mon to in vitro small molecule metabolic reactions, the techniques described may prove equally effective in the study of other substrate-enzyme systems.
LITERATURE CITED (1) D. J. Jenden and A. K. Cho. An.. Rev. Pharmacol., 13, 371 (1973). (2) Presented in part before the 166th National Meeting of the American Chemical Society, Chicago, lii., August 1973 and the 22nd Annual Conference on Mass Spectrometry and Allied Topics, Philadelphia, Pa., May 1974. (3) A. T. Shulgin, T. Sargent, and C. Naranjo, Nature, 221, 537 (1969) and references therein. (4) A. T. Shulgin, J. Pharm. Pharmacol.. 25, 271 (1973). (5) F. Benington, R. D. Morin, J. Beaton, J. R. Smythies, and R. J. Bradley, Nature New Biol., 242, 185 (1973). (6) F. A. B. Aldous, B. C. Barrass, K. Brewster, D. A. Buxton, D. M. Green, R. M. Pinder, P. Rich, M. Skiels and K. J. Tutt. J. Med. Chem., 17, 1100 (1974). (7) D. C. Dyer, D. E. Nichols, D. B. Rusterholz, and C. F. Barfknecht, Life Sci., 13, 885 (1973). (8)J. Harley-Mason, A. H. Laird, and J. R. Symythies, Confina Neuroloqica, . . 18, 152-(1958). (9) W. A. Garland, R. J. Weinkam, and W. F. Trager, Chem. hstrum., 5, 271 11973) - -, (10) i :T. Coutts and J. Malicky, Can. J. Chem., 52, 395 (1974). (11) S.B. Matin, P. S.Callery, J. S.Zweig, A. O'Brien, R. Rapoport, and N. Castagnoli, Jr.. J. Med. Chem., 17, 877 (1974). (12) A. T. Shulgin, U.S. Patent 3547999 (1970). (13) J. S. Zweig, Ph.D. Dissertation, Department of Pharmaceutical Chemistry, University of California, San Francisco, Calif., 1974.
(14) G. W. A. Milne, H. M. Fales, and T. Axenrod, Anal. Chem., 43, 1815 (1971). (15) G. W. A. Milne, H. M. Fales, and R. W. Colburn, Anal. Chem.. 45, 1952 (1973). (16) B. T. Ho, V. Estevez, L. W. Tansey, L. F. Englirt, P. J. Criaven, and W. M. Mclsaac, J. Med. Chem., 14, 158 (1971). (17) H. B. Hucker, B. M. Michniewicz, and R. E. Rhodes, Biochem. Pharmacol., 20, 2123 (1971). (18) M. Goldstein, J. Biol. Chem., 237, 1898 (1968). (19) J. Daly, J. Axelrod, and B. Witkop, Ann. N.Y. Acad. Sci, 06, 37 (1962). (20) A. Klutch and M. Bordun, J. Med. Chem., I O , 860 (1967). (21) M. F. Grostic and K. L. Rinehart, "Mass Spectrometry Techniques and Applications", Wiley-Interscience, New York, N.Y., p 217 (1971) and references therein. (22) D. A. Durden. B. A. Davis, and A. A. Boulton, Biomed. Mass Spectrom., I, 83 (1974) and references therein. (23) R. J. Weinkam, "Mass Spectrometry in Biochemistry and Medicine, 1975", Raven Press, New York, N.Y. in press. (24) J. Gal, L. D. Gruenke. and N. Castagnoli, Jr.. J. Med. Chem.. 18, 683 (1975). (25) J. Gal, N. Castagnoli, Jr., and R. J. Weinkam, unpublished results. (26) C. Mitoma, D. M. Yasada, J. Tagg, and M. Tanabe, Biochem. Biophys. Acta, 136, 566 (1967). (27) D. V. Bowen and F. H. Field, J. Org. Mass Spectrom., 0, 195 (1974). (28) P. McGraw and N. Castagnoli, Jr., unpublished results.
\
RECEIVEDfor review January 16, 1975. Accepted October 6, 1975. This work was supported by an NIMH Research Grant MH 21219, and Research Career Development Award l-K4-GM-00007, (R.J.W.).
Graphite-Epoxy Mercury Thin Film Working Electrode for Anodic Stripping Voltammetry Jeffrey E. Anderson and Dennis E. Tallman' Department of Chemistry, North Dakota State University, Fargo, N.D. 58 102
A graphite-epoxy electrode is described which appears to be a viable alternative to other working electrodes currently used in anodic stripping voltammetry (ASV). Below pH 4, the electrode is essentialiy free of intrinslc electrochemical interference over the potential range of interest in ASV. A comparison is made of results obtained by DPAS analysis of Cd, Pb, and Cu in EPA reference samples wlth published results and with results obtained by flameless atomic absorption in our laboratory. Precision and accuracy of DPAS analysis of these samples wlth the graphoxy electrode Is typically 5-10%. The electrode is easy and inexpenslve to make, is easily polished, and is both physically and electrochemically durable.
Anodic stripping voltammetry (ASV) has proved to be a powerful method for the trace analysis of certain metal ions of environmental concern (1-3). The sensitivity of this technique is inherently associated with the preconcentration step during which metal species are electrochemically deposited a t a working electrode. The various problems and attributes which are associated with the different types of working electrodes have been treated elsewhere (3, 4 ) . We direct our attention here to mercury thin film electrodes (MTFE) which have found widespread use in anodic stripping voltammetry. Of the substrates used for plating these films, carbon appears to best fulfill the modest requirements of 1) conductivity, 2) chemical inertness to solution and to mercury, and 3) electrochemical inertness throughout the potential region of interest. Perhaps the two most widely used electrodes in stripping analysis are the glassy or vitreous carbon electrode and the
wax impregnated graphite (WIG) electrode. There appears, however, to be some controversy over which of these electrodes performs best and, indeed, most workers soon find preference for one over the other. Both of these electrodes appear to fail upon extended use, particularly under the acidic conditions often required to ensure dissociation of the metals from naturally occurring ligands. Modes of electrode failure have been studied by Clem and co-workers (5-7) and two modes appear to exist. One is common to the WIG electrodes in which solution gains access to the interior of the electrode, such as in the event of crystallization and cracking of the wax impregnator ( 5 , 6). Much effort has been devoted to finding appropriate waxes to circumvent this problem; however, wax being a rather complex substance, these attempts have suffered somewhat from irreproducible and inconsistent behavior. The other mode of failure appears to result from changes in the carbon itself involving formation of carboxyl compounds a t the surface of the electrode ( 7 ) .Thus, in acidic solutions, the hydrogen ion concentration a t the surface is effectively increased yielding the first-sign of electrode failure, an apparent decrease in hydrogen overpotential. Eventually the surface becomes unuseable and must be repolished, this problem being common to both WIG and glassy carbon electrodes. Our work began as a result of limited success with wax impregnated graphite electrodes and our desire to find an electrode which would be inexpensive and easy to make, durable, yet not require constant attention. The first electrode which came to mind was the carbon paste electrode used in organic voltammetry, the surface of which is easily renewable in the event of film formation a t the surface (4). If a similar electrode could be produced which would harden, yet still have an easily renewable surface, it may prove ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
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Figure 1. Graphoxy electrode ( a ) Polished surface: (b) graphite-epoxy plug; (c)epoxy body: (d)copper connecting wire, looped slightly in graphite-epoxy plug so as to provide better electrical connection and better compression of plug during preparation
useful for ASV. In this report, we describe a graphite-epoxy or graphoxy electrode which we feel is useful for ASV and which has certain desirable characteristics. The fabrication of an electrode bearing some resemblance to ours has been described previously wherein carbon black and epoxy were used to produce a wafer of 100-0 resistance (8).The wafer was then implanted into an electrode which was subsequently used in voltammetric experiments. However, the application of this electrode to stripping analysis was not considered. By employing a nonviscous epoxy in conjunction with finely powdered graphite, we have fabricated an electrode having a resistance of approximately 10 R. The construction of the graphoxy electrode and its application to the determination of Cu, Cd, and P b are described.
EXPERIMENTAL I n s t r u m e n t a t i o n . A Princeton Applied Research Model 174A
Polaragraphic Analyzer was used in the differential pulse mode (9) with 50 mV/pulse and 5 mV/sec scan rate. T h e sample was contained in a 15 ml ASV ElectRoCell obtained from M P I ( 5 ) .A saturated calomel reference electrode from Radiometer was used in conjunction with a n isolation salt bridge. Atomic absorption analysis was carried out by standard additions using a Perkin-Elmer Model 303 spectrometer equipped with a HGA 2100 Graphite Furnace. R e a g e n t s and M a t e r i a l s . Both KC1 and K N 0 3 have been used as electrolytes. Saturated stock solutions of these salts were prepared from reagent grade chemicals purified via extended controlled potential electrolysis. A 13.5 mg/ml mercury(I1) stock soluStock sotion was prepared from distilled Hg dissolved in "03. lutions of t h e various cations were prepared from t h e corresponding metals dissolved in minimum amounts of "03. Microliter volumes of t h e appropriate stock solutions, or dilutions thereof, were added t o 15 ml of sample. Each sample then contained 18 p p m Hg(I1) and either 0.03 M KC1 or 0.05 M KN03. Outgassing of oxygen was accomplished with nitrogen bubbled first through distilled deionized water, then through a V(I1) solution used as an oxygen scavanger and finally through more water. With the ElectRoCell, effective outgassing could be accomplished in 2 min. T h e isolation salt bridge for the reference electrode was filled with a 1:l dilution of the appropriate saturated electrolyte solution. Distilled deionized water was used throughout. T h e epoxy used for electrode fabrication was Epon 815 from Shell Chemical (Polymer Division, Houston, Texas). Curing was accomplished with reagent grade triethylenetetramine from Eastman. T h e powdered graphite used was obtained from t h e Joseph Dixon Co. (Jersey City, N.J.) with a particle size ranging from 10 t o 300 w , E l e c t r o d e F a b r i c a t i o n . T h e electrodes were molded in plastic soda straws cut t o t h e desired electrode length. T h e plastic straw was found to be more readily removed upon curing of the epoxy t h a n was glass tubing initially used. Epon 815 epoxy resin used for electrode fabrication is a diglycidyl ether of bisphenyl A t o which has been added a diluent. T h e low viscosity of this epoxy facilitates its mixing with large amounts of graphite. T h e tri210
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
E vs SCE
Figure 2. Anodic scan of 0.03 M KCI electrolyte in distilled deionized water ( a ) No Hg present, scan begun after equilibrium at -1.00 V vs. SCE was achieved, pH 5.0; ( b ) 18 ppm Hg(ll) present, 1-min deposition with stirring, 0.3-min wait with no stirring before scan, pH 4.0
ethylenetetramine (TETA) used for curing allowed sufficient time t o work with the epoxy before hardening. T E T A was mixed with the epoxy resin yielding a mixture 11%T E T A by weight. T o a portion of this epoxy-amine mixture was added powdered graphite which was blended into a uniform paste, 50% graphite by weight. Air bubbles trapped in this paste were effectively removed by placing t h e paste in test tubes and centrifuging for approximately 10 minutes in a table-top centrifuge. T h e paste was then placed under vacuum for 10 min after which it was again centrifuged for 10 min. A plastic straw was filled with t h e epoxy-amine mixture and a solid copper wire (19 gauge) was inserted down the center of the straw. This wire protruded from one end of t h e straw so as to permit electrical connection. T h e opposite end of the straw was forced into the epoxy-graphite paste still contained in a test tube, resulting in a %-inch plug of paste in the end of t h e straw. T h e copper wire protruding from the other end was pushed into the epoxygraphite paste establishing electrical contact with t h e graphite and further compressing the paste. After hardening (-5 hr), t h e straw was removed from the electrode and the graphite end of the electrode was dipped into an additional portion of epoxy-amine mixture. This additional sheath of epoxy around the graphite ensures t h a t conduction occurs only a t the polished planar surface of t h e electrode. T h e electrode was placed in a oven a t 'io OC over night to ensure complete curing. T h e electrode tended t o yellow slightly a t this point presumably because of oxidation of excess amine. T h e end of t h e electrode was initially abraded t o remove t h e epoxy coating on the end, exposing graphite. Once this had been accomplished, the surface was easily polished on wet or dry Tri-m-ite paper from the 3M Company. Electrodes fabricated in this manner typically had resistances of approximately 10 R. A completed graphoxy electrode is shown in Figure 1.
RESULTS AND DISCUSSION Several epoxies were employed in our early attempts to fabricate the graphoxy electrode. Most of these epoxies either softened a t the electrode surface upon prolonged contact with acidic sample solutions or exhibited high background current due to intrinsic electroactivity within the potential range of interest. Epon 815 from Shell does not appear to suffer from either of these problems. Figure 2 displays the current-potential curves of the graphoxy electrode scanning anodically from -1.00 V vs. SCE. Curve a corresponds to scans a t pH 5 with no mercury film covering the electrode surface. Under these conditions, some electroactivity is present. Heating the electrode surface in an oven (70 "C) after polishing further reduces this interference. Separate experiments suggest that this electroactivity results from slight excesses of TETA exposed a t the surface upon polishing. The electroactivity is shifted anodically upon lowering the solution pH and in this fashion can be
Pb
n
1 0
08
06
0 4
0 2
I
I
I
1
I
I
1 0
08
06
0 4
02
0 0
-E
0 0
- E vr SCE
Repeatability with EPA Reference Sample No. 2 in 0.05 M K N 0 3 ; same conditions as in Figure 2 ( b )
Figure 3.
moved out of the potential region of interest. Curve b of Figure 2 shows the behavior of the electrode plated with a mercury film in a solution of pH 4. The electroactivity observed in curve a has now been shifted under the mercury peak, and the electrode surface is behaving as a mercury thin film electrode, relatively free from interferences. Preliminary chronoamperometric investigations with lead indicate that approximately 75% of the electrode’s polished surface area is electrochemically active (10). When MTFE’s are used, two approaches may be taken: the Hg film may be plated before the analysis and used continuously for several analyses without removing it, or Hg(I1) may be added to the solution of interest and plated a t the same time as the metals under investigation, stripping the film off a t the end of each scan. The latter method would seem to be more pleasing in that a new film is used each time, and we have found this approach to be more sensitive and yield more reproducible results, as have others ( 1 1 ) . Unfortunately, we have found complications with this co-deposition technique in chloride media. T o get repeatable results, the electrode must be taken to +1.00 V vs. SCE and held a t this potential for several minutes a t the conclusion of each scan. Even then, the electrode exhibits some memory behavior towards mercury as has been observed with WIG electrodes treated with surface active agents ( 5 ) . If the surface of the electrode is wiped after each scan, this memory effect is partially alleviated; however, because of possible contamination and changing of the electrode surface, we have found this approach undesirable. This behavior is probably due in part to formation of a film of mercurous chloride a t the electrode surface since the phenomenon is not observed in a KN03 medium. Furthermore, in chloride media a t potentials more positive than 0 volts vs. SCE, chlorine is generated a t the electrode surface. Since chlorine rapidly attacks all forms of carbon (12), a substantial modification of the electrode surface may occur. For these reasons, the results which follow were obtained with KN03 as the electrolyte. Figure 3 shows repeated scans on EPA Reference Sample No. 2 (trace metals) acidified to p H 3 in 0.05 M KNO:, with a 1-minute deposition time a t -1.00 V vs. SCE. Mercury was co-deposited as discussed above. The electrode used to obtain these results had been used in similar solutions for several days without polishing. Repeatability of peak height from scan to scan is approximately 5%. For the experimental conditions described, the Hg film is sufficiently
VI
SCE
Standard additions of Cd, Pb, and Cu to EPA Reference Sample No. 2 in 0.05 M K N 0 3 ; same conditions as in Figure 2 ( b )
Figure 4.
( a )Sample alone; ( b ) sample
+ 20 ppb; (c)sample + 40 ppb
cu
120
100
BO
60
40
20
C O N C . ADDED
Figure 5.
0
20
40
60
S T A N D A R D (119/1)
Standard additions plot of data from Figure 4
Vertical scale is in arbitrary units of peak height with 1 unit equal to 0.75 p A
removed by scanning to +0.6 volts vs. SCE. We do observe the slow loss of hydrogen overpotential as is evident from Figure 3, but this problem is easily remedied by repolishing the electrode surface. For the analysis of Zn, such repolishing may be required more frequently. We also note the difficulty one may encounter in measuring the Cu peak because of its proximity to the large mercury peak. Because of the additional complication of Zn-Cu intermetallic formation ( 1 3 ) , Zn was not electrodeposited from the solutions used in the present study. However, with a freshly polished electrode, we have been able to obtain well defined zinc peaks in Cu-free solution a t the 10-ppb level a t p H 3.0 after 3-min deposition. Figure 4 displays the scans resulting from 20-ppb standard additions of each Pb, Cd, and Cu to EPA Reference Sample No. 2 and the resulting standard addition plots are shown in Figure 5. As is seen from these figures, the chosen conditions are nearly optimal for the determination of Cd in this particular sample, less so for Cu and Pb. Although additional scans could be made so as to optimize conditions for the determination of P b and Cu, we elected to take advantage of perhaps the most intriguing aspect of ASV, that of simple multielement analysis using a single standard solution containing all the metals of interest. Unless the concentrations of metals in the standard solution are optimized for each individual sample to be analyzed, this apANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
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to prepare several electrodes a t one time, storing them until needed.
Table I. Comparative Analytical Results for the Determination of Cu, Pb, and Cd in Water Samples
CONCLUSIONS
Concentration, pg/l. EPAa
cu
Reference sample 1 Reference sample 2 Tap water
9 67
RS-1 RS-2 Tap water
28 92
DPASVb
AASC
12.0 .i 1.7 55.0 i 3.8 53.5 i 8.5
9.0 58 72
Pb 29 t 2 102 i 15 3.8 i 0.13
29 125 4.0
Cd
2.1 i 0.4 2.0 RS-1 1.8 17.0 16.4 * 2.0 RS-2 16
