Electroanalytical and Spectral Study of Bilirubin in Dimethyl Formamide John D. Van Norman Department of Chemistry, Youngstown State University, Youngstown, Ohio 44503
BILIRUBIN, one of the bilirubinoids, is a member of the class of compounds found in the body called bile pigments. Such compounds are linear tetrapyrroles and as such are derivatives of porphyrins. Bilirubin is formed by the physiological degradation of the porphyrin system, hemoglobin. Because of the great importance of the bile pigments in the body, they have been the focus of a large number of clinical, medical, biological, and chemical investigations. An excellent review of the work in this field covering aspects through 1967 has been written by T. K. With ( I ) . Out of approximately 3,000 references only three, all by B. Tvaroha (2-4), deal with direct electrochemical studies of bilirubinoid systems. One of the greatest problems associated with the analytical chemistry of bilirubin, or for that matter with many biochemicals of clinical interest, is that of the establishment of criteria for purity of clinical standards. Meinke (5) has pointed out this problem and the involvement of the National Bureau of Standards in its solution. In the case of bilirubin, the material is dissolved in chloroform and the spectrum is obtained. If the molar absorptivity is between 59,100 and 62,300, the bilirubin can be certified as a clinical standard. This study was undertaken in an effort to establish an alternate and perhaps more accurate criterion for purity of bilirubin. The inherent accuracy possible in certain electroanalytical techniques such as coulometry suggested this approach ; dimethyl formamide was selected as the solvent system because of the relatively high solubility of bilirubin in it, and the relative ease of handling the solvent for electrochemical studies. EXPERIMENTAL
Chemicals. The bilirubin used, obtained from Sigma Chemical, was of 99% purity and had a measured molar absorptivity in chloroform of 60,100 + 600. The biliverdin used was in the hydrochloride form and was also obtained from Sigma Chemical; its purity was 85%. The dimethyl formamide was of reagent grade and was used directly from pint bottles with no further treatment. Anhydrous sodium perchlorate was used as the supporting electrolyte and was of reagent grade. Argon, used as an inert atmosphere, was treated by passing over copper chips at 600 “C to remove oxygen. Equipment. Electrochemical measurements were taken with a National Instrument Laboratory “Electrolab,” a multi-functional electroanalytical system in conjunction with a ValTech, Model 1024 X-Y recorder. Electrodes used were Sargent-Welch Pt indicator electrodes with an area of approximately 2 cm2, platinum foils, and a saturated aqueous calomel electrode. The electrochemical cell was a Metrohm Model EA 874 titration vessel which had five openings in the upper portion to permit insertion of the appropriate elec(1) T. K. With, “Bile Pigments:
Chemical, Biological and Clinical Aspects,” Academic Press, New York, N. Y . , 1968. ( 2 ) B. Tvaroha, Cus. Lek. Cesk., 100, 27 (1961). ( 3 ) B . Tvaroha, Collect. Czech. Chem. Commun., 26,2271 (1961). ( 4 ) B. Tvaroha, N~irurwisse~rscl~ufterz, 4, 99 (1961). (5) W . Meinke, ANAL.CHEM., 43 (6), 28A (1971).
160
’
I
/ ’
1A 0
100 120
80
t I 1
0’ 0
*0 2
r0 4
-0 v0.ts
E
-0 8 +l 3 SCE
I’
f.
4
“5
Figure 1. Anodic voltammograms obtained at platinum electrode, 1.98 cm2 in area, bilirubin concentration of 2.5 X 10-4Min DMF, 0.1M in NaCIO, Scan rates: A . Solvent only 0.0417 volt per second B. 0.0167 volt per second C. 0.0417 volt per second D . 0.0833 volt per second E . 0.1667 volt per second
trodes and gas bubblers. The voltammetric studies were performed using the platinum indicator electrodes while the controlled potential coulometric measurements were taken using a large platinum foil and a platinum counter electrode in an isolated fritted compartment. In all cases the reference electrode was an aqueous saturated calomel electrode (SCE). All spectral measurements were taken on a Beckman DB spectrophotometer and were checked on either a Beckman DU or a Cary Model 14 spectrometer. RESULTS AND DISCUSSION
Solutions of bilirubin in DMF made 0.1M in sodium perchlorate were either yellow or orange, depending upon the concentration and appeared to be resistant to the photochemically induced oxidation which is well known for the aqueous solutions ( I ) . Anodic voltammograms of solutions 2.5 x 10-4M in bilirubin were obtained at a platinum anode. Figure 1 shows the typical anodic voltammograms obtained at various scan rates. The voltammograms show two major waves with peaks at approximately +0.6 and $0.8 volt cs. the aqueous saturated calomel electrode (SCE), depending upon the scan rate. Cyclic voltammograms obtained by reversing the direction of scan after the first wave or after the second wave, gave no cathodic current due to the reduction of the oxidation product formed, as shown in Figure 2. The oxidation of bilirubin and of the subsequent product appears to be irreversible in this solvent system. In an effort to identify the oxidation products, controlled potential coulometry was performed at a potential of $0.85 volt os. the SCE. The electrolysis was halted at intervals and
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
173
ua
1.
o L
'
360
480 520 560 W A V E L E N G T H (nm)
440
400
600
640
680
720
1
760
Figure 3. Spectra of bilirubin solution electrolyzed at 0.85 V us. the SCE 0
0
+0.2 +0.4 +0.6 V o l t s v s SCE
A . Initial solution-8.91 X 10-6 mole of bilirubin B . 1.0 coulomb passed C. 1.9 coulombs passed
+0.8
Figure 2. Cyclic voltammograms obtained at a scan rate of 0.0417 volt per second. Bilirubin concentrationof 2.5 X 10-4M in DMF, O.1Min NaC104
D. 2.9 coulombs passed strengths. The color changes can be attributed to the following sequence of reactions :
o'a\ ca ac,ao M
H
( B ) Scan reversed after the second wave
(6) B. Zak, N. Moss, A. Boyle, and A. Zlatkis, ANAL. CHEM., 26, 1220 (1954). 174
P
M
M
V
B I I lrubln
( b IIo d Ie n e - o c)
( A ) Scan reversed after the first wave
samples were taken for spectral measurement. Figures 3 and 4 show the results obtained. The spectrum initially obtained was that of bilirubin with a maximum at 460 nm, the same as in chloroform. As coulombs were passed, the peak at 460 diminished and new peaks a t 370 and 645 nm appeared. After passage of sufficient number of coulombs to account for a 2-electron step, no further increase in the 370 or 645 peaks occurred, Curve D in Figures 3 and 4. Passage of more coulombs resulted in the diminution of these two peaks and the appearance of a peak a t 530 nm. The color of the solution went from yellow to a green t o a blue green at n = 2, then into a blue, then purple, and finally in one case to an orange. As will be shown later, a potential of +0.85 volt us. the SCE is sufficiently positive to oxidize not only the bilirubin to biliverdin but the biliverdin formed to further oxidation products. It must be pointed out that the results reported in Figures 3 and 4 are only qualitative as there was no background correction made and care was not taken to obtain 100% current efficiency for a particular reaction. It is interesting to note that there is a parallel between the electrochemical sequence of spectral species and that found chemically. This color sequence is noted in the well-known Gmelin reaction ( I ) wherein bilirubinoids are oxidized in chloroform using fuming nitric acid. Similar spectral changes have been reported by Zak er a/. (6) using oxidizing agents of varying
P
M
H
V
M
H
H
"
P
H
P
Yell0 w
M
H
Purpurin g
(biladlene- ab- one-e)
H
Purple
M
V
M
Ho ~
C
br Ivl= - C H 3
P
n
P ~
V:-CH=CH2
M f;
M
V
t i
~
Choletelln ( bCi l e n e - b - o~n e - a c )
o
Yellow P=-CH2-CH2'COOH
The color change has been attributed to the change in the number of conjugated double bonds going from 5 in bilirubin to 10 in biliverdin to 8 for the purpurin and back to 5 for the appropriate choletelin. The spectrum of biliverdin hydrochloride corresponded in every detail with Curve D in Figures 3 and 4. Thus curves B and C show the build up of biliverdin while curves E, F, and G show the diminution of biliverdin and build up of further oxidation products. Controlled potential electrolysis a t f0.65 volt L'S. the SCE yielded only the shift to Curve D and no further change in either the coulombs which could be passed or in the spectrum. In a similar manner, the change in the voltammetric behavior of a solution of bilirubin electrolyzed a t f0.65 volt
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
H
