End-column Amperometric Detection Research Articles

Determination of trimetazidine in urine by capillary electrophoresis with amperometric detection

In this contribution, nonaqueous capillary electrophoresis with end-column amperometric detection using a platinum ultramicroelectrode is presented along with several extraction procedures, both liquid–liquid extraction (LLE) and solid-phase extraction (SPE), for extracting trimetazidine from urine. Trimetazidine is an anti-ischemic drug, which changes hearth metabolism pathways and is being abused as a doping. Electrochemical detection in a nonaqueous environment provided a stable response with a relative standard deviation of only 3.6% (n = 10) in repeatability measurement at concentration of 50 µg cm−3. LOD and LOQ of the proposed method were determined as 0.054 µg cm−3 and 0.180 µg cm−3, respectively. From the point of view of LLE, the most efficient procedure was the double extraction with ethyl acetate as an extraction agent in combination with prior alkalinization of the sample by Na2CO3. Nevertheless, the extraction efficiency was only around 68%. The most efficient SPE procedure was based on the combination of HLB cartridge and elution with background electrolyte containing 20% of methanol. Its recovery reached up to 92% and 101% in case of 50 µg cm−3 and 5.0 µg cm−3 of trimetazidine in urine, respectively.Graphical abstract

Enhanced sensitivity and resolution for the analysis of paralytic shellfish poisoning toxins in water using capillary electrophoresis with amperometric detection and field-amplified sample injection.

The profiling of the most lethal paralytic shellfish poisoning toxins (PSTs) in freshwater has increased the need to establish an alternative analytical method with high sensitivity and resolution. In this paper, a coupling technique of field-amplified sample injection (FASI) and CE with end-column amperometric detection (CE-AD) was developed to improve the detection sensitivity and separation of PSTs by electrokinetically injecting a water plug of analytes to the capillary filled with a high-conductivity BGE. Parameters affecting FASI and CE process were carefully adjusted to achieve the highest response and resolution. Separation selectivity for PSTs, especially for the analogues and epimers, was greatly enhanced by using 40 mM Britton-Robinson buffer (pH 9.5) as BGE, which altered the EOF and mobility of the analytes that interacted with polyborate ions. Satisfactory linear relationship between peak current and concentration of toxins were gained over a wide range of 1.95-254 μg/L. The detection limits (S/N = 3) for five PSTs ranged from 0.63 to 3.11 μg/L, which are below the health alert level in drinking water. In comparison with the up-to-date reporting chromatographic methods, the FASI-CE-AD method was simple, low-cost, selective, and sensitive enough for direct quantification of PSTs at very low levels, implying a potential for screening and monitoring of PSTs in surface waters.

Analysis of phenolic xenoestrogens by pressurized CEC with amperometric detection

A new method, pressurized CEC with end-column amperometric detection using carbon paste electrode, has been developed for the separation and determination of five phenolic xenoestrogens in chicken eggs and milk powder samples. Efficient separation of five analytes was performed by pressurized CEC using a mobile phase consisting of 60% v/v ACN and 40% v/v Tris buffer (5 mmol/L, pH 8.0), +6 kV of applied voltage and 7.0 MPa of supplementary pressure. Detection limits of 50, 5, 2, 10 and 20 ng/mL for pentachlorophenol, bisphenol-A, 2,4-dichlorophenol, 4-tert-octylphenol and 4-nonylphenol, respectively, were achieved using carbon paste electrode as working electrode and +0.8 V as detection potential. Matrix solid phase dispersion extraction method had been employed during sample preparation procedure, and mean recoveries ranged from 79.2 to 102.6% at different concentrations of phenolic xenoestrogens for spiked egg and milk powder samples were obtained.

Construction and evaluation of a novel end-column amperometric detection system for electrophoresis microchips

A set of integrated end-column amperometric detection system has been developed, onto which an electrophoresis microchip can be conveniently integrated. Finely machined by a piece of transparent organic glass, the system consists of an electrophoresis microchip platform and an amperometric detection reservoir, in which the microchip can be fixed onto the platform by microchip grooves and with stainless steel fixture. Each detection electrode can be directly fixed in the amperometric detection reservoir by screws and nuts. With dopamine as the model analyte, we take platinum disc electrodes with different diameters of 100 μm, 300 μm and 500 μm and a carbon fiber electrode with the diameter of 240 μm as the working electrode, all of which accomplish sensitive detection. The detection parameters of the system are optimized with the carbon fiber electrode. The detection results show that in the electrochemical cleaning procedure, the relative deviations of 3.2% and 0.5% for the peak current and retention time, respectively, can be obtained for the successive detections of 100 μM dopamine, and the limit of detection for dopamine can reach 0.4 μM (S/N = 3). This system is small, but its performance of detection is stable and sensitive, and the replacement of its working electrodes is convenient, so it is very suitable as a universal platform of end-column amperometric detection for electrophoresis microchips.

Comparison of Four Carbon Fiber Electrodes in Microfluidic Chip Integrated With Electrochemical Detector

A microchip electrophoresis system with integrated amperometric detector was developed. Using 0.02 M Tris-HCl (pH = 8) as buffer, at the detection potential of 0.6 V ( vs. Ag/AgCl), dopamine (DA) and isoprenaline (IP) were detected by chip-CE with end-column amperometric detection using a carbon fiber microelectrode (CFME), a carbon fiber nanoelectrode (CFNE), a carbon fiber microelectrode modified by single-walled carbon nanotubes (SWNTs-CFME), and a carbon fiber microdisc electrode (CFMDE). The resolution of DA and IP are 0.64, 1.06, 0.61 and 1.22, and the sensitivity of DA ( S/ N = 3) are 1.7 × 10 −7, 5.9 × 10 −8, 2.3 × 10 −8, and 5.3 × 10 −7, respectively. As the CFNE had high resolution and sensitivity, the nanoelectrode-based microchip CE system was successfully applied to the determination of DA in cultured rat pheochromocytoma (PC12) cells, and the average content of DA in an individual PC12 cell was 0.57 ± 0.07 fmol ( n = 5), which was in good agreement with that reported in the literatures.

Fabrication and performance of poly(methyl methacrylate) microfluidic chips with fiber cores

In this work, a piece of glass fiber was inserted into the channel of a poly(methyl methacrylate) (PMMA) electrophoresis microchip to enhance the electroosmotic flow (EOF) and the separation efficiency. The EOF value of the glass fiber-containing microchannel at pH 8.2 was determined to be 4.17 × 10 −4 cm 2 V −1 s −1. The performance of the new microchip was demonstrated by its ability to separate and detect three purines coupled with end-column amperometric detection. In addition, a piece of trypsin-immobilized glass fiber was inserted into the channel of a PMMA microchip to fabricate a core-changeable microfluidic bioreactor that can be regenerated by changing the fiber. The in-channel fiber bioreactor has been coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the digestion and peptide mapping of bovine serum albumin and myoglobin.

Fabrication and performance of fiber electrophoresis microchips

A method based on the in situ polymerization of methyl methacrylate (MMA) has been developed for the rapid fabrication of a novel separation platform, fiber electrophoresis microchip. To demonstrate the concept, prepolymerized MMA molding solution containing a UV initiator was sandwiched between a poly(methyl methacrylate) (PMMA) cover plate and a PMMA base plate bearing glycerol-permeated fiberglass bundles and was exposed to UV light. During the UV-initiated polymerization, the fiberglass bundles were embedded in the PMMA substrate to form fiberglass-packed microchannels. When the glycerol in the fiberglass bundles was flushed away with water, the obtained porous fiberglass-packed channels could be employed to perform electrophoresis separation. Scanning electron micrographs (SEMs) and microscopic images offered insights into the fiber electrophoresis microchip. The analytical performance of the novel microchip has been demonstrated by separating and detecting dopamine and catechol in connection with end-column amperometric detection. The fiber-based microchips can be fabricated by the new approach without the need for complicated and expensive lithography-based microfabrication techniques, indicating great promise for the low-cost production of microchips, and should find a wide range of applications.

Capillary Electrophoresis-Amperometric Determination of Antioxidant Propyl Gallate and Butylated Hydroxyanisole in Foods

Capillary electrophoretic separation coupled with end-column amperometric detection for the simultaneous quantification of butylated hydroxyanisole (BHA) and propyl gallate (PG) in food was developed. Important factors affecting separation and detection, such as the running buffer, separation voltage, and detection potential, were investigated in detail. An improved working electrode preparation method was used, where a carbon disk of 33 microm in diameter was sealed in a tip and positioned opposite the outlet of a capillary. The experiments indicated that the preparation method was simple, and the obtained electrode exhibited good flexibility and stability for the determination of phenolic antioxidants. The separation was carried out within 5 min using a 50 cm length capillary, with a solution containing 5 mM phosphate and 5 mM borax of pH 8.84 as a separation buffer, and a separation potential of 20 kV. Amperometric detection was achieved with an applied potential of 0.70 V versus Ag|AgCl| saturated KCl. There was excellent linearity between the peak current and the concentrations of the analytes in the range of 1.8 - 180.2 microg/mL for BHA and 10.6 - 212.2 microg/mL for PG, respectively. Relative standard deviations of 4.92% for BHA and 5.27% for PG were obtained, respectively. The developed method was successfully applied for the determination of antioxidants in several commercial foods.

Carbon fiber nanoelectrodes applied to microchip electrophoresis amperometric detection of neurotransmitter dopamine in rat pheochromocytoma (PC12) cells

Microelectrodes have been adopted in electrochemical detection for CE or microchip CE in recent years. In this paper, the use of nanoelectrodes (with tip diameter of 100-300 nm) as the electrochemical detector in microchip CE is firstly reported. The experimental results indicated that both the sensitivity and resolution of microchip CE with the carbon fiber nanoelectrode (CFNE) amperometric detection have been improved markedly comparing with the traditional microelectrodes. The detection limit of dopamine (S/N = 3) is 5.9x10(-8) M, which is one or two orders of magnitude lower than that reported so far, and the resolution of dopamine (DA) and isoprenaline (IP) has also improved from 0.6 (using 7 mum carbon fiber microelectrodes, CFME) to 1.0. We assembled a novel and easily operated microchip CE system with end-column amperometric detection, which allows the convenient and fast replacement of the passivated electrodes. Under the optimized condition, the RSDs of peak height and migration time are 1.47 and 0.31%, respectively (n = 40), indicating that the system displays excellent reproducibility. The nanoelectrode-based microchip CE system has been successfully applied to the determination of DA in cultured rat pheochromocytoma (PC12) cells, and the average content of DA in an individual PC12 cell is 0.54 +/- 0.07 fmol, which is in good agreement with that reported in the literature.

Fabrication of a fiberglass-packed channel in a microchip for flow injection analysis

A novel fiberglass-packed channel in a microchip has been fabricated for flow injection analysis (FIA) based on the in situ polymerization of methyl methacrylate (MMA). MMA prepolymer molding solution containing an ultraviolet initiator was sandwiched between a poly(methyl methacrylate) (PMMA) cover plate and a PMMA base plate bearing a glycerol-permeated fiberglass bundle and exposed to UV light. During the UV-initiated polymerization, the fiberglass bundle was embedded in the PMMA substrate to form a fiberglass-packed microchannel. When the glycerol in the fiberglass bundle was flushed away with water, the obtained porous fiberglass-packed microchannel could serve as an electroosmotic pump and a FIA channel. Scanning electron micrographs offer insights into the fiber-based microchip. The analytical performance of the FIA system has been demonstrated by detecting catechol in connection with end-column amperometric detection. The fiber- based microchips can be fabricated by the new approach without the need for complicated and expensive lithography-based microfabrication techniques, indicating great promise for the low-cost production of microchips, and should find a wide range of applications.

Determination of endogenous norepinephrine levels in different chambers of the rat heart by capillary electrophoresis coupled with amperometric detection

Capillary electrophoresis with end-column amperometric detection (CE-EC) was used to determine the regional distribution of norepinephrine (NE) in the hearts of sympathetically innervated (control) and chemically sympathectomized rats. Key features of the method are (i) the sample preparation and clean-up step that involved the application of off-line solid phase extraction (SPE) with a 95% NE recovery and (ii) the use of a diamond microelectrode for detection. NE was quantified in the left and right ventricle, the ventricular septum, and the left and right atrium. The NE concentration in the atria was three to five times higher than in the ventricles and ventricular septum of control rats. Basal NE levels in the left and right ventricle and the ventricular septum were reduced to below the detection limit (0.034 μg/g tissue) in tissues treated with the neurotoxin, 6-hydroxydopamine (6-OHDA), while only a moderate reduction was observed in the left and right atrium. Importantly, the diamond microelectrode provided low and stable background current and low peak-to-peak noise ≤0.65 pA at a detection potential of +0.86 V versus Ag/AgCl. A reproducible electrode response was observed for multiple injections of tissue homogenates with minimal response attenuation due to electrode fouling.

Micellar electrokinetic capillary chromatography for fast separation and sensitive determination of melatonin and related indoleamines using end‐column amperometric detection

MEKC was used in conjunction with end-column amperometric detection (AD) at a carbon disc electrode (0.3 mm diameter) for the selective and sensitive determination of melatonin and its five related indoleamines including its precursors and metabolites in the pineal gland. The introduction of a sample stacking technique in injection and the buffer additive SDS in the buffer solution system provided the rapid and sensitive analysis. Optimal buffer conditions (10 mmol/L phosphate containing 20 mmol/L SDS, pH 7.2), detection potential (+1.0 V vs. Ag/AgCl), and electrokinetic injection 10 s with the separation voltage of 24 kV were employed to achieve the baseline separation of six pineal hormones within 15 min. The peak currents and the analyte concentrations have a good linear relationship over the range of 6.0 x 10(-8) 6.0 x 10(-5 )mol/L. The detection limits for six pineal hormones by AD are 9.7 to 41.8 nmol/L (equal to 2.0 to 9.7 ng/mL) (S/N = 3), respectively. It is proved to provide about 30- to 250-fold improvement over UV, and be comparable with the sensitive fluorescence detection, which needs pre-column derivatization. The proposed method has been applied for analysis of melatonin and related indoleamines in rat pineal glands. A very simple sample pretreatment procedure, merely involving the homogenization step in perchloric acid, was enough to achieve recoveries in the range of 71 to 127% for all the analytes in the pineal gland.

Fabrication of Poly(dimethylsiloxane)-Based Capillary Electrophoresis Microchips Using Epoxy Templates

A novel method for the fabrication of poly(dimethylsiloxane) (PDMS)-based capillary electrophoresis (CE) microchips using epoxy templates is described. A PDMS plate containing the channel structure was replicated from a hard master with the positive relief of negative photoresist on a silicon wafer by replica molding. It was used as a soft mold to produce the positive relief of a hard epoxy template for molding of the final soft PDMS micro devices. The present hard-to-soft, soft-to-hard, and hard-to-soft micro fabrication method offers a promising way for the mass production of PDMS microchips, epoxy and other hard polymer templates. The epoxy templates are durable for molding PDMS electrophoresis microchips because of its higher mechanical strength. High chip-to-chip reproducibility has been found with relative standard deviations (n = 30) of 3.7 and 4.1% for the widths (49.2 µm) and depths (41.5 µm) of the replicated channels in the final PDMS substrates, respectively. The feasibility and performance of the PDMS microchips fabricated by the new method have been demonstrated by separating and detecting dopamine and catechol in connection to end-column amperometric detection. Factors influencing the analytical performance of the microsystem for the separation of the two analytes have been characterized and optimized.

Determination of the hydrolysis rate constants and activation energy of aesculin with capillary electrophoresis end-column amperometric detection

Aesculetin is the product of the hydrolysis reaction of aesculin. A high sensitivity and good repeatability method based on capillary electrophoresis with amperometric detection (CE-AD) was developed for simultaneous determination of aesculin and aesculetin in the hydrolysate of aesculin. Under the optimum condition: 10 mmol/L KH 2PO 4–5 mmol/L Na 2B 4O 7 (pH 6.0) buffer, separation at 18 kV and +900 mV (versus Ag/AgCl) as the detection potential, the hydrolysis rate constants of aesculin hydrolysis at 25, 30, 35, 40 and 45 °C in 0.1 mol/L KOH were obtained as 1.45 × 10 −2 min −1, 2.01 × 10 −2 min −1, 2.93 × 10 −2 min −1, 3.76 × 10 −2 min −1 and 5.05 × 10 −2 min −1, respectively. It was calculated that the activation energy for aesculin hydrolysis was 49.4 kJ/mol.

Determination of hydrazine, monomethylhydrazine, 1,1‐dimethylhydrazine, and 1,2‐dimethylhydrazine by nonaqueous capillary electrophoresis with amperometric detection

The present study is concerned with the application of nonaqueous capillary electrophoresis (NACE) with electrochemical detection (ED) to the separation and quantitative determination of hydrazine (Hy) and its methyl derivatives. The best performance of NACE-ED was found when using 4 mM sodium acetate/10 mM acetic acid/methanol: acetonitrile = 1:2 as the running buffer, with a bare platinum working electrode set at +1.0 V in an end-column amperometric detection cell. The choice and ratio of suitable solvents for the separation and injection media played an essential role for the performance characteristics of the method. The limits of detection for Hy, methylhydrazine, symmetrical dimethylhydrazine, and unsymmetrical dimethylhydrazine were 5, 2, 12, and 1 ng/mL, respectively. This is between one and two orders of magnitude lower than that achieved by previously reported CE-ED methods in aqueous buffer systems in conjunction with various types of chemically modified electrodes. The practical utility of the new NACE-ED methodology is demonstrated in terms of the determination of traces of Hys in spiked environmental samples containing a wide range of explosives and related compounds.

Low temperature bonding of poly(methylmethacrylate) electrophoresis microchips by in situ polymerisation

A novel method for bonding poly(methyl methacrylate) (PMMA) electrophoresis microchips at the temperature below the glass transition temperature of PMMA based on in situ polymerization has been demonstrated. Methyl methacrylate (MMA) containing initiators was allowed to prepolymerize in an 85 °C water bath for 8 min and 15 min to produce a bonding solution and a dense molding solution, respectively. The channel plate of the PMMA microchip was fabricated by the UV-initiated polymerization of the molding solution between a nickel template and a PMMA plate at room temperature. Prior to bonding, the blank cover was coated with a thin layer of the bonding solution and was bonded to the channel plate at 95 °C for 20 min under the pressure of binder clips. The attractive performance of the PMMA chips bonded by the new approach has been demonstrated by separating and detecting dopamine, catechol, three cations, and three organic acids in connection with end-column amperometric detection and contactless conductivity detection.

A Capillary Electrophoresis End-Column Amperometric Detection System Incorporating Disposable Copper-Plated Screen-Printed Carbon Electrodes

We report here the development of copper-plated screen-printed carbon electrodes (designated as Cu-SPE) to employ as electrochemical detectors for the determination of sugars by capillary electrophoresis (CE). A simple end-column amperometric detection system with easily exchangeable (or even disposable) electrode and capillary in CE is described in this study. A complex alignment procedure was not required in this system based on the end-column electrode arrangement using an 85 cm length and 20 μm (i.d.) capillary. The optimized separation voltage and applied potential were 9 KV and 0.4 V (vs. Ag/AgCl), respectively, for the detection of sugars using the Cu-SPE. Good resolution was obtained by this proposed system with migration times of 28.8, 29.5, 29.9, 30.7, 31.2, and 32.0 min for galactose, glucose, arabinose, fructose, xylose, and ribose, respectively.

Determination of enrofloxacin and its metabolite ciprofloxacin by high performance capillary electrophoresis with end-column amperometric detection

Enrofloxacin (ENR) and its metabolite ciprofloxacin (CIP) were determined by capillary zone electrophoresis (CZE) with end-column amperometric detection. The effect of several factors, such as pH and concentration of running buffer solution, separation voltage, injection time, and working potential, on CZE were investigated to establish the optimal conditions of separation and detection. Under a given set of conditions (pH 8.00 phosphate buffer solution (20 mmol/L); +0.95 V for the working potential; 18 kV for the separation voltage; sample injection at 18 kV for 10 s), the compounds investigated can be well separated and detected within 8 min. Excellent linearity was observed between peak currents and concentration of analytes in the range from 0.034 to 70.0 mg/kg for these two compounds. The detection limits (S/N= 3) for enrofloxacin and ciprofloxacin were 13.68 mg/kg and 14.35 mg/kg, respectively, which were about 7-fold lower than the maximum residue limits (MRLs) established by the European Union. A simple sample pretreatment method was developed and proved to be effective in obtaining good recoveries and short analysis time. The developed CE-AD method was simpler, faster, and less cost intensive than other reported methods, and allows the determination of ENR and its metabolite CIP in contaminated eel liver samples and other animal tissue samples at the required maximum residue limits.

On-line coupling of pressurized capillary electrochromatography with end-column amperometric detection for analysis of estrogens

An improved technique, pressurized capillary electrochromatography (pCEC) coupling with end-column amperometric detection (AD), was developed and used for the separation and determination of estrogens. The effects of pH value, composition of mobile phase, concentration of the surfactant sodium dodecyl sulfate (SDS) and applied voltage on separation were investigated. The electrochemical oxidation of diethylstilbestrol (DES), dienestrol (DE), and hexestrol (HEX) could be reliably monitored with a carbon electrode at 0.9 V (vs. Ag/AgCl). The pCEC analyses were performed on a capillary separation column packed with 3 microm C18 particles with an acetonitrile/water (31%: 69%) mobile phase containing Tris buffer (5 mmol/L, pH 4.5) and 4 mmol/L SDS. High voltage up to 12 kV reduced the retention time dramatically and still provided a baseline resolution. In addition, supplementary pressure prevented bubble formation and provided reliability and reproducibility of the pCEC performance. The detection limits for the three estrogens ranged from 1.2 to 2.2x10(-7) mol/L, about 10 20-fold lower than those obtained with pCEC-UV detection. To evaluate the feasibility and reliability of this system, the proposed pCEC-AD method was further demonstrated with fish muscle samples spiked with estrogens.

Separation of proteins on surface-modified poly(dimethylsiloxane) microfluidic devices.

Separation and detection of proteins have been realized on nonionic surfactant-modified poly(dimethylsiloxane) (PDMS) microfabricated devices with end-column amperometric detection. The hydrophobic PDMS channels are turned into hydrophilic ones after being modified with Brij35 and facilitate the separation of proteins. The coating can remarkably reduce the adsorption of large protein molecules and is stable in the range of pH 6-12. The detection of proteins in such channels needs less rinsing time and thus efficiency is raised. Even large molecules of proteins can also be detected with better reproducibility and enhanced plate numbers. The relative standard deviation (RSD) of the migration time for glucose oxidase (GOD) is 2.2% (n = 19). Separation of GOD and myoglobin has been developed in modified channels. Predominant operational variables, such as the coating conditions, the concentration of surfactant and buffer, are studied in detail.