On-column electrochemical detection for microchip capillary electrophoresis

The development of a cellulose acetate decoupler for on-column electrochemical detection in microchip capillary electrophoresis is presented. The capillary based laser-etched decoupler is translated to the planar format to isolate the detector circuit from the separation circuit. The decoupler is constructed by aligning a series of 20 30-microm holes through the coverplate of the microchip with the separation channel and casting a thin film of cellulose acetate within the holes. The decoupler shows excellent isolation of the detection circuit for separation currents up to 60 microA, with noise levels at or below 1 pA at a carbon fiber electrode. Detection limits of 25 nM were achieved for dopamine. This decoupler design combines excellent mechanical stability, effective shunting of high separation currents, and ease of manufacture.     

Microchip capillary electrophoresis with electrochemical detection

A novel microchip capillary electrophoresis system with electrochemical detection, using the replaceable microelectrode, is first reported. This kind of electrode can be fabricated in general laboratories and can be replaced quickly with electrodes of different materials according to the requirements of experiments. The end-column electrochemical detection on microchip CE was achieved by fixing the working electrode (such as carbon fiber, Pt, or Au, etc.) through a guide tube on the end of the separation channel. The experiment results indicate that the alignment of the electrode with the channel outlet can be carried out accurately and reproducibly, and therefore, the detection device has low noise and good reproducibility. The detection limit of dopamine is 2.4 x 10(-7) M, which is the lowest result reported so far. The separation and detection of dopamine, 5-hydroxytryptamine and epinephrine using carbon fiber and Pt microdisk electrodes within 50 s was successfully performed.     

Rudimentary capillary-electrode alignment for capillary electrophoresis with electrochemical detection

A capillary-electrode holder was constructed for electrochemical detection in capillary electrophoresis (CE). The device allows for positioning of the working electrode at the end of the capillary column without the aid of micropositioners or microscopes. The design facilitates the exchange of electrodes and capillaries without the need of refabricating the entire capillary-electrode setup. The system can be assembled in a very short period of time. Alignment with the self-guided system proved to be reproducible for the electrodes used (carbon, nickel, copper). The advantages of reduced downtime and low cost, make the device very attractive for the routine analysis of electroactive species by CE with electrochemical detection.     

Application of diamond microelectrodes for end-column electrochemical detection in capillary electrophoresis

Highly boron-doped diamond microelectrodes were employed in an end-column electrochemical detector for capillary electrophoresis (CE). The diamond microline electrodes were fabricated from conducting diamond thin films (exposed surface area, 300 x 50 microm), and their analytical performance as CE detectors was evaluated in a laboratory-made CE installation. The CE-ED system exhibited high separation efficiency for the detection of several catecholamines, including dopamine (DA), norepinephrine (NE), and epinephrine (E), with excellent analytical performance, for example, 155,000 theoretical plates for DA. The diamond-based electrochemical detection system also displayed low detection limits (approximately 20 nM for E at S/N = 3) and a highly reproducible current response with 10 repetitive injections of mixed analytes containing DA, NE, and E (each 50 microM), with relative standard deviations (RSD) of approximately 5%. The performance of the diamond detector in CE was also evaluated in the detection of chlorinated phenols (CP). When compared to the carbon fiber microelectrode, the diamond electrode exhibited lower detection limits in an end-column CE detection resulting from very low noise levels and highly reproducible analyses without electrode polishing due to analyte fouling, which makes it possible to perform easier and more stable CE analysis.     

Boron-doped diamond microelectrodes for use in capillary electrophoresis with electrochemical detection

The fabrication and characterization of boron-doped diamond microelectrodes for use in electrochemical detection coupled with capillary electrophoresis (CE-EC) is discussed. The microelectrodes were prepared by coating thin films of polycrystalline diamond on electrochemically sharpened platinum wires (76-, 25-, and 10-microm diameter), using microwave-assisted chemical vapor deposition (CVD). The diamond-coated wires were attached to copper wires (current collectors), and several methods were explored to insulate the cylindrical portion of the electrode: nail polish, epoxy, polyimide, and polypropylene coatings. The microelectrodes were characterized by scanning electron microscopy, Raman spectroscopy, and cyclic voltammetry. They exhibited low and stable background currents and sigmoidally shaped voltammetric curves for Ru(NH3)6(3+/2+) and Fe(CN)6(3-/4-) at low scan rates. The microelectrodes formed with the large diameter Pt and sealed in polypropylene pipet tips were employed for end-column detection in CE. Evaluation of the CE-EC system and the electrode performance were accomplished using a 10 mM phosphate buffer, pH 6.0, run buffer, and a 30-cm-long fused-silica capillary (75-microm i.d.) with dopamine, catechol, and ascorbic acid serving as test analytes. The background current (approximately 100 pA) and noise (approximately 3 pA) were measured at different detection potentials and found to be very stable with time. Reproducible separation (elution time) and detection (peak current or area) of dopamine, catechol, and ascorbic acid were observed with response precisions of 4.1% or less. Calibration curves constructed from the peak area were linear over 4 orders of magnitude, up to a concentration between 0.1 and 1 mM. Mass limits of detection for dopamine and catechol were 1.7 and 2.6 fmol, respectively (S/N = 3). The separation efficiency was approximately 33,000, 56,000, and 98,000 plates/m for dopamine, catechol, and ascorbic acid, respectively. In addition, the separation and detection of 1- and 2-naphthol in 160 mM borate buffer, pH 9.2, was investigated. Separation of these two analytes was achieved with efficiencies of 118,000 and 126,000 plates/m, respectively.     

Cellulose acetate decoupler for on-column electrochemical detection in capillary electrophoresis.

A new decoupler for on-column electrochemical detection in capillary electrophoresis is presented. The decoupler is constructed by etching a series of holes through the side of the separation capillary with a CO2 laser and then coating the holes with cellulose acetate. The decoupler shows isolation of the detection circuit for separation currents up to 30 microA. Detection limits below 1 nM were achieved for four model compounds, including anions, neutrals, and cations, using the laser-etched decoupler. This decoupler design combines excellent mechanical stability, effective shunting of high separation currents, and ease of manufacture.     

Dual-electrode electrochemical detection for poly(dimethylsiloxane)-fabricated capillary electrophoresis microchips

The development of a poly(dimethylsiloxane)-based (PDMS-based) microchip electrophoresis system employing dual-electrode electrochemical detection is described. This is the first report of dual-electrode electrochemical detection in a microchip format and of electrochemical detection on chips fabricated from PDMS. The device described in this paper consists of a top layer of PDMS containing the separation and injection channels and a bottom glass layer onto which gold detection electrodes have been deposited. The two layers form a tight reversible seal, eliminating the need for high-temperature bonding, which can be detrimental to electrode stability. The channels can also be temporarily removed for cleaning, significantly extending the lifetime of the chip. The performance of the chip was evaluated using catechol as a test compound. The response was linear from 10 to 500 microM with an LOD (S/N = 3) of 4 microM and a sensitivity of 45.9 pA/microM. Collection efficiencies for catechol ranged from 28.7 to 25.9% at field strengths between 200 and 400 V/cm. Dual-electrode detection in the series configuration was shown to be useful for the selective monitoring of species undergoing chemically reversible redox reactions and for peak identification in the electropherogram of an unresolved mixture.     

Portable high-voltage power supply and electrochemical detection circuits for microchip capillary electrophoresis

Miniaturized, battery-powered, high-voltage power supply, electrochemical (EC) detection, and interface circuits designed for microchip capillary electrophoresis (CE) are described. The dual source CE power supply provides +/- 1 kVDC at 380 microA and can operate continuously for 15 h without recharging. The amperometric EC detection circuit provides electrode potentials of +/-2 VDC and gains of 1, 10, and 100 nA/V. The CE power supply power is connected to the microchip through an interface circuit consisting of two miniature relays, diodes, and resistors. The microchip has equal length buffer and separation channels. This geometry allows the microchip to be controlled from only two reservoirs using fixed dc sources while providing a consistent and stable sample injection volume. The interface circuit also maintains the detection reservoir at ground potential and allows channel currents to be measured likewise. Data are recorded, and the circuits are controlled by a National Instruments signal interface card and software installed in a notebook computer. The combined size (4 in. x 6 in. x 1 in.) and weight (0.35 kg) of the circuits make them ideal for lab-on-a-chip applications. The circuits were tested electrically, by performing separations of dopamine and catechol EC and by laser-induced fluorescence visualization.     

In-channel electrochemical detection for microchip capillary electrophoresis using an electrically isolated potentiostat

A new electrode configuration for microchip capillary electrophoresis (CE) with electrochemical (EC) detection is described. This approach makes it possible to place the working electrode directly in the separation channel. The "in-channel" EC detection was accomplished without the use of a decoupler through the utilization of a specially designed, electrically isolated potentiostat. The effect of the working electrode position on the separation performance (in terms of plate height and peak skew) of poly(dimethylsiloxane)-based microchip CEEC devices was evaluated by comparing the more commonly used end-channel configuration with this new in-channel approach. Using catechol as the test analyte, it was found that in-channel EC detection decreased the total plate height by a factor of 4.6 and lowered the peak skew by a factor of 1.3. A similar trend was observed for the small, inorganic ion nitrite. Furthermore, a fluorescent and electrochemically active amino acid derivative was used to directly compare the separation performance of in-channel EC detection to that of a widely used laser-induced fluorescence (LIF) detection scheme. In this case, it was found that the plate height and peak skew for both detection schemes were essentially equal, and the separation performance of in-channel EC detection is comparable to LIF detection.     

Fully integrated on-chip electrochemical detection for capillary electrophoresis in a microfabricated device

Microfabricated lab-on-a-chip devices employing a fully integrated electrochemical (EC) detection system have been developed and evaluated. Both capillary electrophoresis (CE) channels and all CE/EC electrodes were incorporated directly onto glass substrates via traditional microfabrication techniques, including photolithographic patterning, wet chemical etching, DC sputtering, and thermal wafer bonding. Unlike analogous CE/EC devices previously reported, no external electrodes were required, and critical electrode characteristics, including size, shape, and placement on the microchip, were established absolutely by the photolithography process. For the model analytes dopamine and catechol, detection limits in the 4-5 microM range (approximately 200 amol injected) were obtained with the Pt EC electrodes employed here, and devices gave stable analytical performance over months of usage.     

Integrated on-capillary electrochemical detector for capillary electrophoresis

An on-capillary electrochemical detector for capillary electrophoresis is described. It consists of a gold wire mounted permanently at the end of the capillary perpendicular to the direction of flow. This mode of detection eliminates the need for the micromanipulators or specially machined cell holders for alignment that are used for in-capillary detection modes. It also makes it possible to perform relatively fast CEEC separations using very short capillaries. The use of this detector for both off-column detection of catecholamines and end-column detection of carbohydrates by CE-PAD is described.     

A universal concept for stacking neutral analytes in micellar capillary electrophoresis

Unlike recent studies that have depended on manipulation of separation buffer parameters to facilitate stacking of neutral analytes in micellar capillary electrophoresis (MCE) mode, we have developed a method of stacking based simply on manipulation of the sample matrix. Many solutions for sample stacking in MCE are based on strict control of pH, micelle type, electroosmotic flow (EOF) rate, and separation-mode polarity. However, a universal solution to sample stacking in MCE should allow for free manipulation of separation buffer parameters without substantially affecting separation of analytes. Analogous to sample stacking in capillary zone electrophoresis by invoking field amplification of charged analytes in a low-conductivity sample matrix, the proposed method utilizes a high-conductivity sample matrix to transfer field amplification from the sample zone to the separation buffer. This causes the micellar carrier in the separation buffer to stack before it enters the sample zone. Neutral analytes moving out of the sample zone with EOF are efficiently concentrated at the micelle front. Micelle stacking is induced by simply adding salt to the sample matrix to increase the conductivity 2-3-fold higher than the separation buffer. This solution allows free optimization of separation buffer parameters such as micelle concentration, organic modifiers, and pH, providing a method that may complement virtually any existing MCE protocol without restricting the separation method.     

Electrokinetic injection for stacking neutral analytes in capillary and microchip electrophoresis

An on-column mechanism for electrokinetically injecting long sample plugs with simultaneous stacking of neutral analytes in capillary electrokinetic chromatography is presented. On-column stacking methods allow for the direct injection of long sample plugs into the capillary, with narrowing of the analyte peak width to allow for an increase in the detected signal. Low-pressure injections (approximately 50 mbar) are commonly used to introduce sample plugs containing neutral analytes. We demonstrate that injection can be accomplished by applying an electric field from the sample vial directly into the capillary, with neutral analytes injected by electroosmotic flow at up to 1 order of magnitude faster than the corresponding pressure injections. Since stacking occurs simultaneously with electrokinetic injection, stacking is initiated at the capillary inlet, resulting in an increased length of capillary remaining for separation. Reproducibility obtained for peak height and peak area with electroosmotic flow injection is comparable to that obtained with the pressure injection mode, while reproducibility of analysis time is markedly improved. Electrokinetic stacking of neutral analytes utilizing electroosmotic flow is demonstrated with discontinuous (high conductivity, high mobility) as well as continuous (equal conductivity, equal mobility) sample electrolytes. Injecting neutral analytes by electroosmotic flow affords a 10-fold or greater decrease in analysis times when capillaries of 50-microm i.d. or smaller are used. This stacking method should be exportable to dynamic pH junction stacking and electrokinetic chromatography with capillary arrays. Equations describing this electrokinetic injection mode are introduced and stacking of a neutral analyte on a microchip by electrokinetic injection using a simple cross-T channel configuration is demonstrated.     

Development of rotating electrochemical detectors for capillary electrophoresis

A rotating disk electrode (RDE) has been evaluated and optimized for the detection of electroactive species separated by capillary electrophoresis (CE). With catechol as a working model, the limit of detection was estimated to be 0.3 microM, i.e., approximately 2.5-fold better than that of the stationary disk electrode (0.7 microM). Separation efficiency was significantly improved as exemplified by an increase of theoretical plates from 26,000 plates/m at 0 rpm to 67,000 plates/m at 500 rpm. Of particular importance was the capability of RDE to alleviate electrode passivation and electrical interference associated with high separation potential fields. Therefore, rotation amperometry was especially useful for analytes such as phenolic compounds that tended to rapidly foul the electrode surface. The RDE/ CE system was capable of separation and determination of pentachlorophenol in contaminated soils, and the result obtained agreed well with conventional liquid chromatography, an EPA recommended procedure.     

Contactless conductivity detection for capillary electrophoresis

A contactless capacitively coupled conductivity detector for capillary electrophoresis is introduced. The detector consists of two electrodes which are placed cylindrically around the outer polyimide coating of the fused-silica capillary with a detection gap of 2 mm. The electrodes form a cylindrical capacitor, and the electric conductivity of the solution in the gap between the electrodes is measured. A high audio or low ultrasonic frequency for coupling of the ac voltage is used in order to minimize the influence of reactance of the liquid. For an improved version of the detector, two syringe cannulas are used as the electrodes and the capillary is simply assembled into the tubing. This allows an easy placement of the detector on various positions along the capillary. The limit of detection of inorganic cations and anions is 200 ppb, as determined for sodium and chloride, respectively.     

Passive conductivity detection for capillary electrophoresis

A passive electrochemical detection principle that can be applied to capillary electrophoresis is presented. The separation electrical field is used to generate a potential difference between two electrodes located along the channel. For constant-current electrophoresis, the generated signal is proportional to the resistance of the solution passing between the two electrodes. Contrary to conductivity detectors that are ac driven and need to be decoupled from the separation field, the passive detection directly takes advantage of the separation field. The signal is simply measured by a high-impedance voltmeter. The detection concept has been validated by numerical simulations showing how the magnitude of the signal is related to the ratio between the electrode distance and the length of the sample plug. As a proof of the principle, this detection concept has been demonstrated by the electrophoretic separation of three alkali ions on a polymer microchip. Based on preliminary results, a detection limit of 20 microM and a dynamic range of up to 3 orders of magnitude have been achieved.     

Separation of serotonin from catechols by capillary zone electrophoresis with electrochemical detection

Capillary zone electrophoresis with electrochemical detection is demonstrated with columns having only 9-microns inner diameter. Amperometric detection limits of 0.7 amol are reported for serotonin. The difficult problem of resolving serotonin and dopamine--two neurotransmitters of interest having similar electrophoretic mobilities--is addressed by chemical means to improve selectivity. These include buffer modification with 2-propanol and a system employing borate complexation of the catechol in combination with sodium dodecyl sulfate micelles.     

Experimental characterization of end-column electrochemical detection in conjunction with nonaqueous capillary electrophoresis

An end-column electrochemical detector arrangement for capillary electrophoresis (CE) based on a 75-microm-i.d. capillary and a 25-microm microdisk electrode is characterized. The investigations were carried out using a nonaqueous (acetonitrile-based) buffer and ferrocene model compounds which offer high reliability for voltammetric measurements. The positioning of the microdisk electrode relative to the capillary outlet is the most important parameter for optimization of detection performance as it determines the characteristics of mass transport toward the electrode and the effect of ohmic potential drop resulting from the electrophoretic current on the actual detection potential. On the basis of spatially resolved studies, it was concluded that for the detection system used the microdisk electrode should be placed in a central position relative to the capillary outlet at a distance within the range of 75-100 microm. The presence of a high-voltage electric field had no negative effect on baseline noise, which was demonstrated by comparison of capillary flow injection based on gravity flow and CE experiments. Even a faster stabilization of the baseline was observed by increasing the separation voltage.     

Carbon film-based interdigitated array microelectrode used in capillary electrophoresis with electrochemical detection

A carbon film based interdigitated ring-shaped array (IDRA) microelectrode was applied to capillary electrophoresis with electrochemical detection to enhance the detection sensitivity on the basis of the redox cycling of electrochemical reversible species at the IDRA microelectrode. We propose a simple capillary-electrode connection device that consists of an X-Y-Z fiber aligner, an electrochemical cell, and a Nafion tubing joint that will enable the detection capillary to be aligned easily on the IDRA microelectrode and isolate the separation voltage from the electrochemical detection system. We used the off-column amperometric detection of aqueous ferrocene and catecholamines by capillary electrophoresis with an IDRA microelectrode to investigate the effects of the capillary-to-electrode distance and the separation voltage on the response currents in single and dual modes and the collection efficiencies (CE) and redox cycles (Rc) at the IDRA microelectrode. The results show that CE and Rc increase when we increase the distance and lower the separation voltage. The limiting currents also increase as the separation voltage decreases in the dual mode. Under optimum conditions, the CE and Rc of catechol, with good reversibility, reach 83.9% and 3.67, respectively. Our results showed that dual-mode detection with the IDRA microelectrode was capable of achieving lower detection limits than single-mode detection.     

CMOS absorbance detection system for capillary electrophoresis

This paper presents a cost-effective portable photodetection system for capillary electrophoresis absorptiometry. By using a CMOS BDJ (buried double p–n junction) detector, a dual-wavelength method for absorbance measurement is implemented. This system includes associated electronics for low-noise pre-amplification and A/D conversion, followed by digital signal acquisition and processing. Two signal processing approaches are adopted to enhance the signal to noise ratio. One is variable time synchronous detection, which optimizes the sensitivity and measuring rate compared to a conventional synchronous detection technique. The other is a statistical approach based on principal component analysis, which allows optimal estimation of detected signal. This system has been designed and tested in capillary electrophoresis conditions. Its operation has been verified with performances comparable to those of a commercialized spectrophotometric system (HP-3D CE). With potential on-chip integration of associated electronics, it may be operated as an integrable detection module for microchip electrophoresis and other microanalysis systems.