MICROCHEMICAL JOURNAL 58, 13–20 (1998)ARTICLE NO. MJ971503

Study of the Electrochemiluminescence Basedon Tris(2,2*-bipyridine) Ruthenium(II) and

Alcohols in a Flow Injection System

Xi Chen,*1 Masanori Sato,† and Yongjing Lin*

*Department of Chemistry, Research Laboratory of SEDC of Analytical Science for Material and LifeChemistry, Xiamen University, Fujian 361005, People’s Republic of China, and †Laboratory of

Analytical Chemistry, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto 606, Japan

Received November 3, 1996; accepted March 18, 1997

The luminescence electrooxidation of monohydric alcohols and polyhydric alcohols in tris(2,2*-bipyridine) ruthenium(II) [Ru(bpy)2/

3 ] alkaline solutions on a glassy carbon electrode has beenstudied. At a potential of /1.30 V (vs Ag/AgCl), Ru(bpy)2/

3 was oxidized to Ru(bpy)3/3 . The

luminescence wavelength of 608 nm could be found because Ru(bpy)3/3 reacted on alkoxide

radical to produce the excited state. Study of the luminescence intensities of monohydric alcoholsshowed that luminescence intensity decreased as alkyl chain length of the molecules increased.With the increase in the number of hydroxyl groups in a molecule, luminescence intensityincreased for polyhydric alcohols. q 1998 Academic Press

Key Words: electrochemiluminescence; alcohols; tris(2,2*-bipyridine) ruthenium(II).

INTRODUCTION

The chemiluminescence of tris(2,2*-bipyridine) ruthenium(II) [Ru(bpy)2/3 ] has been

reported (1–3). Analytes examined include hydrazine (4), oxalate (5), peroxy disulfate(6), aliphatic amines (7), amino acids (8–10), and ascorbic acid (11). Generally, theelectrochemiluminescence (ECL) of Ru(bpy)2/

3 is due to the reaction of its oxidizedstate, Ru(bpy)3/

3 , with reductants to give Ru(bpy)2/*3 as follows:

Ru(bpy)2/3 r Ru(bpy)3/

3 / e,

Ru(bpy)3/3 / reductant r Ru(bpy)2/*

3 / oxidized product,

Ru(bpy)2/*3 r Ru(bpy)2/3 / light.

We recently reported that many kinds of organic acids with hydroxyl groups exhibitthe ECL phenomenon in solutions of Ru(bpy)2/

3 (11). Methanol is commonly used asan eluant in high-performance liquid chromatography (HPLC), but it was found to beunsatisfactory in the Ru(bpy)2/

3 ECL–HPLC system at pH ú 10, because the back-ground signal increased and the S/N ratio decreased with addition of methanol, ac-cording to our studies. This indicates that the higher background signal was causedby the chemiluminescence reaction between methanol and Ru(bpy)3/

3 .This paper describes the behaviors and mechanism of the ECL reaction between

1 To whom correspondence should be addressed.

130026-265X/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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14 CHEN, SATO, AND LIN

FIG. 1. Experimental setup for ECL-FI.

monohydric alcohols and Ru(bpy)2/3 . Factors influencing ECL intensity are also

discussed.

EXPERIMENTAL

Instrumentation

Luminescence intensity was observed by modifying a commercially availablechemiluminescence analyzer (S-3400, Soma Kogaku, Tokyo, Japan). A thin-layerelectrolysis cell for ECL observation was designed in our laboratory. The main bodyof the cell was composed of two pieces of Diflon block tightly fixed to each other.Since luminescence intensity is closely related to the orifice shape and thickness ofthe Teflon spacer inside the cell, a spacer sheet with a thickness of 50 mm was chosen.The volume of the thin layer cell was 1.5 ml. The experimental setup is shown in Fig.1. A three-electrode system was used for potentiostatic control of the electrolyticsystem. The working electrode was a glassy carbon disk (GC, 22.1 mm2). The counter-electrode set at the outlet consisted of a stainless-steel pipe, and the reference electrodewas Ag/AgCl. A potentiostat (Princeton Applied Research, Type 174A) was used forelectrolysis.

Flow injection experiments were performed using a peristaltic pump (Saruki,DM2M-1024) equipped with a SV1-6U7 sample injector (volume, 10 ml). The carriersolution was prepared by dissolving 0.05 mM Ru(bpy)2/

3 in 0.2 M KCl–KOH solution(pH 12.0). The flow rate was 0.4 ml/min. A VP-6537 pen recorder (Matsushita Co.Ltd.) was used to record luminescence intensity. Cyclic voltammertry (CV) experi-ments and luminescence spectra were performed in batch systems; other experimentswere completed in flow injection systems.

Chemicals and Solutions

Ru(bpy)3Cl2r6H2O was purchased from Sigma Chemical Company (St. Louis, MO)and was used without further purification. Monohydric alcohols and D-glucitol werefrom Nacalai Tesque Inc. (Kyoto, Japan), and other polyhydric alcohols were obtainedfrom Tokyo Chemical Industry Company (Tokyo, Japan). 1,2,3-Propanetriol was pur-

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15ELECTROCHEMILUMINESCENCE OF ALCOHOLS

FIG. 2. Voltammetric responses of CV experiment (A) and ECL intensities as a function of electrodepotential (B). (a) 0.5 mM Ru(bpy)2/

3 (/) basal solution; (b) 50 mM methanol / basal solution (---, basalsolution); (c) 50 mM 1-propanol / basal solution; (d) 50 mM 1-pentanol / basal solution; (a*) 0.05 mMRu(bpy)2/

3 / basal solution; (b*) 10 mM methanol / a*; (c*) 10 mM 1-propanol / a*; (d*) 10 mM 1-pentanol / a*. Basal solution: 0.2 M KOH–KCl, pH 12.0, scan rate: 50 mV/s.

chased from Merck (Darmstadt, Germany). All other chemicals were of guaranteedgrade. Twice-distilled deionized water was used throughout. The stock solution (0.1M) and working solutions of all alcohols were prepared by carrier solution.

RESULTS AND DISCUSSION

Cyclic Voltammetry and ECL Intensity as a Function of Applied Potential forMonohydric Alcohols

To determine the optimum applied potential for observation of ECL, CV of somemonohydric alcohols was performed using a GC electrode in 0.2 M KCl–KOH solution(pH 12.0). Figure 2 shows that a reversible anodic wave with Ep Å /1.20 V couldbe obtained in the presence of Ru(bpy)2/

3 on the potential scan in the positive direction.This resulted from oxidation of Ru(bpy)2/

3 to Ru(bpy)3/3 (Fig. 2, a). Irreversible anodic

waves at about /1.28 V for methanol, /1.34 V for 1-propanol, and /1.45 V for 1-pentanol could be attributed to oxidation of these alcohols. No cathodic waves wereobserved among them on the potential scan in the negative direction (Fig. 2, b–d).The widths of these anodic waves were increased and less negative in the solutioncontaining 0.05 mM Ru(bpy)2/

3 . This is because the anodic oxidized potential ofRu(bpy)2/

3 is close to the oxidized potentials of monohydric alcohols, and they cannotbe instrumentally distinguished from each other. In addition, the potentials at whichoxidation of monohydric alcohols occurred became less positive, and the correspond-ing anodic electrolytic currents decreased with the increase in alkyl chain length(Table 1).

ECL intensities (IECL) at the same GC electrode were measured as a function of thepotential, as shown in Fig. 2B. The intensity was low, even when the potential scan

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16 CHEN, SATO, AND LIN

TABLE 1Comparison of Oxidation Potentials and Relative ECL Intensities of Monohydric Alcohols

POX PECL ip IECLa lb IRu

ECLc lc

Compound (V) (V) (mA) (mV) (nm) (mV) (nm)

Methanol /1.28 /1.52 1.25 22 545 256 608Ethanol /1.31 /1.58 1.21 18 550 239 6091-Propanol /1.34 /1.60 1.00 15 558 235 6081-Butanol /1.40 /1.61 0.40 8 572 201 6091-Pentanol /1.45 /1.60 0.12 3 — 122 6081-Hexanol /1.48 /1.65 0.10 1 — 65 608

a In 0.2 M KCl–KOH solution, pH 12.0. Concentration of each alcohol: 0.1 M applied potential: /1.50 V(AgCl/Ag).

b In 0.2 M KCl–KOH solution, pH 12.0. Concentration of each alcohol: 1.0 M, applied potential: /1.50 V(AgCl/Ag).

c In 0.2 M KCl–KOH solution, 0.05 mM Ru(bpy)2/3 , pH 12.0. Concentration of each alcohol: 10 mM,

applied potential: /1.65 V (AgCl/Ag).

was over /1.20 V for Ru(bpy)2/3 solution. Clearly, the addition of alcohol greatly

enhanced the IECL . The IECL peaks were obtained by scanning the applied potentialsto the positive direction beyond /1.52 V for methanol and /1.60 V for 1-propanoland 1-pentanol. In addition, weaker luminescence could also be obtained if 0.1 Mmonohydric alcohols were added to the basal solution. Table 1 shows the potentialsat which the ECL peaks appeared and the relative maximum luminescence wavelengthand IECL; there are no significant relationships between the potentials of ECL peaks

FIG. 3. Effect of solution pH on the luminescence intensity of monohydric alcohols. l, Methanol; s,ethanol; , 1-propanol; m, 1-butanol; l, 1-pentanol; ., 1-hexanol. Concentration of each alcohol: 10 mM.

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17ELECTROCHEMILUMINESCENCE OF ALCOHOLS

and IECL , but IECL also decreased as alkyl chain length increased whether or not thesolution contained Ru(bpy)2/

3 .It has already been reported that the maximal ECL of hydroxyl groups is observed

at the almost same potential in the Ru(bpy)2/3 solution (11). The light emission was

caused by the reaction between the reducing intermediate (hydroxylic radical ions)and Ru(bpy)2/

3 . Studies of oxidation of some aliphatic alcohols on a platinum electrodein nonaqueous solution showed that the oxidized products involved alkoxide radicals[(RCH2O

•), R Å H(CH2)n , n Å 0–5] at a lower electrolytic potential first, and thenaldehyde ion (RCHO0); the last stage included carboxylic ion (RCOO0) at a morepositive electrolytic potential (12–14). Recently, Hitmi et al. analyzed the product ofthe oxidation of ethanol (15). When the potential applied was lower than 0.8V/rhe, the major product of the oxidation of ethanol was acetaldehyde; the oxidationproceeded directly to acetic acid when the potential was greater than 0.8 V/rhe. Theproducts of the electrooxidation of CH3OH in alkaline media were suggested to beHCHO and HCOOH (16). Karatani and Shizuki reported the molar ratio of HCOO0

to HCHO to be about 85:15 at /1.3 V (vs Ag/AgCl), and it increased with thepotential in alkaline solution (17).

The anodic waves obtained for monohydric alcohols are evidence that the anodiccurrent at approximately /1.28 to /1.48 V is triggered by electrolytic oxidation ofmonohydric alcohols to alkoxide radicals and carboxylic ions in alkaline solution:

OH0 / RCH2OH r RCH2O0 / H2O,

RCH2O0

r RCH2O• / e,

RCH2O• / OH0

r RCHO0 / H2O,

RCHO0 / 3OH0r RCOO0 / H2O / 3e.

pH Studies

The chemiluminescence between Ru(bpy)2/3 and some compounds could be obtained

in a wide pH range; for example, tryptophan was at pH 3.0 (18), oxalate at pH 6.0(3), ascorbic acid at pH 7.2 (11), and some amino acids at pH 10 (19). Figure 3illustrates the ECL responses of the monohydric alcohols as a function of pH. Veryweak ECL signals were obtained before the pH of the carrier solution reached 10.ECL responses increased significantly with increase in pH of the solution over 10 andthe slopes decreased beyond pH 11.5.

Iwakura et al. reported that the anodic oxidation of sodium methylate on a platinumelectrode proceeded as follows (12):

CH3OH / OH0r CH3O

0 / H2O,

CH3O0

r CH3O• / e.

At high oxidation potential, the major product of the reaction of methylate ion withhydroxyl ion electrochemically is formate ion:

CH3O0 / 4OH0

r HCOO0 / 2H2O / 4e.

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18 CHEN, SATO, AND LIN

This suggests that the presence of hydroxyl ion greatly affects the anodic oxidationof methylate ion to methylate radical and formate ion. The pH effect seems to indicatethat the higher concentration of OH0 was helpful in producing alkoxide radicals.

Study of Electrolytes and Other Parameters

To obtain the optimum conditions for ECL observation, the choices of optimalparameters (composition of solution and flow rate of carrier solution) should be madein addition to the pH study. ECL intensity is known to be highly dependent on theconcentration of electrolyte in the carrier solution due to the effect on electrolyticefficiency (20). Sasaki et al. reported the effect of changing the supporting electrolyteon the current efficiency of methanol oxidation (13). The order of reactivity of thesalts examined was F0 É CO20

4 ú CN0 É OH0 ú Cl0 É CH3O0 ú Br0. Comparing

the ECL intensities of methanol in K2CO3, NaH2PO4, and KBr solutions (all concentra-tions were 0.2 M, pH 12.0), brighter luminescence was obtained when KCl orCH3COONa was used. Potassium chloride was selected because sodium acetate gavelarger basal luminescence in alkaline solution. The concentration of Ru(bpy)2/

3 affectedthe luminescence intensities of monohydric alcohols; ECL intensity was enhancedeven by addition of micromolar amounts of Ru(bpy)2/

3 . Although a higher concentra-tion of Ru(bpy)2/

3 elicited a large response, it also yielded higher noise. The lumines-cence intensity decreased when the flow rate was higher than 0.45 ml/min. The lumi-nescence intensity was higher at a lower flow rate, but the background signal wasalso increased in reverse. Thus, the optimal flow rate was estimated to be 0.4 ml/min.

Luminescence Spectra

Karatani and Shizuki reported that the ECL spectral maximum of methanol wasabout 550 nm (17). Table 1 shows the spectral maximum shifted from around 545nm toward longer wavelengths for methanol, ethanol, 1-propanol, and 1-butanol.The luminescence intensities of 1-pentanol and 1-hexanol were too weak to measuretheir spectra. Although Ru(bpy)2/

3 alone in alkaline solution turned emitted light onelectrolytic oxidation, its intensity was greatly enhanced by addition of these alco-hols. The maximum emission wavelength is about 608 nm for Ru(bpy)2/

3 andRu(bpy)2/

3 –RCH2OH solution.The mechanisms of the chemiluminescence reaction between Ru(bpy)2/

3 and oxalate(3) and amino acid (8, 10) have been studied. Ru(bpy)2/

3 was oxidized at a GCelectrode to Ru(bpy)3/

3 . At the same time, the strong reducing intermediate (radicalions) produced from oxidized oxalate or amino acid yielded the excited state,Ru(bpy)2/*3 , by an electron transfer reaction with trivalent ruthenium species. Anemission having a maximum at 610 nm was produced when the excited-state moleculereturned to ground state (2, 3). On the other hand, the mechanism for the ECL reactionbetween pyruvate and Ru(bpy)2/

3 in aqueous solution suggested that the intermediateradical, CH3CO•, was a strong enough reducing agent to participate in electron transferreactions with Ru(bpy)3/

3 (3).As for our ECL system, the following mechanism for the reaction of Ru(bpy)2/

3

with the monohydric alcohols is proposed. It is based on the fact that alkoxide radicalswere produced in the first step in alkaline solution (17) and then further oxidized tocarboxylic ions at about /1.30 V, where the electronically excited states of aldehydes

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19ELECTROCHEMILUMINESCENCE OF ALCOHOLS

TABLE 2Comparison of ECL Intensities of Polyhydric Alcoholsa

Luminescence intensityCompound (mV)

1,2-Propanediol 2931,3-Propanediol 2681,2,3-Propanetriol 5991,2-Butanediol 3222,3-Butanediol 2401,4-Butanediol 2631,2-Ethanediol 528Erythritol 379Adonitol 681D-Glucitol 723

a Concentration of each polyhydric alcohol: 10 mM.

could be produced. Ru(bpy)2/3 was also oxidized to Ru(bpy)3/

3 at the applied potential.The result of reaction between alkoxide radicals and Ru(bpy)3/

3 was the excited stateRu(bpy)2/*3 . ECL could not be obtained at lower applied potentials because oxidationof Ru(bpy)2/

3 was necessary. The ECL emission spectra of Ru(bpy)2/3 and of a mixture

of Ru(bpy)2/3 with monohydric alcohols (Table 1) can further confirm this conclusion.

The ECL reaction sequence of monohydric alcohols and Ru(bpy)2/3 is

Ru(bpy)2/3 r Ru(bpy)3/

3 / e

RCH2OH / OH0r RCH2O

0 / H2O

RCH2O0

r RCH2O• / e

RCH2O• / OH0

r RCHO0 / H2O

2RCH2O•r RCHO* / RCH2OH

RCHO0 / 3OH0r RCOO0 / 2H2O / 3e

4OH0 / RCH2O• / 3Ru(bpy)3/

3 r RCOO0 / 3Ru(bpy)2/*3 / 3H2O

Ru(bpy)2/*3 r Ru(bpy)2/3 / light (l Å 608 nm)

Comparison of ECL Intensities for Polyhydric Alcohols

It is of interest to compare the ECL intensities of polyhydric alcohols. As shownin Table 2, it was observed that ECL intensity increased with the number of hydroxylgroups. The orders of ECL intensities are D-glucitolú adonitolú erythritolú 1,2,3-propanetriol ú 1,2-ethanediol and 1,2-ethanediol ú ethanol, 1,2,3,-propanetriol ú1,2-propanediol, ribitol ú 1,2-butanediol, 2,3-butanediol ú 1-butanol. In addition,polyhydric alcohols bearing several adjacent alcoholic hydroxyl groups were weakerlight emitters than those bearing nonadjacent alcoholic hydroxyl groups; for example,their orders of ECL intensities were 1,3-propanediol ú 1,2,3-propanetriol ú 1,2-propanediol and 1,4-butanediol ú ribitol ú 1,2-butanediol, 2,3-butanediol, respec-

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20 CHEN, SATO, AND LIN

tively. This was due to the self-inhibition among the neighboring oxidized andremained unoxidized alcoholic hydroxyl groups because alcoholic hydroxyl groupsusually act as radical scavengers (21). These results are a further step indicatingthat radical intermediates were generated in the luminescence reaction of alcoholsand Ru(bpy)2/

3 .In conclusion, the product of the reducing intermediates, alkoxide radical ions, has

an important role in the luminescence of monohydric alcohols and Ru(bpy)2/3 . Investi-

gations have shown that the luminescence is obtained only in alkaline solution beyondpH 11.0, indicating that methanol is suitable for use as an eluate for HPLC applicationsbelow pH 11.0 in the Ru(bpy)2/

3 –ECL system. Brighter emission was obtained withshorter alkyl chain length because of their less negative oxidized potentials amongthe monohydric alcohols. The supporting electrolytes also affected the electrolyticefficiency of monohydric alcohols, which led to the different luminescence intensities.In addition, polyhydric alcohols with more hydroxyl groups had brighter emission,but those bearing adjacent alcoholic hydroxyl groups yielded weaker luminescence.These results indicate that compounds such as aldehydes, ketones, and organic acidswith OH groups should emit light on reaction with Ru(bpy)2/

3 . Their determinationwill broaden the applicability of ECL detection.

ACKNOWLEDGMENTSWe express our sincere thanks to the Japanese Ministry of Education, Science and Culture for their

donation of the scholarship to Xi Chen. We are also grateful to the State Education Commission of Chinaand the Chinese Postdoctoral Foundation for their support of this report.

REFERENCES1. Hercules, D. M.; Lytle, F. E. J. Am. Chem. Soc., 1966, 88, 4745–4746.2. Tokel, N. E.; Bard, A. J. J. Am. Chem. Soc., 1972, 94, 2862–2863.3. Rubinstein, I.; Bard, A. J. J. Am. Chem. Soc., 1981, 103, 512–516.4. Nonidez, W. K.; Leyden, D. E. Anal. Chim. Acta, 1978, 96, 401–404.5. Rubinstein, I.; Martin, C. R.; Bard, A. J. Anal. Chem., 1983, 55, 1580–1582.6. Ege, D.; Becker, W. G.; Bard, A. J. Anal. Chem., 1984, 56, 2413–2417.7. Noffsinger, J. B.; Danielson, N. D. Anal. Chem., 1987, 59, 865–868.8. He, L.; Cox, K. A.; Danielson, N. D. Anal. Lett., 1990, 23, 195–210.9. Jackson, W. A.; Bobbitt, D. R. Anal. Chim. Acta, 1994, 285, 309–320.

10. Lee, W. Y.; Nieman, T. A. J. Chromatogr. A, 1994, 659, 111–118.11. Chen, X.; Sato, M. Anal. Sci., 1995, 11, 749–754.12. Iwakura, C.; Hayashi, T.; Kikkawa, S.; Tamura, H. Electrochim. Acta, 1972, 17, 1085–1093.13. Sasaki, K.; Urata, H.; Uneyama, K.; Nagaura, S. Electrochim. Acta, 1967, 12, 137–146.14. Sundholm, G. J. Electroanal. Chem., 1971, 31, 265–267.15. Hitmi, H.; Belgsir, E. M.; Leger, J. M.; Lamy, C.; Lezna, R. O. Electrochim. Acta, 1994, 39, 407–415.16. Sundholm, G. In Encyclopedia of Electrochemistry of the Elements: Organic Section (A. J. Bard, Ed.)

Vol. 11, p. 207.17. Karatani, H.; Shizuki, T. Electrochim. Acta, 1996, 41, 1667–1676.18. Uchikura, K.; Kirisawa, M. Anal. Sci., 1991, 7, 971–973.19. Stephen, N. B.; Bobbit, D. R. Anal. Chem., 1992, 64, 166–170.20. Chen, X.; Sato, M. J. Flow Injection Anal., 1995, 12, 54–65.21. Karatani, H. J. Photochem. Photobiol. A Chem., 1994, 79, 71–80.

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