Anal. Chem. 1998, 70, 563-567
Contactless Conductivity Detection for Capillary Electrophoresis Andreas J. Zemann,* Erhard Schnell, Dietmar Volgger, and Gu 1 nther K. Bonn
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria
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. Efficient on-column detection for capillary electrophoresis is aggravated by the small volumes when capillaries with inner diameters between 25 and 75 µm are used. However, ionic species in the injection plug are being concentrated by electrostacking while forming distinctively separated and narrow zones when an electric field is applied. This effect also contributes to the high separation efficiencies of the method. As a consequence, the concentration of the solutes in the respective band zones can be many times higher as compared to the original injection plug, which significantly facilitates on-column detection.1 The most widely used mode of detection is UV absorptivity detection, which requires the presence of chromophoric moieties. However, inorganic cations and anions do not exhibit a sufficiently high absorptivity to be detected by UV absorption at low concentration levels. As a consequence, indirect photometric and fluorometric UV detection modes have been introduced.2-18 * Corresponding author: (Tel) +43-512-507-5180; (Fax) +43-512-5072965; (e-mail)
[email protected]. (1) Jandik, P.; Bonn, G. Capillary Electrophoresis of Small Molecules and Ions; VCH Publishers: New York, 1993; p 118ff. (2) Yeung, E. S. Acc. Chem. Res. 1989, 22, 125. (3) Kuhr, W. G.; Yeung, E. S. Anal. Chem. 1988, 60, 2642. (4) Gross, L.; Yeung, E. S. J. Chromatogr. 1989, 480, 169. (5) Foret, F.; Fanali, S.; Ossicini, L.; Bocek, P. J. Chromatogr. 1989, 470, 299. (6) Yeung, E. S.; Kuhr, W. G. Anal. Chem. 1991, 63, 275A. (7) Vorndran, A. E.; Oefner, P. J.; Scherz, H.; Bonn, G. K. Chromatographia 1992, 33, 163. (8) Beck, W.; Engelhardt, H. Chromatographia 1992, 33, 313. (9) Ba¨chmann, K.; Haumann, I.; Groh, T. Fresenius J. Anal. Chem. 1992, 343, 901. S0003-2700(97)00759-2 CCC: $15.00 Published on Web 02/01/1998
© 1998 American Chemical Society
Another possibility to specifically detect ionic species with a high equivalent conductivity is to measure the differences in conductivity of the solute zones and the separation electrolyte. By using separation electrolytes with a low conductivity, a sensitive detection is thus possible. Several devices have been described that use direct conductivity detection for cations and anions. The detection can be accomplished either by a direct contact of the solution with the electrodes19-26 or by contactless methods which require inductively or capacitively coupled devices using high frequencies.27-35 Most of these instruments were originally intended to work for chromatographic and isotachophoretic purposes; however, on principle they could be used for capillary electrophoresis as well. A certain drawback of instruments working with conductivity detection measured by direct contact of electrodes and sample are the high instrument costs, as the capillary has to be modified to a specific extent and a rather complicated geometry and (10) Ba¨chmann, K.; Boden, J.; Haumann, I. J. Chromatogr. 1992, 626, 259. (11) Xue, Y.; Yeung, E. S. Anal. Chem. 1993, 65, 2923. (12) Shamsi, S. A.; Danielson, N. D. Anal. Chem. 1994, 66, 3757. (13) Buchberger, W.; Cousins, S. M.; Haddad, P. R. Trends Anal. Chem. 1994, 13, 313. (14) Shamsi, S. A.; Danielson, N. D. Anal. Chem. 1995, 67, 4210. (15) Salimi-Moosavi, H.; Cassidy, R. M. Anal. Chem. 1995, 67, 1067. (16) Desbene, P. L.; Morin, C. J.; Monvernay, A. M. D.; Groult, R. S. J. Chromatogr., A 1995, 689, 135. (17) Collet, J.; Gareil, P. J. Chromatogr., A 1995, 716, 115. (18) Weston, A.; Brown, P. R.; Jandik, P.; Heckenberg, A. L.; Jones, W. R. J. Chromatogr. 1992, 608, 395. (19) Huang, X.; Pang, T. K. J.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1987, 59, 2747. (20) Harrold, M.; Stillian, J.; Bao, L.; Rocklin, R.; Avdalovic, N. J. Chromatogr., A 1995, 717, 371. (21) Avdalovic, N.; Pohl, C. A.; Rocklin, R.; Stillian, J. Anal. Chem. 1993, 65, 1470. (22) Huang, X.; Luckey, J. A.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1989, 61, 766. (23) Huang, X.; Zare, R. N.; Sloss, S.; Ewing, A. G. Anal. Chem. 1991, 63, 189. (24) Huang, X.; Zare, R. N. Anal. Chem. 1991, 63, 2193. (25) Avdalovic, N.; Pohl, C. A.; Rocklin, R. D.; Stillian, J. R. Anal. Chem. 1993, 65, 1470. (26) Haber, C.; Jones, W. R.; Soglia, J.; Surve, M. A.; McGlynn, M.; Caplan, A.; Reineck, J. R.; Krstanovic, C. J. Capillary Electrophor. 1996, 3, 1. (27) Alder, J. F.; Drew, P. K. P. Anal. Chim. Acta 1979, 110, 325. (28) Mu ¨ ller, D.; Jelinek, I.; Opekar, F.; Stulik, K. Electroanalysis 1996, 8, 722. (29) Pal, F.; Pungor, E.; Kovats, E. Anal. Chem. 1988, 60, 2254. (30) Alder, J. F.; Drew, P. K. P. Anal. Chim. Acta 1979, 110, 325. (31) Gas, B.; Demjanenko, M.; Vacik, J. J. Chromatogr. 1980, 192, 253. (32) Everaerts, F. M.; Rommers, P. J. J. Chromatogr. 1974, 91, 809. (33) Vacik, J.; Zuska, J.; Muselasova, I. J. Chromatogr. 1985, 320, 233. (34) Decristoforo, G. Thesis, University of Innsbruck, 1979. (35) Schnell, E.; Zemann, A. J.; Volgger, D.; Bonn, G. K. Patent pending (No. A1016/97), 1997.
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construction is required. Furthermore, changes in the cell geometry can be considered disadvantageously.28 As a consequence, for capillary electrophoresis purposes, a detector without galvanic connection of solutes and electrodes with, at the same time, equal sensitivity as compared to a directly connected conductivity detector is a certain demand. An oscillometric detector for ion chromatography has already been described with electrodes being separated from the electrolyte by a silicon lacquer or Teflon coating.29 This device enables a better reproducibility and stability than detectors with a galvanic contact. Generally, with detectors using a high-frequency voltage29,31,34 a signal resulting from both ohmic resistance and reactance of the liquid is recorded. Inductively coupled contactless conductivity detectors have been described as well.27,30 Several attempts have been made to use contactless conductivity detection for capillary isotachophoretic purposes. Instruments consisting of an arrangement of four electrodes placed outside the PTFE capillary have been described which use a highfrequency voltage.32,33 Polarization of the measuring electrodes or undesirable redox reactions are thus prevented. The application of conductivity detection in isotachophoresis is described in detail in various books on isotachophoresis.36-38 The present paper describes the application of a capacitively coupled conductivity detection system (CCCD) which works on a contactless basis for the detection of cationic and anionic compounds after capillary electrophoretic separation. EXPERIMENTAL SECTION Instrumentation. A Quanta 4000 capillary electrophoresis system (Waters Chromatography, Milford, MA) connected with an AD changer (System Interface Module, Waters) and a personal computer was used. Data acquisition and processing was carried out with a commercial chromatography software (Maxima 820, Waters). Fused-silica capillaries (Composite Metal Services, Worchester, U.K.) with an inner diameter of 50 µm and an outer diameter of 375 µm were used. Sample injection was carried out hydrostatically at an elevation of 10 cm. Reagents. All reagents were of analytical grade (SigmaAldrich Handels-Ges.m.b.H, Vienna, Austria, and Merck GmbH, Vienna, Austria). Standard and buffer solutions were prepared by dissolving the respective compounds in ultrapure water (Barnstead/Thermolyne, Dubuque, IA). Electronic Parts. The contactless detection device was built from electronic components which were purchased from local electronic dealers. Two directly coupled transistors (BC 414), which are connected to a potentiometer (100 kΩ) and an integrated circuit (U420B), were used for amplification. The rectification was performed using two diodes (BA4148). The frequency generator that is suitable for a frequency of 40 kHz sinus was built from an LC oscillator circuit with a transistor (BC 337). The 20-kHz rectangular form frequency generator was made from an integrated circuit (Telefunken NE 555). The dc voltage for the amplifier was stabilized to 9 V. For the mains connection (36) Everaerts, F. M.; Beckers, J. L.; Verheggen, Th. P. E. M. Isotachophoresis: Theory, Instrumentation and Application; J. Chromatogr. Library Vol. 6; Elsevier: Amsterdam, 1976. (37) Everaerts, F. M. Analytical Isotachophoresis; Elsevier: Amsterdam, 1981. (38) Bocek, P.; Deml, M.; Gebauer, P.; Dolnik, V. In Analytical Isotachophoresis; Radola, B. J., Ed.; VCH Publishers: Weinheim-Basel, 1988.
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Figure 1. Schematic drawing of the capacitively coupled conductivity detector (CCCD). The electrodes are made of either a conducting silver varnish or of metallic syringe cannulas with the capillary assembled through.
to the power supply, an RFI line filter was used in order to avoid noise from impulses and spikes. RESULTS AND DISCUSSION The conductivity detector described in this paper works on a contactless basis. It is used in connection with a commercial capillary electrophoresis instrument (Waters Quanta 4000) which enables free handling and accessibility of the capillary along almost its complete length. Among other advantages, this system enables an arbitrary placement of the detector as no capillary cartridge is used. Virtually any effective separation length can be adjusted without the need of removing the polyimide coating which is necessary for on-column UV detection. Thus, fragile detection windows are avoided and the risk of breaking the capillary during handling is reduced. Figure 1 depicts a schematic drawing of the CCCD. Two cylindrical conducting surfaces (electrodes) are placed around the polyimide coating on the outside of an uncoated fused-silica capillary. The electrodes can be made either of a conducting silver varnish which is painted around the polyimide coating of the capillary or of two syringe cannulas with the appropriate length where the capillary is passed through. For experimental testing of the detector, 30 mm was chosen as the length of each of the electrodes. This length can be varied from 15 to 50 mm, which then, as a consequence, requires different amplification to obtain equal signal intensities. In fact, changing the length of the electrodes has only little effect. An increase of length to 50 mm renders only slightly differing electropherograms in terms of sensitivity. For shorter electrodes, however, the respective amplification has to be increased which, at this time with the components used in this system, causes the signal-to-noise (S/N) ratio to become worse. The electrodes are connected to an oscillator through a resistor with a resistance of 50 kΩ. Together with the reactance of the shielded cable connection to the detector and the input resistance of the amplifier, a resistance R of approximately 10 kΩ is formed. A voltage drop of 0.2-2 mV is observed along resistance R, which is then amplified and rectified to direct current. The employed amplifier has a variable amplification of 500-1500-fold. After rectification, a direct voltage of 0.1-0.5 V is fed in the input of the AD changer
which itself is part of the data acquisition and processing system. However, any conventional integrator or other AD changer can be used for this purpose. A detection gap between the two electrodes of 2 mm is chosen, which can be increased to 5 mm, depending on the length of the capillary and the required separation efficiency. A distance of approximately 2 mm is sufficiently long with respect to important separation parameters such as resolution and separation efficiency. If the detection gap is too narrow, a capacitive transfer between the electrodes may occur. As a consequence, if resolution between specific solute zones is too low, it is advisable to increase the capillary length instead of narrowing the detection gap. The detection device described in this paper differs significantly from other contactless conductivity detectors for isotachophoresis or capillary electrophoresis purposes. Most of the other contactless conductivity detectors measure the signal radially across the capillary, a principle that suffers from the very short path lengths. Furthermore, the use of a low frequency bears several advantages when compared to high-frequency conductivity detectors. In addition to the ohmic resistance, a considerable dielectric contribution of the reactance can also be assumed when high frequencies are used. As a consequence, whenever the liquid inside of one of the electrodes changes its dielectric properties, which is likely when sample component zones are passing through, a shift of the baseline would be detected before and after the actual signal. Thus, comparable results with galvanically coupled detectors should be obtained in terms of relative signal intensities when low frequencies are used as primarily the ohmic resistance along the detection gap is responsible for the signal. A frequency of 40 kHz sinus form or 20-30 kHz rectangular form is used to keep the influence of reactance considerably low. With the long cylindrical electrodes used in this investigation, this frequency range works well with voltage amplitudes of 7-10 Vpp, preferably with 8 Vpp. A higher oscillator voltage may improve the S/N ratio to a certain extent, however, due to the limitations of the components and parts that build up the present detection device, a significant improvement of the sensitivity is not achieved. The voltage drop on resistance R is below 1 mV. Thus, the detector and all the connections have to be shielded to ground. In order to measure the differences of the conductivity between the electrolyte and the separated ions, an electric zero point depression is recommended as is found in any other instruments using direct conductivity with galvanically connected electrodes or UV detection. The detector presents a series connection of two capacitors and two resistors. The electrodes serve as two independent cylindrical capacitors with the liquid inside the capillary being the corresponding internal wire. The detection gap between the electrodes acts as one of the resistors and resistance R as the other. A reactance between 2 and 4 MΩ at a frequency of 40 kHz is calculated by assuming cylindrical capacitors. Using a buffer electrolyte with a low conductivity, the resistance of the liquid inside the capillary from the inlet to the outlet end is in the range of several gigaohms. Thus, the electrolyte in the detection gap between the electrodes, which measures approximately 0.3% of the total capillary length, exhibits a resistance in the megaohm range. If an ionic solute zone passes through the detection gap, the resistance decreases due to the increasing conductivity. As
Figure 2. Separation of a standard mixture of inorganic cations comparing UV and conductivity detection. Conditions: fused-silica capillary, L ) 60 cm, l1 ) 40 cm to conductivity detector, l2 ) 52.5 cm to UV detector (214 nm), U ) +20 kV, I ) 3.0 µA, and T ) 23 °C; electrolyte, 10 mM lactic acid, 8 mM 4-methylbenzylamine, and 15% methanol, pH 4.9; injection, 30 s; sample concentration, 0.1 mM.
the resistors are connected in series, this causes the voltage of all the other resistors to change significantly. As a consequence, a voltage increase can be measured on resistance R. After amplification and rectification, the signal is fed into the AD changer. The applicability of the developed device for capillary electrophoretic purposes can be demonstrated for the detection of inorganic cations and anions. A comparison of the contactless conductivity detection method with indirect UV detection provides satisfactory results in terms of sensitivity as depicted in Figure 2. The buffer consists of lactic acid, 4-methylbenzylamine, and methanol at a pH of 4.9. This system was originally introduced for indirect UV detection of cations.39 A 5-fold amplification of the UV signal is necessary to achieve comparable peak heights at given system settings. However, for practical purposes, a detector considered easy to use has to meet several basic requirements. Handling and mounting of the detection electrodes should be as easy as possible with only little pretreatment and modifications of the capillary. Although painting of silver conducting varnish on the outside of the capillary is both an inexpensive and easy pretreatment procedure, the electrodes were subject to further improvement of the detector. Two metallic syringe cannulas with an inner diameter slightly larger than the outside diameter of the silica capillary were cut to the appropriate length and used as electrodes. (39) Shi, Y.; Fritz, J. S. J. Chromatogr. 1993, 640, 473.
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Figure 3. Separation of a standard mixture of inorganic cations using conductivity detection. Conditions: fused-silica capillary, L ) 58 cm, l ) 45 cm to conductivity detector, U ) +20 kV, I ) 3.9 µA, and T ) 24 °C; electrolyte, 20 mM MES and 20 mM histidine, pH 6; injection, 10 s; sample concentration, 10 ppm.
Thus, the fused-silica capillary can be simply passed through the tubes. The sensitivity of the signals obtained from the flexible detector is only slightly inferior as compared to the fixed varnish detector. This is due to the fact that a small gap of air between the polyimide coating of the capillary and the metal tubing increases the dielectric volume between the electrodes. This arrangement generally exhibits some significant advantages over detection systems based on photometric absorptivity or end-capillary conductivity detection. For example, more than one conductivity detector can be easily and reversibly placed at any position along the capillary without the need of removing the polyimide coating with the danger of breaking. This is useful, for example, if kinetic phenomena are investigated or if the effective separation length has to be optimized. With the described detection device a simultaneous UV detection is also possible. In Figure 3, the electropherogram of seven inorganic cations using a low-conductivity electrolyte consisting of 20 mM 2-(Nmorpholino)ethanesulfonic acid (MES) and 20 mM histidine at pH 6 recorded with a flexible on-column conductivity detector is shown. A sample concentration of 10 ppm was chosen with an injection time of 10 s. A different amplification was used for the experiments with the flexible detector as compared to Figure 2 which results in increased signal heights. The conductivity detector is not only suitable for the detection of cations but also for inorganic anions. Figure 4 shows the separation of a mixture of inorganic anions at a concentration of 10 ppm with a buffer system consisting of 20 mM MES and 20 566 Analytical Chemistry, Vol. 70, No. 3, February 1, 1998
Figure 4. Separation of a standard mixture of inorganic anions using conductivity detection. Conditions: fused-silica capillary, L ) 70 cm, l ) 57 cm to conductivity detector, U ) -25 kV, I ) 3.9 µA, and T ) 24 °C; electrolyte, 20 mM MES, 20 mM histidine, and 0.0001% hexadimethrine bromide, pH 6; injection, 10 s.; sample concentration, 10 ppm.
mM histidine at pH 6. In order to reverse the electroosmotic flow (EOF) direction, a polycationic surfactant was added to the separation electrolyte (hexadimethrine bromide; HDB).40-43 As the EOF modifier is required only in nanomolar to micromolar concentrations, a significant contribution of this additional ionic buffer component to the background conductivity of the separation electrolyte is not observed. This is advantageous compared to other dynamic EOF modifiers such as the commonly used alkyltrimethylammonium-based modifiers that have to be used at concentrations in the millimolar range, which significantly increases the background conductivity. The limit of detection for the flexible conductivity detector is 200 ppb (S/N ) 3) for cations and anions as determined for sodium and chloride, respectively, with the MES-histidine system. For both species, a linear range of more than 3 orders of magnitude from 0.5 to more than 1000 ppm with a coefficient of correlation superior than 0.9995 (nine concentrations measured three times each) was obtained. An exact determination of the high end of the linear range was not carried out due to the high peak asymmetry at concentrations above 500 ppm where a separation of components is difficult, anyhow. Limits of detection and the linear range for quantitative analysis also depend on the (40) Masselter, S. M.; Zemann, A. J. Anal. Chem. 1995, 67, 1047. (41) Zemann, A. J. J. Capillary Electrophor. 1995, 2, 131. (42) Volgger, D.; Zemann, A. J.; Bonn, G. K.; Antal, M. J., Jr. J. Chromatogr., A 1997, 758, 263. (43) Zemann, A. J.; Volgger, D. Anal. Chem. 1997, 69, 3243.
buffer system as well as on the linearity of the diode rectifier. It has to be emphasized that not only inorganic ions but also organic ions can be detected by conductivity detection. However, limits of detection for organic ions are inferior compared to inorganic species because of the considerably lower ionic equivalent conductivities. The S/N ratio could be further reduced by using better components for the amplifier. Baseline noise is presumably due to the homemade amplifier, which consists of simple electronic components as described in the Experimental Section. Despite the fact that all connections to the detector were well screened and both detector and amplifier are shielded to ground, a considerable part of the noise is caused by components of the device, such as the high-voltage generator of the CE system and the AD changer. For future work, better electronic components and shielding are required to further lower the limits of detection. Furthermore, for specific purposes, an optimization of the buffer composition will also be necessary to increase resolution and separation efficiency.
easy-to-use adjustment of the detector on virtually any position along the capillary. The changes of the electric conductivity in the capillary inside the gap between the electrodes are measured, and by this means, inorganic cations as well as anions can be detected. Detection limits are in the range of 200 ppb for both cations and anions. By improving the electronic components, a further reduction of detection limits is likely. As the device works contactlessly, any cleaning or flushing of the detection cell is not necessary and it can be easily used without modification of the geometry of the capillary or of the polyimide coating.
CONCLUSION Contactless capacitively coupled conductivity detection is a suitable detection method for inorganic anions and cations in capillary electrophoresis. Placement of the capillary into two metal tubings, which act as two cylindrical capacitors, allows a fast and
Received for review July 15, 1997. Accepted October 30, 1997.X
ACKNOWLEDGMENT The authors dedicate this paper to Prof. Karl-Eberhard Schwarzhans on the occasion of his sixtieth birthday. Thanks to Karl Mayrhofer for experimental help, Dr. Gu¨nter Grienberger for fruitful discussions, and DI Friedrich Neumann for advices on the construction of the homemade amplifier and rectifier. Further information on the electronic details of the amplifier and the mechanical construction of the detector is available on request from Prof. Erhard Schnell via the corresponding author.
AC9707592 X
Abstract published in Advance ACS Abstracts, December 15, 1997.
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