Anal. Chem. 1988, 60, 1179-1185
1179
equilibrium concentration dependence on the initial concentration of a depositing metal ion. The present, and more general model, unites those two limiting approaches of the earlier model by means of the single, nonlinear eq 16, applicable for the full range of the initial concentrations, including the intermediate region. It is also postulated that one way of estimation of ( c , ) ~is by means of eq 6, when the mass-transfer coefficient of deposition, k , , and the specific rate of metal dissolution, k2, are separately measured for a single experiment. It seems that the measurement of k z is more difficult experimentally, than the measurement of kl, which is rather straight-forward under usual experimental conditions.
LITERATURE CITED
v
, -12
-11
109 ic,)
-IO
-6
Flgure 1. Calculated dependence of the equilibrium concentrations, c,, on the initial concentrations, co,of metal ion in solution, according to e 16. Concentrations in mol/cm3; are as follows: lo-'' (A), lo-' (B), and lo-'* (C).
9
equal to the chosen values of (c& of eq 6. These equilibrium concentrations are characteristic for an electrode fully covered by deposit. The third region of the curves in Figure 1 is that between approximately 5 x 10-l' and 5 x mol/cm3 initial concentrations (5 X and 5 X lo-' M) of the metal ion, and corresponds to nonlinear solutions of eq 16. In this region, there is a nonlinear transition between (c,), of eq 5 and (c,)~ of eq 6. This region is relatively narrow (over the range of the initial concentrations of approximately one order) and, hence, practically not the most important. Thus, the earlier and simpler model (14, 19) adequately described the two limiting, and most practically important regions of the
(1) Sloda, R. E., Batiey, G. E.; Lund, W.; Wang, J.; Leach, S. C. Talsnta 1986, 33, 421-428. (2) Leyden, D. E.; Wegschneider, W. Anal. Chem. 1981, 53, 1059A1065A. (3) Krasiishchik, V. 2.;Kuzmin, N. M.; Neiman, E. Ya. Zh. Anal. Khim. 1979, 3 4 , 2045-2056. (4) Jerrstad, K.; Salbu, B. Anal. Chem. 1980, 52, 672-676. (5) Holen, B.; Bye, R.; Lund, W. Anal. Chim. Acta 1981, 130, 257-265. (6) Batiey, G. E.; Matousek, J. P. Anal. Chem. 1977, 49, 2031-2035. (7) Sioda, R. E. Talanta 1984, 3 1 , 135. (8) Salin. E. D.; Habib, M. M. Anal. Chem. 1984, 56, 1186-1188. (9) Habib. M. M.; Salin, E. D. Anal. Chem. 1985, 57, 2055-2059. (10) Matusiewicz, H.; Fish, J.; Malinski, T. Anal. Chem. 1987, 59, 2264-2269. (11) Abduliah, M.: Fuwa, K.; Hiroki, H. Appl. Spectrosc. 1987, 4 1 , 715-721, (12) Wang, J. Stripping Analysis; VCH Publishers: Deerfield, FL, 1985. (13) Brooks, E. E.; Mark, H. B., Jr. J . Environ. Sci. Heafih, Part A 1977, A12, 511-521. (14) Anderson, J. L.; Sioda, R. E. Talanta 1983, 3 0 , 627-629. (15) Sioda, R. E. Talanta 1985, 32, 1083-1087. (16) Nicholson, R. S.; Shain, I. Anal. Chem. 1984, 36, 706-723. (17) Bard, A. J.; Fauikner, L. R. Electrochemical Methods; Wiiey: New York, 1980; Chapter 11. (18) Saveant, J. M.; Vlaneiio, E. Electrochlm. Acta 1985, 10, 905-920. (19) Sioda, R. E. Anal. Lett. 1983, 16, 739-746. (20) Rogers, L. B.; Stehney, A. F. J . Nectrochem. SOC. 1949, 95,25-32. (21) Brainina, Kh. 2.;Kiva, N. K.; Belyavskaya, V. B. Elektrokhimiya 1965, 1 , 311-315. (22) Brainina, Kh. 2. Stripping Vofiammetry in Chemical Analysis; Wlley: New York, 1974; Chapter 1. (23) Rubinson, K. A. Chemlcel Analysis; Little, Brown and Co.: Boston, MA, 1967; Chapter 7.
RECEIVED for review October 23, 1987. Accepted February 5, 1988.
Electrochemical Oxidation of Bilirubin and Biliverdin in Dimethyl Sulfoxide Fathi Moussa,*vl Gaoussou KaDoute, Christine Herrenknecht, Pierre Levillain, and Frangois Trivin Laboratoire de Biochimie Appliquee, Laboratoire de Chimie Analytique et d'Electrochimie organique, Centre d'Etudes Pharmaceutiques Paris-Sud, F 92290 Chatenay-Malabry, France The mechanism of the electrochemical oxidation of biiirubln was reexamined to explain the dlfferences in behavior reported in previous studies publlshed by various authors. The findings point to an ECEC mechanism. Only the first step of this mechanism is speciflc to bllirubin, and it may consequently be used as a crRerlon of purHy for this molecule.
In man, bilirubin (BR) is the product of the catabolism of the tetrapyrrolic prosthetic groups, mainly from hemoglobin '
Present address: Hopital Saint-Joseph, Laboratoire de Biochimie, 7, rue Pierre Larousse, 75674 Paris Cedex 14, France.
(Figure 1). It constitutes a biological marker of hemolysis and hepatopathies. Neonatal hyperbilirubinemia can generate kernicterus, an encephalopathy with extremely serious clinical and social consequences that must be prevented. It is therefore important to be able to measure precisely the BR concentration in the blood. Some technical and analytical difficulties for measurement of bilirubinemia have not been solved in a satisfactory manner. Different circulating forms and a number of isomers may explain some of these difficulties. Current methods of determination do not take account of all these forms, and yet the latter do not all have the same physiopathologic significance. Moreover, there seems to be
0003-2700/88/0360-1179$01.50/00 1988 American Chemlcal Soclety
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988
b
v
T
I
k
A
'HJdJkLH i
A
A
Figure 1. (a) Bllirubln: M, -CH,; V, -CH=CH2; P, -CH,CH,COOH. Bilirubin IXaZZ. (c) Blliverdln.
(b)
no specific method for the IXaZZ isomer assumed to be responsible for kernicterus (1). Lastly, BR is an unstable molecule that is easily oxidized, This property explains the poor keeping of BR solutions and the difficulty of establishing a reliable criterion of purity. T o find more specific methods of determination, we undertook the electrochemical study of BR. Given the relative ease of oxidation of this molecule and the recent development in clinical chemistry labs of high-performance liquid chromatography (HPLC), electrochemical detectors with a solid electrode (graphite, vitreous carbon, platinum, gold, etc.) more conducive to oxidation, we decided to focus more closely on the electrochemical oxidation of this molecule. However, among the innumerable publications devoted to BR, only a few deal with its electrochemical properties (2-11). In fact, the electrochemical oxidation of BR has been discussed to date by only two authors (5-8). Van Norman 1973 (5) and Slifstein 1974 (8) studied the irreversible electrochemical oxidation of bilirubin and prop e d their own mechanism. The findings of these two authors show a difference in the behavior of the molecule which needs further investigation. T o further knowledge of the behavior of bilirubin, we studied the electrochemical oxidation of BR and BV (biliverdin) in dimethyl sulfoxide (DMSO). The latter is an excellent solvent of BR and is often used as an accelerator in the diazo reaction of this molecule (12). T o prevent the solution from coming into contact with the oxygen in the air, all the spectra were recorded in situ with an optical fiber spectrophotometer.
EXPERIMENTAL SECTION Reagents. All reagents were used without prior purification. We used BR (Sigma ref B 4126), BV chlorhydrate 80% (Sigma ref B 3753), dimethyl sulfoxide (DMSO) (Merck ref 2950), tetraethylammonium perchlorate (TEAP) (Fluka ref. 86649), and Nitrogen U grade. Apparatus and Procedure. The supporting electrolyte was 0.1 M TEAP in DMSO. The current-voltage curves were plotted by means of a Tacussel polarograph, type PR G5, using as an indicator electrode a platinum rotating disk electrode (RDE) (total
radius, 0.5 cm; radius of the metal disk, 1mm), type Tacussel ED1 33756. All measurements were made at a speed of 60 revolutions/s. As a reference, we used a Tacussel saturated calomel electrode, type C10, placed in a compartment containing the support electrolyte and separated from the solution by a fritted aluminum pellet (all the potentials reported herein are expressed versus the saturated calomel electrode (SCE)). The auxiliary electrode was a Pt coil. Electrolysis was conducted with a Tacussel potentiostat assembly, type PRT 20-2X, integrator,type IG5. The cyclic voltammetric curves were plotted by a Tacussel potentiostat, type PRGB, connected to a Tacussel triangular signal generator, type GSTP3. Voltammograms were traced on a Tektronik CRT oscilloscope, type T5103N. All the solutions were temperature controlled to 20 10.1 "C. After deoxygenation by paassge of nitrogen for 20 min, the working solutions were maintained under a nitrogen atmosphere during the entire operation. The visible spectra were measured directly in the cell containing the working solutions with a Guidedwave 200 spectrophotometer (distributed in France by Jobin Yvon) controlled by an IBM PC (13).The optrode directs the light selected by the monochromator to the BR solution. After having crossed a thin liquid layer, the monochromatic light is reflected by a mirror onto a second fiber which directs it to the detector. Work can be conducted without interruption during the entire coulometry operation thanks to the sturdy and inert optrode material. In addition, the thickness of the liquid layer crossed by the light can be adjusted from 1 to 9 mm, so that by choosing a minimal thickness, it is possible M) required for to work with the concentrated solution electrochemical studies. It must be specified, however, that the linearity of the spectrophotometer is exceeded at such high BR concentrations. We therefore chose to take into consideration only the qualitative aspect of our spectrophotometric results. Finally, this apparatus can trace first, second, third, and fourth derivatives to the experimental spectra.
RESULTS AND DISCUSSION In 1973, Van Norman (5),working in dimethylformamide (DMF) a t a platinum indicator electrode, reported for BR a voltammogram with two anodic waves (Ell2= +0.6 V (SCE) and El12= +0.8 V (SCE)). After a coulometric and visible spectrum study, he proposed for this molecule an electrochemical oxidation mechanism with three irreversible bielectronic steps BR
4 4; 4 Bv
purpurin
2H'
2H'
choletelin
2H'
In 1974, Slifstein (8) working in DMSO at a gold electrode, described a single anodic wave (Ellz = +0.55 V (SCE)) that he attributed to the irreversible oxidation of BR to BV after loss of two electrons and two H+ according to an EEC mechanism le-
BR
-L
1e-
BR+
) .
Bv
2+
2H'
This difference in mechanism may be ascribable a priori to the different operating conditions (electrodes, solvent, supporting electrolyte, etc.). However, two objections can be raised. Firstly, as the spectra of the solutions containing the BR oxidation products were not obtained under an inert atmosphere, we cannot exclude interaction of the oxygen in the air in the oxidation mechanism proposed by Van Norman. Secondly, Slifstein's results in DMSO need to be confirmed, as generally the biliverdin (BV) formed during the oxidation is in turn easily oxidized. This phenomenon is virtually inescapable and occurs under conditions as different as chemical oxidation (14), photochemical oxidation (15, 16), enzymatic oxidation (17, 18), biological oxidation (resorption of hematomas), etc.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988
1181
a
a
A/
B
V(SCE) 0
+0.3
+0.1
+0.5
+0.7
0
A.U.
+O.l
+0.3
+O. 7
+O. 5
A.U.
b
b 4.001
-0.05 340
395
4kQ
505
580
615
Sf0
340
395
450
505
560
615
670
Figure 3. (a) Voltammograms of a solution Initially containing 0.153 mM of BR in DMSO, 0.1 M in TEAP and electrolyzed at + O X V vs SCE Scan rate 10 mV s-’: S, solvent only; A, BR no coubmbs passed; B, 1.010 C passed; C, 1.600 C passed; D, 3.030 C passed. (b) Spectra of BR solution electrolyzed at 0.55 V vs SCE.
0
+0.2
+0.4
+0.6
V(SCE)
Flgue 2. (a) Anodic voltammograms obtained at platinum rotatin disk electrode, vs SCE in DMSO, 0.1 M in E A P , scan rate 10 mV 8- . BR concentratlons: A, 0.153 mM; B, 0.255 mM. (b) Spectra of BR solution. (c) Cyclic vottammogram obtained at a scan rate of 0.100 V s-’ BR concentration of 0.774 mM.
B
Behavior in Pure DMSO. Figure 2a represents the anodic voltammograms obtained for two solutions of BR with different concentrations. Figure 2b represents the visible spectra that were confirmed by their second derivatives. Under such conditions, the BR does effectively seem to be oxidized in a single wave whose halfwave potential (El,z)equals +0.55 V. Study of the curves IL = f d Z (see further on) and IL = f ( c ) shows that the wave is a kinetic one, which agrees with the
results published by Slifstein (8). The study of the current voltage curve by cyclic voltammetry shows that there is no cathodic peak associated with the anodic peak. The reaction is fuUy irreversible at 100 mV/s (Figure 2c). To analyze this wave, a controlled potential coulometry at +0.55 V was undertaken. With the spectrophotometer optrode plunging directly into the electrolytic cell, we were able to follow the solution’s parallel spectral changes. The voltammograms recorded during the experiment show the progressive disappearance of the first wave = +0.55 V) and the progreeaive appearance of a more anodic wave (Ell2 = +0.65 V) (Figure 3a). At the end of the coulometry we find just the latter wave with low limiting current (IL) (3.5 times lower than the IL of the first wave) independent of the speed of rotation of the working electrode ( w ) . Two electrons have been exchanged. Parallel to the electrochemical variations, the color of the solution changes from yellow to green and then to blue a t the end of the coulometry. The spectral study (Figure 3b) shows a gradual reduction of the BR band at 460
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988
nm followed by the appearance of a shoulder around 384 nm and of a new band a t 646 nm. These wavelengths are characteristic of BV (see further on). As the oxidation progresses, the new absorption becomes predominant and continues once the electrolysis is completed. The phenomenon can be pinpointed by tracing the second derivatives to the spectra. The curves thus obtained clearly show the decrease in the BR present, while the concentration of BV increases. At this stage, we can draw the following conclusions: The end product of BR oxidation at 0.55 V is BV. This compound is obtained after the irreversible loss of 2e- and 2H+ by the BR. The wave appearing a t the end of the coulometry a t a more anodic potential (Ell2= +0.65 V) can be attributed to the subsequent oxidation of the BV. This wave is in fact identical with that produced by the oxidation of a solution of BV (see further on). The electrochemical oxidation of BR in DMSO therefore does not stop a t the BV stage. The absence of this wave in the initial voltammograms (linear and cyclic) and its appearance during coulometry, as well as its low limiting current and its independence in relation to wl/z, suggest the existence of a chemical equilibrium following the electronic step of the BR oxidation (19, 20). The controlled potential coulometry at +0.70 V, we then undertook, gave different results. In this case, the single starting wave (Ellz= 0.55 V) gave way to the second wave already observed ( E l j 2= 0.65 V) which in turn disappeared at the end of the coulometry (Figure 4a). At the same time, after the switch from yellow to blue, the color changed to purple. The spectral study (Figure 4b) indicated a regular drop of the BR band (460 nm). That of the BV increased (390 and 646 mm) and then reached a maximum before decreasing, to the benefit of a new band which absorbs at 545 nm (curves E and F, Figure 4b) and is responsible for the purplish color taken on by the medium. It most likely corresponds to the purpurin stage of the Gmelin series (14). It therefore seems that, contrary to Slifstein's EEC assumption, the electrooxidation of BR follows an ECEC mechanism and could be broken down as follows:
BR
-
BR2++ nle
& BV + 2H+
BR2+
Kb
BV
-
n, = 2 with K = K,/Kb
BVn2++ nze
n2 = ?
BVn2+I purpurine (Pu) Kb
+ nH+
(1) (2)
(3) (4)
The absence of the second wave (Elj2= +0.65 V) in the initial voltammogram could be explained by the fact that the BR2+ (or BVH;+) intermediate cannot be electrochemically oxidized (whereas the BV formed in solution after loss of two protons is oxidizable). In equilibrium 2, if K, is small, BV concentration in the neighborhood of the electrode is negligible compared to that of the BR. Under these conditions, it would seem that as regards the limiting current (IL), the only reaction is the first electrochemical step (equilibrium 1). The intensity of the current can be expressed as follows (19, 20): I L = ID = 0.62nlFD2/3CBR"v-1/6,1/2where I L is the actual current measured, ID is the theoretical current of diffusion (Levich equation), nl is the number of electrons taking part (2), F = 1Faraday = 96496, D (cm2-s-l)is the diffusion coefficient of the electroactive species (here BR), C" (mrn~l-cm-~) is the concentration of the electroactive species in the solution far from the electrode, u ( c m 2 d )is the kinematic viscosity, and w (rades-') is the angular speed of rotation. At the end of the coulometry, only BR2+in equilibrium with the BV remains in solution. We are in the presence of a CE
a
I 1 u A
0
+0.3
+0.1
+O. 5
+0.7
- u
b / *.30
i 4
,
h
n
Figure 4. (a) Voltammograms of a solution inltially containing 0.153 mM of BR in DMSO, 0.1 M In TEAP, and electrolyzed at +0.70 V vs the SCE; scan rate 10 mV s-': S,solvent only; A, initial solution; 6, 2.80 C passed; C, 4.20 C passed; D, 5.80 C passed; E, 7.00 C passed; F, 8.20 C passed. (b) Spectra of BR solution electrolyzed at +0.70 V vs SCE.
mechanism. Since K = K,/& is s m d , then IL(BV) is smaller then ID (BV) and is expressed as follows (19, 20): IL
= nFD1/2Kb112KCToTm
where D ( c m 2 d )is the diffusion coefficient of the electroactive species (here BV) and CToT-
= C*"-
+ CBRZ+-
and where I L is independent of high w. This assumption can be verified by shifting equilibrium 2 toward the right, capturing the H+. In theory, this can be achieved by introducing into the medium a proton acceptor (water for example) or by neutralizing the H+ formed during the coulometry with a strong base (NaOH for example). Addition of Water into the Medium. Linear Voltammetry. Adding water to a solution of BR in TEAP-DMSO modifies the aspect of the voltammogram (Figure 5). A more anodic ( E l / z= +0.65 V) second wave appears with an increase in its IL according to the amount of water added (it is difficult to quantify the phenomenon as the limit of solubility of BR is reached very quickly in such a mixture of solvent (DMSO-HzO) when the percentage of water exceeds 25%). The first wave (El/*= +0.55 V) undergoes a cathodic shift
ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988
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I I B I /
/
/
J -d-
