3,5-triazine (RDX), chemical agent dimethyl methylphosphonate (DMMP), a broad range of organic vapors, such as toluene, benzene, ethylene and acetone, and mixtures of these gas-phase organic compounds. We discuss examples of the current trends in microfabricated preconcentrator technology as well as several applications of microfabricated preconcentrators.     

Development of a 6-hydroxychroman-based derivatization reagent: application to the analysis of 5-hydroxytryptamine and catecholamines by using high-performance liquid chromatography with electrochemical detection

We developed a novel derivatization reagent, (2R)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (NPCA), for electrochemical (EC) detection in HPLC. NPCA was synthesized from (R)-(+)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (alpha-CA), which exhibits intense EC response. NPCA successfully yielded alpha-CA derivatives of primary amines by a two-step derivatization procedure. Following pre-column derivatization with NPCA, a simultaneous determination of alpha-CA derivatives of neuroactive monoamines [dopamine (DA), epinephrine, and 5-hydroxytryptamine (5-HT)], their monoamine oxidase metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindole-3-acetic acid) and their catechol-O-methyltransferase metabolites [3-methoxytyramine (3-MT) and normetanephrine (NMN)] was completely achieved using our HPLC-EC method. Using an HPLC equipped with coulometric electrode-array detection system, the resultant alpha-CA derivatives of NMN, 5-HT, DA and 3-MT showed intense EC responses, that were approximately 1.3, 1.4, 1.1 and 1.4-fold higher than the corresponding native forms, respectively. The detection limits were in the range of approximately 16-60 fmol on column (signal-to-noise ratio 3). The proposed HPLC method was applied to determine 5-HIAA, HVA, alpha-CA-5-HT and alpha-CA-DA in rat urine. As a consequence, these analytes were successfully determined with satisfactory precisions.     

Determination of mannitol and three sugars in Ligustrum lucidum Ait. by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection has been developed for the separation and determination of mannitol, sucrose, glucose, and fructose in Ligustrum lucidum Ait. for the first time. Effects of several important factors such as the concentration of NaOH, separation voltage, injection time, and detection potential were investigated to acquire the optimum conditions. The detection electrode was a 300 μm diameter copper disc electrode at a working potential of +0.65 V (versus saturated calomel electrode (SCE)). The four analytes can be well separated within 13 min in a 40 cm length fused-silica capillary at a separation voltage of 12 kV in a 75 mM NaOH aqueous solution. The relation between peak current and analyte concentration was linear over about three orders of magnitude with detection limits (S/N = 3) ranging from 1 to 2 μM for all analytes. The proposed method has been successfully applied to monitor the mannitol and sugar contents in the plant samples at different growth stages with satisfactory assay results.     

Application of a Square‐Wave Potential Program for Time‐Dependent Amperometric Detection in Capillary Electrophoresis

Use of a square‐wave potential program for time‐dependent amperometric detection of analyte zones in capillary electrophoresis (CE) is described. Electrochemical detection for CE requires that the separation field be isolated from that of the electrochemical detection. This is generally done by physically separating the CE separation field from that of the detection. By applying a time variant potential program to the detection electrode, the detector current has a time dependence that can be used to help isolate the electrochemical detection current from that of the separation. When using a 20 μm inner‐diameter capillary, we find that a square‐wave potential program decreases the RMS baseline current from 4.5×10^?10 A, found with a constant potential amperometric detection, to 1.1×10^?10 A when using a square‐wave potential program. With a 75 μm inner‐diameter capillary, the improvement is even more dramatic, from 2.3×10^?9 A with amperometric detection to 2.06×10^?10 A when using a 1 Hz square‐wave potential program. When not using the time‐dependent detection with the 75 μm capillary, the analyte zones were beneath the S/N for the system and not detected. With the square‐wave potential program and time‐dependent detection, however, the analyte zones for an electrokinetic injection of 200 μM solution of 2,3‐dihydroxybenzoic acid were observed with the 75 μm inner‐diameter capillary. The improvement in the ability to discriminate the analytical signal from the background found experimentally is consistent with modeling studies.     

Electrochemical and optical detectors for capillary and chip separations

In separations in capillaries or on chips, the most predominant detectors outside of the field of proteomics are electrochemical (EC) and optical. These detectors operate in the μM to pM range on nL peak volumes with ms time resolution. The driving forces for improvement are different for the two classes of detectors.With EC detectors, there are two limitations that the field is trying to overcome. One is the ever-present surface of the electrode which, while often advantageous for its catalytic or adsorptive properties, is also frequently responsible for changes in sensitivity over time. The other is the decoupling of the electrical systems that operate electrokinetic separations from the system operating the detector.With optical detectors, there are similarly a small number of important limitations. One is the need to bring the portability (size, weight and power requirements) of the detection system into the range of EC detectors. The other is broadening and simplifying the applications of fluorescence detection, as it almost always involves derivatization.Limitations aside, the ability to make detector electrodes and focused laser beams of the order of 1 μm in size, and the rapid time response of both detectors has vaulted capillary and chip separations to the forefront of small sample, fast, low mass-detection limit analysis.