Materials Science and Engineering C 33 (2013) 811–816
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Highly selective and sensitive voltammetric sensor based on modified multiwall carbon nanotube paste electrode for simultaneous determination of ascorbic acid, acetaminophen and tryptophan Mohsen Keyvanfard a,⁎, Razieh Shakeri b, Hassan Karimi-Maleh a, Khadijeh Alizad a a b
Department of chemistry, Majlesi Branch, Islamic Azad University, Isfahan, Iran Department of chemistry, Shahreza Branch, Islamic Azad University, Shahreza, Iran
a r t i c l e
i n f o
Article history: Received 16 March 2012 Received in revised form 12 October 2012 Accepted 3 November 2012 Available online 12 November 2012 Keywords: Ascorbic acid Acetaminophen Tryptophan 3,4-Dihydroxycinnamic acid Voltammetry Multiwall carbon nanotubes
a b s t r a c t A carbon-paste electrode modified with multiwall carbon nanotubes (MWCNTs) was used for the sensitive and selective voltammetric determination of ascorbic acid (AA) in the presence of 3,4-dihydroxycinnamic acid (3,4-DHCA) as mediator. The mediated oxidation of AA at the modified electrode was investigated by cyclic voltammetry (CV), chronoamperommetry and electrochemical impedance spectroscopy (EIS). Also, the values of catalytic rate constant (k), and diffusion coefficient (D) for AA were calculated. Using square wave voltammetry (SWV), a highly selective and simultaneous determination of AA, acetaminophen (AC) and tryptophan (Trp) has been explored at the modified electrode. The modified electrode displayed strong function for resolving the overlapping voltammetric responses of AA, AC and Trp into three well-defined voltammetric peaks. In the mixture containing AA, AC and Trp, the three compounds can well separate from each other with potential differences of 200, 330 and 530 mV between AA and AC, AC and Trp and AA and Trp, respectively, which was large enough to determine AA, AC and Trp individually and simultaneously. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Electroanalytical applications, which utilize chemically modified electrodes (CMEs), should offer a potentially significant efficiency [1]. Recently there has been a considerable effort in the investigation of catalytic function of CMEs on the oxidation of biologically important compounds, such as tryptophan [2], N-acetylcysteine [3], amitrole [4] and 6-thioguanine [5]. It is generally believed that direct redox reactions of these species at bare electrodes are totally irreversible and therefore require a high overpotential. Therefore, there have been numerous attempts to enhance the electrode kinetics using various CMEs [6–8]. Multiwall carbon nanotubes (MWCNTs) are now used extensively in the fabrication of novel nanostructure electrochemical sensors [9–15]. MWCNT-modified electrodes have many advantages over other forms of carbon electrodes due to their small size, high electrical and thermal conductivity, high chemical stability, high mechanical strength and high specific surface area which make them very promising candidates in a wide range of applications [16–20]. Ascorbic acid or vitamin C is distributed widely in both plant and animal kingdoms. In vegetable cells, it bound to protein as ascorbigen. Among animal organs, the liver, leukocytes and anterior pituitary lobe show the highest concentrations of ascorbic acid. Vitamin C also is
⁎ Corresponding author. Tel.: +98 6732054 (cell phone); fax: +98 311 6732054. E-mail address: [email protected] (M. Keyvanfard). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.11.005
present in many other biological systems and multivitamin preparations, which are commonly used to supplement inadequate dietary intake. Nevertheless, it is widely used in foods as an antioxidant for the stabilization of color and aroma with subsequent extension of the storage time of the products [21,22]. The determination of AA has been established by such methods as chromatography [23–26], spectrophotometry [27–29], mass spectrometry [30], flow injection [31,32], chemiluminescence [33] and electrochemical methods [34–37]. Acetaminophen, paracetamol or N-acetyl-p-aminophenol [38,39] is a mild, safe, widely used analgesic and antipyretic substitute of aspirin. However, it causes liver necrosis in humans and experimental animals when high doses are administered [40]. To date, various techniques including titrimetry [41,42], spectrophotometry [43–46], high performance liquid chromatography [47–50], fluorimetry [51] and electrochemical methods [52–54] have been reported for the determination of acetaminophen. In therapeutic applications, acetaminophen is often found in association with ascorbic acid and/or other pharmacologically and biologically active compounds [55]. Tryptophan is a vital amino acid for humans and herbivores. This compound is a precursor for serotonin (a neurotransmitter), melatonin (a neurohormone), and niacin [56]. This compound is sometimes added to dietary, food products, pharmaceutical formulas due to the scarcely presence in vegetables [57]. Different methods such as spectrophotometry, high performance liquid chromatography (HPLC), and electrochemical methods have so far been available for the determination of Trp [58–60].
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High doses of vitamin C may lower the amount of acetaminophen passed in urine, which could cause the levels of this drug in your blood to rise [61]. Lactose and ascorbic acid did not have tryptophan contamination when used as anti-oxidizing reagents. Both improved tryptophan recovery, but ascorbic acid was more effective [62,63]. To the best of our knowledge, most previously published electrochemical studies have dealt with individual determination of AA, AC or Trp utilizing carbon paste electrodes or other kinds of modified electrode. Only one study has been reported on the simultaneous determination of AA, AC and Trp in the presence of each other using modified carbon nanotube paste electrodes [64]. In this study, we proposed 3,4-DHCA as a novel mediator for the rapid, sensitive, and highly selective voltammetric determination of AA on the surface of a multiwall carbon nanotube paste electrode. We also evaluate the analytical performance of the modified electrode for quantification of AA in the presence of AC and Trp. 2. Experimental 2.1. Reagents and apparatus All chemicals used were of analytical reagent grade purchased from Merck (Darmstadt, Germany) unless otherwise stated. Doubly distilled water was used throughout. Ascorbic acid was used from Merck (Darmstadt, Germany), acetaminophen and tryptophan from Fluka, all used as received. Phosphate buffer (phosphoric acid, sodium dihydrogen phosphate; disodium monohydrogen phosphate and trisodium phosphate plus sodium hydroxide, 0.1 mol L −1) solutions with different pH values were used. Spectrally pure graphite powder (particle size b 50 μm) from Merck and multiwall carbon nanotubes (> 90%, MWCNTs, d × l = (100 − 70 nm) × (5 − 9 μm)) from Fluka was used as the substrate for the preparation of the carbon paste electrode. High viscosity paraffin (d = 0.88 kg L − 1) from Merck was used as the pasting liquid for the preparation of the paste electrodes. All the voltammetric measurements were performed using an Autolab PGSTAT 302 N, potentiostat/galvanostat (Utrecht, The Netherlands) connected to a three-electrode cell, Metrohm (Herisau, Switzerland) Model 663 VA stand, linked with a computer (Pentium IV, 1200 MHz) and with Autolab software. A platinum wire was used as the auxiliary electrode. Multiwall carbon nanotube paste electrode (MWCNTPE) and Ag/AgCl/KClsat were used as the working and reference electrodes, respectively. The electrode prepared with carbon nanotubes was characterized by scanning electron microscopy (SEM) (Seron Tech. AIS 2100). A digital pH/mV-meter (Metrohm model 710) was applied for pH measurements. 2.2. Preparation of the electrode Graphite powder (0.900 g) was dispersed in diethyl ether and hand mixed with 0.100 g carbon nanotubes in a mortar and pestle. The solvent was evaporated by stirring. A syringe was used to add paraffin to the mixture, which was mixed well for 40 min until a uniformly wetted paste, was obtained. The paste was then packed into a glass tube. Electrical contact was made by pushing a copper wire down the glass tube into the back of the mixture. When necessary, a new surface was obtained by pushing an excess of the paste out of the tube and polishing it on a weighing paper. 2.3. Preparation of practical samples Urine sample was stored in a refrigerator immediately after collection. Ten milliliters of the sample was centrifuged for 15 min at 3000 rpm. The supernatant was diluted 10 times with a phosphate buffer solution (PBS) (pH 6.0). The solution was transferred into the
voltammetric cell to be analyzed without any further pretreatment. The standard addition method was used for the determination of ascorbic acid in practical samples. For the tablets, an accurately weighed portion of finely powdered sample obtained from five tablets (Daro pakhsh Co.), equivalent to about 50 mg of ascorbic acid dissolved in 100 mL water with ultrasonication. Then, 0.1 mL of the solution plus 9.9 mL of the buffer (pH 6.0) was used for the analysis with standard addition method. For injection solution, an accurate ampoule ((standard content 100 mg per mL AA, 5 mL per injection) (Daro Pakhsh Co.)) volume equivalent to about 50 mg of vitamin C was transferred to a 25 mL flask and diluted to volume with PBS. A 0.5 mL portion of the solution was subjected for voltammetric measurement as described for tablets. Fresh fruit juices were obtained using a mechanical squeezer. The juices obtained were filtered. A 0.5 mL portion of the filtrate juice was added to the supporting electrolyte solution in voltammetric cell. Vegetable juices were obtained using a grater and a centrifuge respectively, a 0.5 mL portion of vegetable juice was subjected for the voltammetric measurement. In all cases the amounts of vitamin C in the samples were evaluated by the standard addition method. 3. Results and discussion 3.1. SEM characterization Fig. 1 shows SEM images for MWCNTPE and carbon paste electrode (CPE). As can be seen at a surface of CPE (Fig. 1A), the layer of irregularly flakes of graphite powder was present and isolated with each other. After the multiwall carbon nanotubes (MWCNTs) were added to the carbon paste, it can be seen that MWCNTs were distributed on the surface of electrode with special three-dimensional structure (Fig. 1B), indicating that the MWCNTs were successfully modified on the MWCNTPE. 3.2. Electrochemistry of 3,4-DHCA The electrochemical behavior of the 3,4-DHCA was characterized by cyclic voltammetry. The experimental results showed well defined and reproducible anodic and cathodic peaks related to 3,4-DHCA(red)/ 3,4-DHCA(ox) redox couple with a quasi reversible behavior and with a peak separation potential of ΔEp = (Epa − Epc) = 100 mV. These cyclic voltammograms were used to examine the variation of the peak currents vs. the square root of potential scan rates. The plot of the anodic peak current was linearly dependent on ν 1/2 with a correlation coefficient of 0.9937 at all scan rates. 3.3. Catalytic effect As shown in Fig. 2, the anodic peak potential for AA oxidation at the MWCNTPE in the presence of 3,4-DHCA (curve a) and CPE in the presence of 3,4-DHCA (curve b) was about 310 mV, while at the MWCNTPE (without mediator) (curve d), the peak potential was about 430 mV. At the unmodified CPE and without mediator, the peak potential was about 450 mV of AA (curve e). From these results, it was concluded that the best electrocatalytic effect for AA oxidation was observed at the MWCNTPE in the presence of 3,4-DHCA (curve a). For example, results show that the peak potential for AA analysis at the MWCNTPE in the presence of 3,4-DHCA (curve a) shifted by about 120 and 140 mV toward negative values when compared with that at the MWCNTPE and CPE without mediator (curve d and e), respectively. Similarly, when comparing the oxidation of AA at the MWCNTPE (curve a) and CPE (curve b) in the presence of mediator, a dramatic enhancement of the anodic peak current at the MWCNTPE relative to that obtained at the CPE in the presence of mediator was observed. In other words, the data clearly show that the combination of multiwall carbon nanotubes and mediator definitely improve the characteristics of AA oxidation. The 3,4-DHCA at a surface of MWCNTPE, in 0.1 M PBS (pH 6.0)
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Fig. 1. SEM image of A) CPE and B) MWCNTPE.
and without AA in solution, exhibited a well-behaved redox reaction (curve c) upon addition of 300 μmol L−1 AA. The process corresponds to an EC' (catalytic) mechanism (Scheme 1), where the electrochemically formed 3,4-DHCA(Ox) reacts chemically with AA diffused toward the electrode surface, while the simultaneous oxidation of the regenerated 3,4-DHCA(Red) causes an increase in the anodic current. For the same reason, the cathodic current of the modified electrode is smaller in the presence of AA. The effect of the scan rate on the electrocatalytic oxidation of 750 μmol L−1 AA at the MWCNTPE was investigated by cyclic voltammetry in the presence of 3,4-DHCA. The oxidation peak potential shifts with increasing scan rates towards a more positive potential, confirming the kinetic limitation of the electrochemical reaction. Also, a plot of peak height (Ip) against square root of scan rate (ν1/2), in range of 3–20 mV s −1, was constructed, which was found to be linear, suggesting that at sufficient overpotential the process is diffusion rather than surface controlled. To obtain information about the rate-determining step, the Tafel plot was drawn, as derived from points in the Tafel region of the cyclic voltammogram. The slope of the Tafel plot was equal to n(1 − α)F/ 2.3RT (where n is number of electron in steady state stage and α is charge transfer coefficient), which came up to 9.95 decade V−1. Therefore, we obtained the value of α equal to 0.41. In addition, the value of α was calculated for the oxidation of AA at pH 6.0 for both the MWCNTPE
in the absence and presence of mediator using the following Equation [65]: α nα ¼ 0:048= EP –EP=2
ð1Þ
where EP/2 is the potential corresponding to IP/2. The values for α were found to be 0.20 and 0.42 at the surface of the MWCNTPE in the absence and presence of mediator, respectively. Those values show that the over-potential of AA oxidation is reduced at the surface of MWCNTPE in the presence of mediator, and also that the rate of electron transfer process is greatly enhanced. This phenomenon is, thus, confirmed by the larger Ipa values recorded during cyclic voltammetry at MWCNTPE in the presence of mediator (Scheme 2). For the determination of the diffusion coefficient and the catalytic reaction rate constant of AA, double potential step chronoamperometry was used with MWCNTPE in the presence of mediator. Fig. 3A shows the current–time curves of MWCNTPE in the presence of mediator by setting the electrode potential at 200 mV (first step) and 450 mV (second step) for different AA concentrations. As can be seen, there is no net anodic current corresponding to the oxidation of the mediator in the presence of AA. On the other hand, the forward and backward potential step chronoamperometry for the mediator in the absence of AA shows symmetrical chronoamperogram with an equal charge consumed for the reduction and oxidation of the mediator at the surface of MWCNTPE (Fig. 3C, a /). Also, the charge value associated with forward chronoamperometry in the presence of AA is significantly greater than that observed for backward chronoamperometry (Fig. 3C, b /–f /). The rate constant for the chemical reaction between AA and mediator at a surface of MWCNTPE, kh, can be evaluated by chronoamperometry according to the method of Galus [66]: 1=2 1=2
IC =IL ¼ π
γ
¼π
1=2
1=2
ðkh Cb tÞ
ð2Þ
where IC is the catalytic current of AA at MWCNTPE in the presence of mediator, IL is the limited current in the absence of AA, and t is the time elapsed (s). The above equation can be used to calculate the rate constant of the catalytic process kh. Based on the slope of the IC/IL vs. t1/2 plots (Fig. 3B), kh can be obtained for a given AA concentration. Based on the values of the slopes, the average value of kh was found to be equal to 2.198×103 mol L−1 s−1. The value of kh explains the sharp feature of the catalytic peak observed for catalytic oxidation of AA at the surface of MWCNTPE in the presence of mediator. 3.4. Calibration plot and limit of detection
Fig. 2. Cyclic voltammograms of (a) 500 μmol L−1 3,4-DHCA + 300 μmol L−1 AA at the surface of MWCNTPE; (b) 500 μmol L−1 3,4-DHCA + 300 μmol L−1 AA at the surface of carbon paste electrode; (c) the 500 μmol L−1 3,4-DHCA at the surface of MWCNTPE in 0.1 mol L−1 PBS (pH 6.0); (d) 300 μmol L−1 AA at the surface of MWCNTPE; (e) 300 μmol L−1 AA at the surface of carbon paste electrode, scan rate of 20 mV s−1.
Since square wave voltammetry (SWV) has a much higher current sensitivity and better resolution than cyclic voltammetry, SWV was used for simultaneous determination of AA, AC and Trp. In order to get the best sensitivity under the specific conditions, an amplitude potential of 50 mV and frequency of 10 Hz were selected, respectively. The plot of peak current vs. AA concentration consisted of two linear
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Scheme 1. Electrocatalytic mechanism for determination of AA at the surface of MWCNTPE in the presence of 3,4-DHCA.
segments with slopes of 37.8710 and 0.1128 μA/μmol L−1 in the concentration ranges of 0.02–0.1 μmol L−1 and 0.1–140.0 μmol L−1, respectively. The decrease in sensitivity (slope) of the second linear segment is likely due to kinetic limitation (Fig. 4A and B). On other hand, the responses were linear with AC concentration in the range from 2.0 to 400.0 μmol L−1 and the current sensitivity was 0.1025 μA/μmol L−1 (Fig. 4C), while the dynamic range was linear with Trp concentration in the range 5.0 to 500 μmol L − 1 and the current sensitivity was 0.0963 μA/μmol L − 1 (Fig. 4D). Detection limits were determined as 9.1 nmol L − 1AA, 0.8 μmol L − 1 AC and 1.0 μmol L − 1 Trp based on YLOD = YB + 3σ [67]. This value of detection limit and the linear dynamic range for AA observed for the MWCNTPE in the presence of mediator are comparable and even better than those obtained for several other modified electrodes (Table 1).
and presence (0.1116 μA μM−1) of AC and Trp are virtually the same, which further indicate that the oxidation processes of AA, AC and Trp at the MWCNTPE in the presence of mediator are independent and therefore, simultaneous measurements of the three analytes are feasible without any interference.
4. Interference study In order to evaluate the selectivity of the proposed method in the determination of AA the influence of various foreign species on the determination of 1.0 μmol L −1 AA was investigated. Tolerance limit was taken as the maximum concentration of foreign substances that
3.5. Simultaneous determination of AA, AC and Trp One of the main objectives of this study was to develop a modified electrode with the capability of separating the electrochemical responses of AA, AC and Trp. Therefore, SWV was used for the simultaneous determination of AA, AC and Trp. Using MWCNTPE in the presence of mediator as the working electrode, the analytical experiments were carried out by varying the concentration of AC, AC or Trp in 0.1 M PBS (pH 6.0). The SW voltammetric results show three well-distinguished anodic peaks at potentials of 400, 600 and 930 mV, corresponding to the oxidation of AA, AC and Trp, respectively, indicating that the simultaneous determination of AA, AC and Trp is possible at the MWCNTPE in the presence of mediator (Fig. 5). The sensitivities of the modified electrode towards AA in the absence (0.1128 μA μM−1)
Scheme 2. The equivalent circuit compatible with the Nyquist diagram recorded in the absence and presence of AA.
Fig. 3. A) Chronoamperograms obtained at the MWCNTPE in the absence a) and in the presence of b) 100, c) 200, d) 300, e) 400 and f) 500 μmol L−1 AA in a buffer solution (pH 6.0). B) Dependence of Ic/IL on the t1/2 derived from the chronoamperogram data. C) The charge-time curves a/) for curve (a); b/) for curve (b); c/) for curve (c); d/) for curve (d); (e/) for curve (e) and (f/) for curve (f).
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Fig. 4. Plots of Ip vs. A) AA concentration in the range 0.02–0.1; B) AA concentration in the range 0.1–140.0 μmol L−1; C) AC concentration in the range 2.0–400.0 μmol L−1; and D) Trp concentration in the range 5.0–500.0 μmol L−1.
caused an approximate relative error of ±5%. The results are shown in Table S1 (Supporting information). 5. Practical samples analysis In order to demonstrate the ability of the modified electrode to determine AA, AC and Trp in practical samples, these compounds were determined in tablet, ampoule, fruit juice, and vegetable juice and urine samples. The results are presented in Table 2. Clearly, modified electrode is capable of voltammetric determination of AA, AC and Trp with high selectivity and good reproducibility.
AA determination compare to other electrochemical sensor. Finally, this modified electrode used for determination of AA in practical samples with satisfactory results. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.msec.2012.11.005.
Acknowledgments The authors wish to thank Shahreza and Majlesi Branch, Islamic Azad University, for their support.
6. Conclusion A new voltammetric sensor developed for the determination of AA is very rapid, reproducible, highly selective and sensitive, and can be used for practical sample analysis. This mediator (3,4-DHCA) shows excellent catalytic effects on the oxidation of AA. The results show that the oxidation of AA is catalyzed at pH 6.0, whereas the peak potential of AA is shifted by 140 mV to a less positive potential at the surface of the MWCNTPE in the presence of mediator. Potential differences of 200, 330 and 530 mV between AA and AC, AA and Trp and AC and Trp were detected, respectively which were large enough to determine AA, AC and Trp individually and/or simultaneously. Results show better linear dynamic range, detection limit or sensitivity for
Table 1 Comparison of the efficiency of some reported electrochemical methods in the determination of AA. Method
pH
Limit of detection (μmol L−1)
Linear dynamic range (μmol L−1)
Sensitivity μA/μmol L−1
Ref.
Amperometry Differential pulse voltammetry Differential pulse voltammetry Differential pulse voltammetry SWV SWV
5.0 6.8
8.0 20.0
8.8–23.2 50.0–740
0.082 –
[68] [69]
7.0
4.2
9.0–350.0
0.0078
[70]
7.0
0.075
0.1–800.0
0.0195
[71]
7.0 6.0
0.01 0.009
0.03–70.0 0.02–140.0
0.1126 37.871
[58] This work
Fig. 5. SWVs of MWCNTPE in the presence of 500 μmol L−1 3,4-DHCA in 0.1 M PBS (pH 6.0) containing different concentrations of AA–AC–Trp in μmol L−1, from 1 to 6: 6.0+20.0+10.0; 30+40.0+50.0; 70.0+100.0+130.0; 90.0+120.0+150.0; 115.0+ 170.0+180 and 140.0+200.0+205.0, respectively.
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Table 2 Determination of AA, AC and TP in practical samples (n = 3). Sample
AA added (μmol L−1)
AC added (μmol L−1)
TP added (μmol L−1)
Found (AA) proposed method (μmol L−1)
Found (AA) reference method (μmol L−1) [68]
Found (AC) proposed method (μmol L−1)
Found (AC) published method (μmol L−1) [10]
Found (TP) proposed method (μmol L−1)
Found (TP) published method (μmol L−1) [2]
Tablet
5.00 15.00 – 6.00 10.00 0.10 0.50 – – – – – – –
– – – 20.00 30.00 – – – – – – – – –
– – – 10.00 20.00 – – – – – – – – –
5.21 ± 0.30 15.30 ± 0.33 bLOD 6.33 ± 0.43 10.45 ± 0.51 0. 11 ± 0.02 0.52 ± 0.03 – 148.35 ± 0.82 75.51 ± 0.85 11.01 ± 0.63 – 43.55 ± 0.71 80.21 ± 1.11
4.72 ± 0.41 15.87 ± 0.95 bLOD 6.58 ± 0.72 10.64 ± 0.68 10.22 ± 0.33 0.54 ± 0.05 – 149.10 ± 1.35 75.91 ± 0.93 11.38 ± 1.01 – 44.51 ± 1.23 81.02 ± 1.25
– – bLOD 20.25 ± 0.31 30.44 ± 0.52 – – – – – – – – –
– – bLOD 20.45 ± 0.50 29.89 ± 0.72 – – – – – – – – –
– – bLOD 10.32 ± 0.41 20.32 ± 0.45 – – – – – – – – –
– – bLOD 10.52 ± 0.63 20.75 ± 0.84 – – – – – – – – –
Urine
Injection Fruit juices Orange Kiwi Apple Vegetable juices Pimento Dill
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Highly selective and sensitive voltammetric sensor based on modified multiwall carbon nanotube paste electrode for simultaneous determination of ascorbic acid, acetaminophen and tryptophan Mohsen Keyvanfard a,⁎, Razieh Shakeri b, Hassan Karimi-Maleh a, Khadijeh Alizad a a b
Department of chemistry, Majlesi Branch, Islamic Azad University, Isfahan, Iran Department of chemistry, Shahreza Branch, Islamic Azad University, Shahreza, Iran
a r t i c l e
i n f o
Article history: Received 16 March 2012 Received in revised form 12 October 2012 Accepted 3 November 2012 Available online 12 November 2012 Keywords: Ascorbic acid Acetaminophen Tryptophan 3,4-Dihydroxycinnamic acid Voltammetry Multiwall carbon nanotubes
a b s t r a c t A carbon-paste electrode modified with multiwall carbon nanotubes (MWCNTs) was used for the sensitive and selective voltammetric determination of ascorbic acid (AA) in the presence of 3,4-dihydroxycinnamic acid (3,4-DHCA) as mediator. The mediated oxidation of AA at the modified electrode was investigated by cyclic voltammetry (CV), chronoamperommetry and electrochemical impedance spectroscopy (EIS). Also, the values of catalytic rate constant (k), and diffusion coefficient (D) for AA were calculated. Using square wave voltammetry (SWV), a highly selective and simultaneous determination of AA, acetaminophen (AC) and tryptophan (Trp) has been explored at the modified electrode. The modified electrode displayed strong function for resolving the overlapping voltammetric responses of AA, AC and Trp into three well-defined voltammetric peaks. In the mixture containing AA, AC and Trp, the three compounds can well separate from each other with potential differences of 200, 330 and 530 mV between AA and AC, AC and Trp and AA and Trp, respectively, which was large enough to determine AA, AC and Trp individually and simultaneously. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Electroanalytical applications, which utilize chemically modified electrodes (CMEs), should offer a potentially significant efficiency [1]. Recently there has been a considerable effort in the investigation of catalytic function of CMEs on the oxidation of biologically important compounds, such as tryptophan [2], N-acetylcysteine [3], amitrole [4] and 6-thioguanine [5]. It is generally believed that direct redox reactions of these species at bare electrodes are totally irreversible and therefore require a high overpotential. Therefore, there have been numerous attempts to enhance the electrode kinetics using various CMEs [6–8]. Multiwall carbon nanotubes (MWCNTs) are now used extensively in the fabrication of novel nanostructure electrochemical sensors [9–15]. MWCNT-modified electrodes have many advantages over other forms of carbon electrodes due to their small size, high electrical and thermal conductivity, high chemical stability, high mechanical strength and high specific surface area which make them very promising candidates in a wide range of applications [16–20]. Ascorbic acid or vitamin C is distributed widely in both plant and animal kingdoms. In vegetable cells, it bound to protein as ascorbigen. Among animal organs, the liver, leukocytes and anterior pituitary lobe show the highest concentrations of ascorbic acid. Vitamin C also is
⁎ Corresponding author. Tel.: +98 6732054 (cell phone); fax: +98 311 6732054. E-mail address: [email protected] (M. Keyvanfard). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.11.005
present in many other biological systems and multivitamin preparations, which are commonly used to supplement inadequate dietary intake. Nevertheless, it is widely used in foods as an antioxidant for the stabilization of color and aroma with subsequent extension of the storage time of the products [21,22]. The determination of AA has been established by such methods as chromatography [23–26], spectrophotometry [27–29], mass spectrometry [30], flow injection [31,32], chemiluminescence [33] and electrochemical methods [34–37]. Acetaminophen, paracetamol or N-acetyl-p-aminophenol [38,39] is a mild, safe, widely used analgesic and antipyretic substitute of aspirin. However, it causes liver necrosis in humans and experimental animals when high doses are administered [40]. To date, various techniques including titrimetry [41,42], spectrophotometry [43–46], high performance liquid chromatography [47–50], fluorimetry [51] and electrochemical methods [52–54] have been reported for the determination of acetaminophen. In therapeutic applications, acetaminophen is often found in association with ascorbic acid and/or other pharmacologically and biologically active compounds [55]. Tryptophan is a vital amino acid for humans and herbivores. This compound is a precursor for serotonin (a neurotransmitter), melatonin (a neurohormone), and niacin [56]. This compound is sometimes added to dietary, food products, pharmaceutical formulas due to the scarcely presence in vegetables [57]. Different methods such as spectrophotometry, high performance liquid chromatography (HPLC), and electrochemical methods have so far been available for the determination of Trp [58–60].
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High doses of vitamin C may lower the amount of acetaminophen passed in urine, which could cause the levels of this drug in your blood to rise [61]. Lactose and ascorbic acid did not have tryptophan contamination when used as anti-oxidizing reagents. Both improved tryptophan recovery, but ascorbic acid was more effective [62,63]. To the best of our knowledge, most previously published electrochemical studies have dealt with individual determination of AA, AC or Trp utilizing carbon paste electrodes or other kinds of modified electrode. Only one study has been reported on the simultaneous determination of AA, AC and Trp in the presence of each other using modified carbon nanotube paste electrodes [64]. In this study, we proposed 3,4-DHCA as a novel mediator for the rapid, sensitive, and highly selective voltammetric determination of AA on the surface of a multiwall carbon nanotube paste electrode. We also evaluate the analytical performance of the modified electrode for quantification of AA in the presence of AC and Trp. 2. Experimental 2.1. Reagents and apparatus All chemicals used were of analytical reagent grade purchased from Merck (Darmstadt, Germany) unless otherwise stated. Doubly distilled water was used throughout. Ascorbic acid was used from Merck (Darmstadt, Germany), acetaminophen and tryptophan from Fluka, all used as received. Phosphate buffer (phosphoric acid, sodium dihydrogen phosphate; disodium monohydrogen phosphate and trisodium phosphate plus sodium hydroxide, 0.1 mol L −1) solutions with different pH values were used. Spectrally pure graphite powder (particle size b 50 μm) from Merck and multiwall carbon nanotubes (> 90%, MWCNTs, d × l = (100 − 70 nm) × (5 − 9 μm)) from Fluka was used as the substrate for the preparation of the carbon paste electrode. High viscosity paraffin (d = 0.88 kg L − 1) from Merck was used as the pasting liquid for the preparation of the paste electrodes. All the voltammetric measurements were performed using an Autolab PGSTAT 302 N, potentiostat/galvanostat (Utrecht, The Netherlands) connected to a three-electrode cell, Metrohm (Herisau, Switzerland) Model 663 VA stand, linked with a computer (Pentium IV, 1200 MHz) and with Autolab software. A platinum wire was used as the auxiliary electrode. Multiwall carbon nanotube paste electrode (MWCNTPE) and Ag/AgCl/KClsat were used as the working and reference electrodes, respectively. The electrode prepared with carbon nanotubes was characterized by scanning electron microscopy (SEM) (Seron Tech. AIS 2100). A digital pH/mV-meter (Metrohm model 710) was applied for pH measurements. 2.2. Preparation of the electrode Graphite powder (0.900 g) was dispersed in diethyl ether and hand mixed with 0.100 g carbon nanotubes in a mortar and pestle. The solvent was evaporated by stirring. A syringe was used to add paraffin to the mixture, which was mixed well for 40 min until a uniformly wetted paste, was obtained. The paste was then packed into a glass tube. Electrical contact was made by pushing a copper wire down the glass tube into the back of the mixture. When necessary, a new surface was obtained by pushing an excess of the paste out of the tube and polishing it on a weighing paper. 2.3. Preparation of practical samples Urine sample was stored in a refrigerator immediately after collection. Ten milliliters of the sample was centrifuged for 15 min at 3000 rpm. The supernatant was diluted 10 times with a phosphate buffer solution (PBS) (pH 6.0). The solution was transferred into the
voltammetric cell to be analyzed without any further pretreatment. The standard addition method was used for the determination of ascorbic acid in practical samples. For the tablets, an accurately weighed portion of finely powdered sample obtained from five tablets (Daro pakhsh Co.), equivalent to about 50 mg of ascorbic acid dissolved in 100 mL water with ultrasonication. Then, 0.1 mL of the solution plus 9.9 mL of the buffer (pH 6.0) was used for the analysis with standard addition method. For injection solution, an accurate ampoule ((standard content 100 mg per mL AA, 5 mL per injection) (Daro Pakhsh Co.)) volume equivalent to about 50 mg of vitamin C was transferred to a 25 mL flask and diluted to volume with PBS. A 0.5 mL portion of the solution was subjected for voltammetric measurement as described for tablets. Fresh fruit juices were obtained using a mechanical squeezer. The juices obtained were filtered. A 0.5 mL portion of the filtrate juice was added to the supporting electrolyte solution in voltammetric cell. Vegetable juices were obtained using a grater and a centrifuge respectively, a 0.5 mL portion of vegetable juice was subjected for the voltammetric measurement. In all cases the amounts of vitamin C in the samples were evaluated by the standard addition method. 3. Results and discussion 3.1. SEM characterization Fig. 1 shows SEM images for MWCNTPE and carbon paste electrode (CPE). As can be seen at a surface of CPE (Fig. 1A), the layer of irregularly flakes of graphite powder was present and isolated with each other. After the multiwall carbon nanotubes (MWCNTs) were added to the carbon paste, it can be seen that MWCNTs were distributed on the surface of electrode with special three-dimensional structure (Fig. 1B), indicating that the MWCNTs were successfully modified on the MWCNTPE. 3.2. Electrochemistry of 3,4-DHCA The electrochemical behavior of the 3,4-DHCA was characterized by cyclic voltammetry. The experimental results showed well defined and reproducible anodic and cathodic peaks related to 3,4-DHCA(red)/ 3,4-DHCA(ox) redox couple with a quasi reversible behavior and with a peak separation potential of ΔEp = (Epa − Epc) = 100 mV. These cyclic voltammograms were used to examine the variation of the peak currents vs. the square root of potential scan rates. The plot of the anodic peak current was linearly dependent on ν 1/2 with a correlation coefficient of 0.9937 at all scan rates. 3.3. Catalytic effect As shown in Fig. 2, the anodic peak potential for AA oxidation at the MWCNTPE in the presence of 3,4-DHCA (curve a) and CPE in the presence of 3,4-DHCA (curve b) was about 310 mV, while at the MWCNTPE (without mediator) (curve d), the peak potential was about 430 mV. At the unmodified CPE and without mediator, the peak potential was about 450 mV of AA (curve e). From these results, it was concluded that the best electrocatalytic effect for AA oxidation was observed at the MWCNTPE in the presence of 3,4-DHCA (curve a). For example, results show that the peak potential for AA analysis at the MWCNTPE in the presence of 3,4-DHCA (curve a) shifted by about 120 and 140 mV toward negative values when compared with that at the MWCNTPE and CPE without mediator (curve d and e), respectively. Similarly, when comparing the oxidation of AA at the MWCNTPE (curve a) and CPE (curve b) in the presence of mediator, a dramatic enhancement of the anodic peak current at the MWCNTPE relative to that obtained at the CPE in the presence of mediator was observed. In other words, the data clearly show that the combination of multiwall carbon nanotubes and mediator definitely improve the characteristics of AA oxidation. The 3,4-DHCA at a surface of MWCNTPE, in 0.1 M PBS (pH 6.0)
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Fig. 1. SEM image of A) CPE and B) MWCNTPE.
and without AA in solution, exhibited a well-behaved redox reaction (curve c) upon addition of 300 μmol L−1 AA. The process corresponds to an EC' (catalytic) mechanism (Scheme 1), where the electrochemically formed 3,4-DHCA(Ox) reacts chemically with AA diffused toward the electrode surface, while the simultaneous oxidation of the regenerated 3,4-DHCA(Red) causes an increase in the anodic current. For the same reason, the cathodic current of the modified electrode is smaller in the presence of AA. The effect of the scan rate on the electrocatalytic oxidation of 750 μmol L−1 AA at the MWCNTPE was investigated by cyclic voltammetry in the presence of 3,4-DHCA. The oxidation peak potential shifts with increasing scan rates towards a more positive potential, confirming the kinetic limitation of the electrochemical reaction. Also, a plot of peak height (Ip) against square root of scan rate (ν1/2), in range of 3–20 mV s −1, was constructed, which was found to be linear, suggesting that at sufficient overpotential the process is diffusion rather than surface controlled. To obtain information about the rate-determining step, the Tafel plot was drawn, as derived from points in the Tafel region of the cyclic voltammogram. The slope of the Tafel plot was equal to n(1 − α)F/ 2.3RT (where n is number of electron in steady state stage and α is charge transfer coefficient), which came up to 9.95 decade V−1. Therefore, we obtained the value of α equal to 0.41. In addition, the value of α was calculated for the oxidation of AA at pH 6.0 for both the MWCNTPE
in the absence and presence of mediator using the following Equation [65]: α nα ¼ 0:048= EP –EP=2
ð1Þ
where EP/2 is the potential corresponding to IP/2. The values for α were found to be 0.20 and 0.42 at the surface of the MWCNTPE in the absence and presence of mediator, respectively. Those values show that the over-potential of AA oxidation is reduced at the surface of MWCNTPE in the presence of mediator, and also that the rate of electron transfer process is greatly enhanced. This phenomenon is, thus, confirmed by the larger Ipa values recorded during cyclic voltammetry at MWCNTPE in the presence of mediator (Scheme 2). For the determination of the diffusion coefficient and the catalytic reaction rate constant of AA, double potential step chronoamperometry was used with MWCNTPE in the presence of mediator. Fig. 3A shows the current–time curves of MWCNTPE in the presence of mediator by setting the electrode potential at 200 mV (first step) and 450 mV (second step) for different AA concentrations. As can be seen, there is no net anodic current corresponding to the oxidation of the mediator in the presence of AA. On the other hand, the forward and backward potential step chronoamperometry for the mediator in the absence of AA shows symmetrical chronoamperogram with an equal charge consumed for the reduction and oxidation of the mediator at the surface of MWCNTPE (Fig. 3C, a /). Also, the charge value associated with forward chronoamperometry in the presence of AA is significantly greater than that observed for backward chronoamperometry (Fig. 3C, b /–f /). The rate constant for the chemical reaction between AA and mediator at a surface of MWCNTPE, kh, can be evaluated by chronoamperometry according to the method of Galus [66]: 1=2 1=2
IC =IL ¼ π
γ
¼π
1=2
1=2
ðkh Cb tÞ
ð2Þ
where IC is the catalytic current of AA at MWCNTPE in the presence of mediator, IL is the limited current in the absence of AA, and t is the time elapsed (s). The above equation can be used to calculate the rate constant of the catalytic process kh. Based on the slope of the IC/IL vs. t1/2 plots (Fig. 3B), kh can be obtained for a given AA concentration. Based on the values of the slopes, the average value of kh was found to be equal to 2.198×103 mol L−1 s−1. The value of kh explains the sharp feature of the catalytic peak observed for catalytic oxidation of AA at the surface of MWCNTPE in the presence of mediator. 3.4. Calibration plot and limit of detection
Fig. 2. Cyclic voltammograms of (a) 500 μmol L−1 3,4-DHCA + 300 μmol L−1 AA at the surface of MWCNTPE; (b) 500 μmol L−1 3,4-DHCA + 300 μmol L−1 AA at the surface of carbon paste electrode; (c) the 500 μmol L−1 3,4-DHCA at the surface of MWCNTPE in 0.1 mol L−1 PBS (pH 6.0); (d) 300 μmol L−1 AA at the surface of MWCNTPE; (e) 300 μmol L−1 AA at the surface of carbon paste electrode, scan rate of 20 mV s−1.
Since square wave voltammetry (SWV) has a much higher current sensitivity and better resolution than cyclic voltammetry, SWV was used for simultaneous determination of AA, AC and Trp. In order to get the best sensitivity under the specific conditions, an amplitude potential of 50 mV and frequency of 10 Hz were selected, respectively. The plot of peak current vs. AA concentration consisted of two linear
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Scheme 1. Electrocatalytic mechanism for determination of AA at the surface of MWCNTPE in the presence of 3,4-DHCA.
segments with slopes of 37.8710 and 0.1128 μA/μmol L−1 in the concentration ranges of 0.02–0.1 μmol L−1 and 0.1–140.0 μmol L−1, respectively. The decrease in sensitivity (slope) of the second linear segment is likely due to kinetic limitation (Fig. 4A and B). On other hand, the responses were linear with AC concentration in the range from 2.0 to 400.0 μmol L−1 and the current sensitivity was 0.1025 μA/μmol L−1 (Fig. 4C), while the dynamic range was linear with Trp concentration in the range 5.0 to 500 μmol L − 1 and the current sensitivity was 0.0963 μA/μmol L − 1 (Fig. 4D). Detection limits were determined as 9.1 nmol L − 1AA, 0.8 μmol L − 1 AC and 1.0 μmol L − 1 Trp based on YLOD = YB + 3σ [67]. This value of detection limit and the linear dynamic range for AA observed for the MWCNTPE in the presence of mediator are comparable and even better than those obtained for several other modified electrodes (Table 1).
and presence (0.1116 μA μM−1) of AC and Trp are virtually the same, which further indicate that the oxidation processes of AA, AC and Trp at the MWCNTPE in the presence of mediator are independent and therefore, simultaneous measurements of the three analytes are feasible without any interference.
4. Interference study In order to evaluate the selectivity of the proposed method in the determination of AA the influence of various foreign species on the determination of 1.0 μmol L −1 AA was investigated. Tolerance limit was taken as the maximum concentration of foreign substances that
3.5. Simultaneous determination of AA, AC and Trp One of the main objectives of this study was to develop a modified electrode with the capability of separating the electrochemical responses of AA, AC and Trp. Therefore, SWV was used for the simultaneous determination of AA, AC and Trp. Using MWCNTPE in the presence of mediator as the working electrode, the analytical experiments were carried out by varying the concentration of AC, AC or Trp in 0.1 M PBS (pH 6.0). The SW voltammetric results show three well-distinguished anodic peaks at potentials of 400, 600 and 930 mV, corresponding to the oxidation of AA, AC and Trp, respectively, indicating that the simultaneous determination of AA, AC and Trp is possible at the MWCNTPE in the presence of mediator (Fig. 5). The sensitivities of the modified electrode towards AA in the absence (0.1128 μA μM−1)
Scheme 2. The equivalent circuit compatible with the Nyquist diagram recorded in the absence and presence of AA.
Fig. 3. A) Chronoamperograms obtained at the MWCNTPE in the absence a) and in the presence of b) 100, c) 200, d) 300, e) 400 and f) 500 μmol L−1 AA in a buffer solution (pH 6.0). B) Dependence of Ic/IL on the t1/2 derived from the chronoamperogram data. C) The charge-time curves a/) for curve (a); b/) for curve (b); c/) for curve (c); d/) for curve (d); (e/) for curve (e) and (f/) for curve (f).
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Fig. 4. Plots of Ip vs. A) AA concentration in the range 0.02–0.1; B) AA concentration in the range 0.1–140.0 μmol L−1; C) AC concentration in the range 2.0–400.0 μmol L−1; and D) Trp concentration in the range 5.0–500.0 μmol L−1.
caused an approximate relative error of ±5%. The results are shown in Table S1 (Supporting information). 5. Practical samples analysis In order to demonstrate the ability of the modified electrode to determine AA, AC and Trp in practical samples, these compounds were determined in tablet, ampoule, fruit juice, and vegetable juice and urine samples. The results are presented in Table 2. Clearly, modified electrode is capable of voltammetric determination of AA, AC and Trp with high selectivity and good reproducibility.
AA determination compare to other electrochemical sensor. Finally, this modified electrode used for determination of AA in practical samples with satisfactory results. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.msec.2012.11.005.
Acknowledgments The authors wish to thank Shahreza and Majlesi Branch, Islamic Azad University, for their support.
6. Conclusion A new voltammetric sensor developed for the determination of AA is very rapid, reproducible, highly selective and sensitive, and can be used for practical sample analysis. This mediator (3,4-DHCA) shows excellent catalytic effects on the oxidation of AA. The results show that the oxidation of AA is catalyzed at pH 6.0, whereas the peak potential of AA is shifted by 140 mV to a less positive potential at the surface of the MWCNTPE in the presence of mediator. Potential differences of 200, 330 and 530 mV between AA and AC, AA and Trp and AC and Trp were detected, respectively which were large enough to determine AA, AC and Trp individually and/or simultaneously. Results show better linear dynamic range, detection limit or sensitivity for
Table 1 Comparison of the efficiency of some reported electrochemical methods in the determination of AA. Method
pH
Limit of detection (μmol L−1)
Linear dynamic range (μmol L−1)
Sensitivity μA/μmol L−1
Ref.
Amperometry Differential pulse voltammetry Differential pulse voltammetry Differential pulse voltammetry SWV SWV
5.0 6.8
8.0 20.0
8.8–23.2 50.0–740
0.082 –
[68] [69]
7.0
4.2
9.0–350.0
0.0078
[70]
7.0
0.075
0.1–800.0
0.0195
[71]
7.0 6.0
0.01 0.009
0.03–70.0 0.02–140.0
0.1126 37.871
[58] This work
Fig. 5. SWVs of MWCNTPE in the presence of 500 μmol L−1 3,4-DHCA in 0.1 M PBS (pH 6.0) containing different concentrations of AA–AC–Trp in μmol L−1, from 1 to 6: 6.0+20.0+10.0; 30+40.0+50.0; 70.0+100.0+130.0; 90.0+120.0+150.0; 115.0+ 170.0+180 and 140.0+200.0+205.0, respectively.
816
M. Keyvanfard et al. / Materials Science and Engineering C 33 (2013) 811–816
Table 2 Determination of AA, AC and TP in practical samples (n = 3). Sample
AA added (μmol L−1)
AC added (μmol L−1)
TP added (μmol L−1)
Found (AA) proposed method (μmol L−1)
Found (AA) reference method (μmol L−1) [68]
Found (AC) proposed method (μmol L−1)
Found (AC) published method (μmol L−1) [10]
Found (TP) proposed method (μmol L−1)
Found (TP) published method (μmol L−1) [2]
Tablet
5.00 15.00 – 6.00 10.00 0.10 0.50 – – – – – – –
– – – 20.00 30.00 – – – – – – – – –
– – – 10.00 20.00 – – – – – – – – –
5.21 ± 0.30 15.30 ± 0.33 bLOD 6.33 ± 0.43 10.45 ± 0.51 0. 11 ± 0.02 0.52 ± 0.03 – 148.35 ± 0.82 75.51 ± 0.85 11.01 ± 0.63 – 43.55 ± 0.71 80.21 ± 1.11
4.72 ± 0.41 15.87 ± 0.95 bLOD 6.58 ± 0.72 10.64 ± 0.68 10.22 ± 0.33 0.54 ± 0.05 – 149.10 ± 1.35 75.91 ± 0.93 11.38 ± 1.01 – 44.51 ± 1.23 81.02 ± 1.25
– – bLOD 20.25 ± 0.31 30.44 ± 0.52 – – – – – – – – –
– – bLOD 20.45 ± 0.50 29.89 ± 0.72 – – – – – – – – –
– – bLOD 10.32 ± 0.41 20.32 ± 0.45 – – – – – – – – –
– – bLOD 10.52 ± 0.63 20.75 ± 0.84 – – – – – – – – –
Urine
Injection Fruit juices Orange Kiwi Apple Vegetable juices Pimento Dill
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