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In situ FT-IR spectroelectrochemical study of electrooxidation of pyridoxol on a gold electrode
Electrochimica Acta 51 (2005) 1059–1068
In situ FT-IR spectroelectrochemical study of electrooxidation of pyridoxol on a gold electrode Mei Ling Wang, You Yu Zhang ∗ , Qing Ji Xie, Shou Zhuo Yao Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China Received 14 May 2005; received in revised form 30 May 2005; accepted 30 May 2005 Available online 5 July 2005
Abstract The electrochemical oxidation of pyridoxol (PN) on a polycrystalline gold electrode was investigated by cyclic voltammetry and in situ Fourier transform infrared spectroscopy (FTIRS). In 0.1 M aqueous NaOH solution, the gold electrode showed a high catalytic activity for the irreversible oxidation process of PN. The individual ionic species and the major tautomeric equilibria of PN molecules in aqueous solutions were evidenced well from the pH-dependent attenuated total reflectance (ATR) spectra, and the results were in good agreement with the voltammetric observations. In situ single potential alteration infrared reflectance spectroscopy (SPAIRS) demonstrated that a lactone form of PN, rather than pyridoxal aldehyde, was likely formed, which was subsequently diffused into the thin layer solution and underwent hydrolysis slowly to pyridoxic acid (PA) as the final product. In addition, the adsorption of PN at Au electrode was characterized by in situ subtractively normalized interfacial Fourier transform infrared reflectance spectroscopy (SNIFTIRS) method, which revealed that the adsorption of deprotonated PN, via nitrogen atom in vertical configuration on electrode surface, occurred from −0.5 V versus Ag|AgCl|KCl(sat), which was much lower than the potential of PN electrooxidation observed from ca. 0 V. © 2005 Elsevier Ltd. All rights reserved. Keywords: In situ FT-IR; Pyridoxol; Vitamin B6; Electrochemical oxidation; Au electrode
1. Introduction The vitamins of B6 group (VB6) that contain a pyridine ring in structure, including pyridoxol (PN), pyridoxal (PL), pyridoxamine (PM) and their phosphate derivatives, are important natural compounds essential in diet needed as the co-factor in some VB6-dependent enzymes involved in the metabolism and synthesis of amino acids and other related compounds for the maintenance of body cells due to their vital roles in various biological processes [1–3]. Since the first work on voltammetric determination of VB6 using a carbon paste electrode [4], the electrochemical detection and polarographic analysis have been widely used because of the sensitive linear voltammetric response for vitamin B6 [5–8], but only very few investigations have been reported ∗
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on the nature of the electrochemical reaction of VB6 [9–10]. Because PL is an oxidative form of PN and pyridoxic acid (PA) is the final product of VB6 metabolism in mammalian tissue (Scheme 1) [11], understanding the electrochemical kinetics and mechanism of VB6 may be helpful for the study of its catabolism process in vivo [12]. Recently, with the wide applications of simultaneous techniques of spectrometric/electrochemical measurement as powerful and informative tools for investigation and further elucidation of the electrochemical reaction mechanism, the electrochemical behaviors and the reaction pathways of PN and other VB6 derivatives on a solid electrode in aqueous medium have received considerable attention [12–16]. A study on the electrooxidation mechanisms of VB6 in different pH phosphate buffers at a pyrolytic graphite electrode was carried out by Zhu et al. using ultra-violet spectroelectrochemistry (UV-SEC) method [13]. Pineda et al. monitored the course of the electroreduction of PA in neutral buffer
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Scheme 1. Chemical structures of pyridoxol (PN), pyridoxal (PL) and pyridoxic acid (PA).
[12] and electrooxidation of PL in alkaline solution [14] on a polycrystalline gold electrode by cyclic voltammetry and UV–vis spectroscopy. Using a fluorescence spectroelectrochemical measurement, Tan et al. reported the electrochemical redox processes of pyridoxine hydrochloride at a glass carbon electrode modified with poly(methylene blue) film in alkaline phosphate buffer [15]. Cao et al. coupled the simultaneous EQCM and fluorescence to examine the adsorption/desorption and oxidation of PN on gold electrode in alkaline medium [16]. However, the intermediate/product of the oxidation of VB6 was not so well identified, and the mechanism for the electrochemical interaction at the electrode interface was not quite clear. Currently, vibrational studies of VB6 [1,3,17,18] have shown Fourier transform infrared spectroscopy (FTIRS) a powerful tool in characterizing chemical structure with reduced interferences from solvent bands using attenuated total internal reflectance (ATR) technique. In situ FT-IR/ATR has become one of the investigative techniques of choice currently available to explore solid/liquid interfacial phenomena in situ, to obtain information on the nature of adsorbed species readily, and to identify the intermediate, adsorbed species and products of electrode reaction, and it has been widely used to study electrocatalytic oxidation mechanism of small organic molecules at noble metal electrode [19–23]. To our knowledge, no in situ IR-spectroscopic investigations have been directed toward the electrochemical properties of VB6 yet, despite the significant technological interests in the elucidation of the reaction mechanism for the electrochemical interactions. In this respect, the electrocatalytic oxidation of PN on gold electrode in alkaline medium was investigated by cyclic voltammetry and in situ FT-IR spectroscopy, and the aim of the present study is to reveal the oxidation mechanism of PN at the molecular level.
2. Experimental The cyclic and rotating disk electrode voltammetries were carried out with a CHI660A electrochemical workstation (CH Instruments Co., USA) controlled by CHI660A software and in a conventional three-electrode electrochemical cell. The working electrode was a polycrystalline gold electrode of 6 mm in diameter. The counter electrode was a platinum sheet electrode. An Ag|AgCl|KCl(sat) electrode served as the reference electrode. For rotating disk electrode voltammetry, the
surface of the working Au disk electrode was polished carefully with emery paper and then sonicated in double-distilled water prior to each potential sweep experiment. Nitrogen atmosphere was maintained over the solution during the measurements. In situ FT-IR spectroscopic measurements were performed using a Nicolet Nexus 670 spectrometer (Nicolet Instrument Co., Madison, WI) equipped with a liquid nitrogen cooled MCT detector. The spectroelectrochemical cell was made of Teflon and provided with a prismatic CaF2 window bellow at 60◦ . A detailed description can be found in previous papers [22,24]. The working electrode was a gold disk with a diameter of 8 mm. Prior to each experiment, it was mechanically polished using alumina powder of 5, 1, 0.3, and 0.05 m then rinsed with double-distilled water. A platinum wire was used as the counter electrode, and the reference electrode was an Ag|AgCl|KCl(sat) electrode. Different IR spectra were obtained using the single potential alteration infrared reflectance spectroscopy (SPAIRS) technique [19,25]. The reference potential (ER ) was chosen at −0.8 V, while the sample potential (ES ) stepped positively in a multiple-step manner. Normalized reflectance spectra were calculated as R/R = [R(ES ) − R(ER )]/R(ER ), where R(ES ) and R(ER ) are the single beam spectra obtained at sample potential ES and the reference potential ER , respectively. Each spectrum resulted from co-addition of 50 interferograms with a resolution of 8 cm−1 . The subtractively normalized interfacial Fourier transform infrared reflectance spectroscopy (SNIFTIRS) technique [26,27] was used to obtain potential difference IR spectra. The reference potential was maintained at −0.8 V, and the reflectivities were obtained using a multiple potential step (MPS) procedure in which the electrode potential was stepped m times between the reference and sample potentials, ER and ES , respectively. During each step, n interferograms with resolution 4 cm−1 were acquired at both potentials. This procedure was repeated to give the total number (N = m × n) interfreograms acquired for each of the two potentials. The values of n and m employed in this study were 100 and 20, respectively. Final spectra were normalized as R/R = [R(ES ) − R(ER )]/R(ER ). In all experiments, each sample was placed on the sampling device and aligned according to the manufacturer’s recommendation; Ominic E. S. P. software of version 6.0a (Nicolet Instrument Co.) was used for processing the spectral data. An attenuated total reflectance (ATR) sampling accessory (Specac Inc., Technology Court, Smyrna, Georgia, USA)
M.L. Wang et al. / Electrochimica Acta 51 (2005) 1059–1068
was employed for ATR-FT-IR experiments. The ATR spectra were obtained for PN or PA in aqueous solutions of different pH values using ZnSe internal reflectance crystal. For each spectrum, 64 interferograms with a resolution 4 cm−1 were co-added. The background spectra were recorded by adding double-distilled water at the same pH value. The net PN spectrum in solution was calculated by subtracted the background spectrum from the solution spectrum. Accurate subtraction was obtained by varying the subtraction factor in an interactive manner until the broad water adsorption around 2100 cm−1 was totally removed to baseline levels. Pyridoxol hydrochloride, pyridoxal hyrochloride and pyridoxic acid were purchased from Sigma and used without further purification. All other chemicals and reagents were of analytical grade. Aqueous solution of PN, PL or PA was prepared daily by dissolving the corresponding solid compound in double-distilled water. The pH values were adjusted by an addition of concentrated NaOH aqueous solution. The solutions were deaerated by bubbling nitrogen gas of high-purity (99.99%) before each IR measurement. All the experiments were carried out at room temperature (25 ± 1 ◦ C).
3. Results and discussion 3.1. Electrochemical studies The typical cyclic voltammograms in the absence and presence of PN for gold electrode in 0.1 M NaOH aqueous solution are shown in Fig. 1(a). In the alkaline supporting electrolyte alone, the curve displays a weak anodic peak with the potential above 0.15 V and a broad peak at 0.2 V, and has a reduction current peak appearing near 0.15 V on its negative side. According to the previous studies [16,23,28], the anodic peaks are associated with a surface oxidation process, i.e., OH− chemisorption on Au surface to form short-lived AuOH species that is subsequently converted to surface oxide, and
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the cathodic peak corresponds to the reduction of the gold oxide formed in the anodic scan. In the presence of PN, the gold shows a pronounced catalytic activity for this aromatic alcohol, and an oxidation reaction begins at approximately −0.2 V with a new anodic peak (pa ) for oxidation of PN at ca. 0.1 V, but no corresponding reductive peak is found during the subsequent cathodic scan. It is interesting to note that during the potential negative sweep, as soon as the surface oxides are reduced, another anodic peak is found at ca. 0.1 V, apparently due to again oxidation of PN on the newly generated electrode surface. This behavior is similar to the electrooxidation of aliphatic alcohol, sugar or pyridoxal in alkaline medium [14,25,29,30]. Fig. 1(b) shows the typical cyclic voltammogram of Au at a scan rate of 2 mV s−1 recorded in spectroelectrochemical cell during the in situ FT-IR acquisition. The anodic peak (pa ) is shifted to more positive potential compared with the voltammogram in Fig. 1(a), which may result from different concentration of PN. With the increase of PN concentration, the oxidation peak potential moves positively [16]. In addition, the pH change and the resistance to transferring reaction species between the thin layer and the bulk solution during the oxidation process of PN in situ IR measurements may also contribute to the shift [31,32]. In the case of the voltammograms shown in Fig. 1, the electrooxidation of PN on polycrystalline gold seems to be irreversible, which is consistent with the results observed by Cao et al. [16]. 3.1.1. Effect of PN concentration To investigate the effect of PN concentration on oxidation current, a series of experiments was performed in 0.1 M NaOH solutions containing different concentrations of PN. The results demonstrate that the anodic peak current linearly increases with the increasing of PN concentration, which varies from 0.5 to 10 mM. At constant potential scan rate of 50 mV s−1 , plots of the anodic peak current density (jpa ) versus PN concentration (c) give a straight line with a slope
Fig. 1. (a) Cyclic voltammograms of a polycrystalline Au electrode in 0.1 M NaOH (· · ·) and in 1 mM pyridoxol + 0.1 M NaOH (—) at scan rate of 50 mV s−1 . (b) Cyclic voltammogram of a polycrystalline Au electrode in 10 mM pyridoxol + 0.1 M NaOH taken in spectroelectrochemical cell at scan rate of 2 mV s−1 .
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of 0.027 A cm−2 mol L−1 , i.e., 0.0956 A mol L−1 for plot of the anodic peak current (ipa ) versus PN concentration in this study, which is identical with the reported value of 0.0954 [16]. In the range of PN concentration investigated, the reaction order estimated from the linear section of plots for log jp versus log c was 0.835 ± 0.05. 3.1.2. Effect of the pH value From previous studies on the electrooxidation of several sugars and alcohols, gold is characterized by its poor catalytic activity of this electrode in acidic medium but conversely proves to be a more effective catalyst for oxidizing these compounds in the case of alkaline solutions [25,29]. In order to investigate the influence of pH on the electrooxidation behavior of PN, the voltammetric response of the polycrystalline gold electrode was studied over a wide pH range from 7 to 13.5 in a 0.1 M phosphate buffer containing 1 mM PN. As has been expected, PN is practically not oxidized on gold at low pH, and the current of the oxidation peak begins to increase with the concentration of OH− when the solution pH is adjusted above 8 (Fig. 2). Results on the electrooxidation of PL [14] at gold as well as the molecular aspects of PN ionization in aqueous solution [33] show that the pH-dependent oxidation phenomenon originates from two contributions: the coverage of Au(OH)ads species on the electrode surface and the formation of pyridoxol anion in solution. It is usually accepted that the existence of a submonolayer of adsorbed hydrous oxide species is necessary as the active intermediate for electrocatalytic oxidation of a certain organic compound on metallic electrode, and the formation of these surface hydroxyl groups is favored in gold in alkaline solutions [34]. In this experiment, the anodic peak current increases with the increasing of solution pH, suggesting that the higher catalytic activity is observed for Au electrode in solutions of higher pH values. Actually, the anodic peak current for 1 mM PN oxidized at the same pH value of 12.8 and sweep rate (50 mV s−1 ) in the 0.1 M phosphate buffer solu-
Fig. 2. The influence of pH on the anodic peak current (ipa ), for the oxidation of 1 mM pyridoxol in 0.1 M phosphate buffer adjusted with concentrated NaOH solution, on a polycrystalline Au electrode at 50 mV s−1 and 25 ◦ C.
tion (Fig. 2) is lower than that in the 0.1 M NaOH solution (Fig. 1). The difference may be related to the various types of phosphate anions of buffer composition as the adsorption species, which may either lower the mediator activity or interfere with the interaction of the catalytic electrode surface with PN molecules [35]. On the other hand, a UV–vis adsorption spectroscopy study by Metzler and Snell on ionization equilibria of pyridoxine under various pH conditions demonstrated that four interchangeable ionic forms would occur corresponding in acidic solution (pH < 5) to the cation, at neutrality (pH 6.8) to neutral form, chiefly the dipolar ion, and in basic solution (pH > 8) to the anion [33]. Scheme 2 denotes the equilibria and the referenced values of dissociation constants (pKa ) between these species in aqueous
Scheme 2. Dissociation equilibria for ionic species and tautomeric forms of pyridoxol in aqueous solution due to the protonazation states of phenolic group and ring nitrogen.
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solution. According to the previous electrooxidation studies on aldehydes and glucose at gold electrode in solutions of high pH [14,28,35,36], the negative-charged anion showed a high electroactivity. In strong alkaline solutions with pH > 9, the deprotonated PN exists as an electroactive anion, and the oxidation is prone to occur in this form. So increasing the pH value is favor of oxidation reaction. In this study, the electrooxidation of PN is carried out in 0.1 M NaOH solution with a pH value of 12.8. Similar to the behavior of pyridoxal anion, the peak current of PN changes with the pH of 7–14 as a sigmoid shaped dissociation curve, i.e., almost no peak occurs at low pH, but at higher pH the current increases with pH until a plateau reaches at pH around 13 [14]. In comparison with the situation of PL, the current for PN electrooxidation is observed coming into existence from pH 8 (Fig. 2), a value more coincident with the pKa2 value of around 9, at which the pyridoxol anion is dissociated from its dipolar form (Scheme 2). It is also interesting to note that a distinct difference should be drawn in the shape of the curve for oxidation peak current versus pH between pyridoxal hemiacetal and PN [14]. Unlike the behavior of PL, an increase of the peak current for hemiacetal oxidation is shown in the pH range of 7–10 with an inflection around pH 8.5, then followed by decrease at pH > 13. This is due to the existence of three species of PL including free aldehyde and two electroreactive forms of hemiacetal and its anion, and the ionic equilibria among these species in strong alkaline solution with pKa values of around 8 and 13, respectively. In the case of PN with one existent dissociated form mediated with regard to oxidation by the Au species at high pH, no equilibria would occur and the kinetics is unlikely to be controlled by the deprotonation reaction, but undoubtedly affected by the electrocatalytic activity of the Au electrode. Since the detailed mechanism for electrooxidation of PN may involve complicated multi-step or multi-electron reactions, the electrochemical behavior may be affected by the oxidation conditions, such as the property of the electrolyte solution, the applied potential parameters, and the nature of electrode material. It has been reported that the anion peak current of pyridoxine oxidation at a copper() hexacyanoferrate(Ш) modified carbon paste electrode shows a bell shaped variation with pH range of 4–8 [9], and the oxidation of PN at a pyrolytic graphite electrode can take place in all phosphate buffers of a wide pH range from 2 to 12 [13]. 3.1.3. Rotating disk electrode (RDE) voltammetry The electrooxidation process of PN on gold electrode was also evaluated using rotating disk electrode (RDE) voltammetry technique. The typical linear sweep voltammograms of an Au disk rotating at different rates in 1 mM pyridoxol + 0.1 M NaOH are shown in Fig. 3. The anodic peak for pyridoxol oxidation appears at 0.1 V for all rotation rates. The peak current (ip ) tends to decrease rather than increase with the increase of the rotating rate of the electrode, suggesting that the mass transport controlled process play no key role and that a cer-
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Fig. 3. Linear-sweep voltammograms of Au disk electrode in 1 mM pyridoxol + 0.1 M NaOH at different rotation rates. The rotation rates were in rpm: (1–9) 200, 400, 600, 800, 1200, 1600, 2000, 2400 and 3000, respectively. Scan rate: 20 mV s−1 . Insert: rotation rate-dependence of the peak current (ip ) of Au disk electrode.
tain hindering process such as adsorption might take place at the gold surface [37,38]. As shown in Fig. 3, the current at lower potentials, independent of PN oxidation, is not affected by electrode rotation. And the lower oxidation peak current at the higher rotating rate should be in relation to the possible PN oxidation mechanisms. This is in accordance with the result of IR spectroscopic studies thereinafter. 3.2. IR spectroscopic studies 3.2.1. ATR spectra of PN at different pH values As has been illustrated above, PN exists as different ionic species and with tautomeric forms, depending on pH, composition and polarity of solution [33]. To further investigate the pioneering findings of ultraviolet spectra, we turn to an attenuated total reflectance (ATR) technique to provide direct molecular level evidence of the individual ionic species in aqueous solution. Fig. 4 presents the ATR spectra of PN in aqueous solutions of various pH values (2.8, 5.0, 7.1, 9.1, 11.2 and 12.8) measured in the range of 900–3000 cm−1 . A concentration of 0.1 M PN is chosen in these spectroscopic studies and similar experiments carried out with 10 mM PN show the same bands but with weaker intensities. It should be noticed that substantial difference appears in the spectra at region of 1600–900 cm−1 of PN in the various solutions, indicating the change in the molecular structure resulting from the variation of the pH. At pH of 2.8, the spectrum shows manifold absorption bands around at 1547, 1464, 1440, 1426, 1386, 1290, 1225, 1090 and 1023 cm−1 . According to a vibrational study of pyridoxine by Cinta et al. [3], the sharp and intense band at
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(Scheme 2). At the same time a new sharp band arises at about 1402 cm−1 with a broad shoulder band, a similar phenomenon observed in previous IR results for pyridoxamine 5 phosphate [18], which is due to the deprotonation of the pyridine nitrogen. The broad shoulder band around at 1398 cm−1 may be associated with the dissolved carbonate anion in the strong alkaline medium [39]. A band at 996 cm−1 seems clearer at high pH, due to ring bending vibration. Similarly, no significant variation in other bands related to the ring structure and aliphatic substitute at 1294, 1234, 1101 and 1023 cm−1 is observed. Thus, the results are in very good agreement with those obtained by UV–vis and other vibrational spectroscopies under all the conditions [3,17,18,33], and the variation of ATR spectra with pH can reflect well the individual ionic species and the major tautomeric equilibria of PN in aqueous solutions.
Fig. 4. The pH influence on the ATR spectra of 0.1 M pyridoxol in aqueous solutions. The pH values are denoted at the corresponding spectra.
1547 cm−1 may be assigned to the in-plane pyridine ring stretching vibration and C N+ H stretching vibration of nitrogen-protonated pyridine ring, the broad bands at 1023 and 1090 cm−1 are due to the ring and C O stretching vibration in CH2 OH group, and the weak band at 1464 cm−1 may be related to C O H bending vibration for phenolic hydroxyl group [3,17]. In addition, the weak bands located at 1440, 1426 and 1386 cm−1 can be ascribed to CH2 scissors vibration, CH3 asymmetrical and symmetrical bending, and the two bands at about 1290 and 1225 cm−1 may correspond to the ring stretching modes [3]. When the pH value is increased to 5.0, the band intensity of 1464 cm−1 is greatly reduced and the band at 1547 cm−1 red shifts with an increase in the bands at 1440, 1426, 1386, 1290 and 1225 cm−1 , but no change is found for the bands at 1090 and 1023 cm−1 . This is due to deprotonation of phenolic hydroxyl group (pKa1 = 5) of the cation form of PN (Scheme 2). However, the fact that the peaks of 1464 and 1547 cm−1 are still present suggests the presence of the dipolar ion as well as the neutral forms in this case. A similar spectrum is observed in the solution of pH 7.1 but the band at 1464 cm−1 almost disappears (Fig. 4), indicating the existence of tautomeric equilibrium of the deprotonated pyridxol at pH of 5–7, and the dipolar ion is the predominant form in aqueous solution. With further increasing of the pH value from 9.1 to 12.3, the band at 1547 cm−1 strongly decreases due to the deprotonation of pyridine nitrogen (pKa2 = 9) in high pH solutions
3.2.2. In situ FT-IR studies on PN electrooxidation at Au in alkaline medium To investigate the electrochemical process and propose a plausible reaction mechanism for PN oxidation on a gold electrode, two in situ infrared reflectance absorption spectroelectrochemical techniques, SPAIRS and SNIFTIRS, have been used to identify the species of reaction intermediate and product adsorbed on the gold surface as well as near the surface in the thin layer solution. A reference potential (ER ) was set at −0.8 V, where no related oxidation is known to take place, and sample potential (ES ) was varied stepwise to positive direction. So in these spectra, positive- and negative-going adsorption bands indicate the consumption and formation of species at ES as compared to ER , respectively. 3.2.2.1. SPAIR spectra. Fig. 5 shows a series of in situ FTIR reflectance spectra obtained for Au electrode in 10 mM PN in 0.1 M NaOH solution (pH 12.8). The sample potential (ES ) was varied stepwise from −0.6 to 0.5 V. When ES ≥ 0 V, several bands would appear on the SPAIR spectra, including negative-going bands at 1739, 1569, 1460, 1423, 1286, 1198 and 1109 cm−1 , and positive-going bands at 1402 and 1296 cm−1 with two bipolar bands denoted as 1402/1423 and 1296/1286 cm−1 , respectively. For the better clarification of the bands observed, the SPAIR spectrum from Fig. 5 is compared with the ATR spectrum of neat PN in alkaline solution of pH 12.8 from Fig. 4, and the result is presented in Fig. 6 and summarized in Table 1. It is clear that all the positive-going bands appearing in the SPAIR spectrum correspond well to the negative-going absorption bands due to PN in the ATR spectrum. The bipolar bands at 1402/1423 cm−1 and 1296/ 1286 cm−1 , in accordance with the literature [3,18,27], are assigned to in-plane stretching mode and in-plane bending mode of the nitrogen pyridine ring, respectively, and their changes in transition dipole moments should be related to molecule adsorption. As a polysubstituted pyridine, PN may have at least three orientations at the surface due to three
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Fig. 5. SPAIR spectra of 10 mM pyridoxol in 0.1 M NaOH solution on a polycrystalline Au electrode at various potentials from −0.6 to 0.5 V (vs. Ag/AgCl). Sample potential indicated for each spectrum. Reference potential was set at −0.8 V.
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Fig. 6. Comparison of (a) ATR spectrum of neat pyridoxol recorded in transmission mode in alkaline solution of pH 12.8 (shown in Fig. 4 with (b) in situ SPAIR spectrum recorded using the sample potential ES = 0.2 V and the reference potential ER = −0.8 V (vs. Ag/AgCl) (shown in Fig. 5).
different adsorption sites of the molecule: the lone pair electrons on nitrogen in the pyridine ring, the -system of the aromatic ring and the nonbonding electrons from the alcoholic groups [40]. In a study on the adsorption of VB6 at silver surface at different pH values by Raman and surface
enhanced Raman spectroscopy (SERS) [3], Cinta et al. proposed a parallel or tilted orientation of the skeletal ring with the respect of the Ag particles, due to the presence of the bands assigned to the ring deformation mode and red shifted after adsorption of deprotonated VB6. However, neither red
Table 1 Comparisons of typical IR vibrational bands in the range of 1800–900 cm−1 for pyridoxol (PN), and pyridoxic acid (PA) in 0.1 M NaOH solution SPAIR spectrum of PN (on Au electrode at ES = 0.2 V)
ATR spectrum of PN Wavenumber – – 1547 – – 1402 1294 1234 – – 1101 1023 996
(cm−1 )
Description
↓ (w) ↓ (s) ↓ (m) ↓ (m) ↓ (w) ↓ (m) ↓ (w, sh)
Wavenumber 1739 1569 1545 1460 1423 1402 1296 1286 1234 1198 1109 1100 1023 –
(cm−1 )
ATR spectrum of PA
Descriptiona
Wavenumber (cm−1 )
↓ (s) ↓ (m) ↑ (w) ↓ (w) ↓ (s) ↑ (s) ↑ (m) ↓ (m) ↑ (w) ↓ (m) ↓ (s) ↑ (w) ↑ (m)
– 1589 – 1487 – 1404 – 1290 – 1219 – – – 1005
The arrows ↓ and ↑indicate negative and positive band, respectively. a Abbreviations for band intensity and shape: s, strong; m, medium; w, weak; sh, shoulder band.
Description ↓ (s) ↓ (w) ↓ (s, sh) ↓ (m) ↓ (m)
↓ (s, sh)
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shift nor band broadening occurs in the ring modes of PN in this work, suggesting that the interaction between the ring -system and Au surface is not important. Moreover, the characteristic of bipolar bands is similar to previous results for pyridine N-bonded to the polycrystalline gold electrode surface [27], and a similar phenomenon can be expected to occur in the case of pyridine derivatives. At high pH values, the participation of the nitrogen of vitamin PP ring to the interaction with silver surface was more probable [40]. So it may conclude that the deprotonated PN is adsorbed via nitrogen atom probably in vertical position on the Au surface. The frequency of the bipolar band of in-plane stretching mode plotted against the electrode potential. A small shift of the frequency is observed with a slope of 5 cm−1 V−1 with our spectral resolution. This is in agreement with the work ¨ and Yeager. They found a slope of 6 cm−1 V−1 for of kOtz pyridine adsorbed on silver [41]. No significant shifts for pyridine adsorbed on Au (210) and Au (1 1 1) were found by Brolo et al. and H˙ebert et al., respectively [42,43]. This small potential dependence of the frequency of the vibration mode is consistent with N-bonded adsorption behavior. A very intense negative-going band centered at 1739 cm−1 is attributed to the vibration bands of reaction products or reaction intermediates, which contain a carbonyl group, aldehyde or carboxyl acid. Previous reports have shown that PN oxidation can convert to PL or PA [13,16]. To check whether the observed band is due to PL, the FT-IR spectra of PL was recorded under the same condition (Fig. 7). It is interesting to note that similar bands are observed for both spectra of PN and PL, i.e., the negative-going bands around 1738, 1582, 1197 and 1109 cm−1 and bipolar bands at 1407/1426 cm−1 and 1333/1287 cm−1 . The result indicates that the presence of PL as the final product in the oxidation of PN is less possible, and that both PL and PN should have the same oxidative product and the similar oxidation mechanism in this condition. As shown in Fig. 7, the bands for PL appear at the sample potential of −0.2 V lower than that of PN. It is noticeable that no positive-going bands reflecting aldehyde group of PL are observed. This is due to the fact that PL exists as its hydrated forms with a very small percentage of the free aldehyde species under this pH condition [14]. On the other hand, Pineda et al. proposed a lactone derivative intermediate for PL electrooxidation at gold electrode in alkaline solution, and PA as a final product, which confirmed by mass spectrometry, UV–vis and fluorescence spectroscopies [14]. According to the literatures [30,44], the band at 1739 cm−1 can be ascribed to the C O stretch in pyridoxic lactone. The other strong bands, which can be assigned to the lactone, are clearly observed at 1198 and 1109 cm−1 in the in situ FT-IR spectra, due to the C O C asymmetric stretch and symmetric stretch of the lactone [30]. In Fig. 8, the peak intensities at the wavenumbers of these bands are plotted versus the sample electrode potentials. At ca. 0.3 V, the intensity of band at 1739 cm−1 reached the maximum then decreased gradually. This phenomenon is in accordance with the result of cyclic voltammogram. Thus, the present IR result demon-
Fig. 7. SPAIR spectra of 10 mM pyridoxal in 0.1 M NaOH solution on a polycrystalline Au electrode at various potentials from −0.6 to 0.5 V (vs. Ag/AgCl). Sample potential indicated for each spectrum. Reference potential was −0.8 V.
Fig. 8. Relationship between the peak intensities of wavenumbers at 1739 cm−1 (), 1569 cm−1 (), 1402 cm−1 (䊉), 1296 cm−1 (), 1109 cm−1 () vs. the electrode potential during the oxidation of pyridoxol (shown in Fig. 5).
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strates lactone as an intermediate in the oxidation process of PN. In addition, two negative-going bands at 1569 and 1460 cm−1 appear in the spectrum at 0.1 V and increase in intensity as potential increases. The bands of 1569 and 1460 cm−1 may be associated with the asymmetric stretching vibration of a carboxylate group [44] and the CH2 scissors vibration, respectively, which were also found in ATR spectrum of PA in alkaline solution (Table 1). It should be mentioned that the carboxylate group also has a band at 1393 cm−1 , due to the symmetric stretching, may be swamped by the positive band at 1402 cm−1 . This indicates that the intermediate lactone formed at the electrode surface was desorbed and underwent hydrolysis to yield pyridoxic anion in the basic solution. It is noted that, however, the intensity ratio of peak at 1569 cm−1 is lower in comparison with the peak at 1739 cm−1 yet (Fig. 5), suggesting that the lactone accumulated at electrode surface is advantageous and the degree of hydrolysis is small, because of the N-bonded adsorption and pH decrease in the thin layer solution due to gradual consumption of OH− anions [45]. 3.2.2.2. SNIFTIR spectra. In order to further investigation of the adsorption behavior of PN on the electrode surface, we performed the SNIFTIRS measurements. Since the oxidation of PN is irreversible, the utilization of the SNIFTIRS over the potential range below the oxidation potential. The reference potential was maintained at −0.8 V, and the reflectivities were obtained using a multiple potential step (MPS) procedure. In
Fig. 9. SNIFTIR spectra recorded for 10 mM pyridoxol in 0.1 M NaOH solution on a polycrystalline Au electrode at various sample potentials (indicated on the corresponding spectrum) with a base potential set at −0.8 V.
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Fig. 9, the spectra mainly display the O H bending band of water and the two bipolar bands as mentioned in SPAIR spectrum. The bipolar bands begin to occur at −0.5 V, which is below the oxidation potential 0 V, indicating that the change of dipole moments is due to the PN adsorbed at the metal surface and the adsorption potential is around −0.5 V. The intensity of the bands is not remarkable probably due to weak adsorption at low potentials, but they can be discerned in these spectra. According the IR selection rules, the spectrum is sensitive to component of the vibration dipole perpendicular to the surface only [27]. So a vertical configuration of the adsorption behavior of PN is more possible.
4. Conclusion The application of simultaneous cyclic voltammetry and in situ Fourier transform infrared spectroscopy (FTIRS) measurement was first investigated for the electrochemical characterization and electrooxidation monitoring of pyridoxol (PN) on a polycrystalline gold electrode. Two in situ infrared reflectance absorption spectroelectrochemical techniques, SPAIRS and SNIFTIRS, were used to monitor the adsorption behavior of reactant and identify the species of intermediate or product generated on the gold surface as well as near the surface in the thin layer solution during PN oxidation in alkaline medium. In 0.1 M NaOH aqueous solution, gold showed a high catalytic activity for the irreversible oxidation of pyridoxol to pyridoxic acid as the final product. Meanwhile, new negative-going bands at 1739, 1569, 1460 and 1109 cm−1 , and new bipolar bands around 1402/1423 cm−1 and 1297/1284 cm−1 appeared under the positive direction of potential of above 0 V. These positivegoing bands appearing in the SPAIR spectrum correspond well to the negative-going absorption bands in the ATR spectrum of PN. The individual ionic species and the major tautomeric equilibria of PN molecules in aqueous solutions were evidenced well from the pH dependence ATR spectra, and the results were in good agreement with the voltammetric observation and those previously obtained by UV–vis and other vibrational spectroscopies. From the negative-going and bipolar bands of pyridoxol and pyridoxal, it was suggested that the two kinds of substances have the similar electrochemical behaviors on gold electrode in alkaline medium. It implied that the intermediate pyridoxic lactone instead of pyridoxal aldehyde likely formed at the electrode surface was desorbed and only a few underwent hydrolysis to yield pyridoxic acid anion in the basic solution. This result strongly supported an electrooxidation of pyridoxol or pyridoxal involving formation of lactone as an intermediate step, seeming to play an essential role at the electrode surface, which was consistent with the findings described previously. The adsorption behavior of pyridoxol was investigated by means of SPAIRS and further studied with SNIFTIRS method. The appearance of the bipolar bands is at −0.5 V which is below the oxidation potential 0 V, indicating that the
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change of dipole moments is due to the molecule N-bonded to the gold surface at potential of around −0.5 V, and subsequently oxidation occurred at 0 V. In situ infrared reflectance absorption spectroelectrochemical method that provided more direct evidence for the oxidation mechanism of pyridoxol was highly recommended for electrochemical studies of pyridoxol and other related vitamin B6 groups and polysubstituted pyridine derivatives. Acknowledgements This work was supported by the National Natural Science Foundation of China (20275010, 20335020) and Hunan Province (02JJY4054), the Basic Research Special Program of the Ministry of Science and Technology of China (2003CCC00700), and the Foundation of the Ministry of Education (MOE) of China (jiaorensi (2000) 26, jiaojisi (2000) 65). References [1] J.M. Sanchez-Ruiz, M. Martinez-Carrion, Biochemistry 25 (1986) 2915. [2] R.A. John, Biochim. Biophys. Acta 1248 (1995) 81. [3] S. Cinta, C. Morari, E. Vogel, D. Maniu, M. Aluas, T. Iliescu, O. Cozar, W. Kiefer, Vib. Spectrosc. 19 (1999) 329. [4] J. Soderhjelm, J. Lindquist, Analyst 100 (1975) 349. [5] E. Jacobsen, T.M. Tommelstad, Anal. Chim. Acta 162 (1984) 379. [6] Y.F. Yik, H.K. Lee, S.F.Y. Li, S.B. Khoo, J. Chromotogr. A 585 (1991) 139. [7] Q. Hu, T.S. Zhou, L. Zhang, H. Li, Y.Z. Fang, Anal. Chim. Acta 437 (2001) 123. [8] S.R. Hern´andez, G.G. Ribero, H.C. Goicoechea, Talanta 61 (2003) 743. [9] M.F.S. Teixeira, A. Segnini, F.C. Moraes, L.H. Marcolino-J´unior, O. Fatibello-Filho, E.T.G. Cavalheiro, J. Braz. Chem. Soc. 14 (2003) 316. ´ [10] M.F.S. Teixeira, G. Marino, E.R. Dockal, E.T.G. Cavalheiro, Anal. Chim. Acta 508 (2004) 79. [11] J.D. Mahuren, T.A. Pauly, S.P. Coburn, J Nutr. Biochem. 2 (1991) 449. [12] T. Pineda, M.I. Lopez-Cozar, J.M. Sevilla, M. Blazquez, J. Electroanal. Chem. 403 (1996) 101. [13] S.M. Zhu, X.Z. Hu, J.M. Zeng, Z.Y. Guo, Acta Pharm. Sinica 31 (1996) 455. [14] T. Pineda, J.M. Sevilla, A.J. Rom´an, M. Bl´azquez, J. Electroanal. Chem. 492 (2000) 38.
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In situ FT-IR spectroelectrochemical study of electrooxidation of pyridoxol on a gold electrode Mei Ling Wang, You Yu Zhang ∗ , Qing Ji Xie, Shou Zhuo Yao Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China Received 14 May 2005; received in revised form 30 May 2005; accepted 30 May 2005 Available online 5 July 2005
Abstract The electrochemical oxidation of pyridoxol (PN) on a polycrystalline gold electrode was investigated by cyclic voltammetry and in situ Fourier transform infrared spectroscopy (FTIRS). In 0.1 M aqueous NaOH solution, the gold electrode showed a high catalytic activity for the irreversible oxidation process of PN. The individual ionic species and the major tautomeric equilibria of PN molecules in aqueous solutions were evidenced well from the pH-dependent attenuated total reflectance (ATR) spectra, and the results were in good agreement with the voltammetric observations. In situ single potential alteration infrared reflectance spectroscopy (SPAIRS) demonstrated that a lactone form of PN, rather than pyridoxal aldehyde, was likely formed, which was subsequently diffused into the thin layer solution and underwent hydrolysis slowly to pyridoxic acid (PA) as the final product. In addition, the adsorption of PN at Au electrode was characterized by in situ subtractively normalized interfacial Fourier transform infrared reflectance spectroscopy (SNIFTIRS) method, which revealed that the adsorption of deprotonated PN, via nitrogen atom in vertical configuration on electrode surface, occurred from −0.5 V versus Ag|AgCl|KCl(sat), which was much lower than the potential of PN electrooxidation observed from ca. 0 V. © 2005 Elsevier Ltd. All rights reserved. Keywords: In situ FT-IR; Pyridoxol; Vitamin B6; Electrochemical oxidation; Au electrode
1. Introduction The vitamins of B6 group (VB6) that contain a pyridine ring in structure, including pyridoxol (PN), pyridoxal (PL), pyridoxamine (PM) and their phosphate derivatives, are important natural compounds essential in diet needed as the co-factor in some VB6-dependent enzymes involved in the metabolism and synthesis of amino acids and other related compounds for the maintenance of body cells due to their vital roles in various biological processes [1–3]. Since the first work on voltammetric determination of VB6 using a carbon paste electrode [4], the electrochemical detection and polarographic analysis have been widely used because of the sensitive linear voltammetric response for vitamin B6 [5–8], but only very few investigations have been reported ∗
Corresponding author. Fax: +86 731 8865515. E-mail address: [email protected] (Y.Y. Zhang).
0013-4686/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2005.05.046
on the nature of the electrochemical reaction of VB6 [9–10]. Because PL is an oxidative form of PN and pyridoxic acid (PA) is the final product of VB6 metabolism in mammalian tissue (Scheme 1) [11], understanding the electrochemical kinetics and mechanism of VB6 may be helpful for the study of its catabolism process in vivo [12]. Recently, with the wide applications of simultaneous techniques of spectrometric/electrochemical measurement as powerful and informative tools for investigation and further elucidation of the electrochemical reaction mechanism, the electrochemical behaviors and the reaction pathways of PN and other VB6 derivatives on a solid electrode in aqueous medium have received considerable attention [12–16]. A study on the electrooxidation mechanisms of VB6 in different pH phosphate buffers at a pyrolytic graphite electrode was carried out by Zhu et al. using ultra-violet spectroelectrochemistry (UV-SEC) method [13]. Pineda et al. monitored the course of the electroreduction of PA in neutral buffer
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Scheme 1. Chemical structures of pyridoxol (PN), pyridoxal (PL) and pyridoxic acid (PA).
[12] and electrooxidation of PL in alkaline solution [14] on a polycrystalline gold electrode by cyclic voltammetry and UV–vis spectroscopy. Using a fluorescence spectroelectrochemical measurement, Tan et al. reported the electrochemical redox processes of pyridoxine hydrochloride at a glass carbon electrode modified with poly(methylene blue) film in alkaline phosphate buffer [15]. Cao et al. coupled the simultaneous EQCM and fluorescence to examine the adsorption/desorption and oxidation of PN on gold electrode in alkaline medium [16]. However, the intermediate/product of the oxidation of VB6 was not so well identified, and the mechanism for the electrochemical interaction at the electrode interface was not quite clear. Currently, vibrational studies of VB6 [1,3,17,18] have shown Fourier transform infrared spectroscopy (FTIRS) a powerful tool in characterizing chemical structure with reduced interferences from solvent bands using attenuated total internal reflectance (ATR) technique. In situ FT-IR/ATR has become one of the investigative techniques of choice currently available to explore solid/liquid interfacial phenomena in situ, to obtain information on the nature of adsorbed species readily, and to identify the intermediate, adsorbed species and products of electrode reaction, and it has been widely used to study electrocatalytic oxidation mechanism of small organic molecules at noble metal electrode [19–23]. To our knowledge, no in situ IR-spectroscopic investigations have been directed toward the electrochemical properties of VB6 yet, despite the significant technological interests in the elucidation of the reaction mechanism for the electrochemical interactions. In this respect, the electrocatalytic oxidation of PN on gold electrode in alkaline medium was investigated by cyclic voltammetry and in situ FT-IR spectroscopy, and the aim of the present study is to reveal the oxidation mechanism of PN at the molecular level.
2. Experimental The cyclic and rotating disk electrode voltammetries were carried out with a CHI660A electrochemical workstation (CH Instruments Co., USA) controlled by CHI660A software and in a conventional three-electrode electrochemical cell. The working electrode was a polycrystalline gold electrode of 6 mm in diameter. The counter electrode was a platinum sheet electrode. An Ag|AgCl|KCl(sat) electrode served as the reference electrode. For rotating disk electrode voltammetry, the
surface of the working Au disk electrode was polished carefully with emery paper and then sonicated in double-distilled water prior to each potential sweep experiment. Nitrogen atmosphere was maintained over the solution during the measurements. In situ FT-IR spectroscopic measurements were performed using a Nicolet Nexus 670 spectrometer (Nicolet Instrument Co., Madison, WI) equipped with a liquid nitrogen cooled MCT detector. The spectroelectrochemical cell was made of Teflon and provided with a prismatic CaF2 window bellow at 60◦ . A detailed description can be found in previous papers [22,24]. The working electrode was a gold disk with a diameter of 8 mm. Prior to each experiment, it was mechanically polished using alumina powder of 5, 1, 0.3, and 0.05 m then rinsed with double-distilled water. A platinum wire was used as the counter electrode, and the reference electrode was an Ag|AgCl|KCl(sat) electrode. Different IR spectra were obtained using the single potential alteration infrared reflectance spectroscopy (SPAIRS) technique [19,25]. The reference potential (ER ) was chosen at −0.8 V, while the sample potential (ES ) stepped positively in a multiple-step manner. Normalized reflectance spectra were calculated as R/R = [R(ES ) − R(ER )]/R(ER ), where R(ES ) and R(ER ) are the single beam spectra obtained at sample potential ES and the reference potential ER , respectively. Each spectrum resulted from co-addition of 50 interferograms with a resolution of 8 cm−1 . The subtractively normalized interfacial Fourier transform infrared reflectance spectroscopy (SNIFTIRS) technique [26,27] was used to obtain potential difference IR spectra. The reference potential was maintained at −0.8 V, and the reflectivities were obtained using a multiple potential step (MPS) procedure in which the electrode potential was stepped m times between the reference and sample potentials, ER and ES , respectively. During each step, n interferograms with resolution 4 cm−1 were acquired at both potentials. This procedure was repeated to give the total number (N = m × n) interfreograms acquired for each of the two potentials. The values of n and m employed in this study were 100 and 20, respectively. Final spectra were normalized as R/R = [R(ES ) − R(ER )]/R(ER ). In all experiments, each sample was placed on the sampling device and aligned according to the manufacturer’s recommendation; Ominic E. S. P. software of version 6.0a (Nicolet Instrument Co.) was used for processing the spectral data. An attenuated total reflectance (ATR) sampling accessory (Specac Inc., Technology Court, Smyrna, Georgia, USA)
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was employed for ATR-FT-IR experiments. The ATR spectra were obtained for PN or PA in aqueous solutions of different pH values using ZnSe internal reflectance crystal. For each spectrum, 64 interferograms with a resolution 4 cm−1 were co-added. The background spectra were recorded by adding double-distilled water at the same pH value. The net PN spectrum in solution was calculated by subtracted the background spectrum from the solution spectrum. Accurate subtraction was obtained by varying the subtraction factor in an interactive manner until the broad water adsorption around 2100 cm−1 was totally removed to baseline levels. Pyridoxol hydrochloride, pyridoxal hyrochloride and pyridoxic acid were purchased from Sigma and used without further purification. All other chemicals and reagents were of analytical grade. Aqueous solution of PN, PL or PA was prepared daily by dissolving the corresponding solid compound in double-distilled water. The pH values were adjusted by an addition of concentrated NaOH aqueous solution. The solutions were deaerated by bubbling nitrogen gas of high-purity (99.99%) before each IR measurement. All the experiments were carried out at room temperature (25 ± 1 ◦ C).
3. Results and discussion 3.1. Electrochemical studies The typical cyclic voltammograms in the absence and presence of PN for gold electrode in 0.1 M NaOH aqueous solution are shown in Fig. 1(a). In the alkaline supporting electrolyte alone, the curve displays a weak anodic peak with the potential above 0.15 V and a broad peak at 0.2 V, and has a reduction current peak appearing near 0.15 V on its negative side. According to the previous studies [16,23,28], the anodic peaks are associated with a surface oxidation process, i.e., OH− chemisorption on Au surface to form short-lived AuOH species that is subsequently converted to surface oxide, and
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the cathodic peak corresponds to the reduction of the gold oxide formed in the anodic scan. In the presence of PN, the gold shows a pronounced catalytic activity for this aromatic alcohol, and an oxidation reaction begins at approximately −0.2 V with a new anodic peak (pa ) for oxidation of PN at ca. 0.1 V, but no corresponding reductive peak is found during the subsequent cathodic scan. It is interesting to note that during the potential negative sweep, as soon as the surface oxides are reduced, another anodic peak is found at ca. 0.1 V, apparently due to again oxidation of PN on the newly generated electrode surface. This behavior is similar to the electrooxidation of aliphatic alcohol, sugar or pyridoxal in alkaline medium [14,25,29,30]. Fig. 1(b) shows the typical cyclic voltammogram of Au at a scan rate of 2 mV s−1 recorded in spectroelectrochemical cell during the in situ FT-IR acquisition. The anodic peak (pa ) is shifted to more positive potential compared with the voltammogram in Fig. 1(a), which may result from different concentration of PN. With the increase of PN concentration, the oxidation peak potential moves positively [16]. In addition, the pH change and the resistance to transferring reaction species between the thin layer and the bulk solution during the oxidation process of PN in situ IR measurements may also contribute to the shift [31,32]. In the case of the voltammograms shown in Fig. 1, the electrooxidation of PN on polycrystalline gold seems to be irreversible, which is consistent with the results observed by Cao et al. [16]. 3.1.1. Effect of PN concentration To investigate the effect of PN concentration on oxidation current, a series of experiments was performed in 0.1 M NaOH solutions containing different concentrations of PN. The results demonstrate that the anodic peak current linearly increases with the increasing of PN concentration, which varies from 0.5 to 10 mM. At constant potential scan rate of 50 mV s−1 , plots of the anodic peak current density (jpa ) versus PN concentration (c) give a straight line with a slope
Fig. 1. (a) Cyclic voltammograms of a polycrystalline Au electrode in 0.1 M NaOH (· · ·) and in 1 mM pyridoxol + 0.1 M NaOH (—) at scan rate of 50 mV s−1 . (b) Cyclic voltammogram of a polycrystalline Au electrode in 10 mM pyridoxol + 0.1 M NaOH taken in spectroelectrochemical cell at scan rate of 2 mV s−1 .
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of 0.027 A cm−2 mol L−1 , i.e., 0.0956 A mol L−1 for plot of the anodic peak current (ipa ) versus PN concentration in this study, which is identical with the reported value of 0.0954 [16]. In the range of PN concentration investigated, the reaction order estimated from the linear section of plots for log jp versus log c was 0.835 ± 0.05. 3.1.2. Effect of the pH value From previous studies on the electrooxidation of several sugars and alcohols, gold is characterized by its poor catalytic activity of this electrode in acidic medium but conversely proves to be a more effective catalyst for oxidizing these compounds in the case of alkaline solutions [25,29]. In order to investigate the influence of pH on the electrooxidation behavior of PN, the voltammetric response of the polycrystalline gold electrode was studied over a wide pH range from 7 to 13.5 in a 0.1 M phosphate buffer containing 1 mM PN. As has been expected, PN is practically not oxidized on gold at low pH, and the current of the oxidation peak begins to increase with the concentration of OH− when the solution pH is adjusted above 8 (Fig. 2). Results on the electrooxidation of PL [14] at gold as well as the molecular aspects of PN ionization in aqueous solution [33] show that the pH-dependent oxidation phenomenon originates from two contributions: the coverage of Au(OH)ads species on the electrode surface and the formation of pyridoxol anion in solution. It is usually accepted that the existence of a submonolayer of adsorbed hydrous oxide species is necessary as the active intermediate for electrocatalytic oxidation of a certain organic compound on metallic electrode, and the formation of these surface hydroxyl groups is favored in gold in alkaline solutions [34]. In this experiment, the anodic peak current increases with the increasing of solution pH, suggesting that the higher catalytic activity is observed for Au electrode in solutions of higher pH values. Actually, the anodic peak current for 1 mM PN oxidized at the same pH value of 12.8 and sweep rate (50 mV s−1 ) in the 0.1 M phosphate buffer solu-
Fig. 2. The influence of pH on the anodic peak current (ipa ), for the oxidation of 1 mM pyridoxol in 0.1 M phosphate buffer adjusted with concentrated NaOH solution, on a polycrystalline Au electrode at 50 mV s−1 and 25 ◦ C.
tion (Fig. 2) is lower than that in the 0.1 M NaOH solution (Fig. 1). The difference may be related to the various types of phosphate anions of buffer composition as the adsorption species, which may either lower the mediator activity or interfere with the interaction of the catalytic electrode surface with PN molecules [35]. On the other hand, a UV–vis adsorption spectroscopy study by Metzler and Snell on ionization equilibria of pyridoxine under various pH conditions demonstrated that four interchangeable ionic forms would occur corresponding in acidic solution (pH < 5) to the cation, at neutrality (pH 6.8) to neutral form, chiefly the dipolar ion, and in basic solution (pH > 8) to the anion [33]. Scheme 2 denotes the equilibria and the referenced values of dissociation constants (pKa ) between these species in aqueous
Scheme 2. Dissociation equilibria for ionic species and tautomeric forms of pyridoxol in aqueous solution due to the protonazation states of phenolic group and ring nitrogen.
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solution. According to the previous electrooxidation studies on aldehydes and glucose at gold electrode in solutions of high pH [14,28,35,36], the negative-charged anion showed a high electroactivity. In strong alkaline solutions with pH > 9, the deprotonated PN exists as an electroactive anion, and the oxidation is prone to occur in this form. So increasing the pH value is favor of oxidation reaction. In this study, the electrooxidation of PN is carried out in 0.1 M NaOH solution with a pH value of 12.8. Similar to the behavior of pyridoxal anion, the peak current of PN changes with the pH of 7–14 as a sigmoid shaped dissociation curve, i.e., almost no peak occurs at low pH, but at higher pH the current increases with pH until a plateau reaches at pH around 13 [14]. In comparison with the situation of PL, the current for PN electrooxidation is observed coming into existence from pH 8 (Fig. 2), a value more coincident with the pKa2 value of around 9, at which the pyridoxol anion is dissociated from its dipolar form (Scheme 2). It is also interesting to note that a distinct difference should be drawn in the shape of the curve for oxidation peak current versus pH between pyridoxal hemiacetal and PN [14]. Unlike the behavior of PL, an increase of the peak current for hemiacetal oxidation is shown in the pH range of 7–10 with an inflection around pH 8.5, then followed by decrease at pH > 13. This is due to the existence of three species of PL including free aldehyde and two electroreactive forms of hemiacetal and its anion, and the ionic equilibria among these species in strong alkaline solution with pKa values of around 8 and 13, respectively. In the case of PN with one existent dissociated form mediated with regard to oxidation by the Au species at high pH, no equilibria would occur and the kinetics is unlikely to be controlled by the deprotonation reaction, but undoubtedly affected by the electrocatalytic activity of the Au electrode. Since the detailed mechanism for electrooxidation of PN may involve complicated multi-step or multi-electron reactions, the electrochemical behavior may be affected by the oxidation conditions, such as the property of the electrolyte solution, the applied potential parameters, and the nature of electrode material. It has been reported that the anion peak current of pyridoxine oxidation at a copper() hexacyanoferrate(Ш) modified carbon paste electrode shows a bell shaped variation with pH range of 4–8 [9], and the oxidation of PN at a pyrolytic graphite electrode can take place in all phosphate buffers of a wide pH range from 2 to 12 [13]. 3.1.3. Rotating disk electrode (RDE) voltammetry The electrooxidation process of PN on gold electrode was also evaluated using rotating disk electrode (RDE) voltammetry technique. The typical linear sweep voltammograms of an Au disk rotating at different rates in 1 mM pyridoxol + 0.1 M NaOH are shown in Fig. 3. The anodic peak for pyridoxol oxidation appears at 0.1 V for all rotation rates. The peak current (ip ) tends to decrease rather than increase with the increase of the rotating rate of the electrode, suggesting that the mass transport controlled process play no key role and that a cer-
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Fig. 3. Linear-sweep voltammograms of Au disk electrode in 1 mM pyridoxol + 0.1 M NaOH at different rotation rates. The rotation rates were in rpm: (1–9) 200, 400, 600, 800, 1200, 1600, 2000, 2400 and 3000, respectively. Scan rate: 20 mV s−1 . Insert: rotation rate-dependence of the peak current (ip ) of Au disk electrode.
tain hindering process such as adsorption might take place at the gold surface [37,38]. As shown in Fig. 3, the current at lower potentials, independent of PN oxidation, is not affected by electrode rotation. And the lower oxidation peak current at the higher rotating rate should be in relation to the possible PN oxidation mechanisms. This is in accordance with the result of IR spectroscopic studies thereinafter. 3.2. IR spectroscopic studies 3.2.1. ATR spectra of PN at different pH values As has been illustrated above, PN exists as different ionic species and with tautomeric forms, depending on pH, composition and polarity of solution [33]. To further investigate the pioneering findings of ultraviolet spectra, we turn to an attenuated total reflectance (ATR) technique to provide direct molecular level evidence of the individual ionic species in aqueous solution. Fig. 4 presents the ATR spectra of PN in aqueous solutions of various pH values (2.8, 5.0, 7.1, 9.1, 11.2 and 12.8) measured in the range of 900–3000 cm−1 . A concentration of 0.1 M PN is chosen in these spectroscopic studies and similar experiments carried out with 10 mM PN show the same bands but with weaker intensities. It should be noticed that substantial difference appears in the spectra at region of 1600–900 cm−1 of PN in the various solutions, indicating the change in the molecular structure resulting from the variation of the pH. At pH of 2.8, the spectrum shows manifold absorption bands around at 1547, 1464, 1440, 1426, 1386, 1290, 1225, 1090 and 1023 cm−1 . According to a vibrational study of pyridoxine by Cinta et al. [3], the sharp and intense band at
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(Scheme 2). At the same time a new sharp band arises at about 1402 cm−1 with a broad shoulder band, a similar phenomenon observed in previous IR results for pyridoxamine 5 phosphate [18], which is due to the deprotonation of the pyridine nitrogen. The broad shoulder band around at 1398 cm−1 may be associated with the dissolved carbonate anion in the strong alkaline medium [39]. A band at 996 cm−1 seems clearer at high pH, due to ring bending vibration. Similarly, no significant variation in other bands related to the ring structure and aliphatic substitute at 1294, 1234, 1101 and 1023 cm−1 is observed. Thus, the results are in very good agreement with those obtained by UV–vis and other vibrational spectroscopies under all the conditions [3,17,18,33], and the variation of ATR spectra with pH can reflect well the individual ionic species and the major tautomeric equilibria of PN in aqueous solutions.
Fig. 4. The pH influence on the ATR spectra of 0.1 M pyridoxol in aqueous solutions. The pH values are denoted at the corresponding spectra.
1547 cm−1 may be assigned to the in-plane pyridine ring stretching vibration and C N+ H stretching vibration of nitrogen-protonated pyridine ring, the broad bands at 1023 and 1090 cm−1 are due to the ring and C O stretching vibration in CH2 OH group, and the weak band at 1464 cm−1 may be related to C O H bending vibration for phenolic hydroxyl group [3,17]. In addition, the weak bands located at 1440, 1426 and 1386 cm−1 can be ascribed to CH2 scissors vibration, CH3 asymmetrical and symmetrical bending, and the two bands at about 1290 and 1225 cm−1 may correspond to the ring stretching modes [3]. When the pH value is increased to 5.0, the band intensity of 1464 cm−1 is greatly reduced and the band at 1547 cm−1 red shifts with an increase in the bands at 1440, 1426, 1386, 1290 and 1225 cm−1 , but no change is found for the bands at 1090 and 1023 cm−1 . This is due to deprotonation of phenolic hydroxyl group (pKa1 = 5) of the cation form of PN (Scheme 2). However, the fact that the peaks of 1464 and 1547 cm−1 are still present suggests the presence of the dipolar ion as well as the neutral forms in this case. A similar spectrum is observed in the solution of pH 7.1 but the band at 1464 cm−1 almost disappears (Fig. 4), indicating the existence of tautomeric equilibrium of the deprotonated pyridxol at pH of 5–7, and the dipolar ion is the predominant form in aqueous solution. With further increasing of the pH value from 9.1 to 12.3, the band at 1547 cm−1 strongly decreases due to the deprotonation of pyridine nitrogen (pKa2 = 9) in high pH solutions
3.2.2. In situ FT-IR studies on PN electrooxidation at Au in alkaline medium To investigate the electrochemical process and propose a plausible reaction mechanism for PN oxidation on a gold electrode, two in situ infrared reflectance absorption spectroelectrochemical techniques, SPAIRS and SNIFTIRS, have been used to identify the species of reaction intermediate and product adsorbed on the gold surface as well as near the surface in the thin layer solution. A reference potential (ER ) was set at −0.8 V, where no related oxidation is known to take place, and sample potential (ES ) was varied stepwise to positive direction. So in these spectra, positive- and negative-going adsorption bands indicate the consumption and formation of species at ES as compared to ER , respectively. 3.2.2.1. SPAIR spectra. Fig. 5 shows a series of in situ FTIR reflectance spectra obtained for Au electrode in 10 mM PN in 0.1 M NaOH solution (pH 12.8). The sample potential (ES ) was varied stepwise from −0.6 to 0.5 V. When ES ≥ 0 V, several bands would appear on the SPAIR spectra, including negative-going bands at 1739, 1569, 1460, 1423, 1286, 1198 and 1109 cm−1 , and positive-going bands at 1402 and 1296 cm−1 with two bipolar bands denoted as 1402/1423 and 1296/1286 cm−1 , respectively. For the better clarification of the bands observed, the SPAIR spectrum from Fig. 5 is compared with the ATR spectrum of neat PN in alkaline solution of pH 12.8 from Fig. 4, and the result is presented in Fig. 6 and summarized in Table 1. It is clear that all the positive-going bands appearing in the SPAIR spectrum correspond well to the negative-going absorption bands due to PN in the ATR spectrum. The bipolar bands at 1402/1423 cm−1 and 1296/ 1286 cm−1 , in accordance with the literature [3,18,27], are assigned to in-plane stretching mode and in-plane bending mode of the nitrogen pyridine ring, respectively, and their changes in transition dipole moments should be related to molecule adsorption. As a polysubstituted pyridine, PN may have at least three orientations at the surface due to three
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Fig. 5. SPAIR spectra of 10 mM pyridoxol in 0.1 M NaOH solution on a polycrystalline Au electrode at various potentials from −0.6 to 0.5 V (vs. Ag/AgCl). Sample potential indicated for each spectrum. Reference potential was set at −0.8 V.
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Fig. 6. Comparison of (a) ATR spectrum of neat pyridoxol recorded in transmission mode in alkaline solution of pH 12.8 (shown in Fig. 4 with (b) in situ SPAIR spectrum recorded using the sample potential ES = 0.2 V and the reference potential ER = −0.8 V (vs. Ag/AgCl) (shown in Fig. 5).
different adsorption sites of the molecule: the lone pair electrons on nitrogen in the pyridine ring, the -system of the aromatic ring and the nonbonding electrons from the alcoholic groups [40]. In a study on the adsorption of VB6 at silver surface at different pH values by Raman and surface
enhanced Raman spectroscopy (SERS) [3], Cinta et al. proposed a parallel or tilted orientation of the skeletal ring with the respect of the Ag particles, due to the presence of the bands assigned to the ring deformation mode and red shifted after adsorption of deprotonated VB6. However, neither red
Table 1 Comparisons of typical IR vibrational bands in the range of 1800–900 cm−1 for pyridoxol (PN), and pyridoxic acid (PA) in 0.1 M NaOH solution SPAIR spectrum of PN (on Au electrode at ES = 0.2 V)
ATR spectrum of PN Wavenumber – – 1547 – – 1402 1294 1234 – – 1101 1023 996
(cm−1 )
Description
↓ (w) ↓ (s) ↓ (m) ↓ (m) ↓ (w) ↓ (m) ↓ (w, sh)
Wavenumber 1739 1569 1545 1460 1423 1402 1296 1286 1234 1198 1109 1100 1023 –
(cm−1 )
ATR spectrum of PA
Descriptiona
Wavenumber (cm−1 )
↓ (s) ↓ (m) ↑ (w) ↓ (w) ↓ (s) ↑ (s) ↑ (m) ↓ (m) ↑ (w) ↓ (m) ↓ (s) ↑ (w) ↑ (m)
– 1589 – 1487 – 1404 – 1290 – 1219 – – – 1005
The arrows ↓ and ↑indicate negative and positive band, respectively. a Abbreviations for band intensity and shape: s, strong; m, medium; w, weak; sh, shoulder band.
Description ↓ (s) ↓ (w) ↓ (s, sh) ↓ (m) ↓ (m)
↓ (s, sh)
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shift nor band broadening occurs in the ring modes of PN in this work, suggesting that the interaction between the ring -system and Au surface is not important. Moreover, the characteristic of bipolar bands is similar to previous results for pyridine N-bonded to the polycrystalline gold electrode surface [27], and a similar phenomenon can be expected to occur in the case of pyridine derivatives. At high pH values, the participation of the nitrogen of vitamin PP ring to the interaction with silver surface was more probable [40]. So it may conclude that the deprotonated PN is adsorbed via nitrogen atom probably in vertical position on the Au surface. The frequency of the bipolar band of in-plane stretching mode plotted against the electrode potential. A small shift of the frequency is observed with a slope of 5 cm−1 V−1 with our spectral resolution. This is in agreement with the work ¨ and Yeager. They found a slope of 6 cm−1 V−1 for of kOtz pyridine adsorbed on silver [41]. No significant shifts for pyridine adsorbed on Au (210) and Au (1 1 1) were found by Brolo et al. and H˙ebert et al., respectively [42,43]. This small potential dependence of the frequency of the vibration mode is consistent with N-bonded adsorption behavior. A very intense negative-going band centered at 1739 cm−1 is attributed to the vibration bands of reaction products or reaction intermediates, which contain a carbonyl group, aldehyde or carboxyl acid. Previous reports have shown that PN oxidation can convert to PL or PA [13,16]. To check whether the observed band is due to PL, the FT-IR spectra of PL was recorded under the same condition (Fig. 7). It is interesting to note that similar bands are observed for both spectra of PN and PL, i.e., the negative-going bands around 1738, 1582, 1197 and 1109 cm−1 and bipolar bands at 1407/1426 cm−1 and 1333/1287 cm−1 . The result indicates that the presence of PL as the final product in the oxidation of PN is less possible, and that both PL and PN should have the same oxidative product and the similar oxidation mechanism in this condition. As shown in Fig. 7, the bands for PL appear at the sample potential of −0.2 V lower than that of PN. It is noticeable that no positive-going bands reflecting aldehyde group of PL are observed. This is due to the fact that PL exists as its hydrated forms with a very small percentage of the free aldehyde species under this pH condition [14]. On the other hand, Pineda et al. proposed a lactone derivative intermediate for PL electrooxidation at gold electrode in alkaline solution, and PA as a final product, which confirmed by mass spectrometry, UV–vis and fluorescence spectroscopies [14]. According to the literatures [30,44], the band at 1739 cm−1 can be ascribed to the C O stretch in pyridoxic lactone. The other strong bands, which can be assigned to the lactone, are clearly observed at 1198 and 1109 cm−1 in the in situ FT-IR spectra, due to the C O C asymmetric stretch and symmetric stretch of the lactone [30]. In Fig. 8, the peak intensities at the wavenumbers of these bands are plotted versus the sample electrode potentials. At ca. 0.3 V, the intensity of band at 1739 cm−1 reached the maximum then decreased gradually. This phenomenon is in accordance with the result of cyclic voltammogram. Thus, the present IR result demon-
Fig. 7. SPAIR spectra of 10 mM pyridoxal in 0.1 M NaOH solution on a polycrystalline Au electrode at various potentials from −0.6 to 0.5 V (vs. Ag/AgCl). Sample potential indicated for each spectrum. Reference potential was −0.8 V.
Fig. 8. Relationship between the peak intensities of wavenumbers at 1739 cm−1 (), 1569 cm−1 (), 1402 cm−1 (䊉), 1296 cm−1 (), 1109 cm−1 () vs. the electrode potential during the oxidation of pyridoxol (shown in Fig. 5).
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strates lactone as an intermediate in the oxidation process of PN. In addition, two negative-going bands at 1569 and 1460 cm−1 appear in the spectrum at 0.1 V and increase in intensity as potential increases. The bands of 1569 and 1460 cm−1 may be associated with the asymmetric stretching vibration of a carboxylate group [44] and the CH2 scissors vibration, respectively, which were also found in ATR spectrum of PA in alkaline solution (Table 1). It should be mentioned that the carboxylate group also has a band at 1393 cm−1 , due to the symmetric stretching, may be swamped by the positive band at 1402 cm−1 . This indicates that the intermediate lactone formed at the electrode surface was desorbed and underwent hydrolysis to yield pyridoxic anion in the basic solution. It is noted that, however, the intensity ratio of peak at 1569 cm−1 is lower in comparison with the peak at 1739 cm−1 yet (Fig. 5), suggesting that the lactone accumulated at electrode surface is advantageous and the degree of hydrolysis is small, because of the N-bonded adsorption and pH decrease in the thin layer solution due to gradual consumption of OH− anions [45]. 3.2.2.2. SNIFTIR spectra. In order to further investigation of the adsorption behavior of PN on the electrode surface, we performed the SNIFTIRS measurements. Since the oxidation of PN is irreversible, the utilization of the SNIFTIRS over the potential range below the oxidation potential. The reference potential was maintained at −0.8 V, and the reflectivities were obtained using a multiple potential step (MPS) procedure. In
Fig. 9. SNIFTIR spectra recorded for 10 mM pyridoxol in 0.1 M NaOH solution on a polycrystalline Au electrode at various sample potentials (indicated on the corresponding spectrum) with a base potential set at −0.8 V.
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Fig. 9, the spectra mainly display the O H bending band of water and the two bipolar bands as mentioned in SPAIR spectrum. The bipolar bands begin to occur at −0.5 V, which is below the oxidation potential 0 V, indicating that the change of dipole moments is due to the PN adsorbed at the metal surface and the adsorption potential is around −0.5 V. The intensity of the bands is not remarkable probably due to weak adsorption at low potentials, but they can be discerned in these spectra. According the IR selection rules, the spectrum is sensitive to component of the vibration dipole perpendicular to the surface only [27]. So a vertical configuration of the adsorption behavior of PN is more possible.
4. Conclusion The application of simultaneous cyclic voltammetry and in situ Fourier transform infrared spectroscopy (FTIRS) measurement was first investigated for the electrochemical characterization and electrooxidation monitoring of pyridoxol (PN) on a polycrystalline gold electrode. Two in situ infrared reflectance absorption spectroelectrochemical techniques, SPAIRS and SNIFTIRS, were used to monitor the adsorption behavior of reactant and identify the species of intermediate or product generated on the gold surface as well as near the surface in the thin layer solution during PN oxidation in alkaline medium. In 0.1 M NaOH aqueous solution, gold showed a high catalytic activity for the irreversible oxidation of pyridoxol to pyridoxic acid as the final product. Meanwhile, new negative-going bands at 1739, 1569, 1460 and 1109 cm−1 , and new bipolar bands around 1402/1423 cm−1 and 1297/1284 cm−1 appeared under the positive direction of potential of above 0 V. These positivegoing bands appearing in the SPAIR spectrum correspond well to the negative-going absorption bands in the ATR spectrum of PN. The individual ionic species and the major tautomeric equilibria of PN molecules in aqueous solutions were evidenced well from the pH dependence ATR spectra, and the results were in good agreement with the voltammetric observation and those previously obtained by UV–vis and other vibrational spectroscopies. From the negative-going and bipolar bands of pyridoxol and pyridoxal, it was suggested that the two kinds of substances have the similar electrochemical behaviors on gold electrode in alkaline medium. It implied that the intermediate pyridoxic lactone instead of pyridoxal aldehyde likely formed at the electrode surface was desorbed and only a few underwent hydrolysis to yield pyridoxic acid anion in the basic solution. This result strongly supported an electrooxidation of pyridoxol or pyridoxal involving formation of lactone as an intermediate step, seeming to play an essential role at the electrode surface, which was consistent with the findings described previously. The adsorption behavior of pyridoxol was investigated by means of SPAIRS and further studied with SNIFTIRS method. The appearance of the bipolar bands is at −0.5 V which is below the oxidation potential 0 V, indicating that the
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change of dipole moments is due to the molecule N-bonded to the gold surface at potential of around −0.5 V, and subsequently oxidation occurred at 0 V. In situ infrared reflectance absorption spectroelectrochemical method that provided more direct evidence for the oxidation mechanism of pyridoxol was highly recommended for electrochemical studies of pyridoxol and other related vitamin B6 groups and polysubstituted pyridine derivatives. Acknowledgements This work was supported by the National Natural Science Foundation of China (20275010, 20335020) and Hunan Province (02JJY4054), the Basic Research Special Program of the Ministry of Science and Technology of China (2003CCC00700), and the Foundation of the Ministry of Education (MOE) of China (jiaorensi (2000) 26, jiaojisi (2000) 65). References [1] J.M. Sanchez-Ruiz, M. Martinez-Carrion, Biochemistry 25 (1986) 2915. [2] R.A. John, Biochim. Biophys. Acta 1248 (1995) 81. [3] S. Cinta, C. Morari, E. Vogel, D. Maniu, M. Aluas, T. Iliescu, O. Cozar, W. Kiefer, Vib. Spectrosc. 19 (1999) 329. [4] J. Soderhjelm, J. Lindquist, Analyst 100 (1975) 349. [5] E. Jacobsen, T.M. Tommelstad, Anal. Chim. Acta 162 (1984) 379. [6] Y.F. Yik, H.K. Lee, S.F.Y. Li, S.B. Khoo, J. Chromotogr. A 585 (1991) 139. [7] Q. Hu, T.S. Zhou, L. Zhang, H. Li, Y.Z. Fang, Anal. Chim. Acta 437 (2001) 123. [8] S.R. Hern´andez, G.G. Ribero, H.C. Goicoechea, Talanta 61 (2003) 743. [9] M.F.S. Teixeira, A. Segnini, F.C. Moraes, L.H. Marcolino-J´unior, O. Fatibello-Filho, E.T.G. Cavalheiro, J. Braz. Chem. Soc. 14 (2003) 316. ´ [10] M.F.S. Teixeira, G. Marino, E.R. Dockal, E.T.G. Cavalheiro, Anal. Chim. Acta 508 (2004) 79. [11] J.D. Mahuren, T.A. Pauly, S.P. Coburn, J Nutr. Biochem. 2 (1991) 449. [12] T. Pineda, M.I. Lopez-Cozar, J.M. Sevilla, M. Blazquez, J. Electroanal. Chem. 403 (1996) 101. [13] S.M. Zhu, X.Z. Hu, J.M. Zeng, Z.Y. Guo, Acta Pharm. Sinica 31 (1996) 455. [14] T. Pineda, J.M. Sevilla, A.J. Rom´an, M. Bl´azquez, J. Electroanal. Chem. 492 (2000) 38.
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