Anal. Chem. 1988, 60, 1710-1713
1710
Stable Modified Electrodes for Flow Injection Amperometry: Application to the Determination of Thiocyanate James A. Cox* and Thomas Gray Department of Chemistry, Miami University, Oxford, Ohio 45056
Krishnaji R. Kulkarni' Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, Illinois 62901
Platinum electrodes that are modified by adsorption of iodine and coated with cellulose acetate and glassy carbon electrodes that are modified by anodlzatlon in a RuCLJK,Ru(CN), mixture are stable amperometrlc indicators for thiocyanate in flow systems. The former yield linear calibration curves over the 5.0 p M to 0.1 mM SCN- range and permit the detection of 100 pg of SCN- In a 20-pL injection. At the latter electrodes a narrower linear dynamic range, 0.2-4.0 pM SCN-, is observed, but the stability is on the order of months rather than days. Throughputs calculated from the half-widths of the peaks and the condition of base-line resolution of Gaussian peaks are 64 injectionslh at 1.0 mL/mln at the modified Pt electrode and 240 injections/h at 1.7 mL/min at the modifled glassy carbon electrode. The method was used for the determlnatlon of SCN- in urine; Interference by uric and ascorbic acids was eliminated by controlled potential electrolysis prior to the flow Injection determination.
The determination of thiocyanate in body fluids is important for various reasons. Thiocyanate is a detoxification product of hydrogen cyanide; hence, determinations of thiocyanate in body fluids have been used to monitor exposure to hydrogen cyanide from tobacco smoke ( I ) , fire atmospheres (2), and certain vegetables that contain cyanogenic glucosides (3). Thiocyanate is also known to block the iodine uptake by the thyroid gland. Determinations of serum thiocyanate have also been advocated for supervising therapy with sodium nitroprusside ( 4 ) , a hypertensive drug that is metabolically converted to cyanide. Numerous methods for the determination of thiocyanate in body fluids have been developed. Spectrophotometric methods that are based on the formation of a red complex with Fe(II1) ions are common, but they are nonselective. Several methods have been based on the Konig synthesis in which the cyanogen bromide produced by the reaction of thiocyanate with bromine reacts to produce a pyridine dye. The Konig method has been modified to improve selectivity (5). To meet the needs of applied laboratories where a large number of samples must be efficiently analyzed, the flow injection method is often used, but few studies on the flow injection determination of thiocyanate have been reported. In one case, Tanaka et al. (6)detected thiocyanate on the basis of its catalysis of the oxidation of As(II1) by Ce(1V). The decrease in absorbance of Ce(1V) measured at 254 nm was proportional to the amount of thiocyanate injected into a carrier stream containing Ce(1V) and As(II1). A detection limit of 0.2 mg of thiocyanate/L was achieved. In another report, Present address: Department of Chemistry, University of Ala-
bama, Birmingham, AL 35294.
0003-2700/88/0360-1710$01.50/0
Alexander et al. (7)employed a copper wire as a potentiometric flow detector. In a carrier stream of pH 4.8, acetate buffer, the copper wire has Cu2+,Cu+, and Cuo at its surface; these species determine the potentiometric response of the electrode. The injected thiocyanate forms a Cu(SCN),- complex that changes the potential as a function of thiocyanate concentration. Anions such as phosphate, chloride, and cyanide interfered. A third report deals with the determination of thiocyanate by its oxidation at a platinum electrode (8). Generally, the determination of thiocyanate at bare metal electrodes has been severely limited by the poisoning caused by its oxidation product. Austin et al. overcame this problem in the determination of thiocyanate and other sulfur-containing compounds at a platinum electrode in a flow system by application of a multistep potential waveform in the 0.5-2 Hz range (8). When the potential of the electrode was held a t -1.0 V vs a saturated calomel electrode (SCE) the platinum oxide present on its surface was reduced to platinum with simultaneous adsorption of thiocyanate. On stepping the potential to 0.6 V, oxidation of thiocyanate, which was induced by anodic formation of an oxide of platinum, was observed. To desorb the oxidation product and to form platinum oxide, the potential was further stepped to 0.7 V. Another approach to elimination of passivation by electrolysis products is to modify electrodes with a film of a catalyst. For example, the adsorption of iodine on platinum (9)yields an indicator electrode for the determination of nitrite that is not limited by adsorption of an intermediate during the oxidation of the analyte (10, 11). Because our goal was to develop an indicator electrode for thiocyanate that operated in the constant potential mode, the P t / I system was tested in this study. Since the envisioned applications were to urine and saliva samples, the electrode was further coated with a cellulose acetate film. Others have demonstrated that this film is permeable to small analytes but blocks the adsorptive interference of macromolecules on the electrolysis (12, 13). Also described are the results of a study of the oxidation of thiocyanate in a flow system with a glassy carbon electrode, which is modified by formation of a thin film during voltammetry of a solution containing RuC13 and K4Ru(CN)6a t pH 2 (14). This modified electrode was known to mediate oxidations at the appropriate potential for that of thiocyanate (ca. 0.8 V vs SCE) and to display long-term stability (at least 2 months in static solution or dry storage).
EXPERIMENTAL SECTION Apparatus. The flow injection system consisted of a carrier solution reservoir with a nitrogen-purging device, a Cole Parmer Master-Flex peristaltic pump, a Rheodyne 7010 (for the modified Pt electrode experiments) or a Rheodyne 7125 (for the modified glassy carbon electrode experiments) six-port injection valve, a Bioanalytical Systems (BAS) TL-5A or TL-1OA flow-through electrochemical cell, either an IBM Instruments EC/230 pof2 1988 American Chemical
Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
tentiostat designed for use with an amperometric liquid chromatography detector or a BAS CV 37 low-current potentiostat, and a strip-chart recorder. A pulse dampener was introduced between the pump and the injector. A short Teflon tube (45-cm length and 0.08-cm inner diameter) was used to connect the injection port to the flow cell. The carrier stream for work with the platinum-iodine system was 0.2 M NaC1, adjusted to pH 4 with HCl; for the modified glassy carbon electrode experiments, it was 0.2 M KzS04, adjusted to pH 2 with HzS04. The BAS TL-5A and TL-1OA flow cells consisted, respectively, of a glassy-carbon-diskworking electrode with a geometrical area of 7.5 mm2 and a platinum-disk working electrode with a geometrical area of 9.1 mm2. The upper block of the cell was made from stainless steel, which served as the auxiliary electrode. A BAS Model RE-1 Ag-AgC1 electrode placed in a downstream compartment served as the reference;the electrode was filled with 1.0 M KCl. The cyclic voltammetry experiments were performed with a BAS 100 Electrochemical Analyzer using a BAS glassy carbon indicator electrode. Reagents. Unless otherwise noted, the chemicals were ACS Reagent Grade and were used without further purification. The RuC13was purchased from Pfaltz and Bauer, and the &Ru(CN), from Alfa Products. The water used was house-distilled, which was further purified by successively passing through Cole Parmer research grade cartridges and a Barnstead Nanopure I1 system. Procedures. Prior to the modification step all working electrodes were polished with 0.1-pm alumina on a Gamal cloth (Fisher Scientific)with water as lubricant. Polishing was generally performed for about 10 min. The polished electrodes were then thoroughly rinsed with water. . The platinum electrodes were further cleaned electrochemically by cycling the potential between 1.4 and -0.2 V vs Ag-AgC1 in deaerated 1 M HzSO4 for 1h. The cycling was terminated at 0.4 V, and the electrode was held at that potential until the background current decayed to a constant value. This step usually took 30-45 min. Modification of the Pt was performed immediately thereafter in the flow cell. In the modification, the clean surface of the electrode was contacted with a freshly prepared, deaerated 2 mM solution of aqueous sodium iodide for 30 min in the flow cell. The cell was then dismantled, and the electrode was rinsed with water. After the surface of the electrode was gently wiped with tissue paper, two 4-pL aliquots of a 2% cellulose acetate solution in 1:l cyclohexanone-acetone were placed onto the surface with a microsyringe. The resulting coating was sufficient to protect the surface; thicker films were not used because of the known effect of thickness on sensitivity (12). The solvent of the first drop was allowed to evaporate before the second was placed thereon. The mass of the cellulose acetate deposit was about 0.16 mg. The electrode was dried in the air for 1h before it was assembled into the flow cell. The modified electrode was stored in a solution of the composition of the carrier stream. The electrode was held at 0.7 V vs Ag-AgC1 for the SCN- determinations. Polished glassy carbon electrodes were modified by cycling the for 40 cycles in potential between 0.50 and 1.00 V at 50 mV a 3 mM RuC13/3 mM K&u(CN), solution at pH 2 in the flow cell ( 2 4 ) . The nature of the resulting film has been elucidated by Kulesza (15). The flow injection determination of thiocyanate with this indicator was performed at 0.85 V vs Ag-AgC1.
RESULTS AND DISCUSSION Initial experiments were performed a t a P t / I electrode without an overlayer of cellulose acetate. When 20-pL injections of 5.0 pM SCN- were introduced into a carrier stream with a flow rate of 1.0 mL min-’, the response gradually decayed. Currents for injection numbers l , 5, 10,20, and 30 were 12.5, 10.2, 8.5, 7.0, and 6.0 nA, respectively. When the overlayer of cellulose acetate (CA) was used, the response of the resulting Pt/I/CA electrode was stable over a t least the 120-injectionset that was employed (Table I). The low results on the initial trials that appear in this set are not typical. The sensitivity of the Pt/I/CA electrode, 58 pA/pM, is much less than the initial sensitivity of the P t / I electrode, 2.5 nA/pM, with a 9.1 mm2 area for each. This is undoubtedly
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Table I. Stability of the Pt/I/CA Electrode during Oxidation of Thiocyanate injection no.a
peak current,bpA
1-5
560 f 5
26-30 51-55 76-80 101-105 116-120
590 f 8
580 f 5 580 f 8 580 f 10 590 4
+
“ A total of 120 samples (20-pL)were injected consecutively at a rate of 40 samples/h with a flow rate of 1.0 mL/min. The peak widths were about 20 s. bCurrents were developed with 10 p M SCN- samples. The calculated relative standard deviations were of the five trials in the reported subsets of the 120-injection experiment.
related to hindered transport of the SCN- through the CA film. The diminished rate of product formation with the Pt/I/CA electrode may be the factor that eliminates the passivation observed with the P t / I system. Thus, the CA film,which was initially employed to prevent passivation by surfactants in the intended samples, has an important role in stabilizing the modified surface during the oxidation of SCN- in general samples. Because the CA coating decreases the sensitivity of the electrode, it is apparent that the response of the Pt/I/CA electrode will change from preparation to preparation because of variations in the thickness of the overlayer. With the 9.1 mm2 geometric surface area, sensitivities in the range 50-100 pA/pM were observed. Further, after a few hundred injections, thinning of the CA film can change the sensitivity. For any given preparation of the Pt/I/CA surface, the flow injection amperometric peak current is directly proportional to thiocyanate concentration over a wide range. For example, with a seven-point calibration curve over the range 5.0 pM-0.1 mM SCN-, the following linear least-squares characteristics are obtained: slope, 93 nA/mM; relative standard deviation of the slope, 0.8%; intercept, -0.30 f 0.04 nA; and correlation coefficient, 0.9992. The detection limit, expressed as the concentration that gives a signal that is 3 times the uncertainty of the measurement of a 5 X lo4 M standard solution, is 100 pg of thiocyanate in a 20-pL sample. This corresponds to 1.2 x M SCN-. Injection of a blank solution does not result in any detectable signal even a t the highest sensitivity of the instrument. The peak widths measured a t half-height for injections of a 20-pL sample containing 5 X lo4 and 1 X lo4 M thiocyanate were 23 and 22 s, respectively, a t a flow rate of 1.0 mL/min. A calculation based on the assumption of a Gaussian peak yields a throughput of 64 injectionslh with base-line resolution of peaks (six standard deviations between the peaks) with a half-width of 22 s. Experimentally, 40 samples/h were routinely run. The Pt/I/CA electrode was stable for several days. To illustrate this point, the same modified electrode that was used in the previous experiment was employed to prepare calibration curves on 6 consecutive days with seven standard solutions of thiocyanate in the concentration range 5 X lo4 to 1 x M. The characteristics of the calibration curves are summarized in Table 11. The relative standard deviations of the reported slopes are less than 1%; therefore, the tabulated differences in the slope are probably related to dayto-day variations in temperature and flow rate. After the sixth day, the sensitivity decreased by about 30%, which indicates that some fouling of the surface occurred; however, the calibration curve was still linear. Although the sensitivity of the Pt/I/CA electrode was constant for 6 days, the detection limit varied from 60 to 200 pg of SCN- in a 20-pL injection because
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
Table 111. Relative Sensitivities of the Pt/I/CA and mvRuCN Electrodes”
Table 11. Day-to-Day Reproducibility of Calibration Curves at a Pt/I/CA Electrode. day
slope, nA/mM
intercept, nA
corr coeff
rsd, %” analyte
1 2 3 4 5 6
93 98 105 91 101 99
-0.3 -0.3 -0.3 -0.2 -0.2 -0.1
0.9992 0.9994 0.9986 0.9998 0.9998 0.9997
0.9 1.8 0.7 1.6 0.5 0.7
Relative standard deviation of five trials with 5 fiM SCN-; flow rate, 1.0 mL/min. The data were obtained with 20-fiL injections of seven standard solutions of SCN- in the range of 5 fiM-O.1 mM.
63.01
3 * 45.01
/”
/
27-01-L.--9. 00
.500
1.50
J,”lm
2.50 3.50 SCN-/L
4.50
Figure 1. Calibration curve for the flow injection determination of thiocyanate at a glassy carbon electrode modified in a RUCl3/K,Ru(CN), mixture. The data were obtained with 50-pL injections into a carrier stream of 0.2 M K,SO, at pH 2 with a flow rate of 1.7 mL/min. The appliec‘ potential was 0.85 V vs Ag-ASCI.
of day-to-day changes in the uncertainty of the current measurement. The electrode that was modified by cyclic voltammetry at glassy carbon in a R u C I ~ / K ~ R U ( Cmixture, N)~ hereafter referred to as the mixed-valent ruthenium oxide-ruthenium cyanide electrode (mvRuCN), was also suitable for the flow injection amperometric determination of thiocyanate. A calibration curve is shown in Figure 1. The order of injection in these and all other experiments involving calibration curves was random in terms of concentration so the nonlinearity is not a reflection of an effect of electrode history. With 50-pL injections the calibration curve is linear only at concentrations below 1KMSCN-. A linear least-squares analysis of the points in the range 0.2-1.0 FM SCN- yields the following: slope, 43 f 1 nA/pM; correlation coefficient, 0.999; and y-intercept, 4 f 1 nA. The background current at the mvRuCN electrode is a function of potential. Under the conditions in Figure 1,except with applied potentials of 0.55, 0.60,0.65,0.70,0.75, 0.80, and 0.85 V vs Ag-AgC1, the steady-state background currents are 1.0, 2.0, 3.0, 5.5, 7.0, 15, and 29 nA, respectively. The trend is indicative of a kinetically controlled oxidation of a component of the carrier; water is the most probable candidate. Since the background current is quite independent of time, it is not deleterious to the determination of SCN- at conM level. centrations in and above the The mvRuCN electrodes are very stable. One of the preparations has been used for several hundred determinations to date; an actual count was maintained until 300 flow injection determinations were made over a 3-month period. The sensitivity, as measured by the slope of the calibration curve in the linear region, remains constant from day to day. For example, the peak currents measured on 6 consecutive days with 50-pL injections of an 0.8 pM SCN- solution were as follows: 42.1 f 0.6, 45 f 3, 42 f 1, 42 f 4, 42 f 1, and 44 f 2 nA. Each value represents the average and standard deviation of five trials. Because an overlayer of cellulose acetate is not necessary to prevent passivation from the products of
uric acid
ascorbic acid thiocyanate uric acid ascorbic acid thiocyanate
concn, fiM
electrode
sensitivity, nA/pM
250 50 5.0 1.0 1.0 1.0
Pt/I/CA Pt/I/CA Pt/I/CA mvRuCN mvRuCN mvRuCN
1.8 x 10-2 0.5 X 7.4 x 18 14 32
Sample size was 20 fiL; conditions for the experiments with the Pt/I/CA electrode are in Table I; conditions for the mvRuCN electrode experiments are in the Experimental Section with the flow rate at 2.8 mL/min. the oxidation of thiocyanate, these sensitivities are much greater than those at the Pt/I/CA electrode. One limitation of the mvRuCN electrode for the flow injection determination of SCN- under the conditions in Figure 1is a narrow linear dynamic range. In an attempt to increase it, the experiments were repeated by using 20-pL injections; the flow rate was 2.8 mL/min. A calibration curve that was linear over the range 0.2-4.0 p M SCN- was obtained. The linear least-squares characteristics were the following: slope, 31.6 f 0.4 nA/KM; correlation coefficient, 0.9997; and y-intercept, 7.3 f 0.7 nA. With the 20-pL samples and a 1.7 mL/min flow rate, the results were similar; the senstivity was 37 nA/pM. Although the linear range is increased when smaller samples are injected, it is still less than that observed with the Pt/I/CA electrode. A second problem is that when the mvRuCN electrode is used without interruption in the flow injection mode, the sensitivity gradually increases. For example, when 150 consecutive injections (50-pL) of SCN- are made, the sensitivity increases from the initial value of 43 to 50 nA/pM. This problem does not occur if pauses of a few minutes are made between subsets of five to 10 samples. Also, the initial sensitivity is restored by allowing the electrode to stand at open circuit. However, because a high sampling frequency is a primary attribute of the flow injection method, an interrupted mode is not desirable. If the ca. 15% error cannot be tolerated, standards must be interspersed with the samples to correct for the change in sensitivity. The calculated throughput of flow injection experiments using a mvRuCN electrode is greater than that with a Pt/I/CA electrode in the amperometric detector because the response of the latter is slowed somewhat by diffusion of the analyte through the CA film. At a 1.7 m l / m i n flow rate, with 50- and 2 0 - ~ Linjections, the half-widths of the peaks were 12 and 6 s, respectively. Under the assumption of a Gaussian peak shape and with the calculation based on base-line resolution of the peaks, the respective throughputs were 120 and 240 injectionslh. With a 2.8 mL/min flow rate and 2 0 - ~ Linjections, the half-widths of the peaks were 5.3 s, and the calculated throughput was 270 injections/h. This value exceeds the capability of manual injection, so faster flow rates were not investigated. A common application of thiocyanate determinations is to measure the level of that ion in urine. Controlled potential amperometry is complicated by the fact that two major components, uric acid and ascorbic acid, are electroactive. The relative sensitivities of the Pt/I/CA and mvRuCN electrodes toward SCN-, uric acid, and ascorbic acid are summarized in Table 111. With both electrodes, the response to SCN- is significantly greater than to either uric or ascorbic acid. Nevertheless, because these acids are a t much higher concentrations than thiocyanate in urine, neither electrode has
ANAL.YTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
