Anal. Chem. 1988, 60, 2158-2161
2158
Ingraham. R. H.; Lau, S.Y. M.; Taneja, A. K.; Hcdges, R . S.J. Chromafogr. 1985, 327, 77-92. Hearn, M. T. W.: Hodder, A. N.; Aguiiar, M. I . J. Chromatogr. 1985, 327. 47-66. Cohen, S. A.; Benedek, K. P.; Dong, S.;Tapuhi, Y.; Karger, B. L. Anal. Chem. 1984, 56, 217-221. Benedek, K.; Dong, S.; Karger, B. L. J. Chromafogr. 1984, 317, 227-243. Saavedra, S. S . Ph.D. Dissertation, Duke University, 1986. Prendergast. F. G.; Meyer, M.; Carlson, G. L.; Iida, S.; Potter, J. D. J. Biol. Chem. 1983, 258, 7541-7544. Laemmll, U. K. Nature (London) 1970, 227, 680-685. Lochmuller. C. H., Colborn, A. S.; Hunnicutt, M. L.; Harris, J. M. Anal. Chem. 1983, 55. 1344-1348. Drenth. J.: Jansonius. J. N.: Koekoek. R.: Wolthers, B. G. A&. Protein Chem. 1971, 25, 79-115. (15) Weber, G.; Farris, F. J. Siochemisfty 1979, 78, 3075-3078. (16) Herskovks. T. T.; Gadegbeku, E.; Jaillet, H. J. Bid. Chem. 1970. 245, 2588-2598. (17) Sadler. A. J.; Micanovic. R.; Katzenstein, G. E.; Lewis, R . V.; Middaugh, C. R. J. Chromafogr. 1984, 377, 93-101.
S. Physicochemical Aspects of Protein Denaturation: Wiiey: New York, 1978; pp 142-156.
(18) Lapanje,
'Present address:
Perkin-Elmer Corp., 761 Main Ave., Norwaik, CT
06859-0088.
Steven S. Saavedra' A. Worth Grobin C. H. Lochmiiller*
P. M. Gross Chemical Laboratory Duke University Durham, North Carolina 27706
RECEIVED for review February 2, 1988. Accepted June 21, lg8&This work was supported, in part, by the National Science Foundation under Grant CHE-8500658.
TECHNICAL NOTES Voltammetric Electrochemical Detection for Normal-Phase High-Performance Liquid Chromatography M. B. Thomas a n d T. A. Last*
Analytical Chemistry Department, Smith Kline and French Labs, King of Prussia, Pennsylvania 19406 Voltammetric electrochemical detection has been employed by various workers (1-5) because of the increased selectivity and additional qualitative information provided by the technique. Most electrochemical detection (including voltammetric) has been performed in aqueous media because of the need for supporting electrolyte and the widespread interest in reversed-phase high-performance liquid chromatography (HPLC). There are, however, certain advantages to be gained by performing nonaqueous electrochemical detection as pointed out by Gunasingham and co-workers (6),and normal-phase HPLC is an important area where electrochemical detection has yet to make a large impact. One of the advantages of nonaqueous electrochemical detection is that the background current is generally reduced and is much more reproducible (6) compared with that for reversed phase mode electrochemical detection. Another advantage is the increased potential range available for electrochemical detection in the normal-phase mode. Most pharmaceuticals, because of their polar nature, are easily amenable to reversed-phase HPLC or paired-ion chromatography. For this reason, most of the recent activity in the field of liquid chromatography for small molecules has been concerned with reversed-phase HPLC. However, it is not unusual for synthetic intermediates or side products to be nonpolar to the extent that they will not elute from a reversed-phase column using typical mobile phase compositions or, if they elute, the retention time is so great that peak shapes and analysis times are unacceptable. Therefore, normal-phase HPLC must be employed for the assay of these nonpolar intermediates and for impurity profiling of the final drug substance if any nonpolar intermediates or impurities might be present, even if the final drug substance is polar. The amperometric mode of electrochemical detection for normal-phase HPLC has been investigated by several workers (6, 7). Gunasingham et al. (8) employed cyclic staircase voltammetry to study the redox behavior of several substituted quinones as they eluted from a silica column. We report here
the use of voltammetric (coulostatic) electrochemical detection to quantitate several substituted benzazepines and several sulfur-containing compounds eluted under both normal-phase and reversed-phase HPLC conditions. EXPERIMENTAL SECTION HPLC Instrumentation. Chromatography was carried out with a Model 8800 gradient liquid chromatographic system (Du Pont Analytical Instrument Division, Wilmington, DE) operated in the isocratic mode. The injector was a Micromeritics Model 725 automatic injector (Micromeritics, Norcross, GA) equipped with a 20-yL sample loop. Chromatographic separations were achieved on a LiChrosorb DIOL column (10-Km particles, E. Merck) operated in the normal phase mode, or a 4.6 mm x 250 mm i.d. Du Pont Zorbax ODS reversed-phase column (5 ym porous support particles). Electrochemical Instrumentation. The detector was a modified version of a coulostatic electrochemical detector, which has been previously described (9),equipped with a glassy carbon electrode. The coulostatic pulse generator is operated under real-time computer control and, therefore, can simulate any desired forcing function. In the voltammetric mode, the instrument is capable of scanning the applied potential at rates up to 3 VIS, while simultaneously recording up to 15 channels of chromatographic data, each corresponding to a different applied potential. A microVAX (Digital Equipment) computer with 2.5 Mbytes of memory was used to generate the necessary signals to control the detector, process the data, store them on the disk, and display them on the graphics terminal. All parameters for the detector are specified from a set-up menu displayed on the terminal. All potentials specified in the text are vs Ag/AgC1(1 M NaCl) for reversed-phase studies and Ag/AgCl (saturated NaCl in methanol) for normal-phase studies. When the electrochemicaldetector was used, the mobile phase was purged vigorously with helium for at least 15 min before the pump was started and purging was continued throughout the experiment. Chemicals and Reagents. The Smith Kline & French (SK&F) compounds were received from within the company. These included fenoldopam (SK&F 82526, 6-chloro-2,3,4,5-tetrahydro-
0003-2700/88/0360-2158$01.50/00 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, OCTOBER 1, 1988 T I
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tions at each ionic strength are plotted on the figure. Fenoldopam concentration was 0.1 mg/mL.
l-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-diol) and its deschloro and N-methyl analogues. The sulfhydryl containing SK&F 102698 (1-(3,5-difluorophenyl)-methyl-1,3-dihydro-W-imidazole-2-thione) , 6-mercaptopurine, and methimazole were also studied. The latter two compounds were purchased from Sigma Chemical Co. (St Louis, MO) and used without further purification. Tetraethylammonium p-toluenesulfonate and dl-10-camphorsulfonic acid were purchased from Aldrich Chemical Co. (Milwaukee, WI). HPLC grade methanol and chloroform were purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ). The tetraethylammonium p-toluenesulfonate solution was prepared by dissolving a known amount in methanol to the desired concentration. The solution was filtered through a 0.45-pm nylon membrane before being used for HPLC. The various mobile phases for the normal-phase HPLC studies were prepared by mixing chloroform with the tetraethylammonium p-toluenesulfonate solution on a volume basis (80/20 (v/v)). The camphorsulfonic acid solution was prepared by dissolving 2.6 g in 1.0 L of distilled/deionized water. The solution was filtered as previously stated. The mobile phases for reversed-phase HPLC studies were prepared by mixing the camphorsulfonicacid solution with methanol on a volume basis (60/40 (v/v)).
RESULTS AND DISCUSSION The effects of ionic strength in the nonaqueous mobile phase were investigated by producing a series of mobile phases where the composition remained invariant except for the amount of tetraethylammonium p-toluenesulfonate (TEAPTS). For each mobile phase, the peak area of the fenoldopam peak was measured by chromatographing solutions of identical concentration (0.1 mg/mL). The results of the experiment are shown in Figure 1. Duplicate injections were performed at each ionic strength, and each injection was plotted on the figure. The detector potential was scanned between 1.0 and 2.0 V and ten channels of chromatographic data were collected; however, only those results corresponding to 2.0 V applied are included in the figure. (The other data channels produced similar results.) I t can be seen from the figure that the response for fenoldopam remains essentially constant above a TEAPTS concentration of 0.01 M. Below this value, the response drops off even though the linearity remains acceptable. The behavior at low ionic strength is what would typically be expected for amperometric detection with uncompensated solution resistance. I t is remarkable to observe this behavior in the present case because the coulostatic technique is generally believed to be immune from the effects of uncompensated solution resistance. This immunity derives from the fact that no current flows in the bulk solution during the time period when the faradaic current is measured.
2159
There are two possible explanations for the behavior at low ionic strength, and both involve the interfacial potential gradient. One possibility is that the coulostatic technique is not actually immune from the effects of the uncompensated solution resistance. We feel this is unlikely for two reasons. First, the linearity of response for fenoldopam remains intact at TEAPTS concentrations as low as 0.0025 M, while Figure 1 indicates that the response has decreased by 50% a t 0.005 M TEAPTS. If uncompensated solution resistance were the cause of the reduced response at low ionic strength, the effect would become more apparent as the concentration of analyte increased. Second, according to the present understanding of how coulostatic pulse generators function, the effects of solution resistance should first become evident as an inability of the pulse generator to deliver the full charge content of the coulostatic pulse. Observations of the pulse generator indicated that charge injection was complete, and the charge pulse amplifier was not saturating during the charge injection, even a t the lowest TEAPTS concentrations. The second possible cause for the behavior exhibited in Figure 1 is the loss of the compact double layer. In the low dielectric mobile phase, a significant portion of the electrolyte is probably associated into ion pairs. Therefore, the ionic strength of the solution is probably significantly lower than the electrolyte concentration. As the ionic strength of the solution decreases, the double layer must become increasingly diffuse. This necessarily leads to a decrease in the potential gradient at the electrode interface and a concomitant decrease in electron transfer rate. We feel that this is the most probable reason for the decrease in response at low ionic strength, and if correct, this means that no known technique would be immune to the behavior exhibited at low ionic strength in the figure. The chromatographic retention time and peak shape were also affected by the ionic strength to some extent. The best peak shape was obtained a t the higher ionic strengths. Therefore, all remaining experiments were performed a t a TEAPTS concentration of 0.04 M. In some of the preliminary experiments, nonlinear calibration curves were occasionally observed at concentrations where the instrument had previously demonstrated linearity. On inspection it was observed that over a period of time the electrochemical response decreased as the electrode surface became contaminated with the reaction products, eventually leading to a condition where the electrode had to be polished. The application of a 1-min cleaning scan between chromatographic runs, where the potential was repeatedly swept between 0 and -1.0 V, was found to alleviate the problem. Catechols. Chromatograms for fenoldopam recorded under both normal- and reversed-phase conditions are shown in Figure 2. The upper trace corresponds to normal-phase HPLC and the lower trace is the reversed-phase chromatogram. In both cases, the detector potential was scanned from 1.0 to 2.0 V and ten channels of chromatographic data were collected. Only one of the ten channels is plotted in each case. Although the fenoldopam concentration was identical in both cases, the normal-phase chromatogram displays approximately a 40% greater response (peak height), even though the retention time is slightly greater for normal phase. In addition, the base line for the normal-phase chromatogram is quieter than that for the reversed-phase chromatogram leading to a 2-fold improvement in signal-to-noise ratio for the normalphase mode. Fenoldopam gave a linear response in both the normal- and reversed-phase mode; however, the point scatter about the line was best for normal phase. For reversed-phase operation, the slope was 8.65 f 0.23 area X mL/mg and the intercept was -0.044 f 0.010 area units. The correlation coefficient was
2160
ANALYTICAL CHEMISTRY, VOL. 60, NO. 19. OCTOBER 1, 1988
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0.9995 and the standard error of estimate was 6.6 X For normal-phase operation the slope was 9.98 f 0.02 area X mL/mg and the intercept was -0.017 f 0.005 area. The correlation coefficient was 0.99999 and the standard error of Therefore, the calibration curves estimate was 8.1 X indicate only a 15% greater response (peak area) for fenoldopam in the normal phase mode, but the quality of the normal phase data is approximately 10-fold better, in terms of the slope standard deviations and the standard errors of estimate. It should be noted that the slope comparison involves peak area while the signal-to-noise ratio comparison involves peak height. Although the cleanliness of the electrode surface affects the response, these data are a strong indication that an improved detection limit for fenoldopam can be obtained by operating in the normal-phase mode. Figure 3 contains a multichannel normal-phase chromatogram for fenoldopam spiked with its N-methyl and deschloro analogues. All of the traces (and seven other channels) were obtained from a single chromatographic injection. The detector voltage was scanned from 1.0 to 2.0 V a t a scan rate equivalent to 2.0 V/s. The top trace in the figure corresponds to an applied potential of 2.0 V, the middle trace to 1.8 V, and the lower trace corresponds to an applied potential of 1.6 V. Although the three components are chromatographically well resolved in the figure, it is clear that some voltammetric
Figure 4. Chromatograms for SK&F 102698 recorded under both normal- and reversed-phase conditions with electrochemical detection: upper trace, normal-phase HPLC; lower trace, reversed-phase HPLC. SK&F 102698 concentration was 0.2 mg/mL.
discrimination is also provided by the detector. In the upper trace (2.0 V applied), the deschloro compound exhibits the greatest response, while for the lower trace (1.6 V applied), the fenoldopam response is greatest. This voltammetric resolution could augment the chromatographic resolution in the case where two components were not cleanly resolved by the column. Sulfhydryl Groups. Electrochemical detection of thiols has generally (10, 11) involved the catalytic oxidation of Hg(SR), on amalgam electrodes. Abounassif and Jefferies (12) showed that thiols could be directly oxidized on glassy carbon; however, the large potentials required (>1 V) promote large background currents and decrease the selectivity offered by amperometric detection. Nonaqueous mobile phases produce a more stable base line, and voltammetric detection provides additional selectivity through peak subtraction (3). Therefore, direct sulfhydryl oxidations on glassy carbon become more attractive for normal-phase HPLC especially when voltammetric detection is employed. Chromatograms for SK&F 102698 under both normal- and reversed-phase conditions are shown in Figure 4. The upper trace is a normal-phase chromatogram and the lower trace corresponds to reversed-phase HPLC. In both cases, the detector potential was scanned from 1.0 to 2.0 V and ten channels of chromatographic data were collected. Only one of the ten channels is plotted in each case. The peak shape is much better for the normal-phase chromatogram. Although the chromatograms represent equivalent concentrations of SK&F 102698 the peak height is 3-fold higher for the normal-phase chromatogram while the peak area is greater for the reversed-phase chromatogram. The signal-to-noise ratio for the normal-phase mode is nearly 4-fold better. The chemical name for SK&F 102698 implies that the molecule contains the thione moiety rather than thiol. In fact, the molecule exists in two tautomeric forms in solution, where a proton is exchanged between the imidazole nitrogen and the sulfur atom. This tautomerization is apparently rapid enough that a significant fraction of SK&F 102698 is available in the thiol form a t the working electrode. SK&F 102698 and 6-mercaptopurine were studied by both reversed-phase and normal-phase HPLC with electrochemical detection. Both were linear in the normal-phase mode but neither was linear over a similar concentration range (i.e., 0.01-0.2 mg/mL) in the reversed-phase mode. SK&F 102698 exhibited a slope of 6.60 f 0.02 area X mL/mg, an intercept of 0.024 f 0.001 area units, a correlation coefficient of 0.9992, The slope for and a standard error of estimate of 6.1 X 6-mercaptopurine was 6.3 h 0.05 area X mL/mg, the intercept
2161
Anal. Chem. 1988, 60, 2161-2162
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equivalent to 2.0 V/s. It can be seen from the figure that some voltammetric discrimination is provided by the detector. The peak height ratio between the two compounds is approximately 3.6 for the upper trace, 3.0 for the middle trace, and only 2.6 for the lower trace. While this would certainly not be a sufficient amount to provide complete resolution without any chromatography, the figure illustrates that voltammetric detection augments the resolution provided by the column. Registry No. TEAPTS, 733-44-8;fenoldopam, 67227-56-9; deschlorofenoldopam, 75510-66-6; N-methylfenoldopam, 115534-32-2;SK&F 102698,95333-81-6;6-mercaptopurine,50-442; methimazole, 60-56-0; DL-lo-camphorsulfonic acid, 5872-08-2.
j/ llil
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LITERATURE C I T E D Samuelson. R. E.; Odea, J.; Ostewouna. . - J. Anal. Chem. 1980, 5 2 ,
+1.6 V I
22 15-22 16.
Kafil, J. B.;Last, T. A. J. Chromatogr. 1985, 348, 397-405. Last, T. A. Anal. Chim. Acta 1883, 155, 287-291. Barnes, A. C.; Nieman, T. A. Anal. Chem. 1983, 55, 2309-2312. White, J. G.; St. Claire, R. L., 111; Jorgenson, J. W. Anal. Chem. 1986. 58. 293-298.
Gunasingham, H.;Tay, B. T.; Ang, K. P. Anal. Chem. 1984, 56, 2422-2426.
Jane, I.; McKinnon, A.; Flanagan, R. J. J. Chromatogr. 1985, 323, 191-225.
Gunasinaham, H.; Tay. B. T.; Ana, K. P. Anal. Chem. 1987, 5 9 , 262-266. Last, T. A. Anal. Chem. 1983, 5 5 , 1509. Rabenstein, D. L.; Saetre, R. Anal. Chem. 1877, 49, 1038-1039. Allison, L. A.; Shoup, R. E. Anal. Cbem. 1983, 5 5 , 8-12. Abounassif, M. A.; Jefferies, T. M. J . Pharm. Biomed. Anal. 1983, 1 , 65-72.
RECEIVED for review December 23, 1987. Accepted May 6, 1988.
Wall-Tube Electrode Cell for Computerized Potentiometric Stripping Analysis Amando F. K a p a u a n
Department of Chemistry, Ateneo de Manila University, P.O. Box 154, Manila, Philippines Computerized potentiometric stripping analysis (PSA) as developed by Jagner et al. provides a rapid determination of electroadive metals in solution at very low concentration levels (1-3). In this technique, the metal is first plated at a constant potential onto a suitable electrode surface, usually mercury film on glassy carbon, and chemically stripped off while the voltage of the electrode is followed as a function of time. The length of time that the potential of the electrode stays in a range characteristic of the metal is a function of the amount plated on and therefore of the original concentration in the solution. Reproducibility of the method is strongly influenced by the hydrodynamic regime at the working electrode and only the rotating disk electrode (RDE) system and Kissinger laminar-flow thin film liquid cells have been found useful for analyses at very low concentrations where stripping plateaus are on the order of a few milliseconds ( 4 , 5 ) . However, the RDE system is difficult to construct and use properly while Kissinger type flow cells, especially with electrodes that use a mercury film, are hard to maintain since they have to be taken apart and reassembled to clean and polish the working electrode. Recently, Albery and Bruckenstein (6) pointed out the complete hydrodynamic equivalence of the wall-tube electrode and the RDE. We have exploited this equivalence with a PSA cell that uses a wall-tube electrode configuration and has a built-in centrifugal pump.
EXPERIMENTAL SECTION Apparatus. The important parts of the wall-tube cell are shown in Figure 1. The motor M drives the 35-mm smooth disk impeller D at 3600 rpm and pumps solution through the tube T at 10.2 mL/s against the glassy carbon disk electrode E. In the wall-tube configuration, the tube's internal bore should be larger than the working electrode's active diameter. Thus the tube in this cell has a 6 mm diameter bore and the working electrode is a 3-mm glassy-carbon rod (Tokai 20) cast in epoxy (AralditeCY230 resin and HY951 hardener, 1O:l). Distance between the tube's end and the surface of the working electrode is fixed at 4 mm. A platinum coil counter electrode and a Ag/AgCl reference electrode are mounted as close as possible to the working electrode. Acrylic plastic is used for all components in contact with the solution. All of the dimensions and operating parameters given above follow closely the limits given by Albery and Bruckenstein as those required for "safe" operation of this electrode system. Solution in a beaker (85 mL minimum) can be introduced and withdrawn from the bottom of the setup. The working electrode's mount allows it to be removed and replaced for cleaning and polishing without difficulty. Data Acquisition System. Since a fairly rapid data acquisition rate is required in millisecond stripping PSA, a computerized system is a practical necessity for the technique. The lab-built data acquisition system (DAS) used here has two 12-bit 25-ps analog-to-digital converters equipped with a teraohm input resistance instrumentation and sample and hold amplifiers, two 12-bit digital-to-analog converters, a 24-bit clock with a 1-MHz crystal time-base, and four independent DPDT reed switches. A
0003-2700/88/0360-2161$01.50/00 1988 American Chemical Society
