January 27, 2014.
In the field of analytical chemistry, one of the most popular and straight forward techniques is the separation analysis. The good example is chromatagraphy, which is the well-known technique conventionally used by chemists and biologists. There is the technique, however, to be used in the separation of chemical analytes benefiting the influence of an external electric field. This technique is called Capillary Electrophoresis (CE). The sub-technique of CE namely Capillary Zone Electrophoresis (CZE) is the focus of our study. Normally, the separation is performed in a 50-100 cm long glass capillary of average internal diameter ranging from 30-100 microns. Therefore, it requires quite high separation voltage and long operation period. On the top of that, the system is larger when it is integrated with the detector, which is usually a light detector (UV absorption or emission). Such a system has to be placed in a laboratory only. Thus, the Microchip Capillary Electrophoresis (MCE) technique, which is the operation of CE on a microfluidic chip, has been proposed. The main advantages of MCE are its portability and high integrability. Low cost and low chemical consumption are also considered in poor countries.
The separation mechanism in CZE can be described based on physics phenomena. The principle composes of two electrokinetic flow, i.e., electro-osmosis and electrophoresis. The first phenomenon is the flow of charged solution along the capillary as a result of the external electric field (Fig. 1(a)). The origin of the flow is the existence of the electrical double layer (EDL) at the capillary wall/solution interface. The EDL composes of the Stern layer, which is a charged layer tightly bounded with the capillary wall due to Coulomb interaction, and the adjacent diffuse layer. The diffuse layer is a moveable layer, which will flow as an influence of the electric force originated by an applied electric field. As the diffuse layer flows, the bulk molecules are dragged along. This leads to the electro-osmotic flow, which the flow velocity is dependent on the EDL potential and external electric field strength. The second effect is electrophoretic flow, which is the movement of the charged particle placed in liquid environment. When the electric field from external source is applied, the charged molecule experiences both electric force and drag force (Fig. 1(b)). At equilibrium, two forces are balanced, and then the electrophoretic velocity of the charged molecule can be calculated. The electrophoretic velocity of different molecules in the same environment is different. It depends on the charge and size of each specific molecule. In real experiments, the two phenomena occurs simultaneously. What we can observe after the solution is driven for a while is the separation of the analytes into zones of different electrophoretic velocity (Fig. 2). Therefore, this principle is used in the separation analysis.
Figure 2 The CZE separation mechanism. The sample plug of mixture of two analytes is initially introduced to the microchannel (a). The two analytes are separated into two zones before they reach the detector (b).
After the analytes are separated into zones, each zone flows toward the detector. The C4D (Capacitively Coupled Contactless Conductivity Detection) technique is adopted from several applicable detection methods. The conductivity of the analytes is detected with respect to the background electrolyte. To avoid the degradation of detection electrodes, the analyte passing through the detection cell (including analyte and electrodes) without touching the metal electrodes in C4D technique. The electrodes used for conductivity detection compose of the excitation and pick-up electrodes. The alternating voltage is generated from the excitation electrode, passing the analyte, and then detected by the pick-up electrode. The acquiured signal is then sent to the amplification part, to display and collect. The impedance of the detection cell can be calculated by using the electronic equivalent circuit, which an induced EDL and the analyte are modeled as the capacitor and resistor, respectively. At high excitation frequency range, the impedance of the detection cell is dominated by the resistance of the analyte, which relates to the conductivity. The varation of the cell impedance leads to the change of current passing between two electrodes. Therefore, the conductivity of the analytes can be measured from the variation of the detected current.
Microfluidic Research Unit of Plasma and Beam Physics Research Facility, under the support of Thailand Center of Excellence in Physics (ThEP Center), has developed the self-made MCE-C4D prototype, which is aimed to be improved as a powerful analytical tool in the future. In this work, the separation microchanel of 200 micrometers wide, 30 micrometers depth, and 45 millimeters long, was fabricated from PDMS (poly-dimethylsiloxane) by the EWBT (Etched Wiring Board Technique). As shown in Fig. 3, the PDMS microfluidic chip was integrated on the PDMS-spin-coated PCB detection platform. The microchannel was aligned on the detection electrodes, which were placed on the detection platform. The integration was achieved by oxygen plasma bonding technique. The detection platform can be simply connected to the detector. The HV power supplies were built for electric field generation along the microchannel in separation process. The HV can be varied ranging from 0 to 2500 V. The C4D detecion unit, as shown in Fig. 4, was designed and built for compatible working with the MCE part. The excitation voltage of this system has the value of 25 V P-P with the adjustable frequency between 0-250 kHz. The detection unit is controlled by the PC through USB connection port, therefore, the acquired data is collected and can be display as a real time observation on the computer monitor.
Figure 3 (a) Shematic drawing for MCE-C4D device fabrication. The PDMS replica is laid over the PDMS-coated platform by carefully align microchannel with the electrodes. Four holes are made on the PDMS replica as 4 reservoirs. The volume of each reservoir is 0.1 mL. (b) A photograph of the device in comparison with the purchased glass chip on the low left corner of the PCB platform.
Figure 4 The C4D system with plug-in MCE-C4D device at the top.
The MCE-C4D was tested by measuring of the signal from three ionic salts, KCl, NaCl, and LiCl. The standard sample solution of the mixture of three ions was prepared with DI water. The background electrolyte was MES/His buffer of pH 6.1 as mentioned in several research papers. The separated three positive ions zones, K+, Na+, Li+, were flow along the separation channel toward the detection cell by the CZE mechanism as mention previously. In this study, three important parameters that indicate the separation efficiency and detection sensitivity were investigated. Background electrolyte concentration was firstly concerned since it vastly affects the separation of the analytes. Different BGE concentration can leads to the different electro-osmotic velocity. Therefore, the BGE concentration has to be the first parameter to be optimized. We found from the experiment that the suitable BGE concentration for our system was 20 mM (see Figs. 5(a) – 5(c)). After the optimization of the BGE concentration, the next topic to be studied can be separation voltage. The separation voltage is the voltage applied across the separation channel in order to generate the electric field. The migration time, which is the time duration used by the analyte zone in travelling from the original position to the detector, can be affected by the electric field strength or the separation voltage. The desirable separation voltage should provide the best separation resolution. To find out the suitable separation voltage for our system, the voltage was varied in different experiments and we found that the best resolution was obtained from 800 V separation voltage (see Figs. 5(d) – 5(f)). The last parameter studied in this work was the analyte concentration. The study of J. G. A. Brito-Neto et al. explains that the conductivity of the analytes depends on the concentration. Therefore, the unequimolar mixture analytes were served as samples in the experiments. The results obviously show that the peak height is dependent on the analyte concentrations (see Figs. 5(g) – 5(h)). These results can be extended to study on the quantitative measurement, which can lead to the improvement of the sensitivity of the detector.
From the experimental results, the MCE-C4D system developed by our group has an acceptable efficiency in sense of separation and detection of standard analyte solution. However, this system is only the first prototype, which needs more study and improvement. By continuously study, we may have a high efficiency and low-cost analytical tool for use in our country. Sustainable development.
Figure 5 (a) to (c) are electropherograms showing the effects of BGE (MES/His) concentration, i.e. 5 mM, 10 mM and 20 mM, respectively, on the MCE separation. (d) to (F) are electropherograms showing the effects of separation voltage, i.e. 400 V, 600 V and 800 V, respectively, on the MCE separation. (g) and (h) are electropherograms showing the effects of analyte concentration, i.e. 5 mM Na+ and 20 mM K+/Li+ and 5 mM K+ and 20 mM Na+/Li+, respectively, on the MCE separation. These also confirm that the three peaks represent, from left to right, K+, Na+ and Li+ zones.
 M. Jitvisate, “Development of Compact System of Microchip Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection for the Detection of Caffeine in Beverages”, M.S. thesis, Chiang Mai University, Chiang Mai, 2013.
 J. G. A. Brito-Neto, J. A. F. da Silva, L. Blanes and C. L. do Lago, “Understanding Capacitively Coupled Contactless Conductivity Detection in Capillary and Microchip Electrophoresis. Part 1. Fundamentals”, Electroanalysis, 17(13), 2005, 1198-1206.
Mr. Monchai Jitvisate and Assoc. Prof. Dr. Somsorn Singkarat
Microfluidic Research Unit, Plasma and Beam Physics Research Facility, Dept. of Physics and Materials Science, Fac. of Science, Chiang Mai University, Chiang Mai- 50200, Thailand
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