(2) (3) (4) (5) (6) (7) (8) (9) (10' (11) (12) (1 3) (14) (15) (16)
Lieberman, S. H.; Zirino, A. Anal. Chem. 1974, 46, 20. Andrews, R. W.; Johnson, D. C. Anal. Chem. 1978, 48, 1056. Wang, J.; Ariel, M , J . Nectroanal. Chem. 1977, 85, 289. Schieffer, G. W.; Blaedel, W. J. Anal. Chem. 1977, 49, 49. Wang, J., Ariel, M. Anal. Chim. Acta 1978, 99, 89. Schieffer, G. W.; Blaedei, W. J. Anal. Chem. 1978, 50, 99. Zirino, A.; Lieberman, S. H., Clavell, C. Environ. Sci. Techno/. 1978, 12,73. Wang, J.; Ariel, M. Anal. Chim. Acta 1978, 701,1. DeAngelii, T. P.; Bond, R. E.; Brooks, E. E.; Heineman, W. R. Anal. Chem. 1977, 49, 1792. Meyer, M. L.; DeAngeiis, T. P.; Heineman, W. R. Anal. Chem. 1977, 49, 602. Yaniv, D.; Ariel, M. J . Electroanal. Chem. 1977, 79, 159. "Reticulated Vitreous Carbon (RVC)", 1978 Chemotronics International Ann Arbor, Mich. 48104. Strohi, A. N.; Curran, D. J. Anal. Chem. 1979, 51,353. Blaedel, W. J.; Wang, J. Anal. Chem. 1979, 51,799. Norvell, V. E.; Mamantov, G. Anal. Chem. 1977, 49, 1470
(17) Strohl, A. N., Curran, D. J. Presented at the 177th National Meeting of the American Chemical Society, Honolulu, Hawaii, 1979. (18) Strohl, A. N.; Curran, D. J. Anal. Chim. Acta, in press. (19) devries, W. T.; van Dalen, E. J . Electroanal. Chem. 1987, 14, 315. (20) Sioda, R. E. Nectrochim. Acta 1970, 15,783. (21) McLaren, K. G.,Batiey, G. E. J . Electroanal. Chem. 1977, 79, 169. (22) Florence, T. M. J. Electroanal. Chem. 1979, 27, 273. (23) Clem, R. G.;Litton, G.; Ornelas, L. D. Anal. Chem. 1973, 4 5 , 1306. (24) Ellis, W. D. J . Chem. Educ. 1973, 50, A131. (25) Clem, R. G. Anal. Chem. 1975, 47, 1778.
RECEIVED for review April 16, 1979. Accepted June 19,1979. This work was funded in part by the University Sea Grant Program under a grant from the Office of Sea Grant, National Oceanic and Atmospheric Administration, U S . Department of Commerce, and by the State of Wisconsin.
Elimination of Intermetallic Compound Interferences in Twin-Electrode Thin-Layer Anodic Stripping Voltammetry Daryl A. Roston, Elwood E. Brooks,' and William R. Heineman" Depaement of Chemistry, University of Cincinnati, Cincinnati, Ohio 4522 1
Anodic stripping voltammetry (ASV) performed in a twlnelectrode thin-layer cell eliminates Cu-Zn and Cu-Cd Intermetallic interferences that are often encountered In ASV. Cu is first exhaustively deposited on one electrode, and then the second electrode is used to complete the Zn and/or Cd determination. Intermetallic interferences are circumvented since the Interfering constituents are deposited on different working electrodes. Effects of increasing Cu concentiatlon on the Cd and Zn stripping peaks are shown. Linear calibration curves for Cd and Zn (13 to 130 ppm) are obtained from standard solutions in which Cu2+ concentratlons vary from 16 to 76 ppm. The feasibility of determining Pb, Cd, Zn, and Cu simultaneously by anodic stripping voltammetry with no Intermetallic interferences is enhanced with the twin-electrode thin-layer cell.
Thin-layer electrochemical cells have been used for studying a variety of electrochemical phenomena: adsorption at electrode surfaces; spectroelectrochemical measurements of biological, inorganic, and organic systems; and kinetics of chemical reactions coupled to electrode processes (1-5). In addition to these applications, the analytical utility of the thin-layer cell has been demonstrated. An early application of the thin-layer cell for analytical measurements was the determination of Cu2+,PbZt, CdZt, and Zn2+by thin-layer coulometry (6). The capability of performing differential pulse anodic stripping voltammetry in a thin-layer cell was recently demonstrated by the determination of Pb2+,Cd2+,CU", and Tl' down to 30 ng/mL (7, 8). Thin-layer differential pulse voltammetry was also shown to be applicable to electroactive organic compounds by the determination of the drug diazepam down to 100 ng/mL. Such techniques combine the smallvolume capability inherent in thin-layer electrochemistry with the low detection limit of differential pulse voltammetry. Present address: Department of Chemistry, H o w a r d University, Washington, D.C. 20059. 0003-2700/79/0351-1728$01.OO/O
An inherent difficulty with stripping voltammetry is the potential interaction between materials which have been preconcentrated into or onto the electrode. One such interaction is the formation of intermetallic compounds by metals deposited into mercury in anodic stripping voltammetry (ASV). Numerous intermetallic compounds have been reported (S12). The formation of such compounds can cause erroneous analytical results since metals in the compounc, ;an oxidize a t different potentials than the individual metals oxidize. This problem has been noted a t the two commonly used electrodes for ASV the hanging mercury drop electrode (HMDE) and the mercury film electrode (MFE) (13). Effects of intermetallic compound formation have also been observed in thin-layer ASV. The Cu-Zn interference in A S ? has been frequently noted ( S l l ,13-17). Under certain conditions, the deposition of Cu and Zn in a mercury film electrode (MFE) or a hanging mercury drop electrode (HMDE) results in formation of a Cu-Zn intermetallic compound, CuZn, where x = 1 , 2 or 3 (13). The intermetallic compound is oxidized from mercury at the same potential as Cu, causing enhancement of the Cu stripping peak and depression of the Zn stripping response. As much as 16% of the Cu and Zn present can be nonelectroactive as a result of Cu-Zn interactions. The Cu-Cd interference has not been as widely reported, and the nature of the interference is less clearly defined. Some authors have not detected any interfering effects between Cu and Cd (14); others have reported substantial interference (18 and references therein). Ostapczuk and Kublik have shown that there are no detectable differences in the Cd stripping responses from a saturated Cu amalgam HMDE and a pure Hg HMDE (18). However, when the solubility of Cu in the HMDE is exceeded, a depression in the Cd stripping wave results. They propose that exceeding the solubility limit of Cu results in other metals plating onto nondissolved Cu atoms on the surface of the mercury, forming binary intermetallic compounds. In a pratical sense, interferences among Cu-Zn and Cu-Cd are particularly important since these metals are frequently determined by ASV. The interferences described above pose 0 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
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E
Flgure 1. Twin-electrode thin-layer cell (A) Volume brace. (B) Wax-impregnated graphite electrode. (C) Reference electrode compartment. (D) Auxiliary electrode compartment. (E) Solution outlet. (F) Glassy carbon electrode. (G) Volume brace
a limitation on the general applicability of ASV since Cu2+ and Zn2+ are invariably found in environmental and food samples. T h e effects of intermetallic compound formation on the practical application of ASV for the determination of Cu, Zn, and Cd have been generally disregarded (13). One approach t o eliminating errors due to a particular intermetallic compound has been the preferential formation of another intermetallic compound. A procedure has been reported whereby the Cu-Zn interference is circumvented by spiking the analyte solution with Ga3+ (11). Since the Cu-Ga intermetallic compound has a higher formation constant than the Cu-Zn compound, the presence of Ga precludes the formation of CuZn,. Copper can then be determined in a separate experiment in which the deposition potential is controlled so t h a t only Cu is deposited into the electrode. Twin-electrode thin-layer electrochemistry offers another method for eliminating intermetallic interactions for thin-layer ASV. The concept of twin-electrode thin-layer electrochemistry (two working electrodes sandwiching the thin solution layer) put forth by Reilley and Anderson has been used for the evaluation of electrode mechanisms and the measurement of diffusion coefficients (3,19). However, this unique variation on thin-layer electrochemistry has seen no application t o voltammetric analysis. The objective of this research is to explore the potentialities of using selective deposition on two different working electrodes to eliminate interferences resulting from intermetallic compound formation in multielement analysis by thin-layer ASV. T h e research focuses on the Cu-Zn and the Cu-Cd interferences. T h e strategy is t o selectively deposit copper on one of the electrodes and zinc and cadmium on the other electrode (8). T h e Cu-Zn and Cu-Cd interferences are avoided by physical separation of the interfering components on different electrodes. Since the diffusion path in a thin-layer cell is relatively short, selective electrolysis of ions from the thin solution layer is achievable in a reasonable period of time with diffusion as the mode of mass transport.
EXPERIMENTAL Apparatus. Figure 1 shows the twin-electrode thin-layer electrochemical cell. The body of the cell was machined from acrylic rod, purchased from Cincinnati Plastics, Inc. Working electrodes were positioned in the cell at a distance of 0.1 mm apart. Braces were placed on the electrodes to ensure that the cell thickness did not vary. The diameter of the cylinder into which the working electrodes were positioned was 0.180 in. Wax-impregnated graphite electrodes (WIGE) were positioned directly into the cell body. The diameter of the glassy carbon electrode (GCE) employed was 0.112 in.; it was inserted into a piece of cylindrical Teflon, 0.180 in. in diameter, which was then fitted into the cell body. A junction between the working electrodes and the reference and auxiliary electrodes was formed by drilling through the body of the cell with a no. 60 drill bit. A no. 80 drill
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bit was used to form the portion of the junction canal immediately adjacent to the solution between the working electrodes. The small diameter of the junction canal (0.0135 in.) minimized diffusion from the solution in the canal to the solution between the electrodes. High supporting electrolyte concentrations were used to minimize resistance of the small-diameter junction canal between the reference and working electrode compartments. The dual working electrode potentiostat was built from a circuit designed for ring-disk electrodes (20). Stripping voltammograms were recorded on a Hewlett-Packard 136A X-Y-Y’recorder. The potentiostat employed to preform the Hg films on the GCE was of conventional operational amplifier design. A Houston Instrument Omnigraphic 2000 X-Y recorder was used to record coulometric stripping responses. Reagents. Standard solutions of Cu2+,CdZ+,F’b2+,and Zn2+ were prepared by diluting the respective Fisher atomic absorption standards (Fisher Scientific Co.) with appropriate amounts of 1.0 M NaC2H302-0.1M HNO, buffer, pH 5.6. Preformed mercury films were deposited from a 0.006 M Hg(NOJ--0.5 M KNO, M Hg2+in 1.0 M NH,C1-NH40H solution. A solution of 3.0 X buffer, pH 8.3, was used in the in situ deposited Hg film studies. Procedures. Electrode and Cell Preparation. Wax-impregnated graphite electrodes (WIGE) were made from POCOFXI spectroscopic graphite rods. They were prepared in a manner reported previously (8). Glassy carbon electrodes (Atomergic Chemicals Co., Plainview, N.J.) were prepared by polishing with successively finer grades of diamond paste (Buehler Ltd., Evanston, Ill.). The thin-layer cell was cleaned in a Harrick Plasma Cleaner (Harrick Scientific Co.) each day before use. A S V Experiments. Glassy carbon mercury film electrodes (GCMFE) were formed in a bulk electrochemical cell. A 0.006 M Hg(N03)2-0.5M KN03solution was purged with Npfor 15 min. The GCE was placed in the stirred solution and held at a potential of -1.2 V for 4 min. This potential was used because it has been shown that film quality improves as the deposit ion potential becomes more negative (21). After the GCMFE had been formed, it was transferred to the thin-layer cell. The WIGE had been placed in the cell prior to the formation of the GCMFE. After injection of the sample, the potential of the WIGE was stepped to -0.500 V while the potential of the GCMFE was kept at +0.050 V. These potential settings ensured that Cu was being deposited on the WIGE and not on the GCMFE. Once the Cu had been exhaustively deposited on the WIGE, the GCMFE: was stepped to a potential of -1.4 V. After 4 min, the potential of the GCMFE was scanned to +0.100 V at a rate of 15 mV/s. Since the Cu2+ had been removed from the solution, the deposition of Cd2+and ZnZ+could be completed without interference from Cu. Whether or not exhaustive Cu deposition on the WIGE had been achieved was determined by stepping the potential of the GCMFE negative enough to cause the deposition of Cu. If after 4 min the scan to f0.10 V indicated that Cu was present in the GCRIFE, a longer Cu collection period was employed. Higher concentrations of Cu required longer times to effect the removal of the metal from the analyte solution. For the standard solutions used in this study, the times required for Cu2+removal varied from 15 to 90 min. Estimating Hg-Film Thickness. Stripping coulometry was employed to estimate the Hg-film thicknesses achieved with the procedure described above. .4fter the 4-min deposition of Hg, the potential of the GCE was stepped from -1.2 V to +0.70 V. The current response resulting from the oxidation of the Hg on the electrode surface was integrated. Several stripping coulometry determinations indicated that a film thickness of ca. 4000 A was achieved.
RESULTS AND DISCUSSION Twin-Electrode Thin-Laqer Cell. The twin-electrode thin-layer cell designed for this study is shown in Figure 1. The thin-solution layer is sandwiched between a wax-impregnated graphite electrode (WIGE) and a glassy carbon mercury film electrode (GCMFE) which are positioned with their electrode surfaces parallel. T h e distance between electrodes is variable; a 0.1-mm solution layer was used in these experiments; 30-40 MLis required t o fill the cell. Since the solution layer is thin, analyte metals can be deposited on either electrode in a short period of time with diffusion as the mode
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979 A
I
0
20
40
68
80
100
W CONC. P T
E V v3i-E
Figure 2. Twin-electrode thin-layer stripping voltarnrnograms. (A) Cu deposited on WIGE prior to deposition of Zn on GCMFE. (E) Cu and Zn codeposited on the GCMFE. (C) Cu deposited on WIGE prior to deposition of Cd on the GCMFE. (D) Cu and Cd codeposited on GCMFE. For A and 8,the standard solution contained 15 pprn Zn" and 30 ppm Cu2+. For C and D, the standard solution contained 30 pprn Cu2+and 45 pprn Cd". Scan rate, 15 rnV/s, pH 5.6 acetate buffer
of mass transport. The cell is easy to construct and to use. Cu-Zn Intermetallic. The problem of Cu-Zn intermetallic compound formation in ASV is illustrated by a thin-layer stripping voltammogram for 30 ppm Cu and 15 ppm Zn. A stripping voltammogram in which both Cu and Zn were deposited into the same MFE is shown in Figure 2B. The potential was first held a t -1.4 V for 4 min to deposit Cu and Zn from the thin solution layer into the GCMFE. As the voltammogram shows, no peak is obtained a t -1.1 V for Zn stripping since the Zn is essentially completely bound in a Cu-Zn intermetallic compound. The sharp stripping peak at +0.15 V is attributed to oxidation of free Cu and both the copper and zinc in the Cu-Zn intermetallic. Consequently, the "copper" peak is enhanced by the zinc component of the Cu-Zn intermetallic. The magnitude of zinc peak depression is determined by the ratio of [Cu]/[Zn] deposited in the mercury film. Curve B in Figure 3 shows peak current for the Zn stripping wave as a function of Cu concentration in solution. As the copper concentration in solution increases, resulting in a greater concentration of Cu in the MFE after deposition, the height of the Zn peak decreases substantially. At a [Cu]/[Zn] ratio of 1.0, a peak for Zn is barely observable; no peak is observable a t a ratio of 2.0 as shown by the voltammogram in Figure 2B. The advantage in using the twin-electrode thin-layer cell is demonstrated by the voltammogram in Figure 2A. In this case, the Cu and Zn were selectively deposited on two different electrodes. Cu ion was first exhaustively removed from the thin solution layer by applying a potential of -0.4 V to the WIGE for 30 min. A potential of -1.4 V was then applied to the GCMFE to deposit Zn into the MFE. The potential of the GCMFE was then scanned positively. The resulting stripping peak for Zn is apparent. The peak is not diminished by the presence of Cu since all of the copper wcs deposited on the other electrode, preventing Cu and Zn from interacting in the same MFE. The Zn peak was not affected by increasing concentration of Cu as shown by curve A in Figure 3. The peak current for Zn stripping was found to respond linearly t o changes in Zn concentration as shown in Figure 4. The calibration curve was recorded for standard solutions of Zn2+containing varying amounts of Cuz+(concentrations of Cu2+are indicated in parentheses). The linearity indicates
Flgure 3. Effect of changing Cu2+ concentration on Cd and Zn stripping responses. (A) Zn stripping peak (30 ppm) height with Cu deposited
on WIGE prior to deposition of Zn on GCMFE. (E) Zn stripping peak (30 ppm) height with Cu and Zn codeposited on GCMFE. (C) Cd stripping peak height (30 pprn) with Cu deposited on WIGE prior to deposition of Cd on GCMFE. (D) Cd stripping peak height (30 pprn) with Cu and Cd codeposited on GCMFE
n"carc.
Pad
Figure 4. Peak height vs. Zn2' (A) and Cd2+ (0)concentration (pprn).
Cu2+ concentrations are indicated in parentheses (ppm)
immunity of the Zn peak to variations in Cu2+concentration. The non-zero intercept of the Zn calibration curve can be attributed to difficulties in determining the background component of the Zn stripping response, a problem due largely to the concurrence of Zn oxidation with H2 evolution. Cu-Cd Interferences. A Cu-Cd interference similar to the Cu-Zn interference is obtained in thin-layer ASV. For the voltammogram in Figure 2D, the potential of the GCMFE was stepped to -0.9 i7 for 4 min, resulting in the simultaneous deposition of Cu and Cd from a solution containing 45 ppm Cu2+and 30 ppm Cd2+. Well-defined stripping peaks occurred for both metals when the electrode potential was scanned to f 0 . 1 V. The same analyte solution and GCMFE was used to obtain the voltammogram depicted in Figure 2C. However, before the deposition of Cd at -0.9 V for 4 min on the GCMFE, the potential of the WIGE was set a t -0.4 V for 45 min, resulting in exhaustive removal of Cu2+from the solution between the electrodes. When the GCMFE potential was scanned to +0.1 V, an enhanced Cd stripping response was observed. Figures 2C and 2D illustrate that suppression of the Cd stripping peak occurs as a result of the codeposition of Cu and Cd on the GCMFE. As with the Cu-Zn interference, the suppression of the Cd stripping response is a function of the relative concentrations of the interfering metals. Curves C and D of Figure 3 show the peak current of the Cd wave for a series of solutions in which Cd2+concentration was held constant while the Cu2+
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
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I
A
/
c .01
-065
-4.4
Zn
I
B
cu E
v "5
scc
.3'a
-061
-14
Figure 5. Twinelectrode thin-layer stripping voltammograms from the same standard solution and GCMFE. (A) Cu deposited on WIGE prior to deposition of Pb, Cd, and Zn on the GCMFE. Potential scan from -1.4 to +0.1 V. (B) Cu, Pb, Cd, and Zn codeposited on GCMFE. Potential scan from -1.4 to +0.18 V. Scan rate, 15 mV/s. 18 ppm Cd2+, Pb2+, Zn2+, and Cu2+, pH 5.6 acetate buffer
concentration increased from 0 to 90 ppm. When the Cu and Cd were codeposited at -0.9 V for 4 min in the GCMFE, the Cd stripping peak declined as Cu concentration increased (curve D). However, when the Cu was exhaustively deposited on the WIGE before the deposition of Cd on the GCMFE, the Cd stripping peak remained independent of Cu concentration (curve C). Although the Cu-Cd interference effect is less pronounced than the Cu-Zn intermetallic problem, it is significant enough to cause substantial error in analysis when varying Cu2+ concentrations are present in andyte solutions containing Cu2+ and Cd2+. It should be noted the Cu2+concentrations in these solutions were high enough that deposition of the metal on the GCMFE resulted in its solubility in Hg being exceeded. This is an important consideration since Ostapzuk and Kublik have shown that the Cu-Cd intermetallic problem arises as a result of Hg-Cu solubility problems (18). The twin-electrode thin-layer cell can be used to eliminate error caused by the codeposition of Cu and Cd. Data represented in Figure 4 indicate that a linear CdZ+concentration vs. peak current response was obtained despite the presence of varying Cu2+levels in the standard solutions. In each case, Cu2+ was removed from the solution between the working electrodes by exhaustive deposition on the WIGE prior to the deposition of Cd on the GCMFE. Simultaneous Determination of Pb, C d , and Zn. The simultaneous determination of Cd, Pb, and Zn in the presence of Cu is a common analytical problem. However, the Cu-Zn and Cu-Cd interferences detract from the feasibility of determining these metals simultaneously with ASV. Figure 5B is a stripping voltammogram a t a GCMFE for a solution containing 18 ppm Pb2+,Cd2+,Zn2+,and Cu2+. The metals were deposited a t a potential of -1.4 V for 4 min. When the potential of the GCMFE was scanned to +0.2 V, the Zn stripping response was barely detectable. However, when the Cu2+in the solution was separated by exhaustive electrolysis in the WIGE prior to the deposition of Pb2+,Cd2+,and Zn2+ on the GCMFE, a much improved voltammogram resulted as shown in Figure 5A. The Zn stripping peak is well-defined and the Cd and P b stripping waves occur on a substantially decreased residual current. The twin-electrode thin-layer cell enhances the analytical reliability of a simultaneous Pb2+, Cd2+,and Zn2+determination by thin-layer ASV. E f f e c t of Cu on t h e Negative Potential Range of a MFE. Besides eliminating the occurrence of Cu-Zn and Cu-Cd interferences, the twin-electrode thin-layer ASV technique alleviates another aspect of the presence of Cu. In addition
i
+ 0.1
- 0.65
b
-14
E,V vs. SCE
Figure 6. Effect of the presence of Cu on the negative potential window of the GCMFE. (A) Potential scan from -1.4 to +0.1 V. The standard solution contained -0.1 ppm Cu2+, which was codeposited with Zn. (B) Scan from -1.4 to 4-0.1 V. The trace of Cu was deposited on WIGE before the deposition of Zn on GCMFE. Scan rate, 15 mV/s. -0.1 ppm Cu2+, -0.1 ppm Cd, and 15 ppm Zn2+, pH 5.6 acetate buffer
to causing suppression in the stripping response of Cd and Zn, the presence of Cu can have a deleterious effect on the negative potential window of a MFE. Figure 6A shows a stripping voltammogram obtained on a GCMFE for a solution containing 15 ppm Zn2+and a trace of (-0.1 ppm) Cu2+. The Zn stripping peak occurs on a large background. When the trace of Cu2+was removed by deposition on the WIGE before the deposition of Zn2+ on the GCMFE, the resulting voltammogram (Figure 6B) had a substantially decreased background and a larger Zn stripping response. As shown in Figure 6, the presence of a relatively small amount of Cu in a MFE can diminish the negative potential range of the MFE. The deposition of larger amounts of Cu in a MFE lead to an increase in the severity of this background problem. This can be seen by comparing the voltammograms in Figures 2 , 5 , and 6. Substantial increases in the background current of all the voltammograms occurred when Cu was present in the MFE. In Situ Deposited MFE. As an alternative to preforming the GCMFE in a bulk solution cell with subsequent transfer to the twin-electrode thin-layer cell, an in situ deposited mercury film was investigated. Standard solutions of Cu2+ and Zn2+ or Cd2+were made 3 X M in Fig2+. By appropriately controlling potentials of the twin electrodes, the Hg2+could be deposited as a MFE on either the WIGE or the glassy carbon electrode or both. Exhaustive removal of Hg2+ from the thin solution layer resulted in film thicknesses of only ca. 10 A. Calibration curves of the type shown in Figure 4 were distinctly nonlinear when obtained on such thin mercury films. This behavior is attributed to Zn and Cd exceeding their mercury solubility when deposited into such a small volume of mercury as described by Stojek et al. (22, 23).
Preforming the GCMFE enabled a film thickness of ca. 4000
A to be easily achieved. Such relatively thick films gave linear responses as shown in Figure 4. However, thinner in situ deposited films should be practicable at lower analyte concentrations which would be accessible with differential pulse voltammetry as the stripping technique (7, 8). Edge Effects. A problem with most thin-layer cells is diffusion into the cell proper from surrounding electrolyte, Le., the edge effect (24). The effects of edge diffusion were
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
apparent in this cell when the Cu2+:M2+ratio was large. In such instances, substantial diffusion from the narrow channel leading to the reference electrode necessitates the use of relatively long deposition times to prevent the deposition of Cu on the GCMFE. Future modifications of the cell design will be directed toward minimizing the effects of Cu2+diffusion by further isolating the GCMFE from the channel.
Oglesby, D. M.; Anderson, L. B.: McDuffie, B.; Reilley, C. N. Anal. Chem. 1965, 37, 1317. DeAngelis, T. P.; Heineman, W. R. Anal. Cbem. 1976, 4 8 , 2262. DeAngelis. T. P.; Bond, R. E.;Brooks, E. E.; Heineman, W. R. Anal. Chem. 1977, 49. 1792. Vydra, F.; Stulik, K.: Julakova, E. "Electrochemical Stripping Analysis"; Halsted Press: New York, 1976; pp 58-66. Barendrecht, E. In "Electroanalytical Chemistry"; Bard, A. J., Ed.; Marcel Dekker: New York, 1967: Vol. 11. Copeland, T. R.; Osteryoung, R. A,: Skogerboe, R. K. Anal. Chem. 1974, 4 6 , 2093. Robbins, D. G.: Enke, C. G. J . Electroanal. Chem. 1969, 23 343. Shuman. M. S.; Woodward, G. P., Jr. Anal. Chem. 1976, 48, 1979. Crosum, S. T.: Dean, J. A,; Stokely. J. R. Anal. Chim. Acta 1975, 75, 421. Kemula, W.; Kublik, Z. Nature 1958, 182, 1228. Stromberg, A. G.; Gorodovykh, V. E. Zh. Neorg. Khim. 1963, 8, 2355. Stromberg, A. G.; Zakharov, M. S.; Mesyots. N. A. Nektrokhimiya 1967, 3 , 1440; 1968, 4 , 987. Ostapczuk, P.; Kublik, Z. J . Nectroanal. Chem. 1977, 83, 1. Anderson, L. B.; Reilley, C. N. J . Electroanal. Chem. 1965, 10, 295, 538. Napp, D. T.: Johnson, D. C.: Bruckenstein, S. Anal. Chem. 1967, 39, 481. Stulikova, M. J . Electroanal. Chem. 1976, 4 8 , 33. Stojek, Z.; Stepnik, B.; Kublik, Z. J . Electroanai. Chem. 1976, 74, 277. Stojek, Z.; Kublik, Z. J . Electroanal. Chem. 1977, 77, 205. McDGffie, B.; Anderson, L. B.; Reilley, C. N. Anal. Chem. 1986, 38, 883. Paul, D. W.: Ridgway, T. H. "Abstracts of Papers", 176th National Meeting of the American Chemical Society, Miami Beach, Fia., Sept. 1978; American Chemical Society: Washington, D.C.; Abstr. COMP 2.
CONCLUSION Selective deposition of interfering metals onto different electrodes is an effective means of avoiding intermetallic interactions in thin-layer ASV as demonstrated here for Cu-Zn and Cu-Cd. Results reported here were obtained in the parts per million concentration range with linear sweep voltammetry as the stripping technique. The development of instrumentation for twin-electrode differential pulse AS\' (2.5') should enable analysis in the parts-per-billion range as has been demonstrated for single-electrode thin-layer ASV ( 7 , s ) . The magnitude of these intermetallic compound interferences in this lower concentration range is being investigated. L I T E R A T U R E CITED (1) Hubbard, A. T.: Anson, F. C. In "Electroanalytical Chemistry"; Bard, A J., Ed.; Marcel Dekker: New York, 1970: Vol. I V , Chapter 2. (2) Hubbard, A. T. CRC Crit. Rev. Anal. Chem. 1973, 3 , 201. (3) Reilley, C. N. Rev. Pure Appl. Chem. 1968, 18, 137. (4) Kuwana, T.; Heineman, W. R. Acct. Chem. Res. 1976, 9, 241. (5) Heineman, W. R. Anal. Chem. 1978, 50, 390A.
R E C E ~ Efor D review March 12,1979. Accepted June 25,1979. The authors gratefully acknowledge support provided by the National Science Foundation.
Computerized Pattern Recognition Applied to Gas Chromatography/Mass Spectrometry Identification of Pentafluoropropionyl Dipeptide Methyl Esters James N. Ziemer and S. P. Perone" Purdue University, Department of Chemistry, West Lafayette, Indiana 47907
R. M. Caprioli* and W. E. Seifert University of Texas Medical School, Department of Analytical Chemistry, Houston, Texas 77025
A promising new technique for the indentification of amino acid sequences in polypeptides involves the enzymatic hydrolysis of intact polypeptides to dipeptides followed by analysis of the products with gas chromatography/mass spectrometry. The feasibllity of this approach for fast on-line analysis was demonstrated here by the use of computerized pattern recognition in the identification process. The fundamental basis for classificationwas the separate identificationof the N- and C-terminal amino acids in the dipeptides using two muiticategory k-nearest neighbor (kNN) analyses. Two sets of ions characteristic of amino acids derived from either the N- or C-terminus were chosen as features for the tests on the basis of similarities in intensities among the members of each class. Features were normalized to the sum of only those ions used in a particular test in order to ensure that the relative ion intensities used were not influenced by the charge retaining characteristics of the amino acid on the other terminus. Classification accuracy for 86 PFP dipeptide methyl esters was 100% in the N-termlnal amino acid test, and 9 7 % in the C-terminal test.
In recent years there has been considerable interest in the development of mass spectrometric methods for the se-
quencing of polypeptides. One such technique which has shown promise involves the enzymatic hydrolysis of intact polypeptides to dipeptides by dipeptidylaminopeptidases (DAP) I and IV, followed by product identification with gas chromatography/mass spectrometry (GC/MS) ( I , 2). The key to implementing this technique on a routine basis lies in the development of a method for identification of the dipeptide mass spectra which is fast, accurate, and largely unaffected by the presence of impurities. This paper reports on the potential use of computerized pattern recognition for the interpretation of these low resolution dipeptide mass spectra. The enzymatic hydrolysis-GC/MS method provides amino acid sequence information from two related mixtures of dipeptides. The two dipeptide mixtures are obtained from two separate enzymatic hydrolyses, one involving the original polypeptide and the other involving the polypeptide whose N-terminal amino acid has been removed via the Edman method. DAP I and IV operate by sequentially cleaving the polypeptide into dipeptide fragments from the N-terminus. Each dipeptide mixture is subjected to acylation of the Nterminal amino acid with pentafluoropropionyl (PFP) anhydride and esterification of the carboxyl groups with methanol prior to separation and identification by GC/MS. Because of the generally nonredundant nature of most polypeptide amino acid sequences, stemming from different 1979 American Chemical Society
