Preliminary electrochemiluminescence study of allantoin in the presence of tris(2,2′-bipyridine)...

Copyright © 2005 John Wiley & Sons, Ltd. Luminescence 2005;20:109–116

Preliminary ECL study of allantoin in the presence of Ru (bpy)32+ ORIGINAL RESEARCH 109 ORIGINAL RESEARCH 109

Luminescence 2005; 20: 109–116Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bio.828 ORIGINAL RESEARCH

Preliminary electrochemiluminescence study of allantoinin the presence of tris(2,2′-bipyridine) ruthenium (II)

Xi Chen,1* Ying Tao,1 Li Zhao,1 Zenghong Xie2 and Guonan Chen2

1The Key Laboratory of Analytical Sciences of MOE and Department of Chemistry, Xiamen University, Xiamen 361005, People’sRepublic of China

2Department of Chemistry, Fuzhou University, Fuzhou, Fujian 350002, People’s Republic of China

ABSTRACT: Electrochemiluminescence (ECL) based on allantoin and tris(2,2′-bipyridine)ruthenium (II) [Ru(bpy)32+] was stud-

ied in aqueous alkaline buffer solution (pH 11.0). In a flowing system, the eluted allantoin was mixed with 1.0 mmol/L Ru(bpy)32+.

When the solution passed through a thin layer flow electrolytic cell equipped with a glassy carbon disc electrode (22.1 mm2), bothhydroxyl groups of allantoin and Ru(bpy)3

2+ were oxidized at the potential of +1.50 V (vs. Ag/AgCl). The luminescence with λmax

610 nm caused by the reaction of electrolytically formed Ru(bpy)32+ with alkoxide radical to generate the excited state of

Ru(bpy)32+*. A possible ECL process of allantoin in Ru(bpy)3

2+ alkaline solution has been discussed. In addition, the factorsaffecting the ECL response of allantoin are also investigated. Copyright © 2005 John Wiley & Sons, Ltd.

KEYWORDS: electrochemiluminescence; allantoin; tris(2,2′-bipyridyl) ruthenium; flow injection analysis

Copyright © 2005 John Wiley & Sons, Ltd.

INTRODUCTION

The electrogenerated chemiluminescence (ECL) methodusing tris(2,2′-bipyridine) ruthenium (II) [Ru(bpy)3

2+]has become an attractive means of detection for bio-chemical substances. Because of ECL’s low detectionlimit and wide linear working range, with relativelysimple instrumentation, it has been reported that ECLhas been used for the determination of numerousreducing amino compounds, such as amines or aminoacids and bioactive alkaloids (1–6). Moreover, somereductants of hydroxyl groups, such as alcohols (7),hydroxyl carboxylic acid (8), sugar (9) and ascorbicacid (10), exhibit ECL in a Ru(bpy)3

2+ alkaline solution.Generally, the ECL of Ru(bpy)3

2+ is caused by thereaction between its oxidized state, Ru(bpy)3

3+, and areductant to give Ru(bpy)3

2+* (see Scheme 1) (1):Since uric acid, the end-product of purine metabolism,

acts as an antioxidant and can react with biologically

*Correspondence to: X. Chen, The Key Laboratory of AnalyticalSciences of MOE and Department of Chemistry, Xiamen University,Xiamen 361005, People’s Republic of China.E-mail: [email protected]

Contract/grant sponsor: National Natural Scientific Foundation,People’s Republic of China; Contract/grant number: NSFC 20375033.

relevant oxidants to form allantoin (Fig. 1), allantoinexcreted in urine can be used as a microbial proteinindicator (11). Allantoin measurements in humans wereproposed as a marker of oxidative stress. Recently,the study of quantitative analysis of urinary allantoinhas become widely attractive. There are several ana-lytical techniques available to determine the compound.Colorimetric assays based on the Rimini–Schryverreaction (12) have been used for quantitation. Theseassays lack specificity, often resulting in overestimationdue to the presence of interfering material (12), and aresensitive to the experimental conditions employed (13).Although chromatographic methods, including liquidchromatography–mass spectrometry (LC–MS) (14),high-performance liquid chromatography (HPLC) (15,16) and capillary electrophoresis (CE) (17) are the mostcurrent methods for analysis, the equipments are expen-sive. Among the methods for the quantitation of urinaryallantoin based on HPLC (mostly reversed-phase), thecapacity factors (k%) in the published methods werevery small on reversed-phase HPLC columns, and resolu-tion was difficult to achieve, due to the extreme polarnature of allantoin. Another attempt by a precolumnderivatization method for allantoin HPLC determination

Figure 1. Chemical structures of allantoin and relatedcompounds.

Ru(bpy)32+ Ru(bpy)3

3+ − e−

Ru(bpy)33+�+�reductant Ru(bpy)3

2+∗+ oxidized product

Ru(bpy)32+∗ Ru(bpy)3

2+ + hν (λmax�=�610 nm)

Scheme 1. ECL reaction processes of reductant andRu(bpy)3

2+.

110 ORIGINAL RESEARCH X. Chen et al.


was reported by Chen et al. (18). In that study allantoinwas sequentially hydrolysed under alkaline and acidicconditions, and the product, glyoxylic acid (GLX),was allowed to react with 2,4-dinitro-phenylhydrazoneto form GLX 2,4-dinitrophenyl-hydrazone, which wasdetected at 360 nm. In the fluorescence or UV-Vismethods, the procedures for allantoin analysis aregenerally lengthy and often involve oxidation methodsto convert the analytes to detectable form (19,20).

In the course of the ECL study, we found thatallantoin would produce luminescence in the presence ofRu(bpy)3

2+. The ECL determination limit of allantoinwas 5 × 10−8 g/mL at S/N 2 in a flow injection systemwithout any pre-derivatizaion, which indicated that thismethod presents a higher sensitivity than the existingmethods. This paper describes a new method for flowinjection analysis of allantoin using an ECL detectionsystem. The behaviors and mechanism of the ECL reac-tion between allantoin and Ru(bpy)3

2+ were studied.Furthermore, factors influencing ECL intensity ofallantoin are also considered.

MATERIALS AND METHODS

Reagents

Ru(bpy)3Cl2·6H2O was obtained from Sigma ChemicalCompany (St. Louis, MO) and was used without furtherpurification. Allantoin was purchased from Bio-productInstitute of Chinese Medicine (Beijing, China). Allthe other chemicals were of guaranteed grade. Twice-distilled deionized water was used throughout. The stocksolution (0.01 g/mL) and working solutions of allantoinwere prepared by solution containing 1.0 mmol/LRu(bpy)3Cl2-50 mmol/L NaOH-NaAc-0.2 mol/L KNO3

(pH 11.0).

Instrumentation and procedure

The instrumentation for flow injection experimentsis showed in Fig. 2. A three-electrode system was usedfor potentiostatic control of the electrolytic system.

The working electrode was a glassy carbon disk (GC,22.1 mm2), the reference electrode was Ag/AgCl, andthe counter-electrode at the outlet consisted of a stain-less-steel pipe. Electrochemical measurements wereperformed by a CHI 800 Electrochemical Analyser(CHI Co., Shanghai, China). A flow system equippedwith a Rheodyne 7125 sample injector (Cotati, CA,20 µl) was used. The reagent solution was preparedby dissolving 1.0 mmol/L Ru(bpy)3Cl2 in 50 mmol/LNaOH-NaAc-0.2 mol/L KNO3 (pH 11.0). The flow ratewas 0.5 mL/min. The light emission was detected usinga photomultiplier tube (R464, Hamamatsu, Japan)and the signal was amplified and recorded using aChromatographic Processor (Echro 98, Yilite Co. Ltd,Dalian, China). The measurement of ECL spectra wascarried out with a set of cut-off filters from 450 nm to650 nm (Aopu Optics, Beijing, China).

RESULTS AND DISCUSSION

Effect of applied potential and cyclicvoltammetry

ECL intensity on a glassy carbon electrode (22.1 mm2)in a flow injection system was measured as a function ofthe applied potential. Sample solution (20 µl) containing1.0 µg/mL allantoin was injected into a carrier stream of1.0 mmol/L Ru(bpy)3Cl2 in 50 mmol/L NaAc-NaOH-0.2 mol/L KNO3, and then passed over the working elec-trode, which was held at a linearly increasing potentialfrom +1.1 to 1.8 V (vs. Ag/AgCl). The luminescence in-tensity of allantoin was measured and plotted against theapplied potential in Fig. 3. The ECL intensity was clearlyobserved when the applied potential was over +1.2 V,and increased as the applied potential increased to+1.5 V.

Since the applied potential affects the ECL beha-viours of Ru(bpy)3

2+ and coexisting compounds, cyclicvoltammetry (CV) was performed in 50 mmol/L NaAc-NaOH-0.2 mol/L KNO3 (pH 11.0). As shown in Fig. 4A,a reversible anodic wave of +1.05 V could be obtainedat the presence of Ru(bpy)3

2+ on the potential scan in the

Figure 2. Experimental set-up for ECL flow-injection application.



positive direction, resulting from oxidation of Ru(bpy)32+

to Ru(bpy)33+. Correspondingly, an irreversible anodic

wave at about +0.91 V was obtained in the allantoinoxidation (Fig. 4B,b). No cathodic wave was observedon the potential scan in the negative direction. It hasalready been reported that allantoin changes to itsanion form in alkaline solution (pH 10.5–11.5), as shownin Scheme 2 (21). Studies of oxidation of some aliphaticalcohols on a platinum electrode showed that theoxidized products involved alkoxide first radicals(RCH2O·) at a lower electrolytic potential, andthen aldehyde ion (RCHO−); the last stage includedcarboxylic ion (RCOO−) (22, 23). The anodic wave ob-tained from allantoin in alkaline NaOH-NaAc solutionprovides an evidence that the anodic current at approxi-mately +0.91 V is triggered by electrolytic oxidation ofallantoin in anion form to alkoxide radical (Scheme 2).Generally, the ECL emission was caused by the reactionbetween oxidized Ru(bpy)3

2+ and the reduced alkoxideradical from hydroxylic ion. According to our previousstudies, it can be concluded that the generating hydro-

xylic ion, which can be transferred to reducing alkoxideradical further, is a key reaction step in the allantoinECL process.

From the relationship between anodic current andapplied potential shown in Fig. 5, the anodic current in-tensity of Ru(bpy)3

2+ increased in the present of allantoinon the potential scan in the positive direction, andanodic current intensity at +1.05 V was changed from9.4 µA for Ru(bpy)3

2+ to 11.2 µA for allantoin addition.This is an evidence that oxidized Ru(bpy)3

2+ reacted withallantoin in alkaline solution, and the addition ofallantoin in Ru(bpy)3

2+ solution obviously increasedthe oxidation rate of Ru(bpy)3

2+ on the glassy carbon,resulting in the increase of ECL intensity. A furtherevidence of the electrolytic efficiency on the increaseof Ru(bpy)3

2+ could be obtained by chronocoulometryas Zhao’s description (24), which shows the effect ofallantoin on the electron-transfer quantity of Ru(bpy)3

2+

on the electrode. As shown in Fig. 6, four approximatelylinear sections were observed in both the forward (F)and reverse (R) directions.

In the presence of Ru(bpy)32+ solution, QF(Ru), QR(Ru)

and t1/2 represent the relationships as follows:

QF(Ru) = −1.3848 × 10−6 − 7.5812 × 10−5 t1/2, QR(Ru)

= −5.8600 × 10−6 − 5.7864 × 10−5 t1/2 (1)

Figure 3. Effect of applied potential on ECL. Experimentalconditions: concentration of sample, 1 µg/mL for allantoin;volume of injection sample, 20 µL; PMT voltage, −900 V;carrier solution, 1.0 mmol/L Ru(bpy)3Cl2 −50 mmol/L NaAc–NaOH–0.2 mol/L KNO3 (pH 11.0); flow rate, 0.5 mL/min.

Figure 4. Cyclic voltammograms of allantoin in alkaline aqueous solution. (A) 1.0 mmol/LRu(bpy)3

2+ + 50 mmol/L NaAc–NaOH–0.2 mol/L KNO3 (pH 11.0) (B) 50 mmol/L NaAc-NaOH–0.2 mol/L KNO3 (pH 11.0) buffer solution (a) and buffer solution + 100 µg/mLallantoin (b).

Scheme 2. Transformation of allantoin to its anion form inalkaline solution.



In the presence of Ru(bpy)32+ and allantoin solution:

QF(AT) = −4.8365 × 10−6 − 1.1839 × 10−4 t1/2, QR(AT)

= −6.2510 × 10−6 − 3.7727 × 10−5 t1/2 (2)

The slope of the linear section indicates the effectiveconcentration of Ru(bpy)3

3+ on the electrode. Before theECL reaction, in the presence of allantoin, the effectiveconcentration of Ru(bpy)3

3+ was 1.56-fold compared withthe values observed in the presence of Ru(bpy)3

2+ solu-tion only. After luminous emission, the concentration ofRu(bpy)3

3+ decreased by 65.2% for allantoin comparedwith that of Ru(bpy)3

2+ solution only. It is obvious thatthe increase of ECL intensity of allantoin is caused

Figure 5. Voltammetric response of CV experiment forallantoin. a, 1.0 mmol/L Ru(bpy)3

2+ + 50 mmol/L NaAc–NaOH–0.2 mol/L KNO3 (pH 11.0); b, 50 µg/mL allantoin +1.0 mmol/L Ru(bpy)3

2+ + 50 mmol/L NaAc–NaOH–0.2 mol/LKNO3 (pH 10.0).

Figure 6. Chronocoulograms of Ru(bpy)32+ and allantoin. a,

1.0 mmol/L Ru(bpy)32+ + 50 mmol/L NaAc–NaOH–0.2 mol/L

KNO3 (pH 11.0); b, 50 µg/mL allantoin + 1.0 mmol/LRu(bpy)3

2+ + 50 mmol/L NaAc–NaOH–0.2 mol/L KNO3 (pH10.0). Initial potential, 600 mV; final potential, 1200 mV; pulsewidth, 0.25 s; step, 2.

by the increase of electrolytic efficiency on the glassycarbon electrode.

pH and electrolytes of carrier solution

The ECL between Ru(bpy)32+ and amines depends on

the pH of the carrier solution. Uchikura (25) reportedthat the suitable pH for the determination of tryptophanwas 3, but pH 8 was good for primary amine afterderivatization with divinysulphone (26). Figure 7 illus-trates the ECL responses of allantoin as a function ofpH. Very weak ECL signal was obtained before the pHof the carrier solution reached 10.5. The strongest lumi-nescence for allantoin in the medium of Ru(bpy)3

2+ wasobserved at pH 11.0, and the slope decreased beyond pH11.5. Iwakura and co-workers (21) reported that theanodic oxidation of sodium methylate on a platinumelectrode proceeded as in Scheme 3. This suggests thatthe presence of hydroxyl ion greatly affects the anodicoxidation of methylate ion to methylate radical. The pHeffect seems to indicate that the higher concentration ofOH− promoted production of alkoxide radicals. Wenote, on the contrary, that the ECL intensity of allantoindecreased at pH values greater than 11.5, as shownin Fig. 7. One of the reasons for the decrease can beconsidered the reduction of the available concentrationof Ru(bpy)3

3+ at the high pH value, owing to the com-petitive reaction between allantoin and OH− ion (27).The tentative explanation of the mechanism was de-scribed by Mertin and co-workers (28), which includedthe presence of reactive intermediates producing OH·

Scheme 3. Anodic oxidation of methanol in alkaline solution.

Figure 7. ECL responses in different pH solutions. A, ECLof Ru(bpy)3

2+ as a function of pH; B, ECL of 20 µL injectionof 1.0 µg/mL allantoin. Other experimental conditions as inFig. 3.



the ECL reaction between ascorbic (H2A) (10) ordehydroascobric (DA) (27) acids and Ru(bpy)3

2+ inaqueous solution suggested that the intermediate radi-cal, HA−· or DA·, was strong enough to reduce and par-ticipate in an electron transfer reaction with Ru(bpy)3

3+.At an applied potential of +1.5 V, the electrochemicaloxidation of allantoin will form a alkoxide cation radi-cal, as shown in Scheme 2, and the radical cation thenreacts with oxidized Ru(bpy)3

3+ to produce light emis-sion. In Ru(bpy)3

2+–50 mmol/L NaOH-NaAc-0.2 mol/LKNO3 solution alone, the emitted light is due toelectro-oxidation of Ru(bpy)3

2+ to Ru(bpy)33+, and then

Ru(bpy)32+ reacts on dissolved oxygen and OH− in the

aqueous alkaline solution to yield a luminescence ata maximum wavelength of 610 nm (Fig. 8). The ECLintensity was enhanced by the addition of allantoin andthe maximum wavelength was kept the same, at 610 nm.As for our ECL system, the following mechanism forthe reaction of Ru(bpy)3

2+ with allantoin is proposed. Itis based the fact that alkoxide radical (A2−·) was pro-duced in alkaline solution (pH > 10) at the potentialof +1.5 V (vs. Ag/AgCl). Ru(bpy)3

2+ was also oxidizedto Ru(bpy)3

3+ at the applied potential. The result of thereaction between A2−· and Ru(bpy)3

3+ was the excitedstate Ru(bpy)3

2+*. ECL could not be obtained at a lowerapplied potential because oxidations of allantoin andRu(bpy)3

2+ were necessary. The ECL emission spectra at

or O2−· in OH− oxidation. The intermediates have a

long lifetime and react with Ru(bpy)33+ (Scheme 4). The

luminescence caused by the reaction of OH’ andRu(bpy)3

3+ made noise increase at higher pH. For theanalytical application, the pH of the solution was keptat 11.0.

ECL intensity is known to be highly dependent on theconcentration of electrolyte in the carrier solution, dueto the electrolytic efficiency (7). Greenway (2) reportedthat the ECL intensity of morphine was affected by theconcentration and species of electrolyte, and poor ECLresponse of morphine could be obtained in carbonatebuffer. Danielson (29) reported that more Ru(bpy)3

2+

could be electrochemically translated into Ru(bpy)33+ ion

and that Ru(bpy)33+ was more stable in acetate solution

compared to sodium sulphate. The luminescence inten-sity of Ru(bpy)3

2+ was increased by changing the sup-porting electrolyte, in the order NO3

−, CH3COO− (F−),CH3O

− (Cl−) and Br−. ECL intensity was also affectedby different kinds of buffer solution at pH 11.0. Compar-ing the ECL intensities of allantoin in NaOH-K2HPO4,NaAc-NaOH, KHCO3-NaOH, Na2B4O7-NaOH, allconcentrations were 50 mmol/L, containing 0.2 mol/LKNO3, pH 11.0, brighter luminescence was elicited inNaOH–NaAc buffer solution. The ECL intensity wasenhanced even by addition of micromolar amounts ofRu(bpy)3

2+; a higher concentration of Ru(bpy)32+ elicited

a large response, and noise was also enhanced simulta-neously. Because Ru(bpy)3

2+ made a higher lumines-

cence background signal, 1.0 mmol/L Ru(bpy)32+

wasused for greater noise/signal ratio. A lower flow rate ofreagent might cause larger background signal. The op-timal flow rate was estimated to be 0.5 mL/min.

Luminescence spectra

It has been clear that the luminescence of Ru(bpy)32+ is

caused by the reaction between the radical intermediateand Ru(bpy)3

3+ to produce an excited state (30,31). Inthe ECL reaction of Ru(bpy)3

2+ and amine, a strong re-duced radical cation intermediate was produced fromthe electron oxidation of amine to yield the exited stateRu(bpy)3

2+*. For an electron transfer reaction, an emis-sion with a maximum wavelength at 610 nm resultedwhen the excited-state Ru(bpy)3

2+* returned to groundstate (30). On the other hand, the mechanism for

Scheme 4. Reaction between Ru(bpy)32+ and OH−.

Figure 8. ECL spectra of Ru(bpy)32+ (A) and allantoin

+ Ru(bpy)32+ (B). Concentrations: Ru(bpy)3

2+, 1 mmol/L;allantoin, 20 µg/mL.



610 nm in Ru(bpy)32+ and in a mixture of Ru(bpy)3

2+ withallantoin solution can further confirm this conclusion. Inaddition, the amine group effects should be taken intoaccount in the ECL reaction of Ru(bpy)3

2+ and allantoin.Knight and Greenway reviewed the effect of a sub-stituent attached to the nitrogen of an α-carbon atom inamines in the ECL of amine and Ru(bpy)3

2+ (4). Gener-ally, the ECL reaction of Ru(bpy)3

2+ and amines involvesa charge-transfer process. The electron-withdrawingsubstituent attached to the nitrogen or α-carbon atomwill destabilize positive or electro-deficient radical ionsof amine, and hence tend to increase its ionizationpotential (IP) and decrease the ECL activity of thecompound (9). In the allantoin molecule, the electron-withdrawing substituent (carbonyl group) attached tothe α-carbon atom caused to change charge from nega-tive to positive. According to B3LYP/6-31 G(d) calcu-lation results, the IP values of N atoms in the allantoinionization form (A3−) are 7.35 eV, 7.40 eV, 7.38 eV and7.36 eV, respectively, indicating that the values increasedobviously more than those of IPs (about 7.05 eV) with-out a carbonyl substituent (9). Since the IPs values oftheophylline (chemical structure is showed in Scheme 1)are 7.38 eV and 7.53 eV for its two nitrogen atoms, re-spectively, the ECL signal of theophylline was too weakto be recorded under the same experimental conditions.The experimental results indicated that the contributionof amine groups in allantoin, which have large IP values,to ECL, could be neglected, due to their inactivity at theapplied potential. Furthermore, the IP values for trans-formation of hydroxyl groups to alkoxide radical (A2−·)in allantoin were around 7.04 eV, indicating that a lowerionic potential is needed in the oxidation of a hydroxylgroup. In order to confirm the main role of hydroxylgroups in the ECL of allantoin, uric acid, with a similarchemical structure to allantoin, was selected. It wasinteresting that almost the same intensity of light emis-sion could be obtained from the uric acid ECL reaction.The maximum emission wavelength was about 608 nmfrom its Ru(bpy)3

2+ solution. Since the carbonyl groupsin uric acid might be converted to hydroxyl groups inthe ionization process in the same alkaline solution, theresult confirmed that hydroxyl groups obviously play akey role in the ECL of allantoin. The ECL of Ru(bpy)3

2+

and allantoin in alkaline aqueous solution is presentedin Scheme 5.

Flow injection analysis

A typical flow injection recording of samples withallantoin concentrations of 0.1 and 1.0 µg/mL is shownin Fig. 9. The best ECL signal was achieved by elutingwith 50 mmol/L NaOH-NaAc (pH 11.0), 0.2 mol/LKNO3 carrier solution and a flow rate of 0.5 ml/min.Under these conditions, the calibration curve was linearfrom 7 × 10−8 to 1 × 10−5 g/mL allantoin. The determina-

tion limit was 5 × 10−8 g/mL at S/N 2, and the determin-ation showed good reproducibility, with RSD value wellbelow 3% at 1.0 µg/mL allantoin.

Some coexisting substances having a –N or –OHgroup, such as amino acids, fructose or glucose, produceECL emission in the NaOH-NaAc buffer (Table 1).Among these, only the secondary amino acids, such ashistidine, proline and hydroproline, gave a stronger lightemission. The weaker luminescence of carbohydratesand acids with –OH groups, such as saccharic acid, malicacid or tartaric acid, was also noted. A stronger light in-tensity was also produced from a few compounds, suchas ascorbic acid and sodium oxalate. Addition of metalion affected alkaloid ECL as well. The ECL intensity of1.0 µg/mL allantoin increased 21.9%, 4.7%, 13.2%,12.5%, 13.0%, 14.2% when 10 µg/L Zn2+, Ca2+, Ag+, Pb2+,Li+ and Mg2+, respectively, were added to the carriersolution. In addition, the ECL responses of VO2

2+ andWO2

2+ were measured in the same medium. A smallamount of VO2

2+ addition greatly increased ECL inten-sity, but 10 µg/mL WO2

2+ drastically quenched the ECLof allantoin.

Figure 9. Typical ECL responses of allantoin in Ru(bpy)32+

alkaline solution. Experimental conditions as in Fig. 3. Con-centration of allantoin: A, 0. 1 µg/mL; B, 1.0 µg/mL.

Scheme 5. ECL reaction sequences of Ru(bpy)32+ with

allantoin (A) or hydroxylic anion (B).



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Table 1. ECL response for co-exiting compounds

Compound ECL intensity/mVa

Histidine 1150Hydroxyproline 850Proline 1230d-Fructose 328d-Glucose 230Mannose 425Methanol 38Ethanol 12Thiamine 87Saccharic acid 276Malic acid 132Citric acid 595Sodium oxalate (2 µmol/L) 59

a At 0.2 mmol/L; other experimental conditions same as Fig. 3.

CONCLUSIONS

This study identified that ECL emission of allantoin wascaused by the reaction between Ru(bpy)3

2+ and itshydroxylic radical ion, generated in its ionization inalkaline solution. The contribution of the carbonyl groupof allantoin to the ECL can be neglected, due to itshigher ionization potential and inactivity on ECL. Inves-tigation results have shown that luminescence at thewavelength of 610 nm was generated by the return ofthe excited state of Ru(bpy)3

2+* to its ground state,Ru(bpy)3

2+. The supporting electrolyte also affected theelectrolytic efficiency of alkaloid, which led to differ-ent luminescence intensity. Flow injection analyticalexperiments showed that the ECL method providedhigher sensitivity for the determination of allantoin.Moreover, the avoidance of possible interferencesshould be considered in their application.

Acknowledgements

This work was supported by the National NaturalScientific Foundation of China (NSFC No. 20375033).We express our sincere thanks to Professor XiaoqingYang, College of Foreign Languages and Culture,Xiamen University, for her kind revisions.

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Preliminary electrochemiluminescence study of allantoin in the presence of tris(2,2′-bipyridine)...

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