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Electrochemical behavior of poly (naphthol green B)-film modified carbon paste electrode and its application for the determination of dopamine and uric acid
Journal of Electroanalytical Chemistry 667 (2012) 66–75
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Electrochemical behavior of poly (naphthol green B)-film modified carbon paste electrode and its application for the determination of dopamine and uric acid S. Chitravathi a, B.E. Kumara Swamy a,⇑, G.P. Mamatha b, B.S. Sherigara a a b
Department of P.G. Studies and Research in Industrial Chemistry, Kuvempu University, Jnana Sahyadri, Shankaraghatta 577 451, Karnataka, India Department of Pharmaceutical Chemistry, Kadur, Kuvempu University, Karnataka, India
a r t i c l e
i n f o
Article history: Received 17 June 2011 Received in revised form 19 October 2011 Accepted 13 November 2011 Available online 6 December 2011 Keywords: Dopamine Uric acid Naphthol green B Carbon paste electrode Cyclic voltammetry Differential pulse voltammetry
a b s t r a c t A polymerized film of naphthol green B was prepared on the surface of a carbon paste electrode in Britton–Robinson (BR) buffer solution by electropolymerization technique. Electrochemical investigation of the resulting film was studied by cyclic voltammetric method. Several factors affecting the electrocatalytic activity of the hybrid material such as, effect of pH, scan rate and concentration were studied. The results suggested that the modified electrode exhibited enhanced sensitivity and selectivity towards the oxidation of dopamine (DA) and uric acid (UA). Well defined and separated oxidation peaks were observed by cyclic voltammetry and differential pulse voltammetry. Linear calibration plots for the oxidation of DA and UA were obtained in the range of 5 106 M to 2.7 104 M for DA and 12.5 106 M to 7.5 104 for UA, with a correlation co-efficient of 0.991 and 0.995, respectively. Detection limits of DA and UA were found to be 0.25 ± 0.05 lM and 5 ± 0.04 lM, respectively. The analytical performance of this sensor has been evaluated for detection of DA and UA in real samples. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Dopamine (DA) is one of the crucial catecholamine neurotransmitter and extracellular messenger distributed in the mammalian central nervous system (CNS) and renal systems. It plays an important role in the function of CNS, renal, hormonal and cardiovascular systems. Thus its deficiency may lead to neurological disorders such as Parkinsonism and schizophrenia [1–4] and to HIV infection [3,5]. Uric acid (UA) (2,6,8-trihydroxypurine) is the primary product of purine metabolism in the human body. Abnormal concentration levels of UA can lead to several disorders such as gout, Lesch–Nyhan syndrome, hyperuricaemia, cardiovascular disease, and multiple sclerosis [6,7]. Thus extreme abnormalities of DA and UA concentrations levels may lead to several diseases and it is essential to develop a simple and rapid method for the determination of both DA and UA for routine analysis. Usually, in the extra cellular fluid of the central nervous system, DA exists in only a nanomolar to micromolar range (0.01–1 lM) [8,9] and the amount of uric acid present in the serum range from 240 to 520 lM and in urine it is estimated to be 250–750 mg/24 h [10]. The adsorption or coating of polymeric species or films onto the surface of conventional substrates has become a preferred approach for the construction of chemically modified electrodes (CMEs). In recent years, polymer film-modified electrodes have at⇑ Corresponding author. Tel.: +91 8282 256225 (O); fax: +91 8282 256255. E-mail address: [email protected] (B.E. Kumara Swamy). 1572-6657/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2011.11.017
tracted great attention, because of their good stability, reproducibility and their wide applications in the fields of chemical sensors and biosensors [11–14]. When polymer modified electrodes (PMEs) were used, carbon-base electrodes have been primarily used compared to metal electrodes due to its biocompatibility with tissue, having low residual current over a wide potential range and minimal propensity to show a deteriorated response as a result of electrode fouling. Up to now different methodologies have been used to prepare polymeric film-modified electrodes. Among them electropolymerization yields a modified electrode with a three-dimensional distribution of mediators. This type of electrodes enhances the sensitivity and improves the catalytic activity than monolayers. A variety of examples of the electrochemical determination of DA and UA along with AA have been proposed. These include a carbon paste electrode using pyrogallol red as a mediator [15], 2-amino-5-mercapto-[1,3,4] triazole self-assembled monolayers gold electrode [16], indole-3-carboxaldehyde glassy carbon electrode [17], cobalt salophen-modified carbon-paste electrode [18], p-aminobenzene sulfonic acid functionalized glassy carbon electrode [19], phosphorylated zirconia-silica composite electrode [20], 2,20 -[1,2-ethanadiylbis(nitriloethylidyne)]-bis-hydroquinone-carbon nanotube paste electrode [21] and poly (caffeic acid)modified glassy carbon electrode [22]. Naphthol green B (1-nitroso-2-naphthol-6-sodium sulphonate ferric salt, NGB) (Scheme 1) is an important commercial green nitroso dye and it is the sodium salt of naphthol green Y. As an anion,
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2.2. Apparatus Electrochemical measurements were carried out with a model201 electrochemical analyzer (EA-201 Chemilink system) in a conventional three-electrode system. The working electrode was a carbon paste electrode, having cavity of 3 mm diameter. The counter electrode was a bright platinum wire with a saturated calomel electrode (SCE) as a standard electrode completing the circuit.
2.3. Preparation of bare carbon paste electrode
Scheme 1. Structure of naphthol green B.
it acts as acid dye and possesses excellent redox characteristics. Such dye is able to undergo electropolymerization from aqueous solution, producing stable redox active layers [23,24]. Thus it can be used as a mediator for electro catalysis of biological compounds because of its high electron transfer efficiency and low cost [25–27]. Few reports have been reported where NGB was used as a mediator for the analysis of some neurotransmitters [28–31]. In the present work, an electropolymerized film of NGB was synthesized, studied and was applied for the determination of DA in presence of UA. These modified electrode allow for better sensitivity and selectivity for the determination of DA in the presence of UA and proved better than unmodified electrode. Thus the fabrication of the modified electrode has been considered in detail. This modified electrode was used for the determination of DA and UA in pharmaceutical and urine samples (see Scheme 2).
The carbon paste electrode was prepared by hand-mixing 70% graphite powder and 30% silicon oil in an agate mortar for about 30 min to get homogeneous carbon paste. The paste was then packed into the cavity of a Teflon tube electrode (3 mm diameter). Before measurement, the modified electrode was smoothened on a piece of transparent paper to get a uniform, smooth and fresh surface.
2.4. Preparation of the NGB polymer film modified carbon paste electrode The polymer film-modified electrode was fabricated by electrochemical polymerization of NGB by cyclic voltammetry in the potential range 500 to 1200 mV at a sweep rate of 100 mV/s in B–R buffer solution (pH 3.2). The monomer concentration was usually 0.5 mM. After 10 cycles, the surface of the electrode was washed with doubly distilled water to remove the physically adsorbed material. This modified electrode was immersed in PBS (pH 7.0) and electrochemical studies were carried out. Poly (NGB) film with different thickness was achieved by altering scan cycles during polymerization process.
2. Experimental 2.1. Reagents and materials
2.5. Electrochemical measurements
DA, UA, NGB were purchased from Himedia Chemicals and were of analytical grade. The electropolymerization of NGB was performed in 0.1 M (B–R) buffer solution. Phosphate buffer solution (0.033 M, pH 7.0) was prepared from KH2PO4 and K2HPO4 and the pH was adjusted with 0.05 M NaOH. Other chemicals used were of analytical grade except for spectroscopically pure graphite powder. All solutions were prepared with doubly distilled water. Freshly prepared DA and UA solutions were used prior to measurements.
Electrochemical determination of DA and UA was carried out in a voltammetric cell with 20 mL of supporting electrolyte solution. Standard stock solution of DA and UA was added to the cell according to the requirement. Cyclic voltammograms and differential pulse voltammograms from 50 mV to +600 mV were recorded in PBS (pH 7.0) at the given scan rate for the determination of DA and UA. The same procedure was applied for the samples analysis and all electrochemical measurements were carried out at room temperature.
Scheme 2. Oxidation mechanisms of DA and UA.
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shown in the figure, the current response is maximum up to scan number 10 and decreases thereafter. However, after 10 cycles, poly (NGB) covers the electrode surface completely and active area does not change significantly thereafter and any further increase in the scan number will result in the decrease in the redox peak current. Hence, we selected 10 cycles as the optimum scan number for the film formation process in this study.
Fig. 1. Continuous cyclic voltammograms for the electrochemical polymerization of 0.5 mM NGB on a CPE in B–R buffer solution (pH 3.2) at the scan rate 100 mV/s.
3. Results and discussion 3.1. Optimum conditions for preparation and other electrochemical activity of poly (NGB)-film 3.1.1. Electropolymerization of NGB on a CPE Electropolymerization of NGB was performed on a CPE. Fig. 1 displays the continuous cyclic voltammetric sweeps of 0.5 mM NGB monomer in B–R buffer solution (pH 3.2) by scanning over the range of 500 to 1200 mV at a scan rate of 100 mV/s for 10 cycles. During the electropolymerization process two oxidation peaks Pa1 and Pa2 at 155 mV and 590 mV respectively were observed in the anodic scan and four reduction peaks Pc1, Pc2, Pc3 and Pc4 at 550, 30, 116 and 250 mV respectively in reverse reduction scan from the first scan onwards due to the formation of poly (NGB). The peaks Pa2 and Pc1 are may be due to Fe2+/Fe3+ couple, because as an Fe(III) compound NGB in aqueous solution undergo reduction from Fe(III) to Fe(II) in analogy to numerous other Fe(III) complexes [32–34]. In the following scans the larger peaks were observed upon continuous growth of the film. It was observed that the film growth was faster for the first 5 cycles. The oxidation and reduction peak currents increased with the increase of cyclic number of scans, indicating that an electroconductive polymer film was formed on the electrode surface. After electropolymerization, the modified electrode was carefully rinsed with doubly distilled water and then stored in pH 7 PBS. The thickness of the poly (NGB) film can be adjusted by controlling the cyclic number of voltammetric scans and the concentration of NGB. Fig. 2 shows the effect of scan number on the oxidation peak current (ipa) of 0.1 mM DA in PBS (pH 7.0). As
3.1.2. Electrochemical properties of the poly (NGB)-film modified electrode The electrochemical properties of poly (NGB)-film were tested through cyclic voltammetric method in PBS (pH 7). Fig. 3a exhibits the typical voltammogram of the poly (NGB) electrode which shows one redox couple with anodic and cathodic peak potentials at 0 mV and 56 mV respectively. The separation of peak potential (DEp = Epa Epc) was 56 mV which is close to 59/n mV at 25 °C which indicates that the equal number of electrons and protons are involved in the reaction. Electrode reaction of poly (NGB) film is a redox process and it can be suggested that the ligand itself may serve as electron donor between NGB/NGB on the surface of the electrode [35]. Fig. 3b shows the cyclic voltammograms of poly (NGB)-film modified electrode in the range of 10–300 mV/s. The redox peak currents obtained in each scan increased linearly with the increase of scan rates (Fig. 3c). The anodic (ipa) and cathodic (ipc) currents were linearly dependent on scan rates with the linear equations: ipa (lA) = 5.397 0.276 m (r2 = 0.998) and ipc (lA) = 16.07 + 0.3375 m (r2 = 0.996). These results showed that the reduction and oxidation of the NGB/NGB- couple is adsorption controlled. An approximate estimation of amount of incorporated NGB was calculated by the amount of surface coverage of the electroactive species on the electrode, and is given by the following equation [36].
ip ¼
n2 F 2 ACm 4RT
where C (M/cm2) represents the surface coverage concentration which is proportional to the peak current (ip), m is the scan rate, A is the geometric surface area of the electrode (0.028 cm2), n is the number of electrons involved in the reaction and R, F, T have their normal meanings. The surface concentration of poly (NGB) was determined to be 0.318 1010 M/cm2. 3.1.3. Stability and reproducibility of poly (NGB)-film modified electrode The main advantage of using the modified electrode is that the electrode surface can be renewed after every use by extrusion of approximately 0.5 mm carbon paste from the cavity of the Teflon rod and replacing it with a new paste. Indeed five successive renewing of a poly (NGB)-film modified CPE resulted in an RSD of 4.91%. The stability of the modified film was evaluated by examining the cyclic voltammetric peak currents of NGB after continuously scaning for 50 cycles in phosphate buffer solution (pH 7.0) (Fig. 4). The percentage degradation of poly (NGB)-film was calculated by the following equation,
%degradation ¼
Fig. 2. Dependence of the oxidation peak current of 0.1 mM DA on the number of voltammetric scans.
ipn 100 ip1
where ip1 and ipn are the first and nth anodic peak currents respectively and is found to be less than 1%, indicating that the modified electrode is stable. The stability of the film was also checked by measuring the current response over a period of 30 days, and it is found that the poly (NGB) electrode maintains 95% of its initial activity even after 30 days.
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Fig. 4. Successive cyclic voltammograms of a poly (NGB)-film electrode in PBS (pH 7.0).
[37,38]. Fig. 5 shows the electrochemical response of 1 mM potassium ferrocyanide at the bare (solid line) and at the poly (NGB)film modified CPE (dashed line) in 1 M KCl. The anodic and cathodic peaks of ferrocyanide shifted towards more positive and more negative respectively giving less reversible behavior at the modified electrode compared to bare electrode. It is apparent that electron transfer of negatively charged probe is partially blocked at modified electrode. This may be due to the repulsion interaction between negatively charged poly (NGB)-film and the negatively charged ferrocyanide redox probe. The current response of potassium ferrocyanide at poly (NGB) film-modified CPE noticeably improved indicating that the surface coverage property of the modified electrode has changed significantly resulting in favorable and stable electrochemical behavior. 3.3. The electrochemical behaviors of DA and UA at poly (NGB) modified CPE
Fig. 3. (a) A typical cyclic voltammogram of the redox peak of poly (NGB) –film electrode (curve ‘b’) with polymer blank (curve ‘a’). (b) Cyclic voltammograms of poly (NGB)-film electrode in the range of 10–300 mV/s in PBS (pH 7.0). (c) Variation of the anodic and cathodic peak currents with the scan rate.
Fig. 6a shows cyclic voltammograms of 0.1 mM DA at the bare (curve ‘a’) and at poly (NGB)-film modified (curve ‘c’), CPE along with poly (NGB) blank (curve ‘b’) in 0.033 M PBS (pH 7.0). DA exhibited poor current response with DEp, the difference between the anodic peak potential (Epa) and the cathodic peak potential (Epc) 64 mV. However a well defined redox wave of DA was observed at the poly (NGB)-film modified electrode with DEp, 38 mV which shows fast electron transfer kinetics. The oxidation peak potential shifted negatively from 185 mV to 174 mV and the reduction peak potential shifted positively from 121 mV to 136 mV. The obvious catalytic reaction accompanied by the increase in the peak current by almost 10 folds may be due to
3.2. Interaction of poly (NGB) film-modified CPE surface with potassium ferrocyanide Fe[(CN)6]3/4 was used as the electrochemical redox probe to investigate interfacial properties of poly (NGB)-film modified electrode. Usually modification of electrodes with charged species has remarkable effects on the electrochemical behavior of redox probe reactions. These effects depend on charge of both electrode surface and redox probe. More reversible behavior is observed for the charged probe redox reactions at the modified electrodes with opposite charge and less reversible behavior for the charged probe redox reactions at the modified electrodes with similar charge
Fig. 5. Cyclic voltammograms of 1 mM potassium ferrocyanide at the bare (solid line) and at the poly (NGB)-film modified CPE (dashed line) in 1 M KCl.
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3.4. Optimization of the experimental conditions 3.4.1. Influence of pH In most cases, the solution pH plays an important influence factor to the electrochemical reaction. The effect of varying of pH (2.0–9.0) towards the redox behavior of DA and UA at the poly
Fig. 6. (a) Cyclic voltammograms of 0.1 mM DA in pH 7.0 PBS at (a) bare (solid line), (c) poly (NGB)-film modified CPE (dashed line) and (b) polymer blank (without DA). (b) Cyclic voltammograms of 0.1 mM UA in pH 7.0 PBS at (a) bare (solid line) and (b) poly (NGB)-film modified CPE (dashed line).
electrostatic interaction between DA cations and anionic NGB, which lead to increase in the concentration of DA around the surface of the modified electrode. This shows that NGB is acting as an efficient electron transfer mediator between the DA and the carbon paste electrode surface. Certainly, the increased surface area due to the presence of polymer film and multielectroactive spots (quinoid structure) inside polymers which is reflected from the increased background in CV after poly (NGB)-film formation also plays an important role in the enhanced current. These results indicated that poly (NGB) film could accelerate the electrode process and facilitate the kinetics of the electron transfer of DA on the surface of the modified electrode. Fig. 6b shows the cyclic voltammograms of 0.1 mM UA at the bare (curve ‘a’) and at poly (NGB)-film modified (curve ‘b’) CPE in 0.033 M PBS (pH 7.0). It is clear from the figure that a small and broad oxidation peak at 320 mV was observed at the bare electrode and a well defined oxidation peak at same potential was observed at the poly (NGB)-film modified electrode. The current enhancement was 10 times higher than that at the bare electrode. It has been previously reported that UA exists in the neutral form in weak acidic buffer solution [39], accordingly in this paper poly (NGB)-film encapsulates UA and catalyzes its oxidation. The enhancement in current of UA reasonable to originate from the multielectroactive spots (quinoid structure) inside the polymers and big surface area due to the presence of poly (NGB)-film which result in the accumulation of UA molecules and improve the oxidation peak current.
Fig. 7. (a) Plot of anodic peak current vs. pH (2.0–9.0) of 0.1 mM DA at the poly (NGB)-film modified CPE. (b) Plot of Epa vs. pH for DA. (c) Plot of anodic peak current vs. pH (2.0–9.0) of 0.1 mM UA at the poly (NGB)-film modified CPE. (d) Plot of Epa vs. pH for UA.
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(NGB)-film modified CPE was studied. Both the anodic and cathodic peak currents of DA first decreased gradually with increasing pH up to pH 5.0, then the peak currents increased drastically from pH 5.0 to 7.0 with increase of pH value. Fig. 7a shows the plot of anodic peak current vs. the pH of the solution. This could be partly explained on the basis of the dissociation ability of ASO3Na group of poly (NGB) in different pH environment. When the solution pH was less than 5.0 the ANH2 group will be in undissociated form and the ASO3Na group of poly (NGB) film could dissociate favorably into a negative charge group ASO 3 . Under this condition the ANH2 group of DA molecule (pKa 8.9) [40] could obtain a proton. The negative charge group ASO 3 on the surface of poly (NGB)-film modified electrode had a well affinity to the DA positive ions. Therefore more DA cations were attracted to the electrode surface and promote oxidation of DA. The DA peak currents increased with
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increase of pH till 7.0 then the protonated degree of DA decreased with the increase of pH, and hence the current. In addition, the solution pH affected the anodic and cathodic peak potentials of DA. With pH increasing, their oxidation peak potentials shift to less positive values, showing that protons take part in their electrode reactions. The anodic peak potential (Epa) of DA was proportional with the solution pH in the range of 2.0–9.0 (Fig. 7b). The linear regression equation for the anodic peak is given by
Epa ¼ 0:5476 þ 0:05117 pH ðr2 ¼ 0:994Þ A slope of 51.17 mV/pH which is close to the theoretical value of 59 mV/pH showed that the equal number of protons and electrons are involved in the chemical reaction. Thus, solution pH 7.0 was taken for the following determination of DA.
Fig. 8. (a) Cyclic voltammograms of 0.1 mM DA at the poly (NGB)-film modified CPE in pH 7.0 PBS at various scan rates. From (a–h): 50, 100, 150, 200, 250, 300, 350 and 400 mV/s. (b) Plot of the anodic peak current of DA as a function of the scan rate. (c) Dependence of the logarithm of anodic peak current on logarithm of scan rate. (d) Cyclic voltammograms of 0.1 mM UA at the poly (NGB)-film modified CPE in pH 7.0 PBS at various scan rates. From (a–f): 100, 150, 200, 250, 300, and 350 mV/s. (e) Plot of the anodic peak current of UA as a function of the scan rate.
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Table 1 Comparison of efficiency of some modified electrodes for the simultaneous determination of DA and/or UA along with AA at different modified electrodes. Electrode
Modifier
Analyte
Detection limit (M) 7
Linear range (M) 6
Refs. 4
Carbon paste electrode
Pyrogallol red
DA UA AA
7.8 10 35 106 –
1.0 10 to 7 10 50 106 to 10 104 –
[15]
Gold electrode
2-Amino-5-mercapto-[1,3,4] triazole
DA UA AA
8 107 1 106 –
2.5 106 to 5 104 5 106 to 1 104 –
[16]
Glassy carbon electrode
Indole-3-carboxaldehyde
DA UA AA
17 107 4.99 106 –
10 106 to 1 104 10 106 to 1 104 –
[17]
Carbon paste electrode
Cobalt salophen
DA UA AA
7 107 – 5 107
1 106 to 1 104 – 1 106 to 1 104
[18]
Glassy carbon electrode
p-Aminobenzene sulfonic acid
DA UA AA
– 7.5 107 12 106
– 50 106 to 2.5 104 35 106 to 1.75 104
[19]
Composite electrode
Phosphorylated zirconia–silica
DA UA AA
1.7 106 3.7 106 8.3 106
6 106 to 1 104 22 106 to 35 105 1 104 to 16 104
[20]
Carbon nanotube
2,20 -[1,2-Ethanediylbis(nitriloethylidyne)]-bis-hydroquinone
DA UA AA
15 106 7.5 108
2 105 to 7 104 7 107 to 8 104
[21]
Glassy carbon electrode
Poly (caffeic acid)
DA UA AA
4 107 – 9 107
1 106 to 4 105 – 2 105 to 1.2 103
[22]
Carbon paste electrode
Poly (naphthol green B)
DA UA AA
2.5 107 5 106 –
5 106 to 2.7 104 12.5 106 to 7.5 104 –
This work
Fig. 7c and d shows the relationship between the pH and anodic peak current and pH and anodic peak potential respectively. From the figure, it was clear that the peak current of UA decreases with the increase of pH in the whole range from pH 2 to 9 and anodic peak potential (Epa) shifted towards the negative direction with increasing of pH up to 7 and becomes constant, which indicated that protons have taken part in electrode process. The linear regression equation for the anodic peak is given by Epa = 649.79– 43.65 pH (r2 = 0.989). The slope of 43.65 was close to the theoretical value indicated that the electrons and protons involved in the redox of UA were equal (1:1). In the view of determination of both DA and UA and also in order to carry out the reaction at physiological environment, pH 7.0 was chosen for further study. 3.4.2. Influence of scan rate Useful information involving electrochemical mechanism usually can be acquired from the relationship between peak current and scan rate. Fig. 8a shows the cyclic voltammograms of 0.1 mM DA at different scan rates from 50 to 400 mV/s at the poly (NGB)-film modified electrode and Fig. 8b shows relationship between scan rate and oxidation peak current at different scan rates. As shown in the figure the oxidation peak currents exhibited a good linear relationship between peaks current and scan rate. The linear regression equation was given by:
ipa ¼ 0:2855m þ 7:989 ðr 2 ¼ 0:999Þ This indicates that the electrode process was controlled by adsorption rather than diffusion. Furthermore the anodic peak potential shifted towards more positive and the cathodic peak potential shifted towards more negative with the increase in scan rate indicating the adsorption taking place at the electrode. The slope value indicates that two electron are involved in the reaction for DA at the poly (NGB)-film modified electrode. This is shown in Scheme 2(a). Further to prove this plot of log m and log ipa illustrated the linear relation (Fig. 8c) with the linear regression equation for anodic peak:
log ipa ¼ 0:860 log m þ 2:42 ðr 2 ¼ 0:999Þ The slope of 0.86 is close to the theoretically expected value of 1.0 for an adsorption controlled process [41]. Fig. 8d illustrated the cyclic voltammograms of 0.1 mM UA at the modified electrode in phosphate buffer (pH 7.0) at various scan rates and Fig. 8e shows relationship between scan rate and oxidation peak current of UA at different scan rates. The results showed that the anodic peak current was proportional to the scan rate in the range of 100– 350 mV/s indicating that the electrode reactions was controlled by adsorption. The linear regression equation is given by:
ipa ¼ 0:0592m þ 4:007 ðr 2 ¼ 0:995Þ
Table 2 Simultaneous determination of DA and UA in injection and human urine mixture sample in PBS (n = 5). Sample no.
1 2 3 4 5
DA added (lM)
0 10 20 30 40
UA added (lM)
0 10 20 30 40
DA
UA
Found (lM)
Recovery (%)
RSD (%)
Found (lM)
Recovery (%)
RSD (%)
39.31 49.53 59.60 68.95 79.25
– 100.44 100.48 99.48 99.92
2.4 1.39 1.43 2.04 2.17
24.82 34.93 44.45 54.75 65.02
– 100.31 99.17 99.87 100.30
1.71 2.27 1.89 2.12 1.85
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The slope value of 59.2 which is close to the theoretical value of 59 mV showed that the equal number of protons and electrons are involved in the chemical reaction. This is shown in Scheme 2(b). The oxidation peak potential shifted towards positive direction. 3.5. Simultaneous determination of DA and UA by cyclic voltammetry Fig. 9a shows the cyclic voltammograms of the mixture containing DA and UA at the bare CPE (curve ‘a’) and at the poly (NGB)-film modified CPE (curve ‘b’) in PBS (pH 7.0). As shown in the figure, at bare CPE DA and UA exhibited poor current response and irreversible electrochemical behaviors but when the modified electrode was used, the mixture displayed anodic peaks with the potential difference of about 138 mV. From the experimental results
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described above, it was known that in the mixture containing DA and UA, the oxidation peaks of the two compounds were clearly separated from each other with 10 folds current enhancement. If the concentrations of DA and UA increased synchronously, the anodic and cathodic peak currents of DA and anodic peak current of UA at the poly (NGB)-film increased synchronously, at the same time the anodic peak potentials of both the peaks shifts towards positive direction. Fig. 9b and c shows the linear relationship between the anodic peak current (ipa) with the concentration for DA and UA respectively. It can be seen that the peak currents for the two increased linearly with their concentrations. Under the optimum detection conditions, the oxidation peak currents were proportional to concentration of DA and UA in the range of 5 106 M to 2.7 104 M and 12.5 106 M to 7.5 104 M, respectively. The linear range and detection limits of this proposed method is compared with other existing methods with different modifiers in Table 1. These values are comparable with those of other researchers using different types of modifiers. The linear regression equations for DA and UA respectively were expressed as
ipa ðlAÞ ¼ 28:4 þ 96C
ðr 2 ¼ 0:997Þ
ipa ðlAÞ ¼ 2:7 þ 3:68C
ðr 2 ¼ 0:999Þ
The detection limit (LOD) of DA and UA at the poly (NGB)-film modified CPE from 5 different calibration curves were found to be 0.25 ± 0.05 lM for DA and 5 ± 0.04 lM for UA respectively [42–44]. The LOD was calculated using the following equation, LOD = 3 S/M where, S is the standard deviation and M is the slope (sensitivity) obtained from the calibration curves [45]. 3.6. Resolution of DA and UA by differential pulse voltammetry (DPV) Differential pulse voltammetry (DPV) was used to investigate the possibility of poly (NGB)-film modified electrode for the simultaneous determination of DA and UA. The current responses of these two changed simultaneously by changing the concentrations of DA and UA in a mixture. As illustrated in Fig. 10 the DPV responses of the modified electrode of DA and UA increased linearly with increase of their concentrations. The linear regression equations for DA and UA in the range of 10–70 lM and 5–35 lM simultaneously are given by,
Fig. 9. (a) Cyclic voltammograms of 0.1 mM DA and 0.1 mM UA mixture in pH 7.0 PBS at (a) bare and (b) at poly (NGB)-film modified CPE. (b) Calibration plot for the determination of DA at the poly (NGB)-film modified CPE in pH 7.0 PBS with the scan rate 100 mV/s. (c) Calibration plot for the determination of UA at the poly (NGB)-film modified CPE in pH 7.0 PBS with the scan rate 100 mV/s.
ipa ðlAÞ ¼ 3:63 þ 4:84C
ðr 2 ¼ 0:999Þ
ipa ðlAÞ ¼ 3:51 þ 4:79C
ðr 2 ¼ 0:998Þ
Fig. 10. Differential pulse voltammograms for the mixture containing DA and UA with different concentrations at the poly (NGB)-film modified CPE. DA concentrations: (a) 10, (b) 20, (c) 30, (d) 40, (e) 50, (f) 60 and (g) 70 lM. UA concentrations: (a) 5, (b) 10, (c) 15, (d) 20, (e) 25, (f) 30 and (g) 35 lM.
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injection USP (specified content of DA is 40 mg/ml) and 1 ml of human urine sample without any pretreatment were diluted to 100 ml with buffer respectively and diluted solutions were pipetted into each of series of 10 ml volumetric flasks and to this, different known standard concentrations of DA and UA solutions were added and diluted to the mark with pH 7.0 phosphate buffer. The cyclic voltammograms were recorded and the peak currents were measured for DA and UA. Fig. 12 shows a typical cyclic voltammogram for the simultaneous determination of 1:500 dilutions of DA and UA in DA hydrochloride injection USP and healthy human urine sample solution respectively. The obtained results were shown in Table 2 and the results were consistent well with the certified values, suggesting that the poly (NGB)-film electrode has a good precision and the proposed method can be efficiently applied to the simultaneous determination of DA and UA. Fig. 11. Differential pulse voltammograms of DA and UA in presence of AA at poly (NGB)-film modified CPE in pH 7.0 PBS with the scan rate 100 mV/s. (a) 0.1 mM DA, (b–e) 0.1 mM DA+ 0.1, 0.2, 0.3 and 0.4 mM AA, and (f) 0.1 mM UA.
Fig. 12. Typical cyclic voltammogram for the simultaneous determination of 1:500 dilution of DA and UA in DA hydrochloride injection USP and healthy human urine sample solution respectively.
3.7. Interference study The possible influence of AA which coexists with DA and UA in body fluids on the electrochemical behavior of DA and UA has been investigated. The effect of increasing concentration of AA on DA and UA at modified electrode was carefully examined by differential pulse voltammetry. Fig. 11 shows the differential pulse voltammograms of mixed solution of 0.1 mM DA, 0.1 mM UA and varying concentrations of AA (from b to e, 0.1, 0.2, 0.3 and 0.4 mM). As can be seen from the Figure, the oxidation peak current of DA changes only ± 8% and the peak potential did not change much. However, when the concentration of AA exceeds 0.4 mM this interference is constant and there is no obvious change in the oxidation currents of DA [46,47]. This means that in the extracellular fluid of central nervous system, where the AA level is usually present more than 100-folds of magnitude larger than DA, the poly (NGB)-film modified electrode can be used for the determination of DA and also in real samples. As shown in the figure the presence of AA has no effect on the UA signal. Moreover, AA shows no reduction peak in pH 7.0 PBS at the modified electrode by CV. This can also be used for the simultaneous determination of DA in presence of AA.
3.8. Analytical application The practical application of the modified electrode was illustrated by simultaneous determination of DA and UA in injection and human urine sample respectively. 1 ml of DA hydrochloride
4. Conclusion In this work, a poly (NGB) film modified CPE was fabricated and the characteristics of the electrode was studied. The modified electrode was used for the simultaneous determination of DA and UA in the PBS (pH 7.0) by CV and DPV techniques. The modified electrode showed good stability and sensitivity. Well-defined and discrete voltammetric oxidation peaks were observed. Linear calibration plots for the oxidation of DA and UA were obtained in the range of 5 106 M to 2.7 104 M for DA and 12.5 106 M to 7.5 104 M for UA with a correlation co-efficient of 0.991 and 0.995, respectively. The detection limits (LOD) of DA and UA at the poly (NGB)-film modified CPE were found to be 0.25 ± 0.05 lM and 5 ± 0.04 lM, respectively The possible interference of AA in the determination of DA and UA was also studied using the modified electrode and the proposed method has been practically and successfully applied for the simultaneous determination of AA and DA in real samples. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29]
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S. Chitravathi et al. / Journal of Electroanalytical Chemistry 667 (2012) 66–75 [30] E. Gurr, Synthetic dyes in biology, Medicine and Chemistry, Academic Press, London, 1971. [31] A. Mohadesi, M.A. Taher, Sens. Actuators B 123 (2007) 733–739. [32] V. Balzani, V. Carassiti, Photochemistry of Coordination Compounds, Academic Press, London, 1970, p. 161. [33] O. Horvath, K.L. Stevenson, Charge Transfer Photochemistry of Coordination Compounds, VCH, New York, 1992. p. 218. [34] J. Sima, J. Makanova, Coord. Chem. Rev. 160 (1997) 161–189. [35] H. Kunkely, A. Vogler, Z. Naturforsch 58 (9) (2003) 922–924. [36] M. Sharp, M. petersson, K. Edstrom, J. Electroanal. Chem. 95 (1979) 123–130. [37] M. Kooshki, E. Shams, Anal. Chim. Acta 587 (2007) 110–115. [38] R. Karimi-Shervedani, M. Bagherzadeh, S.A. Mozaffari, Sens. Actuators B 115 (2006) 614–621. [39] S. Wu, T. Wang, Z. Gao, H. Xu, B. Zhou, C. Wang, Biosens. Bioelectron. 23 (2008) 1776–1780. [40] H. Yao, Y. Sun, X. Lin, Y. Tang, L. Huang, Electrochim. Acta 52 (2007) 6165– 6171.
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[41] R.N. Hegde, N.P. Shetty, S.T. Nandibewoor, Talanta 79 (2009) 361–368. [42] G. Alarcon-Angeles, S. Corona-Avendano, M.T. Ramirez-Silva, A. RojasHernandez, M. Romero-Romo, M. Palomar-Pardave, Electrochim. Acta 53 (2008) 3013–3020. [43] S. Corona-Avendano, M.T. Ramirez-Silva, M. Palomar-Pardave, L. HernandezMartinez, M. Romero-Romo, G. Alarcon-Angeles, J. Appl. Electrochem. 40 (2010) 463–474. [44] S. Corona-Avendano, G. Alarcon-Angeles, M.T. Ramirez-Silva, M. RomeroRomo, A. Cuan, M. Palomar-Pardave, J. Electrochem. Soc. 156 (12) (2009) J375– J381. [45] U. Chandra, B.E. Kumara Swamy, O. Gilbert, B.S. Sherigara, Electrochim. Acta 55 (2010) 7166–7174. [46] Y.H. Xiao, C.X. Guo, C.M. Li, Y.B. Li, J. Zhang, R.H. Xue, S. Zhang, Anal. Biochem. 371 (2007) 229–237. [47] S. Hou, N. Zheng, H. Feng, X. Li, Z. Yuan, Anal. Biochem. 179 (2008) 179–184.
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Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Electrochemical behavior of poly (naphthol green B)-film modified carbon paste electrode and its application for the determination of dopamine and uric acid S. Chitravathi a, B.E. Kumara Swamy a,⇑, G.P. Mamatha b, B.S. Sherigara a a b
Department of P.G. Studies and Research in Industrial Chemistry, Kuvempu University, Jnana Sahyadri, Shankaraghatta 577 451, Karnataka, India Department of Pharmaceutical Chemistry, Kadur, Kuvempu University, Karnataka, India
a r t i c l e
i n f o
Article history: Received 17 June 2011 Received in revised form 19 October 2011 Accepted 13 November 2011 Available online 6 December 2011 Keywords: Dopamine Uric acid Naphthol green B Carbon paste electrode Cyclic voltammetry Differential pulse voltammetry
a b s t r a c t A polymerized film of naphthol green B was prepared on the surface of a carbon paste electrode in Britton–Robinson (BR) buffer solution by electropolymerization technique. Electrochemical investigation of the resulting film was studied by cyclic voltammetric method. Several factors affecting the electrocatalytic activity of the hybrid material such as, effect of pH, scan rate and concentration were studied. The results suggested that the modified electrode exhibited enhanced sensitivity and selectivity towards the oxidation of dopamine (DA) and uric acid (UA). Well defined and separated oxidation peaks were observed by cyclic voltammetry and differential pulse voltammetry. Linear calibration plots for the oxidation of DA and UA were obtained in the range of 5 106 M to 2.7 104 M for DA and 12.5 106 M to 7.5 104 for UA, with a correlation co-efficient of 0.991 and 0.995, respectively. Detection limits of DA and UA were found to be 0.25 ± 0.05 lM and 5 ± 0.04 lM, respectively. The analytical performance of this sensor has been evaluated for detection of DA and UA in real samples. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Dopamine (DA) is one of the crucial catecholamine neurotransmitter and extracellular messenger distributed in the mammalian central nervous system (CNS) and renal systems. It plays an important role in the function of CNS, renal, hormonal and cardiovascular systems. Thus its deficiency may lead to neurological disorders such as Parkinsonism and schizophrenia [1–4] and to HIV infection [3,5]. Uric acid (UA) (2,6,8-trihydroxypurine) is the primary product of purine metabolism in the human body. Abnormal concentration levels of UA can lead to several disorders such as gout, Lesch–Nyhan syndrome, hyperuricaemia, cardiovascular disease, and multiple sclerosis [6,7]. Thus extreme abnormalities of DA and UA concentrations levels may lead to several diseases and it is essential to develop a simple and rapid method for the determination of both DA and UA for routine analysis. Usually, in the extra cellular fluid of the central nervous system, DA exists in only a nanomolar to micromolar range (0.01–1 lM) [8,9] and the amount of uric acid present in the serum range from 240 to 520 lM and in urine it is estimated to be 250–750 mg/24 h [10]. The adsorption or coating of polymeric species or films onto the surface of conventional substrates has become a preferred approach for the construction of chemically modified electrodes (CMEs). In recent years, polymer film-modified electrodes have at⇑ Corresponding author. Tel.: +91 8282 256225 (O); fax: +91 8282 256255. E-mail address: [email protected] (B.E. Kumara Swamy). 1572-6657/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2011.11.017
tracted great attention, because of their good stability, reproducibility and their wide applications in the fields of chemical sensors and biosensors [11–14]. When polymer modified electrodes (PMEs) were used, carbon-base electrodes have been primarily used compared to metal electrodes due to its biocompatibility with tissue, having low residual current over a wide potential range and minimal propensity to show a deteriorated response as a result of electrode fouling. Up to now different methodologies have been used to prepare polymeric film-modified electrodes. Among them electropolymerization yields a modified electrode with a three-dimensional distribution of mediators. This type of electrodes enhances the sensitivity and improves the catalytic activity than monolayers. A variety of examples of the electrochemical determination of DA and UA along with AA have been proposed. These include a carbon paste electrode using pyrogallol red as a mediator [15], 2-amino-5-mercapto-[1,3,4] triazole self-assembled monolayers gold electrode [16], indole-3-carboxaldehyde glassy carbon electrode [17], cobalt salophen-modified carbon-paste electrode [18], p-aminobenzene sulfonic acid functionalized glassy carbon electrode [19], phosphorylated zirconia-silica composite electrode [20], 2,20 -[1,2-ethanadiylbis(nitriloethylidyne)]-bis-hydroquinone-carbon nanotube paste electrode [21] and poly (caffeic acid)modified glassy carbon electrode [22]. Naphthol green B (1-nitroso-2-naphthol-6-sodium sulphonate ferric salt, NGB) (Scheme 1) is an important commercial green nitroso dye and it is the sodium salt of naphthol green Y. As an anion,
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2.2. Apparatus Electrochemical measurements were carried out with a model201 electrochemical analyzer (EA-201 Chemilink system) in a conventional three-electrode system. The working electrode was a carbon paste electrode, having cavity of 3 mm diameter. The counter electrode was a bright platinum wire with a saturated calomel electrode (SCE) as a standard electrode completing the circuit.
2.3. Preparation of bare carbon paste electrode
Scheme 1. Structure of naphthol green B.
it acts as acid dye and possesses excellent redox characteristics. Such dye is able to undergo electropolymerization from aqueous solution, producing stable redox active layers [23,24]. Thus it can be used as a mediator for electro catalysis of biological compounds because of its high electron transfer efficiency and low cost [25–27]. Few reports have been reported where NGB was used as a mediator for the analysis of some neurotransmitters [28–31]. In the present work, an electropolymerized film of NGB was synthesized, studied and was applied for the determination of DA in presence of UA. These modified electrode allow for better sensitivity and selectivity for the determination of DA in the presence of UA and proved better than unmodified electrode. Thus the fabrication of the modified electrode has been considered in detail. This modified electrode was used for the determination of DA and UA in pharmaceutical and urine samples (see Scheme 2).
The carbon paste electrode was prepared by hand-mixing 70% graphite powder and 30% silicon oil in an agate mortar for about 30 min to get homogeneous carbon paste. The paste was then packed into the cavity of a Teflon tube electrode (3 mm diameter). Before measurement, the modified electrode was smoothened on a piece of transparent paper to get a uniform, smooth and fresh surface.
2.4. Preparation of the NGB polymer film modified carbon paste electrode The polymer film-modified electrode was fabricated by electrochemical polymerization of NGB by cyclic voltammetry in the potential range 500 to 1200 mV at a sweep rate of 100 mV/s in B–R buffer solution (pH 3.2). The monomer concentration was usually 0.5 mM. After 10 cycles, the surface of the electrode was washed with doubly distilled water to remove the physically adsorbed material. This modified electrode was immersed in PBS (pH 7.0) and electrochemical studies were carried out. Poly (NGB) film with different thickness was achieved by altering scan cycles during polymerization process.
2. Experimental 2.1. Reagents and materials
2.5. Electrochemical measurements
DA, UA, NGB were purchased from Himedia Chemicals and were of analytical grade. The electropolymerization of NGB was performed in 0.1 M (B–R) buffer solution. Phosphate buffer solution (0.033 M, pH 7.0) was prepared from KH2PO4 and K2HPO4 and the pH was adjusted with 0.05 M NaOH. Other chemicals used were of analytical grade except for spectroscopically pure graphite powder. All solutions were prepared with doubly distilled water. Freshly prepared DA and UA solutions were used prior to measurements.
Electrochemical determination of DA and UA was carried out in a voltammetric cell with 20 mL of supporting electrolyte solution. Standard stock solution of DA and UA was added to the cell according to the requirement. Cyclic voltammograms and differential pulse voltammograms from 50 mV to +600 mV were recorded in PBS (pH 7.0) at the given scan rate for the determination of DA and UA. The same procedure was applied for the samples analysis and all electrochemical measurements were carried out at room temperature.
Scheme 2. Oxidation mechanisms of DA and UA.
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shown in the figure, the current response is maximum up to scan number 10 and decreases thereafter. However, after 10 cycles, poly (NGB) covers the electrode surface completely and active area does not change significantly thereafter and any further increase in the scan number will result in the decrease in the redox peak current. Hence, we selected 10 cycles as the optimum scan number for the film formation process in this study.
Fig. 1. Continuous cyclic voltammograms for the electrochemical polymerization of 0.5 mM NGB on a CPE in B–R buffer solution (pH 3.2) at the scan rate 100 mV/s.
3. Results and discussion 3.1. Optimum conditions for preparation and other electrochemical activity of poly (NGB)-film 3.1.1. Electropolymerization of NGB on a CPE Electropolymerization of NGB was performed on a CPE. Fig. 1 displays the continuous cyclic voltammetric sweeps of 0.5 mM NGB monomer in B–R buffer solution (pH 3.2) by scanning over the range of 500 to 1200 mV at a scan rate of 100 mV/s for 10 cycles. During the electropolymerization process two oxidation peaks Pa1 and Pa2 at 155 mV and 590 mV respectively were observed in the anodic scan and four reduction peaks Pc1, Pc2, Pc3 and Pc4 at 550, 30, 116 and 250 mV respectively in reverse reduction scan from the first scan onwards due to the formation of poly (NGB). The peaks Pa2 and Pc1 are may be due to Fe2+/Fe3+ couple, because as an Fe(III) compound NGB in aqueous solution undergo reduction from Fe(III) to Fe(II) in analogy to numerous other Fe(III) complexes [32–34]. In the following scans the larger peaks were observed upon continuous growth of the film. It was observed that the film growth was faster for the first 5 cycles. The oxidation and reduction peak currents increased with the increase of cyclic number of scans, indicating that an electroconductive polymer film was formed on the electrode surface. After electropolymerization, the modified electrode was carefully rinsed with doubly distilled water and then stored in pH 7 PBS. The thickness of the poly (NGB) film can be adjusted by controlling the cyclic number of voltammetric scans and the concentration of NGB. Fig. 2 shows the effect of scan number on the oxidation peak current (ipa) of 0.1 mM DA in PBS (pH 7.0). As
3.1.2. Electrochemical properties of the poly (NGB)-film modified electrode The electrochemical properties of poly (NGB)-film were tested through cyclic voltammetric method in PBS (pH 7). Fig. 3a exhibits the typical voltammogram of the poly (NGB) electrode which shows one redox couple with anodic and cathodic peak potentials at 0 mV and 56 mV respectively. The separation of peak potential (DEp = Epa Epc) was 56 mV which is close to 59/n mV at 25 °C which indicates that the equal number of electrons and protons are involved in the reaction. Electrode reaction of poly (NGB) film is a redox process and it can be suggested that the ligand itself may serve as electron donor between NGB/NGB on the surface of the electrode [35]. Fig. 3b shows the cyclic voltammograms of poly (NGB)-film modified electrode in the range of 10–300 mV/s. The redox peak currents obtained in each scan increased linearly with the increase of scan rates (Fig. 3c). The anodic (ipa) and cathodic (ipc) currents were linearly dependent on scan rates with the linear equations: ipa (lA) = 5.397 0.276 m (r2 = 0.998) and ipc (lA) = 16.07 + 0.3375 m (r2 = 0.996). These results showed that the reduction and oxidation of the NGB/NGB- couple is adsorption controlled. An approximate estimation of amount of incorporated NGB was calculated by the amount of surface coverage of the electroactive species on the electrode, and is given by the following equation [36].
ip ¼
n2 F 2 ACm 4RT
where C (M/cm2) represents the surface coverage concentration which is proportional to the peak current (ip), m is the scan rate, A is the geometric surface area of the electrode (0.028 cm2), n is the number of electrons involved in the reaction and R, F, T have their normal meanings. The surface concentration of poly (NGB) was determined to be 0.318 1010 M/cm2. 3.1.3. Stability and reproducibility of poly (NGB)-film modified electrode The main advantage of using the modified electrode is that the electrode surface can be renewed after every use by extrusion of approximately 0.5 mm carbon paste from the cavity of the Teflon rod and replacing it with a new paste. Indeed five successive renewing of a poly (NGB)-film modified CPE resulted in an RSD of 4.91%. The stability of the modified film was evaluated by examining the cyclic voltammetric peak currents of NGB after continuously scaning for 50 cycles in phosphate buffer solution (pH 7.0) (Fig. 4). The percentage degradation of poly (NGB)-film was calculated by the following equation,
%degradation ¼
Fig. 2. Dependence of the oxidation peak current of 0.1 mM DA on the number of voltammetric scans.
ipn 100 ip1
where ip1 and ipn are the first and nth anodic peak currents respectively and is found to be less than 1%, indicating that the modified electrode is stable. The stability of the film was also checked by measuring the current response over a period of 30 days, and it is found that the poly (NGB) electrode maintains 95% of its initial activity even after 30 days.
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Fig. 4. Successive cyclic voltammograms of a poly (NGB)-film electrode in PBS (pH 7.0).
[37,38]. Fig. 5 shows the electrochemical response of 1 mM potassium ferrocyanide at the bare (solid line) and at the poly (NGB)film modified CPE (dashed line) in 1 M KCl. The anodic and cathodic peaks of ferrocyanide shifted towards more positive and more negative respectively giving less reversible behavior at the modified electrode compared to bare electrode. It is apparent that electron transfer of negatively charged probe is partially blocked at modified electrode. This may be due to the repulsion interaction between negatively charged poly (NGB)-film and the negatively charged ferrocyanide redox probe. The current response of potassium ferrocyanide at poly (NGB) film-modified CPE noticeably improved indicating that the surface coverage property of the modified electrode has changed significantly resulting in favorable and stable electrochemical behavior. 3.3. The electrochemical behaviors of DA and UA at poly (NGB) modified CPE
Fig. 3. (a) A typical cyclic voltammogram of the redox peak of poly (NGB) –film electrode (curve ‘b’) with polymer blank (curve ‘a’). (b) Cyclic voltammograms of poly (NGB)-film electrode in the range of 10–300 mV/s in PBS (pH 7.0). (c) Variation of the anodic and cathodic peak currents with the scan rate.
Fig. 6a shows cyclic voltammograms of 0.1 mM DA at the bare (curve ‘a’) and at poly (NGB)-film modified (curve ‘c’), CPE along with poly (NGB) blank (curve ‘b’) in 0.033 M PBS (pH 7.0). DA exhibited poor current response with DEp, the difference between the anodic peak potential (Epa) and the cathodic peak potential (Epc) 64 mV. However a well defined redox wave of DA was observed at the poly (NGB)-film modified electrode with DEp, 38 mV which shows fast electron transfer kinetics. The oxidation peak potential shifted negatively from 185 mV to 174 mV and the reduction peak potential shifted positively from 121 mV to 136 mV. The obvious catalytic reaction accompanied by the increase in the peak current by almost 10 folds may be due to
3.2. Interaction of poly (NGB) film-modified CPE surface with potassium ferrocyanide Fe[(CN)6]3/4 was used as the electrochemical redox probe to investigate interfacial properties of poly (NGB)-film modified electrode. Usually modification of electrodes with charged species has remarkable effects on the electrochemical behavior of redox probe reactions. These effects depend on charge of both electrode surface and redox probe. More reversible behavior is observed for the charged probe redox reactions at the modified electrodes with opposite charge and less reversible behavior for the charged probe redox reactions at the modified electrodes with similar charge
Fig. 5. Cyclic voltammograms of 1 mM potassium ferrocyanide at the bare (solid line) and at the poly (NGB)-film modified CPE (dashed line) in 1 M KCl.
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3.4. Optimization of the experimental conditions 3.4.1. Influence of pH In most cases, the solution pH plays an important influence factor to the electrochemical reaction. The effect of varying of pH (2.0–9.0) towards the redox behavior of DA and UA at the poly
Fig. 6. (a) Cyclic voltammograms of 0.1 mM DA in pH 7.0 PBS at (a) bare (solid line), (c) poly (NGB)-film modified CPE (dashed line) and (b) polymer blank (without DA). (b) Cyclic voltammograms of 0.1 mM UA in pH 7.0 PBS at (a) bare (solid line) and (b) poly (NGB)-film modified CPE (dashed line).
electrostatic interaction between DA cations and anionic NGB, which lead to increase in the concentration of DA around the surface of the modified electrode. This shows that NGB is acting as an efficient electron transfer mediator between the DA and the carbon paste electrode surface. Certainly, the increased surface area due to the presence of polymer film and multielectroactive spots (quinoid structure) inside polymers which is reflected from the increased background in CV after poly (NGB)-film formation also plays an important role in the enhanced current. These results indicated that poly (NGB) film could accelerate the electrode process and facilitate the kinetics of the electron transfer of DA on the surface of the modified electrode. Fig. 6b shows the cyclic voltammograms of 0.1 mM UA at the bare (curve ‘a’) and at poly (NGB)-film modified (curve ‘b’) CPE in 0.033 M PBS (pH 7.0). It is clear from the figure that a small and broad oxidation peak at 320 mV was observed at the bare electrode and a well defined oxidation peak at same potential was observed at the poly (NGB)-film modified electrode. The current enhancement was 10 times higher than that at the bare electrode. It has been previously reported that UA exists in the neutral form in weak acidic buffer solution [39], accordingly in this paper poly (NGB)-film encapsulates UA and catalyzes its oxidation. The enhancement in current of UA reasonable to originate from the multielectroactive spots (quinoid structure) inside the polymers and big surface area due to the presence of poly (NGB)-film which result in the accumulation of UA molecules and improve the oxidation peak current.
Fig. 7. (a) Plot of anodic peak current vs. pH (2.0–9.0) of 0.1 mM DA at the poly (NGB)-film modified CPE. (b) Plot of Epa vs. pH for DA. (c) Plot of anodic peak current vs. pH (2.0–9.0) of 0.1 mM UA at the poly (NGB)-film modified CPE. (d) Plot of Epa vs. pH for UA.
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(NGB)-film modified CPE was studied. Both the anodic and cathodic peak currents of DA first decreased gradually with increasing pH up to pH 5.0, then the peak currents increased drastically from pH 5.0 to 7.0 with increase of pH value. Fig. 7a shows the plot of anodic peak current vs. the pH of the solution. This could be partly explained on the basis of the dissociation ability of ASO3Na group of poly (NGB) in different pH environment. When the solution pH was less than 5.0 the ANH2 group will be in undissociated form and the ASO3Na group of poly (NGB) film could dissociate favorably into a negative charge group ASO 3 . Under this condition the ANH2 group of DA molecule (pKa 8.9) [40] could obtain a proton. The negative charge group ASO 3 on the surface of poly (NGB)-film modified electrode had a well affinity to the DA positive ions. Therefore more DA cations were attracted to the electrode surface and promote oxidation of DA. The DA peak currents increased with
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increase of pH till 7.0 then the protonated degree of DA decreased with the increase of pH, and hence the current. In addition, the solution pH affected the anodic and cathodic peak potentials of DA. With pH increasing, their oxidation peak potentials shift to less positive values, showing that protons take part in their electrode reactions. The anodic peak potential (Epa) of DA was proportional with the solution pH in the range of 2.0–9.0 (Fig. 7b). The linear regression equation for the anodic peak is given by
Epa ¼ 0:5476 þ 0:05117 pH ðr2 ¼ 0:994Þ A slope of 51.17 mV/pH which is close to the theoretical value of 59 mV/pH showed that the equal number of protons and electrons are involved in the chemical reaction. Thus, solution pH 7.0 was taken for the following determination of DA.
Fig. 8. (a) Cyclic voltammograms of 0.1 mM DA at the poly (NGB)-film modified CPE in pH 7.0 PBS at various scan rates. From (a–h): 50, 100, 150, 200, 250, 300, 350 and 400 mV/s. (b) Plot of the anodic peak current of DA as a function of the scan rate. (c) Dependence of the logarithm of anodic peak current on logarithm of scan rate. (d) Cyclic voltammograms of 0.1 mM UA at the poly (NGB)-film modified CPE in pH 7.0 PBS at various scan rates. From (a–f): 100, 150, 200, 250, 300, and 350 mV/s. (e) Plot of the anodic peak current of UA as a function of the scan rate.
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S. Chitravathi et al. / Journal of Electroanalytical Chemistry 667 (2012) 66–75
Table 1 Comparison of efficiency of some modified electrodes for the simultaneous determination of DA and/or UA along with AA at different modified electrodes. Electrode
Modifier
Analyte
Detection limit (M) 7
Linear range (M) 6
Refs. 4
Carbon paste electrode
Pyrogallol red
DA UA AA
7.8 10 35 106 –
1.0 10 to 7 10 50 106 to 10 104 –
[15]
Gold electrode
2-Amino-5-mercapto-[1,3,4] triazole
DA UA AA
8 107 1 106 –
2.5 106 to 5 104 5 106 to 1 104 –
[16]
Glassy carbon electrode
Indole-3-carboxaldehyde
DA UA AA
17 107 4.99 106 –
10 106 to 1 104 10 106 to 1 104 –
[17]
Carbon paste electrode
Cobalt salophen
DA UA AA
7 107 – 5 107
1 106 to 1 104 – 1 106 to 1 104
[18]
Glassy carbon electrode
p-Aminobenzene sulfonic acid
DA UA AA
– 7.5 107 12 106
– 50 106 to 2.5 104 35 106 to 1.75 104
[19]
Composite electrode
Phosphorylated zirconia–silica
DA UA AA
1.7 106 3.7 106 8.3 106
6 106 to 1 104 22 106 to 35 105 1 104 to 16 104
[20]
Carbon nanotube
2,20 -[1,2-Ethanediylbis(nitriloethylidyne)]-bis-hydroquinone
DA UA AA
15 106 7.5 108
2 105 to 7 104 7 107 to 8 104
[21]
Glassy carbon electrode
Poly (caffeic acid)
DA UA AA
4 107 – 9 107
1 106 to 4 105 – 2 105 to 1.2 103
[22]
Carbon paste electrode
Poly (naphthol green B)
DA UA AA
2.5 107 5 106 –
5 106 to 2.7 104 12.5 106 to 7.5 104 –
This work
Fig. 7c and d shows the relationship between the pH and anodic peak current and pH and anodic peak potential respectively. From the figure, it was clear that the peak current of UA decreases with the increase of pH in the whole range from pH 2 to 9 and anodic peak potential (Epa) shifted towards the negative direction with increasing of pH up to 7 and becomes constant, which indicated that protons have taken part in electrode process. The linear regression equation for the anodic peak is given by Epa = 649.79– 43.65 pH (r2 = 0.989). The slope of 43.65 was close to the theoretical value indicated that the electrons and protons involved in the redox of UA were equal (1:1). In the view of determination of both DA and UA and also in order to carry out the reaction at physiological environment, pH 7.0 was chosen for further study. 3.4.2. Influence of scan rate Useful information involving electrochemical mechanism usually can be acquired from the relationship between peak current and scan rate. Fig. 8a shows the cyclic voltammograms of 0.1 mM DA at different scan rates from 50 to 400 mV/s at the poly (NGB)-film modified electrode and Fig. 8b shows relationship between scan rate and oxidation peak current at different scan rates. As shown in the figure the oxidation peak currents exhibited a good linear relationship between peaks current and scan rate. The linear regression equation was given by:
ipa ¼ 0:2855m þ 7:989 ðr 2 ¼ 0:999Þ This indicates that the electrode process was controlled by adsorption rather than diffusion. Furthermore the anodic peak potential shifted towards more positive and the cathodic peak potential shifted towards more negative with the increase in scan rate indicating the adsorption taking place at the electrode. The slope value indicates that two electron are involved in the reaction for DA at the poly (NGB)-film modified electrode. This is shown in Scheme 2(a). Further to prove this plot of log m and log ipa illustrated the linear relation (Fig. 8c) with the linear regression equation for anodic peak:
log ipa ¼ 0:860 log m þ 2:42 ðr 2 ¼ 0:999Þ The slope of 0.86 is close to the theoretically expected value of 1.0 for an adsorption controlled process [41]. Fig. 8d illustrated the cyclic voltammograms of 0.1 mM UA at the modified electrode in phosphate buffer (pH 7.0) at various scan rates and Fig. 8e shows relationship between scan rate and oxidation peak current of UA at different scan rates. The results showed that the anodic peak current was proportional to the scan rate in the range of 100– 350 mV/s indicating that the electrode reactions was controlled by adsorption. The linear regression equation is given by:
ipa ¼ 0:0592m þ 4:007 ðr 2 ¼ 0:995Þ
Table 2 Simultaneous determination of DA and UA in injection and human urine mixture sample in PBS (n = 5). Sample no.
1 2 3 4 5
DA added (lM)
0 10 20 30 40
UA added (lM)
0 10 20 30 40
DA
UA
Found (lM)
Recovery (%)
RSD (%)
Found (lM)
Recovery (%)
RSD (%)
39.31 49.53 59.60 68.95 79.25
– 100.44 100.48 99.48 99.92
2.4 1.39 1.43 2.04 2.17
24.82 34.93 44.45 54.75 65.02
– 100.31 99.17 99.87 100.30
1.71 2.27 1.89 2.12 1.85
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The slope value of 59.2 which is close to the theoretical value of 59 mV showed that the equal number of protons and electrons are involved in the chemical reaction. This is shown in Scheme 2(b). The oxidation peak potential shifted towards positive direction. 3.5. Simultaneous determination of DA and UA by cyclic voltammetry Fig. 9a shows the cyclic voltammograms of the mixture containing DA and UA at the bare CPE (curve ‘a’) and at the poly (NGB)-film modified CPE (curve ‘b’) in PBS (pH 7.0). As shown in the figure, at bare CPE DA and UA exhibited poor current response and irreversible electrochemical behaviors but when the modified electrode was used, the mixture displayed anodic peaks with the potential difference of about 138 mV. From the experimental results
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described above, it was known that in the mixture containing DA and UA, the oxidation peaks of the two compounds were clearly separated from each other with 10 folds current enhancement. If the concentrations of DA and UA increased synchronously, the anodic and cathodic peak currents of DA and anodic peak current of UA at the poly (NGB)-film increased synchronously, at the same time the anodic peak potentials of both the peaks shifts towards positive direction. Fig. 9b and c shows the linear relationship between the anodic peak current (ipa) with the concentration for DA and UA respectively. It can be seen that the peak currents for the two increased linearly with their concentrations. Under the optimum detection conditions, the oxidation peak currents were proportional to concentration of DA and UA in the range of 5 106 M to 2.7 104 M and 12.5 106 M to 7.5 104 M, respectively. The linear range and detection limits of this proposed method is compared with other existing methods with different modifiers in Table 1. These values are comparable with those of other researchers using different types of modifiers. The linear regression equations for DA and UA respectively were expressed as
ipa ðlAÞ ¼ 28:4 þ 96C
ðr 2 ¼ 0:997Þ
ipa ðlAÞ ¼ 2:7 þ 3:68C
ðr 2 ¼ 0:999Þ
The detection limit (LOD) of DA and UA at the poly (NGB)-film modified CPE from 5 different calibration curves were found to be 0.25 ± 0.05 lM for DA and 5 ± 0.04 lM for UA respectively [42–44]. The LOD was calculated using the following equation, LOD = 3 S/M where, S is the standard deviation and M is the slope (sensitivity) obtained from the calibration curves [45]. 3.6. Resolution of DA and UA by differential pulse voltammetry (DPV) Differential pulse voltammetry (DPV) was used to investigate the possibility of poly (NGB)-film modified electrode for the simultaneous determination of DA and UA. The current responses of these two changed simultaneously by changing the concentrations of DA and UA in a mixture. As illustrated in Fig. 10 the DPV responses of the modified electrode of DA and UA increased linearly with increase of their concentrations. The linear regression equations for DA and UA in the range of 10–70 lM and 5–35 lM simultaneously are given by,
Fig. 9. (a) Cyclic voltammograms of 0.1 mM DA and 0.1 mM UA mixture in pH 7.0 PBS at (a) bare and (b) at poly (NGB)-film modified CPE. (b) Calibration plot for the determination of DA at the poly (NGB)-film modified CPE in pH 7.0 PBS with the scan rate 100 mV/s. (c) Calibration plot for the determination of UA at the poly (NGB)-film modified CPE in pH 7.0 PBS with the scan rate 100 mV/s.
ipa ðlAÞ ¼ 3:63 þ 4:84C
ðr 2 ¼ 0:999Þ
ipa ðlAÞ ¼ 3:51 þ 4:79C
ðr 2 ¼ 0:998Þ
Fig. 10. Differential pulse voltammograms for the mixture containing DA and UA with different concentrations at the poly (NGB)-film modified CPE. DA concentrations: (a) 10, (b) 20, (c) 30, (d) 40, (e) 50, (f) 60 and (g) 70 lM. UA concentrations: (a) 5, (b) 10, (c) 15, (d) 20, (e) 25, (f) 30 and (g) 35 lM.
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injection USP (specified content of DA is 40 mg/ml) and 1 ml of human urine sample without any pretreatment were diluted to 100 ml with buffer respectively and diluted solutions were pipetted into each of series of 10 ml volumetric flasks and to this, different known standard concentrations of DA and UA solutions were added and diluted to the mark with pH 7.0 phosphate buffer. The cyclic voltammograms were recorded and the peak currents were measured for DA and UA. Fig. 12 shows a typical cyclic voltammogram for the simultaneous determination of 1:500 dilutions of DA and UA in DA hydrochloride injection USP and healthy human urine sample solution respectively. The obtained results were shown in Table 2 and the results were consistent well with the certified values, suggesting that the poly (NGB)-film electrode has a good precision and the proposed method can be efficiently applied to the simultaneous determination of DA and UA. Fig. 11. Differential pulse voltammograms of DA and UA in presence of AA at poly (NGB)-film modified CPE in pH 7.0 PBS with the scan rate 100 mV/s. (a) 0.1 mM DA, (b–e) 0.1 mM DA+ 0.1, 0.2, 0.3 and 0.4 mM AA, and (f) 0.1 mM UA.
Fig. 12. Typical cyclic voltammogram for the simultaneous determination of 1:500 dilution of DA and UA in DA hydrochloride injection USP and healthy human urine sample solution respectively.
3.7. Interference study The possible influence of AA which coexists with DA and UA in body fluids on the electrochemical behavior of DA and UA has been investigated. The effect of increasing concentration of AA on DA and UA at modified electrode was carefully examined by differential pulse voltammetry. Fig. 11 shows the differential pulse voltammograms of mixed solution of 0.1 mM DA, 0.1 mM UA and varying concentrations of AA (from b to e, 0.1, 0.2, 0.3 and 0.4 mM). As can be seen from the Figure, the oxidation peak current of DA changes only ± 8% and the peak potential did not change much. However, when the concentration of AA exceeds 0.4 mM this interference is constant and there is no obvious change in the oxidation currents of DA [46,47]. This means that in the extracellular fluid of central nervous system, where the AA level is usually present more than 100-folds of magnitude larger than DA, the poly (NGB)-film modified electrode can be used for the determination of DA and also in real samples. As shown in the figure the presence of AA has no effect on the UA signal. Moreover, AA shows no reduction peak in pH 7.0 PBS at the modified electrode by CV. This can also be used for the simultaneous determination of DA in presence of AA.
3.8. Analytical application The practical application of the modified electrode was illustrated by simultaneous determination of DA and UA in injection and human urine sample respectively. 1 ml of DA hydrochloride
4. Conclusion In this work, a poly (NGB) film modified CPE was fabricated and the characteristics of the electrode was studied. The modified electrode was used for the simultaneous determination of DA and UA in the PBS (pH 7.0) by CV and DPV techniques. The modified electrode showed good stability and sensitivity. Well-defined and discrete voltammetric oxidation peaks were observed. Linear calibration plots for the oxidation of DA and UA were obtained in the range of 5 106 M to 2.7 104 M for DA and 12.5 106 M to 7.5 104 M for UA with a correlation co-efficient of 0.991 and 0.995, respectively. The detection limits (LOD) of DA and UA at the poly (NGB)-film modified CPE were found to be 0.25 ± 0.05 lM and 5 ± 0.04 lM, respectively The possible interference of AA in the determination of DA and UA was also studied using the modified electrode and the proposed method has been practically and successfully applied for the simultaneous determination of AA and DA in real samples. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29]
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