ANALYST, FEBRUARY 1986, VOL. 111 157 Reduction in Size by Electrochemical Pre-treatment at High Negative Potentials of the Background Currents Obtained at Negative Potentials at Glassy Carbon Electrodes and its Application in the Reductive Flow Injection Amperometric Determination of Nitrofurantoin Ahmad B. Ghawji and Arnold G. Fogg Chemistry Department, Loughborough University of Technology, Loughborough, Leicestershire LEI I 3TU, UK The reduction of dissolved molecular oxygen a t a glassy carbon electrode was shown to be made more difficult on electrochemically pre-treating a newly polished glassy carbon disc electrode (3 mm in diameter) in 0.1 M sulphuric acid solution at -2.7 Vfor 1 min. By this means a background current of only 1 pA was obtained when this electrode was held at -1.05 V in a flow injection system incorporating extensive PTFE transmission tubing and using a deoxygenated pH 7 Britton - Robinson buffer as eluent (flow-rate 6.5 mi min-1).The size of the signal obtained when 100 1-11 of eluent that had not been deoxygenated were injected as the sample blank was only 0.02 FA at -0.7 V. When nitrofurantoin was determined using these latter conditions, this blank signal was equivalent to about 2 x 10-7 M nitrofurantoin. Keywords: Electrochemical pre-treatment; amperometric detection; reduction; flow injection analysis; nitro furantoin determination Increasing attention is being paid to the advantages of electrochemically pre-treating glassy carbon electrodes used for amperometric detection in HPLC and in flow injection analysis.1-8 The studies reported to date have been made to improve the performance of glassy carbon electrodes used for monitoring oxidation processes at positive potentials.In many irreversible oxidation processes, electrochemical pre- treatment first at a high positive potential and then at about -1.0 V reduces the overpotential for oxidation of the determinand such that an improved hydrodynamic voltammo- gram is obtained. Oxidation occurs more completely and at a less positive potential such that a higher and more reproduc- ible signal is obtained. At any particular potential the background signal is also increased, but this does not detract significantly from the technique. An important HPLC method that involves reductive amperometric detection at a glassy carbon electrode held at negative potentials is the determination of vitamin K and its analogues.9JO Hart et ~1.10 determined vitamin K1 in a 95% methanol eluent that was 0.05 M in a pH 3 sodium acetate - acetic acid electrolyte holding the potential of the glassy carbon electrode at - 1.0 V; the eluent was deoxygenated with nitrogen and an all-metal solvent delivery system was used to prevent the re-entry of oxygen.Calibration graphs were obtained by injecting 1-10 ng of vitamin K1. Adsorbed product on the electrode was removed periodically by holding the electrode at +0.7 V, which re-oxidised the product as the reduction process is quasi-reversible. Hanging mercury drop electrodes have been used by other workers, notably by Lloydll-13 for determining explosives residues, as detectors of reductive processes.The rigorous exclusion of oxygen has been an important feature of all of these methods. In the work described in this paper a study was made of the possibility of improving signals for reductive processes at glassy carbon electrodes held at negative potentials by applying electrochemical pre-treatment. Experimental Flow injection analysis was carried out in a single-channel system that has been described previously.14 Eluent flow was produced by means of an Ismatec Mini-S peristaltic pump. Sample (approximately 100 pl) was injected with a Rheodyne 5020 low-pressure injection valve connected to a laboratory- built detector cell by means of 50 cm of 0.58 mm bore PTFE tubing. The detector cell holds the glassy carbon electrode only, eluent being presented to it in a wall-jet configuration.The cell is used partially immersed in an electrolyte having the same composition as the eluent. A counter platinum and a conventional potentiometric calomel reference electrode are placed in the electrolyte to obtain electrical contact with the working electrode. The glassy carbon disc electrode (3 mm diameter) was constructed from Le Carbonne glassy carbon and was mounted in PTFE. An eluent flow-rate of 6.5 ml min-1 was used. The potential of the glassy carbon electrode was controlled by means of a PAR 174 polaro- graphic analyser and current signals were monitored on a Linseis L650 y - t recorder. Linear sweep voltammetry was carried out at a sweep rate of 10 mV s-1 using the same working, counter and reference electrodes immersed in the appropriate measuring solution.Preliminary Linear Sweep Experiments in a Static System During studies of the effect of positive- and negative-potential electrochemical pre-treatments of glassy carbon electrodes on oxidation processes at low positive potentials, it was noticed that electrochemical pre-treatment at high negative potentials was effective in making smaller the background currents obtained at negative potentials. This is clearly illustrated in Fig. 1, in which base-line linear sweep voltammograms obtained with a static electrode system in 0.01 M sulphuric acid before and after electrochemical pre-treatment are shown; the electrode was electrochemically pre-treated in 0.1 M sulphuric acid. Pre-treatment at -3 V in the static mode is seen to remove the oxygen reduction wave most effectively.Further, electrochemical pre-treatment was shown to be effective only when carried out in dilute sulphuric acid; attempts to effect pre-treatment in Britton - Robinson buffer solution of pH between 2 and 8 were unsuccessful. The pre-treatment that had been effected in dilute sulphuric acid, however, was also as effective when the electrode was used in these buffer solutions. Linear sweep voltammograms obtained for the reduction of158 11 8 f . c If 2 3 0 4 0 -0.4 -0.8 PotentialiV Fig. 1. Blank linear sweep voltammograms in 0.01 M sulphuric acid without deoxygenating the solution. A and A’, first and second scans at a newly polished glassy carbon electrode; B, scan after pre- treatment at -2.5 V for 1 min in 0.1 M sulphuric acid; and C, scan after pre-treatment at -3 V for 1 min in 0.1 M sulphuric acid I 4 f .5 0 c L 3 0 8 4 0 I / -0.4 -0.8 Potent i a I N Fig. 2. Linear sweep voltammograms of nitrofurantoin (2 x M) in undeoxygenated pH 7 Britton - Robinson buffer. (a) At a newly polished glassy carbon electrode; and (b) at an electrode pre-treated at -3 V for 1 min. The blank linear sweep voltammograms are given as broken lines in both instances 8 f . c C ? 3 0 4 0 ANALYST, FEBRUARY 1986, VOL. 111 -0.4 -0.8 Potent ia I/V 1.2 Fig. 3. Linear sweep voltammograms of cephalonium (100 pg ml-1) in 0.1 M sulphuric acid. A, Without deoxygenating the solution at a newly polished glassy carbon electrode; B, without deoxygenating the solution at an electrode pre-treated at -3 V for 1 min; and C, after deoxygenating the solution at a newly polished glassy carbon electrode nitrofurantoin, which occurs at about -0.58 V in pH 7 Britton - Robinson buffer, are shown in Fig.2. When the electrode is newly polished a hump due to the reduction of dissolved molecular oxygen is apparent as a post-peak. After pre- treating the electrode at -3 V this hump is no longer apparent. An illustration of the oxygen reduction process occurring before that of a determinand is shown in Fig. 3, in which linear sweep voltammograms for the reduction of the cephalosporin cephalonium are shown. Here also electrochemical pre- treatment at -3 V removes visible signs of the oxygen reduction process. Polarographic methods are available for the determination of nitrofurantoin15 and cephalonium.16 Effect of Electrochemical Pre-treatment at High Negative Potentials in Flow Injection Analysis In using a glassy carbon electrode for amperometric detection in HPLC or flow injection analysis, two characteristics of the system should be considered before the quality of the signal obtained with the determinand is studied. These are the background current associated with the eluent and the blank signal obtained when a control blank is injected. When the eluent is used as the control blank, clearly eluent and sample are the same and no signal should be observed when the control blank is injected, except at high sensitivities owing to disturbance to the flow of eluent caused by the process of injecting the eluent.In determinations made at potentials where oxygen reduction occurs, however, a finite blank signal will be observed if the oxygen contents of the eluent and the blank sample solution differ. A negative signal will be observed if the eluent contains more oxygen than the blank sample. Clearly analytical determinations become very unreli- able when the level of interferent in the solvent system and sample solution have to be balanced, and this is particularly so with dissolved molecular oxygen. In general, with increasing background current the detection limit attainable is increased; with the system used in this work it has generally been observed that if the background current reaches 1 yA then determinations can only be made down to about 5 x 10-6 M and that this also applies at positive potentials where the background current is not associated with the reduction of oxygen.The results reported here for electrochemically pre-treated electrodes were obtained with electrodes pre-treated either atANALYST, FEBRUARY 1986, VOL. 111 3 - f . 5 2 - 4- L J u 1 - 0 - -2.7 V for 1 min in a static system before being inserted into the detector cell or at -3 V for 1 min on-line in 0.1 M sulphuric acid at a flow-rate of 2 ml min-1. These were found to be the optimum off-line and on-line electrochemical pre-treatment conditions. This latter process was readily effected by switch- ing eluents before the pump. The use of higher pre-treatment potentials than those recommended led to higher background noise. The background current levels obtained at various potentials with the flow injection system in which pH 7 Britton - Robinson buffer was used as the eluent are shown in Fig.4. These were obtained for a newly polished electrode and for pre-treated electrodes in all instances with and without deoxygenation of the eluent with nitrogen (it should be borne in mind that the term “deoxygenation,” which is used extensively by polarographers, is misleading in that the oxygen concentration is reduced only to a particular level that is determined by the effectiveness of the “deoxygenation” process and also by the effectiveness of preventing oxygen from re-entering the eluent before the measurement is made). It is clear from Fig. 4 that electrochemical pre-treatment extends the useful range of the electrode to more negative potentials both when the eluent is deoxygenated and when it is not and that the static electrochemical pre-treatment process is more effective than the on-line pre-treatment.In effect, on pre-treatment the overpotential for the reduction of oxygen at the glassy carbon electrode is being increased, i.e., the reduction of oxygen is being made more difficult by the pre-treatment process. Perhaps not surprisingly, electrochemical pre-treatment has a more significant effect on the useful range of the electrode when the oxygen content of the eluent has been reduced to a lower level by deoxygenation. Nevertheless, deoxygenation of eluent and sample solutions is a time-consuming task and there is a distinct advantage to be gained in avoiding the necessity of having to carry it out.Compounds such as vitamin Ks, which can be determined at potentials less negative than -0.5 V, can be determined at low levels even with an unpre-treated electrode without having to deoxygenate the eluent and sample solutions. Nevertheless, even in these instances the background current is reduced and the detection limit should be lowered by using a pre-treated electrode. Electrochemical pre-treatment produces a slight extension of the useful range of the electrode in an eluent that has not been deoxygenated and this should allow other compounds to be determined without the need to deoxygenate the eluent or sample solutions, particularly if determinations are to be made at high concentrations. The extension of the useful range on pre-treating the glassy carbon electrode, however, is much greater for the deoxygen- ated eluent.From Fig. 4 it can be seen that the potential at which a background current of 1 pA is obtained is moved from -0.72 to -1.05 V on pre-treating the electrode at -2.7 V in the static mode. Hence electrochemical pre-treatment should make amperometric detection possible for compounds that are reduced (or oxidised) at these more negative potentials. Again, the added advantage that lower background currents are obtained in determining compounds at lower negative potentials should not be overlooked. Deoxygenation of eluent in a flow injection system by means of nitrogen is readily carried out, and nitrogen can be bubbled continuously through the eluent in the eluent reservoir during determinations with no great inconvenience or loss of time once the initial deoxygenation has been effected.Deoxygenation of every sample solution is extremely time consuming, however, and the need to do this should be avoided if at all possible. The size of signals obtained at various potentials on injecting pH 7 Britton - Robinson buffer that had not been deoxygenated into deoxygenated eluent of the same composition is illustrated in Fig. 5. These results were obtained with both newly polished and electrochemically pre-treated electrodes. The marked effect of electrochemical 15 f 10 2- 4- E 3 0 5 1 0 -0.8 PotentiaW 159 Fig. 4. Background currents obtained with flow injection ampero- metry using pH 7 Britton - Robinson buffer as eluent.A, Without deoxygenating the solution at a newly polished glassy carbon electrode; B, without deoxygenating the solution at an electrode pre-treated at -3 V for 1 rnin on-line; C, after deoxygenating the solution at a newly polished glassy carbon electrode; D, after deoxygenating the solution at an electrode pre-treated at -3 V for 1 rnin on-line; and E, after deoxygenating the solution at an electrode pre-treated at -2.7 V for 1 rnin (in a static system) I 4 t r I -0.5 -0.7 -0.9 PotentiaVV Fig. 5. Blank hydrodynamic voltammograms representing the size of signals obtained on injecting undeoxygenated eluent into deoxy- genated eluent ( H 7 Britton - Robinson buffer). A, At a newly polished electrog; B, at an electrode pre-treated on-line in 0.1 M sulphuric acid at -3 V for 1 min; and C, at an electrode pre-treated in the static mode at -2.7 V for 1 min pre-treatment on the size of the signal obtained can be clearly seen.The potential at which the blank signal due to oxygen in the blank sample reaches 1 pA is moved from -0.77 to -0.95 V on pre-treating the electrode at -2.7 V in the static mode. In Figs. 6 and 7 are shown hydrodynamic voltammograms of nitrofurantoin in pH 7 Britton - Robinson buffer using newly polished and pre-treated electrodes, respectively. In both instances the hydrodynamic voltammograms that are shown were obtained using deoxygenated eluent. The effect of deoxygenating the sample on the hydrodynamic voltammo- grams obtained is also clear. Blank hydrodynamic voltammo- grams in which undeoxygenated eluent was injected intoANALYST, FEBRUARY 1986, VOL.111 D - - T I 0.1 pA & -0.4 -0.8 Potent i a l/V Fig. 6. Hydrodynamic voltammograms obtained at a newly polished electrode for injection of nitrofurantoin (2 x 10-4 M) into deoxygen- ated pH 7 Britton - Robinson buffer. A, Sam le solution undeoxygen- ated; B, sample solution deoxygenated; and 8, undeoxygenated blank injection I 0.1 pA D -Time -0.4 -0.8 PotentialN Fig. 8. Signals obtained near the determination limit for nitrofuran- toin at (a) a newly polished electrode and (b) at an electrode pre-treated at -2.7 V in the static mode for 1 min. Measurement potential = -0.65 V. Nitrofurantoin concentration: A, 0; B, 2 X 10-7 M; C, 5 x M; and D and D’, 10 x 10-7 M. Eluent and sample solution D’, deoxygenated; sample solutions, A-D, undeoxygenated Fig. 7.Hydrodynamic voltammograms obtained at an electrode pre-treated in the static mode at -2.7 V for 1 min for injection of nitrofurantoin (2 x 10-4 M) into deoxy enated pH 7 Britton - Robinson buffer. A, Sample solution uncfeoxygenated; B, sample solution deoxygenated; and C, undeoxygenated blank injection deoxygenated eluent are also shown. It should be noted that the signals due to the reduction of nitrofurantoin are made smaller by the pre-treatment process. Clearly, the reduction of nitrofurantoin is also being inhibited, although not to the same extent as the reduction of oxygen. The precision of the signals, however, remained excellent. The beneficial effect of the electrochemical pre-treatment in making smaller the size of the oxygen signal can be clearly seen in Fig.7. At the current sensitivity used in obtaining the hydrodynamic volt- ammograms shown in Fig. 7 there is no difference in the signal at -0.7 V on deoxygenating the sample solution. Hence it is clear that at these levels of determinand there is no need to deoxygenate the sample solutions. Fig. 8 shows signals obtained near the determination limit both with a newly polished electrode and a pre-treated electrode. The measurement potential used here was -0.65 V to reduce the oxygen blank to an acceptable level for this concentration of determinand. The large-scale removal of background noise on electrochemically pre-treating the elec- trode can be seen clearly. The extensive reduction in the signal from the oxygen dissolved in the sample solution on electro- chemical pre-treatment is also apparent; this blank is equiv- alent to 6 x 10-8 M nitrofurantoin. At significantly higher concentrations determinations would normally be made at -0.7 V where the blank signal is equivalent to 2 X M nitrofurantoin. At levels of nitrofurantoin above 5 X M coefficients of variation for five injections at the same concentration were typically less than 1%.Conclusions Electrochemical pre-treatment at high negative potentials in dilute sulphuric acid is effective in inhibiting the reduction of dissolved molecular oxygen at glassy carbon electrodes and therefore lowers the background currents caused by reduction of dissolved oxygen when such electrodes are used at negative potentials in flow injection analysis and, by extrapolation, in HPLC applications. With nitrofurantoin a slight loss of determinand signal also occurs, but without loss of precision.For most reversible systems it is expected that little or no loss of signal would be experienced, i.e., that the systems would remain reversible. It is further expected that detection limits even for compounds that are determined at low negative potentials, where oxygen is not a major interferent at high determinand concentrations, will be lowered. Further studies are being made on such systems. In flow injection applications using PTFE transmission tubing it is possible to deoxygenate eluents to a sufficiently low level to enable compounds that are reduced at potentials up to about -0.7 V to be determined at a pre-treated electrode without the need to deoxygenate the sample solutions. The authors thank Dr. J. P. Hart for his interest in this work.ANALYST, FEBRUARY 1986, VOL. 111 161 References 1. Blaedel, W. J., and Jenkins, R. A., Anal. Chem., 1975, 47, 1337. 2. Chan, H. K., and Fogg, A. G., Anal. Chim. Acta, 1979, 111, 281. 3. Van Rooijen, H. W., and Poppe, H., Anal. Chim. Acta, 1981, 130, 9. 4. Engstrom, R. C., Anal. Chem., 1982, 54, 2310. 5. Ravinchandran, K., and Ba1dwin;R. P., Anal. Chem., 1983, 55, 1782. 6. Engstrom, R. C., and Strasser, V. A,, Anal. Chem., 1984,56, 136. 7. Fogg, A. G., Fernfindez-Arciniega, M. A., and Alonso, R. M., Analyst, 1985, 110, 851. 8. Fogg, A. G., Fernindez-Arciniega, M. A., and Alonso, R. M., Analyst, 1985, 110, 1201. 9. 10. 11. 12. 13. 14. 15. 16. Ikenoya, S . , Abe, K., Tsuda, T., Yamano, Y., Hiroshima, O., Ohmae, M., and Kawabe, K., Chem. Pharm. Bull., 1979,27, 1237. Hart, J. P., Shearer, M. J., McCarthy, P. T., and Rahim, S., Analyst, 1984, 109,477. Lloyd, J . B. F., J . Chromatogr., 1983, 256, 323. Lloyd, J. B. F., J . Chromatogr., 1983, 257, 227. Lloyd, J. B. F., J . Chromatogr., 1983, 261, 391. Fogg, A. G., and Summan, A. M., Analyst, 1984, 109, 1029. “United States Pharmacopeia,” Twentieth Revision, United States Pharmacopeial Convention, Rockville, MD, 1980, p. 549. Fogg, A. G., Fayad, N. M., Burgess, C., and McGlynn, A., Anal. Chim. Acta, 1979, 108, 205. Paper A51282 Received August Ist, I985 Accepted August 21st, I985