Anal. Chem. 1994,66, 2531-2543
Computer- Interfaced Bipolar Pulse Conductivity Detector for Capillary Systems Satyajlt Kar, Purnendu K. Dasgupta,' Hanghul Llu, and Hoon Hwangt Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409- 106 1
Conductivity detectors sufficiently small for deployment in capillary analytical systems can be fabricated very simply and reproducibly from commonly available micro assemblies of multiconductor ribbons. It results in a unique geometry for the conductivity detection cell where the fluid does not flow Between the electrodes. Interface circuitry for a personal computer (PC)-based bipolar pulse conductance detector is described. The cell can be used with this or commercial detectors. Results obtained with the new cell and electronic design in a suppressed conductometriccapillary electrophoresis separation system are presented. Capillary electrophoresis (CE) is currently one of the most active areas in separation science; citation frequency on this topic is still in the exponential growth phase.' The advantage of this technique over other liquid-phase separation systems is characterized by its high separation efficiency, very high mass sensitivity, and very small sample requirement. However, CE imposes very stringent requirements on the detection system which must detect individual analytes at low concentrations in the presence of a carrier electrolyte. Further, the detection volumes must be small enough to prevent any degradation of the separation already achieved in the capillary without causing any undue flow restriction. Development of sensitiveand affordable detectors for CE, therefore, continues to be an actively pursued area. Although direct UV-visible absorbance or fluorescence detection is commonly used in CE,2 they cannot be used for many inorganic ions or aliphatic carboxylic acids that lack chromophores. Several authors3 have reported the utility of conductivity detectors in CE. Zare et al.4 described an oncolumn conductivity detector that involved a laser drilling 40-pm holes on opposite sides of the capillary walls, followed by the insertion of two 25 pm diameter Pt wires inside the holes to act as electrodes. Subsequently, a simpler grounded end-column arrangement was reported for both conductometric and amperometric detection. The sensing electrode (Pt wire, 50 pm in diameter) in this setup was placed at the outlet of the separation ~ a p i l l a r y .Some ~ further refinements + Permanent address: Department of Chemistry, College of Natural Science, Kangwwn National University, 192-1, Hyoja 2-Dong. Chuncheon, KangwwnDo, South Korea. (1) Albin, M.; Grossman, P.; Moring, S. E. AMI. Chem. 1993,65, 489A-497A. (2) Kuhr, W. G.; Monning, C. A. Anal. Chem. 1992, 64, 389R-407R. (3) (a) Tsuda, T.; Nakagawa, G.; Sato, M.; Yagi, K. J. Appl. Biochem. 1983,5, 330-336. (b) Gebauer, P.; Deml, M.; Bocck, P., Janak, J. J. Chromatogr. 1983, 267,455-457. (c) Deml, M.; Foret, F.; Bocck, P. J. Chromatogr. 1988, 320, 159-165. (4) Huang, X.; Pang, T.-K.; Gordon, M. J.; Zare, R. N . Anal. Chem. 1987, 59, 2747-2749. ( 5 ) Huang, X.; Zare, R. N.; Sloss, S.;Ewing. A. G. Anal. Chem. 1991,63, 189192.
0003-2700/94/0360-2537$04.50/0 0 1994 American Chemlcal Society
of this end column detection arrangement has since been reported.6 Deployed in the conventional manner, conductivity detectors are nonselectivebulk property detectors. An analyte signal arises from the difference in equivalent conductance between the analyte ion and the correspondingly charged carrier electrolyte ion. The limit of detection (LOD) of such a system is rarely better than M. A suppressed conductometric capillary electrophoresis separation system (SUCCESS)has recently been r e p ~ r t e d . ~ ~Much * like its better known counterpart in ion chr~matography,~ this system provides significantly better LODs than nonsuppressed systems by lowering the conductivity of the carrier electrolyte through an ion-exchange process. The SuCCESS for anion analysis uses a tubular cationexchange membrane suppressor, housed in a reservoir of dilute acid regenerant solution, attached to the end of a fused-silica separation capillary. The conductivity detector that follows the suppressor was constructed by inserting two 100 pm diameter Pt wires through the wall of a 190pm i.d. poly(viny1 chloride) (PVC) capillary in parallel and as close to each other as possible. A positive high voltage is applied to the capillary inlet and the regenerant solution is grounded. Thus, the detector is isolated from the electric field, and the electroosmotic flow (EOF) generated in the capillary carries the solution through the suppressor and the detector. The system provides LODs at the tens of micrograms per liter level for a variety of anions without any preconcentration. While the SuCCESS works quite well, because of the difficulty in achieving the same spacing between the wires each time, it is problematic to reproducibly construct conductivity detection cells with similar cell constants. In the original we simply connected the cell to a commercial ion chromatography conductivity detector. In this paper, we describe a novel and simple design of a conductivity detection cell based on bifilar wire electrodes that is rugged and easy to construct. While the cell can be used with commercial conductance detector electronics, a personal computer (PC)based bipolar pulse conductance detection system that significantly exceeds the performance of commercial conductance detectors was also developed and is described here. Results from the deployment of this detector in SuCCESS is reported. (6) Huang, X.; a r e , R. N. AMI. Chem. 1991,63, 2193-2196. (7) Dasgupta, P. K.; Bao, L. AMI. Chem. 1993, 65, 1003-1011. (8) Avdalovic, N.; Pohi, C. A.; Rocklin, R. D.; Stillian, J. R. AMI. Chem. 1993, 65, 1470-1475. (9) Small, H. Ion Chromatography; Plenum: New York, 1989.
AnaljticalChemistry, Vol. 68, No. 15, August 1, 1994 2537
EXPERIMENTAL SECTION Equipment. Electrophoretic experiments were carried out on a Model CES-1 instrument (Dionex Corp., Sunnyvale, CA). Samples were injected by pneumatic pressure (all reported data refer to 2-s injections at 5 psi (ca. 100 nL), except as mentioned). For the commercial detector, the electronics of a Dionex Model CDM-I conductivity detector was used without modifi~ation.~ Except as stated,, thedetector was operated at a sensitivity of 100-nS full scale. A fused-silica capillary (75 pm i.d. X 375 pm o.d., 60 cm long, Polymicro Technologies, Phoenix, AZ) was used with a 2 mM Na2B407 electrolyte at an applied voltage of +20 kV in the constant voltage mode. With the CDM-I, the data output was recorded on a strip-chart recorder and also acquired by a PC using a ACI computer interface and AI 450 software, both from Dionex Corp. With the PC-based detector, software that governs detector operation also provides flexible data acquisition and display capabilities. The tubular cation-exchange membrane (Nafion, 90 pm i.d., 240 pm 0.d.) used as the suppressor was a generous gift of David Leighty, Perma-Pure Products, Tom’s River, NJ. A 0.8-cm segment of the membrane was used constructing the suppressor. Construction detailsand couplingto the separation capillary and the detection cell are the same as that described earlier.7 Construction of Conductivity Cell. Very small multiwire assemblies are used in the microelectronics industry for the constructionof hybrid integrated circuit packages. These are available in a variety of metallic conductors and insulation, both in the twisted and the ribbon format. For the ribbontype wire assemblies, depending on the number of individual conductors, they are termed bifilar, quadrifilar, etc. Insulated bifilar conductors are available down to individual wire diameters of 25 pm (Hudson Wire Co., Atlanta, GA). The results reported in this work were obtained solely with 80 pm diameter Ni conductor 19-pm polyimide insulated bifilar wire electrodes housed in 380 pm i.d. poly(viny1 chloride) (PVC) tubing (Elkay Products, Shrewsbury, MA). First, insulation is removed from all sides of a -30-cm segment of the bifilar wire, but the insulation between the conductors is left intact, as depicted in Figure la, such that the two wires remain electrically isolated. Three different methods were evaluated to accomplish this. In the first approach, the coating material is removed by a surgical scalpel blade under a 20X magnification stereomicroscope. In the second method, the bifilar wire is held within the folds of a fine abrasive paper (1240 mesh), and the wire is pulled slowly back and forth several times. The process is repeated after repositioning the wire between the folds. In the third approach, a device was fashioned to see if the mechanization of the first method can further improve cell reproducibility. After the bifilar wire is held flat on a metal block of the device, it further allows the positioning of the sharp edge of a heavy razor blade over the top side of the bifilar wire in an adjustable manner. When the wire is pulled out manually, the insulation is evenly removed from the side in contact with the blade. Similarly, the coating from the other side of the wire is removed after turning the wire over and repeating the process. Some coating material does remain on the edges, this is then removed manually by a razor blade. All three of the processes leave 2538 AnalyticalChemistry, Vol. 66, No. 15, August 1, 1994
WCCaplllary
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Flgute 1. Schematic diagram of the conductivity cell (not to scale). (a) Bifilar wire after the removal of coating, a = 45 pm, b = 80 pm. (b) A cross-sectional view of the conductivity cell assembly, dl = 380 pm, d2 = 75 pm.
the insulation between the two conductors unaffected. The wire, thus processed, is then cleaned with soapy water and ethanol and dried. Microscopic examination shows that the average width of the insulation left between the two wires is approximately 45 pm. A -2-cm segment of the PVC tube is positioned vertically on a lox magnification microscope platform and focused on the bore, the depth of field is -3 mm. The tubing is then radially punctured all the way through, ca. 2 mm from the top end, with a 26-gauge hypodermic needle. Note that when an elastomeric tubing is punctured with a needle having a tapered point, the cut made has the appearance of a slit. It is important to orient the needle such that the vertical aspect of the slit is parallel to the long axis of the tube. The processed bifilar wire is next inserted through the bore of the needle to a depth of about 1 cm to the other side of the tube, and the needle is then removed carefully while maintaining the longer aspect of the bifilar wire cross section aligned with the longer aspect of the slit. This orientation ensures minimum flow resistance (Figure lb). The wire is then cut off, leaving -2 mm and 3 cm of the wire protruding from the tubing. Both these ends are now folded back over the body of the tubing in opposite directions without the two ends touching each other. Fast curing epoxy adhesive is now applied over the whole area to secure things in place; ca. 25 mm of the wire protrudes out of the assembly. After the adhesive is cured, the terminal end of the wire is exposed to the flame of a butane
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lighter; the insulation between the two conductors burn, and the two leads are separated. These leads are connected to the conductivity detection electronics. Evaluation of Uniformity of Coating Removal. An efficient method for even removal of the insulation is essential to the reproducible fabrication of sensitive detection cells. The different approaches to coating removal were evaluated by affixing a piece ofihe processed bifilar wire (with 1 cm or so of insulation remaining in tact at the bottom end) tautly to a graduated glass rod with a spacer using cotton threads. The two conductors are then separated at the top for connection to the detector electronics. The glass rod/wire assembly is next placed vertically in an empty glass cylinder. The cylinder is filled in steps with 0.1 mM KCl; the conductivity is measured after the addition of each aliquot. Necessary precautions were taken so that the undipped portion of the wire remained dry. Bipolar Pulse Conductance Detector. The bipolar pulse technique for conductance measurements was first described by Johnson and Enke.lo An automated and computerized version of this detection scheme was developed for kinetic measurements by Caserta et al." The circuitry presented here follows the same design philosophy of Caserta et al. However, both electronics and computers have changed sufficiently in the past 15 years so that a major improvement in the overall simplicity, sophistication, and flexibility of the system is possible. Figure 2 shows the schematic diagram of the system, the heart of which is a 80386-based PC with a 80387 coprocessor operating at 33 MHz and housing a DAS~
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(IO) Johnson, D. E.; Enke, C. G. Anal. Chem. 1970, 42, 329-335. ( 1 1) Caserta, K.J.; Holler, F. J.; Crouch, S.R.; Enke, C. G. Anal. Chem. 1978, 50, 1534-1541.
1600 data acquisition board (12 bit, 100 kHz, Keithley/ Metrabyte Corp., Taunton, MA). In the peripheral circuit, U2 and U3 are monolithic CMOS devices (ADGZOlHSKN, Analog Devices, Norwood, MA), each comprising four independently selectable SPST switches with very fast switching speeds (50 ns) and low resistance in the on state (-30 a typical). A brief positive pulse period (typical pulse width, 25-100 ps) during which S1-4 is turned on is followed by a negative pulse period of the same length in which S1-1 is turned on. A bipolar pulse wave form is thus generated. The pulse height is controlled by the D/A output. In the present scheme, pulse height precision is limited by the D/A resolution. A voltage reference source can also be used to govern the pulse height and attain better precision, at the expense of some control flexibility. The pulse frequency (typically 3 kHz) is controlled by the timer on the A/D board. The timer output is connected to the standard digital input port. Each time the computer detects a rising edge from at this input port, it is programmed to generate a bipolar pulse. The potential of one cell electrode is maintained at virtual ground while the potential of the other electrode is controlled by the generated wave form. The current flowing through the feedback resistor (RF) will cause a potential difference across the resistor. This potential difference is processed by amplifier U 1 and appears as the output at its pin 7. During the negative pulse period, the sample/hold amplifier U4 is kept in the sampling mode (OP1 = 0). Exactly at the end of the negative pulse period, U4 is transformed to hold mode (OP1 = 1). Therefore, U1 pin 7 output, as of the exact end of the negative pulse, is stored by U4. U4 (SHC5320-KH, Burr-Brown Corp., Tucson, AZ) is a bipolar monolithic integrated circuit designed for use in precision high speed applications (1.5-ps acquisition time for accuracies to within 0.01%). The output of U4 is digitized by the A/D board. A selectable number of successive readings thus obtained (typically 500) are averaged to improve S/N. Values of the feedback resistor RF are controlled by port A of the programmable peripheral interface (8255 PPI) on the DAS-1600 board. Software written for the purpose automatically selects a suitable feedback resistor (from a bank of five resistors ranging from 1 kQ to 10 Ma). When the system is first turned on, the software performs an initial calibration routine during which the electronic blank signals (cell disconnected or air in cell) are stored for each individual feedback resistor. During operation, this blank value is automatically subtracted from the readings obtained. The software consists of a 25-kbyte source file and a 95-kbyte executable file, written in C, and it is available from the authors on request for noncommercial use. Reagents and Samples. Standards and buffers were made from reagent-grade alkali metal salts or the free acids (Sigma/ Aldrich). Distilled deionized water was used throughout. RESULTS AND DISCUSSION Insulation Removal Uniformity. When the observed conductivity is plotted against the wetted length of the stripped bifilar wire, the degree of linearity is a direct measure of the uniformity with which the insulation is removed across the length of the wire. The slope of the line is largely governed by the width of the insulation left between the conductors. Figure 3 illustrates the results obtained with the three stripping AnalyticalChemlstry, Vol. 66, No. 15, August 1, 1994
2659
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Flguro7. Magnifiedviewoftheresdutionof(a, b)fiuorideandphosphate and (c, d) thiocyanate and azlde (a, c) old cell,' and (b, d) bifilar wire cell. Analyte concentrations: 500 pg/L, lnjectlon 1 s at 5 psi.
Figure 8. Magnlfled view of the asymmetry (at 10% peak helght) of (a, b) sulfamate and (c, d) bromlde using (a, c) the old cell,' and (b, d) the new cell. Improvement is only observed for the early and mideluting Ions. Sample concentratlon and lnjectlon are as in Figure7.
small spacing. The cell constant obtained with the new cells were not as small as those attained in the best of the old style detection cells7(because the latter used larger diameter wires which could be pushed quite close to each other), but in every other respect the new cell design was more attractive. The resistance to fluid flow was smaller, resulting in perceptibly decreasedmigration times and it provided superior separation efficiency both in terms of plate counts (N = 5.54 ( t , / ~ ~ / 2 ) ~ , where N is the plate count for an analyte eluting at time tm with a peak half width of w l p ) and plates per unit time ( N /
these systems, greater plate counts are generally obtained with lower sample concentrations; nevertheless, the new cell performs better at the 200 pg/L level than its previous counterpartat the lo0rg/L level. Note that chloride produces relatively low plate counts because a partial separation of the 3sCl and 37Clisotopes take place.* It should be noted that even the enhanced efficiencies presently reported are still not as good as those typically reported for large molecule CE separations, even after the differences in the diffusion coefficients are taken into account. This may be intrinsic to SUCCESS.' Figure 7 shows expanded views of the partially resolved pairs (a, b) fluoride and phosphate and (c, d) thiocyanate and azide obtained with the old cell (a, c) and the new cell design
tm).
Table 1 reports the relevant data on theobserved separation efficiencies for early eluting (benzoate and fluoride), midregion (thiocyanate), and late eluting (chloride) anions. In 2542
AnalyticaIChem/Stry, Vol. 66, No. 15, August 1, 1994
(c, d). Abscissa scaling is the same in the corresponding cases, smaller peak widths resulting in better resolution is evident in both cases with the new cell. Figure 8 similarly shows peak symmetry obtained with the old and the new cells. For most early and mid-eluting peaks typified by sulfamate, symmetry is markedly improved in the new vs. the old cell (Figure 8a vs 8b). The symmetry of the late eluting peaks such as bromide, however, is dominantly governed by the mobility mismatch between the sample ion and the carrier electrolyte ion, and there is no significant improvement (Figure 8c vs 8d). In summary, it is felt that we have described here a particularly simple and attractive design for sensitive highperformance conductance detection in capillary systems with (13) (a) Baba, N.; Housako, K. US.Patent 4,462,962, 1984. (b) Haddad, P. R.; Jackson, P. E. Ion Chromatography;Elsevier, New York, 1990 pp 258-260.
bifilar wire electrodes. Not only direct extensions to more involvedconductance detection schemes (e.g., a four-electrode c e l P with quadrifilar wires) can be readily envisioned, multifilar wires may be highly useful in other applications, such as very inexpensively devising (semi)microelectrode arrays. ACKNOWLEDGMENT We gratefully acknowledge the initial gift of the bifilar wire samples from David DiMartino (Hudson Wire Co.). This research was supported by cooperative research agreements with Dionex Corp. and Dow Chemical Co. No endorsement of the contents of this article by these organizations should, however, be inferred. Received for review February 7, 1994. Accepted May 6, 1994.' Abstract published in Aduance ACS Abstracts, June 15, 1994.
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