407
Anal. Chem. 1986, 58,407-412
other ions does not-it is possible to enhance the selectivity of the measurement toward lead. Figure 9 illustrates the measurement of 2 X lo-' M lead in the presence of 4 X lo-' M indium at the bare (a) and coated (b) mercury film electrodes. The difference in the peak potentials for these two species is approximately 150 mV, and their peaks are partially merged. Due to the differences in permeability of the two ions at the 15-min hydrolysis coating, the degree of overlap is substantially reduced, resulting in an indium-blead peak ratio of 0.20 compared to 2.0 at the bare electrode. Similar improvement may be observed for other binary systems, e.g., measurements of bismuth in the presence of copper or of lead in the presence of cadmium (using 30- and 10-min hydrolysis times, respectively; see Figure 4).
CONCLUSIONS The above results clearly demonstrate the potential utility of cellulose acetate coated mercury f i i electrodes for stripping voltammetry. The controlled permeability achieved by hydrolyzing this polymeric coating in alkaline media is exploited for minimizing interference such as adsorption of organic surfactants and overlapping stripping peaks. Since such problems are minimized via an in situ separation step-carried out a t the electrode surface-time-consuming sample pretreatment procedures may not be required. Because the response is not affected by external mass transport, stripping measurements can be performed with systems with poorly controlled mass transport (or in the absence of convective transport without any sensitivity loss). Even though the concept is presented here in terms of trace-metal measurements, it could be extended to determination of anions and organic analytes by cathodic stripping and adsorption voltammetric procedures. In future work we will examine the
utility of these electrodes to speciation studies, based on the possible discrimination between sorption interferences and complexation effects. Such discrimination would be extremely useful for the determination of complexing capacity of natural waters. Registry No. Hg, 7439-97-6;Pb, 7439-92-1;Cd, 7440-43-9;Cu, 7440-50-8; cellulose acetate, 9004-35-7.
LITERATURE CITED (1) Wang, J "Stripping Analysis: Principles, Instrumentation, and Applications"; VCH Publishers: Deerfieid Beach/Weinheim, 1985. (2) Roe, D. K.; Toni, J. E. A. Anal. Chem. 1965, 3 7 , 1503. (3) Florence, T. M. J . Electroanal. Chem. 1970, 2 7 , 273. (4) Korfhage, K. M.; Ravichandran, K.; Baidwln, R. P. Anal. Chem. 1984, 5 6 , 1514. (5) Wang. J.; Freiha, B. Anal. Chem. 1984, 56, 2266. (6) Cheek, G. T.; Nelson, R. F. Anal. Lett. 1978, 7 7 , 393. (7) Cox, J.; Kuiesza, P. J. Anal. Chlm. Acta 1983, 754, 71. (8) Wang, J.; Greene, 8.; Morgan, C. Anal. Chim. Acta 1984, 758, 15. (9) Szentirmay, M. N.: Martin, C. R. Anal. Chem. 1984, 56, 1898. (10) Guadaiupe, A. R.; Abruna, H. D. Anal. Chem. 1985, 5 7 , 142. (11) Sittampalam, 0.; Wilson, G. S. Anal. Chem. 1983, 55. 1608. (12) Wang, J.; Hutchins, L. D. Anal. Chem. 1985, 5 7 , 1536. (13) Chien, Y. W.; Oisen, C. L.; Sokoloski, T. 0. J . Pharm. Sci. 1973, 6 2 , 435. (14) Gough, D. A.; Leypoidt, J. K. Anal. Chem. 1979, 57, 439. (15) Stewart, E. E.; Smart, R. B. Anal. Chem. 1984, 56, 1131. (16) Freese, J. W.; Smart, R. B. Anal. Chem 1982, 5 4 , 836. (17) Holtzclaw, H. F.; Robinson, W. R.; Nebergail, W. H. "General Chemistry"; D.C. Heath and Co.: Lexington, MA, 1984. (18) Buffie, J.; Cominoli, A.; Greter, F. L., Haerdi, W. Roc. Anal. Div. Chem. SOC. 1978, 75, 59. (19) Hayashita, T.; Takagi, M. Talanta 1985, 3 2 , 399.
RECEIVED for review August 5, 1985. Accepted October 1, 1985. This work was supported in part by the National Institutes of Health, Grant GM30913-02. The authors acknowledge H. P. Adams (EML, NMSU) for taking the scanning electron microscopy photographs.
Potentiometric Stripping Determination of Heavy Metals with Carbon Fiber and Gold Microelectrodes Andrzej S. Baranski* and Henry Quon Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OW0
The determination of Cd, Pb, and Cu wlth mercury film microelectrodes (4 X IO-' to 2 X lo-' cm2 surface area) was examined. I t was demonstrated that carbon fiber mlcroelectrodes are suitable for multicomponent trace analysis of very small (5-pL) samples. Varlous processes occurring at microelectrodes such as charging of the double layer, poisoning of the electrodes, and nucleation processes are also discussed in terms of their effect on the detection limit and precision of the method.
In recent years a considerable interest in the various applications of microelectrodesmade of a carbon fiber or a thin metal wire has been observed (123). Due to the large edge effect, these electrodes have a unique mass transport characteristic ( 4 , 5 ) (large and steady-state flux) as well as low resistance polarization (6, 7). Under limiting current conditions the current density a t a stationary carbon fiber microelectrode (8 km in diameter) is comparable to one observed at a large disk electrode rotating with a speed of 500 rps (1). 0003-2700/86/0358-0407$01.50/0
In addition, if the rotating disk electrode is, for example, 2 mm in diameter, the resistance polarization of the carbon fiber electrode will be 250 times smaller than the first electrode. This illustrates the obvious advantages of small electrodes in stripping analysis. The enhancement of mass transport may lead to a decrease in the preconcentration time. The steady-state flux during the preconcentration step makes stirring the solution unnecessary. This eliminates one parameter that must be controlled and consequently one source of error. The low resistance polarization makes it possible to carry out determinationswithout the supporting electrolyte, which usually contains interfering impurities. The most serious disadvantage of microelectrodes under voltammetric conditions is a very small current that must be measured. Commercially available instruments do not operate in the proper current range. In addition a noise accompanying the signal of interest is troublesome ( I ) . In this context the potentiometric stripping technique developed by Jagner (8) seems to be an attractive alternative. This technique can accommodate electrodes of any size. The response of the electrode does not need to be amplified, thereby a lower noise 0 1986 American Chemical Society
408
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2,FEBRUARY 1986
level can be anticipated. In most applications simple instrumentation consisting of a voltage follower with high input impedance and a strip-chart recorder is sufficient. Under potentiometric stripping conditions, fist, metal amalgams are plated onto an inert electrode from a solution containing metal ions of interest and a known concentration of Hg(I1). Then the circuit is opened, and the electrode potential is recorded as a function of time. At this stage metals with lower formal potential than the formal potential of the Hg(II)/Hg couple are replaced by mercury according to the reaction
2M(Hg) + nHg2+
+
2Mn+ nHg
-+
do Mn+DMn+
= 2c0Hg2+DHgz+ td
(1)
where and DHgz+ are the diffusion coefficients, coM"t and C0ez+are the bulk concentrations of the metal ions of interest and mercuric ions, respectively, and t d is the deposition time. Some theoretical aspects of the potentiometric stripping technique, including potentiometric stripping from microelectrodes have recently been discussed by Hussam and Coetzee (10).
EXPERIMENTAL SECTION Reagents. Analytical grade (BDH chemicals) Hg(CH3C00)2, Pb(CH&00)2 Cd(CH3C00)2, C U ( N O ~ )CH3COONa, ~, and CH3COOH were used. Although potentiometric stripping determination of metals with microelectrodes can be carried out without the supporting electrolyte, in this work a 0.01 M CH3COONa/0.01M CH3COOHbuffer was used in order to ensure well-defined mass transport conditions for all species involved in the electrode process. A 1 M stock solution of this buffer was electrolyzed for 24 h at a large mercury cathode at -1 V vs. SCE in order to remove heavy metal impurities. All solutions were prepared by using Millipore water. cm in diameter, Electrodes. Carbon fibers, (7.0 0.3) X (Union Carbide, Thornell 300, grade WYP 30 1/0) were washed with 1 M HCl and distilled water, dried, and heated for 30 min in a quartz tube under argon at 600 "C in order to remove organic impurities. A single fiber (2-3 cm long) was connected into a thin copper wire with silver epoxy and inserted into a polyethylene tube (0.52 mm i.d., 2.9 mm 0.d.). The polyethylene tube was wrapped tightly with Teflon tape and aluminum foil. Then this assembly was heated up to about 200 "C under vacuum in order to melt the polyethylene and seal the fiber completely in the plastic. Before use the polyethylene tube was cut perpendicular to its length to expose the carbon fiber surrounded by polyethylene. If such an electrode became contaminated during its use, the end of the electrode was cut off to expose a fresh surface of the carbon fiber. Gold microelectrodeswere made of a 50 wm diameter gold wire (Aesar) sealed into soft glass. The electrode surface was polished with an extra-fine carborundum sandpaper and 0.3-fim alumina. A saturated calomel electrode was used as the reference electrode unless otherwise specified. ElectrochemicalMeasurements. Potentiometric stripping experiments were carried out with a home-built apparatus consisting of a 1012-Qinput impedance voltage follower and a simple circuit allowing polarization of the working electrode with preselected potentials. The voltage follower input was connected to the electrodes with a 7 cm long coaxial cable (16.5 pF/ft capacitance). The reference electrodewas grounded. Potential-time curves were recorded with a Cole-Parmer Model 8036 X - Y / t recorder. After each stripping cycle a +1 V potential was applied to the microelectrode in order to remove the deposited mercury. Alternatingcurrent impedance and voltammetricmeasurements were done with equipment described elsewhere (11). A standard
*
b
c
d
e
> \
;r' -0.5 I
t
+ W 0 a 0
When the concentration of M at the electrode surface drops to zero, a sharp change of the electrode potential occurs. It is possible to relate the time at which this change takes place (the transition time, 7)to the concentration of metal ions in solution by knowing the mass transport characteristics of the species involved in the process. In most cases the following equation is obeyed (9): TM
-1.0 . a
Figure 1. Stripping potentiograms obtained with carbon fiber (curves a, b, c, and d) and gold (curve e) microelectrodes for 0.01 M (1:l) acetic buffer SOluMOn contalning (a) M Hg2+,(b) 5 X lo-' M Pb2+ and M Hg2+,(c) 5 X lo-' M Pb2+, 5 X lo-' M Cd2+,and M Hg2+, (d) and (e) 5 X lo-' M Pb2+, Cd2+, and Cu2+ each and M Hg2+. Preconcentration conditions are 1 min at -1.1 V.
electrochemicalcell (20 mL volume) was used in all experiments unless otherwise specified. The cell was located in a faraday cage. All experimentswere performed at room temperature, 25 f 2 "C. High-purity argon was bubbled through the solutions for 10 min before each experiment in order to remove oxygen.
RESULTS AND DISCUSSION
General Studies. Studies of the behavior of carbon fiber and gold microelectrodes under potentiometric strippingconditions were conducted in solutions containing Cd2+,Pb2+, and Cu2+,5 X lo* M each, as well as M Hg2+in a 0.01 M (1:l) acetic buffer. Stripping potentiograms recorded after 1-min preconcentration time with carbon fiber (curves a, b, c, and d) and gold (curve e) microelectrodes are shown in Figure 1. The P b oxidation wave is approximately the same with both electrodes, however, in the case of the gold microelectrode the Cd wave is missing, and the Cu wave is distorted. This is not surprising because gold is known to form difficult-to-oxidize intermetallic compounds with most metals including cadmium and copper (12). The gold electrode under these conditions can be used only for the determination of Pb, T1, and Bi since amalgams of these metals do not react with Au (12). Therefore our further studies were focused on the behavior of carbon fiber electrodes, which have more general applications. The stripping potentiograms for Cd, Pb, and Cu (each in separate solutions) were recorded seven times with the same carbon fiber microelectrode and with five different statistically chosen carbon fiber microelectrodes. The standard deviations of the transtion times were 10% (21701, 5.3% (12.5%), and 7.5% (35%) for Cd, Pb, and Cu, respectively (values in parentheses represent the standard deviation for different electrodes). In addition the average ratios of 7MCo&z+/tdCoM"+ for these three elements were compared with values of DMnt/DHga+. The diffusiod coefficients of metal ions were estimated on the basis of chronoamperometric experiments performed with the hanging mercury drop electrode. According to eq 1TMCoHg2+/tdCoMM"t = DM"+ = DMnt/DHg2+ for divalent metals. However, it was found that the average 7MCo&2+/t,$"M"+ was equal to 0.52 f 0.13,0.83 f 0.1, and 1.7 f 0.6 for Cd, Pb, and c u , respectively. The DMn+/DHg2+ was equal to 0.99 0.06, 1.1f 0.06,and 0.9 h 0.05, respectively. The discrepancies as well as the large standard deviation of the transition times can be attributed to the mechanism of the electrochemical processes occurring at the electrode surface. Equation 1was derived assuming that the geometry of mass transport is the same for all ions involved in the process in
*
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986
I
409
I
0
2
4
6
Figure 2. Typical stripping potentiograms of lead obtained with a carbon fiber microelectrode for 0.01 M (1:l) acetic buffer solution containing various concentrations of Hg": (a) M, (b) 3 X M; CDpb4CoHp2+ = 0.1. M, (c) M, (d) 3 X lo-' M, (e) Preconcentration conditions are 30 s at -1.0 V. both preconcentration and stripping cycles. However, deposition of mercury on carbon electrodes results in a coating of mercury droplets rather than a continuous liquid film (13, 14). Since the surface area of a carbon fiber is extremely small, it may easily happen that only a few mercury droplets are present on the electrode surface (15);as a consequence the surface of the electrode is heterogeneous. The reduction of metal ions on such electrodes may occur on both carbon surface and mercury droplets or exclusively on mercury droplets depending on whether or not the potential of the electrode exceeds the nucleation overpotential of a given metal. Therefore the effective surface area of the electrode depends on the potential, the kind of metal, and the distribution of mercury droplets. Since the distribution of mercury droplets on a carbon fiber microelectrode is not reproducible, the standard deviations of the transition times for the oxidation of metals are large, particularly when different electrodes are compared. The validity of eq 1was examined in a more detailed way in the case of lead, which yields the most reproducible results. Effect of Hg2+Concentration. According to eq 1 the transition time is independent of the concentration of mercury ions as long as CoM"t/CoHgZt is constant. This relation was verified in an experiment in which coHgZt was varied from to M, and Copb2+/CoHg2+ = 0.1 as well as t d = 30 s were kept constant. Some E-t curves recorded during this experiment are shown in Figure 2. In fact the transition time was found constant for the constant ratio C o p b 2 t / CoHg2t(7pbCoHgi+/tdCopb2+ = 0.94 f 0.1 for 12 measurements); however the slope of E-t curves after the oxidation of lead decreases with a decrease in the Hg2+concentration. In addition, for a given concentration of mercuric ions the slopes of E-t curves vary in a wide range for different electrodes made in the same manner with the same kind of carbon fiber and for the same electrodes with time. The curves in Figure 3 are typical for an average electrode. Usually freshly prepared electrodes exhibit sharper transients of potential; however the slope of the E-t curves decreases with time, and after a few (or in some cases several) hours the electrode is no longer suitable for potentiometric stripping experiments. E-t curves recorded for fresh (A) and old (B)carbon fiber electrodes in a solution containing only Hg2+ions are shown in Figure 3. The slope of the E-t curves in the absence of metals other than mercury depends on the double-layercapacitance of the electrode and on the rate of mercury ion reduction
5
0
TIMEIS
10
15
TIME/s
Flgure 3. € 4 curves of fresh (A) and old (B)carbon fiber microeM Hg2+ and 0.01 M (1:l) acetic lectrodes in a solution containing buffer. Preconcentration conditions are 1 min at -1.1 V.
O t
I . 10
100
1000
10000
FREQUENCY/ Hz
Figure 4. The differential double layer capacitance of fresh (A) and old (6)carbon fiber microelectrodes in a 0.01 M (1:l) acetic buffer at -0.3 V VS. SCE. where iHg2t is the current of Hg2+reduction, r is the radius of the electrode, and c d l is the specific double-layer capacitance. In the case of a diffusion-controlled process and in the absence of phenomena associated with the nucleation of mercury, iHg2+ at a small disk electrode can be described by the well-known equation (5) ~ H ~= z +4
n F r D ~ ~ ~ t C ~ ~ ~ z t (3)
Consequently
dEc/di! = 8 F D H g 2 t c o H g 2 t /?rrCdl
(4)
The double-layer capacitance of the two electrodes used in the previous experiment was determined from the ac impedance measurements performed at -0.3 V vs. SCE in a 0.01 M (1:l) acetic buffer solution. (the methodology of such measurements is discussed elsewhere (11)). The double-layer capacitance of both electrodes was frequency dependent (Figure 4). This phenomenon can be attributed to the porosity of carbon fiber microelectrodes (10). The slope of the E-t curve for the electrode A is comparable with the value predicted by eq 4 if a low frequency value of c d l is taken into account. However, in the case of electrode B, which has a lower c d ] and a much lower slope, the discrepancy is very large. It seems to be likely that the aging of carbon fiber microelectrodes is caused by the adsorption of organic impurities, which decreases the double-layer capacitance of the electrodes and on the other hand inhibits the charge transfer processes. This hypathesis was confirmed by a voltammetricexperiment performed in a solution containing
410
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986
A
I
1 L
0.5
I 0
-0 5
-1
POTENTIAL / V
current-potential curve for the reduction of 5 X lo-' M Hg2+from a 0.01 M (1:d)acetic buffer on fresh (A) and old (B) carbon fiber microelectrodes. Preconcentratlon conditions are 1 min at -1 .O Figure 5. A
V.
5X M Hg2+in (1:l)5 X M acetic buffer. In this experiment a potential of -1 V was applied to the electrode for 1 min (to initiate the nucleation of mercury), then the potentials were scanned in a positive direction. The current-potential curves recorded for both electrodes, A and B, are shown in Figure 5. At electrode B the current decreases with a decrease in the overpotential indicating an irreversibility of the charge transfer process. Effect of Deposition Time. According to eq 1the transition time should be proportional to the deposition time if all other parameters are kept constant. This relation was confirmed by an experiment performed with a solution containing 3 X lo4 M Pb2+and M Hg2+in a 0.01 M (1:l) acetic buffer. The deposition time was varied from 3 to 1000 s. The average value of 7pbC0H$+/tdCopb2+ was equal to 0.955 f 0.054 for 13 measurements taken during this experiment. Effect of Pb2+Concentration. The relation between the transition time and the concentration of Pb2+was studied in a solution containing M Hg2+and a 0.01 M (1:l)acetic buffer. The concentration of lead ions was varied from 2.5 X M. The deposition time was 10 min. The to 1 X correlation coefficient for log 7 vs. log CoPbz+dependence was 0.999, and the slope was 0.867 f 0.012. Microanalysis. Probably the most important advantage of microelectrodes in stripping analysis is the possibility of performing multicomponent determinations in extremely small volumes of solution. These possibilities were demonstrated by using a specially designed cell shown in Figure 6. A previously described carbon fiber microelectrode was used as the working electrode. The reference electrode was made of an amalgamated gold wire (5 mm long, 50 pm diameter). A 5-pL sample was placed directly on the top of the working electrode; the reference electrode was immersed in the sample; and argon saturated with water vapor was passed through the cell for 5 min. Then after 10 min of preconcentration at -1.4 V vs. Hg2+/Hga stripping potentiogram was recorded. "ypical E-t curves obtained in this experiment are shown in Figure 7. As expected these E-t curves are undistinguishable from ones obtained for large volumes of solutions. At the lowest concentration the sample analyzed in this experiment contained 2.8 X g of cadmium, but only 3.4 X g of cadmium were deposited on the microelectrode after 10 min of preconcentration; therefore depletion of the solution did not occur. The cell shown in Figure 6 is very simple and convenient to use. The deoxygenation is fast due to the small volume of the solution, and in addition, the probability of contamination of the electrode with organics is lower since the elec-
Y
The electrochemical cell used in microanalysis: (a)working electrode (carbon fiber sealed in polyethylene),(b) analyzed solution, (c) reference electrode (amalgamated goid wire). Figure 6.
L O t TIME
Figure 7. Typical €4curves obtained during potentiometric stripplng analysis of 5-pL samples containin (a) 5 X 1O-' M Cd2+, 1O-' M Pb2+, 2 X lo-' M Cu2+, and M HgW in 0.01 M (1:l) acetic buffer; (b) Cd2+, Pb2+, and Cu2+ 5 X lo-' M each and 5 X M Hg2+ in 0.01 M (1:l) acetic buffer. Preconcentration conditions are 10 min at -1.33 V vs. Hg2+/Hg.
trode is in contact only with a very small volume of solution. Contamination of analyzed solutions by the electrochemical cell is very unlikely because the solution is in contact only with electrodes, which are easy to clean and besides can be used as disposable ones (the cost of material for the more expensive reference electrode is less than $0.10). Although the surface area of the reference electrode is very small cm2),it is about 20000 times larrrger than the surface area of the working electrode. Therefore polarization of the reference electrode does not occur. The position of the working electrode ensures that even large droplets of deposited mercury do not lose contact with the carbon fiber. This should improve the reproducibility of results. Such an improvement has been demonstrated in an experiment in which mercury was deposited on the electrode for 30 min from a 5-pL solution containing Cd2+,Pb2+,and Cu2+,5 X lo4 M each, as well as lo4 M Hg2+. Then the electrode was used (without oxidation of the mercury) in seven consecutive potentiometric stripping determinations of Cd, Pb, and Cu involving a 1-min preconcentration time. The standard deviation of the transition time in this experiment was less than 2.5% for all three metals. Detection Limit. The detection limit of stripping methods can be discussed only in relative terms since it is inversely proportional to the preconcentration time, which can be as long as 16 h (16) if one finds it to be practical. In addition the detection limit in the potentiometric stripping mode depends on the method of data acquisition (17). In this work
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986
the determination of metal ions at concentrations lower than 2.5 X M was not examined because of the limitation caused by the slow response of the X-Y recorder. However, such limitations can be easily overcome by employing a computer system capable of fast data acquisition. In such a case the ultimate limitation of the detectability would arise due to the nonspecific (background) response of the electrode, which is caused mostly by the charging of the double layer. Arbitrarily it can be assumed that a wave associated with the stripping of a metalis noticeable when the slope of a hypothetical EM-t curve due to oxidation of the metal in the absence of capacitance is at some potential (E) lower or equal to the slope of a background curve (E,-t) observed in the absence of the metal
(d t ) E U M
(5)
(%)E
This condition means that when both curves are added the slope of the resulting curve in the plateau region will be at least 2 times lower than in regions preceding and following the plateau, therefore condition 5 is not very rigorous. When a digital data processing system capable of subtracting the background curve is employed condition 5 can be replaced by
where s is the relative standard deviation of (dE,/dt)E. The numerical coefficient was calculated for the 97.5% confidence level assuming that random errors associated with both experimental curves (background and background plus signal of interest) are comparable with respect to their absolute values but otherwise independent. Since s can be estimated only from experimental data obtained with a particular instrument, eq 6 will not be used in this work. According to Hussam and Coetzee (10) the potential-time dependence for stripping of metals from microelectrodes under potentiometric conditions is given by r
nrn
1
(7) where E'is a time-independent parameter and T is the transition time described by eq 1. The lowest slope of the curve occurs a t EM= E'
The expression describingthe detection limit can be obtained from eq 1, 4, 5, and 8
Equation 9 predicts the detection limit independent of mercury ion concentration as long as eq 1and 4 are valid (Le., complete oxidation of metals from the amalgams takes place and the reduction of metal ions is not restricted by nucleation kinetics). In practice C H ~ should Z+ be between and 2 X M. The lower limit is particularly important when mercury is deposited in situ (too low concentration of Hg2+ may result in a very poor coverage of the electrode with mercury). Similarly it can be shown that the presence of other oxidants such as O2 should not affect the detection limit. Equation 9 also predicts a decrease in the detection limit with a decrese in the radius of the electrode. This conclusion is valid only if a leakage of current through the input of a measuring device can be neglected and if a sufficiently large number of atoms is involved in the preconcentration process.
411
In practice electrodes with a radius less than 0.1 pm probably cannot be used. It should also be emphasized that eq 9 is applicable only to determinations performed in unstirred solutions. It is not valid under conventional stripping conditions: macroelectrodes and a forced mass transport. In addition, this equation is not applicable to microelectrodes that are blocked by adsorbed organics. On such electrodes mercury droplets are probably deposited on the top of the organic monolayer, and the exchange of electrons between the mercury and the fiber occurs most likely via the electron tunneling mechanism. The electrical response of such systems is not well-understood, although an increase in the detection limit is obvious. According to eq 9 the detection limit at carbon fiber microelectrodes used in this work should be about M for a 10-min preconcentration period. This relatively high value is caused by an unusually high capacitance of carbon fiber microelecrodes (250 pF cm-2 at low frequencies). The large capacitance is most likely due to the porosity of the fiber or improper sealing of the fiber into the insulator. If one could develop microelectrodes with reasonable capacitance of 10-20 pF cm-2 the detection limit would be lowered significantly. The lowest detection limit of the potentiometric stripping technique with standard size electrodes (M for 4-min preconcentration period) was reported by Graneli et al. (17). The authors used a computer system able to subtract the capacitance background. One could speculate that if a similar system is combined with a properly sealed submicrometer-sizeelectrode, a similar detection limit can be obtained after a 10-20-s preconcentration period. This would create a super-fast technique for metal trace analysis since a computerized version of the potentiometric stripping technique does not require deoxygenation of the sample (17). Precision. The typical precision of potentiometric stripping analysis with standard size electrodes expressed as the relative standard deviation is 6% (16). This value was obtained when a glassy carbon electrode with predeposited mercury film was used for the determination of metal ions at a concentration of 25 fig L-I (-2 X lo-' M). In this work a relative standard deviation equal to 5-10% (Cw+ = 10-'-i06 M) was found for microelectrodes with mercury droplets deposited in situ and removed after each stripping cycle. It was also found that the relative standard deviation decreases to about 2.5% when a microelectrode with predeposited mercury is used. These last results show that the elimination of convective mass transport indeed decreases random errors in stripping analysis. However, mercury deposits on carbon fibers are rather unstable, and great care must be exercised in handling such electrodes. Most recently this difficulty was overcome by Wehmeyer and Wightman (18). The authors developed a mercury microelectrode (a mercury droplet deposited on a platinum microelectrode) and used it for the determination of lead under stripping voltammetry conditions. A relative standard deviation of 0.5-1.5% was reported for Pb2+concentrations of lo-' to 7 x M. Registry No. Cd, 7440-43-9;Pb, 7439-92-1;Cu, 7440-50-8; Hg, 7439-97-6;Au, 7440-57-5.
LITERATURE CITED (1) Wightman, R. M. Anal. Chem. 1981, 53, 1125A-1134A. (2) Dayton, M. A.; Ewing, A. G.; Wightman, R. M. J. Nectroaqal. Chem. 1983, 146, 189-200. (3) Cushman, M. R.; Bennett, 6. G.; Anderson, C. W. Anal. Chlm. Acta 1981, 730. 323-327. (4) Heinze, J. J. Electroanal. Chem. 1981, 724, 73-86. (5) Aoki, K.; Osteryoung, J. J . Nectroanal. Chem. 1984, 760, 335-339. (6) Howell, J. 0.; Wightman, R. M. Anal. Chem. 1984, 56. 524-529. (7) Bond, A. M.; Fleishmann, M.; Robinson, J. J. Nectroanal. Chem. 1984, 180, 257-263. ( 8 ) Jagner, D.; Graneli, A. Anal. Chim. Acta 1976, 83, 19-26. (9) Coetzee, J. F.; Hussam, A.; Petrick. T. R. Anal. Chem. 1983, 55, 120-122. (10) Hussam, A,; Coetzee, J. F. Anal. Chem. 1985, 5 7 , 581-585. (11) Baranskl, A. S., submltted for publication in J. Electrochem. SOC.
Anal. Chem. 1986, 58, 412-415 Galus, 2. CRC Crit. Rev. Anal. Chem. 1974, 4 , 359-422. Stullkova, M. J. Electroanal. Chem. 1973, 48. 33-45. Clem, R. S.; Lmon, S.; Ornelas. L. D. Anal. Chem. 1973, 4 5 , 1306-13 17. Hills, 0.; Pour, A. K.; Scharifker, B. Electrochim. Acta 1983, 28, 89 1-898. Jagner, D. Anal. Chem. 1978, 50, 1924-1929. Graneii, A.; Jagner, D.; Josefson, M. Anal. Chem. 1980, 52, 2220-2223.
(18) Wehmeyer, 1989-1 993.
K. R.; Wightman, R. M. Anal. Chem. 1985,
57,
RECEIVED for review July 8, 1985. Accepted September 12, 1985. This work was supported by the Natural Sciences and Engineering Research ~ o u n c iof l Canada (NSERC) through the operating grant.
Immobilization of Lactate Dehydrogenase on a Pyrolytic Carbon Fiber Microelectrode M. F. Suaud-Chagny* and F. G. Gonon INSERM U 171, Hopital Ste. EugBnie, Pavillon 4H, 1, avenue Georges ClBmenceau, 69230 S t . Genis Laual, France
An lmmoblllzed enzyme carbon fiber microelectrode (12 pm Ld., 500 pm) Is perfected in thls work. The prlnclple of measurement Is based on the voltammetrlc detectlon of NADH. Thls detection Is Improved by electrochemical treatment of the electrode. The oxldatlon current Is proportlonai to NADH from I O J to I O 9 M. The detectlon limit Is 10 pM. The enzyme Is lmmoblilzed by lmpregnatlon in an Inert proteln sheath that has first been electrochemlcally deposited around the actlve tlp of the electrode. The lactate dehydrogenase (LDH) lmmoblllzatlon leads to an electrode sensitlve to pyruvate. For each concentratlon of cofactor the response curve of the electrode exhlbtls a hear range for the substrate. With 3 X IO-’ M NADH the detection limlt for pyruvate is lower than 1 pM. Thls LDH-lmmobliized mlcroelectrode allows us to estimate that pyruvate concentratlon In a rat cerebrosplnal fluld sample of 50 pL Is IO-‘ M.
adapted to carbon microelectrode characteristics. The principle of this method of immobilization is based on the association of the enzyme with an inert porous film immobilized around the active tip of the electrode. For this purpose the carbon is coated with an inert, electrochemically obtained protein sheath (bovine serum albumin, BSA) a few micrometers thick. Then the sheath around the fiber is impregnated with the enzyme (Figure 1). The efficiency of this procedure can be verified by the lactate dehydrogenase immobilization, which leads to an electrode sensitive to pyruvate. The presence of enzyme is tested by the catalysis of its enzymatic reaction. The treated carbon fiber electrode allows us to measure NADH in voltammetry. Consequently, after addition of pyruvate to the solution, the LDH-immobilized microelectrode measures a decrease in the NADH oxidation current (Figure 2) due to the local consumption of NADH (eq la). It is then possible to measure pyruvate concentrations.
+ LDH
This work falls within the scope of measuring nonelectroactive substances by voltammetric techniques. One way of overcoming this problem is to use an immobilized enzyme electrode able to detect the cofactor implied in the metabolism of the product. The development of this type of tool must overcome two difficulties: First, the cofactor must be electrochemically detectable, and second, the enzyme must be immobilized on the electrode. The nicotinamide adenine dinucleotide (NAD/NADH) is a very important cofactor in many enzymatic reactions. In its presence, energy transfer by reversible oxido-reduction reaction of the nicotinamide portion is possible. Moreover, it has been shown that pyridine dinucleotides can be detected by voltammetric methods (1-8). Carbon seems to be a well-adapted material for the study of oxidation in the anodic field. The carbon fiber microelectrode developed by Gonon e t al. (9) presents great interest considering its size for an intratissular implantation and for measurements in microvolume. Gonon et al. (10)have pointed out the importance of the electrochemical treatment of these electrodes on their performances. Thus, this work shows an account of the electrochemical treatment of the carbon microelectrode that greatly improves NADH detection. Many enzyme immobilization methods have been developed (for review see ref 11-13), but few of them are adapted for a covalent linkage to carboneous support (1,14-25) or for a transposition to our electrodes with regard to their size. This work presents a new procedure of enzyme immobilization 0003-2700/86/0358-04 12$01.5010
pyruvate
+ NADH + H+
lactate
+ NAD+
(1)
EXPERIMENTAL SECTION Apparatus. The electrochemical system was composed of a conventional three-electrode assembly. The working electrode was a pyrolytic carbon fiber microelectrode with an active length of 500 fim (12 wm id.) prepared according to Gonon et al. (9). The reference electrode was an AgC1-coated silver wire, and the auxiliary electrode was a platinum wire. These three electrodes were connected to a two-function voltammetric system (BIOPULS, Solea-Tacussel,Villeurbanne, France). The function “measure” was used as the differential normal pulse voltammetry (DNPV). The function “treatment” was used to treat the electrodesby applying continuouspotentials or triangular wave potentials with varying frequencies. Reagents. Pyruvate (Sigma, St. Louis, MO) and NADH (Boehringer, Mannheim, FRG) were dissolved in a phosphatebuffered saline (PBS) solution (KC10.2 g/L, Na2HP041.44 g/L, KHzPOl 0.2 g/L, NaCl8 g/L, pH 7.4). The coating of bovine serum albumin (BSA, Sigma) was prepared from various concentrations of BSA solutions in a PBS solution. The concentrations are expressed in percent (wt/vol). The solution of LDH from hog muscle (10 mg/mL) in 50% glycerol (Boehringer) had a specific activity of 5.5 IU/pL at 25 “C with pyruvate as the substrate. The glutaraldehyde solution (1.25%) was prepared from an aqueous solution at 25% (Sigma) diluted in a PBS solution. Immobilization of the Enzyme (Standard Procedure). Standard Treatment. An alternating potential of a triangular wave form from 0 to +2.6 V (70 Hz) was applied for 20 s to the 0 1986 American Chemical Society
