Electrochemical and Spectroelectrochemical Studies on Pyridoxine Hydrochloride Using a Poly(methylene blue) Modified Electrode

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1 1592 _ Full Paper Electrochemical and Spectroelectrochemical Studies on Pyridoxine Hydrochloride Using a Poly(methylene blue) Modified Electrode Liang Tan, Qingji Xie,* Shouzhuo Yao* Chemical Research Institute, Hunan Normal University, Changsha , P. R. China * tlg0686@163.com; xieqj@hunnu.edu.cn; szyao@hunnu.edu.cn Received:July 29, 2003 Final version:november 25, 2003 Abstract The electrochemical redox processes of pyridoxine hydrochloride (VB 6 ) at a poly(methylene blue) film modified glass carbon electrode (PMBE) in a phosphate buffer solution (PBS, ph 8.0) were studied by cyclic voltammetry. The VB 6 electrode reaction with quasi-reversible characteristics was diffusion-controlled at low scan rates and adsorptioncontrolled at high scan rates. The anodic peak current positive to 0.6 V (vs. SCE) was found to be proportional to the concentration of VB 6 in the range of to 1.03 mg ml 1 with a detection limit of 1.34 mg ml 1. Fluorescence and UV-vis absorption spectroelectrochemical measurements suggest that the pyridine ring was not destroyed over the potential range from 0.8 to 1 V (vs. SCE), and the electrocatalytic generation of pyridoxal was anodically started at 0.57 V. Keywords: Pyridoxine hydrochloride, Spectroelectrochemistry, Poly(methylene blue), Chemically modified electrode 1. Introduction Spectroelectrochemistry is one of the most active fields in electrochemistry and electroanalytical chemistry of late years. The electrochemical and spectroscopic signals can be acquired simultaneously in one spectroelectrochemical experiment. The spectroscopic techniques involved in spectroelectrochemistry included UV-vis absorption spectra [1], fluorescence spectra [2], infrared spectra [3], Raman spectra [4], etc., and spectroelectrochemistry has been widely used for the study of inorganic, organic and biological systems [5 6]. Pyridoxine hydrochloride (i.e., vitamin B 6 ) plays an important role in the synthetic and decomposing processes of amino acids. It may be converted to pyridoxal phosphate, which acts as a coenzyme in many metabolism processes. In the absence of VB 6, human bodies may suffer from nerve excited, nervous and/or gloomy symptoms, and even nerval pathological changes and twitch appear. In addition, vitamin B 6 is generally used to treat puke and neuritis. Many methods for studies of pyridoxine hydrochloride have been carried out, for example, differential pulse polarography [7], voltammetry [8], flow injection [9], spectrophotometry [10], high-performance liquid chromatography [11], fluorescence [12], capillary electrophoresis [13], etc. Recently, Yi et al. reported on the electrocatalytic behavior of poly(methyl red) film modified glass carbon electrode (PMRE) to pyridoxine hydrochloride [14]. Until now there have been no reports on the study of pyridoxine hydrochloride electrochemistry with spectroelectrochemical methods. In this paper, the electrochemical behavior of pyridoxine hydrochloride was studied by cyclic voltammetry at a poly(methylene blue) film modified glass carbon electrode (PMBE) in phosphate buffer solution (ph 8.0). We used a 1- cm quartz cell, where a reflexible PMBE was set, to investigate fluorescence spectroelectrochemical characteristics of vitamin B 6. UV-vis absorption spectroelectrochemical measurements were also carried out. 2. Experimental 2.1. Chemicals and Solutions Pyridoxine hydrochloride (biochemical grade) was purchased from Guangshen Chemical Company (Shanghai). Other chemicals were of analytical reagent grade, and all aqueous solutions were prepared in distilled water, which was produced with a double-distillation quartz set. A pyridoxine hydrochloride stock solution (10.28 mg ml 1 ) was prepared by dissolving g of pyridoxine hydrochloride into 100 ml of water and conserved in dark. This solution was further diluted as required. A ph 8.0 buffer solution was prepared by mixing 5.30 ml of 0.2 mol L 1 NaH 2 PO 4 with ml of 0.2 mol L 1 Na 2 HPO Apparatus Cyclic voltammetry and potential-step experiments were performed with an EG&G PAR 283 Potentiostat/Galvanostat. Fluorescence spectra were collected in situ with a Hitachi F-4500 fluorescence spectrophotometer. A TU PUXI General UV-vis spectrophotometer (Beijing, China) was used to collect UV-vis spectra. DOI: /elan

2 Studies on Pyridoxine Hydrochloride Using a Poly(methylene blue) Modified Electrode 1593 Fig. 1. Fluorescence spectroelectrochemical cell:1) 1 cm quartz cell; 2) polyethylene supporting plate; 3) poly(methylene blue) film modified glass carbon working electrode (PMBE); 4) Pt counter electrode; 5) SCE reference electrode. Fig. 2. Consecutive voltammetric curves during the electrochemical growth of a poly(methylene blue) film at a bare glass carbon electrode. Scan rate:100 mv s 1. Solution:0.1 mol L 1 PBS (ph 8.0) þ mol L 1 methylene blue þ 0.1 mol L 1 Na 2 SO 4. A 1-cm quartz cell, which contained the reflex mode electrodes, was used for fluorescence spectroelectrochemical measurements, as shown in Figure 1. A poly(methylene blue) film modified glass carbon working electrode (PMBE) and a Pt counter electrode were fixed on the two opposite sides of a polyethylene supporting plate. The supporting plate was inserted vertically into the quartz cell along the diagonal of the cross section. The incident beam was conducted to the PMBE surface at an angle of 458, and the reflected beam from the electrode surface was collected and guided to the optical detector of the fluorescence spectrophotometer. The KCl-staturated calomel electrode (SCE) served as the reference electrode, and all potentials were reported relative to it Procedure Preparation of the PMBE working electrode After being polished by coated abrasive, the bare glass carbon electrode was ultrasonically treated in 2 mol L 1 NaOH solution, 1:1 HNO 3 solution, absolute ethyl alcohol and distilled water in turn (ca. 3 minutes for each). The bare glass carbon electrode was then subjected to a 20-cycle cyclic voltammetric treatment, between 0.8 V and 1.2 V and at 100 mv s 1, in an aqueous solution containing mol L 1 methylene blue (MB), 0.1 mol L 1 sodium phosphate buffer (ph 8.0) and 0.1 mol L 1 Na 2 SO 4, allowing the deposition of poly(methylene blue) film on it. The poly- (methylene blue) film modified electrode was aged in electrolyte for 24 hours prior to use Electrochemical and Spectroelectrochemical Measurements The tested solutions were transferred into the quartz cell equipped with the devised electrodes, as shown in Figure 1. Electrochemical and fluorescence responses were monitored simultaneously during experiments. For fluorescence spectral measurements, a maximal excitation wavelength of 340 nm was used. After the tested solutions were electrolyzed at a steady potential for a specific time, the fluorescence or UV-vis absorption spectrum was collected in situ. 3. Results and Discussion 3.1. Growth of Poly(methylene blue) Film on a Glass Carbon Electrode The cyclic voltammogram obtained during the electrochemical growth of poly(methylene blue) film on a glass carbon electrode substrate are shown in Figure 2. The voltammetric profile is similar to that in reference [15]. The primal signals at 0.2 V (vs. SCE) decreased and the height of peaks at 0.2 V (vs. SCE) increased with successive cycles, suggesting the continuous growth of the poly(methylene blue) film on the working electrode Electrochemical Measurements for Pyridoxine Hydrochloride In order to evaluate the electrochemical catalytic properties of the PMBE, cyclic voltammetric analyses were conducted at the bare glass carbon electrode and PMBE in a PBS (ph 8.0) containing mg ml 1 pyridoxine hydrochloride. The representative cyclic voltammogram during the third

3 1594 L. Tan et al. Fig. 3. Cyclic voltammograms of mg ml 1 VB 6 in 0.1 mol L 1 PBS (ph 8.0) at PMBE (solid line) and the bare glass carbon electrode (broken line). Scan rate:100 mv s 1. Fig. 4. The peak current of anodic peak 3 versus the square root of scan rate. Inset b:versus the scan rate. The test solution: 0.41 mg ml 1 VB 6 in 0.1 mol L 1 PBS (ph 8.0). scan cycle (at steady state) is shown in Figure 3. Obviously, the bare glass carbon electrode is hardly responsive for VB 6, but redox signals of VB 6 are visible at the PMBE. Besides a couple of redox peaks, at about 0 and 0.4 V (vs. SCE), due to the poly(methylene blue) film itself, three anodic current peaks (peaks 1, 2 and 3) and a cathodic current peak (peak 4) are seen at about 0.12, 0.44, 0.68 and 0.60 V for the a curve, respectively, suggesting the facile occurrence of VB 6 oxidation at the PMBE and a complicated reaction mechanism. To make certain the correlation among current peaks, CV experiments reversing the potential scan after 0.30 V and 0.57 V were performed. The results are shown as b and c in Figure 3, respectively. We can not find the existence of peaks 1 and 2 in b and c. It indicates that peaks 1 and 2 are related to peaks 3 and 4. It may be deduced that 1 and 2 are the oxidation peaks of the reduction product represented by peak 4. Because the potential was not over than 0.57 V, the onset of the peak 3, pyridoxine hydrochloride could not be oxidized and further reduced. As a result, peaks 1 and 2 could not arise. The dependence of the peak current of anodic peak 3 on scan rate is shown in Figure 4. At low scan rates (v 100 mv s 1 ), the peak height (I) is linear with the square root of scan rate (v), being the characteristic of diffusion-controlled electrochemical reaction [16]. However, a linear relationship between the peak height (I) and the potential scan rates (v) was obtained at scan rates over 100 mv s 1, demonstrating that the electrode reaction was an adsorption-dominated process at high scan rates [16]. After measuring the pyridoxine hydrochloride PBS, the PMBE was subjected to CV treatment in the reagent blank solution (PBS ph 8.0). The peak current remained although the signal was relatively reduced. It further proved the adsorption action of VB 6 in the PMBE. It is obvious that the control over the electrode reaction is a transition from diffusion to adsorption with the increase of scan rates. Since the anodic peak 3 shifted positively but the cathodic peak 4 negatively with the increase of the scan rate, and the heights of peaks 3 and 4 were not equal, the electrode reaction of VB 6 should be a quasi-reversible process. Cyclic voltammetric experiments were performed in the pyridoxine hydrochloride PBS (ph 8.0) at different concentrations, and the results are shown in Figure 5a. Both anodic peaks 2 and 3 were heightened with the increase of VB 6 concentration, and the latter was more conspicuous. In addition, the anodic peak 3 shifted positively at a higher concentration of VB 6. Figure 5b shows that the anodic peak 3 current was linearly proportional to the VB 6 concentration from to 1.03 mg ml 1, with a detection limit of 1.34 mg ml 1, revealing the feasibility for the cyclic voltammetric assay of trace VB 6 at the PMBE Spectroelectrochemical Measurements for Pyridoxine Hydrochloride Figures 6a and b show the fluorescence spectra in situ of 0.41 mg ml 1 pyridoxine hydrochloride in PBS (ph 8.0) after being electrolyzed for 5 minutes at different potentials. From 0.8 to 1.4 V, the fluorescence peak can be observed at 395 nm. The fluorescence intensity at 395 nm increased continuously from 0.8 to 0.57 V, then decreased from 0.57 to 1.4 V, as shown in Figure 6c. The inflexion potential at 0.57 V, which is also the onset of the anodic peak 3 in Figure 3, is regarded as the initial potential for the electrocatalytic oxidation potential of VB 6, and the fluorescence intensity started its decrease at this potential when VB 6 oxidation was initiated. Simultaneously recorded current, fluorescence intensity at 395 nm during the potential cycling experiment in PBS

4 Studies on Pyridoxine Hydrochloride Using a Poly(methylene blue) Modified Electrode 1595 Fig. 5. a) Cyclic voltammograms of PMBE (broken line) and VB 6 (solid lines) at PMBE in 0.1 mol L 1 PBS (ph 8.0). Scan rate: 100 mv s 1.VB 6 concentration:0.010, 0.021, 0.082, 0.164, 0.411, and mg ml 1 from A to G. b) The peak current of anodic peak 3 as a function of the concentration of VB 6. Fig. 6. a) and b) fluorescence spectra of 0.41 mg ml 1 VB 6 in 0.1 mol L 1 PBS (ph 8.0) at various applied potentials: 0.80, 0.40, 0, 0.57, 0.70, 0.90, 1.00 and 1.40 V from A to H. c) Potential effects on the fluorescence intensity at 395 nm of 0.41 mg ml 1 VB 6 in 0.1 mol L 1 PBS (ph 8.0).

5 1596 L. Tan et al. Fig. 7. Cyclic voltammogram a) of mg ml 1 VB 6 in 0.1 mol L 1 PBS (ph 8.0) and simultaneously recorded fluorescence intensity at 395 nm b). Scan rate:100 mv s 1. Fig. 8. Absorption spectra a) and fluorescence spectra b) of mg ml 1 VB 6 in 0.1 mol L 1 PBS (ph 8.0) after 1.0 V (vs. SCE) electrolysis for 0 (A), 1.0 (B), 2.0 (C), 3.0 (D) and 4.0 (E) hours. (ph 8.0) containing mg ml 1 pyridoxine hydrochloride are shown in Figure 7. The anodic peak 3 was shortened continuously during the potential cycling, resulting most likely from the significant depletion of VB 6 during its oxidation but with a very low recovery of VB 6 in the cathodic process, rather than the immediate activity loss of the PMBE during the potential cycling. As a new experiment after the test solution was renewed the height of anodic peak 3 in the first cycle almost recovered here. The simultaneously recorded fluorescence intensity also decreased gradually. There are two inflexion points in Figure 7b, corresponding to 0.57 and 0.70 V, respectively, which are the starting potentials for the anodic peak 3 and the cathodic peak 4. Since the height of anodic peak 3 was much higher than the cathodic peak 4, the more facile oxidation of VB 6 induced a somewhat irreversible conversion of VB 6 to its oxidation products with a lower fluorecence activity cycle by cycle during the potential cycling, leading to the net decrease in the fluorescence intensity. Series steady-state electrolysis experiments at 1.0 V were conducted in PBS (ph 8.0) containing mg ml 1 pyridoxine hydrochloride, and then UV-vis absorption and fluorescence spectra were collected after the specified electrolysis time. The results are shown in Figure 8. It is seen that the absorbance at ca. 323 nm decreased and the one at ca. 251 nm increased with the increase of electrolysis time. The decrease of the fluorescence intensity at 395 nm with the increase of electrolysis time was also observed. The findings may be understood as follows. The absorption peak at ca. 220 nm originates from the p! p* transition of pyridine ring ( 1 La band), while the absorption peak at ca. 323 nm originates from the n! p* transition of pyridine ring connecting with the hydroxyl group ( 1 Lb band) [17, 18]. The absorbance decrease at ca. 323 nm proved that VB 6 is

6 Studies on Pyridoxine Hydrochloride Using a Poly(methylene blue) Modified Electrode 1597 oxidized at the hydroxyl group, rather than the pyridine ring itself, as the intrinsic absorbance for pyridine ring at ca. 220 nm remained unchanged during the VB 6 oxidation electrolysis. The absorption peak at ca. 251 nm results from the p! p* transition of aldehyde group in conjugation with the C¼C double bond [17, 18]. The increase of the absorbance at ca. 251 nm reveals the generation of aldehyde group during VB 6 oxidation. At an electrolysis potential above 0.57 V, pyridoxine was continuously oxidized to pyridoxal that possesses a lower fluorescence activity, so the fluorescence intensity was decreased continuously with electrolysis time, which also qualitatively coincides with the responses shown Figure 7b. 4. Conclusions Electrochemical studies on the redox processes of pyridoxine hydrochloride using PMBE as the working electrode revealed that the electrode reaction was a quasi-reversible process, being diffusion-controlled at low scan rates and adsorption-controlled process at high scan rates. The electrocatalytic current for VB 6 oxidation at the PMBE was found to be linear with the VB 6 concentration in PBS (ph 8.0). Fluorescence spectroelectrochemical studies revealed that the initial potential for the electrocatalytic generation of pyridoxal is at 0.57 V, and UV-vis absorption spectroelectrochemical studies revealed that pyridoxal was generated during the oxidation of VB 6 and the pyridine ring was not destroyed. 6. References [1] Ten-Chin Wen, C. Sivakumar, A. Gopalan, Spectrochim. Acta Part A 2002, 58, 167. [2] M. J. Simone, W. R. Heineman, G. P. Kreishman, Anal. Chem. 1982, 54, [3] S. P. Best, R. J. H. Clark, R. C. S. Mcqueen, S. Joss, J. Am. Chem. Soc. 1989, 111, 548. [4] E. S. Brandt, Anal. Chem. 1985, 57, [5] Y. W. Xie, S. J. Dong, Theories and Applications of Spectroelectrochemical Methods (Chinese), Jilin Science & Technology Press, Changchun [6] R. J. Gale, Spectroelectrochemistry: Theory and Practice, Plenum Press, New York [7] E. Jacobsen, T. M. Tommelstad, Anal. Chim. Acta 1984, 162, 379. [8] J. Soderhjelm, J. Lindquist, Analyst 1975, 100, 349. [9] R. T. Perez, L. C. Martinez, Analyst 1994, 119, [10] B. Morelli, Fresenius J. Anal. Chem. 1996, 354, 97. [11] J. Zempleni, W. Kubler, Clin. Chim. Acta 1994, 229, 27. [12] H. T. Gao, X. G. Chen, Chinese J. Anal. Chem. 1990, 18, 337. [13] S. Boonkerd, M. R. Detaevernier, Y. Michotte, J. Chromotogr. A 1994, 670, 209. [14] Q. S. Yi, Q. J. Wan, J. H. Yu, N. J. Yang, X. P. Zou, J. Chinese J. Anal. Sci. 2001, 17, 379. [15] C. M. A. Brett, G. Inzelt, V. Kertesz, Anal. Chim. Acta 1999, 385, 119. [16] Y. Liu, Y. X. Jiang, W. B. Song, N. Lu, M. Z. Zou, H. D. Xu, Z. X. Yu, Talanta 2000, 50, [17] Y. Z. Shi, X. Z. Sun, Y. H. Jiang, Spectral and Chemical Identification of Organic Compounds (Chinese), Jiangsu Science & Technology Press, Nanjing [18] H. T. Tang, Spectral Identification of Organic Compounds (Chinese), Beijing University Press, Beijing Acknowledgements Financial supports from the National Natural Science Foundation of China and the Foundations of the Ministry of Education of China are gratefully acknowledged.