E L S E V I E R Journal of Electmanalytical Chemistry 438 (1997) 113- ! 19

Redox reaction of benzoquinone on a lipid coated glassy carbon electrode l

Hyun Park, Jang-Su Park, Yoon-Bo Shim * Department of Chemistry. Pusan National University. Pusan 609-735, South Korea

Received I August 1996; received in revised form 7 October 1996

Abstract

Benzoquinone reduction on a lipid coated glassy carbon electrode has been studied in aqueous solutions employing spectroelectro- chemical techniques. The intermediate species, benzoquinone anion radical (BQ~), thus far believed to be unstable in aqueous has been shown to be rather stable in a lipid layer on a glassy carbon electrode, especially in a buffered solution. The kinetic parameters D O and /'o lot the oxidation reaction of benzoquinone anion radical (BQ ~) on a phosphafidylcholine (PC) coal~ electrode were determined to be 8.1 × I0-6 cm 2 s - ' mid 3.2 × 10-2 cm s - i respectively, by cluenoconlometry. The electrochemical data showed that the reduction products of BQ are incorporated inlo the PC layer. The interaction of the reduction intermediates with PC was investigated in detail with FT-IR and NMR spectroscopy, which showed that the reduction product, the anion radical, was bound to the h - ~ bonded phosphate group in the PC molecule. © 1997 Elsevier Science S.A.

Keywords: Benzoquinone: Lipid: Anion radical of benzoquinone: Spectmelectrocheraistry

1, Introduct ion

Quinones are biologically important molecules because of their function of transferring electrons in lipid layers. Thus, it is important to develop a good understanding of their electrochemical behavior on lipid coated electrodes to elucidate the roles of quinone groups in the lipid layer. A lipid layer loaded on an electrode allows the redox active amphiphiles to interact with the lipid layer, which exhibits peculiar electrochemical reactions in an aqueous solution. Kaifer and coworkers [1] reported the electrochemical behavior of tile electrodes ¢~ ted with a layer of phos- phatidylcholine (PC). They reported that the more hy- drophilic redox-active amphiphilic compounds were re- jected by the lipid layer. Tanaka and Tamamushi [2] also reported that hydrophobic layers hindered the electrochem- ical reaction of hydrophilic species substantially. In our preliminary experiments, the cyclic voitammograms of Ru(NH3 )3+ and Fe(CN) 3- showed lower currents at the

" Corresponding author. Fax: +82-51-516-7421; E-mail: ybshim@hy- owon.cc.pusan.ac.kr.

t This paper was presented at the lntematioual Symposium on Electron Transfer in Protein and Supramolecular Assemblies at Interfaces held in Shonan Village, Xanagawa, Japan on 17 to 20 March 1996.

0022-0728/97/$17.~ © 1997 Elsevier Science S.A. All rights t~served. Pll S0022-0728(96)04984-4

PC coated glassy carbon (GC) electrodes compmed with those measured at a bare GC electrode during the redox. These results are in agreement with the observation of Kalfer and coworkers [1]. We also found that p-henzo- quinone (BQ) incorporates with the PC layer on the GC electrode and exhibits a new redox wave in aqueous media, which was not observed on the bare GC electrode in the same solution.

It is well known [3] that BQ undergoes an elecwochemi- cal reduction to hydroqulnone (H2Q) in aqueous media by the reaction

BQ + 2H++ 2e-~ H2Q (I)

A thorough understanding of the redox reaction mecha- nism for reaction (1) is important in interpreting many biological reactions [3]. To date, a number of studies on react/on ( I ) have been conducted in aqueous media [4-9]. The ,~:duction mechanism of BQ involves two types of react,on scheme, namely e - H + e - H + and H+e-H+e - mechanisms, which depend on experimental cor~litions. Here e - and H + represent electron transfer and protoua- tion steps respectively [7]. Because of the cumplexity of the mechanism and the instability of the intermediate species in aqueous solutions, most studies of the reaction mechanisms used indirect electrochemical techniques to

114

show a specific reaction path under a given experimental condition. Both anion radicals and dianions are reasonably stable in aprotic solvents [10-14]. The electronic absorp- tion spectra of anion radicals have been obtained in aque- ous solutions by using pulse radiolysis [15-17], with the exception of a report by Shim and Fark [18] wtio used a platinum electrode to reduce BQ to the anion radical BQ ~ in unbuffered solutions.

In the present study, we investigated the spectroelectro- chemical and electrochemical behavior of BQ in aqueous solutions at different pH values to characterize the new redox wave of BQ that appeared on the lipid coated GC electrode. Cyclic voltammetry, in situ UV-vis spectroelec- trochemistry, FT-IR and FT-NMR spectroscopy were em- ployed to investigate the electrochemical reaction of BQ and the interaction between BQ and lipid layers. The kinetic parameters were determined by chronocoulometry.

2. Experimental

2.1. Materials

H. Park et al. /Journal of Electroanalytical Chemistry 438 (1997) 113-119

W.¢:,

C,E, ""

Personal O i i]] I I Computer Xenon Flash pttoal I I I L_ I Lamp Fiber I I I

Interface ' ~ ' ~ _ .1 _ ..Stopper ~ o uuart;z Cell

Fig. 2. Block diagram for the UV-v is speetroclectrochemical system with an IBM PC.

p-Benzoquinone (BQ, Aldrich, 98%) was purified by a sublimation method after recrystallization in ethanol. Hy- droquinone (H2Q, Fluka, 99%) was purified by recrystal- lization in ethanol. Fig. 1 shows molecular structures of the lipids used in the present study. PC was purified by silicic acid column chromatography [19]. Phosphatidic acid (PAt was prepared from PC by hydrolysis in the presence of cabbage phospholipase D [20,21]. The purity of PA was confirmed by silicic acid thin layer chromatography (TLC). Distilled water (18Mfl cm) was obtained from a Milli-Q system. Buffer solutions were prepared using citric acid + sodium citrate and NaH2PO 4 + Na2HPO 4 mixtures. All other reagents were of the best commercial quality avail- able.

2.2. Electrodes and equipment

Before each experiment, the GC electrode (Tokay Co., GC-30S) was polished mechanically with 0. ! Ixm alumina powder to a mirror finish and rinsed with ethanol. The PC

a.

(CH3hN+--CH2--CH2 --O--P--O--CH2--CH--O---C--R 2

tol I CH2--O--C--R!

--O--P--O--CH2--CH--O--C--R2

CH2--O--C--RI

Fig. I. Molecular structure of (at phosphatidylcholine and (b) phospha- tidie acid.

was dissolved in chloroform solution to an adequate con- centration (10 mg ml- i ). A 3.0 I ~1 aliquot of the solution was dropped onto the surface of the GC electrode. The solvent was then evaporated while slowly rotating the electrode (200rev min -~ ) to increase the homogeneity of the resulting cast lipid layer. The electrode potentials given in the present paper were measured with respect to an AgIAgCI[KCI~ t electrode. The counter electrode was a Pt wire. The temperature of the solution was 250(2. A Pine Instrument Co. model AFRDE4 bipotentiostat and an EG &G PAR model 273 potentiostat/galvanostat were used to record cyclic voltammograms and to control the electrode potentials during the spectroelectrochemical measure- ments. In situ spectroelectrochemical measurements were made with an Ocean Optics Co. model SI000 spectrograph in reflectance mode coupled with a CCD array detector, a xenon lamp, and an optical fiber probe. As shown in Fig. 2, the bifurcated fiber optical beam probe was located on the window of the quartz electrochemical cell above the working electrode. The illumination area through the fiber bundle was smaller than that of the electrode. The spectro- electrochemical system in the refl ,etion mode is similar to one previously described in another communication [22]. The spectra and DCVA signals were plotted employing the software Jandel Scientific Co. SigmaPlot, after data acqui- sition was achieved from the system. IR and NMR spectra were obtained with a Mattson Polaris FF-IR spectrometer and a Varian Unity Plus 300MHz NMR spectrometer respectively.

3. Results and discussion

3.1. Cyclic voltammetry o f BQ at GC electrodes

Fig. 3 shows the cyclic voltammograms recorded for the redox reactions of 1.0mM of (at BQ and (b) H2Q at bare GC electrodes (dashed lines) and PC modified dec- trod*s (solid lines) in a 0.1 M phosphate buffer solution (pH 7.0). In CVs using the PC modified GC electrode, two pairs of CV peaks were observed at - 0 . 1 7 6 / - 0 . 2 3 2 V

H. Park et aL / Journal of Electronnalytical Chemistry 438 ( i 997) ! 13- ! 19 i ii!ii~!!

115

.,,w, . . . . I'

ilo , \ . :

t . | . i . t . , ! , ,

-0.6 .0.4 -0.2 0.0 012 0.4 016 0'.8

E/V vs. AglAgCI

Fig. 3. Cyclic voltammograms of 1.0mM (a) BQ and (b) H2Q in O.IM phosphate buffer solution at pH 7.0 at bare (dashed line) and PC coated GC electrodes (solid line). Scan rate 20raV~-I.

T 1

w | ! ! | t t

-0.7-0.6-0.5-0.4-0.3-0.2.0.1 0.0 0.1

E / V vs. Ag[AgCl

Fig. 4. Time dependence of the newCV peak reconted at a PC coati GC electrode inunersed in a l.OmM BQ+O.IM plmsplu~ buffer ([gt 7.0) solution. Scan rate IOmVs -I.

vs. Ag[AgCI ( A E p - - 5 6 m Y ) which, m our knowledge, have not been reported in the literature, and at + 0 . 2 4 1 / - 0.110V (AEp =- 351 mV), which must correspond to reac- tion (1) (Fig. 3(a)). The new redox wave at - 0 . 1 7 6 / - 0.232 V vs. AgIAgCI appeared only after holding at - 0 . 5 V for 3 min at PC modified electrodes, while the CVs did not show the new redox wave during a scan from a positive or zero potential to a negative one. Thus, all the CVs in our experiment were taken after the potential of the PC coated CJC electrode was maintained at - 0 . 5 V in a buffer solu- tion containing BQ for 3 min. The new redox wave, which appeared at a more negative potential than the reduction of BQ to H2Q, appears reversible under the experimental conditions used here. On the contrary, the new redox wave was not observed at an unmodified GC electrode in BQ solution, and it was not observed in the H2Q solution although using the PC coated GC electrode after holding the potential at - 0.5 V. ~ihe redox waves of both BQ and H2Q originating from reaction (1) at the PC coated GC electrode are of the same shape, but the peak heights are smaller compared with those observed at a bare GC elec- trode.

Fig. 4 shows the time dependence of the new CV peaks recorded with the PC coated electrode immersed in a 1.0 mM BQ + phosphate buffer solution of pH 7.0. As the number of potential cycles between - 0 . 6 and + 0 . 0 V was

increased, the peak currents of the new teciox wave be- came higher and reached a steady current after 10 cycles. This result shows that the reduction intermediate of BQ was accumulated in the PC layer from the bulk o f the solution during the potential cycling. The peak separation of the new redox wave was about 55 mV, implying that the number of electrons participating in this reduction reaction may be one.

We plotted the peak current of the new redox wave versus scan rate, obtained in a 1.0 mM BQ solution with a PC coated electrode in the phosphate buffer solution of pH 7.0. From this plot (not shown), the anodic and cathodic peak currents were linearly proportional to the scan rate from lOmV up to 50mV s -~. However~ the .r-~k c u r r e ~ were p ro lg~ona i to the square root of the scan rate over 70mV up to 200mVs - t . This observation suggests that a

Table 1 CV parameters for BQ on the PC coated GC elecm3de in various pH media

pH Redox wave 1 Redox wave 2

Epa/V Epc/V AEp/mV Epa/V F_~/V AEp/mV

3 +0.473 +0.0¢,0 422 5 +0.340 +0.005 345 -0.147 -0.2.50 103 7 +0.241 -0.110 351 -0.176 -0.232 56 8 +0.21~ -0.064 279 -0.176 -0.23! 55

1 1 6 H. Park et aL / Jou~na! of Electroanalytical Chemistry 438 (1997) !13-119

a

b

. . . . . * . . . . ! i , i i i i , , , |

-1.0 -0.5 0.0 0.5 1.0

EA r vs. AglAgCI

Fig. 5. Cyclic voltammograms of 1.0mM BQ with a PC coated GC electrode recorded in various pH media: (a) 3.0, (b) 5.0, (c) 7.0 and (d) 8.0. The scan rate in all cases was 20mVs -t.

f'mite diffusion model is operative during the lower scan rates, whereas a semi-infinite model takes ovel at higher scan rates. This means that the diffusion layer thickness is comparable with that of the lipid layer, through which the electroactive compound, a reduction intermediate of BQ incorporated into the PC layer, must diffuse into exchange electrons at the electrode. This is similar to the charge transport, via physical diffusion of electroactive reac'wmts themselves, where the electroactive reactants incorporated in the films are attached to functional groups present in polymer films [23].

Fig. 5 shows cyclic voltammograrns of 1.0 mM BQ at the PC coated electrode recorded at various pH values. After the reduction of BQ at - 0.5 V for 3 rain at various pH values, an anodic shoulder and a single pair of reduc- tion/oxidation peaks corresponding to reaction (1) with a large peak separation (about 400mV) were observed at + 0.473 and + 0.050 V respectively at pH about 3. Also, a small anodic peak corresponding to the new reaction is observed. However, two pairs of CV peaks are clearly observed at pI-I above 4. With an increase in pH of the

solution, the new wave becomes more pronounced. The peak separation of this redox peak decreased with increas- ing pH, then it became constant (about 55 mV) at above pH 7. The parameters for the two reduction waves are summarized in Table 1.

The above results for the new redox wave of BQ appearing at the PC modified electrode suggest that the two waves must be involved in different processes. Also, for the reaction at a more negative potential, the number of electrons involved appears to be one. The peak potential of the new wave is constant regardless of the pH of the solution above pH 5. It is quite likely that the new redox wave on the more negative potential is

BQ + e- ~ BQ ~ ( 2 )

because the reaction is independent of proton concentra- tion. No potential shift at the PC coated electrode was observed above pH 5. In other words, the anion radical BQ ~ is reasonably stable in the PC layer. These observa- tions suggest that the reduction pathway of BQ at a PC coated electrode is different from that observed at a bare electrode. In the case of the electrochemical reaction at lipid modified electrodes, the reduction intermediate species was observed even in a well-buffered solution because local proton exhaustion gave rise to an increase in pH in the lipid layer. This behavior is similar to that observed in an unbuffered solution, or the reaction in solutions at higher pH values. Thus, the PC layer stabilizes the reduction intermediates BQ ~.

3.2. Kinetic studies

Since a semi-infinite diffusion prevails on a short exper- imental timescale, in which the diffusion layer thickness is less than the film thickness, conventional electrochemical analysis procedures are applicable [23]. Thus, we calcu- lated kinetic parameters of this experimental system with :hronocoulometry. In the chronocoulometric experiments, the electrode potential was stepped from - 3 1 0 to 0 mV. A plot of the charge Q with respect to t I/2 (not shown) was linear, satisfying the equation Q = 4.18 p,C - 42.9t t/2 (correlation coefficient 0.998) in 1.0mivi BQ at a PC coated GC electrode. From this data, the diffusion coeffi- cient D o was calculated from

2nFADIo/2Co t'/2 Q ¢rl/2 q" Qd.I + nFAFo

2nFAD~/2Co slope = wt/2 (3)

where F is Faraday's constant, A is the area of the electrode (0.139cm2), c~ is the concentration of the bulk solution (1.OmM BQ), and n is the number of electrons involved in the electrochemical reaction. The calculated diffusion coefficient of the reduction product BQ ~, D o, was 8.1 x 10-6 cm2 s -I at pH 7.0.

H. Park el aL / Journal of Electroanalytical Chemistry 438 (1997) 113-119

Although the peak separation of the new redox wave is 56mV at a scan rate of 2 0 m V s - ' , the peak separation was broadened gradually by more than 60mV at scan rates over 40mVs - I , indicating that the process was not fully reversible under our experimental conditions. Thus, the exchange rite constant k o for the oxidation of BQ ~ at the PC coated GC electrode was evaluated from Eq. (4) for the quasi-reversible reaction

~b= A~r I/2 = ( D ° / D R ) a / 2 k ° (4)

[ Doer v ( n F / R r ) ] , /2

where 0 is an equivalent parameter, D O and D a are diffusion coefficients which axe assumed to be approxi- mately the same, v is the scan rate, R is the gas constant, and T is the thermodynamic tempera,~ure [24]. The rate constant was determined from the slope of a ~ vs. l / v ~/2 plot. The values of k o in pH 5.0, 7.0 and 8.0 b, tffe: solutions were 1.95 x 10 -2, 3.18 x 10 -2 and 4.76 X 10- tcm s- t respectively.

3.3. In situ U V - v i s spectroelectrochemical studies

The spectroelectrochemical experiments at the PC mod- ified and bare GC electrodes were mac in aqueous solu- tions. A new absorption band appeared, attributed to an intermediate species at a PC coated GC electrode during applying the reduction potential to - 0.5 V. Meanwhile, no bands attributable to the intermediate species were de- tected at a bare GC electrode in phosphate buffer solution at any pH value, regardless of whether BQ was reduced or H2Q was oxidized. The assignments of the bands were made by comparing the spectra recorded from authentic compounds, i.e. henzoquinone and hydroqalnone.

In unbuffered solutions (0.1 M KCI, pH 5.5) with a bare GC electrode the spectra of 1.0mM BQ showed two

o.oo2 [- i I / .o I

o o.s ° ~ L " i;' I

.0.006 i. | . . i . . i , . ! .i 0 -0.4 0.0 0.4 0,8

~ 0 . 4 E/V vs.AglAgCl

0.2

o.o t

I , I , I , ! i

250 300 !;50 400 450

Wavelength/rim

Fig. 6. In sire UV-vis spectra of 1.0nliM BQ+0.1M KCI (pH 5.5) solution on a PC coated GC electrode at (a) 0,2V, (b) 0.OV anti (c) -0.2V vs. Ag[AgCI and the DCVA curves (inset) determined at 315nm (dashed line) and 415nm (solid line).

|!7

intense bands at 250 and 315rim conesponding to an absorption band assigned to BQ and H2Q respectively. Upon the reduction of BQ at - 0 .5V , the absorbunce at 25Onto decreased while that at 315nm increased. The band assigned to the anion radical was not observed at the bare GC electrode. However, at the PC covered electrode in the same solution the new absorption band a m u ~ 415nm increased when the applied potential was from 0.2 to - 0 . 2 V, as shown in Fig. 6. The absorption peaks at 315 and 250nm showed the same character upon the reduction of BQ at the PC coated electrode as those observed at tbe bare GC electrode. Both bands at 315 and 415nm are generated during the reduction of BQ and disappear during the re-oxidation of its ~ reduc- tion I ~ u c ' . , ~ can be seen in the DCVA curve (inset to Fig. 6). The DCVA signal ~ at 315nm (dashed line) appeared and disappeared reversibly upon the reduc- tion and re-oxidation of BQ in the potential range between +0.8 and -0 .5V. The signal obtained at 415rim ( ~ line) showed similar heha'/ior to that observed at 315nm. However, the signal intensity at 415nm is much than that at 315nm. Thus, the DCVA signals at 415 anti 315nm are assigned to the immediate electron transfer product BQ ~ and the final reduction product H2Q respec- t ively. The absorption bands were very shnilar m that of the BQ reduction product observed in n o n - ~ solu- tions, except the band positions, which are shifted as much as 20 to 35 nm towards shorter wavelength. This shift may he due to the protonafion of the anion radical form in the aqueous medium, which is responsible for raising the transition energy levels of anion radicals [22,25].

At the bare GC electrode in a phosphate buffer solution (pH 7.0), an absorpfon band assigned to H2Q was ob- served having an a ~ o n maximum at 29Ore, n, together with the band assigned to BQ at 245 run (not shown). The wavelengths of these bands were shifted to the shorter

8.e-5

6 5

4.e-5

2.~-5

-2.e-5

-4.C-5

-6.e-5

-8.c-5 P I ~ t ~ ~ - o . 6 ~ . 5 ~ . , - o ~ - o . t o.o o.1 o.2

E/V ~s. ~ J ~ , C l

Ng. 7. DCVA curve at 415rim (solid line) ~ cyclic (dashed line) of I.OmM BQ+phosl~ beffer (pH 7.0) at a PC GC electrode.

118 H. Park et al. / Journal of Electroanalytical Chemistry 438 (1997) 113-119

1217.16 , I , I ,

2000 1500 1000 500 I

Wavenumber/cm

Fig. 8. IR spectra of (a) phosphatidylcholine and (b) a PC film mixed with a reduction intermediate obtained by reduction of BQ with Zn powder.

values compared with those recorded in unbuffered solu- tions due to the solvent effect of the phosphate medium. The new band appeared at 415nm when the electrode potential was applied from +0.65 to -0 .49 V, the inten- sity of which was rather weaker than that in the unbuffered solution. Derivative cyclic voltabsorptometric (DCVA) ex- periments [26] were also conducted at the phosphate buffer medium to observe the potential sweep rate dependence of the absorption bands at 415 nm (Fig. 7). The CV (dashed line) is shown along with the derivative absorbance ( d A / d t ) curve recorded at 415 nm (solid line). In DCVA experiments, the d A / d t signal is recorded at a given wavelength as a function of the potential. The curve at 415 nm agreed with the cyclic voltammogram except for a hysteresis between 0.0 and + 0.1 V. A similar curve was obtained at 290nm, an absorption wavelength correspond- ing to hydroquinone, confirmed with an authentic sample under the same experimental conditions.

3.4. FT-IR and NMR studies for the interaction between BQ ~ and lipid

In order to confirm the interaction position between the reduction intermediate of BQ and the lipid layer, FF-IR measurements were made on BQ, H2Q, and reduced BQ incorporated into a PC or a PA with the NaCI crystal. Fig. 8 shows IR spectra of (a) phosphatidylcholine and (b) a PC n'txed with the reduction products BQ ~ obtained by the reduction of BQ with Zn powder. The reduction product of BQ must not be H2Q, because there is no change of the absorption band of HeQ in the lipid layer. In the spectrum

of (b), the -PO~ group of the PC compound shows two absorption bands at 1217.16cm-i (asymmetric stretching) and 1084.06cm -I (symmetric stretching). These bands were shifted to higher frequencies compared with those of a pure PC molecule, which exhibits bands at 1236.30 and 1088.75cm-'. These bands correspond to the absorption region of a hydrogen bonded phosphate group in the PC molecule. Most P=O compounds have a band in the region 1320 to l l 4 0 c m - ' , but the hydrogen bonded types such as (HO)P=O exhibit a broad absorption as low as 1085cm -I [27]. In addition, the IR spectra of the PC molecules prepared in this study show bands in the region 3200 to 3600cm-i , suggesting that the PC molecule may contain hydroxy groups.

The frequency of the stretching band for -CH 2- N + (CH 3)3 in the PC molecule is 1467.92 cm- I, which did not shift upon addition of the reduction products of BQ to the PC layer. The electrochemical and IR measurements were made for the redox reaction of BQ at the PA modi- fied GC electrode to elucidate the binding site of the reduction intermediates of BQ to a -PO~ or -CH 2- N+(CH3)3 in the PC molecule. The PA molecule contains a -PO~ group whereas PC contains -CH2-N+(CH3)3 in its place. When the BQ was reduced at the PA covered GC electrode, we also observed the new redox wave at - 0 . 1 6 0 / - 0 . 2 2 3 V in the CV, indicating the incorpora- tion of the reduction intermediate with the lipid layer. According to these results, the interaction site of the reduction intermediate of BQ in PC could be a hydrogen bonded type -PO~ or - (HO)P=O group.

t H NMR measurements were also made to investigate the interaction sites between PC and BQ for (a) PC and (b) BQ mixed with PC in CDCI 3 solution. Addition of BQ to the PC + CDCI3 solution may affect the chemical shift of the ethylene group attached to -PO~ or -N+(CTI3)3 in the PC molecule if there is an interaction between BQ and PC (see the structure in Fig. 1). The chemical shift of CH2OPO in the BQ + PC mixture was 3.99ppm, while that of PC itself was 4.27 ppm. In contrast, the chemical shift of -N+(CH3)3 was not influenced by addition of BQ to PC. These observations clearly indicate that BQ was incorporated into the PC layer through the interaction between the hydrated -PO~ group and BQ. The I H NMR signals of PC in CDCI 3 were unaffected by addition of H2Q. This result suggests that H2Q does not interact with the -PO~ group in the PC molecule but BQ does. We conclude from the present investigation that the lipid layer stabilizes the reduction intermediate of BQ through inter- action with each other.

4. Conclusions

We have shown that the reduction intermediate of BQ is incorporated into the PC layer through the interaction with

H. Park et al. / Journal of Electroanalytical Chemistry 438 (1997) 113-I 19 | t 9

a PO 2 group and stabilizes BQ = in neutral solutions at the PC coated GC electrode. It is clearly shown that the electrochemical reduction of BQ proceeds through the formation of an anion radical at the PC layer in unbuffered KCI as well as buffered phosphate solutions at pH above 4. This is due to: (i) the hydrophobic nature of the PC layer, in which the activity of water is low so that the pH value in the lipid layer ".'ncreases with progress of the reduction of BQ; (ii) the formation of hydrogen bonding between the reduction product of BQ, BQ ~ and the hydrogen bonded phosphate group - ( H O ) P = O in the PC molecule; and (iii) stabilization of the reduction intermediate in the lipid film.

Acknowledgements

Grateful acknowledgment is made to Professor K. Niki, who has retired from the Yokohama National University (Japan) and is currently researching at Iowa State Univer- sity (USA), for his kind discussions and correction of the manuscript. This research was supported by the Korean Science and Engineering Foundation (Grant No. 96-0501- 05-01-3) and the Ministry of Education of Korea through the BSRI program (BSRI-96-3410) to Pusan National Uni- versity.

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