Anal. Chem. 2003, 75, 4475-4479
Movable Contactless-Conductivity Detector for Microchip Capillary Electrophoresis Joseph Wang,* Gang Chen, and Alexander Muck, Jr.
Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003
A new movable contactless-conductivity detection system for microchip capillary electrophoresis is introduced. Such a versatile system relies on positioning the detector at different points along the separation channel via “sliding” the electrode holder. The new movable microchip detection system offers distinct improvements compared to common fixed-location conductivity detectors. For example, placing the detector at different locations along the microchannel offers useful insights into the separation process. Three-dimensional plots of resolution/channel length/separation voltage can be used for optimizing the separation process and selecting the analysis time. The system enables rapid switching between “total” (unresolved) and “individual” (resolved/fingerprint) signals on the basis of placing the detector at the beginning and end of the separation channel, respectively. By moving the detector to a shorter effective separation length, after eluting fast-migrating ions, shorter analysis times can be achieved (through faster detection of late-eluting analytes). These and other improvements in the analytical performance and insights into the separation process are illustrated in connection with the detection of low-energy ionic explosives and nerve agent degradation products. Microfabricated (Lab-on-a-Chip) analytical devices represent the fastest developing field in analytical chemistry.1,2 With the rapid development of microfluidic systems, there are growing demands for compatible detection schemes. Electrochemical detection has attracted considerable attention for capillary electrophoresis (CE) microchips owing to its inherent miniaturization and compatibility with micromachining protocols.3,4 Current, potential, and conductance signals have thus been monitored in connection to various on- and off-chip amperometric, potentiometric, and conductivity detectors. Conductivity detection is increasingly being used for chip-based CE microsystems.5,6 Such a detection scheme monitors differences in the conductivity of analyte zones and the background electrolyte (in the detector volume) and, hence, can sense all ionic species. Conductivity detection can be accomplished * Corresponding author. E-mail:
[email protected]. (1) Reyes, D. R.; Iossifidis, D.; Auroux, P. A.; Manz, A. Anal. Chem. 2002, 74, 2623. (2) Figeys, D.; Pinto, D. Anal. Chem. 2000, 72, 330A. (3) Wang, J. Talanta 2001, 56, 223. (4) Lacher, N.; Garrison, K.; Martin, R. S.; Lunte, S. M. Electrophoresis 2001, 22, 2526. (5) Tanyaniwa, J.; Hauser, P. C. Anal. Chem. 2002, 74, 6378. (6) Zemann, A. J. Trends Anal. Chem. 2001, 20, 346. 10.1021/ac030122k CCC: $25.00 Published on Web 07/26/2003
© 2003 American Chemical Society
either by a direct contact of the run buffer and the sensing electrodes or by a contactless mode in which the electrodes do not contact the solution. The contactless detection mode offers several distinct advantages, such as the elimination of surface fouling or bubble formation, a simplified construction and alignment of the detector, and effective isolation from high separation voltages.7-10 This article describes a novel movable contactless-conductivity detector for micromachined CE microchip systems. So far, contactless-conductivity detectors (CCD) have been fixed at a given location close to the end of the separation channel. In principle, however, the CCD detector can be placed at any location along the separation channel. There are very few examples of moving detectors in conventional CE systems.11-13 These moving devices have used optical detection, involving primarily spatialscanning fluorescence detectors in connection to fiber opticcoupled lasers and translational tables. A semitubular CCD with an easily exchangeable capillary has been described for conventional CE.14 To the best of our knowledge, movable detection schemes have not been used in connection with microchip devices. Our new movable contactless-conductivity CE-microchip detector is based on changing the detector position along the separation channel by “sliding” the sensing electrodes along the length of the microchip (Figure 1). Such a mobile detector system consists of the aluminum film sensing electrodes (Figure 1C,k) mounted on a thin polymer plate that is clipped on both sides of the PMMA separation chip (Figure 1E). Movement of the conductivity detector to different locations along the separation channel is shown below to offer several important advantages over fixeddetector formats. These include convenient visualization of the separation progress and improved optimization of the separation process, shorter analysis time (higher sample throughput), convenient switching between “total” and “individual” (fingerprint) assays modes in the same channel, and faster detection of lateeluting compounds (in connection to repositioning the detector (7) Pumera, M.; Wang, J.; Opekar, F.; Jelı´nek, I.; Feldman, J.; Lo ¨we, H.; Hardt, S. Anal. Chem. 2002, 74, 1968. (8) Lichtenberg, J.; de Rooij, N. F.; Verpoorte, E. Electrophoresis 2002, 23, 3769. (9) Tanyaniwa, J. S.; Hauser, P. C. Anal. Chem. 2002, 74, 6378. (10) Laugere, F.; Guilt, R. M.; Bastemeejer, J.; der Steen, G. V.; Berthold, A.; Baltussen, E.; Sarro, P.; van Dedem, G. W.; Vellekoop, M.; Bossche, A. Anal. Chem. 2003, 75, 306. (11) Johansson, J.; Witte, D. T.; Larsson, M.; Nillson, S. Anal. Chem. 1996, 68, 2766. (12) Beale, S. C., Su ¨ dmeier, S. J. Anal. Chem. 1995, 67, 3367. (13) Olivares, J. A.; Stark, P. C.; Jackson, P. Anal. Chem. 2002, 74, 2008. (14) Tu˚ma, P.; Opekar, F.; Jelı´nek, I. Electroanalysis 2001, 13, 989.
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Figure 1. Schematic diagrams of the microchip electrophoretic system with the movable contactless-conductivity detector (A) along with a detailed design of the movable electrode system: top (B) and bottom (C) views, as well as cross-sectional views without (D) and with (E) the PMMA separation chip. (a) Run buffer reservoir, (b) sample reservoir, (c) unused reservoir, (d) movable electrodes, (e) separation channel, (f) sample waste reservoir, (g) PMMA chip, (h) conductive silver epoxy, (i) PVC clamps, (j) copper wires, (k) aluminum foil electrodes, and (l) Plexiglas plate.
during the run). The detailed characterization and attractive analytical performance of the new moving CCD microchip detection system are reported in the following sections in connection to the detection of low-ionic explosives and nerve-agent degradation products. EXPERIMENTAL SECTION Apparatus. The plastic microchip, shown in Figure 1A, consisted of two sealed PMMA plates (70 × 24 mm) with a 50mm-long separation channel (between the injection cross and the channel outlet) and a 18-mm-long injection channel (between the sample and unused reservoirs). The channels had a 50 × 50 µm squared cross section. The PMMA microchips were manufactured at the Institute for Microtechniques of Mainz (IMM, Mainz, Germany) and were described earlier.15 The homemade high-voltage power supply had an adjustable voltage range between 0 and +5000 V. A Plexiglas holder was fabricated for accommodating the separation chip. Short pipet tips were inserted into each of the four holes on the PMMA chip for providing solution contact between the channel on the chip and the corresponding reservoir on the chip holder. The electronic circuitry of the conductivity detector was placed on the top of the chip. Electrode Fabrication. A schematic of the experimental setup is shown in detail in Figure 1. The rectangularly shaped electrodes (0.8 × 24 mm) were fabricated from two 10-µm-thick aluminumfoil strips (Figure 1C,k). The electrodes were fixed around two sides of a 1-mm-thick, 26 × 10 mm-sized PMMA plate of the detector using a quick-setting epoxy (Figure 1B,h), at a distance (15) Wang, J.; Pumera, M.; Chatrathi, M. P.; Escarpa, A.; Konrad, R.; Griebel, A.; Do ¨rner, W.; Lo ¨we, H. Electrophoresis 2002, 23, 596.
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of 3.8 mm from the longer edge of the plate and with a distance of 800 µm distance between them (Figure 1C). The electrodes were placed in an antiparallel orientation to reduce the stray coupling between them. The detector plate was equipped with two poly(vinyl chloride) (PVC) clip-like open rectangular holders (Figure 1B,i) from both shorter ends of the plate to hold the microchip (Figure 1E,g). The cover plate of the chip (125 µm thick) was thus mechanically pressed toward the detection electrodes without an adhesive. The top view of the microchip/ detector setup is displayed in Figure 1A. The position of the detection electrodes could be changed by “sliding” the detector holder along the separation channel. Thin copper wires (Figure 1B,j) were attached to the electrodes (Figure 1C,k) on the top of the detector plate using a conducting epoxy (Chemtronics, Kennesaw, GA) and were tin-soldered to the detector electronics. The length of these wires was minimized to prevent induction of electric noise. The whole detector was then attached by its top plane to the detection circuitry box by plastic tapes. Electronic Circuit. The electronic circuit of the contactless detector was designed in accordance with a previously reported scheme.16 The circuit was completed by adding a passive RC filter (time constant, 0.01s) followed by a voltage follower (LF 356) to the circuit output for noise suppression. This setup allows convenient interfacing to the data acquisition system (chart recorder or computer DAQ). The electronic components were purchased from local suppliers. A HP 8116A function generator (Hewlett-Packard, Palo Alto, CA) was used for generating the sinusoidal signal (usually with a frequency of 200 kHz with peakto-peak amplitude of 5 V). The electronic circuit was placed in a shielding box to protect the electronics from external electric fields. One side of the box was open; this side was placed as close as possible to the detector plate so that the box acted also as a shield for the electrodes. To further minimize the noise, the chip (along with the printed electronic board) was secured from possible mechanical vibrations. Reagents. Histidine (His), 2-(N-morpholino)ethanesulfonic acid (MES), potassium nitrate, methylammonium, sodium chloride, potassium chloride, ammonium chloride, potassium perchlorate, and ammonium nitrate were purchased from Sigma. Methylphosphonic acid (MPA), isopropyl methylphosphonic acid (IMPA), and pinacolyl methylphosphonic acid (PMPA) were obtained from Aldrich. The MES/His run buffer (20 mM each, pH 6.1) was prepared by dissolving MES and His in deionized water. Stock solutions of the target cations (potassium, sodium, ammonium, and methylammonium, at 100 mM) and anions (nitrate, chloride, and perchlorate, 100 mM) were prepared by dissolving the corresponding salts in the run buffer. All chemicals were used without any further purification. Electrophoretic Procedure. The channels of the plastic chip were treated before use by rinsing with deionized water for 10 min. After mounting the detector holder onto the microchip, reservoirs (a), (c), and (f) (Figure 1A) were filled with the electrophoretic run buffer solution, while reservoir (b) was filled with the sample mixture (target ions dissolved in the run buffer). After its initial loading in the injection channel, the sample was injected by applying a potential of +500 V (for cations) or -500 V (for anions) for 2 s between the sample reservoir (b) and the (16) da Silva, J. A. F.; do Lago, C. L. Anal. Chem. 1998, 70, 4339.
Figure 2. Electropherograms for a mixture solution containing 1 mM ammonium (a), methylammonium (b), and sodium (c) ions obtained by placing the mobile contactless-conductivity detector at different distances from the injection cross: (A) 0.5, (B) 1, (C) 2, (D) 3, (E) 4, and (F) 4.8 cm. Operation conditions: separation voltage, 1500 V; injection voltage, 500 V; injection time, 2 s; run buffer, MES/ His (20 mM, pH 6.1); sinus waveform with frequency of 200 kHz; and a peak-to-peak voltage of 5 V.
grounded outlet reservoir (f). This drove the sample plug into the separation channel through the intersection. The analytical separation proceeded by applying the separation potential to the run buffer reservoir (a) with the outlet reservoir (f) grounded and all other reservoirs floating. Safety Considerations. The high-voltage power supply should be handled with extreme care to avoid electrical shock. Alkyl methylphosphonic acids and methylammonium are toxic substances; other used chemicals are irritants. Skin or eye contact and accidental inhalation or ingestion should be avoided. RESULTS AND DISCUSSION The ability to change the effective length of the separation channel allows monitoring of the separation progress and optimization of the analytical performance. Creating an image of the separation along the microchannel allows monitoring of the separation progress and termination of the separation at optimal time (through a judicious adjustment of the effective channel
length). Such visualization of the separation process over the entire channel length is presented in Figure 2. The figure displays electropherograms for a mixture of ammonium (a), methylammonium, (b) and sodium (c) ions detected at (A) 0.5-, (B) 1.0-, (C) 2.0-, (D) 3.0-, (E) 4.0-, and (F) 4.8-cm distances from the injection cross. As expected, the separation efficiency improves upon increasing the effective length of the separation channel; baseline resolution (with a minimal change in sensitivity) is attained using separation-channel lengths longer than 3 cm. Shorter distances result in an insufficient resolution. The 3.0-, 4.0-, and 4.8-cm detector positions require 25, 32, and 38 s analysis times, respectively. Positioning of the detector at 3 cm (from the injection cross) thus allows utilization of the shortest channel length sufficient for resolving the three analytes and, hence, attaining the shortest analysis time. This reduces the overall analysis time by 35% compared to the end-channel (4.8 cm) detection. Note also that the shortest (0.5 cm) detector position results in a single peak for the three cations, reflecting their “total” content (A). The implications of such “total” assays are discussed below. Such an “early” run also allows for a fast determination of the quality of the injection and availability of detector signals.13 By offering deeper insights into the separation process, the movable detection system facilitates the optimization of the microchip operation. Optimization methods used for assessing the separation processes are often based on using the resolution of two adjacent peaks as a criterion for evaluating the effect of individual or multivariate factors.17,18 Such optimization is commonly carried out using a single separation length (in view of the fixed detector position). Adding the parameter of “effective length” (based on the actual detector position) further enhances the ability to optimize the separation. This feature of the moving microchip detector was illustrated using the ammonium/methylammonium cation pair and the nitrate/perchlorate anion pair. Figure 3 displays three-dimensional plots of resolution/channel length/separation voltage for these cation (A) and anion (B) pairs. Such profiles offer detailed information on the efficiency of the separation process along the channel length. The most favorable resolution of the cations (R ) 2.57) is represented by a wave maximum (M) at a separation voltage of 1500 V in connection to a detector position of 4.0 cm. The optimal resolution for the anions (R ) 2.18) is observed using a separation voltage of 1000 V and a channel length of 4.8 cm. Such optimization capability could be
Figure 3. Plots of resolution R1,2 of the ammonium1 and methylammonium2 cation peaks (A) and the nitrate1 and perchlorate2 anion peaks (B) as a function of the separation voltage and effective separation channel length. M, point of maximum resolution. Operation conditions: injection time, 2 s; injection voltage, 500 V (A); injection voltage, -500 V (B); other conditions as in Figure 2.
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Figure 4. Electropherograms showing the separation of explosiverelated anions (A) and cations (B) using the mobile conductivity detector located at the beginning (a, 0.8 cm to the injection cross) and the end (b, 4.8 cm to the injection cross) of the separation channel. (A) Mixture containing 1 mM chloride (1), nitrate (2), and perchlorate (3) ions. (B) Mixture containing 0.7 mM ammonium (4), methylammonium (5), potassium (6), and sodium (7). Conditions: separation voltage, -1500 V; injection voltage, -500 V; running buffer, MES/His (20 mM, pH 6.1) (A); separation voltage, 1000 V; injection voltage, 500 V; running buffer, MES/His (20 mM, pH 6.1) containing 7.5 mM 18-crown-6-ether (B); Other conditions as in Figure 2.
programmed in a hyphenated “learning” software of future analytical systems. The versatility of the movable contactless-conductivity detector adds new capabilities to the operation of the microchip. For example, the system allows convenient switching between (unresolved) “total” and “individual” (resolved/fingerprint) signals by placing the sensing electrodes at the beginning or the end of the separation channel, respectively. This is illustrated in Figure 4 for measurements of several explosive-related anions (A) and cations (B). As expected (from Figure 2A), these mixtures display single peaks (reflecting their “total” contents) at short “effective lengths”. “Total” assays of these explosive-related ions can thus be performed within 13 s (anions) and 11 s (cations). A rapid switching of the detector position to the end of the separation channel leads to three well-resolved anion peaks and four cation peaks, with migration times of the last peak of 47 and 50 s, respectively (i.e., ∼3.6 and 4.5-fold slower than the corresponding “total” assays). Assays rates of up to ∼300 and 70/h can thus be realized for the “total” screening and “individual” measurements, respectively. Such convenient distinction between the “total” and “individual” contents of ionic explosives facilitates chip-based single-channel “flow-injection” (fast-screening) and “separation” (fingerprintidentification) operation modes. Analogous switching between “total” and “individual” amperometric measurements was accomplished by replacing the run buffer in the separation channel. The new (movable detector) approach greatly simplifies the switching procedure, in view of its speed, because it does not require a lengthy (2 min) wash cycle (as in switching-buffer “total”/“individual” protocols).19 The success of such an operation (17) Introduction to Modern Liquid Chromatography; Snyder, L. R., Kirkland, J. J., Eds.; Wiley: New York, 1979. (18) Persson, K.; A° stro ¨m, O. J. Chromatogr., B 1997, 697, 207. (19) Wang, J.; Pumera, M.; Chatrathi, M. P.; Escarpa, A.; Musameh, M.; Collins, G.; Mulchandani, A.; Yuehe, L.; Olsen, K. Anal. Chem. 2002, 74, 1187.
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Figure 5. Portions of electropherograms displaying the precision of switching between “total” (A, C, E) and “individual” (B, D, F) assays. A mixture containing 1 mM ammonium (a), methylammonium (b), and sodium (c) ions. Conditions: separation voltage, 1500 V; injection voltage, 1500 V; injection time, 1s; other conditions as in Figure 2.
Figure 6. Electropherograms for a mixture containing (A) 1 mM chloride ion (a), 0.5 mM perchlorate ion (b), 200 ppm methylphosphonic acid (MPA, c), 200 ppm isopropyl methylphosphonic acid (IMPA, d), and 400 ppm pinacolyl methylphosphonic acid (PMPA, e) using a fixed detector position of 4.8 cm; and (B) the same mixture upon moving CCD, after elution of the ionic explosives, to an effective separation length of 2.5 cm; Conditions: separation voltage, -800 V; injection voltage, -800 V; injection time, 1 s; peak-to-peak voltage, 10 V; other conditions as in Figure 2.
depends on the reproducibility of the detector repositioning step. Such reproducibility was examined from eight cycles of “total” (A,C,D) and “individual” (B,D,F) cation assays (corresponding to “short” and “long” detector positions, respectively). Portions of electropherograms depicting the precision of switching are displayed in Figure 5. The RSD for the “total” assay peak is 3.0%, whereas those of the “individual” ammonium (a), methylammonium (b), and sodium (c) signals are 4.1, 3.7, and 4.5%, respectively. Note also the reproducible migration times of both the “total” and “individual” peaks. Overall, these data indicate that the detector positioning is highly reproducible. Another advantage of the movable CCD system, involving changing the detector position during the analysis for decreasing the assay time, is demonstrated in Figure 6 in connection with simultaneous measurements of low-energy ionic explosives and nerve-agent degradation products. Using the fixed detectorposition format, the separation of a mixture containing chloride
Figure 7. Application of movable CCD to simultaneous detection of cationic and anionic species. Electropherograms for a mixture solution containing 2 mM ammonium (a), 1 mM methylammonium (b), 1 mM sodium (c), 1 mM chloride (d), 1 mM nitrate (e), and 1 mM perchlorate (f) with the distance between the movable CCD and anode cross of 4.1 (A), 3.4 (B), and 2.7 (C) cm, respectively. Operation conditions: separation voltage, +1000 V; dual injection voltage, 500 V; other conditions as in Figure 6. A 84 × 21 mm PMMA separation chip was used with a 66-mm-long separation channel (defined by the two injection crosses on both sides of the chip) and intersected by two 10-mm-long injection channels (between the sample and unused buffer reservoirs on both sides of the chip). The same sample was placed in the two sample reservoirs located on opposite side of the separation channel.
and perchlorate anions and methylphosphonic acid (MPA), isopropyl methylphosphonic acid (IMPA), and pinacolyl methylphosphonic acid (PMPA) requires an analysis time of 3.5 min (Figure 6A). By moving the detector to a shorter effective separation length (2.5 cm) during the run, after eluting the fastmigrating chloride and perchlorate ions, the analysis can be carried out in
