Anal. Chem. 1999, 71, 2523-2527
Quenching of Electrogenerated Chemiluminescence by Phenols, Hydroquinones, Catechols, and Benzoquinones J. McCall, C. Alexander, and M. M. Richter*
Department of Chemistry, Southwest Missouri State University, Springfield, Missouri 65804
Efficient quenching of Ru(bpy)32+ (bpy ) 2,2′-bipyridine) electrogenerated chemiluminescence has been observed in the presence of phenols, catechols, hydroquinones, and benzoquinones. In most instances, quenching is observed with 100-fold excess of quencher over Ru(bpy)32+, with complete quenching observed between 1000- and 2000fold excess. The mechanism of quenching is believed to involve energy transfer from the excited-state luminophore to benzoquinone. In the case of phenols, catechols, and hydroquinones, quenching is believed to occur via a benzoquinone derivative formed at the electrode surface. Photoluminescence and UV-visible experiments coupled with bulk electrolysis support the formation of benzoquinone products upon electrochemical oxidation. A wide variety of methods exist for the detection of chemical and biological analytes of interest. One of the most versatile, and one being commercially developed for the clinical diagnostic market,1 is electrogenerated chemiluminescence or electrochemiluminescence (ECL). ECL is a means of converting electrical energy into light (radiative energy). It involves the formation of electronically excited states by energetic electron-transfer reactions of electrochemically generated species.2-4 Traditionally, ECL was generated via annihilation, where the electron-transfer reaction is between an oxidized and reduced species, both of which are generated at an electrode by alternate pulsing of the electrode potential.4 Of more interest to practical applications, ECL can also be generated in a single step utilizing a coreactant (i.e., a species capable of forming strong oxidants or reductants upon bond cleavage).5-8 For example, in the previously studied Ru(bpy)32+/C9H21N system (C9H21N ) tri-n-propylamine),7 ECL is produced upon concomitant oxidation of Ru(bpy)32+ and C9H21N: (1) IGEN International Inc.; Boehringer Mannheim Corp.; Perkin-Elmer Corp.; Organon Teknika Ltd. (2) Tokel, N.; Bard, A. J. J. Am. Chem. Soc. 1972, 94, 2862. (3) Glass, R. S.; Faulkner, L. R. J. Phys. Chem. 1981, 85, 160. (4) Faulkner, L. R.; Bard, A. J. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1977; Vol. 10, pp 1-95. (5) Rubinstein, I.; Bard, A. J. J. Am. Chem. Soc. 1981, 103, 512. (6) White, H. S.; Bard, A. J. J. Am. Chem. Soc. 1982, 104, 6891. (7) Leland, J. K.; Powell, M. J. J. Electroanal. Chem. 1991, 318, 91. (8) (a) Chang, M.-M.; Saji, T.; Bard, A. J. J. Am. Chem. Soc. 1977, 99, 5399. (b) Rubinstein, I.; Bard, A. J. J. Am. Chem. Soc. 1981, 103, 512. (c) Rubinstein, I.; Martin, C. R.; Bard, A. J. Anal. Chem. 1983, 55, 1580. (d) Ege, D.; Becker, D. W.; Bard, A. J. Anal. Chem. 1984, 56, 2413. 10.1021/ac981322c CCC: $18.00 Published on Web 05/15/1999
© 1999 American Chemical Society
Ru(bpy)32+ - e- f Ru(bpy)33+ C9H21N - e- f [C9H21N•]+ f C9H20N• + H+ C9H20N• + Ru(bpy)33+ f *Ru(bpy)32+ + products
where *Ru(bpy)32+ is the electronically excited species capable of undergoing emission. The ECL mechanism and subsequent signal generation in this system is believed to occur through an “oxidative-reductive” pathway. This involves production of a strong reducing agent (presumably C9H20N•)9 by an initial oxidation sequence. This radical can then reduce Ru(bpy)33+ to *Ru(bpy)32+.7,10 Alternately, C9H20N• may reduce Ru(bpy)32+ to Ru(bpy)31+ followed by annihilation:6,11,12
Ru(bpy)31+ + Ru(bpy)33+ f *Ru(bpy)32+ + Ru(bpy)32+
ECL has become increasingly attractive for the detection of numerous chemical and biological molecules, and many of its known applications have been reviewed.13,14 Our own interest stems from a desire to understand the fundamental mechanisms of ECL and to develop new ECL-based applications. The ECL of Ru(bpy)32+ in solution, specifically the emission of the metal-to-ligand charge transfer (MLCT) excited state, is very well-known2-8 and it serves as a model for transition-metalbased ECL sensitizers. Also, the excited states of ruthenium and osmium polypyridyl systems (e.g., Ru(bpy)32+) are sensitive to subtle changes in solution composition.15 By changing the solvent (e.g., H2O, CH3CN, and their mixtures) or electrolyte or adding chemical species to solution, variations in the ECL emission efficiency (photons emitted per redox event) and wavelength maximum can be observed. For example, the ECL intensity of (9) Smith, P. J.; Mann, C. K. J. Org. Chem. 1969, 34, 1821. (10) McCord, P. M.; Bard, A. J. J. Electroanal. Chem. 1991, 318, 91. (11) Richter, M. M.; Debad, J. D.; Striplin, D. R.; Crosby, G. A.; Bard, A. J. Anal. Chem. 1996, 68, 4370. (12) (a) Boletta, F.; Rossi, A.; Balzani, V. Inorg. Chim. Acta 1981, 53, L23. (b) Boletta, F.; Ciano, M.; Balzani, V.; Serpone, N. Inorg. Chim. Acta 1982, 62, 207. (13) Knight, A. W.; Greenway, G. M. Analyst 1994, 119, 879. (14) (a) Yang, H.; Leland, J. K.; Yost, D.; Massey, R. J. Biotechnology 1994, 12, 193. (b) Blackburn, G. F.; Shah, H. P.; Kenten, J. H.; Leland, J.; Kamin, R. A.; Link, J.; Pterman, J.; Powell, M. J.; Shah, A.; Talley, D. B.; Tyagi, S. K.; Wilkins, E.; Wu, T.-G.; Massey, R. J. Clin. Chem. 1991, 37, 1626.
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Ru(bpy)32+ increased in the presence of benzene,16 and this has been suggested as a potential means of detecting aromatic hydrocarbon pollutants. In an effort to expand on this earlier work and develop analytically important sensors, we were interested in testing other aromatic derivatives for both ECL activity and their effect on the ECL excited state. Here we report on the use of phenolic compounds (e.g., phenols, catechols, hydroquinones, and benzoquinones), a class of pharmaceutically and environmentally important molecules. EXPERIMENTAL SECTION Materials. Ru(bpy)3Cl2 (98%, Strem Chemical Inc., Newbury Port, MA), phenol (99.99%, Aldrich Chemical Co., Milwaukee, WI), catechol (99%, Aldrich), hydroquinone (99+%, Aldrich), benzoquinone (99.5%, Fluka, Milwaukee, WI), potassium phosphate monobasic hydrate (99.0%, EM Science, Gibbstown, NJ), tetrabutylammoniumhexafluorophosphate (Aldrich), 2,3-dichloro-5,6dicyano-1,4-benzoquinone, 1,2,3,4-tetrafluoro-5,8-dihydroxyanthraquinone (Aldrich), 2,5-dibromo-1,4-benzoquionone (Aldrich), 2-methoxy-3-methyl-1,4-naphthoquinone (Aldrich), anthroquinone1,5-sulfonic acid (Aldrich), and tri-n-propylamine (C9H21N, 98%, Avocado Research Chemicals, Ward Hill, MA) were used as received. Acetonitrile was spectroquality (Burdick & Jackson). Potassium phosphate buffer solutions, 0.20 M KH2PO4‚7H2O, were prepared with doubly deionized water. Buffer solutions containing C9H21N (0.05 M) were prepared similarly except that it was necessary to stir vigorously to completely dissolve the amine. The pH of these solutions was adjusted to 7.5 ((0.1) with either concentrated HCl or 6 M NaOH. Methods. ECL intensity vs potential profiles were monitored using a photomultiplier tube (Hamamatsu HC 135) in conjunction with a CH Instruments electrochemical analyzer (Cordova, TN). Solutions were 0.3-30 µM Ru(bpy)32+, 0.05 M C9H21N, 0.2 M potassium phosphate buffer, and 0-10 mM phenol, catechol, hydroquinone, or benzoquinone. The potential was cycled from 0.0 to +1.8 to 0.0 V vs Ag/AgCl at 0.1 V/s and the light intensity recorded every 0.01 V. Maximum intensities were obtained at +1.3 V vs Ag/AgCl (( 0.1V) and these values used to generate intensity vs concentration profiles. The experiments employed a conventional three-electrode configuration. The cell was designed to fit in front of the photomultiplier tube and held a total volume of 50-75 mL. A platinum gauze electrode (6 × 9 mm gauze flag) was employed as the working electrode, with a coiled Pt wire auxiliary (0.5 mm diameter) and Ag/AgCl gel reference (E° ) 0.226 V vs NHE17). The working electrode was cleaned prior to each experiment by repeated cycling (+2.0 to -2.0 V vs Ag/AgCl at 0.2 V/s) in dilute (15) See, for example: (a) Kober, E. M.; Sullivan, B. P.; Meyer, T. J. Inorg. Chem. 1984, 23, 2098. (b) Caspar, J. V.; Meyer, T. J. J. Am. Chem. Soc. 1983, 105, 5583. (c) van Houten, J.; Watts, R. J. J. Am. Chem. Soc. 1975, 97, 3843. (d) van Houten, J.; Watts, R. J. J. Am. Chem. Soc. 1976, 98, 4853. (e) Kitamura, N.; Kim, H.-B.; Kawanishi, Y.; Obata, R.; Tazuke, S. J. Phys. Chem. 1986, 90, 1986. (f) Barigelletti, F.; Juris, A.; Balzani, V.; Belser, P.; von Zelewsky, A. J. Phys. Chem. 1987, 91, 1095. (g) Sun, H.; Hoffman, M. Z. J. Phys. Chem. 1993, 97, 11956. (16) Dixon, B. G.; Sanford, J.; Swift, B. W. In Principles and Practices for Petroleum Contaminated Soils; Calabrese, E. J., Kostecki, P. T., Eds; Lewis Publishers: New York, 1993; pp 85-99. (17) Faulkner, L. R.; Bard, A. J. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1977; Vol. 10, pp 1-95.
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sulfuric acid, followed by immersion in concentrated nitric acid and rinsing in distilled water. Controlled-potential coulometry experiments (bulk electrolysis) were performed using a standard three-electrode system available from Bioanalytical Systems Inc. (West Lafayette, IN). A reticulated vitreous carbon or platinum mesh working electrode biased to an appropriate potential was used to effect electrolysis, with an Ag/AgCl gel electrode used as reference. A Pt wire counter electrode was separated from the working solution via a porous Vycor frit and immersed in an appropriate electrolyte solution. Fluorescence experiments were run in potassium phosphate buffer using a Shimadzu RF-5301 PC fluorometer. Excitation was at 452 nm, the lowest energy absorption for the Ru(bpy)32+ luminophore, with detection between 550 and 650 nm (λem ) 620 nm). Absorption spectra were recorded with an Hitachi U-2001 spectrophotometer. All solutions were composed of micromolar concentrations of Ru(bpy)3Cl2 and 0.05 M C9H21N in water or acetonitrile (0.2 M potassium phosphate and 0.1 M tetrabutylammonium hexafluorophosphate as electrolytes, respectively). Phenol, catechol, hydroquinone, or benzoquinone 100 mM stock solutions were prepared by dissolving an appropriate amount of compound in 10 mL of ethanol and then diluting with the Ru(bpy)32+/C9H21N solution. Working solutions were then prepared by diluting aliquots of these stock solutions with the aqueous or acetonitrile Ru(bpy)32+/Pr3N solutions. RESULTS AND DISCUSSION Experiments have shown that concentrations of phenol greater than 1 mM effectively quench solution ECL in the Ru(bpy)32+/ C9H21N reaction sequence. This is surprising, since the photoluminescence efficiency of Ru(bpy)32+ is known to increase slightly upon addition of phenol.18 However, the ECL of codeine and related pharmaceuticals decreased in the presence of compounds containing phenolic functional groups (e.g., acetaminophen).19 The mechanism of quenching was not studied in detail, but it was proposed that the amine radical might be interacting with the phenolic moiety prior to reaction with Ru(bpy)32+.19 Quenching is observed in the present work with 100-fold excess of quencher (as compared to ECL luminophore), with complete quenching observed between 1000 and 2000-fold excess. Similar results were obtained using oxalate (C2O42-) as coreacant.20 The Ru(bpy)32+/ C9H21N system shows ∼5% higher quenching efficiency, which is within the reproducibility of our experimental results ((10%). Therefore, the quenching of the ECL excited state is not coreactant dependent. Several mechanisms for quenching are possible. The first involves direct quenching of the ECL excited state (e.g., *Ru(bpy)32+) by phenol. However, this seems less likely since fluorescence studies show no excited-state quenching.18 Second is the possibility that the tripropylamine radical (C9H20N•) is being intercepted prior to reaction with Ru(bpy)32+.19 This is also less likely due to the relative concentrations of the species in solution (∼50-fold excess of coreactant vs phenol). The third involves formation of a quenching species upon oxidation of phenol. (18) Li, C.; Sun, H.; Hoffman, M. Z. J. Photochem. Photobiol. A. 1997, 108, 129133. (19) Greenway, G. M.; Knight, A. W.; Knight, P. J. Analyst 1995, 120, 2549. (20) Oxalate, like C9H21N, is an “oxidative-reductive” coreactant. Upon oxidation, it is believed to undergo bond cleavage to form a strong reductant (presumably CO2•).8
Figure 1. Fluorescence spectra of Ru(bpy)32+ (30 µM) in aqueous buffer media containing 0.05 M C9H21N and 0.06 M phenol (λexc ) 452 nm). The potential was stepped to +1.4 V vs Ag/AgCl to effectively oxidize Ru(bpy)32+. C9H21N, and phenol (E° ) 1.3, 0.5, and 1.0 V, respectively). Bulk electrolysis was performed for 3 h with samples taken at 30-min intervals. The spectra reported above are for the following times: (A) 0 min, no electrolysis; (B) 30 min; (C) 2 h; and (D) 3 h.
In a previous study, Hoffman and co-workers18 observed no photoluminescence quenching in the Ru(bpy)32+ emission spectrum in aqueous media with 0.2-0.6 M phenol present. Therefore, to determine whether electrolysis was required for quenching under conditions necessary for ECL, phenol was added in appropriate concentrations to an aqueous buffer solution containing Ru(bpy)32+ and C9H21N. The photoluminescence was then measured with no electrolysis performed. Varying concentrations from 0 to 0.3 M showed that photoluminescence increased slightly as the concentration of phenol was increased, opposite that observed during ECL. Also, the quenching effect observed during electrolysis/ECL is more dramatic than the enhancement observed during photoexcitation (95%) loss of the 620nm emission band of Ru(bpy)32+ is observed in the presence of 0.1 M BQ, emphasizing the efficiency of BQ as an excited-state quencher. Coupling bulk electrolysis with photoluminescence for CAT and HQ resulted in complete loss of photoluminescence with the concomitant formation of a reddish-brown solution. This change in color from clear to reddish-brown is indicative of the formation of benzoquinone species. These results parallel those observed with phenol and indicate that CAT, HQ, and phenol are converted to products at the electrode surface that are then capable of quenching excited states. As noted above, CAT does quench
indicate that benzoquinone or a benzoquinone derivative is formed during electrolysis. We have also observed ECL quenching with derivatives of phenol substituted in the 2-, 3-, and 4-positions (e.g., o-, m-, and p-fluorophenol). The magnitude of quenching is a function of both the nature of the substituent group and its position within the ring. A detailed study of fluorescence, UV-visible, NMR, and ECL properties of luminophores with substituted phenols will be the subject of a forthcoming report. Figure 5. Electronic absorption spectra for (9) phenol with no electrolysis performed (time 0) (2) phenol after passage of 3.016 × 102 C of charge (time 6200 s) and (b) benzoquinone with no electrolysis performed.
Ru(bpy)32+ photoluminescence. However, at comparable concentrations to the photoluminescence study, the product of CAT oxidation quenches the photoluminescent excited state more efficiently than CAT itself. With BQ, on the other hand, little to no enhanced quenching of the photoluminescence is observed upon bulk electrolysis. In fact, a slight increase in luminescence intensity was observed, indicating that upon prolonged oxidation BQ starts to decompose to nonquenching products. On the time frame of an ECL experiment (2 h), the formation of nonquenching benzoquinone species is expected to be minimal. UV-visible spectroscopy was used to investigate the nature of the color change observed during bulk electrolysis. Figure 5 depicts a typical spectrum comparing phenol with benzoquinone and the products of oxidation. The absorbance spectrum of the phenol after bulk electrolysis resembles the spectrum of benzoquinone. CAT and HQ display similar trends. Therefore, fluorescence and UV-visible experiments coupled with bulk electrolysis
CONCLUSIONS In all instances, the presence of phenolic compounds leads to the efficient quenching of an electrochemiluminescent excited state. The differences between the ECL and photoluminescence data suggest that these types of interactions could be very important in probing energy- and electron-transfer processes of Ru(II) sensitizers in solution and at charged interfaces. Also, ECL quenching may find use in the environmental analyses of phenolic compounds as well as in biotechnology and pharmaceutical applications. ACKNOWLEDGMENT We gratefully acknowledge the financial support of Southwest Missouri State University. We also thank Drs. Rich Biagioni and Michelle Driessen for helpful comments in the preparation of the manuscript.
Received for review December 1, 1998. Accepted April 5, 1999. AC981322C
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