Capillary electrophoresis system using electrochemical detection

Journal of Chemistry, Vol. 43 (4), P. 520 - 523, 2005<br /> <br /> <br /> Capillary electrophoresis system using<br /> electrochemical detection<br /> <br /> Received 6th -Oct.-2004<br /> Vu Thi Thu Ha1, In-Ja Lee1, Le Quoc Hung2<br /> 1<br /> Dept of Chemistry, Dongguk University, Gyeongju, Gyeongbuk, 780-714, Korea<br /> 2<br /> Institute of Chemistry, Vietnamese Academy of Science and Technology<br /> <br /> summary<br /> Micellar Electrokinetic Capillary Chromatography (MEKC) was employed to the analysis of<br /> aniline, phenol and 2,5-dinitrophenol using an end-column amperometric detector with a carbon<br /> cloth electrode. The current condition of separation is a solution of 25 mM Na2HPO4, 25 mM<br /> Na2B4O7, and 50 mM SDS (pH 9.11); applied voltage, 10 kV; hydrodynamic injection for 15 s at<br /> 15 cm; applied potential for electrochemical detection, 500 mV. Absorbance electropherograms<br /> is also recorded for comparison.<br /> <br /> <br /> i - Introduction different migration time. Detection method is<br /> one of the most important parameter in CE. A<br /> Nowadays, application of capillary variety of detectors used for CE, including<br /> electrophoresis (CE) for the separation on UV/vis absorbance, fluorescence, laser-induced<br /> analysis in different samples has become fluorescence, mass spectrometric, conductivity,<br /> increasingly widespread because of its minimal amperometric, radiometric and refractive index.<br /> sample volume requirement (µl), short analysis In this paper, amperometric detection has been<br /> time and high separation efficiency [1]. used and UV/vis absorbance is used for<br /> Electrophoresis is the movement of electrically comparison.<br /> charged particles or molecules in a conductive Amperometric detection measures the<br /> liquid medium, usually aqueous, under the current that results from oxidation or reduction<br /> influence of an electric field. Capillary of electroactive solutes at a working electrode.<br /> electrophoresis is typically performed using fuse A potential is applied across a supporting<br /> silica capillary that are 100 cm in length, 50 or electrolyte between a working and reference<br /> 75 µm inner diameter (i.d) and 375 µm outer electrode and this causes the oxidation or<br /> diameter (o.d) with voltages of up to 30 kV. The reduction of solutes [2]. Most detectors have a<br /> principle of CE system with electrochemical third electrode, an auxiliary electrode, which is<br /> detection is shown in figure 1. used to control the potential between working<br /> electrode and reference electrode. The current<br /> When the voltage is applied, positive charge that flow through the working electrode is<br /> cations will migrate toward the negative charged proportional to the number of electron transfers<br /> electrode, the cathode, and the negative charged taking place and, therefore, the concentration of<br /> anions will migrate toward the positively solutes. In CE, Electrochemical detection<br /> charged electrode, the anode. The rates at which typically operated in the amperometric mode<br /> they migrate depend on their charge-to-size can be coupled with CE to provide high<br /> ration. From that compounds are separated in sensibility and selectivity for the determination<br /> 520<br /> of electroactive substances [3, 4]. This mode is the working electrode at a fixed value and<br /> usually performed by controlling the potential of monitoring the current as a function of time [5].<br /> <br /> <br /> <br /> <br /> Figure 1: Schematic representation of electrophoresis system<br /> 1. Capillary 5. Sample Vial 9. Computer<br /> 2. High Voltage Supply 6. Waste Vial WE: Working electrode<br /> 3. Rinsing Vial 7. Destination Vial/Elect. Cell CE: Counter Electrode<br /> 4. Source Vial 8. Analytical equipment RE: Reference Electrode<br /> <br /> <br /> ii - Experimental Exposure working surface electrode was made<br /> by first polishing with fine sand paper and then<br /> Apparatus and electrodes by aluminum oxide powder (used for<br /> chromatography, particle size < 10).<br /> All electrochemical measurements were<br /> A 55 cm fused silica column was used for<br /> performed in the three-electrode mode using a<br /> electrophoresis separation with UV detection<br /> homemade electrochemical analyzer connected<br /> (45 cm from one end to UV detector) and<br /> to a computer (Manufactured in Lab of<br /> electrochemical detection. This capillary had an<br /> Computer Application to Chemical Research,<br /> Institute of Chemistry, Vietnamese Academy of inner diameter 75 µm and outer diameter 365<br /> Science and Technology). A silver wire and a µm (provided by Polymicro Technologies, Inc.<br /> platinum wire were employed as pseudo- Phoenix, AZ). Surfactant SDS was added in to<br /> reference and counter electrode respectively. All buffer in order to improve the separation<br /> potential given in this work were measured with efficiency and Micellar Electrokinetic Capillary<br /> respect to this reference system. Experiments Chromatography (MEKC) was conducted.<br /> were conducted at room temperature applying Before each run, the capillaries were flushed<br /> the desired working potential. Working with doubly distilled water, NaOH 0.1 M and<br /> electrodes were tested from three different then water again for 10 minutes, and finally<br /> materials: platinum microelectrode (CHI 107, filled with the buffer.<br /> 10 µm diameter), glassy carbon disk made in Reagents<br /> lab with 3 mm in diameter and Teflon body (8<br /> mm outer diameter) and carbon cloth. Carbon Phenol, aniline and 2,5-dinitrophenol were<br /> cloth were inserted into a desired plastic tube purchased from Aldrich. Stock solutions were<br /> and filled with epoxy. One copper wire was prepared with 10-2 M by doubly distilled water<br /> used for establishing electrical contact. for each and kept separately. Working solutions<br /> <br /> 521<br /> then were prepared daily by diluting stock to study the behaviors of electroactive compounds<br /> solutions with buffer solution in proper ratios. [4]. It can be used for determination of the applied<br /> Other chemicals for buffer were analytical potential on working electrode. Since the potential<br /> reagent grade. Buffer had 25 mM Na2HPO4, 25 applied to the working electrode directly affects<br /> mM Na2B4O7, 50 mM Sodium Dodecyl Sulfate the sensibility and detection limits of this method.<br /> (SDS-Aldrich) and pH = 9.11. Buffer and It is therefore necessary to determine the<br /> samples were microfiltered through a 0.45 µm maximum potential at that oxidation/reduction<br /> Type HA. (Advantec MFS, Inc. USA) reactions occur. Different materials for working<br /> electrode were examined and carbon cloth was<br /> iii - Results and Discussion chosen for further measurement since its<br /> sensibility is rather high. Figure 2 is cyclic<br /> Electrochemical measurement voltamograms of aniline (A), phenol (B) and 2,5-<br /> Cyclic voltammetry is a suitable technique dinitrophenol (C) in buffer.<br /> <br /> A B C<br /> 0.07<br /> 0.05<br /> 0.06 0.045 0.14<br /> 0.04<br /> 0.05 0.12<br /> 0.035<br /> 0.03 0.1<br /> i(mA/cm^2)<br /> <br /> <br /> <br /> <br /> 0.04<br /> i(mA/cm^2)<br /> <br /> <br /> <br /> <br /> i(mA/cm^2)<br /> 0.025 0.08<br /> 0.03<br /> 0.02<br /> 0.06<br /> 0.02 0.015<br /> 0.01 0.04<br /> 0.01<br /> 0.005<br /> 0.02<br /> 0 0<br /> -0.005 0<br /> <br /> -0.2 0 0.2 0.4 0.6 0.8 -0.2 0 0.2 0.4 0.6 0.8 -0.2 0 0.2 0.4 0.6 0.8 1 1.2<br /> U(v) U(v) U(v)<br /> <br /> <br /> Figure 2: Cylic voltammograms of 5.0x10-4 M of aniline (A), phenol (B) and 2,5-dinitrophenol (C) in<br /> 25 mM Na2HPO4, 25 mM Na2B4O7, 50 mM SDS, pH 9.11 at carbon cloth, scan rate: 50 mV/s<br /> <br /> It is seen that in operating system, aniline, sample peaks are clearly resolved and third peak<br /> phenol and 2,5-nitrophenol were oxidized and for 2,5-dinitrophenol is not seen. They are<br /> maximum peak currents were observed at about 380 eluted at 12 min (aniline), 13.5 min (phenol),<br /> mV, 280 mV and 820 mV (versus Ag wire), respectively. However, figure 3B shows all three<br /> respectively. However, the signal for 2,5- peaks at 11 min, 12 min and 22 min, which<br /> dinitrophenol is rather weak. In this experiment, correspond to aniline, phenol and 2,5-<br /> separation was conducted for 5x10-4 M of aniline, dinitrophenol, respectively. On-column UV<br /> phenol and 2,5-dinitrophenol. For compromising, detector used here consists of a D2 lamp, a<br /> the applied potential for end-column electro- chopper and a Lock in amplifier (SR510).<br /> chemical detection was chosen and maintained at The possible reason for these observations<br /> 500 mV. CE data using electrochemical detection can be attributed to the relatively weak peak<br /> can be presented in figure 3. currents of 2,5-dinitrophenol in the CV and to<br /> Figure 3 represents our preliminary data of the cell design, especially in the alignment of<br /> the obtained electropherorams of aniline, phenol the working electrode and the capillary. In order<br /> and 2,5-dinitrophenol in buffer using a post- to overcome these problems, we are presently<br /> column amperometric detector (A) and on- working on the optimizing the buffer condition<br /> column UV absorbance detector (B) for for the amperometric detection and on<br /> comparison. Fig. 3-(A) shows that only two modifying the cell design.<br /> <br /> <br /> 522<br />  A B<br /> <br /> <br /> <br /> <br /> Fig. 3. Electropherograms of aniline (1), phenol (2) and 2,5-dinitrophenol (3).<br /> (A) and (B) were obtained, using a post-column amperometric detector and a on-column UV absorbance<br /> detector (215 nm), respectively.<br /> The concentration of each species was 5.0x10-4 M.<br /> MEKC conditions: run buffer, a solution of 25 mM Na2HPO4, 25 mM Na2B4O7, and 50 mM SDS (pH<br /> 9.11); applied voltage, 10 kV; hydrodynamic injection for 15 sec at 15 cm; applied potential for electro-<br /> chemical detection, 500 mV<br /> <br /> iv - Conclusions at Gyeongju, Korea. Manufactoring Electro-<br /> chemical equipment was supported by<br /> The capillary electrophoresis for substance Vietnamese Fundamental Research Foundation.<br /> separations with electrochemical detection has<br /> been developed in Vietnam just recently. To References<br /> improve the sensibility and reproducibility, it is<br /> essential to optimize buffer condition and 1. Dale R. Baker. 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