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A pre-anodized inlaying ultrathin carbon paste electrode for simultaneous determination of uric acid and folic acid
Electrochimica Acta 89 (2013) 600–606
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A pre-anodized inlaying ultrathin carbon paste electrode for simultaneous determination of uric acid and folic acid Jing’e Huo, Enbo Shangguan, Quanmin Li ∗ College of Chemistry and Environmental Science, Henan Normal University, Henan Key Laboratory for Environmental Pollution Control, Xinxiang, Henan 453007, PR China
a r t i c l e
i n f o
Article history: Received 20 July 2012 Received in revised form 25 October 2012 Accepted 17 November 2012 Available online 27 November 2012 Keywords: Pre-anodized inlaying ultrathin carbon paste electrode Nichrome Uric acid Folic acid
a b s t r a c t A pre-anodized inlaying ultrathin carbon paste electrode (PAIUCPE) was prepared by electrochemical pretreatment. The scanning electron microscope (SEM) was applied to characterize the surface morphology of PAIUCPE and the performance of the electrode was characterized by cyclic voltammetry (CV). The results indicated that PAIUCPE displayed excellent electrocatalysis for the oxidation of uric acid (UA) and folic acid (FA). The separated extent between the two oxidation peaks of UA and FA was 324 mV, which was enough for the simultaneous detection. In 0.10 mol/L PBS (pH 6.00), the linear scan voltammetry (LSV) response of UA and FA increased linearly with the concentration in the range of 4.0 × 10−6 –3.5 × 10−4 mol/L and 3.0 × 10−6 –2.0 × 10−4 mol/L with the detection limits of 1.1 × 10−7 mol/L and 1.5 × 10−7 mol/L, respectively. It was successfully used to determine UA and FA in human urine simultaneously. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Uric acid (UA, 2,6,8-tridroxypurine) is the main end product of purine metabolism in the human body [1]. Excess urinary UA secretion may lead to urinary calculi or gout. Folic acid (FA) is a form of the water-soluble vitamin Bc widely found in some vegetable and food [2]. A lack of FA gives rise to gigantocytic anaemia, associated with leukopaenia. In recent years, various techniques have been employed for quantification of UA or FA, including fluorescence [3,4], spectrophotometry [5,6], high-performance liquid chromatography (HPLC) [7,8], flow injection [2,9], capillary electrophoresis [10,11], and electrochemical method [12–15]. Because UA and FA always coexists in the human body fluids, it has significance for the simultaneous determination of UA and FA. Carbon paste electrode (CPE) was first reported in 1958 by Adams [16]. It has become widely used in electrochemical research due to its often-cited advantages of low ohmic resistance, large potential window, and ease of modification [14,17,18]. Ease of modification is one of the most valuable features of CPE. This is due to the well-developed surface of CPE, which has a high adsorptivity for substances [19]. To a large extent, it enhanced the sensitivity and selectivity of CPE. Beitollahi et al. introduced two methods of the simultaneous determination of UA and FA that applied carbon nanotube modified carbon paste electrode as a working electrode [20,21]. However, one required the more
∗ Corresponding author. Tel.: +86 373 3326336; fax: +86 373 3326336. E-mail address: [email protected] (Q. Li). 0013-4686/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.electacta.2012.11.073
expensive carbon nanotube and dioxo-molybdenum (VI) complex [Mo(C10 H11 NO2 )O2 (CH3 OH)] [22] synthesized in the laboratory as the material of modified electrode [20]. The other needed synthesize 5-amino-3 ,4 -dimethoxy-biphenyl-2-ol (5ADMB) in addition to carbon nanotube to modify the carbon paste electrode [21]. What is more, the preparation of 5ADMB was cumbersome and adopted Pd(PPh3 )4 as the catalyst. The progress of reaction was heated at about 90 ◦ C for at least 12 h and monitored by TLC. The crude product needed to be further recrystallized to afford pure 5ADMB. Ardakani et al. reported the electrocatalytic and simultaneous determination of UA and FA by using a 2,2-[1,2 buthanediylbis(nit-riloethylidyne)]-bis-hydroquinone (BQ)/TiO2 nanoparticles modified carbon paste electrode (BQTMCPE) [23]. But the synthesis of BQ was a complicated procedure and it took 10 h or more to prepare TiO2 nanoparticles. While, the simultaneous determination of UA and FA was also achieved at the chloranil modified carbon nanotube paste electrode (CAMCNPE) [24]. Thus it can be seen that the above modified electrodes used to determine UA and FA are all composite materials. Obviously, the disadvantages of corresponding electrodes are high cost, numerous of process, longer reaction time. In particular, these electrodes use some harmful organic reagents, for example, chloroform [21,23] and chloranil [24]. Therefore, these modified electrodes will not obtain the widespread popularization and application in routine laboratories. To the best of our knowledge, no study has reported the simultaneous determination of UA and FA by using the pre-anodized inlaying ultrathin carbon paste electrode (PAIUCPE). In the present work, the nichrome was adopted as a substrate and its one end was
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Fig. 1. SEM images of the bare nichrome electrode (A), IUCPE (B) and PAIUCPE (C).
rubbed in the carbon paste to fabricate an inlaying ultra-thin carbon paste electrode (IUCPE). The prepared IUCPE was anodized by successive cyclic voltammetry to fabricate the PAIUCPE. It exhibited an excellent electrocatalytic activity for the oxidation of UA and FA, and their oxidation peak potential difference was about 324 mV that was greater than 280 mV [20] and quite close to 330 mV [21] and 340 mV [23]. Compared to the above modified electrodes, the PAIUCPE was made by the simple and fast electrochemical pretreatment of carbon paste electrode that was used conventional preparation methods.
2. Experimental 2.1. Apparatus and reagents All electrochemical measurements of cyclic voltammetry were performed on a CHI832C electrochemical workstation (Shanghai Chenhua Instrument Company, China) controlled by a microcomputer with CHI832 C software. A PFS-80 digital pH meter (Shanghai Dazhong Analysis Instrument Company, Shanghai, China) was used for the preparation of buffer solutions. A three-electrode electrochemical cell was employed. A saturated calomel electrode (SCE), a platinum wire electrode and a PAIUCPE were served as the reference, auxiliary and working electrode, respectively. All reagents were of analytical reagent grade and used without further purification. UA and FA were both purchased from Shanghai Chemical Reagent Co., Ltd. A 5.00 × 10−3 mol/L stock UA standard solution was prepared in 0.10 mol/L NaOH. A 5.00 × 10−3 mol/L stock FA standard solution was prepared by dissolving FA with 0.5% ammonia and diluting to the mark with double distilled deionized water. Graphite powder (the purity is 99.85%, purchased from Shanghai, China) and paraffin oil (C.P purchased from Xinxiang, China) were used as binding agents for the graphite pastes.
The supporting electrolyte used for all experiments was 0.10 mol/L PBS (KH2 PO4 - K2 HPO4 ) in the pH range from 4.00 to 9.00. 2.2. Preparation of the pre-anodized inlaying ultrathin carbon paste electrode A nichrome rod with 4 mm diameter (0.126 cm2 ) and a known length was sealed in a plastic tube of matching length. One end was used as the electrode connection held out of the plastic tube, the other as the working electrode surface. The surface was polished with alumina slurry (0.05 m), washed with 1:1 nitric acid, absolute ethanol and double distilled deionized water in an ultrasonic bath subsequently and allowed to dry in the air. A 1:0.25 (w/w) mixture of graphite powder and paraffin was blended in an agate mortar and pestled for 20 min until a homogeneous paste was obtained. Then the pretreated nichrome substrate was rubbed in the carbon paste to fabricate an inlaying ultrathin carbon paste electrode (IUCPE) with a layer thickness of about 100 nm carbon paste film. The carbon paste out of the tube was cleaned and rinsed with double distilled deionized water. The prepared IUCPE was anodized by successive cyclic voltammetry for 35 cycles from −0.30 V to +1.50 V with a scan rate of 100 mV/s in NaOH (0.20 mol/L). After the pre-anodization, the electrode was thoroughly rinsed with double distilled deionized water and dried in the air. 2.3. Experimental procedure A certain volume of UA and FA standard solution were transferred into a cell containing 10 mL of 0.10 mol/L PBS (pH 6.00), and the three-electrode system was installed on the cell. The voltammograms were recorded between 0.10 V and 0.90 V at a scan rate of 100 mV/s. The quantitative determination was carried out by
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Fig. 2. Cyclic voltammograms of K4 [Fe(CN)6 ] at different electrodes. (a) The bare nichrome electrode, (b) IUCPE, and (c) PAIUCPE; c(K4 [Fe(CN)6 ]) = 1.0 × 10−3 mol/L; supporting electrolyte: 0.10 mol/L H2 SO4 ; scan rate: 100 mV/s.
recording the oxidation peak current of linear scan voltammetry. All experiments were done at room temperature. 3. Results and discussion 3.1. The surface morphology characteristic of PAIUCPE The surface morphology of PAIUCPE, IUCPE and bare nichrome electrode were characterized by scanning electron microscope (SEM). There were long and narrow grooves on the surface of bare nichrome electrode (Fig. 1A). However, this much porosity was enough for carbon paste inlay to fabricate an inlaying ultrathin carbon paste electrode (IUCPE) with a layer thickness of carbon paste film at nanometer level (Fig. 1B). The calculation indicated that the thickness of the carbon paste film was about 100 nm. The SEM of PAIUCPE (Fig. 1C) revealed that flake structures of graphite got smaller and some chips of graphite form three-dimensional gaps with multi-hole, which could effectively increase the apparent electroactive surface area of the electrode and significantly improve the electrochemical performance on analyte. 3.2. The electrochemical properties of PAIUCPE The electrochemical properties of PAIUCPE were investigated using potassium ferrocyanide (K4 [Fe(CN)6 ]) as an electrochemical probe. The grams of PAIUCPE characterized by cyclic voltammetry (CV) in 1.0 × 10−3 mol/L of K4 [Fe(CN)6 ]/0.10 mol/L H2 SO4 solution at a scan rate of 100 mV/s were shown in Fig. 2. There were no oxidation–reduction peaks at the bare nichrome electrode (curve a). The peak separations were found to be 102 mV and 66 mV at the IUCPE (curve b) and PAIUCPE (curve c), respectively. Moreover, the peak currents were both higher at the PAIUCPE than that of the IUCPE. It indicated that a reversible electron transfer process occurred at the PAIUCPE, and the PAIUCPE could greatly improve the electron transfer rate of electrochemical reaction and the apparent electroactive surface area of the electrode was increased after electrochemical pretreatment [25]. This may be due to fact that many negatively charged oxygen-containing functional groups would be produced on carbon surface during the process of pre-anodization, which exhibited excellent electrocatalytic activity towards adsorption and electron transfer kinetics [26]. 3.3. Voltammetric behaviours of UA and FA Fig. 3 depicts the cyclic voltammetric responses from the electrochemical oxidation of 5.0 × 10−5 mol/L UA and FA at the PAIUCPE (curve d), IUCPE (curve c) and bare nichrome electrode (curve b). As seen, UA and FA overlapped to form a weak and wide oxidation
Fig. 3. Cyclic voltammograms of 5.0 × 10−5 mol/L UA and FA at different electrodes. (a) PAIUCPE in supporting solution, (b) the bare nichrome electrode, (c) IUCPE, and (d) PAIUCPE; supporting electrolyte: PBS (pH 6.00); scan rate: 100 mV/s.
peak at the bare nichrome electrode. At the IUCPE and PAIUCPE, the behaviours of UA and FA were both irreversible. At the IUCPE, two broad and weak oxidation peaks appeared at 0.398 V and 0.694 V, corresponding to the oxidation of UA and FA. The separated extent between the two peaks was 296 mV. While the anodic peak potential for the oxidation of UA and FA at the PAIUCPE were about 0.362 V and 0.686 V, and their anodic potential difference was about 324 mV. Moreover, the peak currents were higher than that of curve c, and there were no obvious voltammetric signals at the PAIUCPE in pH 6.00 PBS buffer (curve a), showing that it had no electrochemical response in the range of the scanning potential. Clearly, it was concluded that the best electrocatalytic effect for UA and FA oxidation was observed at the PAIUCPE, which indicated that the PAIUCPE exhibited excellent electrocatalytic activities towards the oxidation of UA and FA in PBS (pH 6.00). 3.4. Selection of experimental conditions 3.4.1. Selection of pre-anodized conditions In order to prepare a PAIUCPE with better electrochemical performance, the prepared IUCPE was anodized in 0.20 mol/L H2 SO4 , 0.20 mol/L NaOH and 0.20 mol/L NaCl solution to fabricate the PAIUCPE, respectively. In addition, cycles of cyclic voltammetry (20–40 cycles) and the potential scope (−0.30 V to +1.50 V) were also studied. The results showed that the voltammetric response of UA and FA became maximal when the electrode was anodized by successive cyclic voltammetry for 35 cycles from −0.30 V to +1.50 V with a scan rate of 100 mV/s in NaOH (0.20 mol/L). This may be attributed to the fact that the PAIUCPE exposed a very clean pyrolytic carbon film during pre-anodization in basic solution. Moreover, many microfabricate reactive sites were fabricated on the electrode surface when PAIUCPE was activated in higher pH. Therefore, the PAIUCPE can accelerate electron transfer and improve the electrocatalytic activity [26]. 3.4.2. Discussion of the mechanism of the effect of pH on peak current and peak potential From the study of the oxidation peak currents of UA and FA at the PAIUCPE in 0.10 mol/L PBS buffer solution (pH 6.00), sodium citrate-HCl and NaAc-HAc, it was found that the PBS buffer solution exhibited the maximum current response, so it was used as the supporting electrolyte. The variation of peak potential (Ep ) and peak current (ip ) with pH in 0.10 mol/L PBS buffer solution are shown in Figs. 4 and 5. Within the range of pH 4.00–pH 9.00, the peak potential of UA and FA shifted negatively gradually with the increasing pH, representing the participation of protons in their electrochemical oxidation process. But their peak currents increased gradually until attaining a maximum at about pH 6.00, and then decreased. The possible
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Fig. 5. The effect of pH on peak current. c(UA) = c(FA) = 5.0 × 10−5 mol/L; supporting electrolyte: PBS; scan rate: 100 mV/s. Fig. 4. The effect of pH on peak potential. c(UA) = c(FA) = 5.0 × 10−5 mol/L; supporting electrolyte: PBS; scan rate: 100 mV/s.
reasons for this phenomenon were as follows. As seen in Fig. 3, because UA and FA were oxidized at the PAIUCPE, some species should be reduced at the platinum electrode (the auxiliary electrode) at the same time. Furthermore, only H+ or oxygen (O2 ) dissolved in the solution could get electrons to be reduced. But there was no hydrogen (H2 ) appeared at the platinum electrode in the experiments since a higher overpotential was required for hydrogen evolution. Thus, a little of oxygen that was not completely removed in the solution should get electrons to be reduced at the platinum electrode. In view of the above-mentioned facts and the literature [12,27], the reasonable reaction mechanisms at the anode and cathode were given in Scheme 1, respectively. According to the fact that the oxidation peak potential (Ep ) shifted negatively with the increase of pH (4.00–6.00), showing that the higher the pH value, the more easily the UA and FA lost electrons to be oxidized, so their oxidation peak currents enhanced
correspondingly. When the supporting electrolyte (PBS) changed from acidic (pH = 6.00) to neutral (pH = 7.00) or basic (pH > 7.00), the cathodic reaction of oxygen transferred from formulas (1-1) to another (1-2) [28]. O2 + H+ + 2e → H2 O2 O2 + H2 O + 2e → 2OH−
E1 0 = 0.69 V E2 0 = 0.218 V
(1-1) (1-2)
It was not easy for the oxygen to get electrons in neutral or basic media for the reason that E2 0 was less than E1 0 , and the amounts of oxygen that was to be reduced at the cathode decreased. Accordingly, the amounts of oxidized UA and FA also decreased at the anode based on the electrons getting-losed conservation in the oxidation–reduction reactions. As a result, the oxidation peak currents of UA and FA decreased when the pH of the solution exceeded 6.00. Hence, pH 6.00 was selected for their determination at the PAIUCPE.
Scheme 1. Reaction mechanism.
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Fig. 6. The relationships between oxidation peak current and the square roots of scan rate. c(UA) = c(FA) = 5.0 × 10−5 mol/L; supporting electrolyte: PBS (pH 6.00).
3.4.3. The effect of scan rate The effect of scan rate on the electrocatalytic oxidation of UA and FA at the PAIUCPE was investigated by LSV. As can be observed in Fig. 6, the oxidation peak currents (ip ) of UA and FA were linear with the square root of scan rate (1/2 ) in the range of 50–250 mV/s and 50–350 mV/s, respectively, which was ˇ cik equation (ip = 2.69 × 105 in accordance with the Randles-Sevˇ n3/2 ACD1/2 1/2 ) [29]. A good linear regression equation of UA and FA were ip (A) = −1.81932 + 33.753851/2 (R = 0.9985), ip (A) = −12.79849 + 85.492891/2 (R = 0.9970), suggesting that the oxidation process was diffusion at low scan rate. When the scan rate of UA exceeded 250 mV/s and FA exceeded 350 mV/s, curves deviated from the straight to the bend downward. The reason may be that the reaction rate of UA and FA was fast on the electrode interface while the diffusion rate was relatively slow. So a amount of UA and FA on the electrode surface could not be supplied sufficiently quickly. Although their oxidation peak currents were both enhanced with the increase of scan rate, the background current increased and the baseline was unstable correspondingly. So the scan rate of 100 mV/s was chosen as the optimum condition. 3.4.4. The influence of accumulation time The influence of different accumulation mode on the oxidation peak currents of UA and FA was investigated. It was found that closed-circuit accumulation was better than open-circuit accumulation. Consequently, the closed-circuit accumulation ranging from 0 s to 50 s was studied, which is shown in Fig. 7. The peak currents of UA and FA both reached maximum at t = 30 s, which indicated that the concentration of UA and FA became saturated on the surface of PAIUCPE within 30 s [30]. It was probably because the PAIUCPE had relatively strong capability of adsorbing UA with increasing accumulation time, which caused the transfer of electrons to be blocked, the peak current of UA decreased after 30 s. Taking sensitivity and
Fig. 7. The relationships between oxidation peak current of UA and FA vs. the accumulation time. c(UA) = c(FA) = 5.0 × 10−5 mol/L; supporting electrolyte: PBS (pH 6.00); scan rate: 100 mV/s.
efficiency into account, the accumulation time was set to 30 s in the following experiments. 3.5. Interference of coexisting components Keeping the concentration of UA and FA (5.0 × 10−5 mol/L) unchanged, a systematic study on the influence of foreign species was carried out under the optimal experimental conditions. The criterion for interference was a relative error of less than ±5% within analytical determination. The experimental results indicated that 200-fold of dextrose and soluble starch, 50-fold of Al3+ , 400-fold of Ca2+ and SO4 2− , 800-fold of Na+ , K+ , NO3 − , NH4 + and Cl− had no interferences with the determination of UA and FA. 3.6. Linear range and detection limit Remarkable study was performed by recording LSV curves at different UA concentration (4.0 × 10−6 –3.5 × 10−4 mol/L) with 5.0 × 10−5 mol/L fixed FA concentration (Fig. 8A). The peak current increased linearly with UA concentration and the linear regression equation is ip (A) = 2.05486 + 0.16463c (mol/L) with the linearly correlation coefficients of 0.9994. And that of FA was ip (A) = 10.48283 + 0.10222c (mol/L) in the range of 3.0 × 10−6 –2.0 × 10−4 mol/L with the linearly correlation coefficients of 0.9978, which was obtained in the presence of UA (5.0 × 10−5 mol/L) (Fig. 8B). The detection limits of UA and FA were 1.1 × 10−7 mol/L and 1.5 × 10−7 mol/L, respectively. In order to further verify the feasibility of the PAIUCPE for simultaneous determination of UA and FA, the mixed solution was quantitative determined by LSV when the concentration of UA and FA changed simultaneously. For UA and FA, the oxidation peak currents were linear to the concentrations
Fig. 8. Linear sweep voltammograms for the simultaneous determination of UA (A) and FA (B) at the PAIUCPE in PBS (pH 6.00). (A) Linear sweep voltammograms of a mixture of UA (from a to j: 4, 10, 25, 50, 100, 200, 250, 280, 310, 350 mol/L) with 5.0 × 10−5 mol/L FA. Insert: linear relationship of ip vs. c(UA). (B) Linear sweep voltammograms of a mixture of FA (from a to j: 3, 10, 25, 50, 70, 100, 125, 150, 170, 200 mol/L) with 5.0 × 10−5 mol/L UA. Insert: linear relationship of ip vs. c(FA).
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Table 1 Determination of UA and FA in human urine simultaneously. Samples
Detected content (mol/L)
R.S.D. (%)
Added (mol/L)
Found (mol/L)
Recovery (%)
8.86 11.85 18.09
97.3 98.5 101.3
6.22 13.64 22.96
97.8 101.4 102.2
UA
5.94
2.41
3.0 6.0 12.0
FA
4.45
2.56
1.81 9.06 18.12
at the range of 3.0 × 10−6 –2.2 × 10−4 mol/L and 3.0 × 10−6 – 1.3 × 10−4 mol/L. The linear regression equation of UA and FA were ip (A) = 3.46041 + 0.11722c (mol/L) (R = 0.9970) and ip (A) = 6.85604 + 0.14295c (mol/L) (R = 0.9972). Although competitive adsorption between UA and FA was existed on the electrode surface, the oxidation peak currents increased linearly with their concentration in a certain range, which once again showed that PAIUCPE was successfully used to determine UA and FA simultaneously. 3.7. The reproducibility and stability of the PAIUCPE In order to characterize the reproducibility of the PAIUCPE, repetitive measurements were done for UA (5.0 × 10−5 mol/L) and FA (5.0 × 10−5 mol/L) mixture. The relative standard deviations (R.S.D.) of oxidation peak currents for them were 1.13% and 2.51% (n = 7), respectively, which indicated that the PAIUCPE had a better reproducibility. It was attributed to the thin reaction interface of the PAIUCPE, which made it difficult for the analyte to penetrate into the membrane, so it had no memory effect [31]. After the PAIUCPE was stored at room temperature for 9 days, the oxidation peak currents decreased less than 5% (n = 7) of the initial signal which revealed a long-term stability. 3.8. Real samples analysis For the purpose of proving its practical applications, the PAIUCPE was used to determine the content of UA and FA in the human urine using CV. A standard addition method was adopted to estimate the accuracy and the measurement results were shown in Table 1. The recovered ratios of UA and FA were 97.3–101.3% and 97.8–102.2%, respectively, suggesting the good accuracy of the method. 4. Conclusions In this paper, the PAIUCPE was served as the working electrode, and used for the investigation of electrochemical behaviours and the reaction mechanisms of UA and FA. The anodic potential difference of UA and FA was about 324 mV and the simultaneous determination of UA and FA was achieved based on it. Moreover, the PAIUCPE exhibited low cost, reliable performance, long service life and non-pollution on environment. The proposed method was of high sensitivity as well as a wide linear region that made it used for the simultaneous determination of UA and FA in human urine with satisfactory results. References [1] E. Andreadou, C. Nikolaou, F. Gournaras, M. Rentzos, F. Boufidou, A. Tsoutsou, C. Zournas, V. Zissimopoulos, D. Vassilopoulos, Serum uric acid levels in patients with Parkinson’s disease: Their relationship to treatment and disease duration, Clinical Neurology and Neurosurgery 111 (2009) 724. [2] C.B. Huang, H.W. Chen, Q.H. He, Flow injection on line photochemically spectrofluorimetric determination of folic acid in pharmaceutical preparations, Chinese Journal of Analytical Chemistry 31 (2003) 229.
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
A pre-anodized inlaying ultrathin carbon paste electrode for simultaneous determination of uric acid and folic acid Jing’e Huo, Enbo Shangguan, Quanmin Li ∗ College of Chemistry and Environmental Science, Henan Normal University, Henan Key Laboratory for Environmental Pollution Control, Xinxiang, Henan 453007, PR China
a r t i c l e
i n f o
Article history: Received 20 July 2012 Received in revised form 25 October 2012 Accepted 17 November 2012 Available online 27 November 2012 Keywords: Pre-anodized inlaying ultrathin carbon paste electrode Nichrome Uric acid Folic acid
a b s t r a c t A pre-anodized inlaying ultrathin carbon paste electrode (PAIUCPE) was prepared by electrochemical pretreatment. The scanning electron microscope (SEM) was applied to characterize the surface morphology of PAIUCPE and the performance of the electrode was characterized by cyclic voltammetry (CV). The results indicated that PAIUCPE displayed excellent electrocatalysis for the oxidation of uric acid (UA) and folic acid (FA). The separated extent between the two oxidation peaks of UA and FA was 324 mV, which was enough for the simultaneous detection. In 0.10 mol/L PBS (pH 6.00), the linear scan voltammetry (LSV) response of UA and FA increased linearly with the concentration in the range of 4.0 × 10−6 –3.5 × 10−4 mol/L and 3.0 × 10−6 –2.0 × 10−4 mol/L with the detection limits of 1.1 × 10−7 mol/L and 1.5 × 10−7 mol/L, respectively. It was successfully used to determine UA and FA in human urine simultaneously. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Uric acid (UA, 2,6,8-tridroxypurine) is the main end product of purine metabolism in the human body [1]. Excess urinary UA secretion may lead to urinary calculi or gout. Folic acid (FA) is a form of the water-soluble vitamin Bc widely found in some vegetable and food [2]. A lack of FA gives rise to gigantocytic anaemia, associated with leukopaenia. In recent years, various techniques have been employed for quantification of UA or FA, including fluorescence [3,4], spectrophotometry [5,6], high-performance liquid chromatography (HPLC) [7,8], flow injection [2,9], capillary electrophoresis [10,11], and electrochemical method [12–15]. Because UA and FA always coexists in the human body fluids, it has significance for the simultaneous determination of UA and FA. Carbon paste electrode (CPE) was first reported in 1958 by Adams [16]. It has become widely used in electrochemical research due to its often-cited advantages of low ohmic resistance, large potential window, and ease of modification [14,17,18]. Ease of modification is one of the most valuable features of CPE. This is due to the well-developed surface of CPE, which has a high adsorptivity for substances [19]. To a large extent, it enhanced the sensitivity and selectivity of CPE. Beitollahi et al. introduced two methods of the simultaneous determination of UA and FA that applied carbon nanotube modified carbon paste electrode as a working electrode [20,21]. However, one required the more
∗ Corresponding author. Tel.: +86 373 3326336; fax: +86 373 3326336. E-mail address: [email protected] (Q. Li). 0013-4686/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.electacta.2012.11.073
expensive carbon nanotube and dioxo-molybdenum (VI) complex [Mo(C10 H11 NO2 )O2 (CH3 OH)] [22] synthesized in the laboratory as the material of modified electrode [20]. The other needed synthesize 5-amino-3 ,4 -dimethoxy-biphenyl-2-ol (5ADMB) in addition to carbon nanotube to modify the carbon paste electrode [21]. What is more, the preparation of 5ADMB was cumbersome and adopted Pd(PPh3 )4 as the catalyst. The progress of reaction was heated at about 90 ◦ C for at least 12 h and monitored by TLC. The crude product needed to be further recrystallized to afford pure 5ADMB. Ardakani et al. reported the electrocatalytic and simultaneous determination of UA and FA by using a 2,2-[1,2 buthanediylbis(nit-riloethylidyne)]-bis-hydroquinone (BQ)/TiO2 nanoparticles modified carbon paste electrode (BQTMCPE) [23]. But the synthesis of BQ was a complicated procedure and it took 10 h or more to prepare TiO2 nanoparticles. While, the simultaneous determination of UA and FA was also achieved at the chloranil modified carbon nanotube paste electrode (CAMCNPE) [24]. Thus it can be seen that the above modified electrodes used to determine UA and FA are all composite materials. Obviously, the disadvantages of corresponding electrodes are high cost, numerous of process, longer reaction time. In particular, these electrodes use some harmful organic reagents, for example, chloroform [21,23] and chloranil [24]. Therefore, these modified electrodes will not obtain the widespread popularization and application in routine laboratories. To the best of our knowledge, no study has reported the simultaneous determination of UA and FA by using the pre-anodized inlaying ultrathin carbon paste electrode (PAIUCPE). In the present work, the nichrome was adopted as a substrate and its one end was
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Fig. 1. SEM images of the bare nichrome electrode (A), IUCPE (B) and PAIUCPE (C).
rubbed in the carbon paste to fabricate an inlaying ultra-thin carbon paste electrode (IUCPE). The prepared IUCPE was anodized by successive cyclic voltammetry to fabricate the PAIUCPE. It exhibited an excellent electrocatalytic activity for the oxidation of UA and FA, and their oxidation peak potential difference was about 324 mV that was greater than 280 mV [20] and quite close to 330 mV [21] and 340 mV [23]. Compared to the above modified electrodes, the PAIUCPE was made by the simple and fast electrochemical pretreatment of carbon paste electrode that was used conventional preparation methods.
2. Experimental 2.1. Apparatus and reagents All electrochemical measurements of cyclic voltammetry were performed on a CHI832C electrochemical workstation (Shanghai Chenhua Instrument Company, China) controlled by a microcomputer with CHI832 C software. A PFS-80 digital pH meter (Shanghai Dazhong Analysis Instrument Company, Shanghai, China) was used for the preparation of buffer solutions. A three-electrode electrochemical cell was employed. A saturated calomel electrode (SCE), a platinum wire electrode and a PAIUCPE were served as the reference, auxiliary and working electrode, respectively. All reagents were of analytical reagent grade and used without further purification. UA and FA were both purchased from Shanghai Chemical Reagent Co., Ltd. A 5.00 × 10−3 mol/L stock UA standard solution was prepared in 0.10 mol/L NaOH. A 5.00 × 10−3 mol/L stock FA standard solution was prepared by dissolving FA with 0.5% ammonia and diluting to the mark with double distilled deionized water. Graphite powder (the purity is 99.85%, purchased from Shanghai, China) and paraffin oil (C.P purchased from Xinxiang, China) were used as binding agents for the graphite pastes.
The supporting electrolyte used for all experiments was 0.10 mol/L PBS (KH2 PO4 - K2 HPO4 ) in the pH range from 4.00 to 9.00. 2.2. Preparation of the pre-anodized inlaying ultrathin carbon paste electrode A nichrome rod with 4 mm diameter (0.126 cm2 ) and a known length was sealed in a plastic tube of matching length. One end was used as the electrode connection held out of the plastic tube, the other as the working electrode surface. The surface was polished with alumina slurry (0.05 m), washed with 1:1 nitric acid, absolute ethanol and double distilled deionized water in an ultrasonic bath subsequently and allowed to dry in the air. A 1:0.25 (w/w) mixture of graphite powder and paraffin was blended in an agate mortar and pestled for 20 min until a homogeneous paste was obtained. Then the pretreated nichrome substrate was rubbed in the carbon paste to fabricate an inlaying ultrathin carbon paste electrode (IUCPE) with a layer thickness of about 100 nm carbon paste film. The carbon paste out of the tube was cleaned and rinsed with double distilled deionized water. The prepared IUCPE was anodized by successive cyclic voltammetry for 35 cycles from −0.30 V to +1.50 V with a scan rate of 100 mV/s in NaOH (0.20 mol/L). After the pre-anodization, the electrode was thoroughly rinsed with double distilled deionized water and dried in the air. 2.3. Experimental procedure A certain volume of UA and FA standard solution were transferred into a cell containing 10 mL of 0.10 mol/L PBS (pH 6.00), and the three-electrode system was installed on the cell. The voltammograms were recorded between 0.10 V and 0.90 V at a scan rate of 100 mV/s. The quantitative determination was carried out by
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Fig. 2. Cyclic voltammograms of K4 [Fe(CN)6 ] at different electrodes. (a) The bare nichrome electrode, (b) IUCPE, and (c) PAIUCPE; c(K4 [Fe(CN)6 ]) = 1.0 × 10−3 mol/L; supporting electrolyte: 0.10 mol/L H2 SO4 ; scan rate: 100 mV/s.
recording the oxidation peak current of linear scan voltammetry. All experiments were done at room temperature. 3. Results and discussion 3.1. The surface morphology characteristic of PAIUCPE The surface morphology of PAIUCPE, IUCPE and bare nichrome electrode were characterized by scanning electron microscope (SEM). There were long and narrow grooves on the surface of bare nichrome electrode (Fig. 1A). However, this much porosity was enough for carbon paste inlay to fabricate an inlaying ultrathin carbon paste electrode (IUCPE) with a layer thickness of carbon paste film at nanometer level (Fig. 1B). The calculation indicated that the thickness of the carbon paste film was about 100 nm. The SEM of PAIUCPE (Fig. 1C) revealed that flake structures of graphite got smaller and some chips of graphite form three-dimensional gaps with multi-hole, which could effectively increase the apparent electroactive surface area of the electrode and significantly improve the electrochemical performance on analyte. 3.2. The electrochemical properties of PAIUCPE The electrochemical properties of PAIUCPE were investigated using potassium ferrocyanide (K4 [Fe(CN)6 ]) as an electrochemical probe. The grams of PAIUCPE characterized by cyclic voltammetry (CV) in 1.0 × 10−3 mol/L of K4 [Fe(CN)6 ]/0.10 mol/L H2 SO4 solution at a scan rate of 100 mV/s were shown in Fig. 2. There were no oxidation–reduction peaks at the bare nichrome electrode (curve a). The peak separations were found to be 102 mV and 66 mV at the IUCPE (curve b) and PAIUCPE (curve c), respectively. Moreover, the peak currents were both higher at the PAIUCPE than that of the IUCPE. It indicated that a reversible electron transfer process occurred at the PAIUCPE, and the PAIUCPE could greatly improve the electron transfer rate of electrochemical reaction and the apparent electroactive surface area of the electrode was increased after electrochemical pretreatment [25]. This may be due to fact that many negatively charged oxygen-containing functional groups would be produced on carbon surface during the process of pre-anodization, which exhibited excellent electrocatalytic activity towards adsorption and electron transfer kinetics [26]. 3.3. Voltammetric behaviours of UA and FA Fig. 3 depicts the cyclic voltammetric responses from the electrochemical oxidation of 5.0 × 10−5 mol/L UA and FA at the PAIUCPE (curve d), IUCPE (curve c) and bare nichrome electrode (curve b). As seen, UA and FA overlapped to form a weak and wide oxidation
Fig. 3. Cyclic voltammograms of 5.0 × 10−5 mol/L UA and FA at different electrodes. (a) PAIUCPE in supporting solution, (b) the bare nichrome electrode, (c) IUCPE, and (d) PAIUCPE; supporting electrolyte: PBS (pH 6.00); scan rate: 100 mV/s.
peak at the bare nichrome electrode. At the IUCPE and PAIUCPE, the behaviours of UA and FA were both irreversible. At the IUCPE, two broad and weak oxidation peaks appeared at 0.398 V and 0.694 V, corresponding to the oxidation of UA and FA. The separated extent between the two peaks was 296 mV. While the anodic peak potential for the oxidation of UA and FA at the PAIUCPE were about 0.362 V and 0.686 V, and their anodic potential difference was about 324 mV. Moreover, the peak currents were higher than that of curve c, and there were no obvious voltammetric signals at the PAIUCPE in pH 6.00 PBS buffer (curve a), showing that it had no electrochemical response in the range of the scanning potential. Clearly, it was concluded that the best electrocatalytic effect for UA and FA oxidation was observed at the PAIUCPE, which indicated that the PAIUCPE exhibited excellent electrocatalytic activities towards the oxidation of UA and FA in PBS (pH 6.00). 3.4. Selection of experimental conditions 3.4.1. Selection of pre-anodized conditions In order to prepare a PAIUCPE with better electrochemical performance, the prepared IUCPE was anodized in 0.20 mol/L H2 SO4 , 0.20 mol/L NaOH and 0.20 mol/L NaCl solution to fabricate the PAIUCPE, respectively. In addition, cycles of cyclic voltammetry (20–40 cycles) and the potential scope (−0.30 V to +1.50 V) were also studied. The results showed that the voltammetric response of UA and FA became maximal when the electrode was anodized by successive cyclic voltammetry for 35 cycles from −0.30 V to +1.50 V with a scan rate of 100 mV/s in NaOH (0.20 mol/L). This may be attributed to the fact that the PAIUCPE exposed a very clean pyrolytic carbon film during pre-anodization in basic solution. Moreover, many microfabricate reactive sites were fabricated on the electrode surface when PAIUCPE was activated in higher pH. Therefore, the PAIUCPE can accelerate electron transfer and improve the electrocatalytic activity [26]. 3.4.2. Discussion of the mechanism of the effect of pH on peak current and peak potential From the study of the oxidation peak currents of UA and FA at the PAIUCPE in 0.10 mol/L PBS buffer solution (pH 6.00), sodium citrate-HCl and NaAc-HAc, it was found that the PBS buffer solution exhibited the maximum current response, so it was used as the supporting electrolyte. The variation of peak potential (Ep ) and peak current (ip ) with pH in 0.10 mol/L PBS buffer solution are shown in Figs. 4 and 5. Within the range of pH 4.00–pH 9.00, the peak potential of UA and FA shifted negatively gradually with the increasing pH, representing the participation of protons in their electrochemical oxidation process. But their peak currents increased gradually until attaining a maximum at about pH 6.00, and then decreased. The possible
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Fig. 5. The effect of pH on peak current. c(UA) = c(FA) = 5.0 × 10−5 mol/L; supporting electrolyte: PBS; scan rate: 100 mV/s. Fig. 4. The effect of pH on peak potential. c(UA) = c(FA) = 5.0 × 10−5 mol/L; supporting electrolyte: PBS; scan rate: 100 mV/s.
reasons for this phenomenon were as follows. As seen in Fig. 3, because UA and FA were oxidized at the PAIUCPE, some species should be reduced at the platinum electrode (the auxiliary electrode) at the same time. Furthermore, only H+ or oxygen (O2 ) dissolved in the solution could get electrons to be reduced. But there was no hydrogen (H2 ) appeared at the platinum electrode in the experiments since a higher overpotential was required for hydrogen evolution. Thus, a little of oxygen that was not completely removed in the solution should get electrons to be reduced at the platinum electrode. In view of the above-mentioned facts and the literature [12,27], the reasonable reaction mechanisms at the anode and cathode were given in Scheme 1, respectively. According to the fact that the oxidation peak potential (Ep ) shifted negatively with the increase of pH (4.00–6.00), showing that the higher the pH value, the more easily the UA and FA lost electrons to be oxidized, so their oxidation peak currents enhanced
correspondingly. When the supporting electrolyte (PBS) changed from acidic (pH = 6.00) to neutral (pH = 7.00) or basic (pH > 7.00), the cathodic reaction of oxygen transferred from formulas (1-1) to another (1-2) [28]. O2 + H+ + 2e → H2 O2 O2 + H2 O + 2e → 2OH−
E1 0 = 0.69 V E2 0 = 0.218 V
(1-1) (1-2)
It was not easy for the oxygen to get electrons in neutral or basic media for the reason that E2 0 was less than E1 0 , and the amounts of oxygen that was to be reduced at the cathode decreased. Accordingly, the amounts of oxidized UA and FA also decreased at the anode based on the electrons getting-losed conservation in the oxidation–reduction reactions. As a result, the oxidation peak currents of UA and FA decreased when the pH of the solution exceeded 6.00. Hence, pH 6.00 was selected for their determination at the PAIUCPE.
Scheme 1. Reaction mechanism.
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Fig. 6. The relationships between oxidation peak current and the square roots of scan rate. c(UA) = c(FA) = 5.0 × 10−5 mol/L; supporting electrolyte: PBS (pH 6.00).
3.4.3. The effect of scan rate The effect of scan rate on the electrocatalytic oxidation of UA and FA at the PAIUCPE was investigated by LSV. As can be observed in Fig. 6, the oxidation peak currents (ip ) of UA and FA were linear with the square root of scan rate (1/2 ) in the range of 50–250 mV/s and 50–350 mV/s, respectively, which was ˇ cik equation (ip = 2.69 × 105 in accordance with the Randles-Sevˇ n3/2 ACD1/2 1/2 ) [29]. A good linear regression equation of UA and FA were ip (A) = −1.81932 + 33.753851/2 (R = 0.9985), ip (A) = −12.79849 + 85.492891/2 (R = 0.9970), suggesting that the oxidation process was diffusion at low scan rate. When the scan rate of UA exceeded 250 mV/s and FA exceeded 350 mV/s, curves deviated from the straight to the bend downward. The reason may be that the reaction rate of UA and FA was fast on the electrode interface while the diffusion rate was relatively slow. So a amount of UA and FA on the electrode surface could not be supplied sufficiently quickly. Although their oxidation peak currents were both enhanced with the increase of scan rate, the background current increased and the baseline was unstable correspondingly. So the scan rate of 100 mV/s was chosen as the optimum condition. 3.4.4. The influence of accumulation time The influence of different accumulation mode on the oxidation peak currents of UA and FA was investigated. It was found that closed-circuit accumulation was better than open-circuit accumulation. Consequently, the closed-circuit accumulation ranging from 0 s to 50 s was studied, which is shown in Fig. 7. The peak currents of UA and FA both reached maximum at t = 30 s, which indicated that the concentration of UA and FA became saturated on the surface of PAIUCPE within 30 s [30]. It was probably because the PAIUCPE had relatively strong capability of adsorbing UA with increasing accumulation time, which caused the transfer of electrons to be blocked, the peak current of UA decreased after 30 s. Taking sensitivity and
Fig. 7. The relationships between oxidation peak current of UA and FA vs. the accumulation time. c(UA) = c(FA) = 5.0 × 10−5 mol/L; supporting electrolyte: PBS (pH 6.00); scan rate: 100 mV/s.
efficiency into account, the accumulation time was set to 30 s in the following experiments. 3.5. Interference of coexisting components Keeping the concentration of UA and FA (5.0 × 10−5 mol/L) unchanged, a systematic study on the influence of foreign species was carried out under the optimal experimental conditions. The criterion for interference was a relative error of less than ±5% within analytical determination. The experimental results indicated that 200-fold of dextrose and soluble starch, 50-fold of Al3+ , 400-fold of Ca2+ and SO4 2− , 800-fold of Na+ , K+ , NO3 − , NH4 + and Cl− had no interferences with the determination of UA and FA. 3.6. Linear range and detection limit Remarkable study was performed by recording LSV curves at different UA concentration (4.0 × 10−6 –3.5 × 10−4 mol/L) with 5.0 × 10−5 mol/L fixed FA concentration (Fig. 8A). The peak current increased linearly with UA concentration and the linear regression equation is ip (A) = 2.05486 + 0.16463c (mol/L) with the linearly correlation coefficients of 0.9994. And that of FA was ip (A) = 10.48283 + 0.10222c (mol/L) in the range of 3.0 × 10−6 –2.0 × 10−4 mol/L with the linearly correlation coefficients of 0.9978, which was obtained in the presence of UA (5.0 × 10−5 mol/L) (Fig. 8B). The detection limits of UA and FA were 1.1 × 10−7 mol/L and 1.5 × 10−7 mol/L, respectively. In order to further verify the feasibility of the PAIUCPE for simultaneous determination of UA and FA, the mixed solution was quantitative determined by LSV when the concentration of UA and FA changed simultaneously. For UA and FA, the oxidation peak currents were linear to the concentrations
Fig. 8. Linear sweep voltammograms for the simultaneous determination of UA (A) and FA (B) at the PAIUCPE in PBS (pH 6.00). (A) Linear sweep voltammograms of a mixture of UA (from a to j: 4, 10, 25, 50, 100, 200, 250, 280, 310, 350 mol/L) with 5.0 × 10−5 mol/L FA. Insert: linear relationship of ip vs. c(UA). (B) Linear sweep voltammograms of a mixture of FA (from a to j: 3, 10, 25, 50, 70, 100, 125, 150, 170, 200 mol/L) with 5.0 × 10−5 mol/L UA. Insert: linear relationship of ip vs. c(FA).
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Table 1 Determination of UA and FA in human urine simultaneously. Samples
Detected content (mol/L)
R.S.D. (%)
Added (mol/L)
Found (mol/L)
Recovery (%)
8.86 11.85 18.09
97.3 98.5 101.3
6.22 13.64 22.96
97.8 101.4 102.2
UA
5.94
2.41
3.0 6.0 12.0
FA
4.45
2.56
1.81 9.06 18.12
at the range of 3.0 × 10−6 –2.2 × 10−4 mol/L and 3.0 × 10−6 – 1.3 × 10−4 mol/L. The linear regression equation of UA and FA were ip (A) = 3.46041 + 0.11722c (mol/L) (R = 0.9970) and ip (A) = 6.85604 + 0.14295c (mol/L) (R = 0.9972). Although competitive adsorption between UA and FA was existed on the electrode surface, the oxidation peak currents increased linearly with their concentration in a certain range, which once again showed that PAIUCPE was successfully used to determine UA and FA simultaneously. 3.7. The reproducibility and stability of the PAIUCPE In order to characterize the reproducibility of the PAIUCPE, repetitive measurements were done for UA (5.0 × 10−5 mol/L) and FA (5.0 × 10−5 mol/L) mixture. The relative standard deviations (R.S.D.) of oxidation peak currents for them were 1.13% and 2.51% (n = 7), respectively, which indicated that the PAIUCPE had a better reproducibility. It was attributed to the thin reaction interface of the PAIUCPE, which made it difficult for the analyte to penetrate into the membrane, so it had no memory effect [31]. After the PAIUCPE was stored at room temperature for 9 days, the oxidation peak currents decreased less than 5% (n = 7) of the initial signal which revealed a long-term stability. 3.8. Real samples analysis For the purpose of proving its practical applications, the PAIUCPE was used to determine the content of UA and FA in the human urine using CV. A standard addition method was adopted to estimate the accuracy and the measurement results were shown in Table 1. The recovered ratios of UA and FA were 97.3–101.3% and 97.8–102.2%, respectively, suggesting the good accuracy of the method. 4. Conclusions In this paper, the PAIUCPE was served as the working electrode, and used for the investigation of electrochemical behaviours and the reaction mechanisms of UA and FA. The anodic potential difference of UA and FA was about 324 mV and the simultaneous determination of UA and FA was achieved based on it. Moreover, the PAIUCPE exhibited low cost, reliable performance, long service life and non-pollution on environment. The proposed method was of high sensitivity as well as a wide linear region that made it used for the simultaneous determination of UA and FA in human urine with satisfactory results. References [1] E. Andreadou, C. Nikolaou, F. Gournaras, M. Rentzos, F. Boufidou, A. Tsoutsou, C. Zournas, V. Zissimopoulos, D. Vassilopoulos, Serum uric acid levels in patients with Parkinson’s disease: Their relationship to treatment and disease duration, Clinical Neurology and Neurosurgery 111 (2009) 724. [2] C.B. Huang, H.W. Chen, Q.H. He, Flow injection on line photochemically spectrofluorimetric determination of folic acid in pharmaceutical preparations, Chinese Journal of Analytical Chemistry 31 (2003) 229.
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