Anal. Chem. 1996, 68, 2488-2493
Integrated On-Capillary Electrochemical Detector for Capillary Electrophoresis Min Zhong† and Susan M. Lunte*
Departments of Chemistry and Pharmaceutical Chemistry and Center for Bioanalytical Research, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047
An on-capillary electrochemical detector for capillary electrophoresis is described. It consists of a gold wire mounted permanently at the end of the capillary perpendicular to the direction of flow. This mode of detection eliminates the need for the micromanipulators or specially machined cell holders for alignment that are used for incapillary detection modes. It also makes it possible to perform relatively fast CEEC separations using very short capillaries. The use of this detector for both off-column detection of catecholamines and end-column detection of carbohydrates by CE-PAD is described.
Electrochemical detection (EC) has been shown to be a highly selective method for use with capillary electrophoresis.1-3 Detection limits in the nanomolar range have been obtained for many electroactive compounds. The low sample volume requirements, high separation efficiencies, and excellent sensitivity and selectivity make capillary electrophoresis/electrochemistry (CEEC) an ideal method for the analysis of small-volume samples, such as microdialysis samples4-7 and the contents of single cells.8-10 The major obstacles to general acceptance of CEEC as an analytical technique are difficulties associated with minimizing the effect of the separation voltage on the noise at the detector and alignment of the working electrode with the end of the separation capillary. Two different schemes have been employed in CEEC for minimizing noise at the detector.3 In the first, termed off-column detection, a fracture (often called a decoupler or joint) is made in the capillary and grounded prior to the detection cell.11,12 This fracture is frequently covered with a conductive material to minimize loss of analyte at low electroosmotic flows.13 A very small
segment of capillary that extends past the decoupler serves as the detection capillary. Analyte bands are pushed to the detection cell by the electroosmotic flow generated in the separation capillary. In the second approach, termed end-column detection, a microelectrode is placed at the end of separation capillary, eliminating the need for a decoupler.14,15 In general, this method requires the use of capillaries less than 25 µm in diameter.16 In this case, the resistance is high, so most of the voltage drop occurs across the capillary. The potential field at the end of the capillary decays very rapidly; therefore, it is possible to measure the small currents at the working electrode with minimal interference from the separation voltage. Another significant challenge in electrochemical detection is alignment of the microelectrode with the end of the capillary.16-18 For off-column detection, carbon fibers and small-diameter metal wires inserted directly into the capillary are frequently employed as working electrodes. Disk electrodes are used in both configurations but have become very popular for end-column detection.15 This is primarily due to the difficulty of aligning a carbon fiber or metal wire 1-10 µm in diameter with the end of a capillary that is less than 25 µm in diameter. Disk electrodes employed in endcolumn detection are generally much larger than the internal diameter of the capillary, making exact centering of the electrode unnecessary. The major disadvantage of end-column compared to off-column detection is that there is a 1-2 order of magnitude loss of sensitivity with the former.3,15,16 Most of the original work in CEEC involved the use of micromanipulators to align the electrode with the fused silica capillary.3 More recently, a significant amount of attention has been focused on developing more robust electrochemical detection cells that do not require optical alignment.19-22 The first two reported cell designs were developed for off-column detection. Tudo¨s and co-workers described an electrochemical cell consisting of a tee where a carbon fiber bundle disk electrode was juxtaposed with the capillary using PEEK tubing and fittings.19 The third arm of the tee was used for the reference electrode. The problem with this configuration was the dead volume between the capillary
† Department of Chemistry and Center for Bioanalytical Research. (1) Yik,Y. F.; Li, S. F. Y. Trends Anal. Chem. 1992, 11, 325-333. (2) Lunte, S. M.; O’Shea, T. J. Electrophoresis 1994, 15, 79-86. (3) Ewing, A. G.; Mesaros, J. M.; Gavin, P. F. Anal. Chem. 1994, 66, 527A536A. (4) O’Shea, T. J.; Weber, P. L.; Bammel, B. P.; Lunte, C. E.; Lunte, S. M. J. Chromatogr. 1992, 608, 189-195. (5) Malone, M. A.; Zuo, H.; Lunte, S. M.; Smyth, M. R. J. Chromatogr., A 1995, 700, 73-80. (6) Lunte, S. M.; Malone, M. A.; Zuo, H.; Smyth, M. R. Curr. Sep. 1994, 13, 75-79. (7) Lunte, S. M.; Lunte, C. E. In Advances in Chromatography, Vol. 36; Brown, P. R., Grushka, E., Eds.; Dekker: New York, 1995; pp 383-432. (8) Ewing, A. G. J. Neurosci. Methods 1993, 48, 215-224. (9) Hayes, M. A.; Gilman, S. D.; Ewing, A. G. In Capillary Electrophoresis Technology; Guzman, N. A., Ed., Dekker: New York, 1993; pp 753-793. (10) Sloss, S.; Ewing, A. G. In Handbook of Capillary Electrophoresis; Landers, J. P., Ed.; CRC: Boca Raton, FL, 1994; pp 391-417. (11) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1987, 59, 1762-1766. (12) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1989, 61, 98-100. (13) O’Shea, T. J.; Greenhagen, R. D.; Lunte, S. M.; Lunte, C. E.; Smyth, M. R.; Radzik, D. M.; Watanabe, N. J. Chromatogr. 1992, 593, 305-312.
(14) Huang, X.; Zare, R. N.; Sloss, S.; Ewing, A. G. Anal. Chem. 1991, 63, 189192. (15) Ye, J.; Baldwin, R. P. Anal. Chem. 1993, 65, 3525-3527. (16) Lu, W.; Cassidy, R. M.; Baranski, A. S. Anal. Chem. 1994, 66, 200-204. (17) Sloss, S.; Ewing, A. G. Anal. Chem. 1993, 65, 577-581. (18) Lu, W.; Cassidy, R. M.; Baranski, A. S. J. Chromatogr. 1993, 640, 433440. (19) Tudo ¨s, A. J.; Van Dyck, M. M. C.; Poppe, H.; Kok, W. Th. Chromatographia 1993, 37, 79-85. (20) Kuhr, W. G. U.S. Patent 5,244,560, 1993. (21) Guo, Y.; Colon, L. A.; Dadoo, R.; Zare, R. N. Electrophoresis 1995, 16, 493497. (22) Chen, M.-C.; Huang, H.-J. Anal. Chem. 1995, 67, 4010-4104.
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© 1996 American Chemical Society
and the working electrode that led to a loss in separation efficiency. A second type of electrochemical cell for off-column CEEC has been described by Kuhr.20 In his design, the carbon fiber electrode is inserted into the capillary using a guide tube and is then permanently fixed to the end of the capillary. This is an integrated detector design and is similar in concept to the configuration described in this paper. More recently, cells have been designed specifically for CEEC with end-column detection. Guo and co-workers positioned both the capillary and a disk electrode on a glass substrate, using epoxy to hold them in place.21 By utilizing capillaries and electrodes of the same outer diameter, accurate positioning of the two electrodes was not difficult. Most recently, Chen and Huang reported a modification of the original Tudo¨s design, employing a relatively large Pt disk electrode (50 µm) butted against the end of a 5 µm fused silica separation capillary.22 As CEEC has evolved, the nomenclature for various detection modes in CEEC has become very confusing. To accurately describe the instrumental setup, one must specify both the mode of decoupling and the configuration between the working electrode and the separation flow profile. Therefore, for simplicity, in this paper we will use in-capillary detection to refer to that case where the electrode is inserted inside the separation capillary. On-capillary detection will refer to the new configuration reported in this paper, where the electrode is affixed to the end of the fused silica capillary perpendicular to the direction of flow. This new configuration does not require micromanipulators or specially machined cells for alignment. End-column and off-column detection will refer to the absence or presence of a decoupler, respectively, as described in the original papers by Ewing and co-workers. EXPERIMENTAL SECTION Reagents. All catecholamines, carbohydrates, and zwitterionic buffers (3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) and 2-[N-morpholino]ethanesulfonic acid (MES)) were purchased from Sigma (St. Louis, MO). Semiconductor-grade sodium hydroxide pellets were also from Sigma. All standards were made fresh daily in the CE run buffer and filtered prior to injection. Stock solutions (∼1.0 mM) were prepared, and calibration standards were made by successive dilution of these solutions. CEEC System. The construction of the CEEC system has been described previously.13 A three-electrode configuration was employed in all experiments with a Ag/AgCl reference electrode and a platinum auxiliary electrode completing the electrochemical cell. The working electrode in these experiments was a gold wire 25 µm in diameter unless otherwise indicated. A modified BAS LC-4C potentiostat (Bioanalytical Systems, West Lafayette, IN) was employed for direct amperometric detection. Electropherograms were recorded with a Model BD-41 dual-pen strip chart recorder (Kipp & Zonen). A laboratory-built pressure injection system was used for sample introduction, and the injection volume was calculated in a continuous-fill mode by recording the time required for the sample to reach the detector. In-Capillary Detection. The construction of the gold wire microelectrode used in these studies has been reported previously.23 For in-capillary detection, the gold wire microelectrode was placed inside a 93-cm-long capillary (50 µm i.d. × 360 µm (23) O’Shea, T. J.; Lunte, S. M.; LaCourse, W. R. Anal. Chem. 1993, 65, 948951.
Figure 1. On-capillary electrochemical detector for CEEC.
o.d.). The joint was placed 2 cm from the end of this capillary (separation capillary, 91 cm; joint, 2 cm; total length, 93 cm). To evaluate the effect of electrode alignment on detector response, a micropositioner (Newport, Fountain Valley, CA) was used to hold and adjust the relative position of the gold wire microelectrode with respect to the end of the detection capillary. Initially, the tip of the gold wire was placed 110 µm inside the end of the detection capillary. The electrode was slowly pulled outside the detection capillary in ∼20 µm steps, and the response for a standard solution of hydroquinone was measured. The separation was performed using a pH 5.5 run buffer (10 mM MES) and a separation voltage of +30 kV. Detection of hydroquinone was accomplished at +800 mV vs Ag/AgCl. On-Capillary Detection. A schematic diagram of the oncapillary electrochemical detector is shown in Figure 1. To create the integrated detector, a 2-cm length of gold wire 25 µm in diameter (Goodfellow Corp., Malvern, PA) was mounted onto the end of the 50-µm-i.d., 360-µm-o.d. fused silica capillary perpendicular to the direction of flow. This was accomplished by gluing one end of the gold wire to one side of a capillary with 5-min epoxy (Ace Hardware, Oak Brook, IL), leaving about 8 mm unattached. The gold wire was then pulled across the center of the end bore of the capillary, bent to the other side of the capillary, and glued. Electrical contact could be made at either side of the gold wire using a piece of copper wire of approximately the same dimension and silver epoxy (Ted Pella, Redding, CA). Finally, a nonconductive epoxy (Epoxy 907, Mill-Stephens, Danbury, CT) was used to cover those portions of the gold wire that were not involved in detection (including the electrical contacts that were glued onto the side wall of the capillary end). Only the surface of the gold wire that was across the end of the capillary remained exposed. Careful application of the epoxy to the capillary end is critical because blockage of the end of the capillary or contamination of the electrode surface will result if the glue spreads. Separation of Catecholamines Using On-Capillary Detection. Fused silica capillaries (50 µm i.d. × 360 µm o.d.) 100 cm in total length (separation capillary, 98 cm; joint, 2 cm) (Polymicro Technologies, Phoenix, AZ) were used in these experiments. Separation of catecholamines was accomplished using a run buffer of 20 mM sodium citrate, pH 2.5. The use of 100 mM MES, pH 5.5, was also investigated for the determination of the LOD for dopamine. The detection potential was +800 mV vs Ag/AgCl. When necessary, cleaning of the electrode was accomplished by application of a square wave (1.3 to -1.3 V) at 50 Hz. Electrodes could also be sonicated in water if the capillary became plugged. On-Capillary (End-Column) Pulsed Amperometric Detection of Carbohydrates. For pulsed amperometric detection (PAD), a 72-cm-long, 21-µm-i.d. × 360-µm-o.d. fused silica capillary Analytical Chemistry, Vol. 68, No. 15, August 1, 1996
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Figure 2. Schematic diagram of fast CEEC system.
was used with the on-capillary electrochemical detector. Because end-column detection was employed, no decoupler was needed. An EG&G Princeton Applied Research (Princeton, NJ) Model 400 electrochemical detector was used to generate the PAD waveform. Carbohydrates were separated using a run buffer consisting of 10 mM NaOH-8 mM Na2CO3, pH 12. The separation voltage was 25 kV in these experiments. The following PAD waveform was employed: E1 (detection) ) +325 mV, t1 ) 199 ms; E2 (oxidative cleaning) ) +800 mV, t2 ) 166 ms; E3 (reactivation) ) -600 mV, t3 ) 249 ms applied. All potentials are vs Ag/AgCl. Fast CEEC System. Figure 2 shows the schematic design of a fast CEEC system using the on-capillary gold wire electrode. This system employs the on-capillary detector as described above with a Nafion joint. A 5-cm length of 50-µm-i.d. × 360-µm-i.d. fused silica capillary was used for the separation. The fracture for offcolumn detection was made ∼5 mm from the end of the capillary (total length, 5.5 cm). A plastic (PMM) plate with all the necessary features of a conventional CEEC system setup was constructed to house the separation capillary with the electrochemical detector. Two holes were drilled in the plastic, one for the cathode and the other for the detection cell. A channel was made through the center of the plate, penetrating the two reservoirs. The cap of a sample vial was then fixed at the end of the plastic plate with cyanoacrylate adhesive. This sample vial containing the run buffer was the anode buffer reservoir. Platinum wires (Goodfellow) of 1-mm diameter were connected to the reservoirs and fixed onto the plate to act as anode, cathode, and counter electrode. A Ag/AgCl reference electrode was also connected to the detection cell and fixed onto the plate. The separation capillary containing the oncapillary electrochemical detector with the Nafion joint was implanted in the plastic plate, and the buffer reservoirs were isolated from each other using epoxy. Sample injection was accomplished by changing the composition of the sample vial between the run buffer and the sample buffer with electrokinetic injection. For the separation of dopamine and hydroquinone, a run buffer of 10 mM CAPS, pH 10, was employed. The detection potential was +800 mV vs Ag/AgCl, and the applied voltage was 5.5 kV. RESULTS AND DISCUSSION Reproducible positioning of the electrode in the detection capillary is critical for in-capillary electrochemical detection. Changes in position of the electrode due to vibrations can affect the S/N, the separation efficiency, and the day-to-day precision of the detector. To demonstrate this, the effect of the position of the working electrode on detector response (peak height and peak area) was examined for the in-capillary detector in the off-column 2490 Analytical Chemistry, Vol. 68, No. 15, August 1, 1996
Figure 3. Effect of alignment on detector response using in-capillary CEEC with off-column detection. Detection of 0.1 mM hydroquinone. (9) Peak height vs alignment; (O) peak area vs alignment. CE separation conditions: 10 mM MES, pH 5.5; applied voltage, 30 kV; capillary, 50 µm i.d. × 93 cm; E ) +800 mV.
configuration (Figure 3). As the electrode was moved from 110 µm inside the capillary to -20 µm outside of the capillary (0 ) the place where the end of the capillary bore and the electrode are at the same point), both peak height and area decreased. These results are similar to those obtained by Lu et al.18 for cylindrical electrodes and end-column detection. It was also found that the electrode must be properly centered in order to ensure a reproducible detector response. This can be a particular challenge with metal electrodes, since they tend to bend easily. A tilted or skewed electrode can dramatically affect both the detection sensitivity and the separation efficiency. On-Capillary Detection. On-capillary detection was developed in our laboratory in order to simplify the CEEC system. To produce the on-capillary detector, a gold working microelectrode is mounted directly onto the end of the capillary perpendicular to the direction of fluid flow. Figure 4 shows a SEM of an on-capillary electrode constructed in our laboratory using a 25-µm gold wire and a 50-µm-i.d. capillary. The performance of the on-capillary gold electrode relative to the in-capillary configurations in particular, the effect of flow velocity on coulometric efficiency and peak heightswas examined for both configurations (Figures 5 and 6). Coulometric efficiency was calculated using hydroquinone as a standard since it undergoes a well-studied two-electron oxidation. The flow velocity was determined by the applied electrophoretic voltage, which was changed from 10 to 30 kV in 5-kV steps. For the conventional in-capillary detector system, it was found that coulometric efficiency decreased with increasing flow velocity. There was a decrease from 23% to 11%, with the linear velocity going from 0.05 to 0.18 cm/s. In contrast, the coulometric conversion efficiency of the on-capillary electrode did not change significantly with flow velocity and maintained a value between 20% and 25% over the range of flow velocities studied. The relationship between peak height and flow velocity for both detector configurations was also investigated. The results are shown in Figure 6. The peak height increased with increasing flow velocity over the range investigated for both electrode configurations. However, the on-capillary detector was found to be more sensitive to changes in flow velocity than was the incapillary detector.
Figure 6. Effect of linear flow velocity on peak height by (9) incapillary and (O) on-capillary detection for CEEC. Conditions are the same as in Figure 3.
Figure 4. Scanning electron micrograph of the on-capillary microelectrode.
Figure 7. Separation of catecholamines by CEEC with an oncapillary microelectrode. Detection of 20 µM dopamine (DA), norepinephrine (NE), and epinephrine (E). CE separation conditions: 20 mM sodium citrate, pH 2.5; applied voltage, 30 kV; capillary, 50 µm i.d. × 100 cm; E ) +800 mV. Figure 5. Effect of linear flow velocity on coulometric efficiency for (9) in-capillary and (O) on-capillary configurations. Detection of 0.1 mM hydroquinone. CE separation conditions: 10 mM MES, pH 5.5; applied voltage, 10-30 kV; capillary, 50 mm i.d. × 90 cm; E ) +800 mV.
The performance of the on-capillary CEEC system was evaluated for the determination of catecholamines. Figure 7 shows an electropherogram obtained for an equimolar (20 µM) mixture of dopamine, norepinephrine, and epinephrine. The three compounds were well resolved, and the column efficiency for dopamine was 138 300 theoretical plates based on the half-peak width. Using the same capillary and joint with in-capillary detection, the
efficiency for dopamine was found to be 80 064 theoretical plates. The response was linear between 1.0 mM and 0.5 µM for both configurations, with correlation coefficients of 0.9997 and 0.9991 (n ) 6) for the on-capillary and in-capillary configurations, respectively. The sensitivity of the on-capillary detector was slightly higher, with a slope of 17.5 pA/µM compared to 13.4 pA/ µM for the in-capillary configuration. The LODs obtained for the two detection configurations were also compared. To eliminate the possible effect of using different capillaries and joints for the two systems, the same capillary and joint were used for both studies. Low-conductivity zwitterionic buffers were also employed to minimize noise in the detector cell. Analytical Chemistry, Vol. 68, No. 15, August 1, 1996
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The LODs for dopamine were 0.12 and 0.50 µM for the on-capillary and in-capillary detectors, respectively (S/N ) 2). The 5-fold difference is believed to be due to the higher background noise with the in-capillary configuration. If grounding were incomplete in that system, then a working electrode placed inside the capillary would pick up more noise from the separation voltage. Reproducibility of construction for the on-capillary configuration was investigated using three fused silica capillaries of the same dimensions with a gold wire microelectrode. Flow injection analysis (FIA) rather than CE was used for these experiments in order to eliminate the effect of sample loss at the joint on the peak height. A 0.1 mM hydroquinone solution was injected into each capillary at 10 psi for 2 s, and the detector response was compared by FIA. The electrodes were pretreated before each injection. For three separate CEEC electrodes, the relative standard deviation was found to be 5%. Three injections on the same on-capillary CEEC system yielded a precision of better than 2%. The results clearly showed that, for on-capillary detection, highly reproducible measurements can be achieved, and alignment with micropositioner is not necessary. The electrodes were very rugged. In all cases, the capillary needed to be replaced before the electrode. PAD Detection of Glucose and Carbohydrates. PAD is frequently performed using gold microelectrodes24 and has become the method of choice for determination of carbohydrates. PAD at noble metal electrodes exploits surface-catalyzed oxidation of various functional groups. The high electrocatalytic activity of gold electrodes for a constant applied potential is often accompanied by fouling of the electrode surface. PAD overcomes this problem by combining amperometric detection with attenuated anodic and cathodic polarizations to clean and activate the surface. PAD utilizes a multistep potential-time wavelength to maintain uniform and reproducible electrode activity. The performance of the on-capillary gold wire electrode for PAD detection of carbohydrates was evaluated. In this case, a smaller diameter capillary and end-column detection with no joint were employed. Glucosamine, glucosaminic acid, and glucosamine 6-sulfate were used as representative compounds since they had been detected previously by CE-PAD using the in-capillary configuration.23 In that report, glucosaminic acid and glucosamine 6-sulfate were not baseline resolved. Figure 8 shows the separation obtained with the on-capillary electrode configuration. Glucosaminic acid and glucosamine 6-sulfate are baseline resolved under these conditions, possibly due to the use of the smaller inner diameter capillary (21 vs 75 µm) for this separation. Lower separation efficiencies were observed for the carbohydrates than for the catecholamines, which could be the result of the Joule heating produced by the hydroxide electrolyte. Detection limits were found to be about an order of magnitude higher than those obtained in the in-capillary configuration with a decoupler.23 Fast Separation of Catechols by CEEC. The on-capillary configuration makes it possible to employ very short capillaries for CEEC separations. For conventional in-capillary CEEC, the shortest length of capillary that can be used for the separation is equal to the shortest possible distance between the anode and the micropositioner, usually ∼20 cm. The feasibility of using an on-capillary electrode for “fast” CEEC was investigated for the separation of two model analytes, hydroquinone and dopamine. Using a 5.5-cm capillary (5.0-cm separation capillary, 0.5-cm joint), (24) LaCourse, W. R.; Johnson, D. C. Anal. Chem. 1993, 65, 50-55.
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Figure 8. Separation of 1.0 mM (0.24 pmol) (1) glucosamine, (2) glucosaminic acid, and (3) glucosamine-6-sulfate. CE conditions: 10 mM NaOH-8 mM Na2CO3, pH 12; applied voltage, 25 kV; capillary, 21 µm i.d. × 70 cm. PAD waveform as described in Experimental Section.
Figure 9. Fast separation of 0.1 mM dopamine (DA) and hydroquinone (HQ) by “fast” CEEC. CE conditions: 10 mM CAPS, pH 10; applied voltage, 5.5 kV; E ) +800 mV.
the two compounds could be separated within 20 s with baseline resolution (Figure 9). Although this is still much slower than the millisecond separations reported by the groups of Jorgenson,25 Manz26 and Ramsey27 using glass substrates with optical detection, the ability to perform subminute CEEC separations will enable (25) Monnig, C. A.; Jorgenson, J. W. Anal. Chem. 1991, 63, 802-807. (26) Effenhauser, C. S.; Paulus, A.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 2949-2953. (27) Jacobson, S. C.; Hergero ¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 1114-1118.
rapid on-line separation of electroactive analytes following microdialysis sampling.28,29 The major problem that occurred with the present design of the “fast” CEEC system was a loss of separation efficiency caused by excessively large sample injection. This problem, which involves the injection mode rather than the separation/detection system, also has been observed for CE on microchips and was described in detail by Manz et al.26 Future work will involve improvement of sample introduction and the evaluation of electrode materials other than gold. The ultimate goal is miniaturization of the entire device.
ACKNOWLEDGMENT
(28) Hogan, B. L.; Lunte, S. M.; Lunte, C. E.; Stobaugh, J. F. Anal. Chem. 1994, 66, 596-602. (29) Zhou, S.; Zuo, H.; Stobaugh, J. F.; Lunte, C. E.; Lunte, S. M. Anal. Chem. 1995, 67, 594-599.
AC951238M
The authors acknowledge the Kansas Technology Enterprise Corporation (KTEC), the Center for Bioanalytical Research (CBAR), and Bioanalytical Systems (BAS) for financial support. They also thank Professor Steve Weber for helpful discussions and Nancy Harmony for assistance in the preparation of the manuscript.
Received for review December 22, 1995. Accepted May 14, 1996.X
X
Abstract published in Advance ACS Abstracts, June 15, 1996.
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