Anal. Chem. 1982, 5 4 , 221-223
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Anodic Stripping Voltammetry at Graphite-Epoxy Microelectrodes for in Vitro and in Vivo Measurements of Trace Metals Joseph Wang Department of Chemistry, New Mexlco Stare Unlversity, Las Cruces, New Mexlco 88003
Anodic strlpping voltammetry at microelectrodes has been evaluated in vitro toward its exploltation for in VIVO measurements of trace metals. Well-defined stripping voltammograms are obtained at the nanomolar concentratlon level using shod (1-5 mln) deposition periods. The stripping performance at a bare carbon surface is compared to that of a prepiated mercury film. The effect of dissolved oxygen upon the stripping voltammograms Is evaluated. Results obtained with and wlthout forced convection are compared. High precision of results is malntained on the same mercury film.
In recent years the analysis of trace metals in human body fluids has become a subject of high priority (I). Excess or deficiency of metals is associated with various diseases and thus their monitoring is of great importance. With the growing interest in in vivo monitoring of electroadive species using microvoltammetric electrodes (2), it is important to examine the feasibility of applying voltammetric techniques in vivo for trace metal analysis. Anodic stripping voltammetry (ASV) is the most sensitive voltammetric technique. It has been widely used for the determination of trace metals in biological matrices (3);in addition to its inherent sensitivity and selectivity it may provide information regarding the distribution (i.e., speciation) of the metal in question, which may account for various pathological situations. The use of microelectrodes for performing sensitive ASV analyses is important also for the adaptation of the technique for microliter analysis in cases where very small volumes are available; it is also advantageous in large-scale ASV experiments where iR and/or depletion problems have to be minimized. Before ASV is applied in the unique environment of the body fluids, and for the other applications discussed above, it is necessary to evaluate its response characteristics at microelectrodes. The feasibility of performing ASV measurementa at microelectrodes, made of graphite-epoxy, has been demonstrated recently (4). As applied to in vivo ASV measurements we have to consider experimental conditions which differ significantly from those commonly employed in conventional ASV. Among these are the inability of forcing the convection transport, of removing dissolved oxygen, or of using the hanging mercury drop or the Florence (in situ plated) mercury film electrodes. In addition, the significantly decreased surface area of microelectrodes may result in a different ASV response as compared to that observed at conventionally sized electrodes. The properties and behavior of ASV microelectrodes utilizing such conditions are explored in vitro for better understanding and improving the ASV response of microelectrodes to be applied in vivo.
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EXPERIMENTAL SECTION Apparatus. The preparation of the graphite-epoxy microelectrode has been described previously (4). In the present work the mixed resin-accelerator paste was packed in a glass capillary, resulting in a disk-shaped surface of 140 Mm radius. The use of 0003-2700/82/0354-0221$01.25/0
a glass sleeve instead of the Teflon tube (used in ref 4)simplified the polishing procedure required in ASV experiments. A Bioanalytical System Model VC-2 electrochemicalcell was employed. The working electrode, reference electrode (Ag/AgCl, Model RE-1, Bioanalytical Systems), platinum wire auxiliary electrode, and the nitrogen delivery tube joined the cell through holes in its Teflon cover. The cell was placed on a magnetic stirrer (Sargent-Welch, No. 76490) and a 2.2 cm long magnetic bar was placed in the center of the cell bottom. Current-voltage data were recorded with a Princeton Applied Research Model 364 polarographic analyzer and a Houston Omniscribestrip-chart recorder. Reagents. Chemicals and reagents have been described previously (4). Procedure. The mercury film was deposited at the beginning of each day. Before the film was plated, the graphite-epoxy surface was polished with alumina slurry. A 9.5-mL portion of M mercury the 0.1 M KNOBsolution and 0.5 mL of the 1 X solution were introduced to the cell (final mercury concentration -5 X M). The mixture was deaerated for 6 min, while the working electrode was kept at +0.3 V. Then a potential of -1.0 V was applied and the solution was stirred at 450 rpm. After 10 min the potential was switched to 0.0 V and held there for 1 min. Following this, the mercury plating solution was replaced with 10 mL of the supporting electrolyte solution. This exchange, which involves momentary breaking of the circuit, must be done as rapidly as possible in order to minimize possible changes in the film during this break. Following 5 min of deaeration, background and sample measurementswere carried out successively as follows: A deposition potential was imposed on the working electrode and the solution was stirred. The potential was chosen according to the cations to be determined and was maintained for a period depending on their sought-for concentration levels (1-5 min, for M to 1 X lo-* M). At the concentrations ranging from 2.5 X end of the deposition period, the solution stirring was stopped, and after a 15-5 rest period the metals were stripped from the mercury film by applying an anodic potential scan, either in the linear mode or in the differential pulse mode. The scan was stopped at +0.05 V, and this potential was maintained for 30 s before the next measurement was performed. The mercury film was removed at the end of a day’s work by wiping the surface with a soft tissue wet with 2 M nitric acid, and the electrode was rinsed with deionized water. Some experiments were performed by using different procedures; these involved the use of a quiescent solution during the deposition, of bare carbon surfaces, or in the presence of dissolved oxygen, as will be described later in the paper. RESULTS AND DISCUSSION Figure 1illustrates typical anodic stripping voltammograms for some common cations, present at the 1-5 ppb &g/L) level, obtained at the preplated mercury film microelectrode. Both stripping modes employed, linear scan (A) and differential pulse (B), perform well (i.e., defined and separated peaks), and only a slightly improved sensitivity is observed using the differential pulse mode. On the other hand, a slightly improved resolution is obtained in the linear scan mode. The background currents are low and the hydrogen overvoltage is high. The copper peak is resolved from the mercury oxidation current but shows another peak added on as a shoulder. Such behavior was postulated to be due to the different size 0 1982 American Chemical Society
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E ,V Flgure 1. Characteristic stripping voltammograms for 1.2 X lo-' M cadmium, 2.5 X lo-' M lead, and 7.5 X lo-' M copper: 5-min depositionsat -1.15 V; stirring rate, 450 rpm; supporting electrolyte, 0.1 M KNO,: (A) linear scan ASV, 50 mV/s; (B) differential pulse ASV, amplitude 50 mV, scan rate 5 mV/s.
of mercury droplets plated on the carbon substrate (5). In addition, the coverage of the carbon surface may not be complete, resulting with an electroactive surface which is a composite of mercury and carbon sites. Similar sensitivity was obtained in 0.1 M HCl as the supporting electrolyte, with steeper background current around -1.0 V due to lower hydrogen overvoltage. The linear scan limits of detection (based on the signal-to-noise characteristics) are around 2 X lo* M lead and cadmium and 5 X copper, while those of the differential pulse mode are 1 X lo+' M lead, 2 X lo* M cadmium and 4 X lo* M copper. Still lower detectability is obtainable by using deposition periods longer than the 5 min employed in Figure 1. At the lo-' M concentration level (at which some important metals are present in body fluids) a 1-min deposition suffices. The effect of replating the stripped metals during the waiting time between pulses, an advantage of .the differential pulse mode at conventially sized electrodes (6),decreases at microelectrodes; as the surface area decreases significantly a larger fraction of the stripped metal "escapesn by diffusion from the vicinity of the surface before the successive pulse is applied (the present surface area is not regarded as minimal; voltammetric electrodes of 10 pm radius have recently been fabricated (2)). Forced convection (e.g., stirring the solution or rotating the electrode) is commonly used in ASV measurements for transporting the metal ions to the electrode surface during the deposition step. However, in the case of in vivo analysis forcing the convection transport is a difficult task and thus it would be important to examine the ASV response of the microelectrode in a quiescent solution. Figure 2 compares differential pulse stripping voltammograms for lead and copper, employing different stirring rates. Voltammogram A indicates the feasibility of detecting cations at the parts per billion level in a quiescent solution and using short (2 min) deposition periods. The lead peak current obtained under these condtions is 73% and 79% smaller than the currents obtained by forcing stirring rates of 270 (B)and 450 (C) rpm, respectively. Mass transport to a stationary disk in a quiescent solution is by diffusion (perpendicular and edge); as the disk area decreases enhanced mass transport is expected due to thinner diffusion layer (7) and increased edge effects. Compared to the steady-state deposition current for the forcedconvection conditions, commonly employed in ASV, the current in a quiescent solution (due to a potential step to the diffusion-controlled region) may exhibit chronoamperometric behavior (Cottrell equation modified to account for the increased effects of nonlinear (steady-state) diffusion at mi-
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E ,V Flgure 2. Effect of stirring rate upon anodic stripping vottammograms: 3.0 X lo-' M lead and 9.5 X lo-' M copper in 0.1 M KNO,; 2-min depositions at -1.0 V; differential pulse amplitude and scan rate as in Flgure 1 6 stirring rates, 0 (A), 270 (B), and 450 (C) rpm.
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E,v Figure 3. Stripping peaks at the bare carbon surface (A-C) and at the mercwy-coated electrode (D). 2-min deposition at -1.15 V; stirring rate, 450 rpm; (A) linear scan (50 mV/s) for 5 X lo-' M lead in 0.1 M KNO,, (B) as for (A) using the differential pulse mode (50 mV amplitude, 5 mV/s scan rate), (C) as for (B) plus 8 X lo-' M cadmlum, (D) as for (C) but at the mercury-coated electrode.
croelectrodes). Such a Cottrell current transient would yield nonlinear dependence of the peak current on the deposition time. However, study of this dependence in quiescent solution gave a highly linear plot (not shown) for the deposition of lead for periods ranging from 1to 7 min. This may be due to the fact that for an electrode of the size described herein, and for the long time scale of the stripping experiment, the linear Cottrell term contribution to the total deposition current is around 10%. In addition, at times longer than 0.5-2 min natural convection (due to density gradients or vibrations) is contributing to the transport of electroactive species (8). In biological systems, on the other hand, variable rates of natural convection may occur and affect the precision of results. In order to simplify the analytical procedure and to avoid the use of mercury electrodes in the body fluids it would be desirable to examine the ASV response of bare carbon microelectrodes. Figure 3A,B shows such response (linear scan and differential pulse modes, respectively) for 5 X lo-' M lead, employing 2-min depositions. Well-defined sharp peaks are obtained in both stripping modes, with negative peak potential
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E,V Flgure 4. The effect of dissolved oxygen on anodic stripping voitamM lead, and 2.3 X mograms: 1.8 X lo-’ M Cadmium, 2.8 X M copper in 0.1 M KNO,: 2-min depositions at -1.15 V; stirring rate, 450 rpm; (A, C) differential pulse mode as in Figure 16,(6, D) linear scan mode as In Flgure 1 A (A, 6)oxygen removed prior to depositlon, (C, D) dlssolved oxygen present.
shifts of about 80 mV compared to those observed a t the mercury-coated microelectrode (Figure 1). The background current of both stripping modes a t the bare carbon surface is very low (with high hydrogen overvoltage) and compares favorably with that of the mercury-coated microelectrode. Addition of 8 x lo-’ M cadmium to the lead solution results in a combined lead-cadmium peak (at -0.50 V), 90% greater than the lead peak (Figure 3C). When the solution of Figure 3C is determined at the mercury-coated electrode the combined peak is resolved into two defined cadmium and lead peaks, as expeted for mercury films (Figure 3D). In addition a copper peak (with its characteristic shoulder), not observed at the bare carbon surface, appears indicating poor sensitivity of the bare surface toward copper. Repetitive measurements at the bare carbon electrode gave reproducible peak currents when the scan was stopped at +0.35 V, and this potential was maintained for 2 min. Such a procedure was found to be necessary to clean the surface from the metals not stripped during the potential scan. Results similar to those of Figure 3A-C were obtained at bare carbon paste microelectrodes (150 pm radius) that were used, unsuccessfdly,to improve the lead-copper resolution. In some biological matrices (e.g., human serum) lead is present at concentrations much higher (10-20-fold) than cadmium, and thus the error in measuring lead (due to its overlapped peak with cadmium) may be minimal. The measurement of trace metals by ASV is usually done in the absence of oxygen. Dissolved oxygen affects ASV through high background current (due to its reduction), chemical oxidation of metals in the electrode amalgam, or precipitation of metal hydroxides by hydroxyl ions (formed during the reduction of oxygen). The removal of dissolved oxygen (aside from being time-consuming) is impossible in cases such as microelectrodes implanted in vivo. (The problem of oxygen in vivo may not be as severe as in vitro results imply, because oxygen levels are often low in vivo.) Batley has examined recently (9) the feasibility of performing ASV measurements in the presence of oxygen using conventionally sized electrodes. It is desirable, therefore, .to examine this feasibility in the case of microelectrodes. Figure 4 illustrates the effect of dissolved oxygen for a sample containing cadmium, lead, and copper ions. In both stripping modes, differential pulse (C) and linear scan (D), analysis a t the 10 ppb level is not feasible (using 2-min depositions) due to a highly sloping background current. The lead and copper stripping peaks are totally obscured by this oxygen reduction current, while that
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Flgure 5. Precision obtained for successive stripping measurements M lead in 0.1 M KNO,: 1-mln depositions at -1.0 V; of 2.9 X
stirring rate, 450 rpm; linear scan mode, 50 mVls. The peak at -0.55 V is due to cadmium present in the blank solution. The number on top of each peak represents the run number.
for cadmium is only partly obscured and may be measured in the linear scan mode. The cadmium peaks are about 15% lower than those observed in the absence of oxygen (A and B). This diminution is related to effects of metal hydroxide precipitation and chemical oxidation discussed earlier (the first effect my be smaller as the electrode area decreases due to enhanced removal, by diffusion, of hydroxyl ions from the vicinity of the electrode). Lead and copper at the 10 ppb level are detected, in the presence of oxygen, using longer deposition periods that increase the stripping currents without changing the time-independent oxygen background. Improved sensitivity and detectability in the presence of oxygen may be obtained by adapting stripping modes such as substractive (IO) or potentiometric (11)ASV, that are much less affected by dissolved oxygen. Another parameter that is essential for in vivo measurements is the reproducibility and stability of the results. A series of 12 successive determinations of 2.9 X lo-’ M lead was carried out on the same mercury film; six of these measurements are represented in Figure 5. The mean peak current found was 16.3 nA with a range of 15.4-17.6 nA. The relative standard deviation over the complete series was 4.4%. In conclusion,the graphite-epoxy microelectrode appears to be an effective substrate for mercury film used for sensitive ASV measurements. However, major experimental difficulties remain before applying ASV for analytical and mechanistic characterization of trace metals in vivo. In order to make direct ASV measurements in biological systems more reliable, we are presently examining the effects (e.g., complexation, adsorption) of various proteins on ASV peak currents and the corresponding background current. speciation studies for defining the metallic forms measured by direct ASV (as compared to the “total” content measured following a wet digestion procedure) are in progress.
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8)
Dulka, J. J.; Rlsby, T. H. Anal. Chem. 1978, 48, 640 A-653 A. Wightman, R. M. Anal. Chem. 1981, 53, 1125 A-1134 A. Lund, W.; Eriksen, R. Anal. Chim. Acta 1979, 107, 37-46. Wang, J. Anal. Chem. 1981, 53, 2280-2283. Laser, D.; Arlel, M. J. Elecffoanal. Chem. 1974, 4 9 , 123-132. Osteryoung, R. A.; Christie, J. H. Anal. Chem. 1974, 48, 351-355. Saito, Y. Rev. Poiarogr. 1968, 15, 178-187. Bard, A. J.; Faulker, L. R. “Electrochemical Methods, Fundamental and Appllcatlons”; Wlley: New York, 1980; p 144. (9) Batley, G. E. Anal. Chim. Acta 1981, 724, 121-129. (10) Wang, J.; Arlel, M. Anal. Chlm. Acta 1981, 128, 147-152. (11) Jagner, D.; Josefson, M.;Westerlaun, S.; Aren. K. Anal. Chem. 1981, 53. 1406-1410.
RECEIVED for review September 21,1981. Accepted November 2, 1981. J.W. is the recipient of the Starter Grant (Award) from the Society of Analytical Chemists of Pittsburgh.
