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Spectroelectrochemical study of biliverdin produced by the electrooxidation of bilirubin
345
Bioelectrochemistry and Bioenergetics, 26 (1991) 345-350 A section o f J. Electroanal. Chem., and constituting Vol. 321 (1991) Elsevier Sequoia S.A.. Lausanne
J E C BB 01385 Short c o m m u n i c a t i o n
Spectroelectrochemical study of biliverdin produced by the electrooxidation of bflirubin Guangiin Cheng, Yi Y a n g a n d S h a o j u n D o n g * Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China) (Received 6 N o v e m b e r 1990; in revised form 25 March 1991)
INTRODUCTION
For a long time now bilirubin (BR) in m a m m a l s has a t t r a c t e d considerable attention. This is because its structure and metabolic process are closely c o n n e c t e d to huraan health a n d also because it is involved in i m p o r t a n t aspects of the catabolic chemistry of heine. H e m o g l o b i n generally is considered to be catabolized i n t o heine first. The m e t h y n e ( = C H - ) bridge of heme is oxidized, a n d b r o k e n to release CO, resulting in biliverdin (BV). U n d e r enzymatic action, it is reduced i n t o BR[1,2]. BR is liposoluble in physiological conditions; in aqueous solution, i n c l u d i n g physiological conditions, the solubility is very small. Therefore the chemical p r o p e r ties of BR and its analogs are usually investigated in organic solvent. The eleetrooxidation process of B R was first studied b y V a n N o r m a n . H e proposed that BR can be oxidized to BV irreversibly in dimethylfo~n~amide ( D M F ) [3]. Later, Slifstein a n d Ariel [4], M o u s s a et al. [5] a n d P r a d k o et al. [6] studied the mechanism of the oxidation of B R into BV, using different electrochemical a n d spectroscopic techniques. It is k n o w n t h a t BV can be reduced into B R by the action o f BV reductase in the body. However, no r e p o r t s on the m e c h a n i s m of the electrochenfical r e d u c t i o n process of BV have been published. In this note, the e!ectroreduction process o f BV produced by the electrooxidation o f B R is investigated using spectroe'Jectrochemical techniques. R a p i d scan techniques are used to d e m o n s t r a t e that a reactive intermediate is involved in the process. Also, the rate c o n s t a n t of the s u b s e q u e n t chemical reaction is determined. * T o whom correspondence should be addressed. 0302-4598/91/$03.50
© 1991
E l s e v i e r Sequoia S.A. All fights reserved
346
EXPERIMENTAL
Reagents BR, 99% purity, was purchased from Sigma Chemical C o m p a n y . D M F was reagent grade and disti!l~d from calcium h y d r i d e before use. T e t r a b u t y l a m m o n i u m perchlorate (TBAP) was o b t a i n e d from F l u k a C o m p a n y , Switzerland, a n d recrystallized from acetylacetate. In order to protect the B R solution from o x i d a t i o n in air, it was prepared j u s t before use a n d kept under a nitrogen a t m o s p h e r e d u r i n g experiments.
Instruments T h e spectroelectrochemical e x p e r i m e n t s were p e r f o r m e d with a P A R e M o d e l 370 electrochemistry system a n d a T r a c o r N o r t h e r n T N - 6 5 0 0 R a p i d Scan Spectrometer, which makes a h o l o g r a p h i c scan in a very short period of time. It took 5.15 ms to acquire one h o l o g r a p h i c spectrum. T h e optically t r a n s p a r e n t t h i n - l a y e r cell was m a d e in our laboratory. T h e v o l u m e of the t h i n - l a y e r cavity was 10.6 /~1, a n d its thickness was 0.2 mm. A p l a t i n u m minigrid was used as w o r k i n g electrode, a p l a t i n u m foil as auxiliary electrode a n d A g / A g C I (1 M K C l ) as reference electrode. RESULTS AND DISCUSSION
Thin-layer cyclic voltammetric behaoiour In D M F , 02 can be reduced to O~- at --0.86 V (vs. s e E ) . By exhaustive electrolysis at --0.90 V u n d e r a nitrogen a t m o s p h e r e , the residual 02 c a n be c o m p l e t e l y removed from the thin-layer solution. T h e electrochemical e x p e r i m e n t s were carried out after the a d d i t i o n of B R to the d e a e r a t e d D M F . W h e n the p o t e n t i a l is first s c a n n e d in the negative direction, n o r e d u c t i o n peak a p p e a r s on the cyclic v o l t a m m o g r a m (CV) from 0.0 to --0.5 V. U p o n c o n t i n u e d s c a n n i n g in the negati~-e direction, a couple o f redox p e a k s appears. A t a scan rate of 10 m V / s , the c o r r e s p o n d i n g p e a k potentials, Ep¢ a n d Epa, are --0.86 a n d --0.68 V, respectively (see Fig. 1A). A t a scan rate of 2 m V / s , AEp ---- 60 inV. W e suggest that, because the a p p a r e n t resistence of the t h i n - l a y e r cell is 230 ~. B R u n d e r g o e s a quasi-reversible reaction at the electrode. It is evident t h a t the p e a k at --0.86 V is the reduction peak for B R itself. However, if the scan is initiated in the positive direction, B R is oxidized into BV[3,5,6]. A n a n o d i c p e a k appears on the CV at 0.65 V. S u b s e q u e n t l y s c a n n i n g in the negative direction reveals a r e d u c t i o n p e a k at --0.30 V (see Fig. 1B). T h e c o r r e s p o n d i n g o x i d a t i o n peak, Ep.~, is at --0.20 V at a scan rate of 30 m V / s . W i t h decreasing scan rate, ipc/ip~ increases. W h e n the scan rate is 2 m V / s , the o x i d a t i o n p e a k almost disappears. This result shows that a s u b s e q u e n t chemical reaction takes place coupled with the r e d u c t i o n product. T h e scan c o n d i t i o n s in Fig. 2A are the s a m e as those in Fig. 1B, except t h a t the negative scan is extended to --1.6 V. It c a n be seen t h a t the o x i d a t i o n p e a k at --0.20 V disappears. This is p r o b a b l y due to the c o n t i n u e d reaction of the electroreduction p r o d u c t of BV.
347
(A)
I
I 0
10 .,,.:A
I -0.4
I -0.8
I -1.2
1
(B)
•
,
J
0.8
0.4
o
I
-0.4
I
-0.8
i
-1.2
ElVCvs. ~ 1 ~ c l ) Fig. 1. Thin-layer cyclic voltammogram of 1 x l 0 -4 M B R + 0 . 1 M T B A P in D M F solution saturated with N 2. Scan rate: (A) 10 m v / s . (B) (1) 30; (2j 20; (3) 10; (4) 2 m V / s .
F i g u r e 2B s h o w s the C V for t h e c o n t i n u e d o x i d a t i o n o f B R i n t o p u r p u r i n w i t h a n Ep., of 0.84 V [3,5,6]. I n a d d i t i o n to t h e t w o r e d u c t i o n p e a k s a t Ep~ ---- - - 0 . 3 V a n d Ep,. -- --0.84 V, a n e w c a t h o d i c p e a k a p p e a r s at - - 1 . 1 4 V, c o u p l e d t o a n o x i d a t i o n p e a k at Epa = - - 0 . 8 0 V. It is c l e a r t h a t this p a i r o f p e a k s is r e l a t e d t o t h e c o n t i n u e d o x i d a t i o n o f BR. T h e s e e x p e r i m e n t s i n d i c a t e t h a t the r e d o x b e h a v i o u r o f B R is r a t h e r c o m p l i c a t e d .
Thin-layer spectroelectrochemistry T h e t h i n - l a y e r cell u s e d w a s m a d e f r o m a glass t u b e b y s i n t e r i n g , w h i c h is especially s u i t a b l e for t h e i n v e s t i g a t i o n o f s p e c t r o e l e c t r o c h e m i c a l b e h a v i o u r i n
348
A
11
I
I
0.8
0.4
I
I
0
-0.4 S/VCvs.
! ........ -0.8
I -1.2
I -1.6
~/,e~¢Cl )
Fig. 2. Thin-layer cyclic voltammograms of 1 × 1 0 -4 M B R + 0 . I M TBAP in D M F saturated with S c a n rate: 10 m V / s .
(A) 0.0 V --, 0.'/0 V ~
-- 1.6 V ~ 0.0 V; (B) 0.0 V --, 0.90 V ~
N 2.
-- 1.6 V ---*0.0 V.
o r g a n i c s y s t e m s . T h e A E (ca. 1 V ) b e t w e e n t h e o x i d a t i o n p e a k o f B R ( E p a = 0 . 6 5 V ) a n d t h e r e d u c t i o n p e a k o f B V (Eeoc - - 0 . 3 0 V ) is v e r y l a r g e , a s d e s c r i b e d a b o v e . This may be linked with the time needed to investigate the kinetics and the intermediate of the electrode reaction with spectroelectrochemical techniques. Therefore, a rapid scan spectrometer (TN-6500) was used. The instantaneous change in t h e s p e c t r a w a s r e c o r d e d d u r i n g t h e p o t e n t i a l s t e p a n d s a t i s f a c t o r y r e s u l t s w e r e obtained. I n D M F , B V h a s t w o s t r o n g a b s o r p t i o n p e a k s a t 3 8 0 a n d 6 5 0 n m [4,5,6]. F i g u r e 3 shows the absorption spectrum of BV obtained during a potential step from 0.0 to
400
500
600 : 700 800 W a v e l e n g t h Inm Fig. 3. Single-potential-step chronoabsorpiometric spectra. 1 × 1 0 -4 M B R + 0 . I M TBAP in D M F saturated with N 2. (1) 0; (2) 15; (3) 25; (4) 45; (5) 65; (6) 120, (7) 480 s.
349 0.6 V ( c u r v e 1), a n d also the s p e c t r a l c h a n g e w i t h t i m e u p o n a p o t e n t i a l s t e p f r o m 0.6 to --0.45 V ( c u r v e s 2 - 7 ) . W h e n t ---- 120 s, a l a r ge c h a n g e has t a k e n p l a c e i n t h e s p e c t r a (see c u r v e 6 in Fig. 3). T h e p e a k shifts o c c u r at 380 a n d 650 n m , a n d t w o n e w a b s o r p t i o n p e a k s a p p e a r at 418 n m a n d 635 n m . T h r e e w e l l - d e f i n e d i s o s b e s t i c p o i n t s in the s p e c t r a i n d i c a t e t h a t at this t i m e t h e r a t i o b e t w e e n r e a c t a n t a n d p r o d u c t is o n e to o n e , i n d i c a t i n g that no subsequent chemical reaction takes place. T h e isosbestic p o i n t s d i s a p p e a r c o m p l e t e l y , w h e n t ---- 480 s. A t t h e s a m e t i m e , t h e s p e c t r u m c h a n g e s i n t o t h a t o f B R w i t h a c h a r a c t e r i s t i c a b s o r p t i o n p e a k at 449 n m (see c u r v e 7 in Fig. 3). I n t he e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d a r e t h e s a m e as in Fig. 3, b u t t h e c i r c u i t is o p e n e d a t t = 120 s, w e c a n r e c o r d a n i n s t a n t a n e o u s c h a n g e in t h e s p e c t r a ( t h e s a m e as in Fig. 3). It is c l e a r t h a t this s h o u l d b e a s c r i b e d t o a s u b s e q u e n t c h e m i c a l r e a c t i o n . T h i s r e s u l t is in a g r e e m e n t w i t h t h a t o f t h e t h i n - l a y e r c y c l i c v o l t a m m e t r y . T h e a b o v e a n a l y s i s i n d i c a t e s t h a t t h e e l e c t r o r e d u c t i v e m e c h a n i s m o f BV m i g h t b e as follows: '3V is r e d u o , ' d b y t w o e l e c t r o n s i n t o a n i n t e r m e d i a t e R t h e n R u n d e r g o e s a n irreversible c h e m i c a l r e a c t i o n to p r o d u c e B R . R is a l s o a n o x i d i z a b l e e l e c t r o a c tive species: BV 2 e - > R 2 e - >BR
-2.68
8
-2.76
I
-2.84
-2.92 |
120
|
|
140
|
~
160
l
|
180
time/s Fig. 4. Kinetic plot of single-l~tential-step absorbance-time data at 635 nm from Fig. 3. I x 10 -4 M BR + 0.1 M TBAP in DMF saturated with N z.
350
The kinetics o f B V in the reductioe process It is a d v a n t a g e o u s to use t h i n - l a y e r s p e c t r o e l e c t r o c h e m i s t r y to s t u d y a n e l e c t r o d e reaction coupled w i t h a chemical r e a c t i o n of w h i c h t h e r e a c t i o n rate c o n s t a n t is to be d e t e r m i n e d [7,8]. S i n g l e - p o t e n t i a l - s t e p c h r o n o a b s o r p t o m e t r y t h e r e f o r e w a s emp l o y e d to s t u d y the s u b s e q u e n t c h e m i c a l r e a c t i o n of the r e d u c t i o n p r o d u c t of BV. W h e n the p o t e n t i a l was s t e p p e d f r o m 0.6 to --0.45 V, the r a p i d c h a n g e s in t h e s p e c t r a s h o w e d t h a t BV was r e d u c e d i n t o species R. T h e p r o g r e s s i v e d e c r e a s e of the a b s o r p t i o n intensities in the s p e c t r a a n d t h e d i s a p p e a r a n c e of the isosbestic p o i n t s d e m o n s t r a t e t h a t species R forms B R b y a s u b s e q u e n t c h e m i c a l reaction. S u p p o s i n g t h a t it is a p s e u d o - f i r s t - o r d e r r e a c t i o n , t h e n
In( A, - Aoo) = -- k t + I n ( A 0 -- Aoo) v,'here k is p s e u d o - f i r s t - o r d e r rate c o n s t a n t ( s - * ) , A 0 the initial a b s o r b a n c e (A0 = b~x.R[R]), A,o the final a b s o r b a n c e ( A ~ = bEX.BR[BR]), A, the a b s o r b a n c e a t t i m e t (At----b(Cx.R[R] + CX.BR[BR])). Selecting the c h a n g e s in a b s o r b a n c e at a w a v e l e n g t h o f 635 n m , a n d p l o t t i n g l n ( A t - - A ~ ) a g a i n s t t, a s t r a i g h t line w i t h relative coeffic i e n t 0.999 is o b t a i n e d , as s h o w n in Fig. 4. A s u b s e q u e n t c h e m i c a l r e a c t i o n rate c o n s t a n t of 5.0 × 1 0 - 3 s-~ is o b t a i n e d f r o m the slope. ACKNOWLEDGEMENT
T h e s u p p o r t of this s t u d y b y the N a t i o n a l N a t u r a l Science F o u n d a t i o n o f C h i n a is gratefully a c k n o w l e d g e d . REFERENCES 1 2 3 4 5 6
R. Broderson, CRC Crit. Rev. Clin. Lab. Sci., 11 (1979) 305. K.P.M. Heirwegh and S.B. Brown (Eds.), Bilirubin, Vol. 2, CRC, Boca Raton (FL), 1982. .I.D. Van Norman, Anal. Chem., 45 (1973) 173. C. Slifstein and M. Ariel, J. Electroanal. Chem., 48 (1973) 447. F. Moussa, G. Kanoute, C. Herrenknecht, P. Levillain and F. Trivin, Anal. Chem., 60 (1988) 1179. J.R. Pradko, S. Pons, S. Bandyopdhyay, J.F. MeAleer and A.S. Hinman, Bioelectrochem. Bioenerg., 13 (1984) 267. 7 W.R. Heineman, F.N. Hawkridge and H.B. Btount in A J . Bard (Ed.), Electroanalytical Chemistry, Vol. 13, Marcel Dekker, New York, 1983o pp. 1-104. 8 Dong Shaojun, Anal. Chem. (Chinese), 13 (1985) 70.
Bioelectrochemistry and Bioenergetics, 26 (1991) 345-350 A section o f J. Electroanal. Chem., and constituting Vol. 321 (1991) Elsevier Sequoia S.A.. Lausanne
J E C BB 01385 Short c o m m u n i c a t i o n
Spectroelectrochemical study of biliverdin produced by the electrooxidation of bflirubin Guangiin Cheng, Yi Y a n g a n d S h a o j u n D o n g * Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China) (Received 6 N o v e m b e r 1990; in revised form 25 March 1991)
INTRODUCTION
For a long time now bilirubin (BR) in m a m m a l s has a t t r a c t e d considerable attention. This is because its structure and metabolic process are closely c o n n e c t e d to huraan health a n d also because it is involved in i m p o r t a n t aspects of the catabolic chemistry of heine. H e m o g l o b i n generally is considered to be catabolized i n t o heine first. The m e t h y n e ( = C H - ) bridge of heme is oxidized, a n d b r o k e n to release CO, resulting in biliverdin (BV). U n d e r enzymatic action, it is reduced i n t o BR[1,2]. BR is liposoluble in physiological conditions; in aqueous solution, i n c l u d i n g physiological conditions, the solubility is very small. Therefore the chemical p r o p e r ties of BR and its analogs are usually investigated in organic solvent. The eleetrooxidation process of B R was first studied b y V a n N o r m a n . H e proposed that BR can be oxidized to BV irreversibly in dimethylfo~n~amide ( D M F ) [3]. Later, Slifstein a n d Ariel [4], M o u s s a et al. [5] a n d P r a d k o et al. [6] studied the mechanism of the oxidation of B R into BV, using different electrochemical a n d spectroscopic techniques. It is k n o w n t h a t BV can be reduced into B R by the action o f BV reductase in the body. However, no r e p o r t s on the m e c h a n i s m of the electrochenfical r e d u c t i o n process of BV have been published. In this note, the e!ectroreduction process o f BV produced by the electrooxidation o f B R is investigated using spectroe'Jectrochemical techniques. R a p i d scan techniques are used to d e m o n s t r a t e that a reactive intermediate is involved in the process. Also, the rate c o n s t a n t of the s u b s e q u e n t chemical reaction is determined. * T o whom correspondence should be addressed. 0302-4598/91/$03.50
© 1991
E l s e v i e r Sequoia S.A. All fights reserved
346
EXPERIMENTAL
Reagents BR, 99% purity, was purchased from Sigma Chemical C o m p a n y . D M F was reagent grade and disti!l~d from calcium h y d r i d e before use. T e t r a b u t y l a m m o n i u m perchlorate (TBAP) was o b t a i n e d from F l u k a C o m p a n y , Switzerland, a n d recrystallized from acetylacetate. In order to protect the B R solution from o x i d a t i o n in air, it was prepared j u s t before use a n d kept under a nitrogen a t m o s p h e r e d u r i n g experiments.
Instruments T h e spectroelectrochemical e x p e r i m e n t s were p e r f o r m e d with a P A R e M o d e l 370 electrochemistry system a n d a T r a c o r N o r t h e r n T N - 6 5 0 0 R a p i d Scan Spectrometer, which makes a h o l o g r a p h i c scan in a very short period of time. It took 5.15 ms to acquire one h o l o g r a p h i c spectrum. T h e optically t r a n s p a r e n t t h i n - l a y e r cell was m a d e in our laboratory. T h e v o l u m e of the t h i n - l a y e r cavity was 10.6 /~1, a n d its thickness was 0.2 mm. A p l a t i n u m minigrid was used as w o r k i n g electrode, a p l a t i n u m foil as auxiliary electrode a n d A g / A g C I (1 M K C l ) as reference electrode. RESULTS AND DISCUSSION
Thin-layer cyclic voltammetric behaoiour In D M F , 02 can be reduced to O~- at --0.86 V (vs. s e E ) . By exhaustive electrolysis at --0.90 V u n d e r a nitrogen a t m o s p h e r e , the residual 02 c a n be c o m p l e t e l y removed from the thin-layer solution. T h e electrochemical e x p e r i m e n t s were carried out after the a d d i t i o n of B R to the d e a e r a t e d D M F . W h e n the p o t e n t i a l is first s c a n n e d in the negative direction, n o r e d u c t i o n peak a p p e a r s on the cyclic v o l t a m m o g r a m (CV) from 0.0 to --0.5 V. U p o n c o n t i n u e d s c a n n i n g in the negati~-e direction, a couple o f redox p e a k s appears. A t a scan rate of 10 m V / s , the c o r r e s p o n d i n g p e a k potentials, Ep¢ a n d Epa, are --0.86 a n d --0.68 V, respectively (see Fig. 1A). A t a scan rate of 2 m V / s , AEp ---- 60 inV. W e suggest that, because the a p p a r e n t resistence of the t h i n - l a y e r cell is 230 ~. B R u n d e r g o e s a quasi-reversible reaction at the electrode. It is evident t h a t the p e a k at --0.86 V is the reduction peak for B R itself. However, if the scan is initiated in the positive direction, B R is oxidized into BV[3,5,6]. A n a n o d i c p e a k appears on the CV at 0.65 V. S u b s e q u e n t l y s c a n n i n g in the negative direction reveals a r e d u c t i o n p e a k at --0.30 V (see Fig. 1B). T h e c o r r e s p o n d i n g o x i d a t i o n peak, Ep.~, is at --0.20 V at a scan rate of 30 m V / s . W i t h decreasing scan rate, ipc/ip~ increases. W h e n the scan rate is 2 m V / s , the o x i d a t i o n p e a k almost disappears. This result shows that a s u b s e q u e n t chemical reaction takes place coupled with the r e d u c t i o n product. T h e scan c o n d i t i o n s in Fig. 2A are the s a m e as those in Fig. 1B, except t h a t the negative scan is extended to --1.6 V. It c a n be seen t h a t the o x i d a t i o n p e a k at --0.20 V disappears. This is p r o b a b l y due to the c o n t i n u e d reaction of the electroreduction p r o d u c t of BV.
347
(A)
I
I 0
10 .,,.:A
I -0.4
I -0.8
I -1.2
1
(B)
•
,
J
0.8
0.4
o
I
-0.4
I
-0.8
i
-1.2
ElVCvs. ~ 1 ~ c l ) Fig. 1. Thin-layer cyclic voltammogram of 1 x l 0 -4 M B R + 0 . 1 M T B A P in D M F solution saturated with N 2. Scan rate: (A) 10 m v / s . (B) (1) 30; (2j 20; (3) 10; (4) 2 m V / s .
F i g u r e 2B s h o w s the C V for t h e c o n t i n u e d o x i d a t i o n o f B R i n t o p u r p u r i n w i t h a n Ep., of 0.84 V [3,5,6]. I n a d d i t i o n to t h e t w o r e d u c t i o n p e a k s a t Ep~ ---- - - 0 . 3 V a n d Ep,. -- --0.84 V, a n e w c a t h o d i c p e a k a p p e a r s at - - 1 . 1 4 V, c o u p l e d t o a n o x i d a t i o n p e a k at Epa = - - 0 . 8 0 V. It is c l e a r t h a t this p a i r o f p e a k s is r e l a t e d t o t h e c o n t i n u e d o x i d a t i o n o f BR. T h e s e e x p e r i m e n t s i n d i c a t e t h a t the r e d o x b e h a v i o u r o f B R is r a t h e r c o m p l i c a t e d .
Thin-layer spectroelectrochemistry T h e t h i n - l a y e r cell u s e d w a s m a d e f r o m a glass t u b e b y s i n t e r i n g , w h i c h is especially s u i t a b l e for t h e i n v e s t i g a t i o n o f s p e c t r o e l e c t r o c h e m i c a l b e h a v i o u r i n
348
A
11
I
I
0.8
0.4
I
I
0
-0.4 S/VCvs.
! ........ -0.8
I -1.2
I -1.6
~/,e~¢Cl )
Fig. 2. Thin-layer cyclic voltammograms of 1 × 1 0 -4 M B R + 0 . I M TBAP in D M F saturated with S c a n rate: 10 m V / s .
(A) 0.0 V --, 0.'/0 V ~
-- 1.6 V ~ 0.0 V; (B) 0.0 V --, 0.90 V ~
N 2.
-- 1.6 V ---*0.0 V.
o r g a n i c s y s t e m s . T h e A E (ca. 1 V ) b e t w e e n t h e o x i d a t i o n p e a k o f B R ( E p a = 0 . 6 5 V ) a n d t h e r e d u c t i o n p e a k o f B V (Eeoc - - 0 . 3 0 V ) is v e r y l a r g e , a s d e s c r i b e d a b o v e . This may be linked with the time needed to investigate the kinetics and the intermediate of the electrode reaction with spectroelectrochemical techniques. Therefore, a rapid scan spectrometer (TN-6500) was used. The instantaneous change in t h e s p e c t r a w a s r e c o r d e d d u r i n g t h e p o t e n t i a l s t e p a n d s a t i s f a c t o r y r e s u l t s w e r e obtained. I n D M F , B V h a s t w o s t r o n g a b s o r p t i o n p e a k s a t 3 8 0 a n d 6 5 0 n m [4,5,6]. F i g u r e 3 shows the absorption spectrum of BV obtained during a potential step from 0.0 to
400
500
600 : 700 800 W a v e l e n g t h Inm Fig. 3. Single-potential-step chronoabsorpiometric spectra. 1 × 1 0 -4 M B R + 0 . I M TBAP in D M F saturated with N 2. (1) 0; (2) 15; (3) 25; (4) 45; (5) 65; (6) 120, (7) 480 s.
349 0.6 V ( c u r v e 1), a n d also the s p e c t r a l c h a n g e w i t h t i m e u p o n a p o t e n t i a l s t e p f r o m 0.6 to --0.45 V ( c u r v e s 2 - 7 ) . W h e n t ---- 120 s, a l a r ge c h a n g e has t a k e n p l a c e i n t h e s p e c t r a (see c u r v e 6 in Fig. 3). T h e p e a k shifts o c c u r at 380 a n d 650 n m , a n d t w o n e w a b s o r p t i o n p e a k s a p p e a r at 418 n m a n d 635 n m . T h r e e w e l l - d e f i n e d i s o s b e s t i c p o i n t s in the s p e c t r a i n d i c a t e t h a t at this t i m e t h e r a t i o b e t w e e n r e a c t a n t a n d p r o d u c t is o n e to o n e , i n d i c a t i n g that no subsequent chemical reaction takes place. T h e isosbestic p o i n t s d i s a p p e a r c o m p l e t e l y , w h e n t ---- 480 s. A t t h e s a m e t i m e , t h e s p e c t r u m c h a n g e s i n t o t h a t o f B R w i t h a c h a r a c t e r i s t i c a b s o r p t i o n p e a k at 449 n m (see c u r v e 7 in Fig. 3). I n t he e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d a r e t h e s a m e as in Fig. 3, b u t t h e c i r c u i t is o p e n e d a t t = 120 s, w e c a n r e c o r d a n i n s t a n t a n e o u s c h a n g e in t h e s p e c t r a ( t h e s a m e as in Fig. 3). It is c l e a r t h a t this s h o u l d b e a s c r i b e d t o a s u b s e q u e n t c h e m i c a l r e a c t i o n . T h i s r e s u l t is in a g r e e m e n t w i t h t h a t o f t h e t h i n - l a y e r c y c l i c v o l t a m m e t r y . T h e a b o v e a n a l y s i s i n d i c a t e s t h a t t h e e l e c t r o r e d u c t i v e m e c h a n i s m o f BV m i g h t b e as follows: '3V is r e d u o , ' d b y t w o e l e c t r o n s i n t o a n i n t e r m e d i a t e R t h e n R u n d e r g o e s a n irreversible c h e m i c a l r e a c t i o n to p r o d u c e B R . R is a l s o a n o x i d i z a b l e e l e c t r o a c tive species: BV 2 e - > R 2 e - >BR
-2.68
8
-2.76
I
-2.84
-2.92 |
120
|
|
140
|
~
160
l
|
180
time/s Fig. 4. Kinetic plot of single-l~tential-step absorbance-time data at 635 nm from Fig. 3. I x 10 -4 M BR + 0.1 M TBAP in DMF saturated with N z.
350
The kinetics o f B V in the reductioe process It is a d v a n t a g e o u s to use t h i n - l a y e r s p e c t r o e l e c t r o c h e m i s t r y to s t u d y a n e l e c t r o d e reaction coupled w i t h a chemical r e a c t i o n of w h i c h t h e r e a c t i o n rate c o n s t a n t is to be d e t e r m i n e d [7,8]. S i n g l e - p o t e n t i a l - s t e p c h r o n o a b s o r p t o m e t r y t h e r e f o r e w a s emp l o y e d to s t u d y the s u b s e q u e n t c h e m i c a l r e a c t i o n of the r e d u c t i o n p r o d u c t of BV. W h e n the p o t e n t i a l was s t e p p e d f r o m 0.6 to --0.45 V, the r a p i d c h a n g e s in t h e s p e c t r a s h o w e d t h a t BV was r e d u c e d i n t o species R. T h e p r o g r e s s i v e d e c r e a s e of the a b s o r p t i o n intensities in the s p e c t r a a n d t h e d i s a p p e a r a n c e of the isosbestic p o i n t s d e m o n s t r a t e t h a t species R forms B R b y a s u b s e q u e n t c h e m i c a l reaction. S u p p o s i n g t h a t it is a p s e u d o - f i r s t - o r d e r r e a c t i o n , t h e n
In( A, - Aoo) = -- k t + I n ( A 0 -- Aoo) v,'here k is p s e u d o - f i r s t - o r d e r rate c o n s t a n t ( s - * ) , A 0 the initial a b s o r b a n c e (A0 = b~x.R[R]), A,o the final a b s o r b a n c e ( A ~ = bEX.BR[BR]), A, the a b s o r b a n c e a t t i m e t (At----b(Cx.R[R] + CX.BR[BR])). Selecting the c h a n g e s in a b s o r b a n c e at a w a v e l e n g t h o f 635 n m , a n d p l o t t i n g l n ( A t - - A ~ ) a g a i n s t t, a s t r a i g h t line w i t h relative coeffic i e n t 0.999 is o b t a i n e d , as s h o w n in Fig. 4. A s u b s e q u e n t c h e m i c a l r e a c t i o n rate c o n s t a n t of 5.0 × 1 0 - 3 s-~ is o b t a i n e d f r o m the slope. ACKNOWLEDGEMENT
T h e s u p p o r t of this s t u d y b y the N a t i o n a l N a t u r a l Science F o u n d a t i o n o f C h i n a is gratefully a c k n o w l e d g e d . REFERENCES 1 2 3 4 5 6
R. Broderson, CRC Crit. Rev. Clin. Lab. Sci., 11 (1979) 305. K.P.M. Heirwegh and S.B. Brown (Eds.), Bilirubin, Vol. 2, CRC, Boca Raton (FL), 1982. .I.D. Van Norman, Anal. Chem., 45 (1973) 173. C. Slifstein and M. Ariel, J. Electroanal. Chem., 48 (1973) 447. F. Moussa, G. Kanoute, C. Herrenknecht, P. Levillain and F. Trivin, Anal. Chem., 60 (1988) 1179. J.R. Pradko, S. Pons, S. Bandyopdhyay, J.F. MeAleer and A.S. Hinman, Bioelectrochem. Bioenerg., 13 (1984) 267. 7 W.R. Heineman, F.N. Hawkridge and H.B. Btount in A J . Bard (Ed.), Electroanalytical Chemistry, Vol. 13, Marcel Dekker, New York, 1983o pp. 1-104. 8 Dong Shaojun, Anal. Chem. (Chinese), 13 (1985) 70.