A kinetic study of NAD+ reduction on a ruthenium modified glassy carbon electrode

Journal of

Electroanalytical Chemistry Journal of Electroanalytical Chemistry 568 (2004) 301–313 www.elsevier.com/locate/jelechem

A kinetic study of NADþ reduction on a ruthenium modified glassy carbon electrode Felise Man, Sasha Omanovic

*

Department of Chemical Engineering, McGill University, 3610 University Street, Montreal, Que., Canada H3A 2B2 Received 17 September 2003; received in revised form 9 February 2004; accepted 13 February 2004 Available online 20 March 2004

Abstract The kinetics of NADþ reduction was investigated on a ruthenium modified glassy carbon electrode (RuGC) in wide polarization rate, concentration, temperature, and pH ranges using the electrochemical techniques of cyclic and differential pulse voltammetry, chronoamperometry and chronopotentiometry. It was shown that the modification of GC by a sub-monolayer of Ru can provide an electrode surface capable of reducing NADþ directly to NADH, avoiding the formation of a dimer. The reaction is highly irreversible, and occurs at high negative overpotentials, where the reaction rate is controlled by the surface diffusion of electroactive species. The reaction is pH independent. It was postulated that Ru sites have a bifunctional role, serving as both proton-providing sites, and as a possible physical barrier for dimerization of NAD-free radicals. A set of kinetic and thermodynamic parameters was calculated and verified independently using various experimental techniques: the standard heterogeneous electron-transfer rate constant, the apparent transfer coefficient, the apparent diffusion coefficient, the reaction order, and the standard Gibbs energy of activation. Ó 2004 Elsevier B.V. All rights reserved. Keywords: NADþ ; Reduction; Glassy carbon (GC); Ruthenium (Ru); Nano-particles; Kinetics; Cyclic voltammetry; Differential pulse voltammetry; Pulse-techniques

1. Introduction Nicotinamide adenine dinucleotide NAD(H) (Scheme 1) is a cofactor that plays the role of electron and hydrogen shuttle in most of the biochemical reactions catalyzed by redox enzymes (dehydrogenases or oxidoreductases). In its reduced and enzymatically active form (1,4-NADH), the molecule transfers two electrons and a proton to a substrate in the presence of a suitable enzyme to form NADþ . The high cost associated with the stoichiometric feed of NADH to a bioreactor has been the major motivation for research in the development of in-situ NADH regeneration techniques. Chenault and Whitesides [1], in their review paper on regeneration of NADH for use in organic synthesis, summarized a number of different techniques used to

*

Corresponding author. Tel.: +1-514-398-4273; fax: +1-514-3986678. E-mail address: [email protected] (S. Omanovic). 0022-0728/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2004.02.006

regenerate NADH, and discussed factors influencing the stability and lifetime of NADH in a solution, as well as process considerations that are relevant to the use of NADH in synthesis. It was shown that electrochemistry offers a viable alternative for NADH regeneration. Elving et al. [2] reported on the kinetics and mechanistic studies related to both electrochemical reduction of NADþ and oxidation of NADH. They showed that the direct reduction of NADþ on unmodified metallic electrodes results mostly in formation of an enzymatically inactive dimer NAD2 , which can be only partially protonated and further reduced to both 1,4- and 1,6NADH at significantly higher negative overpotentials [3]. They showed that the separation between the current peaks related to the first and second electron transfer is rather large on a Hg electrode, i.e. between 500 and 700 mV depending on the conditions applied, thus indicating that the second electron transfer step requires a much larger energy input. In addition, the polarographic wave of the second reduction step was observed only in the presence of a surface-active electrolyte.

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Scheme 1. Nicotinamide adenine dinucleotide in its oxidized form (NADþ ), and its reduction to enzymatically active 1,4 NADH. ADPR stands for adenosine diphosphoribose.

Reduction of NADþ (i.e. regeneration of NADH) has also been investigated on chemically modified metallic electrodes. Since a very low yield of enzymatically active 1,4-NADH is obtained when NADþ is reduced on unmodified metallic electrodes, a bare metallic electrode surface has to be modified in order to increase either the kinetics of the second electron transfer and/or protonation, or to prevent dimerization. Most of the studies dealing with this approach have relied on the modification of the electrode by adsorbed organic layers, or by adsorbed/immobilized biological molecules (enzymes, lipids). Recently, Long and Chen [4] modified a silver electrode with covalently adsorbed L -histidine, which resulted in the reduction of NADþ to NADH, characterized by only one cathodic peak in cyclic voltammetry measurements. No formation of the dimer was reported. Similarly, Baik et al. [5] performed reduction of NADþ using both unmodified and cholesterol-modified gold and platinum electrodes. Reduction of NADþ on a gold-amalgam unmodified electrode gave only ca. 10% enzymatically active NADH, while the yield in active NADH increased to 50% when an unmodified platinum electrode was used. However, when the gold-amalgam electrode was modified with cholesterol, the yield of active NADH increased to ca. 75%. The cholesterol layer attached to the electrode surface probably served as a physical barrier that prevented dimerization of the radicals formed. The enzymatically catalyzed (electro)reduction of NADþ has also attracted considerable attention, for both biosensor development and larger-scale applications (e.g. bioreactors). A number of studies have been reported, and a large majority of these relies on an electron-mediator assisted reduction mechanism. War-

riner et al. [6] modified the surface of a platinum electrode with a poly(3-methylthiophene):poly(phenol red) film, which served as an electron mediator for reduction of NADþ to NADH. They showed that this configuration, developed as a biosensor configuration, is capable of regenerating a certain amount of enzymatically active NADH. However, they did not report the yield of active NADH. Fry et al. [7–9] have published a series of papers on the reduction of NADþ to NADH on glassy carbon and vitreous carbon electrodes modified by a layer of immobilized methyl viologen and lipoamide dehydrogenase. They showed that the enzyme catalyzed reduction of NADþ on such modified electrodes results in the production of enzymatically active NADH, but the amount of active NADH produced was not explicitly reported. A detailed kinetic study on the interaction of methyl viologen and diaphorase enzyme for the electrocatalytic reduction of NADþ using gold-amalgam electrode has been reported in [10,11]. Beley and Collin [12] also showed that NADþ could be reduced to enzymatically active NADH when a reticulated vitreous carbon electrode was covered by a layer of polypyrrole rhodium bis-terpyridine. Although enzymatic modification of an electrode surface can give encouraging results in the reduction of NADþ to enzymatically active 1,4-NADH, this approach results in a rather complex electrode system due to difficulties related to immobilization of an enzyme and electron mediator at the electrode surface, lost of the enzyme activity, electron mediator leakage, and slow NADH regeneration rate. Therefore, there is a need to develop a simple electrode surface that would allow direct electrochemical regeneration of an enzymatically active form of NADH. As previously discussed, the use of unmodified metal electrode surfaces is not possible due to formation of an inactive dimer, a result of the slow kinetics of the second reaction step (electrontransfer and hydrogen addition). As already outlined, this could be partially avoided by physical prevention of the dimer formation at a molecular level [5], by chemically modifying the electrode surface (e.g. using self-assembled-monolayers). However, such modified electrodes lack stability and durability due to the loss of the modification layer under the applied reduction reaction conditions. Therefore, it would be desirable to design a whole-metallic electrode that would have longterm stability and offer high efficiency in the reduction of NADþ to enzymatically active NADH. In this paper we report our results on a project which focused on the development of an electrode surface that would enhance the rate of the second reduction step (electron and hydrogen transfer), by modifying a glassycarbon (GC) electrode with a sub-monolayer of deposited ruthenium. A GC surface was chosen since it offers a high hydrogen evolution overpotential, a condition necessary in most reduction reactions occurring at po-

F. Man, S. Omanovic / Journal of Electroanalytical Chemistry 568 (2004) 301–313

tentials more negative than the potential of the reversible Hþ /H2 couple [2]. In addition, it offers a high stability, reproducibility and, due to its low cost, high suitability for a possible industrial application. On the other hand, ruthenium was chosen due to its ability to adsorb hydrogen at low negative (over)potentials relative to the NADþ reduction potential, and also its ability to form a H–Ru bond of an intermediate strength, thus ensuring high coverage with hydrogen at low overpotentials while still allowing it to desorb without investing a considerable amount of energy. This is also the major requirement in electrochemical hydrogen evolution catalysis [13]. Here we discuss results related to the kinetics of the NADþ reduction reaction on a RuGC electrode, obtained using electrochemical techniques of cyclic voltammetry (CV), differential pulse voltammetry (DPV), chronoamperometry (CA) and chronopotentiometry (CE). In a forthcoming paper [14] we will report our results related to the investigations of the electrode surface morphology, interface charge distribution, and electrolysis (regeneration) of NADH in an electrochemical batch reactor using the newly developed RuGC electrode. As will be shown, the RuGC electrode is capable of reducing NADþ directly to NADH at a very high yield (96%) of enzymatically active 1,4-NADH.

2. Experimental The kinetics of the reduction of NADþ using a Rumodified GC electrode was studied in 0.05 M phosphate buffer solution at various pHs and in a wide temperature range, from 295 to 331 K. The buffer was prepared by dissolving monobasic KH2 PO4 (Sigma Chemical Co., P5379) in conductivity water (resistivity 18.2 MX cm) and adding 0.10 M sodium hydroxide (made from concentrated volumetric solution, ACP Chemical Inc.) or H3 BO3 to adjust the pH of the solution. A standard three-electrode, two compartment cell was used in all experiments. The counter electrode was a large-area platinum electrode of high purity (99.99%, Johnson– Matthey), which was degreased by refluxing in acetone, sealed in soft glass, electrochemically cleaned by potential cycling in 0.5 M sulfuric acid, and stored in 98% sulfuric acid. During the measurement, the counter electrode was separated from the main cell compartment by a glass frit. The reference electrode was a commercially available saturated calomel electrode (SCE), to which all potentials in this paper are referred. The working electrode was a Ru-modified glassy carbon electrode (RuGC). Prior to Ru electrodeposition, the surface of the two-dimensional GC electrode (BAS Instruments Ltd., diameter 6 mm) was polished with a diamond paste down to 0.03 lm, followed by degreasing

303

with ethanol in an ultra-sound bath and electrochemical activation in 0.5 M H2 SO4 by potentiodynamic polarization between )1.0 and 1.5 V vs. SCE, 40 cycles, scan rate 100 mV s
1 . A sub-monolayer of ruthenium was potentiostatically deposited onto the prepared GC electrode at )0.09 V vs. SCE from a 1 mM RuCl3 solution in 0.5 M H2 SO4 . From scan-dependant CV measurements made in the double-layer region in phosphate buffer pH 7.0, the real total surface area of the RuGC electrode exposed to the electrolyte was estimated to be 1 cm2 [15–17]. All the measurements were carried out in an oxygenfree solution, which was achieved by continuous purging of the cell with argon gas. After oxygen was completely removed from the solution, the bubbler was pulled out of the electrolyte surface, and the inert atmosphere was maintained by saturating the cell space above the electrolyte with argon. Thus, all the measurements were made in an unstirred (quiescent) solution. The stock NADþ solution was prepared in a separate container using phosphate buffer and was allowed to equilibrate for at least 30 min in the constant-temperature bath, fitted with a temperature regulator, at the same temperature as the electrochemical cell. Before measurements in a NADþ (or in some cases NADH)-containing solution, a background response of the electrode was recorded in a phosphate buffer solution. Aliquots of NADþ were then added to the electrochemical cell and the electrochemical measurements were repeated with each aliquot. CV, DPV, CE, CA and capacitance techniques were employed using an Ecochemie Autolab potentiostat/galvanostat PGSTAT30/FRA2, handled by GPES/FRA v.4.9 software. In order to determine the potential-of-zero-charge (pzc) of the RuGC electrode in the given electrolyte, the differential capacitance measurements were done in an O2 -free sodium phosphate buffer solution pH 7 at various concentrations (50, 10 and 5 mM), and at 25 Hz with an ac amplitude of 5 mV by polarizing the electrode potentiostatically in a negative direction.

3. Results and discussion 3.1. Cyclic voltammetry Fig. 1 shows a scanning electron microscope (SEM) image of a glassy carbon (GC) electrode modified by a sub-monolayer of electrodeposited ruthenium (Ru). It can be seen that ruthenium forms well-separated and dispersed islands on the electrode surface. The size distribution of Ru islands is rather wide, between ca. 10 and 40 nm, and the separation distance between islands varies from ca. 20 nm to over 200 nm. In order to characterize the electrochemical behavior of the RuGC surface in a phosphate buffer solution, a cyclic

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Fig. 1. Scanning electron microscope (SEM) image of a RuGC electrode surface obtained after electrodeposition of Ru on a freshly prepared and electrochemically activated GC electrode surface.

voltammogram was recorded in a wide potential region, between hydrogen and oxygen evolution (Fig. 2, dashed curve a). The electrode was polarized at )1.35 V for 10 min prior to the positive polarization scan in order to remove Ru-oxide surface film formed on Ru sites of the RuGC surface, followed by positive polarization starting from )1.5 V. At this starting potential, a significant hydrogen evolution reaction (her) current was recorded. Further in the positive region, two well defined anodic peaks (A1, A2), followed by a weakly defined shoulder (A20 ) overlapping with peak A2 were recorded. These peaks correspond to desorption of hydrogen adsorbed on Ru sites of the electrode (A1), and oxidation of metallic Ru to RuOH and RuOx (A2, A20 ), which is, after adjustment for the

A2

100

A2'

A3 A3"

(d)

A1 50

I / µA

(c)

A3'

(b) 0

(a) C2' C1 C2

-50 C3 -1.5

-1.0

-0.5

0.0

0.5

1.0

E / V vs. SCE

Fig. 2. Cyclic voltammograms of (a) RuGC electrode in a phosphate buffer solution pH 7.0, (b) RuGC electrode in phosphate buffer containing 725 lM of NADþ , (c) GC electrode in phosphate buffer containing 720 lM of NADþ , and (d) RuGC electrode in phosphate buffer containing 200 lM of 1,4-NADH. Scan rate, sr ¼ 500 mV s
1 . Temperature, T ¼ 295 K.

difference in pH, in accordance with the literature [18,19]. In the reverse scan, three cathodic peaks were recorded, corresponding to the reduction of Ru-oxides formed in the positive scan (C20 , C2), and adsorption of hydrogen on Ru sites of the electrode (C1). By polarization of the electrode further into the negative region, a her current is recorded at a potential negative of ca. )1.35 V. For a rough estimate of the ruthenium coverage we have measured the oxidation charge of the ruthenium sub-monolayer (after subtraction for the double-layer charge) in the potential range of )0.27 to 0.02 V (12.4 lC cm
2 ), referenced to a single electron monolayer charge. Assuming the reaction Ru + H2 O ! RuOH + Hþ + e
to account for the peak A2 [20,21], and taking the charge related to the formation of a monolayer of RuOH as 249 lC cm
2 [18,19], the value of the surface coverage calculated is 5%. A similar value (4%) was obtained by integration of the hydrogen desorption peak A1 (8.6 lC cm
2 ), assuming that a full monolayer of adsorbed hydrogen would give a charge of 210 lC cm
2 [22]. When the previous experiment was repeated in a solution containing 725 lM of NADþ , a new peak A3 appeared in the positive scan at a potential of ca. 0.55 V (Fig. 2, curve b). We ascribed this peak to the oxidation of NADH formed by the reduction of NADþ in the negative region at )1.35 V before the polarization scan started. Aizawa et al. [23] reported the same positive potential value in their study of oxidation of NADH on a platinum electrode in phosphate buffer pH 7.0. More recently, Katekawa et al. [24] studied the oxidation of NADH on a bare glassy carbon electrode using DPV, and reported an NADH oxidation peak potential of ca. 0.6 V in phosphate buffer, while the value of 0.55 V was reported on gold modified with ternary substituted selfassembled-monolayers [25]. In the negative scan, a new, well defined cathodic peak (C3), corresponding to the reduction of NADþ to NADH was recorded at ca. )1.2 V, which is in close agreement with the values obtained on a cholesterol-modified gold-amalgam electrode [5], and a basal pyrolytic graphite electrode [26]. The voltammogram (b) also shows that the charge related to anodic peaks A1 and A2 decreased when NADþ was present in the solution. It appears that NADH produced in the negative region prior to the positive scan sweep, at )1.35 V, adsorbs on the electrode surface, thus partially blocking the Ru sites exposed to the solution. In this way, the adsorbed NADH diminishes the access of OH-species to the surface, which are necessary for the oxidation of Ru. On the other hand, the hydrogen desorption peak charge (A1) decreased due to the lower amount of hydrogen adsorbed at Ru sites, which could be a result of the protonation of an NAD-radical by adsorbed hydrogen. This point will be discussed later in the text when we discuss a possible

F. Man, S. Omanovic / Journal of Electroanalytical Chemistry 568 (2004) 301–313

separation between the A3 and C3 peaks is ca. 1.8 V. Such a high value indicates a very high degree of irreversibility of the NADþ /NADH redox reaction. Elving et al. [2] reported an even higher peak separation, ranging from ca. 2.0 V, and over 2.3 V, and 2.6 V for GC, Pt and Au electrodes, respectively. However in their paper, a half-wave potential for the reduction of NADþ completely to NADH, which was actually the potential for the reduction of the free radical NAD
to NADH, was reported to be )1.6 V, i.e. ca. 400 mV more negative of our NADþ reduction peak potential. Also, if we compare independently an NADþ peak reduction potential and NADH oxidation peak potential reported in [5,23,24], we will see that the reduction–oxidation potential separation is comparable to that observed in Fig. 2. Fig. 3 shows a set of cyclic voltammograms recorded with increasing scan rate in the potential region of NADþ reduction. It is evident that the peak position (potential) shifts towards more negative potential values with the increase in scan rate, thus indicating that the NADþ reduction reaction is under diffusion control [31]. In addition, as already discussed in the previous paragraph, the NADþ reduction reaction has been shown to be highly irreversible, which can also be seen from the potential difference between the peak position in Fig. 3 and a formal potential for the NADþ /NADH couple, E0 ¼
0:556 V vs. SCE [2], resulting in an overpotential of more than )600 mV. Also, when the peak current is presented versus peak potential, the curve obtained is linear (R2 ¼ 0:9899) with a slope of 1173 V A
1 cm2 ¼ X cm2 (not shown here). This slope value represents a pseudo-resistance for the reduction of NADþ (which includes both electron-transfer and mass-transfer rates).

0 Ep / V vs. SCE

(a) -10

-1.18 -1.19 -1.20 -1.21

(b)

-1.22

-20

-1.5

-2

-30

-40

-1.2

-0.9 -1

-0.6

log(sr / V s )

50

jp / µA cm

j / µA cm-2

reduction reaction mechanism. It is also apparent that cathodic peaks C10 , C1 and C2 decreased after NADH was oxidized in the positive scan. This could be due to the fouling of the electrode by products of NADH oxidation, as reported in the literature [27,28]. Although we ascribed peak A3 to the oxidation of cathodically produced NADH, and peak C3 to the reduction of NADþ directly to NADH, two subsequent experiments were performed in order to verify these conclusions and dismiss the doubt that the peaks A3 and C3 are related to the oxidation and production of the dimer NAD2 , respectively. In the first experiment, we used a pure unmodified GC electrode and repeated the previous experiment with the NADþ containing solution. It is known from the literature [2,29] that the reduction of NADþ on GC results in the formation of the dimer, and that the oxidation potential of the dimer is more negative than that of NADH. In our experiment, the oxidation peak (A30 ) recorded on the bare GC electrode shifted to more negative potentials for ca. 370 mV, compared to the peak on RuGC (A3) (Fig. 2, curve c). Since the oxidation process related to peak A30 is the oxidation of the dimer, this shows that peak A3, recorded on the RuGC electrode, is indeed related to the oxidation of cathodically formed (regenerated) NADH, not to the oxidation of the dimer. Further, in the second experiment we used our RuGC electrode and performed the oxidation of commercially available and enzymatically active NADH. Curve (d) in Fig. 2 shows that the NADH oxidation peak (A300 ) recorded in this experiment lies in the same potential region as peak A3, which is related to the oxidation of regenerated (by our electrode) NADH. Hence, the results presented in Fig. 2 show that the RuGC electrode is able to reduce NADþ directly to NADH (Scheme 1), characterized by one cathodic peak C3, avoiding the formation of the dimer NAD2 . A similar mechanism was also reported by Katz et al. [30] in their study of reduction of pyrroloquinoline quinone, which was covalently immobilized as a monolayer onto a cystamine-modified gold electrode. Before continuing the discussion, we would like to emphasize that in this paper we do not discuss the capability of the RuGC electrode to produce the enzymatically active form of NADH. This will be discussed in our subsequent paper [14], where it will be shown that our RuGC electrode is capable of producing a 96% yield of enzymatically active 1,4-NADH by the reduction of NADþ at )1.2 V vs. SCE. In this paper, which presents our kinetic studies on reduction of NADþ , it is rather important to notice that the reaction discussed proceeds not to the formation of the dimer NAD2 , but further to NADH, with the exchange of two electrons and one hydrogen per one NADþ molecule (Scheme 1). Another feature that is visible from the cyclic voltammogram recorded in a NADþ containing solution using the RuGC electrode (curve b, Fig. 2) is that the

305

40 30 20

(c) 10 0.1

0.2

0.3

sr

1/2

/V

0.4 1/2

0.5

-1/2

s

-50 -1.3

-1.2

-1.1

-1.0

-0.9

-0.8

-0.7

E / V vs. SCE

Fig. 3. (a) Cyclic voltammograms of a RuGC electrode in phosphate buffer pH 7.0 containing 725 lM of NADþ recorded at various scan rates. The voltammograms were normalized for the background current. The scan rate increases in the direction of the peak increase as 30, 50, 70, 100, 150, 200, and 300 mV s
1 . Temperature, T ¼ 295 K. Dependence of the (b) peak potential, and (c) peak current on scan rate.

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This is a rather high value, but is in accordance with the relatively low peak current densities (<)50 lA cm
2 ) and high overpotential values observed. However, in this paper it is not our intention to evaluate and discuss the catalytic properties of the RuGC electrode on the basis of overpotential and peak current values, but rather on the basis of the RuGC electrode ability to reduce NADþ directly to enzymatically active NADH, instead of the enzymatically inactive NAD2 obtained by direct reduction on most (chemically and/or enzymatically) unmodified electrodes. In order to estimate a value of an apparent transfer coefficient a, a peak potential (Ep ) versus logarithm of scan rate (sr) dependence was plotted as inset to Fig. 3. The observed dependence is linear, and using n ¼ 2, from the slope of the line and relation for irreversible electrochemical reaction at 295 K [31,32]: dEp 0:029 ; ¼ an d logðsrÞ

ð3:1Þ

the transfer coefficient value calculated is a ¼ 0:44. A similar value (a ¼ 0:46  0:03) was obtained from the shape factor equation, jEp
Ep=2 j ¼ 0:0472=an [31]. In order to calculate a diffusion coefficient value, the peak current density (jp ) versus square root of scan rate dependence was plotted (Fig. 3(c)). The graph shows that the peak current density value varies linearly with the square root of scan rate, thus indicating that the reaction is under the mass-transport, i.e. diffusion control under the potentiodynamic conditions applied [26,27]. However, according to the CV theory, it has to be noted that the observed mass-transport control applies to the potential region more negative to, and including the cathodic peak potential, E 6 Ep , while at potentials more positive than Ep , the reaction is most likely under mixed mass-transfer and electron-transfer control. The observed mass-transport control is also in agreement with the peak potential shift recorded on the voltammograms in Fig. 3(a). The log jp vs. log(sr) dependence resulted in a straight line (R2 ¼ 0:9995, the graph not shown here), and the slope was 0.441, thus also indicating mass-transport control. Using the equation that relates the peak current density to scan rate and concentration [31,33,34]: jp ¼ 2:99  105 nðanÞ

1=2

cD1=2 ðsrÞ

1=2

;

ð3:2Þ

the diffusion coefficient value calculated from the slope of line in Fig. 3(c) is D ¼ 4:47  10
8 cm2 s
1 . In Eq. (3.2), and also Eqs. (3.7) and (3.8) used later in the text, n represents the number of electrons transferred up to, and including, the rate determining step [31], which is the transfer of the second electron [1,3,5]. Hence, in the above quoted equations, and also in all other equations in which a number of electrons is used as a required quantity, the value of n is taken as n ¼ 2. The diffusion coefficient value calculated above seems to be rather low

for diffusion in a solution, where common values for molecules of this size are around 10
6 to 10
5 cm2 s
1 . In order to calculate the theoretical value of a diffusion coefficient for NADþ in an aqueous solution, the Wilke– Chang correlation together with corresponding parameters was used [35]: D ¼ 1:173  10
16 ðuMwater Þ

0:5

T : 0:6 lwater VNAD þ

ð3:3Þ

The value of the diffusion coefficient calculated at 293 K is D ¼ 3:3  10
6 cm2 s
1 . Aizawa et al. [23] obtained a similar value experimentally for diffusion of NADH in phosphate buffer at 298 K, D ¼ 2:4  10
6 cm2 s
1 . If we compare the value obtained from our experimental data to the theoretical and experimental values obtained for the diffusion of NADþ or NADH in the solution, respectively, we can see that our value is two orders of magnitude lower. This indicates that the diffusion of NADþ most likely does not occur in the solution, but rather could be considered as the surface diffusion. Similar or even lower values, related to the surface diffusion of electroactive species, are quite common in the fuel cell electrocatalysis on Pt–Ru surfaces [36–38], where CO adsorbed on Pt sites is being oxidized by oxygen-containing species diffusing from neighboring Ru sites, or in electrocrystallization of new phases [39– 42]. More similarly to the NADþ molecule, surface diffusion of N -butyl-3-(hydroxynonyl)pyridinium was recorded as a slow step in adsorption on a platinum electrode [43], while a value of 4.5  10
8 cm2 s
1 was reported for diffusion of tetramethylsilane on Ru [44]. Also, surface diffusion of hydrogen on Ru/Cu was studied by Brown et al. [45], and they reported a similar value, 3.0  10
8 cm2 s
1 . Hence, from the data presented in Fig. 3 it is not quite clear if the surface diffusion observed on RuGC is related to diffusion of NADþ , NAD-intermediates (NAD
and/or NAD
) or hydrogen adsorbed on Ru sites. However, since in the time interval of CV experiments presented in Fig. 3 we did not observe any adsorption of NADþ on the electrode surface (which would presumably result in ‘‘immobilization’’ of the NADþ molecule at a particular electrode site), it is most likely that the diffusion observed is related to diffusion of NAD molecules at the surface, rather than diffusion of hydrogen adsorbed on Ru sites. It is known that the driving force for diffusion represents a concentration gradient, and it should be noted that in the case of NADþ reduction on RuGC the gradient is located on the electrode surface, not in the solution. However, since the bulk NADþ concentration is used in the calculations throughout the text, the calculated diffusion coefficient should rather be regarded as an apparent diffusion coefficient. Nevertheless, it has to be emphasized that the surface NADþ concentration is linearly proportional to the bulk NADþ concentration in the NADþ concentration region investigated and

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under the experimental conditions applied (observe the linearity in Figs. 5 and 7), which justifies the use of the approach employed to calculate the apparent diffusion coefficient. The approach used in the text to describe the mass-transfer and obtain corresponding parameter values is also based on the assumption that the NADþ diffusion length is considerably larger than the NADmolecule size (ca. 0.92, 1.2 or 1.4 nm for the folded configuration perpendicular to the surface, folded configuration parallel to the surface, and extended configuration, respectively [2]). As will be shown later in the text, the Ru sites are fully saturated with hydrogen, and are not available to transfer electrons to NAD-species. Hence, the reduction (electron transfer) and diffusion of NAD-species occurs only on the GC part of the surface, while the protonation occurs at a RujGC boundary interface. Fig. 1 shows that the Ru site separation varies from 20 nm to more than 200 nm, and therefore, the corresponding average diffusion length is considerably larger than the molecule size dimensions. Hence, one would expect the measured response to reflect a response of a linearly mass-transfer controlled reaction, not a thin-layer response (i.e. surface controlled reaction) which would be expected if the Ru-site separation distance were of the NAD-molecule size dimensions. This is evident experimentally from a number of independent experiments discussed in the text (Figs. 3–5 and 7). Chronoamperometric and chronopotentiometric experiments performed on the same system (Fig. 4) also indicated that the NADþ reduction reaction is masstransport controlled in the potential region of interest. Fig. 4(a) shows that the chronoamperometric curve decays with time, which indicates mass-transport control

E / V vs. SCE

-70

-60

-50

-1.0 -1.1 -1.2

-2

0

10

20

-10

-40

30 time / s

40

50

60

1.2

1.4

-2

-15 j / mA cm

j / mA cm

(c)

-1.3

-30

-20 -25 -30 -35

(b) 0.4

-20

0.6

(a) 0

2

4

6

0.8 1.0 -1/2 (time / s)

8

10

12

time / s

Fig. 4. (a) Current transient recorded on a RuGC electrode at )1.15 V vs. SCE in phosphate buffer pH 7.0 containing 725 lM of NADþ . (b) The current transient from (a) presented in a linearized form according to the Cottrell equation. Symbols are experimental values and the line is the best fit according to the Cottrell equation. (c) Potential transient recorded on a RuGC electrode in phosphate buffer pH 7.0 containing 725 lM of NADþ at current density of )10 lA cm
2 . Temperature, T ¼ 295 K.

307

[31]. If we use the well-known Cottrell equation that describes non-steady state mass-transport by linear molecular diffusion, j ¼ nFcðD=ptÞ1=2 [31,33], we can normalize the transient in Fig. 4(a) to present it as a j vs. t
1=2 dependence in order to determine if the observed reaction is entirely mass-transfer controlled. Fig. 4(b) shows that the experimental data (symbols) agree very well with the linearized Cottrell equation (line), and from the slope of the line, the apparent diffusion coefficient has been calculated, D ¼ 5:75  10
8 cm2 s
1 . The calculated value is very close to the value obtained from the CV measurements (D ¼ 4:47  10
8 cm2 s
1 ). Another indication of mass-transfer control is presented in Fig. 4(c), which shows a chronopotentiometric curve recorded on the same system. The presence of a potential plateau between 1 s and ca. 15 s (the so-called transition time, s), is clear evidence that the reaction is predominantly mass-transport controlled [31,33]. Due to difficulties related to the precise determination of the transition time (Fig. 4(c)), which is one of the major problems in analysis of chronopotentiometric curves, an accurate calculation of the apparent diffusion coefficient was not possible. Using the software supplied with the instrument used in the measurements (Ecochemie Autolab General Purpose Electrochemical Software), the transition time has been estimated to be ca. 14 s, and then using the Sand equation [31,33], jjs1=2 j ¼ 1=2 1 , the apparent diffusion coefficient was ap2 nFcðDpÞ proximated to be ca. 9  10
8 cm2 s
1 . This value is slightly higher than the two values calculated previously, but is still of the same order of magnitude. Fig. 5(a) shows a set of CVs recorded at various concentrations of NADþ in the solution. The concentration range investigated is rather wide, spanning over two concentration decades. As expected, the peak current (and corresponding charge) increased with increase in NADþ concentration, and the observed dependence presented in inset (b) to Fig. 5 shows very good linearity. This is in accordance with Eq. (3.2), which allowed us to calculate the apparent diffusion coefficient, D ¼ 4:51 10
8 cm2 s
1 . It is worth noting that, although the surface mass-transport (i.e. surface diffusion) is the rds in the NADþ reduction reaction at the high overpotentials investigated, the amount of NADþ present at the surface at a distance out of the diffusion length, is directly proportional to the NADþ concentration in the bulk electrolyte in the time interval of the experiments performed and the concentration range investigated, as seen in Fig. 5(b). The same conclusion was obtained on the basis of chronocoulometric results done at various concentrations and potentials [14]. The corresponding concentration gradient representing the driving force for the surface mass flux, i.e. mass-transport, is located on the electrode surface rather then at the electrodejelectrolyte interface, as in the case of diffusion in the solution.

F. Man, S. Omanovic / Journal of Electroanalytical Chemistry 568 (2004) 301–313

It is clear that the apparent diffusion coefficient values calculated from three independent (separate) sets of measurements (Figs. 3–5) are very similar. This verifies the approaches used to calculate the diffusion coefficient, and also reveals high reproducibility of the experiments performed. Before proceeding further with the discussion, it is worth to note that the four CV dependences discussed in the previous sections (cathodic-to-anodic peak potential separation, Eq. (3.1), the shape factor equation, and Eq. (3.2)) all fulfil the criteria used to characterize an irreversible electrochemical system [31]. The results presented in Figs. 3–5 show that the NADþ reduction reaction is highly irreversible. Hence, at a formal potential of an NADþ /NADH couple ()0.556 V vs. SCE at pH 7.0), the corresponding standard heterogeneous electron-transfer rate constant, k 0 , is expected to be quite low. In order to calculate the value of k 0 , cyclic voltammograms obtained over the wide scan rate and concentration ranges investigated (Figs. 3 and 5) were fitted by a model for an irreversible electrochemical reaction using the commercially available software [46]. As an example, Fig. 6(a) shows the NADþ reduction peak, where both the simulated (solid line) and experimental (symbols) voltammograms are presented. The potential axis was normalized versus the formal potential of the NADþ /NADH couple. A very good agreement between the two curves can be noted, and the mean value of k 0 calculated is (2.9  2.0)  10
9 cm s
1 , which indicates very slow kinetics of the reaction at the formal potential. The fitting procedure also gave the transfer coefficient value of a ¼ 0:43  0:02, which is in close agreement with the values obtained from the

(5) (6)

-16

-10 10 0

-20

-25

-30

(c)

400

600

[NAD+] / µM

3.4

(1) (2)

3.2

(3)

-30

3.0

-1.1

-40

-1.0

-0.9

-0.8

313 K

E / V vs. SCE

3.2

324 K

3

x 10 / K

3.3

3.4

-1

-20

(a)

-30

331 K

-0.7

-50 -1.4

--1

-10

-0.8

-0.7

3.1

0

307 K

E / V vs. SCE -1.2

(c)

-19

295 K 304 K

319 K

2.8

-1.3 -1.2 -1.1 -1.0 -0.9 -0.8 -0.7 -0.6

(a)

-18

T

3.0

2.6

-1.3

-20

-17

j / µA cm-2

-2

(8)

200

-2

0 3.6

j / µA cm

(7) -15 C25Hz / µF cm

j / µA cm

-2

-10

(b)

20

-1

-2

(4)

0

(b)

30

o

-5

(1) (2) (3)

jp / µA cm

0

peak potentials discussed previously in the paper. Standard heterogeneous electron-transfer coefficient values of the same order of magnitude have also been reported in the literature for various redox systems [47,48]. As an example, a value of 6  10
10 cm s
1 was obtained in the case of the reduction of p-nitrotoluene [49] and 1.89  10
9 cm s
1 of thionyl chloride [34] on a GC electrode. Elving et al. [2] postulated that in an aqueous (i.e. polar) solution and in the absence of specific enzymes, the NADþ molecule (Scheme 1) exists in a folded conformation, which brings the nicotinamide and adenine rings together, with the pyrophosphate group as a hinge. They also concluded that both rings are parallel with the electrode surface (Hg in their case), with the adenine ring oriented in a flat position to the surface. Our differential capacitance measurements in phosphate buffer (inset (c) to Fig. 5) showed that the point-of-zerocharge (pzc) of a RuGC electrode is at ca. )1.07 V vs. SCE. Hence, at the formal potential of an NADþ / NADH couple ()0.556 V vs. SCE) the RuGC electrode is positively charged, which results in repulsion between a positively charged nicotinamide ring of NADþ and the electrode surface, thus facilitating the orientation of the adenine ring part of NADþ towards the surface. Therefore, a long electron-tunneling (or hopping) path between the electrode and the nicotinamide ring could account for the sluggish kinetics (i.e. a very low k 0 value) observed at the formal potential. The model in [2] also suggests that the adenine ring is involved in the electrontransfer process between the electrode and the nicotinamide ring, i.e. it serves as a mediator for nicotinamide reduction. In order to increase the kinetics of the reac-

ln(k / cm s )

308

-1.3

-1.2

-1.1

-1.0

-0.6 -0.5 E - Eo / V vs. SCE

-0.9

-0.8

-0.4

-0.7

-0.6

E / V vs. SCE

Fig. 5. (a) Cyclic voltammograms of a RuGC electrode in phosphate buffer pH 7.0 containing various concentrations of NADþ : (1) 50, (2) 100, (3) 150, (4) 225, (5) 320, (6) 410, (7) 550, and (8) 725 lM. Scan rate, sr ¼ 100 mV s
1 . The voltammograms were normalized for the background current. (b) Dependence of the peak current on NADþ . Temperature, T ¼ 295 K. (c) Capacitance of a RuGC electrode measured in oxygen-free phosphate buffer at various concentrations (1) 0.05 M, (2) 0.01 M, and (3) 0.005 M, and at a frequency of 25 Hz and ac amplitude of ±5 mV. The electrode was polarized potentiostatically going in a negative direction. Temperature, T ¼ 295 K.

Fig. 6. (a) Experimental (symbols) and simulated (line) cyclic voltammogram recorded at a scan rate of 100 mV s
1 in phosphate buffer pH 7.0 containing 725 lM of NADþ at 295 K. (b) Cyclic voltammograms of a RuGC electrode in a phosphate buffer pH 7.0 containing 725 lM of NADþ recorded at various temperatures. Scan rate, sr ¼ 100 mV s
1 . The voltammograms were normalized for the background current. (c) Dependence of the standard heterogeneous electron-transfer rate constant obtained by fitting of cyclic voltammograms shown in (b).

F. Man, S. Omanovic / Journal of Electroanalytical Chemistry 568 (2004) 301–313

In order to verify the kinetics and thermodynamics parameters obtained on the basis of CV experiments, we repeated the concentration and temperature dependent measurements discussed in the previous section using a DPV technique. DPV was chosen as an independent experimental technique offering well developed mathematical kinetic models that could help us to obtain and verify the same set of parameters already discussed in the first part of the paper. In addition, the DPV technique offers higher concentration-dependent sensitivity compared to CV. Fig. 7 shows a set of differential pulse voltammograms recorded in a wide range of concentrations of NADþ in the phosphate buffer solution. It is worthwhile to note that concentrations as low as 25 lM were detected with high accuracy using the RuGC electrode, indicating that the electrode could also be used as an electrochemical analytical tool (i.e. sensor) for NADþ determination. However, due to its non-specific selectivity for NADþ , its analytical application would depend greatly on both the redox potential of other species present in the solution, and their possible effect on the electrode surface (i.e. adsorption, fouling, etc.). Fig. 7 0

(a)

-2

(b)

16

(d)

-4

2.5

18

(c)

2.0

14

(e)

12

-6 (f)

1.5

10

-8 (g)

8

1.0

6

-10

4

-12

(h)

0.5

2

-14

0.0

0 0

-16 -1.2

200

400

600

800

+

[NAD ] / µM

(i) -1.3

-2

the standard Gibbs energy for reduction of NADþ was calculated to be DG0act ¼ 59:7 kJ mol
1 . This value is very close to the value of 60 kJ mol
1 obtained for the oxidation of NADH on glassy carbon [2] or the oxidation of methanol on a Pt–Ru catalysts [50,51]. However, it

3.2. Differential pulse voltammetry

Qp / µC cm

and assuming that a is independent of (over)potential, which is the case for a slow reaction like NADþ reduction [31], the electron-transfer constant value can be calculated at any (over)potential. Using the a and k 0 values previously calculated, the heterogeneous electron-transfer rate constant, k, calculated at the potential of the cathodic peak in Fig. 3, e.g. at )1.2 V (g ¼
0:65 V) is 4.85 cm s
1 , which is an increase of nine orders of magnitude compared to the standard heterogeneous electron-transfer rate constant k 0 . Besides the electrochemical effect related to the increase in the kinetics of the NADþ reaction by increasing the overpotential (Eq. (3.4)), an additional effect related to the change in the orientation of the molecule at the surface can be expected to contribute partially to the increase in the reaction kinetics. Namely, at potentials more negative than the pzc ()1.07 V vs. SCE), the electrode surface is negatively charged. Therefore, in this potential range, we could expect the NADþ molecule to reorient at the surface, now with the positively charged nicotinamide ring oriented towards the negative surface. This would result in a decrease in the electron tunneling distance, and thus, partially contribute to the increase in the reaction kinetics, i.e. electron-transfer kinetics. This configuration would also facilitate protonation of the molecule by hydrogen adsorbed at Ru sites. However, this possible (reorientation) effect is not included in Eq. (3.4), and hence, only the influence of the overpotential on the value of k can be considered using the equation. The effect of temperature on the reduction of NADþ was also investigated. Fig. 6(b) shows a set of cyclic voltammograms obtained in a wide temperature range. With the increase in temperature, the kinetics of the NADþ reduction reaction increase, which results in a substantial increase in the peak current. All the voltammograms were fitted in order to calculate the standard heterogeneous electron-transfer rate constant k 0 . The dependence of ln k 0 on the inverse of temperature is presented as an inset to Fig. 6(c), and using the Arrhenius equation [31,52]:

DG0act k 0 ¼ A exp ; ð3:5Þ RT

-2

ð3:4Þ

jp / µA cm

anF ðE
E0 Þ; RT

-2

ln k ¼ ln k 0


has to be emphasized that our value refers to the formal potential of the NADþ /NADH couple, while in the last two papers cited [50,51], the activation energy was calculated at a specific overpotential, which resulted in a lower value compared to the standard Gibbs energy of activation. In conclusion, the calculated standard activation energy value shows that the reduction of NADþ is only moderately temperature dependant.

jp / µA cm

tion, a large negative overpotential has to be applied. As previously shown, the reaction proceeds at a satisfactory rate only if a negative overpotential larger than )600 mV is applied. Using the equation that relates the k value to the overpotential [31]:

309

-1.1

-1.0

-0.9

-0.8

-0.7

-0.6

E / V vs. SCE

Fig. 7. Normalized differential pulse voltammograms for reduction of various concentrations of NADþ on a RuGC electrode in phosphate buffer pH 7.0. Concentrations of NADþ : (a) 25, (b) 50, (c) 100, (d) 150, (e) 225, (f) 320, (g) 410, (h) 550, and (i) 725 lM. Modulation time: 70 ms; modulation amplitude: 50 mV; interval time: 0.2 s; step potential: 1.95 mV. Scan rate, sr ¼ 9:75 mV s
1 . Temperature, T ¼ 295 K. Inset: Dependence of the (s) peak current and (M) charge on NADþ concentration.

F. Man, S. Omanovic / Journal of Electroanalytical Chemistry 568 (2004) 301–313

3:52RT ; ð3:6Þ anF the mean transfer coefficient value was calculated to be a ¼ 0:49  0:04. This value is in close agreement with the values obtained from the CV results. The value of the apparent diffusion coefficient was then calculated using the equation that relates the current of the DPV peak to the concentration of the electroactive species in the solution [33]:
 nFAD1=2 c 1
r jp ¼ pffiffiffiffiffiffiffi ; ð3:7Þ ptm 1þr W1=2 ¼

where for an irreversible electrochemical reaction [54]:
 anF DE : ð3:8Þ r ¼ exp 2RT The apparent diffusion coefficient value calculated, D ¼ 1:71  10
8 cm2 s
1 , is also in close agreement with the values calculated from the CV results. The apparent transfer coefficient value a was also calculated by applying the logarithmic analysis of the voltammograms in Fig. 7. For that purpose, the DPV voltammograms from Fig. 7 were integrated to give the response of a normal pulse voltammogram (Fig. 8, circles). Symbols in the figure represent the experimental data, while the solid line was obtained by a fitting procedure using the wave-shape equation for an irreversible electrochemical reaction [55]:
 RT jd
j E ¼ E1=2 þ ln ; ð3:9Þ anF j which was modified to give: j  d : j¼ 1 þ exp anF ðE
E1=2 Þ RT

ð3:10Þ

The graph show that the agreement between the model and experimental data is very good, and the value of the half-wave potential is calculated to be E1=2 ¼
1:126 V vs. SCE. Also, the value of the transfer coefficient a ¼ 0:48 obtained is in agreement with previously calculated values. The same values were obtained when the data were treated using the logarithmic analysis, Eq. (3.9), where the slope of the line (Fig. 8,

2.0

6

4 1.5

-2

2

1.0

0

ln[(jd - j) / j]

shows that with the increase in NADþ concentration, the DPV peak current also increased. This dependence is presented as an inset to Fig. 7, and shows that the peak current varies linearly with concentration. The inset also shows that the peak charge (triangles), obtained by the integration of the DPV peaks presented in Fig. 7, depends linearly on concentration. In order to calculate the value of the apparent transfer coefficient a, the width of the peak at its half height, W1=2 was determined at each concentration, and then using the equation for an irreversible electrochemical reaction [53]:

-j / µA cm

310

-2 0.5 -4

E1/2 0.0 -1.4

-1.3

-1.2

-1.1

-1.0

-0.9

-6 -0.8

E / V vs. SCE

Fig. 8. (s) Normal pulse voltammogram obtained by integration of DPV voltammogram (i) from Fig. 7. Symbols are experimental values and the solid line represents values obtained by fitting of the experimental values using Eq. (3.10). (M) Example of the logarithmic analysis of a DPV (i) from Fig. 7 using Eq. (3.9).

triangles) drawn through the linear region of the experimental curve is defined as RT =anF , from which the value a was calculated, and the abscissa intercept at the ordinate value of zero gave the half-wave potential E1=2 . In order to obtain the value of Gibbs energy of activation for the reduction of NADþ on a RuGC electrode, DPV measurements were done in a wide temperature range. Using Eq. (3.6), the mean value of the transfer coefficient was first calculated to be a ¼ 0:46  0:04, which is in very close agreement with previously discussed values. Then, from the DPV peak values the apparent reaction rate rNADþ was calculated following the equation: rNADþ ¼ jp =nF [31], and presented in Fig. 9 (squares) as a function of inverse temperature, according to the Arrhenius equation. The Gibbs activation energy value calculated from the slope of the line is 6.6 kJ mol
1 . However, since this value is related to the reaction rate occurring at DPV peak potential values (ca. )1.126 V vs. SCE), i.e. at an overpotential of ca. )570 mV, where the reaction rate is significantly higher than at the formal potential of the NADþ /NADH couple, it is quite reasonable to expect a significant drop in the activation energy compared to the standard Gibbs energy reported previously in Section 3.1. Hence, in order to compare the two values obtained from two different and independent sets of measurements, the above value had to be corrected for the overpotential using equation [31,33]: DGact ¼ DG0act þ anF ðE
E0 Þ:

ð3:11Þ

Using the transfer coefficient value calculated from the temperature dependent DPVs, the extrapolation of the Gibbs energy value to the formal potential of )0.556 V vs. SCE gave the standard Gibbs energy value of DG0act ¼ 57:2 kJ mol
1 . If we compare this value to the DG0act value calculated from the CV measurements, we can see that the difference is less than 5%. This also

F. Man, S. Omanovic / Journal of Electroanalytical Chemistry 568 (2004) 301–313

3.05

3.10

3.15

3.20

3.25

3.30 -23.3

-23.2 -10.0

-23.1

-10.5

-11.0

ln (rNAD+ / mol s-1 cm-2 )

log (rNAD+ / mol s-1 cm-2 )

-9.5

-23.0

-11.5

-7.6

-7.2

-6.8 +

-6.4

-22.9 -6.0

-3

log ([NAD ] / mol cm )

Fig. 9. Dependence of the reaction rate on concentration obtained by analysis of current peaks of the (s) cyclic voltammograms in Fig. 5, and (M) differential pulse voltammograms in Fig. 7. () Dependence of the reaction rate on temperature obtained by analysis of current peaks of the differential pulse voltammograms recorded in phosphate buffer solution pH 7.0 containing 725 lM of NADþ . Parameters related to DPVs are the same as those in Fig. 7.

-9.9 -10.0 -10.1 -10.2

-2

-10.4 -1.09

-1

-10.3

log(rNAD+ / mol s cm )

verifies both approaches used to calculate the Gibbs energy value, and shows high reproducibility of the experiments performed. Using classical kinetic theory for heterogeneous reactions [52,56], the rate law for the NADþ reduction reaction presented 0 in Scheme 1 can be written as 0 rNADþ ¼ k 0 ½NADþ a [Hþ ]b , where a0 and b0 are the partial reaction orders with respect to NADþ and Hþ , respectively, rNADþ is the apparent reaction rate previously defined as rNADþ ¼ jp =nF [31] and k 0 is the overall (apparent) heterogeneous reaction rate constant. Fig. 9 (circles and triangles) shows the dependence of apparent reaction rate values on NADþ concentration for both techniques used (the concentration of hydrogen, i.e. pH was kept constant). The observed log–log dependence is linear, with a slope of a0app ¼ 0:99 and 1.00 for the CV and DPV results, respectively. This indicates that the observed reaction of reduction of NADþ on a RuGC is pseudo first order with respect to NADþ . In cases where diffusion limitations are present (like in the NADþ peakpotential region in both CV and DPV measurements), the measurement of the true reaction rate could be affected. In that case, Fogler [52] relates an apparent reaction order, a0app to true reaction order, a0 , as a0app ¼ ð1 þ a0 Þ=2. However, when comparing the a0app values calculated from the results in Fig. 9 to the a0 values calculated using FoglerÕs approach, we can see that the difference between them is insignificant (ca. 1% for the CV measurements, and zero for the DPV measurements). As discussed previously, the purpose of modifying a GC electrode with Ru was to provide hydrogen needed in the NADH regeneration reaction by adsorption on Ru sites, i.e. in close proximity to the surface sites where the transfer of electrons to the NADþ molecule occurs,

and in that way to increase the rate of the protonation step, thereby avoiding (or at least reducing) the formation of the dimer. If the proton participating in the NADþ reduction reaction is supplied from the solution, then the CV/DPV peak potential should shift by 59.1 mV towards more negative potentials for each unit increase in pH [1,2,23]. Taking the position of the NADþ reduction peak in Figs. 3, 5 and 7 as a reference, this would result in the position of NADþ reduction peak potentials in the range between ca. )1.05 and )1.30 V vs. SCE for a pH range between 5.5 and 10.2, respectively. This potential region is ca. 460 mV more negative than the potential region where Ru sites are already saturated with hydrogen [57], which thus, ensures that hydrogen is already present at the electrode surface in the potential window of NADþ reduction, and that its surface concentration (i.e. surface coverage) is maximum and constant. Therefore, if the proton participating in the NADþ reduction reaction is supplied by Ru sites, we should not see potential–pH dependant behavior. In order to test our hypothesis, that the proton involved in the reaction is supplied by Ru sites, not from the solution, we performed both CV and DPV measurements in a wide pH range, from 5.5 to 10.2, and the results are presented in Fig. 10. The observed Ep vs. pH dependence is linear, with the slope of 6 mV/pH unit and 7 mV/pH unit for the CV and DPV measurements, respectively. These slopes are ten times lower that the value expected for the solution-assisted protonation mechanism (59.1 mV/pH unit [2]), which confirms our hypothesis about the role of Ru sites as hydrogen-supplying sites. Also, we can apply the kinetic theory approach, previously used to calculate the reaction order with respect to NADþ , to evaluate the reaction order with respect to hydrogen, b0 . Using Scheme 1 and the rate law for the NADþ reduction reaction rNADþ ¼

CV

-1.10

Ep / V vs. SCE

T-1 x 103 / K-1 3.00 -9.0

311

DPV

-1.11 -1.12 -1.13 -1.14 -1.15 -1.16 5

6

7

8

9

10

11

pH = -log[H+]

Fig. 10. Dependence of the reaction rate and peak potential on pH obtained by analysis of current peaks of (s) cyclic voltammograms, and (M) differential pulse voltammograms recorded in phosphate buffer solution containing 725 lM of NADþ . Parameters related to CVs and DPVs are the same as those in Figs. 5 and 7, respectively. For CV measurements, Ep on the graph represents Ep=2 values. Temperature, T ¼ 295 K.

312

F. Man, S. Omanovic / Journal of Electroanalytical Chemistry 568 (2004) 301–313 0

0

k 0 ½NADþ a [Hþ ]b , one would expect the change in pH to influence the measured reaction rate significantly if the participating proton came directly from the solution. However, the reaction rate in both CV and DPV measurements was found to be almost pH independent, see Fig. 10 (note that the concentration of NADþ was kept constant). The apparent reaction order with respect to proton was found to be very close to zero, b0 ¼ 0:04 in DPV and b0 ¼ 0:03 in CV measurements, i.e. the NADþ reduction reaction is practically of zero order with respect to proton. This is a characteristic of surface reactions where a reactant of interest (proton here) has reached a saturated surface coverage [56], and further supports our previous conclusions on the role of Ru sites as proton-providing sites. Hence, the rate law for the NADþ reduction reaction on RuGC would be more 0 appropriately written as rNADþ ¼ k 0 ½NADþ a hH , where the hydrogen surface coverage hH is essentially constant in the potential region of NADþ reduction on RuGC and in the pH range studied. From the previous kinetic experiments it could be concluded that the total reaction order for the NADþ reduction on RuGC, under the experimental conditions applied, is equal to one. Based on the results discussed in the paper, one of the possible reaction mechanisms for reduction of NADþ on a RuGC electrode could the following: a first step involves transfer of an electron to NADþ to form a free radical NAD
, which is immediately further reduced to unstable NAD
that reacts with hydrogen at the neighboring Ru sites giving NADH. The electron transfer occurs on a GC part of the RuGC surface, while the protonation occurs at the GCjRu interface boundary. Since the electrolysis of NADþ at )1.2 V vs. SCE [14] resulted in the yield of 1,4-NADH of 96%, and since only one cathodic peak was observed in CV and DPV measurements discussed previously in the paper, the second electron step, which involves the transfer of an electron to an NAD-radical, is not energetically significantly different than the first electron-transfer step, which supports the above mechanistic scheme as a possible reaction mechanism. Also, besides enhanced electron and hydrogen transfer kinetics on the RuGC electrode, compared to GC, Pt, Au and some other non-modified electrodes, an additional possible reason for avoiding the dimer formation, which allows then for the formation of NADH, could also be that free NAD-radicals formed are separated one from the other by Ru sites (i.e. Ru ‘‘islands’’) distributed on the GC surface (Fig. 1), thus physically preventing the fast dimerization of free radicals.

4. Conclusions The kinetics of NADþ reduction was investigated on a ruthenium modified glassy carbon electrode (RuGC) in wide polarization rate, concentration, temperature,

and pH ranges using the electrochemical techniques of CV and DPV, CE and CA. It was shown that the modification of GC by a sub-monolayer of Ru can provide an electrode surface capable of reducing NADþ directly to NADH, avoiding the formation of a dimer. The reaction is highly irreversible, with separation between the NADþ reduction and NADH oxidation peaks of ca. 1.8 V. The reduction of NADþ on RuGC commences at high negative overpotentials, ca. )0.576 V compared to the formal potential. The high irreversibility and slow kinetics of the NADþ reduction reaction at the formal NADþ /NADH potential is characterized by a very low value of the standard heterogeneous 0 electron-transfer rate constant, kmean ¼ 1:27  10
9
1 cm s . The apparent transfer coefficient was also calculated, amean ¼ 0:48. It was shown that under the experimental conditions employed, the reduction of NADþ is a diffusionally controlled first order reaction, and is of the first order with respect to NADþ and zero order with respect to hydrogen. The low value of the apparent diffusion coefficient, Dmean ¼ 4:1  10
8 cm2 s
1 , indicates that the diffusion of electroactive species occurs on the electrode surface, rather than in the solution. The temperature dependent measurements provided information about the standard Gibbs energy of activation of NADþ reduction, 58.4 kJ mol
1 . It was also shown that the reaction is pH independent, indicating that hydrogen participating in the reaction is supplied by Ru sites uniformly distributed on the electrode surface. Besides serving as proton-providing sites, Ru sites could also serve as a physical barrier that prevents the formation of NAD2 dimers. It was shown that the electrochemical experimental techniques used are useful for studying the kinetics of NADþ reduction. All the techniques used provided independent results that allowed us to calculate and verify kinetic and thermodynamic parameters. List of symbols A pre-exponential factor in the Arrhenius equation, cm s
1 c concentration, mol cm
3 D diffusion coefficient, cm2 s
1 Ep peak potential, V Ep=2 half-peak potential, V E1=2 half-wave potential, V E0 formal potential, V DE modulation amplitude, V F Faraday constant, 96485 C mol
1 0 DGact standard Gibbs energy of activation, J mol
1 jp peak current density, A cm
2 jd diffusion limited current density, A cm
2 0 k standard heterogeneous electron-transfer rate constant, cm s
1 k heterogeneous electron-transfer rate constant, cm s
1

F. Man, S. Omanovic / Journal of Electroanalytical Chemistry 568 (2004) 301–313

k0

W1=2

overall (apparent) heterogeneous reaction rate constant, cm s
1 molar mass, g mol
1 number of electrons standard gas constant, 8.314 J mol
1 K
1 scan rate, V s
1 modulation time, s temperature, K solute molar volume at the boiling point, m3 mol
1 width of a DPV peak (at half height), V

Greek a a0 b0 g u l

transfer coefficient partial reaction order with respect to NADþ partial reaction order with respect to Hþ overpotential, V association parameter of the solvent, 2.6 for water viscosity, kg m
1 s
1

M n R sr tm T V

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