Polarographic catalytic hydrogen waves in aquocobalamin and

and nonbuffered HCI solutions and for methylcobalamin In chloroacetlc acid ... of the various catalytic hydrogen waves caused by B12 com- pounds has n...
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1716

Anal. Chem. 1984, 56, 1716-1722

Polarographic Catalytic Hydrogen Waves in Aquocobalamin and Methylcobalamin Solutions Ronald L. Birke,* Ren-Ao Gu,l Jin-Min Yau, and Myung-Hoon Kim2

Department of Chemistry, The City College of The City University of New York, New York, New York 10031

The catalytic hydrogen process has been investigated for vitamin B,2a, aquocobalamin, in phosphate buffer (pH 6.8), chioroacetlc acid buffer (pH 2.87), sulfate buffer (pH 1.97), and nonbuffered HCI solutions and for methyicobalamin in chloroacetic acid buffer (pH 2.87), sulfate buffer (pH 1-97), and nonbuffered HCI solutions. The proposed mechanisms of these processes all involve protonation of the cobalamin specles, lrreverslble reduction of the protonated specles, and a following redox reaction whlch regenerates a precurser cobalamin specles and produces molecular hydrogen. Depending on the solution condltlons the regeneratlon reaction can take place in a reaction layer in solution or with an adsorbed catalyst and a diffuslng species. Rate constants are estimated for the catalytic rate determining steps and are within a few orders of magnitude of diffusion limlted rates leading to very large catalytic currents and the posslbiiity of uitratrace electroanalysis.

Although many aspects of the electrochemistry of vitamin Blz compounds have been thoroughly studied, the mechanism of the various catalytic hydrogen waves caused by Blz compounds has not been completely worked out. The first polarographic study of these waves was by Abbott (1) with cyanocabalamin (vitamin B12)where two different catalytic hydrogen waves were found, one in phosphate buffer (pH 5.5-7.4) with a peaked-shaped wave at -1.6 V vs. SCE and the other in dilute HC1 and 0.1 M KC1 with an irreversible wave with Ellz = -1.2 V vs. SCE. The latter wave was attributed to a catalytic redox process involving reduction of vitamin BlZr,Co(II), to vitamin BlZs,Co(I), with regeneration of B12rby the reaction of H+ and B12*yielding B12? and H2. The reaction of BlZsand H+ had been directly studied earlier and found to be nonstoichiometric (2). The wave in phosphate buffer was thought to involve an adsorbed catalytic species but detailed mechanistic studies in either the phosphate buffer or the HC1 medium were not undertaken ( I ) . The catalytic reduction of H+ in the presence of vitamin BlZa,aquocobalamin, and various conjugate acid anions has also been studied by coulometric measurements at a mercury pool electrode ( 3 , 4 ) . The results were interpreted as a catalytic reaction involving B1%,H+,and the conjugate acid anion. The cobalt(1) hydride was implicated as an intermediate but in the pH range studied (pH 7.1-10.4) this species is at extremely low concentrations since its pK, = 1 (5). This electrode process was recently reexamined by Schmidt and Swofford (6) in the course of a study of the adsorption of BIZ species a t a mercury electrode. It was pointed out that the process above pH 4.5 with a peak potential of -1.6 V vs. SCE cannot involve a simple redox cycle of the Blzr/BlZscouple (Ell2= -0.85 V vs. SCE), and a Mairanovsky type catalytic hydrogen process (7) with BlZs as a proton acceptor was Present address: Department of Chemistry, Suzhou University, Suzhou, Jiangnsu, People’s Republic of China. 2Present address: Department of Chemistry,New York University, New York, NY 10003. 0003-2700/84/0356-17 16$01.50/0

proposed (6). For the pH region below 4.5, Schmidt and Swofford again proposed a Mairanovsky type catalytic hydrogen process with adsorbed Bla as the proton acceptor. On the other hand, Lexa and Saveant concluded from cyclic voltammetry data in very strong acid medium that the cobalt(1) hydride species was evolving H2 in a redox catalytic mechanism (8). As the literature shows, it is apparent that the nature of catalytic hydrogen waves with Blz compounds is still unresolved. Because of this fact and because cobalamins are a good model for bioinorganic macrocyclic metal complexes, which in general show hydrogen catalytic waves, we undertook further polarographic investigations of this process with aquocobalamin and methylcobalamin. The results to be presented, although containing features of the earlier mechanisms, indicate new mechanisms for the catalytic hydrogen waves and allow in some cases measurement of the rate constants for the catalytic rate determining step. EXPERIMENTAL SECTION Apparatus. A Tacussel PRG-5 polarographic instrument was used to make the polarographicmeasurements which were all in the DC mode. Initial potentials were measured with a Keithley 160B digital multimeter. The current-potential curves were recorded on an MFE Model 815 plotamatic x-y recorder. A three-electrodeconfiguration was used with a DME as the working electrode, a Pt counterelectrode, and a commerical fiber plug saturated calomel electrode (SCE) as the reference. All electrode potentials are quoted vs. the SCE. The cell was a Metrohm EA-76 (BrinkmanInstruments) cell which was at ambient temperatures. With the column height at 86 cm, the DME had mass flow rates of 1.50 and 1.29 mg s-l for the capillaries used. The pH was measured with an Orion Model 801A digital pH meter. Prepurified nitrogen gas was used to remove oxygen from all experimental solutions. Chemicals and Reagents. Aquocobalamin and methylcobalamin were purchased from Sigma Chemical Co., the former in the form of hydroxycobalamin hydrochloride,crystalline. Both compounds were 99% grade. Triton X-100 and Triton QS-30 surfactants were also obtained from Sigma Chemical Co. All other chemicals used were ACS certified reagent grade. Phosphate buffers were prepared by mixing equal moles of Na2HP04and NaH2P04,sulfate buffers were prepared by mixing equal moles of NaHS04and Na2S04,and chloroacetic and acetic acid buffers were prepared by mixing equal moles of the acid and the sodium salt of its conjugate base. When buffer concentrations are given, they are to be taken as equimolar concentration of each conjugate species unless otherwise noted. In some instances the pH values of the sulfate buffers were adjusted by additions of 0.1 M NaOH and H2S04,and acetic acid buffers were adjusted by addition of concentrated acetic acid. Also, several studies were made in nonbuffered HC1 solution with a constant KCl electrolyte concentration. RESULTS AND DISCUSSION Catalytic Process for Vitamin BlZain Neutral Phosphate Buffer, In phosphate buffer solutions, peaked polarographic current-potential curves (Figure l) were found with a maximum at ca. -1.6 V as previously observed (1, 6). A plot of the peak current as a function of vitamin BlZaconcentration in 0.06 M phosphate buffer, pH 6.8, is not linear (Figure 2). The curve bends over but does not become independent of Blzaconcentration even at concentration of about 0 1984 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 56,NO. 9, AUGUST 1984

5- -1.4

1717

E (Volts vs SCE) -15

-1.6

-1.7

-1.8

E(Volts vs SCE)

Flgure 1. Polarographic catalytic hydrogen waves in 0.06 M phosphate buffer (pH 6.8) at various vltamln B,, concentrations: (a) 6.0 X lo-' M, (b) 1.1 X 10" M, (c) 1.7 X 10" M, (d) 2.3 X 10" M, (e) 2.8 X 10" M, (f) 3.4 X 10" M; current oscillations not shown; m = 1.50 mg Si;

drop time = 2.5 s.

'v

o L IO0

200

I

VITAMIN

E,

400

300

CONCENTRATION ( M

Figure 2. Peak current vs. vitamin B,, phosphate buffer (pH 6.8).

x

XXI

IO')

concentration in 0.06 M

M which is similar to results reported for cyanocobalamin, vitamin Blz (1). The log i, vs. log [Blza] plot for the conM to 1 X M BIZa has a slope centration range of 1X of 0.91. Thus at low concentrations the catalytic hydrogen depends nearly on the first power of catalyst concentration but deviates from this dependency as the concentration increases. A plot of l/i, vs. l/[Blza] in 0.06 M phosphate buffer, pH 6.8, shows good linearity over the concentration range 5 X lo4 M to 1 X lo4 M. A least-squares analysis of these data gives the equation I./& = (1.07 X lo4) + (5.97 X 10-4)[B1za]-1 with a standard error in the slope of 0.10 X and a linear regression coefficient of 0.9989. In this equation the peak current is an amps and the BlZaconcentration in moles per cubic centimeter. The variation of the peak current with buffer concentration at a constant Bl, concentration was also investigated. At a low catalyst concentrations,1X lo4 M B1,, the peak current was found to be linear with the square root of the buffer concentration, whereas, at high catalyst concentrations, 1 X M B12a,the peak current was found to be linear with the first power of buffer concentrations from 0.01 M to 0.7 M. At low catalyst concentrations, 1 X lo4 M BIZa, in 0.06 M phosphate buffer, the peak current was independent of the height of the mercury column indicating a kinetically controlled electrode process, whereas, at high B12a concentrations, 1.0 X M, the peak current was dependent on the mercury column height to the 0.50 power indicating

Figure 3. Polarographic current vs. potential curves in 0.06 M sulfate buffer (pH 1.97). Current oscillations are not shown. Vitamin Blpa concentrations are as follows: (a) 2.0 X lo-' M, (b) 1.0 X lo-' M, (c) background current; (A) In the absence of Triton X-100, (B) in the presence of 0.01% Triton X-100. m = 1.29 mg s-I, drop time = 1.0 5.

a diffusion controlled process. The effect of a nonionic surfactant, Triton X-100, on the current-potential curve was also investigated. The addition of 0.3 mL of 0.5% surfactant to 10.0 mL of solution caused the current maximum to shift to -1.7 V in a 4.8 X lo6 M Bl, solution in 0.06 M phosphate buffer (pH 6.8), and the peak height was reduced to 60% of its former value. One millimeter of the surfactant caused the peak to completely disappear. Similar results were observed with the anionic surfactant QS-30. These observations are interpreted as the result of the displacement of the catalyst from the surface of the surfactant clearly indicating that the catalytic hydrogen mechanism involves an adsorbed catalyst; however, the above concentration dependencies show that the rate determining step is a homogeneous reaction. Catalytic Hydrogen Process for Vitamin B,, in Acidic Media. As the pH is lowered to acid values, a new catalytic hydrogen process appears in the polarography of cobalamins (1,6). The current-potential characteristics of vitamin BlZa in 0.060 M sulfate buffer, pH 1.9, show a wave (curve a, Figure 3A) which rises in an irreversible fashion and reaches a diffusion limited plateau at -1.4 V with an Eljz value of -1.2 V. This is clearly a BlZacatalyzed wave since in the absence of Blzathe current does not begin to rise until after -1.5 V (curve c, Figure SA). There is also a maximum on the plateau of the current-potential curves (Figure 3A) which shifts toward negative potentials as the concentration of vitamin B12ais lowered (curve b, Figure 3A). In the presence of Triton X-100, the maxima disappear (Figure 3B). The limiting current on the plateau of the wave varies linearly with the Blzaconcentration at very low concentrations and becomes virtuaJly independent of concentrationabout loa M (Figure 4). In 0.06 M sulfate buffer the limiting current becomes constant at a value of 290 pA (Figure 4A). Similar behavior is observed in 0.06 M chloroacetic acid buffer (pH 2.87) where the limiting current becomes independent of catalyst concentration at ca. 5 X M with a value of 95 pA (Figure 4B). On a linear concentration scale, the plots of catalytic current vs. BIZaconcentration have the form of a Langmuir isotherm, and when the data are plotted as l / i vs. 1/[BlZa], a linear plot is obtained with linear correlation coefficients, r, of 0.9973 and 0.9996 for the sulfate and chloroacetic acid buffer media, respectively (Table I). The fact that the maximum limiting current is much larger in the lower pH medium shows that the pH of the solution is also an important factor in the kinetic process in addition to the concentration of the conjugatic acid.

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ANALYTICAL CHEMISTRY, VOL. 56, NO. 9, AUGUST 1984

Table I. Regression Analysis for i / i L vs. l/[B,,,] Plots in Acidic Media system

intercept (1/A)

slope (M/A)

std error

r

B12ain sulfate buffer (pH 1.97) Blh in chloroacetic acid

3.46 x 103

6.13 x 10-4

0.15 x 10-4

0.9973

1.51 x 104

4.86 x 10-3

0.05 x 10-3

0.9996

7.3 x 103

1.88 x 10-3

0.03 x 10-3

0.9999

7.4 x 104

9.29 x 10-3

0.08 x 10-3

0.9997

2.97 x 103

3.40 x 10-4

0.2 x 10-4

0.9911

8-02 x 103

1.45 x 10-3

0.01 x 10-3

0.9997

6.71 x 103

1.49 x 10-3

0.04 x 10-3

0.9977

7.49 x 104

3.55 x 10-2

0.16 X

0.9919

buffer (pH 2.87) BlZsin HC1, 0.12 M KCl (pH 2.0) BIZsin HC1, 0.12 M KCl (pH 2.9) BIZrnin sulfate buffer

(pH 1.97) BlZmin chloroacetic acid buffer (pH 2.87) BlZmin HC1, 0.12 M (PH 2.0) Blzmin HC1, 0.12 M KCl (pH 2.9)

2oool

t

".1

I

l

-8

6

-5

-4

-8

1500

/

4 -7

I

d

BO

20

t

d

-7

-6

/

-5

-4

IOq {Bizd

Figure 4. Limiting polarographic current vs. -logarithm of vitamin B,2a concentrations: (A) In 0.06 M sulfate buffer (pH 1.97) with TrLon X-100 measured at -1.45 V; (B) in 0.06 M chloroacetic acid buffer (pH 2.87) with Triton X-100 measured at -1.45 V. Other conditions are given in Figure 3.

The dependence of the limiting current on the sulfate buffer concentration at constant pH was investigated at values of BlZaconcentration above and below the break point on the current vs. B12aconcentration curve. At a B12aconcentration of 1 X M, the limiting current is directly proportional to the buffer concentration over the range 0.02 M to 0.1 M (Figure 5), whereas at a BlZaconcentration of 1 X lo4 M the limiting current is proportional to the square root of the buffer concentration (Figure 5). Similar behavior was found in the chloroacetic acid buffer. This behavior of the limiting current with buffer concentration is consistent with an electrode mechanism involving a homogeneous rate determining step in which the conjugate acid of the buffer participates. Further evidence of the catalytic nature of the process is found in the dependence of limiting current on the height of the mercury column, h. For 1 X M BlZasolution in 0.06 M sulfate buffer, the log i vs. log h plot was linear with a slope of 0.47, and for 1 X lo4 M Blzasolution in 0.06 M chloroacetic acid buffer, this plot had a slope of 0.49. Both of these results indicate a diffusion controlled process at high B12aconcentrations as previously observed in acetic acid buffer (6). However, at a B12aconcentration of 1 X lo4 M both 0.06 M sulfate and 0.06 M chloroacetic acid buffers show a limiting current which is virtually independent of column height indicating complete kinetic control of the process. A semilogarithmic analysis of the current-potential curve of 7 X lod M Blzaconcentration in 0.06 M sulfate buffer gives a straight line with an El,:value of -1.2 V and an (Y = 0.47 for n = 1 indicating an irreversible polarographic wave. Lowering the vitamin Blzaconcentration causes a shift of Ellz to more negative values (curve b, Figure 3A). Addition of low

[SULFATE BUFFER]

( M ) and [SULFATE BUFFER]'

( M ")

Figure 5. Limiting polarographic current vs. the sulfate buffer concentration and the square root of the sulfate buffer concentration: (a) i L vs. C"2, at 1.0 X 10" M B12a(A);(b) iL vs. Csulfale at 1.0 X M B12a(0). Other conditions are given in Figure 3.

concentrations of Triton X-100 (Figure 3B) did not affect the slope or the plateau current of the polarographic wave in buffered media but did eliminate the maximum on the diffusion current plateau. This maximum appears to be a polarographic maximum of the first kind. Some experiments were made with an acetic acid-acetate buffer for comparison with an earlier study (6). At a pH 14.5, an irreversible wave with ElI2of -1.2 V and a diffusion controlled limiting current, as reported earlier (6) only developed for Blzaconcentrationslarger than 1 X M. This wave had nearly the same characteristics as those for similar B12aconcentration in sulfate and chloroacetic acid buffers. However, at lower concentrations the wave in acetic acid buffer starts at -1.5 V and rises in a nearly linear manner out to -2.0 V. This current-potential curve appears to be following an Ohm's law behavior with a reciprocal slope of 7.0 X lo2 Q . As the pH is lowered to 2.3 by addition of acetic acid, a maximum develops (1.1 X lo4 M B12,) with a peak at -1.5 V on top of the ohmic behavior. Because this catalytic wave showed ohmic behavior below 1 X M BIZ*,its electrode process was not studied in further detail. Since the pH of the buffer media has a considerable effect on the magnitude of the catalytic current, a set of experiments was carried out in a nonbuffered media of 0.12 M KC1 with 0.01% Triton X-100 (to eliminate maxima on the limiting currents) and HC1 at various concentrations. At a pH of 1.97 and 2.87 the current, measured at -1.40 V with background subtraction, as a function BIzaconcentration had the form of

ANALYTICAL CHEMISTRY, VOL. 56, NO. 9, AUGUST 1984

1719

I

a

32001

t I

80

:50tI 0

1,

5

-08

I

& - -

-10

-12

-14

-16

-18

-20

E (Volts vs SCE)

5 y , -6

-7

,

,

-6

-5

, V I -4

-6

-7

,

,

-6

-5

-4

log(0loi

M meFigure 6. Polarographic current vs. potential for 5.0 X thyicobalamin in 0.06 M chloroacetic acid buffer (pH 2.87). Current oscillations are not shown: (-) wtthout Triton X-100, (---) with Triton background with Triton X-100. Other conditions are given X-100, in Figure 3.

Figure 7. Polarographic current vs. -logarithm of methylcobalamin

a Langmuir isotherm with the current becoming independent of the B12aconcentration above 4.0 X lo4 M. Linear plots of l / i vs. l/[Blza]are obtained (Table I) with linear correlation coefficients of 0.9999 (pH 1.97) and 0.9997 (pH 2.87). At a constant BlZaconcentration, the current was found to be linear with hydrogen ion activity over the range 0.1 mM to 10 mM as measured from the pH. Thus for B12aconcentrations of 5.0 X M and 1.0 X 10" M, the plots of corrected catalytic current in microamperes vs. hydrogen ion activity in millimolar units had linear regression expressions of i = 9.28(H+) - 0.68 and i = 12.21(H+)- 0.46, respectively, both measured a t -1.40 V. The linear correlation coefficients for these plots were 0.9990 and 0.9995 showing nearly perfect linearity. The log i vs. log h plot for the catalytic current in 0.1 M KC1 was linear with a slope of 0.47 at 1.0 X M B12a concentration (pH 2.0) and the limiting current was independent of column height at 5.0 X lo-' M BIzaconcentration (pH 2.0). These measurements again indicate a diffusion controlled process above the break point of the i vs. [B12,] curve and a kinetically controlled process below the break point of this curve. However, the observations that the break point comes at a ten times lower concentration of BlZathan in buffered acidic media and that the limiting current is directly proportional to hydrogen activity at both high and low [B12,] indicate that a different type of catalytic process is taking place than in the case of buffered media. Studies of the effect of Triton X-100 concentration on the catalytic current in nonbuffered medium show a linear decrease of the catalytic current with addition of aliquots of 0.5% surfactant to 10.0 mL of solution. The current changes from 159 pA with 0.3 mL to 63 PA with 1.3 mL of surfactant at 1.4 V. All of the above facts suggest a catalytic process which involves a surface reaction rather than a homogeneous rate determining catalytic step as found in the buffered media. Catalytic Hydrogen Process for Methylcobalamin. Methylcobalamin (Blzm)is a vitamin B12species analogous to aquocobalamin (B12a)in which the substituted upper axial ligand is a methyl group and the compound contains a Co-C bond. We have recently studied (9) the electroreduction on mercury of methylcobalamin in basic media (pH 11.7) where two main reduction waves are observed in the presence of Triton X-100 at E1j2values of -1.20 V and -1.50 V vs. SCE followed by a catalytic hydrogen wave at ca. -1.6 V vs. SCE (9). In acidic media the main reduction waves are obscured by the catalytic hydrogen process and only the catalytic wave is observed (Figure 6). The catalytic wave in 0.06 M chloroacetic acid buffer (pH 2.87) is irreversible with a = 0.42 as calculated from Figure 6. A maximum develops on the plateau

of the wave which is suppressed by 0.01% Triton X-100. As the concentrationof methylcobalamin is lowered the Elj2 value was found to shift from -1.30 V at M to -1.44 V at lo-' M. Because of this shift in the wave position as the concentration of BIzmis lowered, catalytic currents as a function of BIZMwere measured at -1.4 V in order to keep the background current as low as possible. This catalytic current is linearly dependent on the logarithm of BlZmconcentration until ca. lo4 M where it becomes independent of catalyst concentration (Figure 7). Plotted on a linear concentration scale, these curves again have the form of a Langmuir isotherm. This form of the curve is also indicated by the linearity of the l / i vs. l/[Blzm] plots which had linear correlation coefficients of 0.9997 and 0.9911 for the two acid buffers (Table I). In 0.06 M sulfate buffer the catalytic current levels off at 1 X lo4 M with 314 pA (Figure 7A), whereas in 0.06 M chloroacetic acid buffer the catalytic current becomes independent of B12mconcentration at 2 x lo4 M with 111PA of current (Figure 7B). In both of the buffer media the background current in the absence of methylcobalamin is only a few microamps. The maximum catalytic currents (above the break point) with B12mare very close to those found with Blza in the same sulfate, pH 1.9, and chloroacetic acid, pH 2.87, buffer again indicating that the pH of the solution affects the catalytic current. A plot of the logarithm of limiting catalytic current vs. the logarithm of mercury column height for both buffer systems below the break point of Figure 7 at 3 X M methylcobalamin had a zero slope (kinetic control), while above the break point at 1 X 10" M methylcobalamin the slope was 0.56 (diffusion control). In addition the plot of catalytic current vs. buffer concentration was found to be linear at methylcobalamin concentrations both below and above the break point. In nonbuffered media the peak current at constant methylcobalamin concentration was found to vary linearly with hydrogen ion activity over the pH range 2 to 4. Thus for methylcobalamin concentrations of 5.0 X M and 1.0 x M, the plot of corrected catalytic current in microamps VS. hydrogen ion activity, millimolar, had linear regression expressions of i = 10.6(H+)- 0.56 and i = 12.1(H+) 0.09, respectively, with a linear correlation coefficient of 0.999 for each. These current vs. hydrogen activity plots are very similar for both methylcobalaminand aquocobalaminin nonbuffered medium (0.1 M KCl) which indicates that the same electrode mechanism is taking place in both cases. Mercury column height studies in nonbuffered pH 2 media show a diffusion controlled process at 1 X loW4M catalyst and a kinetically controlled process at 5 X M catalyst. Also the plot of

(-a-)

concentration: (A) in 0.06 M sulfate buffer (pH 1.96) with Triton X-100 measured at -1.40 V; (B) in 0.06 M chloroacetic acid buffer (pH 2.87) with Triton X-100 measured at -1.40 V. Other conditions are given in Figure 3.

+

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catalytic current vs. BIZ,,, concentration in the nonbuffered media has a Langmuir isotherm shape becoming independent of catalyst concentration above 2.0 X lo4 M in both pH 2.0 and pH 2.87 conditions. Again plots of l / i vs. l/[Blzm]are nearly perfect straight lines with linear correlation coefficients of 0.9977 and 0.9919 at both pH values of 1.97 and 2.87, respectively. Thus for methylcobalamin, unlike the case of aquocobalamin in acid media where two mechanisms are evident, the catalytic hydrogen process in both buffered and nonbuffered media now shows the same diagnostic criteria. This process appears to involve a surface reaction as the rate determining step leading to a linear dependence of the catalytic current on either conjugate buffer acid concentration or hydrogen ion activity at all concentrations of the cobalamin species. Finally it should be mentioned that catalytic current studies with only the nucleotide base, 5,6-dimethylbenzimidazole, of the cobalamin side chain give virtually no catalytic current in the chloroacetic acid and sulfate buffer systems indicating that the entire cobalamin molecule and thus the redox center is necessary to support the catalytic process. Mechanisms and Rate Constant Calculations. For the hydrogen catalytic wave in vitamin BlZasolution with phosphate buffer (pH 6 8 , the current maximum and the effect of surfactant on the maximum indicate that the electroactive species is adsorbed on the DME. The effect of the mercury column height on the limiting current shows that the process is kinetically controlled a t low catalyst concentrations and diffusion controlled at high catalyst concentrations. Furthermore, the change in the catalytic current dependence with the conjugate acid concentration at constant pH from a square root dependence at low BlZaconcentrations to a linear dependence a t high BlZaconcentrations clearly shows that a homogeneous solution reaction is the rate determining step. Both vitamin BlZrand its protonated form are known to be strongly adsorbed on Hg (6, 10) while B12s,a negatively charged species, appears to be less strongly adsorbed at the negative potentials where it is produced. However, protonated BlZsmight be an adsorbed species. Schmidt and Swofford (6) assign the catalytically active species to vitamin B1&produced by reduction of adsorbed Blp They postulate that this species resides on the surface long enough to produce the catalytic current. This mechanism would include a surface protonation of Blls which does not fit the data. The protonated BlZs species, which is uncharged and produced by a homogenous step in the reaction layer, is a more likely candidate for an adsorbed electroactive species. Thus we postulate this adsorbed species as an intermediate in the electrode process. A reaction mechanism which is consistent with the experimental data in phosphate buffer can be written as follows:

+ HA

k2

k-2

B12,H

+ A-

where HA is the conjugate acid (H2P04-)of the buffer medium. This mechanism is not the typical Mairanovsky catalytic hydrogen process since it involves a homogeneous catalytic redox reaction (5) which regeneratesthe electroactive precursor to the homogeneous protonation step 2. In a Mairanovsky mechanism (7) two BlZsH- molecules would combine to give molecular hydrogen and B12;; however, such a step is highly unlikely for bulky BlZsH-molecules. Indeed, there is no experimental evidence for a bimolecular reaction

step at low catalyst concentrations (1X M) where such a mechanism should predominate. Electrode reaction 1is reversible and occurs at a potential of -0.85 V vs. SCE. Thus BlZrsurface concentration at the much more negative potential of the catalytic wave should be negligible. The electrode reaction 4 represents the reduction of hydrogen ion activated by being bound to a conjugate base (5,6-dimethylbenzimidazole) on the nucleotide side chain of cobalamin,and this step which reduces the hydrogen overpotential is taken as the potential determining reaction of the catalytic process. The sharp decay of the maximum beyond -1.6 V indicates that desorption cuts off the catalytic process. On the limiting current peak the rate of the overall process under steady-state conditions must be determined by either reaction 2 or 5. A reaction layer treatment can be used to express the peak catalytic limiting current as

iL = nFAkip,C,'CHA'

(6)

where the subscript i denotes either reaction 2 or 5, the superscript r indicates concentrations within the BrdickaWeisner reaction layer thickness, p, and the other symbols have their usual meaning. The concentration of the catalyst species, C,' (which can be either B12; or BlzSH-.),is related to the bulk concentration of vitamin B12afor the steady-state limiting situation since the diffusion coefficients of all B12 species are virtually equal. Thus C,' = KAcB12:, and KA depends on whether reaction 2 or 5 is rate controlling. If the reaction layer concentration of the conjugate acid species, HA, is approximated by the expression C H A=~ C H A-~iL/nFAkD

(7) where k D is the mass transfer coefficient (kD = D/6 with 6 as the diffusion layer thickness), the limiting current expression can be obtained from eq 6 and 7. The limiting expression can be written in the reciprocal form

i/;= ~ ~ / ? Z F A ~ D C+ HA 1/nFAk,K>piCHABCB12: ~

(8)

where K A = 1 if reaction 2 is rate limiting and KA = &/(l + K2)if reaction 5 is rate limiting. Equation 8 illustrates that a plot of l / i Lvs. 1/CB12aB will be linear as was found experimentally. On the other hand, a plot of i~ vs. CB12: will have the form of a Langmuir isotherm as in Figure 2. Equation 8 shows that a large enough vitamin B12aconcentrations, the peak limiting current should become independent of BIZa concentration. As Figure 2 illustrates, this limit is not completely reached in phosphate buffer. This fact may be accounted for if the actual catalyst concentrationat the electrode surface does not become equal to the bulk concentration of the catlyst because desorpton occurs before the limiting condition is fully reached. Calculation of the second-order rate constant, k , depends on the form of pi, i.e., whether reaction 2 or 5 is rate determining. For the case where reaction 2 is the rate determining step, H~ = (D/k..ZCA-B)1/2 where D is the diffusion coefficient of vitamin BIzsH. This expression can be also written in the form p2 = (DK2/k2CA-B)1/2, where K2 = k z / k - 2 is the equilibrium constant of reaction 2. For the case where reaction 5 is rate determining pug= (D/k6CHAB)112, where D is the diffusion coefficient of BlZsH-.It should be pointed out that we have made the simplifying assumption that p is independent of the catalytic current, i.e., CA-r= CA-B.The slope of the l/i, vs. l/[Bl2*]expression is 5.97 x (mol s)/(C cm3)which can be used to calculate k , using a diffusion coefficient of 3.7 X lo4 cm2 s-l (11) and a calculated area of 0.0205 cm2 with n = 2 for the overall reaction. For the phosphate buffer, CwB = [H2P04-]and CA-B= [HPO:-] both assumed to be 0.06 M. When reaction 2 is rate determining, a value of k2 = 1.6 X 10' M-I s-l is found using K2 = 5.0 X This value of K2 is given

ANALYTICAL CHEMISTRY, VOL. 56, NO. 9, AUGUST 1984

by the ratio of the second acid dissociation constant of phosphoric acid (pK&= 7.0) and the acid dissociation constant of vitamin BlZsH (pK, = 4.7) (12). On the other hand if reaction 5 is rate determining, then a value of 1.7 X lo8 M-' s-l is found. Because the reaction mechanism is a set of consecutive steps, it is not possible to conclude from the experimental data whether reaction 2 or 5 is the rate determining step. However, since the protonation of bases similar to the 5,6-dimethylbenzimidolesuch as imidazole is diffusion controlled with k = 1.5 X 1OO ' M-I s-l (13), it is probably not reaction 2 but more likely reaction 5 which is the rate controlling step. Equation 8 with either p2 or p5 shows that at low vitamin Blzaconcentrationsthe limiting current should be proportional to the square root of buffer concentration, whereas, at high Blzaconcentration the limiting current should become directly proportionalto buffer concentration. The experimentalresults as previously mentioned are in agreement with these predictions. Equation 8 also indicates that at low BIZa concentrations the catalytic limiting current should be independent of the column height, and at high BI2, concentrations, the catalytic should become dependent on the square root of the column height (diffusion controlled). Both of these conditions are found experimentally. For the case of the vitamin BlZacatalytic mechanism in acidic media, for the sulfate buffer (pH 1.9) and chloroacetic acid buffer (pH 2.9), the limiting current does become independent of vitamin Blzaconcentration at concentrationsabove 1 X lo4 M (Figure 4) unlike the phosphate case and is directly proportional to the square root of column height and the first power of the buffer concentration in agreement with eq 8. Furthermore, at low concentrations of vitamin B12,, the limiting current is directly proportional to vitamin BlZaconcentration, independent of column height, and directly proportional to the square root of buffer concentration in perfect agreement with eq 8. Although eq 8 appears to describe the limiting current in both the phosphate and the two more acidic buffers, the shape and position of the polarographic waves are different in the two cases indicating that the catalytic species involved in the mechanisms are different. In the more acid media the wave has an El,, = -1.2 V compared with a maximum at -1.6 V for the phosphate buffer medium. In a previous study (6),vitamin BlZrwas implicated as the catalytically active species for the catalytic wave below pH 4.5. However, in acidic media at pH 2.9, this species should be protonated (pK, = 2.9 (5, IO)), and on the observed limiting current plateau it would be in very low concentrations since its Ell2 = -0.74 vs. SCE (5). Another possibility for the catalytically active species is the so-called Co(1) hydride, vitamin BlZsH2+,which was invoked as the catalyst in the hydrogen catalytic process in very acidic medium, pH 1 (8). Here a mechanism involving a pure redox catalytic process was suggested which should occur on the reversible reduction wave of B1zrH++ e- = Blz,H at -0.74 V with a regeneration reaction involving the B1zsH2+ species. The fact that the catalytic wave is irreversible with a Ellzof -1.2 V suggests that another mechanism is taking place in the pH region (2-3) studied in this investigation. We postulate for this acidic medium a reaction mechanism similar to the case in phosphate buffer but with a new intermediate, the cobalt(1) hydride (vitamin BlZsH2+) BIZrH++ e-

- BlzSH

BlzsH + HA = B12,HZ+

B1zsHz' + eB1zsHz + HA

4

(9)

+ A-

BizsHz

B1zrH++ H2

(10) (11)

+ A-

(12)

1721

The electrode reaction 11 with BlZsH2+as the electroactive species is now taken as the potential determining step of the process, and this is irreversible with an Ellz = -1.2 V. The equilibriumreaction 10 lies to the left since the pK, of vitamin BlZ8H2+ is 1.0 (5). Thus when HS04-is HA (K, = 1.2 X lo-'), the equilibrium constant of reaction 10 is calculated to be 1.2 the X and for chloroacetic acid as HA (K, = 1.4 X equilibrium constant of reaction 10 would be 1.4 X One fact which is not account for by the above electrode mechanism is that the catalytic current depends not only on the Blz species and HA but also on the H'. This is shown by the large increase in the catalytic current when HS04- is the conjugate acid buffer species as oppossed to chloroacetic acid, and also by the fact that hydrochloric acid supports a sizable catalytic current in the 0.1 M KC1 nonbuffered medium. Thus in acidic buffered media the catalytic current appears to be composed of the sum of two electrode processes. One process involving the conjugate acid species in a homogeneous step and the other process in nonbuffered medium involving the hydrogen ion in a surface catalytic step. In the latter case the linear relationship between catalytic current and hydrogen activity at low catalysts concentration, where the current is kinetically controlled, indicates the rate controlling step is a surface catalytic step. Thus the catalytic current mechanism in the nonbuffered acidic media is postulated to consist of reaction steps similar to eq 9-12 except that H+ replaces HA and the regeneration step 12 involves H+ reacting with an adsorbed B12sH2species. In order to calculate a rate constant for the homogeneous catalytic regeneration reaction, we assume that the contribution to the catalytic current in buffered solution due to the surface regeneration step can be given by the catalytic current in nonbuffered solution (iH). Thus a plot of i~ - iH vs. CB12aB, where iT is the total catalytic current in buffered solution, should obey the following expression at low B1%concentration

iT - iH = nFA(D12,CHAB)1/2CB1ZaB (13) M and The slope of this plot between M BlZais 5.15 X lo5 (C cm3)/(mols). On the other hand the reciprocal plot of l / i -~i ~vs.) 1/cBl2: according to eq 8 has a slope of (1.88 f 0.02) X lo4 (mol s)/(C cm3) with a linear correlation coefficient of 0.995 for BlZaconcentrations between 1 X M and 2 x 10" M. The reciprocal slope of the latter plot is 5.32 X lo5 (C cm3)/(mols) which is very close to the slope of the former plot. This good agreement shows that the simplifying assumption in which the reaction layer thickness, p, is assumed to be independent of catalytic current, that is CB~so4= CHSO~-, is quite accurate. By use of the parameters n = 2, A = 0.011 cm2,D = 3.7 X lo* cm2 s-l, KA= 0.091, and CB~so4= 0.06 M, a homogeneous second-order rate constant of 2.9 X 10l2M-ls-l is calculated for the rate determining step 12. Such a large value is certainly inaccurate indicating that the contribution to the total current from the surface regeneration step may change in the presence of buffer and that other corrections such as the dynamic double layer effect may be necessary. None-the-lessthe data definitely show that the homogeneous solution regeneration reaction in highly acidic media is near the diffusion controlled limit. Finally we consider the surface regeneration reaction which appears to be taking place with aquocobalamin in acidic nonbuffered medium and with methylcobalamin in both buffered and nonbuffered acid media. For the case where the limiting catalytic current under steady-state conditions is controlled by a surface reaction, we can write the approximate catalytic current equation as

i, = nFAk&HT, (14) where k , is the rate constant for the surface reaction, CHris

1722

ANALYTICAL CHEMISTRY, VOL. 56, NO. 9, AUGUST 1984

the concentration at the electrode of the diffusing hydronium ion or conjugate acid species, and rR is the surface concentration in moles per square centimeter of the reduced adsorbed catalytic species. This species is assumed to be vitamin BlzsH2 in all cases. Assuming under limiting current steady state conditions that BlzsH2+in solution is related to the bulk B12a or BIZrnconcentration (BIZ,), we can express the surface concentration of B1ZsH2in terms of a Langmuir adsorption isotherm

+ P’CB12xB)

(15) where p’ is the adsorption coefficient of the BlZsH2species multiplied by an appropriate factor containing the acid dissociation constant and rSis the saturated surface concentration. Equations 7, 14, and 15 can now be combined and expressed in the form l/ic = l/nFAk,CHBrS l/nFAkHDCHB 1/nFAkc.’rsCHBCBlaxB (16) r R = r s P’CB12xB/(1

+

+

Thus plots of l / i c vs. 1/CB12xB should give straight lines and both the intercept and the slope of these plots should be dependent on hydrogen ion activity (concentration). These predications from eq 16 are found experimentally (Table I). Also since at high concentration of the BlZxspecies, mercury column height studies show that the currents are diffusion controlled, the condition kcFs >> kHDmust be obeyed. This condition allows us to put a lower bound on k , for the case of a nonbuffered medium. Calculating the value of kHD from the intercepts in nonbuffered media (Table I) gives an average value of 6 X c m / s and assuming that rsfor both BlZaand BlZmis close to that of cyanocobalamin which is reported to be 1 X mol/cm2 (14),we obtain the result that k , > 6 x IO7 cm3 mol-l s-’. Also, the value of kHDcalculated from the intercept gives a diffusion coefficient of 5 X cmz/s which is not unreasonable for H+. These results are again consistent with the observation that the catalytic current is controlled by the diffusion of H+ to the surface at high catalyst concentrations and that at low catalyst concentrations, where the current is kinetically controlled, the regeneration reaction is very fast. CONCLUSION The mechanisms for the catalytic hydrogen processes which we have proposed all have the common feature of an irreversible reduction of a hydrogen ion bound to a cobalamin species followed by a redox reaction which produces molecular hydrogen and a precurser cobalamin species which again enters the catalytic cycle. The mechanistic features are all consistent with experimental observations and differ in detail from the previous explanations ( I , 4 , 6, 8) of cobalamin catalytic hydrogen waves which were not based on detailed experimental results. The basic mechanism differs from the general Mairanovsky catalytic hydrogen process (7) in that it involves a catalyst which can enter a redox reaction with a conjugate acid species or a hydrogen ion. Experiments without the cobalt redox center, using only the nucleotide base, show that the catalytic process is not supported. The rate constants which have been estimated for the proposed rate determining step in the catalytic processes

involve a Blzscatalytic species and are d very large. The large regeneration rates lead to measurable currents at concentrations as low as 1 X M. When the data are plotted using reciprocal variables, linear plots are obtained with linear regression coefficients close to unity for plots made over 4 orders of magnitude in concentration. Such plots should have analytical applications especially for large biological molecules. For example, vitamin Blz species have molecular weights around 1350 amu and diffusion coefficients around 3 X lo4 cm2s-l which makes determination of concentrations below 1X M very difficult using voltammetry methods for a direct noncatalytic reduction process. Although catalytic hydrogen currents are subject to the same general disadvantages of any kinetically controlled current, they are worthy of consideration for electroanalytical determinations of large biomolecules because of the wide spread occurrence of the catalytic hydrogen process for these species and the high sensitivity of the method. In this study which was aimed at a general elucidation of the mechanisms of the vitamin Blz catalytic hydrogen process, no special precautions were taken for the precise control of temperature, buffer concentrations, and other variables which might affect the catalytic current magnitude. However, the present results indicate that hydrogen catalytic systems, where conditions are accurately controlled, could be used for ultratrace electroanalytical determinations of biologically significant molecules. Such methods for more complex biological molecules should be much more general than for the case of pure redox catalytic currents and worth the extra experimental precaution when noncatalytic electrolysis methods are impossible. Registry No. Aquocobalamin, 13422-52-1;methylcobalamin, 13422-55-4. LITERATURE CITED Abbott, J. C. Ph.D. Dissertation, Ohio State University, 1964, University Microfilms, Inc., Ann Arbor, MI. Tacket, S. L.; Coilat, J. W.; Abbot, J. C. Blochemistry 1963, 2 , 919-923. Das, P. K.; Hili, H. A. 0.; Pratt, J. M., Williams, R. P. J. J. Chem. SOC. A 1966, 1261-1264. Das, P. K. J. Chem. SOC.,Dalton Trans. 1974, 23, 2475-2479. Lexa, D.; Saveant, J.-M. J. Chem. SOC.,Chem. Commun. 1975, 872. Schmidt, C. L.; Swofford, H. S. Anal. Chem. 1979, 51, 2026-2033. Mairanovsky, S. “Catalytic and Kinetic Waves in Polarography”; Pienum Press: New York, 1968; p 261. Lexa, D.; Saveant, J. M. J. Am. Chem. SOC.1976, 98, 2652-2658. Kim, M.-H.; Birke, R. L. J. Nectroanal. Chem. 1883, 144, 331-350. Birke, R. L.; Venkatesan, S. J. Necfrochem. SOC. 1981, 128, 984-991. Schmid, C. L.; Koiphin, C. F.; Swofford, H. S.,Jr. Anal. Chem. 1981, 53, 41-47. Pratt, J. M. “Inorganic Chemistry of Vitamin B,,”; Academic Press: New York, 1972; p 158. Eigen, M.; Hammes, G. G.; Kustin, K. J. Am. Chem. Chem. 1960, 82, 3482.

imhiff, D. W. Ph.D. Dissertation, Ohio State University, 1966, University Microfilms, Inc., Ann Arbor, MI.

RECEIVED for review January 30, 1984. Accepted April 17, 1984. The authors are indebted to the CUNY (PSC-BHE) Faculty Award Program (RF-13909,RF-13699) and the National Institutes of Health under HEW-PHS Grant 5R01AM18440, USPHS Grant 2-SO7-RR07132,and MBRS Grant RR-08168 for financial assistance.