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Borate Interference in Surface-Enhanced Raman Spectroscopy of Amines Tonya M. Heme1 and Robin L. Garrell*B1 Department of Chemistry, Chevron Science Center, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Interference from borate Is observed In surface-enhanced Raman (SER) spectra d lysine and p"hobtalned wHh borohydrlde-reduced silver collokls. Borate bands are also observed In the spectra of other basic analytes, as well as when certaln varlatkns are made In the silver cdlokl prepar a m . TM relathre lntensitks of the analyte and borate bands depend on the pH of the colkkl, the extent d oxklation of the cdldd surface, and the relatlve adsorptlvltles of the analyte and borate. Benzylamlne adsorbs more readlly than propylamlne and also competes more effectively wlth borate for adsorption Snes. On the other hand, borate virtually exdudes lysine from the surface when the solution pH Is 28. The formation of dlver oxlde In badfled colloids may facllltate borate adwptkn. For some barlc analytes, elhnlnatlng the Mkorptbn of borate bn and the multlng spectral Interference may requlre uslng alternatlve SERS substrates.
INTRODUCTION Surface-enhanced Raman spectroscopy (SERS) has been used extensively to investigate adsorbate-surface interactions. On the basis of the SER spectrum of an adsorbed polyfunctional molecule, the relative proximities of functional groups of the adsorbate to the surface can be deduced (1-6). The orientation of functional groups can also be determined qualitatively by comparing the relative intensities and positions of bands in the SER spectrum with those in the Raman spectrum of the pure or solvated analyte (7,8). For these analyses, it is essential to have reproducible spectra that are free from spurious features and from spectral interference that might arise from species that compete with the analyte for adsorption sites. Borohydride-reduced silver colloids are commonly used as the substrate for SERS (1,9,10). Recently, it was noted that a SER spectrum of borate (B(OH),-) is obtained when the water used to make the colloid contains metal ions such as Ca2+or Mg2+(11,12). Borate vibrations were also observed in the SER spectrum of 2,2'-bipyrimidine when an excess of sodium borohydride was used to prepare the colloid (13,14); the bands were attributed to borohydride "byproducts" masking active sites (14). We report here that borate can present a severe and general interference in SER spectroscopy of basic analytes such as aliphatic amines and basic amino acids. This can be a problem not only in the interpretation of SER spectra of known analytes, but could be a serious complication when SERS is used as the detection method in the direct analysis of mixtures or of unknown species separated chromatographically. The conditions under which the interference is observed and methods for avoiding the interference are described. EXPERIMENTAL SECTION Reagents and Solutions. Lysine (Sigma), propylamine (Aldrich, 99+ % purity), and benzylamine (Aldrich, 99% purity) were used as received. All solutions were made with doubly Present address: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90024
distilled deionized water. All other compounds were ACS analytical grade reagenta. Where indicated, the pH of the colloid was adjusted with NaOH or HNO* Apparatus and Procedures. Sodium borohydride reduced silver colloids were prepared as described in ref 15. The citrate colloids were prepared by adding 2.5 mL of sodium citrate (3.4 x M) to 50 mL of boiling silver nitrate solution (5 X lo-' M). The resulting colloid was a murky yellowish green. Aqueous solutions of the analyte were added to the borohydride-reduced silver colloid to give a final concentration of 1.0 X la-' M for lysine, 0.19 M for propylamine, and 2.0 X lo9 M for benzylamine. The final concentration of propylamine in the citrate colloid was 2 x 10-3 M. The W a n and SER spectra were obtained with a SPEX 1403 scanning double monochromator equipped with 1800 groove/" holographic gratings, an RCA C31034 PMT detector,and a SPEX DMlB computer. A Cooper Lesersonia Model 150 argon ion laser ( h = 514.5 nm) was used as the excitation source for the normal Raman and colloid SER spectra. All of the SER spectra shown are single scans obtained at a rate of 1 cm-'/0.5 s, except that of propylamine in the citrate colloid (1 cm-*/s). The Raman spectra of aqueous lysine and aqueous propylamine were obtained by using a scan rate of 1 cm-'/2 s. The spectral band-pass was about 3 cm-' for the SERS experiments. RESULTS AND DISCUSSION Numerous SERS experiments have revealed the tendency for amines to interact with aqueous colloidal silver through the amine nitrogen, even when other functional groups such as carboxylate are present that can complex with silver (3,6, 16-18). The results of SERS studies of di- and tripeptides indicate that the deprotonated amine terminus has a high affinity for the slightly positively charged colloidal silver surface (6,181. On the basis of these results, we predict that lysine will adsorb readily on colloidal silver, and that it will interact with the surface through one or both of the amine groups, depending on the pH. The SER spectrum of lysine is therefore expected to show intense amine group vibrations. The spectrum obtained by adding lysine to a sodium borohydride reduced silver colloid at pH 7 (no pH adjustment) is shown in Figure 1, along with the Raman spectrum of aqueous 1.0 M lysine at pH = 12.5. The lysine-plus-colloid spectrum bears little resemblance to the normal Raman spectrum of lysine. The broad background, which has wide peaks centered a t -1390 and 1580 cm-', is attributable to scattering from graphitic carbon formed by laser-induced degradation of the analyte on the colloid surface (1!+21). The two strong bands at 614 and 925 cm-' in the SER spectrum have no counterparts in the normal Raman spectrum, suggesting that these bands may be due to some other species present in the sample. Lysine ia a basic amino acid with pK, = 10.5. We considered the possibility that an increase in the pH of the colloid upon the addition of lysine might affect the interfacial behavior of ions in the colloidal suspension, ions that do not adsorb a t neutral pH. To test this hypothesis, a very simple control experiment was performed. Sodium hydroxide was added to the silver colloid to final pH values 29.5, and SER spectra were obtained. Figure 2 shows the spectrum obtained at pH 10.5 along with the spectrum of a silver colloid to which no base was added. The band at 472 cm-' in the spectrum of the
0003-2700/Q1/0363-2290$02.50/0 @ 1991 Arnerlcan Chemical Society
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RAMAN SHIFT (cm.’) Flgure 1. (a) Raman spectrum of 1.0 M lyslne. (b) SER spectrum of
sodium borohydride reduced silver colloid to which lysine was added (final concentration 1.0 X lo4 M).
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Flgure 2. (a) SER spectrum of a borohydride-reduced silver colloid to which NaOH was added to glve a final pH of 10.5. (b) Spectrum of
the borohydrlde-reduced colloid as inltlally prepared.
basified colloid is attributable to Ago, which forms readily a t pH >9. This band was reported by Iwasaki et al. in their SER spectrum of a silver electrode in 0.5 M NaOH (22). The 472-cm-’ band is not observed in our lysine SER spectrum (Figure 1). In the spectrum of the pH 10.5 colloid, we also see the same two bands at 615 and 930 cm-’ as were observed in the spectrum of the colloid to which lysine was added. In fact, the intensity of these bands increases as the pH is increased from 9.5 to 10.0 but then begins to decrease at pH 10.5. The decrease occurs because the colloid becomes unstable at high pH and begins to precipitate. We conclude that the adsorbed species in the lysine SER spectrum and in the Ag colloid + NaOH spectrum apparently is/are identical.
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As prepared, the surface of the colloidal silver particle has a slight positive charge, with a potential at the surface of -200 mV vs SCE, compared with the potential of zero charge of --700 mV (23-25). The only ions present in the colloidal suspension without pH adjustment are Na+, NO3-, and B(OH)4-. Na+ would not be expected to adsorb onto the positively charged surface. The nitrate ion vibrations in the normal Raman spectrum of aqueous AgN03 are at 715 cm-’ (weak)and at 1046 cm-’ (very strong) and are clearly unrelated to the 615- and 930-cm-’ bands observed in our basic colloid and “lysine” SER spectra (Figures 2a and lb, respectively). By process of elimination, the latter two bands must be due to adsorbed borate. The infrared spectra of borate salts of lanthanum, scandium, and indium have bands at -613 and 940 cm-’ that have been assigned to the symmetric stretching and degenerate in-plane bending modes of borate ion (26). We conclude that the bands in the basic colloid and “lysine” SER spectra derive from adsorbed (surface-enhanced) borate rather than borate in solution because Raman scattering from 1 mM aqueous borate (without silver colloid) is too weak to be observed under the same spectral acquisition conditions. Similarly,we do not observe the 880-cm-’ borate band reported by Rubim and Diinnwald because that band is due to polymeric borate, which forms only at much higher solution borate concentrations (27,28). Other research groups have also observed borate bands in SER spectra obtained with borohydride-reduced silver colloids as the substrate. Zhang et al. reported spectral interference from both NO, and B(OH), in their SER spectra of tap water added to a silver colloid (11). Zhang et al. suggested that NO, in the colloid complexes with the divalent metal ions present in tap water, and that these complexes chemisorb onto the colloidal silver. The chemisorption of such complexes causes the colloid particles to aggregate, thereby enhancing the intensity of the nitrate and borate Raman bands. Borate vibrations were also observed by Yu et al. in their SER spectra of pyridine, 2,2’-bipyridine, and 1,lO-phenanthroline (12). Sbrana et al. and Kim and Itoh independently reported spectral interference from borate in their spectra of 2,2’-bipyrimidine on colloidal silver (13, 14). In the SER spectra of sulfa drugs containing a terminal amine group (sulfadiazine, sulfamerazine, and sulfamethazine) obtained by Sutherland et al., two bands between 600 and 1100 cm-’ were attributed to contaminants (29). Comparison of their spectra to our SER spectra of borate reveals that, almost certainly, their bands are also attributable to adsorbed borate. Interference from borate has apparently resulted in the misinterpretation of some published SER spectra. For example, the SER spectra of glycine, valine, and leucine obtained by Chumanov et al. are dominated by scattering from graphitic carbon, and by two bands a t 615-625 and 925-932 cm-’, both of which were assigned to the carboxylate bending mode (30). Our results suggest that these assignments may be erroneous and that the bands are attributable instead to vibrations of adsorbed borate. Furthermore, in the SER spectrum of sodium acetate reported by the same authors, only two bands are present, a t 622 and 930 cm-’, both of which were again assigned to carboxylate deformation modes. That spectrum is virtually identical with the spectrum in Figure lb, which leads us to conclude that the purported spectrum of acetate is actually that of borate. The addition of acetate, like lysine, increases the pH of the colloid, shifting the borate ion formation and adsorption equilibria to the right, as shown in the following simplified expressions: H3BO3 + OH- + B(OH)4B(OH)4-(solution)
B(OH),-(surface)
(1)
A similar process is probably responsible for the observation
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of borate bands in the spectrum of carbonate obtained by Kai et al. (31). The SER spectra obtained by Matsuta et al. of vacuumdried sodium borohydride reduced silver colloids exposed to water vapor are also identical with the borate SER spectra shown here (32). It is likely that the strong bands at 637 and 930 cm-' are due to adsorbed borate rather than surface oxidea. Surface enhancement of vibrations of adsorbed borate may also explain the differences between the sulfite and sulfate spectra obtained by Matsuta et al., and those obtained by Dorain et al. with colloidal substrates prepared with hydrazine or glucose as the reducing agent (33). We do not believe that the borate interference we observe is due solely to the analyte-induced increase in the degree of aggregation of the silver colloid, as was suggested by Zhang et al. (11,12). In SERS experiments in which we added other amino acids to aliquots of the same colloid as was used in the lysine experiments, borate vibrations were not observed. While the amino acids other than lysine did cause the colloid to aggregate, the SER spectra were of high quality and free from borate bands. Increasing the concentration of these analytes increased the degree of aggregation without causing borate bands to appear in the SER spectra. Instead, we suggest that the presence of borate bands in the SER spectra of basic analytes results in part from a small analyte-induced increase in the concentration of B(OH),- in solution. This alone is not sufficient to give rise to the appearance of borate bands in the SER spectrum, however. Adding sodium borate to our silver colloid to a final concentration of up to 0.012 M resulted in only weak bands a t 618 and 907 cm-'. Kai et al. observed no borate bands even when the concentration of sodium borate was 0.02 M (31). Adding NaBH, directly to the colloid also yields a null spectrum. It will be shown that only when the silver surface is partially oxidized, and/or when basic analytes (such as amines) are present with which borate can complex, does borate adsorb onto the colloidal silver surface. Furthermore, borate bands are observed only if borate can compete effectively with the analyte for adsorption sites. The relative surface coverage of the analyte and borate thus depend on the chemistry of the surface, and on the solution concentrations and relative adsorptivities of the analyte and borate. The influence of each of these factors is discussed below. On the basis of the borate ion formation and adsorption equilibrium expressions in eq 1, we expect the intensity of borate bands in SER spectra to be greater at higher pH, a t least until the surface is saturated with adsorbed borate. A significant complication, however, is that in alkaline solutions in the presence of an oxidant (e.g. 02),the silver surface becomes coated with oxide (34). As noted in the discussion of Figure 2, the broad band at 472 cm-' in the spectrum of the colloid to which base has been added is due to the Ag-0 stretching vibration of silver oxide, Ago (22). It is known that silver ion can readily form insoluble complexes with borate under basic conditions (35),and also that borate can complex with silver oxide in water even when both species are fairly dilute (36);borate or borate complex adsorption on the colloidal silver oxide surface would therefore not be surprising. Such a process could account for the experimental results in the following way: at alkaline pH, oxidation of the silver colloid surface, combined with a slightly higher borate concentration (still millimolar), leads to increased borate adsorption. This gives rise to more intense borate bands in the SER spectra than are observed at pH 6-7, even at borate concentrations that are 10 times higher. Borate bands dominate the SER spectrum of the lysine + silver colloid sample, shown in Figure Ib. In order to learn more about the relative adsorptivities of borate and lysine,
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Flgurr 9. (a) SER spectrum of 1.4 X lo4 M lysine adsorbed on collddal sllver, flnal pH 8. (b) SER spectrum of 2.7 X lod M lyskre adsorbed on colloidal silver, pH -7.
we decreased the pH of the lysine solution before adding it to the colloid. According to eq 1, this should have the effect of decreasing the borate concentration, which should permit lysine to compete more effectively for surface sites. Shown in Figure 3a,b are SER spectra of lysine obtained when the final pH of the colloid was 8 and 7, respectively. The lysine spectral features are more intense at pH 7 than at pH 8, both absolutely, and relative to the borate bands. The intensity of the 479-cm-' silver oxide band relative to the borate bands is also lower in the pH 7 spectrum, as expected. (The overall absolute intensity of the two spectra cannot be compared, as no internal or external standard was used.) The greater intensity of the lysine bands at pH 7 compared with pH 8 certainly suggesta that there is more lysine on the surface a t pH 7. We cannot determine from this experiment whether this is due to less surface oxidation, a lower borate concentration in solution, or both. The SER spectrum of lysine at pH 7 is quite different from the Raman spectrum of aqueous lysine shown in Figure lb, but nevertheless providea some insights into the lysine-surface interactions. In the SER spectrum, bands due to adsorbed borate (at 613 and 928 cm-') and graphitic carbon (the two broad bands centered at 1391 and 1579 cm-9, as well aa lysine, are present. The latter include bands at 1035,1137,1202,and 1273 cm-'. The first two bands are assigned to the C-N stretching and NH2 twisting vibrations. Suh and Moskovits observed these modes in their SER spectra of glycine, a-and @alanine, and 6-aminocaproicacid (3). The enhancement of the two amine modes is evidence for strong amine groupsurface interactions (3,6,18). Further experiments aimed a t determining whether both of the lysine amine groups interact with the surface are under way. The bands at 779,850, and 906 cm-' in the Raman spectrum of lysine are not observed in the SER spectrum; these bands may be obscured by the strong borate band at 928 cm-'. The CH2group vibrations observed in the Raman spectrum of aqueous lysine at 1319, 1349, and 1442 cm-', as well as the carboxylate stretching vibration at 1410 cm-', if present in the SER spectrum, are obscured by strong scattering from graphitic carbon. Evidence for the competitive adsorption of borate and basic M 2,2'-bianalytes is found in the SER spectra of
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(a)Spectrum of cltratweduced silver colloid aggregated wlth sodium perchbate (final concentration 1.2 X lo-* M). (b) 1.0 X 10" M sodium borate in coiloM prepared as in (a). Spectrum has been smoothed with a five-point polynomial smoothing functlon. Figure 5.
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Flgure 4. (a) Raman spectrum of 0.97 M propylamine, pH 12.5. (b) SER spectrum of 0.19 M propylamine adsorbed on colloidal silver prepared by sodkm borohyblde reductlon of silver nitrate, pH 11.5. (c) SER spectrum of 2 X lo4 M propylamine adsorbed on coiioMal silver prepared by cltrate reductlon of silver nitrate. (d) SER spectrum of 2.0 X lo4 M benzylamine adsorbed on colloidal silver prepared by sodium borohydride reduction of silver nitrate.
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pyrimidine obtained by Sbrana et al. (13) and of 10" M 2,2'-bipyrimidine obtained by Kim and Itoh (14). The spectra of Sbrana et al. contain both borate and 2,2'-bipyrimidine bands, indicating that the two species coadsorb. The intensity of the borate bands relative to the 2,2'-bipyrimidine bands decreased with time, and the borate bands disappeared when M chloride was added. Kim and Itoh found that chloride, bromide, and iodide all displaced the "byproducts" from the colloid preparation (14). It is known that chloride, bromide, and iodide adsorb strongly on silver, displacing even readily adsorbed analytes such as pyridine (37). The analyte band positions and relative intensities observed in the presence of chloride were the same as those observed in the presence of borate; from this we infer that 2,2'-bipyrimidine does not interact strongly with coadsorbed borate. In order to determine whether competitive adsorption of borate is a general problem in SERS of basic analytes such as amines, we examined the behavior of propylamine and benzylamine. Shown in Figure 4a is the normal Raman spectrum of 0.97 M propylamine a t pH 12.5, and in Figure 4b the SER spectrum of 0.19 M propylamine at pH 11.5. Borate bands are observed a t 613 and 923 cm-', just as in the
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lysine SER spectrum. Most of the propylamine bands in the solution Raman spectrum are discernible in the SER spectrum, superimposed on a background between 1300 and 1700 cm-l that arises from rather intense scattering from graphitic carbon formed on the surface (1S21).The band at 1138 cm-' is the most intense feature in the SER spectrum, and is assigned to the z NH2 vibration of the amine group (3,6). The intensity of this band indicates that the amine group is either close to, or interacts directly with, the silver surface. The SER spectrum of propylamine obtained with a citrate-reduced silver colloid 88 the substrate is shown in Figure 4c. This spectrum contains even more intense background scattering from graphitic carbon, but the propylamine features are still distinguishable. The bands at 507, 882, 1003, 1041, 1070,1136, and 1447 cm-' in the spectrum obtained with the citrate-reduced colloid correspond to the bands at 505,879, 1004,1037,1066,1138, and 1445 cm-' obtained with the borohydride-reduced colloid. The band at 613 cm-' and a large portion of the band at 923 cm-' observed with the borohydride-reduced colloid are absent in the spectrum obtained with the citrate colloid, confirming that these bands are due to adsorbed borate and not to the analyte (see also discussion of Figure 5, below). The similarities in the propylamine vibrational frequencies and relative intensities obtained with the two colloids indicate that propylamine adsorbs similarly on the two substrates. This supports the idea that amines and borate can both be adsorbed onto silver without interacting in such a way that the vibrational spectrum or surface interactions of the amine are perturbed. Borate does not interfere in the SER spectroscopy of all amines, as is evident from the spectrum of benzylamine shown in Figure 4d. No borate bands appear in the benzylamine SER spectrum under any conditions employed. This is because benzylamine adsorbs more readily than borate, and so competes more effectively for surface sites. Indirect evidence for the higher adsorptivity of benzylamine compared with propylamine, and hence for benzylamine competing more effectively for surface sites, is the fact that a lower threshold concentration is needed to obtain a good quality SER spectrum of benzylamine (2.0 mM) than propylamine (190 mM). In order to obtain further confirmation of the assignment of the 613- and 923-cm-' bands as borate vibrations, we ob-
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tained SER spectra of an aggregated citrate colloid with and without added borate, shown in Figure 5. As noted by Nabiev, it was necessary to add NaCIOl (final concentration 1.2 X lo-* M) in order to obtain SER spectra with the citrate colloid (38). The spectra have been d e d so that the 1025-cm-' band is approximately the same intensity in each spectrum. The spectrum of the aggregated colloid prior to borate addition (Figure 5a) is very similar to that obtained by Siiman et al. (39). All of the bands have been assigned to adsorbed citrate, including the bands a t 613 and 920 cm-' (39). Adding borate to the aggregated colloid (Figure 5b) leads to an increase in the intensity of the bands at 610 and 920 cm-' relative to the 1025-cm-' citrate band. These spectra clearly support our assignment of the 613- and 923-cm-' bands, observed in some SER spectra obtained with borohydride-reduced silver colloids, as borate vibrations. It is coincidental that similar bands appear in SER spectra obtained with citrate colloids. We have occasionally observed borate interference in S E W studies of nonbasic analytes such as 6-aminocaproic acid (40), but not of the L-amino acids. Although the a,@-aminoacids and amino acids contain a basic functional group, they exist as zwitterions in solution and do not substantially alter the pH of the colloid when present at concentrations of 104-109 M. We therefore would not expect the borate concentration to be substantially increased when a dilute amino acid is added to the colloid. Ultimately, we determined that small differences in individual experimenters' procedures for preparing the silver colloids caused the differences in the behavior of borate in the a,@-aminoacid and L-amino acid experiments. We found that minimizing the time during which the sodium borohydride is exposed to air, and adding the aqueous silver nitrate to the borohydride solution immediately after the borohydride solution is prepared, eliminate the borate bands from the crpamino acid SER spectra (40). This rapid handling procedure was not effective in eliminating borate bands from the SER spectra of basic analytes, however. For analytes such as amines, using a different substrate such as Carey-Lea colloids, citrate colloids, or roughened electrodes may be necessary.
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CONCLUSIONS SER spectra of basic analytes obtained with sodium borohydride reduced silver colloids may exhibit severe interference from adsorbed borate, as shown here for lysine and propylamine. A number of published spectra of diverse analytes also show bands that are probably attributable to borate. The relative surface coverage of the analyte and borate depend on the pH of the colloidal suspension, the solution concentrations of the analyte and borate, and the relative adsorptivities of the analyte and borate. The last factor is particularly important. For example, borate does not compete effectively with benzylamine for adsorption sites, but it coadsorbs with propylamine and virtually excludes lysine from the surface at pH 8. The oxidation of sodium borohydride during the preparation of the colloid and the formation of silver oxide when the colloid is made basic also facilitate borate adsorption. For some basic analytes, eliminating this interference may require using alternative SERS-active substrates. This is likely to be particularly important when SERS is used in the direct analysis of mixtures (41) and in chromatographic applications where SERS is used to detect and identify complex molecules (42). The propylamine results presented here suggest that citrate colloids, in addition to being more stable than colloids prepared by borohydride reduction, provide the additional advantage of eliminating the borate interference problem. Citrate colloids may therefore be suitable for a wider range of analytes in flow injection analysis and chromatographic applications (43, 44). The disadvantages are that citrate, like borate, may compete effectively with some analytes
for adsorption sites, and bands due to adsorbed citrate may then overlap analyte bands of interest. ACKNOWLEDGMENT We thank Katherine Kuhar and Eve Sullenberger for helpful discussions. LITERATURE CITED Moskovits, M. Rev. Mod. Fhys. 1985. 54. 783-828. Moskovits, M.; Suh, J. S. J . A m . Chem. SOC. 1985, 107, 6826-6829. Suh, J. S.; Moskovits, M. J . Am. Chem. SOC. 1988, 108, 4711-4718. Crelghton, J. A. In Specboscopy of Swfaces; Clark, R. S. H., Hester, R. E., Eds.; John Wlley and Sons: New Ywk, 1988; Chapter 2. Creighton, J. A. Swf. Sci. 1983. 124, 209-219. Herne, T. M.: Ahern. A. M.: Garrell. R. L. J . Am. Chem. Soc. 1991. 113, 846-854. Cotton. T. M.; U P ~ ~ UR.S A.; . Mijbiw, D. J . Fhys. Chem. 1988. 90. 6071-6073. Kovacs, E. J.; L o w , R. 0.;Vlncett, P. S.; Jennings, C.; Aroca, R. L a m u & 1988, 2 , 689-694. Creighton, J. A.; Blatchford, C. 0.; Albrecht, M. 0. J . Chem. Soc., Faraday Trans. 2 1979, 75, 790-798. Garrell, R. L. Anal. Chem. 1989, 61, 401A-411A. Zhang, C.P.; Yu, F.Q.; Zhang, G.-Y. J . Raman Spcbusc. 1989, 20, 431-434. Yu, F.-Q.; Zhang, C.P.; Zhang, G.-Y. J . Raman Speclrosc. 1989, 20, 435-438. Sbrana. G.; Neto, N.; Munlz-Miranda, M.; Nocentini. M. J . Fhys. Chem. 1990, 94, 3706-3710. Kim, M.; Itoh, K. J . Pnys. Chem. 1987, 91, 126-131. Ahern, A. M.; Garrell, R. L. Langmuk 1988, 4 , 1162-1168. Park, H.; Lee, B. S.; Kim, K.; Kim, M. S. J . Fhys. Chem. 1990, 94, 7576-7580. . -. - . - - -. Ono, C.; de Mul, F. F. M.; Huizinga, A.; Greve, J. J . phvs. Chem. 1988, 92, 1239-1244. Heme, T. M.; Ahern, A. M.; Garrell, R. L. AMI. chkn.Acta 1991, 246, 75-84. Mernaugh, T. P.; Cooney. R. P.; Turner, K. E. Chem. Fhys. Lett. 1984, 110, 536-541. Bowling, R. J.; Packard, R. T.; McCreery, R. L. J . Am. Chem. SIX. 1989, 1 1 1 , 1217-1223. Bowlina R.; Packard. R. T.; McCreery, R. L. L a m & 1989, 5, 683-688. Iwasakl, N.; Sasakl, Y.; Nishina, Y. J . Elec~mhsm.Soc. 1988, 135, 2531-2534. Wetzel. H.; Gerischer. H.; Pettinger. B. C t " . Fhys. Lett. 1982. 85, 187- 189. Wetzel, H.; Oetlscher, H. Chem. Fhys. Len. 1980, 76, 460-464. Lekis, D. I.; Rybalka, K. V.; Sevastyanov, E. S.; Frumkin, A. N. J . Electroa~l.Chem. 1973, 46, 181-169. S t d % W. C.; DeClUS, J. C. J . Chem. FhyS. 1958, 2 5 , 1184-1168. Rubim, J. C.; Diinnwald. J. J . Electroanal. Chem. IntwfechlEkCbp&em. 1989, 258, 327-344. Maya, L. Imrg. Chem. 1978, 15, 2179-2184. Sutherland, W. S.; Laserna, J. J.; Angebranndt, M. J.; Winefordmn, J. D. Anal. Chem. 1990, 62, 689-693. Churnanov, 0. D.; Efremov, R. G.; Nabiev, I.R. J . Ramen speclrosc. inno. .- - -, 21.43-48. - . .- .- . Kal, S.; Chauzhl, W.; Guangzhl, X. Spectrochlm. Acta 1989, 45A, 1029-1032. M%utai H;: Hkokawa, K. Appl. Speclrosc. 1989, 43, 239-245. Dorain, P. B.; Von Raben, K. U.; Chang, R. K.; Laube. B. L. Chem. phvs. Len. 1981, 84.405-409. b l a h a y , P.; PWrbalx. M.; Van Rysselberghe, P. J . €/"&em.Soc. 1951, 98. 65-67. Hickling, A.; Taylor, D. Dlscws. Farady Soc. 1947, 1 , 277-285. Sadeghi, N. Ann. Chim. 1987, 2 , 123-131. Garrell, R. L. Ph. D. Thesis, Unlversity of Michigan, 1984. Nabiev. I . Private Anrll 1991. - .cammunlcatbn. . - _S i i i n , ' 0.;Bumm, L. A.; Callaghan,rR.; Bitchford, C. G.; Kerker, M. J . Fhvs. Chem. 1983. 87. 1014-1023. KU&, K.; Garreii, R. L. work presented at the XVII FACSS meetlng. Cleveland, OH, Oct 1990 Abstr 689; Appl. Specmc., in prow. Lasema, J. J.; Camplglla, A. D.;Winefordner, J. D. AMI. CY". 1989, 61, 1697-1701. SBquaris, J.-M. L.; Koglin. E. Anal. Chem. 1987, 59, 527-530. Freeman, R. D.; Hammaker, R. M.; Meloan, C. E.; Fateby, W. G. Appl. SpeclrOSC. 1988, 42, 456-460. Shen. R.; NI, F.; Cotton, T. M. Anal. Chem. 1991, 63, 437-442.
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RECEIVED for review November 19,1990. Accepted July 18, 1991. We gratefully acknowledge support of this work by a National Science Foundation Presidential Young Investigator Award (R.L.G.), a graduate fellowship from the United S t a h Department of Education (T.M.H.), and grants from the National Institutes of Health, the Eastman Kodak Co., and BP America.
