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Wall-jet conductivity detector for microchip capillary electrophoresis
Talanta 78 (2009) 207–211
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Talanta journal homepage: www.elsevier.com/locate/talanta
Wall-jet conductivity detector for microchip capillary electrophoresis Joseph Wang a,∗ , Gang Chen b,∗ , Alexander Muck c,∗ a
Department of Nanoengineering, University California San Diego, La Jolla, CA 92093, USA School of Pharmacy, Fudan University, Shanghai 200032, China c Mass Spectrometry Group, Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, 07745 Jena, Germany b
a r t i c l e
i n f o
Article history: Received 27 August 2008 Received in revised form 24 October 2008 Accepted 31 October 2008 Available online 11 November 2008 Keywords: Capillary electrophoresis Conductivity detection Hybrid detection electrodes Microchip Miniaturization Wall-jet
a b s t r a c t A new end-column ‘hybrid’ contactless conductivity detector for microchip capillary electrophoresis (CE) was developed. It is based on a “hybrid” arrangement where the receiving electrode is insulated by a thin layer of insulator and placed in the bulk solution of the detection reservoir of the chip, whereas the emitting electrode is in contact with the solution eluted from the channel outlet in a wall-jet arrangement. The favorable features of the new detector including the high sensitivity and low noise, can be attributed to both the direct contact of the ‘emitting’ electrode with the analyte solution as well as to the insulation of the detection electrode from the high DC currents in the electrophoretic circuit. Such arrangement provides a 10-fold sensitivity enhancement compared to currently used on-column contactless conductivity CE microchip detector as well as low values of noise and easy operation. The new design of the wall-jet conductivity detector was tested for separation of explosive-related methylammonium, ammonium, and sodium cations. The new detector design reconsiders the wall-jet arrangement for microchip conductivity detection in scope of improved peak symmetry, simplified study of inter-electrode distance, isolation of the electrodes, position of the wall-jet electrode to the separation channel, baseline stability and low limits of detection. © 2008 Elsevier B.V. All rights reserved.
1. Introduction There has been a considerable advance in development of detection techniques for micro total analysis systems (-TAS) in the past few years [1]. Among techniques currently employed, laser-induced fluorescence detection (LIF) still dominates [2–4]. However, its sensitivity and selectivity often requires derivatization procedures, complicated focusing of optical microscope detection systems and expensive instrumentation. Accordingly, some advantageous features of these microanalytical systems are compromised. Therefore, there has been a considerable activity towards the development of alternative detection methods [5]. Considerable attention has been given to electrochemical detection modes such as conductivity or amperometry [6–8]. Conductivity detection is a universal detection technique based on measuring the conductivity between two electrodes through which a high frequency alternating current is passed. Conductiv-
Abbreviations: C4 D, combined contact/contactless conductivity detector; CCD, contactless conductivity detector; CD, contact conductivity detector; CE, capillary electrophoresis; PMMA, poly(methylmethacrylate). ∗ Corresponding author. Fax: +1 858 534 9553. E-mail addresses: [email protected] (J. Wang), [email protected] (G. Chen), [email protected] (A. Muck). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.10.055
ity detection has been widely used for capillary electrophoresis (CE) microchip systems [9,10] allowing the monitoring of ionic species down to the micromolar level. Such detectors in CE electrophoretic separation systems are commonly designed as “oncolumn” devices and less often in “end-column” modes [11–14]. Considering the connection of the sensing electrodes and the electrophoretic buffer, the “contact” mode, utilizing direct galvanic link to the solution, and capacitively coupled “contactless” mode have been used. In the latter, the electrodes are electrically isolated from solution. It can offer several advantages over the contact mode, including the absence of electrolytic reactions, effective isolation from high separation voltages or a simplified alignment of the detector. Most conventional CE systems are based on two or three electrode on-column contactless conductivity measurements with tubular or semitubular electrodes placed over the separation capillary [15–17]. Contact on-column arrangement was reported by Zare and coworkers [18] and the first contact end-column “wall-jet” setup was published by the same group later [19]. In their work, the platinum wire sensing electrode was mounted directly in the end of electrophoretic capillary. The conductivity was measured between the sensing and ground electrodes, which also acted to complete the electrophoretic circuit. Another example of an end-column conductivity detector employed a conductive epoxy sensing electrode placed by the side of the capillary end with a hydrophillic polymer strip connecting the capillary outlet with the solution in the
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outlet vessel. Improved baseline and effective shielding from the high-voltage circuitry was reported, yielding submillimolar limits of detection [20]. All previous arrangements have been already employed for the capillary format. For microchip arrangement, only on-column contact (CD) [21–23] or contactless conductivity detection (CCD) [24–26] modes have been reported. While different on-column conductivity electrode geometries have been already compared for integrated planar CE systems [27], the potential of the end-column conductivity detectors has not been investigated. This article reports for first time on an end-column ‘hybrid’ capacitively coupled C4 D for microchip CE demonstrating the detection of explosive-related ions, which earn a high priority in the view of the current danger of terrorist activities [28,29]. Our new detection system brings new insights into contact/contactless conductivity sensing in a wall-jet format, which has been preferred by many other electrochemical sensing schemes in CE microchip methods. The present design utilizes the inherent advantages of both the contactless conductivity detection, such as low noise and isolation from the high-voltage electrophoretic circuit, with the higher sensitivity and simplified geometry of the contact mode measurements in an alternative system where the receiving electrode is isolated by a thin layer of insulator and placed in the bulk solution of the detection reservoir of the chip while the emitting electrode is in contact with the solution in a wall-jet orientation to the separation channel outlet. Such arrangement assures high signal to noise ratios and an easy operation. The attractive performance characteristics of the hybrid wall-jet capacitively coupled microchip conductivity detector are reported in the following sections. To the best of our knowledge, no such detector arrangement has been reported for conductivity detection in both capillary and microchip systems. 2. Experimental 2.1. Apparatus A top view of the CE microchip with the wall-jet CCD is given in Fig. 1. The (70 mm × 24 mm) PMMA microchip (manufactured at the Institut für Mikrotechnik Mainz (IMM, Mainz, Germany)) had a 50 mm long separation channel (between the injection cross and the channel outlet reservoir) and an 18 mm long injection channel (between the sample and unused reservoirs); the channels crossed each other halfway between the sample and the unused reservoirs 9 mm from the run buffer reservoir, and had a 50 m × 50 m square cross section [30]. The end of the chip was cut off as shown in Fig. 1. The PMMA chip was fixed in a laboratory-built plexiglas holder with silicone grease providing proper sealing [31]. Short pipet tips were inserted into the fluidic ports of the chip to connect to the injection (Fig. 1B), running buffer (Fig. 1C) and unused (Fig. 1D) reservoirs on the holder to establish solution contact between the channel on the chip and corresponding reservoirs on the chip holder. The homemade high-voltage power supply had an adjustable voltage range between 0 and +4000 V; platinum wires were used to connect the high-voltage electrodes in the solution reservoirs (Fig. 1G) and the electronic circuitry of the conductivity detector (placed on the top of the chip using a laboratory stand). 2.2. Electrode fabrication The wall-jet ‘emitting’ electrode (Fig. 1E) consisted of a platinum wire (300 m diameter) inserted axially in the plastic screw of the system using an epoxy resin. The disc end of the electrode was polished with 0.05 m alumina slurry. The position of the “wall-jet”
Fig. 1. Schematic diagram of the CE microchip coupled with wall-jet CCD detection. Acrylic holder with PMMA microchip (A), sample reservoir (B), run-buffer (C) and unused (D) reservoirs, plastic screw with the wall-jet emitting electrode (E) in the detection reservoir, receiving electrode (F), and platinum electrodes (G) for the highvoltage circuit.
electrode could be changed by turning the plastic screw by a desired angle. A 9◦ turn resulted in 30 m shift between the channel outlet and the electrode along the separation channel. A thin insulated copper wire (0.3 mm × 50 mm) served as the “receiving” electrode (Fig. 1F). To provide proper isolation of the end of the electrode from the DC currents in the detection reservoir, the PVC insulation was pulled over the cut end of the wire and sealed by pressing the excess of plastic in heat. For the full contactless arrangement, the emitting electrode was insulated by casting 2 L of 5% (w/v) polystyrene in toluene on the disc electrode and dried in air. The detection reservoir was equipped by a poly(vinylchloride) (PVC) L-shaped 125 m thick rectangular spacer across the whole width of the detection reservoir, to hold the receiving electrode. The position of this electrode could be adjusted by placing its end into 1 mm holes arranged in the shorter arm of the spacer, with a 1.2 mm distance between them. Thus, the distance between the sensing electrodes could easily be controlled. The ‘emitting’ electrode wire was tin-soldered directly to the detector electronics; the wall-jet electrode was connected to the detector using a thin copper wire. The length of the wires was minimized to prevent induction of electric noise. 2.3. Electronic circuit The electronic circuit of the contactless detector was designed according to a previously reported scheme [32]. The circuit was equipped with a passive RC filter (time constant, 0.01 s) followed by a voltage follower (LF 356) to the circuit output to minimize electrical noise and to allow for convenient data reading. A HP 8116A function generator (Hewlett-Packard, Palo Alto, CA, USA) was used for generating the sinusoidal signal (usually with a frequency of 200 kHz with peak-to-peak amplitude of 10 V). The
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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 detection reservoir, to act also as a shield for the sensing electrodes. For data collection, a PCI-6035E 200 kS/s, 16 bit A/D board with NI-DAQ driver software for WIN 2000/NT and a program written in LabView 6.0.2 Full Development Version software were used (all National Instruments, Austin, TX, USA). The electronic components were purchased from local suppliers. 2.4. Reagents Histidine (His), 2-(N-morpholino)ethanesulfonic acid (MES), methyl ammonium, ammonium chloride, and sodium chloride were purchased from Sigma. The run buffer (20 mM, pH 6.1) was prepared by dissolving MES and His in deionized water. Stock solutions of the target cations (ammonium, methylammonium, and sodium at 100 mM concentrations) were prepared daily by dissolving the corresponding salts in the run buffer. All chemicals were used without any further purification. 2.5. Electrophoretic procedure The channels of the plastic chip were treated before use by rinsing with deionized water for 10 min. Reservoirs (C) and (D) on the microchip holder (Fig. 1) 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 an initial 20 s electrokinetic sample loading in the injection channel, the sample was injected by applying a potential of +1000 V for 1 s between the sample reservoir (B) and the grounded outlet reservoir. This drove the sample “plug” into the separation channel through the intersection. The analytical separation was preceded by switching the high-voltage contacts, the separation potential was subsequently applied to the run buffer reservoir (C) with the outlet reservoir grounded and all other reservoirs floating. Safety considerations. The high-voltage power supply can cause electrical shock. It should be handled with extreme care. Methyl ammonium is a toxic substance and other used chemicals are irritants. Skin or eye contact and accidental inhalation or ingestion should be avoided. 3. Results and discussion The new detection system brings new insights into contact/contactless conductivity sensing in a wall-jet format, which has been preferred by many other elelctrochemical sensing schemes in CE microchip methods. The performance of the new hybrid wall-jet CCD detector was evaluated for electrophoretic separation of ammonium, methylammonium, and sodium ions. Electropherograms obtained with the ‘hybrid’ detector design (Fig. 2C) (in which the emitting electrode was bare while the receiving electrode was insulated to sense only capacitive changes of the AC signal) have been compared to those obtained at the walljet ‘full’ contactless (Fig. 2B) and on-column contactless (Fig. 2A) conductivity detection schemes. In such arrangement, the new wall-jet configuration exhibits over 10-fold sensitivity enhancement compared to the wall-jet contact conductivity detector. The direct contact of the ‘emitting’ electrode with solution avoids the additional capacitive effects of the insulator and the double layer capacitance of the solution-capillary wall on the emitted signal, which is present in the on-column contactless arrangement. Such effects observed even at optimum frequencies for the ‘full’ contactless wall-jet arrangement (with the emitting electrode coated
Fig. 2. electropherograms showing the separation of 1 mM ammonium (a), methyl ammonium (b), and sodium (c) using on-column contactless conductivity detector (A) and wall-jet disc electrodes with (B) and without (C) polystyrene membrane coated. Operation conditions: (A) separation voltage, +1000 V; injection voltage, +1000 V; injection time, 1 s; frequency, 200 kHz; peak-to-peak amplitude, 10 V; sinusoidal ac waveform; running buffer, MES/HIS (20 mM, pH 6.1); the distance between the emitting (wall-jet disc) and receiving electrodes, 3.6 mm; the distance between wall-jet disc electrode and the outlet of separation channel, 30 m.
with polystyrene membrane) which displays smaller signals and a higher noise (Fig. 2B). The exact reason for the increase sensitivity associated with the bare emitting electrode in the hybrid system is still not fully understood, however, we assume that placing the bare emitting electrode near the high-voltage ground electrode and the channel outlet also levels off potentials generated between the separation channel and the detector. This is expected to improve the detector performance [6]. Although the wall-jet configuration results in some degree of extracolumn band-broadening, such arrangement provided significant peak shape improvements and a baseline resolution compared to the on-column design. The migration times of the analytes in the on-column contactless arrangement are slightly shorter due to the shorter length of the separation channel (45 mm vs. 50 mm in the case of end-column arrangement, respectively). The arrangement using the wall-jet electrode as the ‘emitting’ one was compared to a reversed scheme with the wall-jet electrode set as ‘receiving’ (Fig. 3). The system in which the bare emitting electrode is combined with an insulated receiving electrode (Fig. 3B) provides approximately 100 times higher signal-to-noise characteristics compared to an arrangement with an insulated emitting and bare wall-jet receiving electrodes (Fig. 3A). Such behavior is attributed to the fact that the high-voltages and DC currents at the channel outlet directly effect the input signal of the detection circuit, resulting in increased noise and inferior signal. The favorable features of the new detector, including the high sensitivity and low noise, can be attributed to both the direct contact of the ‘emitting’ electrode with the analyte solution as well as to the insulation of the detection circuitry by insulating the receiving electrode from the high DC currents in the electrophoretic circuit. Thus, no electrophoretic currents pass through the detector. Several experimental parameters affecting the response were examined and optimized. The distance between the two sens-
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Fig. 3. Electrophoregrams showing the separation of 1 mM ammonium (a), methyl ammonium (b), and sodium (c) using bare wall-jet disc electrode as ‘receiving’ electrode (A) and ‘emitting’ electrode (B). Other conditions, as in Fig. 2.
ing electrodes was adjusted using a plastic spacer. The holder enabled to vary the distance of the ‘receiving’ electrode vs. the fixed-positioned ‘emitting’ electrode over the 1.2–6 mm range. The dependence of the signal on the electrode distance was found to increase slowly with maximum response obtained for 3.6 mm distance (Fig. 4A). Further increase in electrode distance led to increased electronic noise and more rapid decrease of the signal. The influence of the distance between the emitting electrode and the channel outlet on the peak height was studied over the 15–75 m range (Fig. 4B). The response was nearly unaffected by changing the distance between 15 and 30 m, decreased rapidly between 30–60 m, and then more slowly. Most favorable S/N characteristics were obtained for 30 m.
As expected, the response of the wall-jet C4 D is strongly influenced by the operation frequency and the amplitude of the applied voltage. The influence of the applied frequency is shown in Fig. 5A. For ammonium, methylammonium and sodium, the signal was initially increasing with increase of the frequency. The curves were recorded pointwise over the 50–800 kHz range. The response increases slowly in the region of 50–300 kHz, then more rapidly with a peak at 500 kHz and decreasing in the same manner to 800 kHz. However, unstable and distorted peaks were observed for operation frequencies higher than 300 kHz, probably due to the characteristics of the operational amplifier [13]. The response of all ions increased linearly with the peak-to-peak amplitude of the alternating voltage between 2.5 and 10 V, and then more slowly (Fig. 5B). Optimal response (S/N characteristics) was obtained for peak-to-peak voltage of 10 V. All subsequent work employed a frequency of 200 kHz and amplitude of 10 Vpp . Fig. 6 shows the influence of the separation voltage upon the detector performance. As expected, increasing the separation voltage from +600 to +2500 V significantly decreases the migration times of the test ions from 56 to 26 s, from 80 to 30 s and from 97 to 35 s, for ammonium, methylammonium and sodium, respectively (A–E). The shorter migration times observed at higher electrical fields are coupled to substantially sharper peaks. However, separation voltages over +2000 V caused higher influence of the detection circuit and resulted in lower stability of the baseline. Over the studied voltage range, the separation voltage had a negligible influence on the baseline noise. Also, minimum peak broadening should be noted, which corresponds to other data reported on end-column conductivity detectors. The end-column wall-jet conductivity microchip detector displays a well-defined concentration dependence. Fig. 7 displays
Fig. 4. Effect of (A) the distance between the ‘emitting’ and ‘receiving’ electrodes and (B) the space between wall-jet disc electrode and the channel outlet upon the response for 0.5 mM ammonium ion. Other conditions, as in Fig. 2.
Fig. 5. Influence of (A) the applied ac voltage frequency and (B) the applied ac voltage upon the detector output. (A) Sample containing 1 mM ammonium (a), methyl ammonium (b), and sodium (c); peak-to-peak amplitude, 5 V. (B) Sample containing 0.5 mM ammonium (a), methyl ammonium (b), and sodium (c). Other conditions, as in Fig. 2.
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measurements which provided relative standard deviation values as low as 4.1% for sodium, 4.7% for methylammonium and 5.2% for ammonium ions. 4. Conclusions The analytical performance of the CE microchip with a new combined contact/contactless end-column conductivity detector clearly demonstrates the advantages of the new design. A great improvement in signal-to-noise ratio was achieved, with limits of detection as low as 3 and 5 M (for ammonium and sodium, respectively). The new setup offers improved Gaussian peak shapes in comparison to the contactless modes. The simplified construction of the detector allows for convenient replacement, positioning and alignment of the sensing electrodes as well as spatial integration with amperometric detection in commercial applications. In comparison to previously reported end-column capillary systems, the baseline stability was dramatically improved due to effective isolation of the detector circuit from the high-voltage. Such conductivity CE microsystem can be used in a wide range of analytical applications, providing a versatile alternative to UV and other optical means of detection. Further improvements of the detector sensitivity are expected by application of high-voltage conductivity detection schemes and new detection geometries. Fig. 6. Influence of the separation voltage upon the response for 0.5 mM ammonium (a), methyl ammonium (b), and sodium (c). Other conditions, as in Fig. 2.
Fig. 7. Electrophoregrams for mixtures containing increasing levels of ammonium (a) and sodium (b) in increments of 0.2 mM (A–E). Also shown (as insets) are the resulting calibration plots. Other conditions, as in Fig. 2.
electropherograms for sample mixtures containing increasing levels of ammonium (a) and sodium (b) ions in 200 m steps. A linear range of two orders of magnitude (correlation coefficients higher than 0.996) was obtained for ammonium and sodium. Detection limits of 5 M ammonium and 3 M sodium were estimated based on the response characteristics of a mixture containing 10 M of these cations (S/N = 3, not shown). The improved sensitivity was coupled to very good reproducibility and stability. No decrease in response sensitivity was observed for series of eight consecutive
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Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Wall-jet conductivity detector for microchip capillary electrophoresis Joseph Wang a,∗ , Gang Chen b,∗ , Alexander Muck c,∗ a
Department of Nanoengineering, University California San Diego, La Jolla, CA 92093, USA School of Pharmacy, Fudan University, Shanghai 200032, China c Mass Spectrometry Group, Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, 07745 Jena, Germany b
a r t i c l e
i n f o
Article history: Received 27 August 2008 Received in revised form 24 October 2008 Accepted 31 October 2008 Available online 11 November 2008 Keywords: Capillary electrophoresis Conductivity detection Hybrid detection electrodes Microchip Miniaturization Wall-jet
a b s t r a c t A new end-column ‘hybrid’ contactless conductivity detector for microchip capillary electrophoresis (CE) was developed. It is based on a “hybrid” arrangement where the receiving electrode is insulated by a thin layer of insulator and placed in the bulk solution of the detection reservoir of the chip, whereas the emitting electrode is in contact with the solution eluted from the channel outlet in a wall-jet arrangement. The favorable features of the new detector including the high sensitivity and low noise, can be attributed to both the direct contact of the ‘emitting’ electrode with the analyte solution as well as to the insulation of the detection electrode from the high DC currents in the electrophoretic circuit. Such arrangement provides a 10-fold sensitivity enhancement compared to currently used on-column contactless conductivity CE microchip detector as well as low values of noise and easy operation. The new design of the wall-jet conductivity detector was tested for separation of explosive-related methylammonium, ammonium, and sodium cations. The new detector design reconsiders the wall-jet arrangement for microchip conductivity detection in scope of improved peak symmetry, simplified study of inter-electrode distance, isolation of the electrodes, position of the wall-jet electrode to the separation channel, baseline stability and low limits of detection. © 2008 Elsevier B.V. All rights reserved.
1. Introduction There has been a considerable advance in development of detection techniques for micro total analysis systems (-TAS) in the past few years [1]. Among techniques currently employed, laser-induced fluorescence detection (LIF) still dominates [2–4]. However, its sensitivity and selectivity often requires derivatization procedures, complicated focusing of optical microscope detection systems and expensive instrumentation. Accordingly, some advantageous features of these microanalytical systems are compromised. Therefore, there has been a considerable activity towards the development of alternative detection methods [5]. Considerable attention has been given to electrochemical detection modes such as conductivity or amperometry [6–8]. Conductivity detection is a universal detection technique based on measuring the conductivity between two electrodes through which a high frequency alternating current is passed. Conductiv-
Abbreviations: C4 D, combined contact/contactless conductivity detector; CCD, contactless conductivity detector; CD, contact conductivity detector; CE, capillary electrophoresis; PMMA, poly(methylmethacrylate). ∗ Corresponding author. Fax: +1 858 534 9553. E-mail addresses: [email protected] (J. Wang), [email protected] (G. Chen), [email protected] (A. Muck). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.10.055
ity detection has been widely used for capillary electrophoresis (CE) microchip systems [9,10] allowing the monitoring of ionic species down to the micromolar level. Such detectors in CE electrophoretic separation systems are commonly designed as “oncolumn” devices and less often in “end-column” modes [11–14]. Considering the connection of the sensing electrodes and the electrophoretic buffer, the “contact” mode, utilizing direct galvanic link to the solution, and capacitively coupled “contactless” mode have been used. In the latter, the electrodes are electrically isolated from solution. It can offer several advantages over the contact mode, including the absence of electrolytic reactions, effective isolation from high separation voltages or a simplified alignment of the detector. Most conventional CE systems are based on two or three electrode on-column contactless conductivity measurements with tubular or semitubular electrodes placed over the separation capillary [15–17]. Contact on-column arrangement was reported by Zare and coworkers [18] and the first contact end-column “wall-jet” setup was published by the same group later [19]. In their work, the platinum wire sensing electrode was mounted directly in the end of electrophoretic capillary. The conductivity was measured between the sensing and ground electrodes, which also acted to complete the electrophoretic circuit. Another example of an end-column conductivity detector employed a conductive epoxy sensing electrode placed by the side of the capillary end with a hydrophillic polymer strip connecting the capillary outlet with the solution in the
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J. Wang et al. / Talanta 78 (2009) 207–211
outlet vessel. Improved baseline and effective shielding from the high-voltage circuitry was reported, yielding submillimolar limits of detection [20]. All previous arrangements have been already employed for the capillary format. For microchip arrangement, only on-column contact (CD) [21–23] or contactless conductivity detection (CCD) [24–26] modes have been reported. While different on-column conductivity electrode geometries have been already compared for integrated planar CE systems [27], the potential of the end-column conductivity detectors has not been investigated. This article reports for first time on an end-column ‘hybrid’ capacitively coupled C4 D for microchip CE demonstrating the detection of explosive-related ions, which earn a high priority in the view of the current danger of terrorist activities [28,29]. Our new detection system brings new insights into contact/contactless conductivity sensing in a wall-jet format, which has been preferred by many other electrochemical sensing schemes in CE microchip methods. The present design utilizes the inherent advantages of both the contactless conductivity detection, such as low noise and isolation from the high-voltage electrophoretic circuit, with the higher sensitivity and simplified geometry of the contact mode measurements in an alternative system where the receiving electrode is isolated by a thin layer of insulator and placed in the bulk solution of the detection reservoir of the chip while the emitting electrode is in contact with the solution in a wall-jet orientation to the separation channel outlet. Such arrangement assures high signal to noise ratios and an easy operation. The attractive performance characteristics of the hybrid wall-jet capacitively coupled microchip conductivity detector are reported in the following sections. To the best of our knowledge, no such detector arrangement has been reported for conductivity detection in both capillary and microchip systems. 2. Experimental 2.1. Apparatus A top view of the CE microchip with the wall-jet CCD is given in Fig. 1. The (70 mm × 24 mm) PMMA microchip (manufactured at the Institut für Mikrotechnik Mainz (IMM, Mainz, Germany)) had a 50 mm long separation channel (between the injection cross and the channel outlet reservoir) and an 18 mm long injection channel (between the sample and unused reservoirs); the channels crossed each other halfway between the sample and the unused reservoirs 9 mm from the run buffer reservoir, and had a 50 m × 50 m square cross section [30]. The end of the chip was cut off as shown in Fig. 1. The PMMA chip was fixed in a laboratory-built plexiglas holder with silicone grease providing proper sealing [31]. Short pipet tips were inserted into the fluidic ports of the chip to connect to the injection (Fig. 1B), running buffer (Fig. 1C) and unused (Fig. 1D) reservoirs on the holder to establish solution contact between the channel on the chip and corresponding reservoirs on the chip holder. The homemade high-voltage power supply had an adjustable voltage range between 0 and +4000 V; platinum wires were used to connect the high-voltage electrodes in the solution reservoirs (Fig. 1G) and the electronic circuitry of the conductivity detector (placed on the top of the chip using a laboratory stand). 2.2. Electrode fabrication The wall-jet ‘emitting’ electrode (Fig. 1E) consisted of a platinum wire (300 m diameter) inserted axially in the plastic screw of the system using an epoxy resin. The disc end of the electrode was polished with 0.05 m alumina slurry. The position of the “wall-jet”
Fig. 1. Schematic diagram of the CE microchip coupled with wall-jet CCD detection. Acrylic holder with PMMA microchip (A), sample reservoir (B), run-buffer (C) and unused (D) reservoirs, plastic screw with the wall-jet emitting electrode (E) in the detection reservoir, receiving electrode (F), and platinum electrodes (G) for the highvoltage circuit.
electrode could be changed by turning the plastic screw by a desired angle. A 9◦ turn resulted in 30 m shift between the channel outlet and the electrode along the separation channel. A thin insulated copper wire (0.3 mm × 50 mm) served as the “receiving” electrode (Fig. 1F). To provide proper isolation of the end of the electrode from the DC currents in the detection reservoir, the PVC insulation was pulled over the cut end of the wire and sealed by pressing the excess of plastic in heat. For the full contactless arrangement, the emitting electrode was insulated by casting 2 L of 5% (w/v) polystyrene in toluene on the disc electrode and dried in air. The detection reservoir was equipped by a poly(vinylchloride) (PVC) L-shaped 125 m thick rectangular spacer across the whole width of the detection reservoir, to hold the receiving electrode. The position of this electrode could be adjusted by placing its end into 1 mm holes arranged in the shorter arm of the spacer, with a 1.2 mm distance between them. Thus, the distance between the sensing electrodes could easily be controlled. The ‘emitting’ electrode wire was tin-soldered directly to the detector electronics; the wall-jet electrode was connected to the detector using a thin copper wire. The length of the wires was minimized to prevent induction of electric noise. 2.3. Electronic circuit The electronic circuit of the contactless detector was designed according to a previously reported scheme [32]. The circuit was equipped with a passive RC filter (time constant, 0.01 s) followed by a voltage follower (LF 356) to the circuit output to minimize electrical noise and to allow for convenient data reading. A HP 8116A function generator (Hewlett-Packard, Palo Alto, CA, USA) was used for generating the sinusoidal signal (usually with a frequency of 200 kHz with peak-to-peak amplitude of 10 V). The
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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 detection reservoir, to act also as a shield for the sensing electrodes. For data collection, a PCI-6035E 200 kS/s, 16 bit A/D board with NI-DAQ driver software for WIN 2000/NT and a program written in LabView 6.0.2 Full Development Version software were used (all National Instruments, Austin, TX, USA). The electronic components were purchased from local suppliers. 2.4. Reagents Histidine (His), 2-(N-morpholino)ethanesulfonic acid (MES), methyl ammonium, ammonium chloride, and sodium chloride were purchased from Sigma. The run buffer (20 mM, pH 6.1) was prepared by dissolving MES and His in deionized water. Stock solutions of the target cations (ammonium, methylammonium, and sodium at 100 mM concentrations) were prepared daily by dissolving the corresponding salts in the run buffer. All chemicals were used without any further purification. 2.5. Electrophoretic procedure The channels of the plastic chip were treated before use by rinsing with deionized water for 10 min. Reservoirs (C) and (D) on the microchip holder (Fig. 1) 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 an initial 20 s electrokinetic sample loading in the injection channel, the sample was injected by applying a potential of +1000 V for 1 s between the sample reservoir (B) and the grounded outlet reservoir. This drove the sample “plug” into the separation channel through the intersection. The analytical separation was preceded by switching the high-voltage contacts, the separation potential was subsequently applied to the run buffer reservoir (C) with the outlet reservoir grounded and all other reservoirs floating. Safety considerations. The high-voltage power supply can cause electrical shock. It should be handled with extreme care. Methyl ammonium is a toxic substance and other used chemicals are irritants. Skin or eye contact and accidental inhalation or ingestion should be avoided. 3. Results and discussion The new detection system brings new insights into contact/contactless conductivity sensing in a wall-jet format, which has been preferred by many other elelctrochemical sensing schemes in CE microchip methods. The performance of the new hybrid wall-jet CCD detector was evaluated for electrophoretic separation of ammonium, methylammonium, and sodium ions. Electropherograms obtained with the ‘hybrid’ detector design (Fig. 2C) (in which the emitting electrode was bare while the receiving electrode was insulated to sense only capacitive changes of the AC signal) have been compared to those obtained at the walljet ‘full’ contactless (Fig. 2B) and on-column contactless (Fig. 2A) conductivity detection schemes. In such arrangement, the new wall-jet configuration exhibits over 10-fold sensitivity enhancement compared to the wall-jet contact conductivity detector. The direct contact of the ‘emitting’ electrode with solution avoids the additional capacitive effects of the insulator and the double layer capacitance of the solution-capillary wall on the emitted signal, which is present in the on-column contactless arrangement. Such effects observed even at optimum frequencies for the ‘full’ contactless wall-jet arrangement (with the emitting electrode coated
Fig. 2. electropherograms showing the separation of 1 mM ammonium (a), methyl ammonium (b), and sodium (c) using on-column contactless conductivity detector (A) and wall-jet disc electrodes with (B) and without (C) polystyrene membrane coated. Operation conditions: (A) separation voltage, +1000 V; injection voltage, +1000 V; injection time, 1 s; frequency, 200 kHz; peak-to-peak amplitude, 10 V; sinusoidal ac waveform; running buffer, MES/HIS (20 mM, pH 6.1); the distance between the emitting (wall-jet disc) and receiving electrodes, 3.6 mm; the distance between wall-jet disc electrode and the outlet of separation channel, 30 m.
with polystyrene membrane) which displays smaller signals and a higher noise (Fig. 2B). The exact reason for the increase sensitivity associated with the bare emitting electrode in the hybrid system is still not fully understood, however, we assume that placing the bare emitting electrode near the high-voltage ground electrode and the channel outlet also levels off potentials generated between the separation channel and the detector. This is expected to improve the detector performance [6]. Although the wall-jet configuration results in some degree of extracolumn band-broadening, such arrangement provided significant peak shape improvements and a baseline resolution compared to the on-column design. The migration times of the analytes in the on-column contactless arrangement are slightly shorter due to the shorter length of the separation channel (45 mm vs. 50 mm in the case of end-column arrangement, respectively). The arrangement using the wall-jet electrode as the ‘emitting’ one was compared to a reversed scheme with the wall-jet electrode set as ‘receiving’ (Fig. 3). The system in which the bare emitting electrode is combined with an insulated receiving electrode (Fig. 3B) provides approximately 100 times higher signal-to-noise characteristics compared to an arrangement with an insulated emitting and bare wall-jet receiving electrodes (Fig. 3A). Such behavior is attributed to the fact that the high-voltages and DC currents at the channel outlet directly effect the input signal of the detection circuit, resulting in increased noise and inferior signal. The favorable features of the new detector, including the high sensitivity and low noise, can be attributed to both the direct contact of the ‘emitting’ electrode with the analyte solution as well as to the insulation of the detection circuitry by insulating the receiving electrode from the high DC currents in the electrophoretic circuit. Thus, no electrophoretic currents pass through the detector. Several experimental parameters affecting the response were examined and optimized. The distance between the two sens-
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Fig. 3. Electrophoregrams showing the separation of 1 mM ammonium (a), methyl ammonium (b), and sodium (c) using bare wall-jet disc electrode as ‘receiving’ electrode (A) and ‘emitting’ electrode (B). Other conditions, as in Fig. 2.
ing electrodes was adjusted using a plastic spacer. The holder enabled to vary the distance of the ‘receiving’ electrode vs. the fixed-positioned ‘emitting’ electrode over the 1.2–6 mm range. The dependence of the signal on the electrode distance was found to increase slowly with maximum response obtained for 3.6 mm distance (Fig. 4A). Further increase in electrode distance led to increased electronic noise and more rapid decrease of the signal. The influence of the distance between the emitting electrode and the channel outlet on the peak height was studied over the 15–75 m range (Fig. 4B). The response was nearly unaffected by changing the distance between 15 and 30 m, decreased rapidly between 30–60 m, and then more slowly. Most favorable S/N characteristics were obtained for 30 m.
As expected, the response of the wall-jet C4 D is strongly influenced by the operation frequency and the amplitude of the applied voltage. The influence of the applied frequency is shown in Fig. 5A. For ammonium, methylammonium and sodium, the signal was initially increasing with increase of the frequency. The curves were recorded pointwise over the 50–800 kHz range. The response increases slowly in the region of 50–300 kHz, then more rapidly with a peak at 500 kHz and decreasing in the same manner to 800 kHz. However, unstable and distorted peaks were observed for operation frequencies higher than 300 kHz, probably due to the characteristics of the operational amplifier [13]. The response of all ions increased linearly with the peak-to-peak amplitude of the alternating voltage between 2.5 and 10 V, and then more slowly (Fig. 5B). Optimal response (S/N characteristics) was obtained for peak-to-peak voltage of 10 V. All subsequent work employed a frequency of 200 kHz and amplitude of 10 Vpp . Fig. 6 shows the influence of the separation voltage upon the detector performance. As expected, increasing the separation voltage from +600 to +2500 V significantly decreases the migration times of the test ions from 56 to 26 s, from 80 to 30 s and from 97 to 35 s, for ammonium, methylammonium and sodium, respectively (A–E). The shorter migration times observed at higher electrical fields are coupled to substantially sharper peaks. However, separation voltages over +2000 V caused higher influence of the detection circuit and resulted in lower stability of the baseline. Over the studied voltage range, the separation voltage had a negligible influence on the baseline noise. Also, minimum peak broadening should be noted, which corresponds to other data reported on end-column conductivity detectors. The end-column wall-jet conductivity microchip detector displays a well-defined concentration dependence. Fig. 7 displays
Fig. 4. Effect of (A) the distance between the ‘emitting’ and ‘receiving’ electrodes and (B) the space between wall-jet disc electrode and the channel outlet upon the response for 0.5 mM ammonium ion. Other conditions, as in Fig. 2.
Fig. 5. Influence of (A) the applied ac voltage frequency and (B) the applied ac voltage upon the detector output. (A) Sample containing 1 mM ammonium (a), methyl ammonium (b), and sodium (c); peak-to-peak amplitude, 5 V. (B) Sample containing 0.5 mM ammonium (a), methyl ammonium (b), and sodium (c). Other conditions, as in Fig. 2.
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measurements which provided relative standard deviation values as low as 4.1% for sodium, 4.7% for methylammonium and 5.2% for ammonium ions. 4. Conclusions The analytical performance of the CE microchip with a new combined contact/contactless end-column conductivity detector clearly demonstrates the advantages of the new design. A great improvement in signal-to-noise ratio was achieved, with limits of detection as low as 3 and 5 M (for ammonium and sodium, respectively). The new setup offers improved Gaussian peak shapes in comparison to the contactless modes. The simplified construction of the detector allows for convenient replacement, positioning and alignment of the sensing electrodes as well as spatial integration with amperometric detection in commercial applications. In comparison to previously reported end-column capillary systems, the baseline stability was dramatically improved due to effective isolation of the detector circuit from the high-voltage. Such conductivity CE microsystem can be used in a wide range of analytical applications, providing a versatile alternative to UV and other optical means of detection. Further improvements of the detector sensitivity are expected by application of high-voltage conductivity detection schemes and new detection geometries. Fig. 6. Influence of the separation voltage upon the response for 0.5 mM ammonium (a), methyl ammonium (b), and sodium (c). Other conditions, as in Fig. 2.
Fig. 7. Electrophoregrams for mixtures containing increasing levels of ammonium (a) and sodium (b) in increments of 0.2 mM (A–E). Also shown (as insets) are the resulting calibration plots. Other conditions, as in Fig. 2.
electropherograms for sample mixtures containing increasing levels of ammonium (a) and sodium (b) ions in 200 m steps. A linear range of two orders of magnitude (correlation coefficients higher than 0.996) was obtained for ammonium and sodium. Detection limits of 5 M ammonium and 3 M sodium were estimated based on the response characteristics of a mixture containing 10 M of these cations (S/N = 3, not shown). The improved sensitivity was coupled to very good reproducibility and stability. No decrease in response sensitivity was observed for series of eight consecutive
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