Bilirubin-Metal Ion Complexes

John D. Van Norman and Michelle M. Humans Department of Chemistry. Youngsto wn State University. Youngsto wn. Ohio 44503

Velapoldi and Menis ( I ) have shown tha t bilirubin, a bile pigment, forms definite, strong metal-ion complexes in chloroform-ethanol mixtures and have studied the spectral shifts of the bilirubin absorption as a function of added metal ion. They concluded that bilirubin com- plexes with a variety of metal ions and those metal ions which form strong square planar complexes promote oxi- dation of bilirubin by placing a strain on the bilirubin structure.

Van Norman ( 2 ) has studied the electrochemical oxida- tion of bilirubin in N,N-dimethylformamide and found that controlled potential coulometry could be used as a possible alternate method of checking the purity of biliru- bin.

This present investigation is concerned with the electro- chemical behavior of bilirubin and bilirubin-metal ion complexes in aqueous media. Since bilirubin is fairly in- soluble in acidic media, all measurements were made in a phosphate buffer of pH 7.8 or 8.0. The use of this pH also minimizes the problem of intra- and intermolecular hy- drogen bonding interfering with complex formation.

EXPERIMENTAL Equipment ; All electrochemical measurements were obtained

with a National Instrument Laboratories “Electrolab” (3) , a mul- tifunctional electroanalytical system in conjunction with a Valtec Model 1024 X-Y recorder. All pH measurements were taken using either a Chemtrix Type 40E p H meter or a Sargent Model LS pH meter. The electrochemical cell used was a Metrohm Model EA 874 titration vessel and the indicator electrode was a Metrohm Hanging Mercury Drop Electrode (HMDE), Type E410, commer- cially available. A micrometer dial a t the top of the HMDE as- sembly permits formation of mercury drops of known and repro- ducible size. All measurements were taken with a n electrode area of 0.0222 f 0.0007 cm2. The other two electrodes in the three-elec- trode system were a Sargent platinum electrode which served a s the counter electrode and a Sargent saturated calomel electrode (SCE) which was the reference electrode. All experiments were performed a t ambient temperature (22-23 “C).

Reagents. The bilirubin used was obtained from Sigma Chemi- cal and was of 99% purity and had a measured molar absorptivity in chloroform of 60,100 f 600. It was stored in the freezer com- partment of a laboratory refrigerator. The buffer solutions pre- pared and used were checked against standard buffer solutions furnished by Sargent. All were 0.05M in phosphate ion. The metal ion solutions were prepared by acid dissolution of the pure metal in the cases of zinc, copper, nickel, and iron. The cobalt(I1) solutions were obtained by dissolving cobaltous chloride (CoClZ. 6HzO) in distilled water, the cadmium solution by dissolving cadmium acetate (Cd(CzH302j2.2Hz0) in distilled water, and the mercury(I1) solution by dissolving mercuric chloride (HgC12) in distilled water. Both argon and nitrogen gases were used as inert atmospheres in this study. The argon was passed over clean cop- per chips a t 600 “C to remove any traces of oxygen.

Procedure. The following procedure was followed for obtaining the cathodic or reductive voltammograms of bilirubin. A buffer of 7.8 or 8.0 was deoxygenated by passage of argon gas through it for 1 hour. A background reductive voltammogram was obtained for the buffer by sweeping the potential from 0.0 volts us. the SCE to -2.0 volts a t different scan rates. The current (i) between the hanging mercury drop electrode (HMDEj and the counter electrode ( P t j was measured a s a function of applied potential ( E ) between the HMDE and the reference electrode ISCE). The

(1) R A Velapoldi, and 0. Menis, Clfn Chem.. 17, 1165 (1971) (2) J D Van Norman, Anal Chem 45, 173 (1973) (3) G. W Ewing.J. Chem Educ 46, A717 (1969)

resulting i-E plots were recorded on the X-Y recorder. Using the three-electrode system minimized effects due to solution resis- tance. A measured amount of a bilirubin master solution (in 0.1N NaOH) was then added to the known volume of buffer, the solu- tion again deoxygenated for 10 minutes, and more reductive scans were taken a t a variety of scan rates. The pH of the solutions were always checked after addition of the bilirubin master solu- tion.

The procedure for studying the bilirubin-metal ion complexes was similar. Again, a background was obtained on the deoxygen- ated buffer. Then a n aliquot of metal ion solution was added, argon bubbled again, and more reductive scans were taken. Then small known volumes of the bilirubin solution were added to give different bilirubin concentrations. Voltammograms were obtained a t each concentration of bilirubin (Note: the metal ion concentra- tion remains constant).

RESULTS AND DISCUSSION In voltammetry, the height of the peak is a function of

concentration, scan rate, and other factors as shown below in the Randles-Sevcik equation: i, = 269 X 10’ nJ’’ AD”’ C LJ’? for reversible waves (1)

or i, = 2.98 X 10j n(cu n)’I2 AD1’? C ul’? for irreversible

waves ( 2 ) where i, is the peak current in amperes, C is the concen- tration of electroactive species in moles per cubic centi- meter, L: is the scan rate in volts per second, A is the elec- trode area in square centimeters, D is the diffusion coeffi- cient in cm2/sec, n is the number of electrons transferred per molecule and a is defined as the degree of irreversibil- ity and can be evaluated from:

0.04771 o n = E , 2 - E , ( 3 )

where E,, is the peak potential and E,, 2 is the potential where the current has the value &/2 .

It should be noted tha t Equation 1 is for a plane linear- diffusion electrode. For a spherical electrode such as the Hanging Mercury Drop Electrode (HMDE), the correct expression is

where r is the radius of the drop, 0.042 cm in this in- stance. For the operating conditions of this investigation such as scan rate, drop size, and diffusion coefficient, the correction to the Randles-Sevcik equation, Equation 1, is less than 5% and, for the sake of simplicity, the correction was omitted. For a more complete discussion of voltam- metry, one is referred to Meites (4) . Thus, one can use Equations 1-3 to evaluate diffusion coefficients for either reversible or irreversible systems. If a metal ion and its complex reduce reversibly to the

amalgam, then measurements of the shift in half wave or full wave potential (E,> 2 or I?,]) to more negative poten- tials with an increase in ligand concentration can be ap- plied to determine the overall stability constant. When a fairly stable complex exists in the solution and the com-

( 4 ) L Meites Polarographic Techniques second ed revised Inter- science New York N Y 1965 p p 4 1 3 - 1 5

926 ANALYTICAL CHEMISTRY, VOL 46, NO. 7, JUNE 1974

Table I. Diffusion Coefficients for Bilirubin. Do, crnt/sec

Scan rate, Reduction (V, rnin) potential, V Peak current, pA aNn, calcd

0 . 5 - 1 . 3 7 0 . 4 0 1 . 2 6 1 . 0 - 1 . 3 7 0 . 5 3 1 . 2 6 2 . 5 - 1 . 3 8 0 . 8 0 1 .19 5 . 0 - 1 . 3 9 1 . 1 3 0 .95

[BRI = 1 . 1 X 10-'M: supporting electrolyte, phosphate buffer, pH 7 . 8 .

Reversible Irreversible

5 . 9 4 x 10-6 8 . 4 9 X 6 . 0 0 X 6 . 9 1 X 6 . 8 5 X 7 . 9 2 X 6 . 0 3 X 10 .0 x 10-6

Av 6 . 2 1 =t 0 . 3 2 X 10-6 8 . 3 6 + 0 . 8 4 X

- E ( V O L T S ) V S SCE

Figure 1. Cathodic voltammograms of bilirubin (1 X 10-4M) in a phosphate buffer of p H 7.8

( A ) Scan rate of 0.5 V;min. Ep = -1 39 V ; (6) Scan rate of 1.0 V/min, E, = -1.40 V; (C) Scan rate of 2.5 V/min, E p = 1.40 V; ( D ) Scan rate of 5 0 V,/min, E p = 1.40 V: (E) Buffer only, scan rate of 2 5 Vlm in

plexing agent is in fair excess, the following relationship holds ( 5 ) :

where and are peak potentials for the simple and complexed ion, respectively, p is the number of lig- ands coordinated to the metal ion (one in the case of bili- rubin) and the Pp is the overall stability constant and C is the ligand (bilirubin) concentration. If we let AE be (E,,)s - (Ep)c, a plot of AE us. log C should be a straight line with a slope of 0.059 plm and an intercept of 0.059jn log P P .

Electrochemical reduction a t the Hanging Mercury Drop Electrode (HMDE) was accomplished for a bilirubin solution 1.0 x 10-4M a t a potential of -1.4 volts us. the SCE. The voltammograms obtained a t various scan rates are shown in Figure 1. Diffusion coefficients were calcu- lated using Equations 1 and 2 for reversible and irreversible systems and the values obtained as shown in Table I. The bilirubin system was experimentally found to be irrevers- ible as the reversal of the voltage scan after reduction gave no anodic wave due to the re-oxidation of the re- duced species. Irreversibility could also be inferred from the spreading out of the voltammograms. Although irre- versible, the reductive process was highly reproducible. The peak potential, E,, of -1.40 volts us. an SCE in the phosphate buffer of pM 7.8 compares favorably with the value reported by Tvaroha (6) for a half wave potential of -1.48 volts in 0.1M NaOH. ( 5 ) J B Headridge. "Electrochemical Techniques for Inorganic Chem-

ists, ' Academic Press. London, 1969, pp 32-33 (6) 8 Tvaroha. Naturwissenschaften. 48,99 (1961)

h

3 1.0 .-

0.5

0 1.10 1.14 1.18 1.22 1.26

- E (VOLTS) vs SCE

Figure 2. Cathodic voltammograrns of 1 X 10-4M Z n ( l l ) solu- tion in a 0.04M phosphate buffer of pH 8.0 as a function of added b i I ir u b i n ( A ) [Bilirubin] = 2.5 X 10-5M. E, = -1.16 V; ( E ) [Bilirubin] = 5 0 X 10-5M. E, = -1.17 V; (C) [Bilirubin] = 6.0 X 10-5M E,, = - 1 18 V; ( D ) [Bilirubin] = 7.5 X 10-5M El, = -1.20 V: ( E ) [Bilirubin] = 4 2 X

Note: [Bilirubin] = 6.0 X 10-4M. same as ( E ) 1 0 - 4 ~ . E,, = -1.21 v.

Table 11. Zinc (11)-Bilirubin Complex Reduction" Zn(I1)

reduction (El>)5 - [BR] moles/l. potential, V V 8P

0 - 1 . 1 2 . . . . . . 2 . 5 x 10-5 - 1 . 1 6 0 . 0 4 1 . 0 x 106 5 . 0 x 10-5 - 1 . 1 7 0 . 0 5 1 . 0 x 1 0 6

6 . 0 X 10-6 - 1 . 1 8 0 . 0 6 2 . 0 x 106 7 . 5 x 10-5 - 1 . 2 0 0 . 0 8 7 . 0 X 106 4 . 0 x 10-4 - 1 . 2 1 0 . 0 9 3 . 0 X lo6 6 . 0 X 10-4 -1 .215 0,095 3 . 0 X 106

Av 2 . 8 * 1 . 5 X lo6 [Zn(II)I = 1.0 X lO-AM, supporting electrolyte, 0 . 0 5 M phosphate

buffer, pH 8 . 0 , scan rate = 2 . 6 V;min.

Diffusion coefficients were calculated for a theoretically reversible system and for the actual irreversible system. Values obtained were 6.21 & 0.32 X cm2/sec for the reversible case and 8.36 f 0.84 x cm2/sec for the ac- tual system. These results compare quite favorably with values found for other large organic species in aqueous so- lution (7).

A zinc ion solution, 1.0 x 10-4M, was reduced a t the HMDE and a wave was obtained a t -1.12 volts us. the SCE. The reduction potential, E,, shifted negative as a function of added bilirubin as shown in Figure 2. Stability or formation constants for the reaction Zn(I1) + BR = ZnBR where BR represents the bilirubin species in solu- tion and ZnBR, the complex without reference to charges, were calculated using Equation 5. The results of these cal-

(7) R . N Adams, "Electrochemistry at Solid Electrodes, Marcel Dek- ker, New York, N.Y , 1961, pp 220-22

A N A L Y T I C A L C H E M I S T R Y , V O L . 46, NO. 7 , JUNE 1974 927

lo- I__ 10-61

0 0 32 0 04 0 . 0 6 0 . 0 8 0 . 1 9 0 . 1 2 0. A E ( v o l t s )

Figure 3. Plot of [BR] vs. A€

1

Table 111. Cadmium (11)-Bilirubin Complex Reductioncz

Cd(I1) [Bilirubin], reduction (E,,)? -

moles 1. potential, V (El,)c, V P P

0 -0 .71 . . . . . . 5 . 0 X 10-j -0 .73 0 . 0 2 1.0 x 105 6 . 0 x 10-5 -0.74 0 .03 1.8 x 105 7.0 X 10- j -0.75 0 .04 3 . 0 x 105 4 . 0 x 10-4 -0.76 0 .05 1.0 x 105 5 . 0 x 10-4 -0.76 0 .05 1.0 x 105

Av 1 . 6 = 0 . 7 X lo5 [Cd(I I ) ] = 1 . 0 X 10-’M, supporting electrolyte, 0 . 0 5 M phosphate

buffer, pH 8 . 0 , scan rate, 2 5 V I min.

culations are summarized in Table 11. This table is the re- sult of point by point calculation. The data were also ana- lyzed graphically. Figure 3 shows a plot of concentration of bilirubin, [BR], us. AE plotted with the ordinate on a logarithmic scale. This manner of presentation is conve- nient from the point of view of discussion. This plot gives a straight line with the exception of the point a t a [BR] of

Equation 5 holds strictly, when the ligand concentration is in great excess. The reason for this is that when the metal ion complex is reduced to the metal a t the surface of the electrode, the ligand is released resulting in a local excess a t the electrode surface. When the ligand concen- tration is large compared to the metal ion, the effect of the local excess on the potential shift is small; and when the ligand is small compared to the metal ion, there is not enough to complex a significant portion of the remaining free metal ion. It is when the metal ion and ligand con- centrations are similar that a maximum effect would be expected, as is the case in the present study. However, it must be recognized that the points obtained a t the lower concentrations are not as significant as the points ob- tained with the ligand in excess. Therefore to extrapolate to zero concentration would unfortunately magnify any er- rors made a t lower concentrations. For example, there is a trend noticeable in Table I1 toward lower p’s a t lower con- centrations. Extrapolation of a plot of AE GS. log [BR] (not shown) yielded a Pp of 4.2 X lo5 in contrast to the average of point by point calculations of 2.8 x 106.

Obviously the best solution would have been to work only at higher ligand concentrations, but unfortunately the solubility of bilirubin under these conditions is just above the highest point measured. Attempts to study zinc ion a t significantly lower concentrations resulted in the experimental inability to measure potential shifts accu- rately enough to make meaningful calculations. Thus, the measurements are a compromise between the solubility limits of bilirubin and the experimental determination of potential shifts.

The slope of the plot of S E GS. log [BR] gave a slope of 0.040 V which gives a value of 1.3 for “p”, interpreted as only one ligand. The trend a t higher ligand concentrations is toward a lower slope; toward p = 1. Experimental evi- dence for the existence of bilirubin complexes with metal ions with more than one bilirubin per metal ion is not available. There is, however, evidence that more than one metal ion may complex with bilirubin, a t least in non- aqueous media ( 8 ) . Whether this could occur in aqueous solution is a t the moment conjectural.

( 8 ) C C Kuenzle. R P Pelloni, M H Weibel, and P Hemmerich, BJO-

1.5 x 10- 5 ~ .

chem J 130. 1147 11972)

Table IV. Iron (II)-Bilirubin Complex Reductions

Fe(II1, reduction (E,) -

[BR], moles ‘I. potential, V (ED)c, V PI,

0 - 0 . 4 0 , . . . . . 3 . 0 x 10-5 -0 .48 0 .08 2 .0 x 10’ 5 . 0 x 10-5 -0.49 0.09 2 . 2 x 107 7 . 0 X 10-5 -0.50 0.10 4 . 0 x 107 9 . 0 x 10-5 -0 .51 0.11 6 . 0 x 107

6 . 0 X 10-4 -0 .53 0 .13 4 . 0 x 107 4 . 0 x lo-‘ -0.52 0 .12 3 . 0 X 10‘

AV 3 . 5 * 1.1 x 107

[Fe( I I ) ] = 1 . 0 x 10-1, supporting electrolyte, 0 . 0 5 M phosphate buffer, pH 8 .0 , scan rate, 2 5 V /min.

The reduction wave of cadmium(I1) solution, 1.0 x 10-4M, occurred a t a peak potential of -0.71 volt us. the SCE. The voltammetric waves were quite similar to those of Zn(1I) except for the potential. Bilirubin-cadmium ion complex formation constants were evaluated by the shift in reduction potential and the results are shown in Table 111.

The plot of AE L‘S log [BR] for the cadmium(I1) system had considerable scatter. Extrapolation to 1 E = 0 of vari- ous “best straight lines” gave a range in value of Pp from 2 X lo5 to 5 X lo5 compared to the average of 1.6 X lo5 in Table 111. Slopes varied between 0.027 and 0.038 V indicat- ing once again one ligand. As was the case for the zinc(I1) system, the point where the ligand and metal ion concen- trations were similar, was well off the straight line.

Iron(I1) solution, 1.0 x 10-4M, gave a reduction poten- tial a t -0.40 volt cs. the SCE. The results for the evalua- tion of the formation constant of the iron(I1)-bilirubin complex are summarized in Table IV. The plot of A E us. log [BR] gave a slope of 0.033 V, indicating one ligand, and a JP of 1.25 x lo7 compared to the value in Table IV of 3.5 X 10:.

Unsuccessful attempts were made to study the forma- tion of bilirubin complexes with copper(II), mercury(II), nickel(II), and cobalt(I1). Copper(I1) reduced a t -0.46 volt but addition of bilirubin did not change the potential; however, the bilirubin yellow color changed rapidly to a green color which was attributed to the oxidation of the bilirubin to the green biliverdin. Mercury(I1) reduction wave could not be obtained with the HMDE under these conditions, and bilirubin was oxidized. Kickel(I1) waves could not be obtained either. The cobalt(I1) wave occurred a t -1.27 volts but addition of bilirubin did not shift the wave a t all.

The reduction potential of the metal ions studied all were well within the range noted for each ion in a variety of solutions as reported in the literature (4 ) . From the

928 ANALYTICAL CHEMISTRY. VOL. 46. N O 7. J U N E 1974

shifts in the potentials as a function of added bilirubin, the formation constants for bilirubin complexes with zinc- (11), cadmium(II), and iron(I1) were evaluated. The values are; for zinc(I1)-bilirubin, /? = 2.8 f 1.5 X lo6; for cadmium(I1)-bilirubin, /3 = 1.6 f 0.7 X lo5; for iron(I1)- bilirubin, /? = 3.5 f 1.1 X lo7. Since there are no reported values for these formation constants in the literature, no comparison is possible.

The precision of the values for the formation constants was generally 15070, fairly typical for this type of mea- surement where a small error in measuring potential shifts results in a large error in the value. In each case, the value obtained for the formation constant a t approximate- ly equal metal ion and bilirubin concentrations was signif- icantly larger than those obtained with low bilirubin con- centrations and with bilirubin in excess. This is probably due to local excesses of bilirubin a t the surface of the elec- trode following the metal ion reduction. This value was, however, included in the overall calculation of the aver- age. The use of plots of LE us. log [BR] gave values of the same order of magnitude, but it is felt tha t extrapolation

may overemphasize errors a t lower concentrations of lig- and. The results obtained indicate an order of complex formation of metal ion with bilirubin of Fe(I1) > Zn(I1) > Cd(I1).

It should be pointed out that the formation constants measured in this study are “effective” or “conditional” formation (stability) constants as their numerical value depends upon the solution conditions, pH, and composi- tion. Bilirubin, however, probably does not form strong metal ion complexes in more acidic aqueous solution due to inter- and intramolecular hydrogen bonding, making measurements in acidic solution even more difficult.

ACKNOWLEDGMENT The authors would like to acknowledge helpful discus-

sions with L. B. Spiegel and T . N. Dobbelstein.

Received for review November 21, 1973. Accepted Febru- ary 27, 1974.

Induced Colorimetric Method for Carbon Monoxide

Jack L. Lambert and Robert E. Wiens’ Depar tment of Chemistry, Kansas State University, Manhattan, Kansas

Methods in common use for carbon monoxide in air in- clude a colorimetric method which employs silver p-sul- faminobenzoate complex in alkaline solution ( I ) , infrared spectrometry at 4.67 p (2), and nondispersive infrared ad- sorption ( 3 ) . The method described here is the second to be reported in which a soluble colored compound is formed in aqueous solution a t room temperature; the first was a method which employed tetrachloropalladate(II), ethylenediaminetetraacetatoferrate(III), and 1.10-phenan- throline and produced red-orange t r i s ( 1,lO-phenanthro- line)iron(II) cation ( 4 , 5 ) .

In the reaction described here, a reagent consisting of tetrachloropalladate(II), iodate, and leuco crystal violet (4,4’,4”-methylidynetris(N,N-dimethylaniline) produces crystal violet in proportion to the concentration of carbon monoxide and the time of reaction. At pH 3.1, the reac- tion between iodate and leuco crystal violet is kinetically inhibited, but the sequential reduction of palladium(I1) to palladium(0) followed by reduction of iodate by palladi- um(0) produces hypoiodous acid which rapidly oxidizes leuco crystal violet to crystal violet. As palladium(I1) is produced in the reduction of iodate by palladium(0). no metallic palladium appears in the solution. Iodide pro-

Present address, Norden Laboratories, Lincoln, Neb. 68501

( 1 ) R . G . Smith, R. J Bryan, M Feldstein, B Levadie, F. A Miller, E. R Stephens, and N G . White (Subcommittee 4 of the Intersociety Committee), Health Lab. Sci.. 7 , 75 (January Supplement) (1970)

(2) Ibid., p 78. ( 3 ) Ibid., p 81 ( 4 ) J. L . Lambert and P A Hamlin, Anal. Lett.. 4, 745 (1971) (5) J L . Lambert, R R Tschorn, and P A. Hamlln, Anal. Chem.. 44,

1529 (1972)

duced by the reduction of hypoiodous acid apparently can act as a catalyst by reacting with iodate to produce hy- poiodous acid, but the catalysis reaction apparently is of minor importance in the production of crystal violet.

EXPERIMENTAL All solutions were prepared from deionized xvater with the pur-

est chemicals available from commercial sources. Prepare leuco crystal violet solution, 6.85 x 10 - 4 .Id by dissolving 0.256 gram of 4,4’,4’‘-methylidynetri~(~Y,~’v‘-dimethylani!ine) (Eas tman Kodak Co.) in 200 ml of water containing 2.5 ml of’ 8<5C/c phosphoric acid and diluting to exactly 1 liter. Prepare sodium tetrachloropallad- ate(I1) solution, 5.00 x 10-3M, by dissolving stoichiometric quan- tities of palladium(I1) chloride and sodium chloride in the appro- priate quantity of water; for 1 liter of solution, 0.887 gram of pal- ladium(I1) chloride and 0.584 gram of sodium chloride are re- quired. Iodate solution. 0.100,1.1, contains 21.4 grams of potassium iodate per liter of solution. Prepare sodium monohydrogen phos- phate solution, 0.100M, by dissolving 26.8 grams at’ sodium mo- nohydrogen phosphate heptahydrate. XazHPO4.7Hz0. to make l liter of solution. Measure absorbances for Reagent A at 587 nm in 1-cm cuvettes in a spectrophotometer.

Leuco Crys ta l Violet Reagent (Reagent A ) , Pipet 1.00 ml each of leuco crystal violet solution. potassium iodate solution, and sodium tetrachloropalladate solution into 25.00 nil of water. The pH is adjusted to 3.10 f 0.02 with the sodium monohydrogen phosphate solution. Freshly made reagent had an absorbance of 0.02, compared to water. which increased to 0.07 after 2 hours without exposure to carbon monoxide.

Si lver p-Sulfaminobenzoate Reagent (Reagent €3). Mix 20.00 ml each of 0.100M p-sulfaminobenzoic acid and 0.lOO.W silver ni- t ra te solutions, and add 10.00 ml of 1 . O M sodium hydroxide with shaking, as recommended in the tentative method by Subcom- mittee 4 of the Intersociety Committee ( I ) . Measure absorbances a t 425 nm in a 1-cm cuvette for concentrations in the 0-400 ppm range (above 400 ppm to 1800 ppm, absorbances are measured a t 600nm) .

ANALYTICAL CHEMISTRY, VOL. 46, NO 7. JUNE 1974 929