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Electrochemical Oxidation of Adenine: A Mixed Adsorption and Diffusion Response on an Edge-Plane Pyrolytic Graphite Electrode Luı´s M. Gonc¸alves,† Christopher Batchelor-McAuley,‡ Aquiles A. Barros,† and Richard G. Compton*,‡ Requimte, Department of Chemistry and Biochemistry, Faculty of Science, UniVersity of Porto, Rua do Campo Alegre, no. 687, 4169-007 Porto, Portugal, and Department of Chemistry, Physical and Theoretical Chemistry Laboratory, Oxford UniVersity, South Parks Road, Oxford OX1 3QZ, United Kingdom ReceiVed: May 21, 2010; ReVised Manuscript ReceiVed: July 15, 2010
Understanding the oxidation of the purines adenine and guanine is primary to improving electrochemical methods of DNA detection and analysis. Adenine in the solution phase is reported to undergo a complex electrochemical oxidation mechanism that is overall a -6H+, -6e- process, involving irreversible chemical steps. The observed voltammetry associated with the oxidation of adenine is strongly dependent upon the electrode used; this is a reflection of both the kinetics of oxidation and strength of bonding to the electrode surface, both of which are surface specific. Two main cases are presented within the article, one in which the adsorption of adenine to the electrode surface is strong, as is the case with gold and one in which the bonding is weaker, as found with graphitic surfaces. In the case of gold, adsorption is strong enough to prevent the adenine oxidation to be observed within the electrochemical window and further to this the formed monolayer prevents oxidation of the gold surface. With graphitic surfaces adsorption is weaker and as such oxidation of adenine is observed, this oxidative signal is demonstrated to be due to the oxidation of both surface bound and solution phase species. A generic method for analyzing the peak currents for reversible electron transfers coupled with an irreversible chemical process is presented. The analysis is dependent upon knowledge of the number of electrons transferred prior to the first chemically irreversible process and the total number of electrons transferred during the redox process. In addition to this, the analysis provides a description for the peak height of voltammetric waves that have contributions from both adsorbed and solution phase species, with these two processes being resolved through their differing dependencies with scan rate. The methodology is then used for the analysis of the oxidation of adenine on an edge-plane pyrolytic graphite electrode, where the influences on peak current from adsorption and diffusion are demonstrated. The diffusion coefficient for adenine is found to be (1.25 ( 0.2) × 105 cm s-1, which is in close agreement to that found by independent measurements reported in the literature. The adsorption of adenine at low concentrations to the electrode surface, is shown to exhibit a linear dependence of coverage with the solution phase concentration, where the surface coverage is given by Γ ) KC* with a measured K value of (1.7 ( 0.1) × 10-3 cm and C* is the solution phase concentration. Introduction Adenine is one of the five main nucleobases and is fundamental to the coding of genetic information within biological systems. Along with guanine, adenine is of particular interest to electrochemists due to the relative ease with which it may be oxidized. As a consequence, a number of electrochemical analytical techniques for the quantification of DNA have arisen, one of the first examples was provided by Palecˇek et al. who studied the adsorption and oxidation of DNA on a mercury electrode.1 This work has been subsequently enhanced with the attainment of much lower levels of detection and improved methodologies allowing differentiation between single and double stranded DNA.2,3 The study of the oxidation of DNA is not limited to mercury electrodes, with work also focusing on the use of both graphitic4 and carbon nanotube5 based electrodes. The electrochemical oxidation mechanism of free adenine base has been shown to follow a complex mechanistic pathway * Corresponding author. Fax: +44 (0) 1865 275410. Tel: +44 (0) 1865 275413. E-mail:
[email protected]. † University of Porto. ‡ Oxford University.
involving the loss of six protons and six electrons;6 this is schematically shown in Figure 1. Furthermore, it is known that the products of the first two oxidations (E1 and E2) are more easily oxidized than the parent molecule.7 Previous work has used a variety of carbon electrode substrates for the study of this process, namely, glassy carbon,7,8 carbon nanotubes,9 pyrolytic graphite,6,10 carbon paste,11 and sol-gel carbon composite,12 among others. Clearly, carbon provides a suitable substrate for the study of adenine, but the work done is generally qualitative in nature with little or no effort to ascribe physical reasoning to the observed voltammetric differences. Work by Banks et al. has demonstrated that in many cases the electroactive sites for oxidation at a graphitic electrode are often the edge-plane defects;13 subsequent to this, recent work by Li et al. studied the voltammetry of guanine on a number of carbon substrates and demonstrated how the observed voltammetry may be explained in terms of the varying densities of basal and edge plane sites present for the adsorption and electro-oxidation of the species respectively.14 For both of the purines, adenine and guanine, adsorption to the electrode surface plays a crucial role, as such the majority
10.1021/jp1046672 2010 American Chemical Society Published on Web 07/30/2010
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Gonc¸alves et al. oxidation of adsorbed and solution phase species. Through appropriate analysis of the peak current it is possible to resolve these two contributions and demonstrate that under these conditions the adsorption has a linear dependency with concentration. At higher concentrations, self-inhibition alters the observed voltammetry and the formation of a less electro-active layer upon the electrode substrate causes a shift in peak position. The following article presents a generic method for the analysis of systems involving weakly adsorbed surface species in which both the adsorbed and solution phase species contribute to the observed current. This analysis allows physical insight into the complex nature of the electron transfer processes occurring within the adenine oxidation system. Experimental Procedure
Figure 1. Reaction mechanism for adenine oxidation, in aqueous media.6
of the previous work with adenine on carbon allows some level of accumulation time so as to “optimize” the peak signal, thus allowing lower limits of detection to be attained. On highly ordered pyrolytic graphite the adsorption and surface structure of adenine has been studied using both STM and AFM, where the adsorbed monolayer is found to be composed of adsorbate molecules lying parallel with the surface.15-17 This adlayer is found to be potential dependent, where between +0.1 and -0.4 V (vs Ag/AgCl saturated KCL) a well-organized condensed structure is formed, at +0.25 V a phase change occurs, likely resulting in the desorption of some species from the surface.15 The formation of adenine monolayers on gold is also well studied, the formation of which is found to occur spontaneously with the bases being strongly adsorbed.18-20 In systems in which the reactant may be adsorbed to the electrode surface, the observed voltammetry can vary depending upon the strength of the binding of species to the surface. Wopschall and Shain theoretically described systems in which there is a mixed response, i.e., where both diffusional and adsorbed species contribute to the observed current. Of interest is the case in which the reactant is weakly adsorbed; this leads to an increase in peak height from that expected from a diffusion-only response.21 The peak current for reversible electron transfer is described by the well-known RandlesSˇevcˇ´ık equation,22 but direct application of this to more complex systems, i.e., involving chemical process, must be approached with caution. It is assumed by the Randles-Sˇevcˇ´ık equation that all subsequent electron transfers are highly driven; this is not necessarily the case and, as will be shown, knowledge of which step is rate determining in nature is essential for a more accurate description of the peak current. In this work, the voltammetric response of a number of electrodes is studied in the presence of adenine; the influence of strength of adsorption is clearly highlighted where in the case of gold no current relating to the oxidation of the adenine species is observed and the oxidation of the gold surface is also inhibited. On carbon based substrates, the situation is markedly different, weaker adsorption allows the oxidation process to be observed and the surface structure influences the voltammetric response. The best resolved peak for the oxidation of adenine is found to occur on an edge-plane pyrolytic graphite electrode. The system is found to exhibit one oxidation peak at low concentrations, which is demonstrated as being related to the
Reagents and Equipment. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) at the highest grade available and used directly without any further purification. All solutions were prepared with deionized water of resistivity not less than 18.2 Ω cm-1 at 298 K (Millipore, Billerica, MA). The pH was measured using a Hannah (Padova, Italy) pH 213 pH meter. All voltammetric measurements were recorded using an Autolab PGSTAT20 computer controlled potentiostat (EcoChemie, Utrecht, Netherlands). Cyclic voltammetric experiments were run in aqueous electrolytic solutions containing 0.05 M K2HPO4, 0.05 M KH2PO4, and 0.1 M KCl. A standard threeelectrode configuration was used throughout, consisting of a working electrode (see below), a platinum wire (99.99%, GoodFellow, Cambridge, U.K.), and a saturated calomel electrode (SCE, Radiometer, Copenhagen, Denmark) acting as the counter and the reference electrodes, respectively. Two stock solutions of adenine were kept at 4 °C in the absence of light, and they were used throughout all experiments: 3.6 and 0.5 mM in phosphate buffer solution (PBS), pH 6.85. Experimental Procedure and Working Electrode Preparation. Adsorptive stripping voltammetry experiments were performed, unless stated otherwise, by using cyclic voltammetry swept from +0.4 to +1.3 V. An accumulation time of 1 min was applied under an open circuit prior to each experiment. Macro-glassy carbon (GC, area 0.0707 cm2, BASi, West Lafayette, IN) and boron-doped diamond (BDD, area 0.0707 cm2, Windsor Scientific, Slough Berkshire, U.K.) electrodes were polished using diamond spray (1.0-0.3 µm, Kemet, Kent, U.K.). Edge-plane pyrolytic graphite (EPPG, area 0.196 cm2, Le Carbone, Sussex, U.K.), gold (Au, area 0.0314 cm2, BASi), and platinum (Pt, area 0.00785 cm2, BASi) electrodes were polished using alumina (1.0, 0.3 µm, Buehler, Lake Bluff, IL), while basal-plane pyrolytic graphite (BPPG) was prepared by renewing the surface with cellotape (i.e., repeatedly pressing cellotape onto the electrode’s surface and removal of the same leave a freshly cleaved surface). All electrodes were rinsed with deionized water prior to be immersed in the sample. Results and Discussion Theory. For a reversible multiple “n” electron diffusion-only process, in which no chemical steps are present and all electron transfers subsequent to the first are highly driven, the current peak height of a linear sweep voltammogram may be described by the equation22
ip ) (2.69 × 105)AD0.5C*ν0.5n1.5
(1)
Electrochemical Oxidation of Adenine
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TABLE 1: Parameters Used To Produce the Simplified EECirrev within DIGISIM electrochemical step
potential/V
electron transfer rate constant/cm s-1
E11 E12 Ex
0.5 0.7 0.2
10 10 10
where ip is the peak current in amperes, A is the area of the electrode surface in cm2, D is the diffusion coefficient of the species in cm2 s-1, C* is the bulk concentration of the species in mol cm-3, and ν is the scan rate in V s-1. Tafel analysis of the voltammetry of such a system (i.e., a plot of ln |I| vs V) would yield a gradient of nF/RT; here F is the Faraday constant, R is the gas constant, and T is the temperature. It was highlighted by Batchelor-McAuley et al. how in a two-electron process where both electron transfers are reversible but the second electron is not fully driven, insofar that it possesses a similar or higher standard potential as compared to the first electron then, although a single peak may be observed, the peak potential is substantially less than that given by the above equation.23 This is in part due to the increased probability of intermediate species being able to diffuse away from the surface without having undergone full oxidation. The situation is further complicated when the two-electron transfer is followed by an irreversible chemical step; here the presence of the chemical step decreases the equilibrium potential required for the second electron transfer. Such a system leads to the rate-determining step being potential dependent and Tafel analysis of such systems leads to the measurement of gradient of nappF/RT, where in this system napp can take a value between 1 and 2; this value does not represent the transfer coefficient (R), as in both cases the electron transfers are fully reversible. To exemplify the above case, a simulation was produced using the commercially available software DIGISIM (version 3.0, BASi Technicol, USA). Digisim is based on a fully implicit finite difference (IFD) method as proposed by Rudolph.24,25 An EECirrev mechanism was set up as given by the equations below and with the parameters shown in Table 1.
A h B + e-
(E11)
B h C + e-
(E12)
CfD The chemical step has been set as being an irreversible unimolecular reaction with a fast forward rate constant (Keq ) 1 × 1010, kf ) 1 × 1010 s-1). The Tafel analysis of the resulting voltammogram is shown in Figure 2 (solid black line), also depicted are the Tafel plots expected for a reversible oneelectron process and a reversible two-electron process (the dotted and dashed lines, respectively). It should be noted that the standard potential for the reversible two-electron process has been lowered so to account for the shift in peak potential caused by the chemical step and therefore allow a direct comparison to be made between the reversible two-electron process and the EECirrev mechanism. It is clearly seen that the Tafel plot resulting from the analysis of the simulated EECirrev is an intermediate mixture of the one- and two-electron Tafel plots. The measurement of the gradient associated with EECirrev mechanism would yield an apparent n value (napp) of approximately 1.3. Due to the Tafel plot for the EECirrev process not being a perfectly
Figure 2. Tafel plot of a simulated reversible one-electron process (dotted blue line), a reversible two-electron process (dashed red line), and the EECirrev mechanism (solid black line). The inlay depicts the simulated cyclic for the EECirrevE mechanism with varying numbers of electrons after the chemical step; +0e- black line, +2e- red line, and +4e- blue line.
straight line, which is a result of the change in rate-determining step, there is some dependence upon the measured napp with the values between which a line of best fit is measured but this error is within that found experimentally. It is noted that upon reaching the peak the second electron is the rate-determining step and, as a consequence, the measured peak current is the same as would be expected for a fully reversible two-electron process. The measured value for napp is dependent upon the difference in potential between the first and second electron, lowering of the potential of the second electron causes it to be more highly driven in nature and as a consequence, this leads to a value of napp closer to 2, as would be expected for a fully reversible two-electron transfer. The above may be further developed by considering the effect of further reversible electron transfers occurring after the irreversible chemical step, i.e., an EECirrevE, where the final electrochemical step after the irreversible chemical reaction may be composed of multiple electrons, the scheme for which is shown below, the parameters for which are shown in table 1.
A h B + e-
(E11)
B h C + e-
(E12)
CfD D h E + ne-
(Ex)
The chemical step effectively makes the second electron transfer rate determining in nature; as a consequence, the peak current will not vary with n1.5, as is the case shown in eq 1. Instead, the further electron transfers present in step Ex will act to scale the peak current with θ, where θ is the total number of electrons (i.e., sum of the electrons transferred in steps E11, E12, and Ex) divided by n′, where n′ is the number of electrons involved in and up to the rate-determining step (i.e., the sum of the electrons transferred in steps E11, E12). The simulations of this EECirrevE process are depicted within the inlay of Figure 2, with varying
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numbers of electrons occurring in step Ex, n ) 0 (black line), n ) 2 (red line), and n ) 4 (blue line). The peak current for the n ) 2 and n ) 4 extra electron transfers are 2 and 3 times larger, respectively, than the n ) 0 response. This analysis gives the following equation for the peak potential for a multielectron diffusion-only process involving a chemically irreversible step:
ipD ) (2.69 × 105)AD0.5C*ν0.5n'1.5θ
(2)
where ipD is the peak current for a diffusional species. This only holds if the chemical step is fast and irreversible and the electron transfer prior to the chemical step does not have a standard potential more than approximately 200 mV greater than that of the first electron transfer. A similar analysis of the form above may be applied to that found for a surface bound species. For an “n” electron reversible surface bound process the peak current may be described by the following equation:
ip )
n2F2 νAΓ 4RT
(3)
where all constants are the same as before but here Γ is the surface coverage with units of mol cm-2. Due to the species being bound to the electrode surface the peak potential more closely represents the associated standard potentials, it should be noted that as a direct consequence of this, the shift in peak potential and increase in peak height caused by the addition of the same unimolecular chemical step is greater for a surface bound species compared to that of the solution phase. As a result, the Tafel analysis on the same system as shown in Table 1 but for a surface bound species will result in an “napp” value of 2. Although a larger shift in peak potential for a given set of standard potentials is observed for a surface bound species, this is in reality counteracted by the stabilization of the species upon the surface and therefore effectively increases the standard potential relative to that of the solution phase. The magnitude of the stabilization is dependent upon the strength of the bonding of the species to the electrode surface. In a method analogous to that above, an improved description of the peak height of an “n” surface bound process that involves an irreversible chemical step is described by the equation
ipS )
n'2θF2 νAΓ 2.718RT
(4)
where ipS is the peak current for a surface bound species involving multiple electrons and a chemically irreversible process, n′ is the number of electrons transferred prior to the chemically irreversible step, θ is the total number of electrons transferred in the redox process divided by n′, F is the Faraday constant, ν is the scan rate, A is the area of the electrode surface, Γ is the surface coverage, R is the gas constant, and T is the temperature. It should be noted that the denominator has changed from 4 in eq 3 to 2.718 in eq 4, this reflects the effect of the chemical step upon the voltammetric wave shape, where the voltammetric feature is now asymmetric, hence leading to a greater peak current. This form of the equation is comparable to that found for an irreversible surface bound species.22,26 For a given voltammetric peak which has both surface bound and solution phase contributions to its current, it is possible to separate the two responses as a result of their different
Figure 3. Overlaid cyclic voltammograms of aqueous PBS (pH 6.85) containing adenine, 0.1 mM, on several electrodes: EPPG (a), Au (b), GC (c), BPPG (d) BDD (e), and Pt (f). Cyclic voltammetry was run between +0.4 and +1.3 V at 50 mV s-1.
dependencies with scan rate. The assumption must be made that the measured peak current is a sum of the two responses, i.e., itotal ) ipD + ipS; this is a reasonable approximation if only one peak is observed under all conditions, as is found for the oxidation of adenine on an EPPG electrode. Oxidation of Adenine. The cyclic voltammetry of 0.1 mM adenine in pH 6.85 PBS was recorded at 50 mV s-1 on a variety of electrodes, namely, gold, platinum, glassy carbon, edge-plane pyrolytic graphite, basal-plane pyrolytic graphite, and borondoped diamond, as shown in Figure 3. Note that the voltammetry has been plotted in terms of current density so as to allow direct comparison between electrodes of differing sizes. It is clearly apparent that the best resolved signal is found to be the EPPG electrode. Comparison of the BPPG and EPPG responses is particularly informative, with the response for the BPPG electrode being significantly smaller than that found for the EPPG. Furthermore, the peak potential for oxidation on BPPG is substantially more positive, with the measured peak far broader than that found for the EPPG. This broadening may be attributed to the presence of two sites present upon the BPPG electrode surface, where the shoulder at lower potentials is common to both the EPPG and BPPG surface. This leads to the possible conclusion that the edge-plane sites are the dominant electro-active sites for the oxidation of adenine, as was found to be the case for guanine.14 The voltammetric responses for glassy carbon and BDD also show peak signals that are more positive than for EPPG. Again this suggests that these electrodes are less electro-active toward the oxidation of adenine. The inlay of Figure 3 shows the comparisons of the responses for an EPPG and Pt electrode. It was not possible to resolve any features on the cyclic voltammogram run on Pt that could be ascribed as being due to the oxidation of adenine. This does not necessarily imply it is not occurring, but it may be obscured by the large current associated with the breakdown of solvent. The voltammetric response of the Au electrode in the presence of adenine is of particular interest when compared to the response in the absence of adenine. This data are shown in Figure 4. In the absence of adenine a large voltammetric feature corresponding to the oxidation of the gold surface is observed at +1.10 V with the corresponding reduction wave found at +0.46 V (vs SCE). In the presence of adenine this peak is far smaller, suggesting that the adsorption of adenine to the gold surface inhibits the oxidation process. Given that the adsorption
Electrochemical Oxidation of Adenine
Figure 4. Overlaid cyclic voltammograms of aqueous PBS (pH 6.85), with adenine 0.1 mM (a) and without adenine (b) on a gold macroelectrode. Cyclic voltammetry was run between +0.4 and +1.3 V at 50 mV s-1.
Figure 5. pH dependence of the adenine voltammetric signal at an EPPG electrode. Cyclic voltammetry was run between +0.4 and +1.3 V at 50 mV s-1 in aqueous PBS (from pH 5.85 to 7.85) containing adenine, 0.1 mM.
of adenine to gold surfaces is reported in the literature to be relatively strong, this inhibition may be due to the stabilization of the gold surface.18-20 Alternatively, the adenine layer may be blocking solvent molecules binding to the oxidized surface and therefore causing a larger overpotential to be required.27,28 As a further influence of the strong adsorption of adenine to the gold electrode, no oxidation signal corresponding to the adenine oxidation is observed as a result of the stabilization of the reactant upon the surface and therefore increasing the potential required for oxidation. Due to the markedly improved voltammetric signal observed on the EPPG electrode for the oxidation of adenine, this system was further investigated. The variation of the adenine peak potential was investigated between pH 6 and 8, as shown in Figure 5. This pH study was performed across a limited range so as not to be affected by the two adenine pKa (25 °C) values of 4.2 and 9.8,29 as deprotonation/protonation may cause a change in the observed reaction mechanism. As can be seen from the inlay of Figure 5, the peak potential varies linearly with pH, with a value of -58 mV/pH. This shift in peak potential is related to the change in standard potential for the rate-determining process as given by the Nernst equation. For
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Figure 6. Shift in the voltammetric signal shape with increasing adenine concentration. Cyclic voltammetry was run between 0.0 and +1.3 V at 100 mV s-1, in aqueous PBS (pH 6.85) containing K4[Fe(CN)6] (peak I), 5 mM, and adenine (peak II): 0 (a), 0.025 (b), and 1.8 mM (c). Inlay: cyclic voltammetry was run between +0.4 and +1.3 V at 50 mV s-1, in aqueous PBS (pH 6.85) containing different concentrations of adenine from 0.0125 to 3.6 mM.
a system in which the number of protons transferred is equal to the number of electrons, one would expect a shift of 59 mV/ pH (25 °C), this value is very close to that obtained experimentally. As a consequence, it may be concluded that for the oxidation of adenine the number of protons and electrons transferred prior to the chemically irreversible step is equal; this is in agreement with the literature.6,12,30,31 Through Tafel analysis of the voltammetry (not shown) it was found that the observed value of napp was 1.3. Due to the value of napp being greater than 1 we infer that the rate-determining step occurs after the first electron transfer but that the second electron transfer is likely at a more positive potential than the first, with the assumption that both electron transfers are reversible in nature. This leads us to conclude that the rate-determining step for the oxidation of adenine may be viewed as being EECirrev in nature. After the chemically irreversible step the system will be insensitive to the effects of a further chemical process and, as shown in the previous section, the observed peak height will be merely scaled by the number of subsequent electron transfers in terms of θ. It must be highlighted that even though it would be possible to obtain the same gradient (1.3) on a Tafel plot from a system in which the first electron transfer was reversible and the second irreversible, this situation is not physically realistic. Such a system would require the potential of the second electron to be far less than that of the first, this constraint is a result of the large overpotential imposed by the slow kinetics. In the case of adenine oxidation, the second electron will require a higher standard potential to be removed but the presence of the irreversible chemical step will lower the equilibrium potential for this redox couple to a value similar to that found for the first electron. The chemical step leads to the addition of a ketonal group to the purine system, it is the presence of the p-orbitals up on this added oxygen group that make the subsequent product more easily oxidizable. The voltammetric response of the oxidation of adenine on an EPPG electrode was studied with varying concentrations of analyte, the results of which are within Figure 6. Increasing the adenine concentration increases the peak current but at higher concentrations (above 0.5 mM) there is a major change in the voltammetric peak shape with the presence of a second peak
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potential, the position of which is concentration dependent. The origins of this change signal were investigated. If this second peak was due to the domination of the diffusional response, it would be expected for this peak to vary linearly with the concentration of adenine; this was not found to be the case. Furthermore, the shift in peak potential is suggestive of a change in the surface structure and as such an altering of the kinetics at the electrode surface. Through the use of the redox indicator ferrocyanide, it was possible to probe any changes to the electrode surface caused by the oxidation of adenine. This was achieved through running the cyclic voltammetry of an EPPG electrode in pH 6.85 PBS containing 5 mM K4[Fe(CN)6], with adenine concentrations of 0, 0.025, and 1.8 mM. In the absence of adenine the K4[Fe(CN)6] shows a reversible redox signal at around +0.2 V (vs SCE), upon addition of 0.025 mM adenine the oxidation of adenine is clearly visible but this has little influence upon the ferrocyanide redox signal, suggesting there was little or no modification of the electrode surface. At the higher concentration of 1.8 mM adenine, the adenine peak is observed to exhibit a shoulder at the same potential (+0.9 V vs SCE) as that found for the lower concentration adenine peak. Here the reverse scan of the ferrocyanide redox process is diminished, thus suggesting that the electrode surface has been modified. It may be concluded that at higher adenine concentrations polymerization upon the electrode surface is a significant mechanistic process. Formation of this polymer layer decreases the electro-activity of the electrode, and as such, the adenine exhibits this second oxidation peak, which is a result of the slower electron transfer kinetics. The oxidative peak current for adenine was studied as a function of scan rate at low adenine concentration (0-0.05 mM). It was found that the peak current did not vary linearly either with the square root of the scan rate or with the scan rate, as would be expected respectively for a purely diffusion or adsorptive response. A plot of ip/ν0.5 vs ν0.5 yielded a straight line. This suggested that the peak was a result of both diffusion and adsorption. Given that from the literature the overall oxidation of adenine is known to be a -6H+, -6e- process and that within the article it has already been ascertained that the rate-determining step involves the transfer of -2H+ and -2e- so that in terms of the theory presented earlier n′ ) 2 and θ ) 3. Hence the following equation describes the observed peak current for the oxidation of a low concentration of adenine at pH 6.85 at a EPPG electrode, where it is assumed that the two processes occur in parallel:
ip ) (2.69 × 105)AD0.5C*ν0.5n'1.5θ +
n'2θF2 νAKC* 2.718RT
(5) where the first term is related to the peak current for the diffusional process and the second term is related to that of the adsorbed species. Note that the second term has been adapted from that shown in eq 4, so that the surface coverage is now described as having a linear dependency on the concentration of adenine in solution, Γ ) KC*, where K is a constant. This simple adsorption isotherm is only valid for low bulk concentrations of adenine; at higher concentrations a more complex analysis would be required, but as previously demonstrated for this system, it is not possible to measure the response at higher levels due to the inhibition of the surface. The assumption that the two process may be viewed as being independent (as in eq 5) is valid for this system; this too is a result of the low concentration of preadsorbed adenine present. In addition to this,
Figure 7. Peak current dependence with the square root of scan rate for the average experimental response normalized with respect to the concentration: theoretical adsorptional (green line) and diffusional (blue line) contributions to the peak current and the total peak current (red line). Inlay: peak current per square root of scan rate dependency with the square root of the scan rate for the three lowest experimental adenine concentrations in aqueous PBS (pH 6.85), 0.050 (a), 0.025 (b), and 0.0125 mM (c). Squares represent the experimental data and the lines are their theoretical fitting.
as the edge plane and basal plane sites are likely responsible for the electro-oxidation and adsorption, respectively,14 this would also allow the two terms to be viewed as occurring in parallel. Fitting of eq 5 to the experimental data for the variation in peak height as a function of concentration and scan rate was undertaken, where for this experimental system θ has a value of 3 and n′ is equal to 2. This is depicted in the inlay of Figure 7. Even given the experimental variation in peak height, a good fit of eq 5 to the data is obtained by assuming a linear dependency of the surface coverage on solution concentration, and as such, an associated value of (1.7 ( 0.1) × 10-3 cm was obtained for K. The magnitude of the diffusion coefficient is assessed through measurement of the intercept, which is equal to (2.69 × 105)AD0.5C*n′1.5θ. Such analysis is consistent with a diffusion coefficient of (1.25 ( 0.2) × 105 cm s-1; this value is comparable to that found within the literature from independent measurements, leading to the confirmation that the analysis is of the correct form.32,33 The error associated with the diffusion coefficient is a result of the experimental variation in the measured peak height; this variation was greater at higher concentrations and is likely a result of the potential dependence of the surface structure.15 Figure 7 shows the average result for all three concentrations plotted as peak current against the square root of the scan rate. The resolved influences of both the adsorption (green line) and diffusion (blue line) can clearly be seen, with the total peak current being a sum of the two responses (red line). Conclusions This work highlighted how for complex reaction mechanisms, in which an irreversible chemical step is present, the peak current may be deconvoluted to reflect both the solution phase and adsorbed species. Such an analysis requires knowledge of the total number of electrons involved and the number of electrons transferred prior to the irreversible chemical step. In demonstration, a simulation of an EECirrev process was developed and it was shown how the Tafel analysis of such a system may lead
Electrochemical Oxidation of Adenine to the measurement of an apparent number of electrons between 1 and 2, depending upon the relative potentials associated with the first and second electron transfer. This napp does not directly represent the true transfer coefficients (R) associated with the electrochemical process, as in all cases the electron transfers are fully reversible in nature but arise from the presence of the closely linked formal potentials for the two-electron transfers. The oxidation mechanism of adenine is known to be complex in nature and is likely overall the transfer of 6H+ and 6e- and involves a number of chemically irreversible processes. The oxidation of this molecule was studied upon a number of electrode surfaces at pH 6.85 where it was demonstrated how adsorption of the species to the surface affects the observed voltammetry. In the case of Au the binding of adenine to its surface is strong enough that the oxidation is not apparent within the electrochemical window. Furthermore, the adenine adlayer blocks the oxidation of the gold surface. On carbon based electrodes the oxidation of adenine is far better resolved, likely as a result of the weaker binding to the electrode surface. With graphitic surfaces the density of edge-plane sites influenced the oxidative voltammetry, with the EPPG electrode exhibiting the highest peak current. The oxidation of adenine was further investigated on the EPPG electrode, showing that the peak current shifted with 58 mV/pH at pHs close to that being studied, thus suggesting that the rate-determining step for the oxidation of adenine involves an equal number of protons and electrons. At a higher concentration of adenine, a less electro-active layer was formed as a result of polymerization on the electrode surface. Having provided a method for the analysis of the peak current for complex electrochemical pathways involving an irreversible chemical step, this was further developed to demonstrate how these equations may be used to assess systems involving mixed adsorptional and diffusional responses. The cyclic voltammetry of adenine upon an EPPG electrode was used as a representative case study. The influence of the two contributions to the peak current was resolved as a result of their differing dependencies with the scan rate. A diffusion coefficient for adenine was found to be (1.25 ( 0.2) × 105 cm s-1, which is close to that found from independent studies within the literature. Futhermore, the adsorption of adenine at low concentration (below 0.05 mM) to an EPPG surface was shown to exhibit a linear dependence with the solution phase concentration such that Γ ) KC*, with the value of K found to be (1.7 ( 0.1) × 10-3 cm. Acknowledgment. L.M.G. (SFRH/BD/36791/2007) acknowledges Portuguese Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT) for his Ph.D. studentship.
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