determination of trace amounts of sulfamethizole using a multi-walled carbon nanotube modified...

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Determination of Trace Amounts ofSulfamethizole Using a Multi-WalledCarbon Nanotube Modified Electrode:Application of Experimental Design inVoltammetric StudiesSayed Mehdi Ghoreishi a b , Mohsen Behpour a , Asma Khoobi a &Zohreh Moghadam aa Department of Analytical Chemistry , Faculty of Chemistry,University of Kashan , Kashan , Islamic Republic of Iranb Deylaman Institute of Higher Education , Lahijan , Islamic Republicof IranAccepted author version posted online: 20 Aug 2012.Publishedonline: 02 Jan 2013.

To cite this article: Sayed Mehdi Ghoreishi , Mohsen Behpour , Asma Khoobi & Zohreh Moghadam(2013) Determination of Trace Amounts of Sulfamethizole Using a Multi-Walled Carbon NanotubeModified Electrode: Application of Experimental Design in Voltammetric Studies, Analytical Letters,46:2, 323-339, DOI: 10.1080/00032719.2012.718831

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Electrochemistry

DETERMINATION OF TRACE AMOUNTS OFSULFAMETHIZOLE USING A MULTI-WALLEDCARBON NANOTUBE MODIFIED ELECTRODE:APPLICATION OF EXPERIMENTAL DESIGN INVOLTAMMETRIC STUDIES

Sayed Mehdi Ghoreishi,1,2 Mohsen Behpour,1 Asma Khoobi,1

and Zohreh Moghadam1

1Department of Analytical Chemistry, Faculty of Chemistry, University ofKashan, Kashan, Islamic Republic of Iran2Deylaman Institute of Higher Education, Lahijan, Islamic Republic of Iran

The present paper is the first report for the simultaneous investigation of various factors

affecting the oxidation peak currents of sulfamethizole (SM) in a 0.20M Britton-Robinson

(B-R) buffer solution. Experimental factors (pH, multiwalled carbon nanotube amount,

scan rate, step potential, and modulation amplitude) on voltammetric determination of

SM were optimized simultaneously by a central composite rotatable design (CCRD). Then,

in the optimized conditions, differential pulse voltammetry (DPV) was applied for selective

determination of SM at the surface of a multi-walled carbon nanotube modified carbon paste

electrode (MWCNT/CPE).The modified electrode showed enhanced effect on the oxidation

peak current of SM. In these conditions the linear dynamic range for SMwas from 0.10 up to

131.94lM and the detection limit was found to be 14.31 nM. The characterization of

MWCNT/CPE was carried out bycyclic voltammetry (CV) and electrochemical impedance

spectroscopy (EIS). Also, linear sweep voltammetry (LSV) and chronocoulometry of SM

were studied. Using these techniques, the diffusion coefficient (D¼ 4.91� 10�5 cm2 s�1) and

the kinetic parameters such as the electron transfer coefficient (a¼ 0.79) and exchange

current density (j0¼ 2.78� 10�12lA cm�2) for SM were calculated. The proposed method

was then successfully applied to the determination of SM in plasma samples.

Keywords: Central composite rotatable design; Modified carbon paste electrode; Multi-wall carbon nano-

tube; Sulfamethizole; Voltammetric analysis

INTRODUCTION

In recent years, chemometric methods have been frequently used for optimizingand affecting parameters in analytical procedures. On the other hand, chemometricmethods have advantages such as a decrease of the number of experiments that need

Received 21 June 2012; accepted 31 July 2012.

The authors are grateful to the University of Kashan for supporting this work byGrant No.15919513.

Address correspondence to Sayed Mehdi Ghoreishi, Department of Analytical Chemistry, Faculty

of Chemistry, University of Kashan, Kashan, I.R. Iran. E-mail: [email protected]

Analytical Letters, 46: 323–339, 2013

Copyright # Taylor & Francis Group, LLC

ISSN: 0003-2719 print=1532-236X online

DOI: 10.1080/00032719.2012.718831

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be implemented resulting in lower time and reagent consumption. Also, these methodsallow the development of mathematical models that allow valuation of the relevance aswell as statistical significance of the factor effects being studied and elucidation of theinteraction effects between the parameters. When there are significant interactioneffects between factors, the optimal conditions introduced by the univariate methodswill be different from the correct results of the multivariate optimization. Thus, theunivariate procedure may fail as the effect of one variable can be dependent on thelevel of the others involved in the optimization. There are several multivariate optimi-zation methods such as factorial design, Doehlert matrix, central composite designs(CCD), and Box-Behnken design (BBD) (Ferreira, Bruns et al. 2007; Box, Hunter,and Hunter 2005; Bruns, Scarminio, and Neto 2006; Massart et al. 1977).Among thesemethods, the CCD is a better alternative to the others as it needs a smaller number ofexperiments while providing comparable results. Thus, this has been the most acceptedexperimental design for second-order models. The total number of design pointsneeded is determined by the points (Tarley et al. 2009). The CCD was introducedby Box and Wilson (1951). A CCD combines a two-level full or fractional factorialdesign with additional points (star points) and at least one point at the center of theexperimental region. This point is chose to obtain several properties, such as orthogon-ality or rotatability, in order to fit the quadratic polynomials (Ferreira, Korn et al.2007; Ghoreishi, Behpour, and Khoobi 2012).

Sulfamethizole [4-amino-N-(5-methyl-1,3,4-thiadiazol-2-yl)- benzene sulfona-mide (SM)] (Scheme 1) is a class of sulfonamides (sulfa drugs). Sulfonamides are asynthetic antibiotic category that is widely used to treat diseases for both humansand livestock since their development in 1968 (Littlefeild et al. 1990).The develop-ment of sulfonamides has been one of the most important studies in medicinal chem-istry. However, Domagk was the pioneer who showed that a chemotherapeutic agent(prontosil) was able to control the evolution of a bacterial infection (Souza et al.2008). Prontosilis a prodrug that is inactive in vitro, but when metabolized in vivo,is changed into an active product and is called sulfanilamide. Subsequently, varioussulfonamides were used for many applications, including antibacterial action. Thechemotherapeutic activity of the sulfonamides is accumulated with their competitionwith p-amino benzoic acid in the synthesis of folic acid, which is necessary for thegrowth of both mammalian cells and bacteria (Wormser and Keusch 1979). Also,these drugs are used in the treatment of pneumocystis pneumonia, urinary-tractinfections, chronic bronchitis, acute otitis, meningococcal meningitis, and toxoplas-mosis. Various analytical methods, such as titrimetric-assay (Tseng and Smith 1994),photometric (Hassouna 1997), potentiometric (Nazer, Shaber, and Riyazuddin2001), capillary electrophoresis (You, Yang, and Wang 1998), high-performanceliquid chromatography (HPLC) (Amini and Ahmadiani 2007), and gas chromato-graphy (Assassi, Tazerouti, and Canselier 2005)are used for the determination of

Scheme 1. Structure of sulfamethizole.

324 S. M. GHOREISHI ET AL.

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different sulfonamides in pharmaceuticals and residues in food products. Thesemethods are suitable for determination of sulfonamides. However, only a few elec-trochemical methods have been reported for the quantification of sulfonamide drugs(Msagati and Ngila 2002; Abdullin, Chernysheva, and Budnikov 2002; Sabry 2007;Rao et al. 2000; Preechaworapun et al. 2006;Ozkorucuklu,Sahin, and Alsancak2008;Sabriye,Ozkorucuklu, and Alsancak 2011), most due to the deactivation orfouling of electrode by adsorbing of oxidation=reduction products on the surfaceof the electrodes. Despite this defect, electrochemical methods based on carbon pasteelectrodes offer certain advantages, such as savings in time, no sample preparationrequirement, and a dynamic range and sensitivity that is comparable to other ana-lytical techniques.

Carbon paste electrodes are the most commonly used electrodes in voltam-metric techniques because of their low cost, low electrical resistances, wide potentialwindows, ability in surface renewal, and versatility of chemical modification. Amongmodified electrodes, carbon nanotubes (CNTs) modified carbon paste electrodes arenew kinds of carbon nanostructure materials possessing properties such as high sur-face area, high electrical conductivity, chemical stability, and significant mechanicalstrength (Umasankar et al. 2012). CNTs have been used as single-walled carbonnanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs). They canbe applied to promote electron transfer reactions when applied as electrode materialsin electro chemical devices (Yang et al. 2005).CNTs are novel and interestingmembers of nanostructure materials that can be used as a support for immobiliza-tion different electron transfer mediators onto electrode surfaces and making idealminiaturized sensors.

To the authors’ knowledge, no method using voltammetry based on MWCNTmodified electrode has been reported for determination of SM. The aim of the paperis to develop a voltammetric method suitable for the sensitive analysis of SM. In thepresent work, we prepare a sensitive and simple multi-walled carbon nanotubes modi-fied carbon paste electrode (MWCNT=CPE) for determination of SM in biologicalsamples with differential pulse voltammetry in Britton-Robinson (B-R) buffer sol-ution. Furthermore, we calculated kinetic parameters of SM with some voltammetricmethods. The novelty of this paper also includes using central composite rotatabledesign (CCRD) for optimization of effective factors in determination of SM.

EXPERIMENTAL

Reagents

Sulfamethizole were purchased from Sigma-Aldrich. Multi-walled carbonnanotubes (O.D¼ 10�20 nm, L< 1�2 mm,>95% pure) were obtained from the Chi-nese Academy of Science and were purified using nitric acid treatment. Pure finegraphite powder (Merck) and paraffin oil (density¼ 0.88 g cm�3, DC 350, Merck)were used as binding agents in graphite pastes. All solutions were freshly preparedwith deionized water. A 0.20M of B-R buffer solution was applied as supportingelectrolyte and prepared from an acidic solution of 0.20M CH3COOH, H3PO4,and H3BO3, by adjusting the pH with a saturated solution of NaOH. All otherreagents were of analytical grade from Merck.

DETERMINATION OF SULFAMETHIZOLE 325

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Instrument and Electrochemical Measurements

The cyclic voltammetry (CV), differential pulse voltammetry (DPV), chrono-coulometry, and linear sweep voltammetry (LSV) measurements were carried outusing a Sama 500 potentiostat (Isfahan, Iran). Electrochemical impedance spec-troscopy (EIS) experiments were carried out by an Autolab potentiostat-galvanostatPGSTAT 35 (Eco chemie Utrecht, Netherlands), equipped with NOVA 1.6software.A rotating electrode system, from Pine instrument, was employed. A Pine InstrumentCompany (Grove City, PA, USA) AFMSRX 1270 rotator and MSRX speed control-ler were used. A personal computer (Pentium IV) was applied for data storage andprocessing. A digital pHmeter (Metrohmmodel 691) was used when preparing buffersolutions that served as the supporting electrolyte in the electrochemical measure-ments. An ultrasound bath (Bandelin Sonorex, Germany) at a constant frequencyof 35 kHz was used for dispersing of MWCNT during experiments. The electrochemi-cal cell was equipped with a three-electrode system: the modified carbon paste as theworking electrode, a platinum as the counter electrode, and a silver=silver chloride[Ag=AgCl =KCl(sat)] as the reference electrode. The body of the carbon paste work-ing electrode was a polyethylene tube with a rod (2.00mm diameter and 5.00mmdeep) bored at one end and filled with paste. A copper wire was placed through thecenter of the rod to make contact. The working electrode was pretreated by pushingpaste out of the tube, removing the excess, and mechanically polishing the surfacewith weighing paper. All electrochemical experiments were done at 25.0� 0.5�C.

Preparation of CPE and MWCNT/CPE

The bare carbon paste electrode was obtained by thoroughly hand-mixing0.50 g graphite powder with approximately 0.20mL of paraffin oil in a pestle mortar.For preparation the MWCNT modified CPE after purring of them by nitric acid,6.00-mg (optimal amount) of MWCNT was added to 5.00ml ethanol and sonicatedfor 30min with an ultrasonic bath to get a homogeneous and stable suspension. Thissuspension was added into 0.50 g graphite powder in a small mortar, and ethanol wasallowed to evaporate at room temperature in air. Then, about 0.20mL of paraffin oilwas added to the aforementioned mixture and mixed for 30min until a uniformlywetted paste was formed. After that, the paste was pressed manually in the cavityof the electrode body, and the surface was smoothed against clean paper. Unlessotherwise stated, the paste was carefully removed prior to pressing a new portion intothe electrode after every measurement.

Experimental Design and Data Analysis

Response surface methodology (RSM) is a collection of statistical and math-ematical techniques that are suitable for analysis and modeling of problems when aresponse is influenced by several factors. Therefore the optimization of effectivechemical and instrumental variables on the determination of SM was carried out byRSM.CCRD is a useful method for optimization of the effective factors and analyzethe interaction between the variables with a minimum number of experiments. Thisdesign consists of 3 parts: (1) a full or fractional factorial design; (2) an additionaldesign, usually a star design in which experimental points are at a distance a from


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its center; and (3) a central point. Full uniformly routable central composite designspresent the following characteristics:(1) Require an experiment number according toN¼ 2fþ 2f þr, where f is the number of factors and r is the replicate number of thecentral point; (2) a -values depend on the number of variables and can be calculatedaccording to a¼� 2f=4; and (3) All factors are studied in five levels. The experimentswere completed in randomized order to reduce the effects of unexplained variability inthe real responses due to extraneous parameters (Brereton 2007). The responsebehavior could be related to the selected independent variables by a second-orderpolynomial. The generalized response surface model is shown as follows:

Y ¼ b0 þX5

i¼1bi Xi þ

X5

i¼1biiX

2iþR

X5

i<j¼1bi jXiXj ð1Þ

where Yi is the predicted response; Xi is the independent variables;b0 is intercept(constant); and bi, bii, and bij are the linear, quadratic, and interaction coefficients,respectively.

In the present work, we used CCRD for simultaneous investigation of fiveeffective factors on the DPV response, that is, pH, MWCNT amount, scan rate, steppotential, and modulation amplitude. MINITAB Release 16, developed by MinitabInc. (USA), a statistical software package, was applied for data processing andstudying linear terms, squared terms, and interaction between the variables. Thesignificance level 95% was set for the mathematical model and surface response.

RESULTS AND DISCUSSION

Response Surface Study: CCRD

CCRD was used for investigation of five effective factors on the DPV response,that is, pH (2.00–10.00, X1), MWCNT amount (2.00–10.00mg, X2), scan rate (0.02–0.18V s�1, X3), step potential (0.001–0.009V, X4), and modulation amplitude (0.01–0.09V, X5). Then five levels of each factor to covering the experimental conditionswere selected. Thus, experiments were based on a five-factor central composite rotat-able design with a¼� 2.38 and three replicates (r) of the central point. Therefore,f¼ 5, a¼� 2.38, N (number of experiments)¼ 45 (for r¼ 3) and levels (in codedvalues) were: �2.38, �1, 0, þ1, þ2.38. These coded levels corresponded to 2.00,4.00, 6.00, 8.00, and 10.00 for pH; 2.00, 4.00, 6.00, 8.00, and 10.00mg for MWCNTamount; 0.02, 0.06, 0.10, 0.14, and 0.18V s�1 for scan rate; 0.001, 0.003, 0.005, 0.007,and 0.009V for step potential; and 0.01, 0.03, 0.05, 0.07, and 0.09V for modulationamplitude. Also, the scheme of CCRD is similar to our previous work (Ghoreishiet al. 2012). Thus, we calculated optimum value of each variable with MINITABRelease 16 software for 70.00 mM of SM in 0.20M B-R buffer solutions, and deter-mination of SM was achieved by selecting the predicted conditions. Therefore,the coded optimized values for pH, MWCNT amount, scan rate, step potential,and modulation amplitude were obtained as 0.0651, 0.1682mg, 1.1291V s�1,�0.8409V, and �0.7448V, respectively. According to the levels of factors, thesecoded optimized values corresponded to 6.11, 6.30mg, 0.1390V s�1, 0.0035V, and0.0371V, respectively.


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Voltammetric Investigation of the Modifier Effect

The differential pulse voltammogram responses of SM with bare CPE andMWCNT=CPE were studied. Figure 1 shows the voltammograms of 50.00 mM SMon the surface of bare CPE and MWCNT=CPE in 0.20M B-R buffer solution ofpH 6.00.The SM shows a relatively broad and weak oxidation peak at 867mV(Fig. 1b). However, with the MWCNT=CPE (Fig. 1b), the oxidation peak becomeswell-defined and sharp, with a magnification 2.67 times greater than bare CPE and anegative potential shift of 26.00mV. These results indicate that MWCNT promotesand facilities electron transfer reactions.

Electrochemical Impedance Spectroscopy of the Bare CPE andMWCNT/CPE

Electrochemical impedance spectroscopy (EIS) is a well-known and powerfultechnique for studying of the surface properties of the modified electrode. Figure 2shows the Nyquist plot of the bare CPE and MWCNT=CPE in 0.20M B-R buffersolution (pH¼ 6.00) containing 5.00� 10�3M [Fe(CN)6]

3�=4�. It can be seen thatimpedance spectrum of bare CPE electrode exhibits two distinct parts: (1) a semi-circle, related to charge-transfer process; and (2) a line defining a region ofsemi-infinite diffusion of species in the electrode. The value of the electron transferresistance (Rct, semicircle diameter) depends on the dielectric and insulating featuresat the electrode=electrolyte interface (Luo et al. 2005). Significant difference of Rct

was observed upon the stepwise formation of the modified electrode. The Rct valuesfor the bare CPE and MWCNT=CPE were 5.58 kX and 1.91 kX respectively. Thismight be due to the presence of the MWCNT on the CPE that accelerate the transferof the electrons at the surface of electrode.

Figure 1. Differential pulse voltammograms: (a) MWCNT=CPE in B-R buffer solution (pH¼ 6.00), (b)

CPE in the presence of 50.00mM SM, and (c) as (b) at the surface of MWCNT=CPE. (Figure available

in color online.)


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Kinetic Studies of the Modified Electrode

For electrochemical investigations of modified electrode the cyclic voltammo-grams of the electrode in the presence of Feþ2=Feþ3 (probes solution) in pH 6.00of 0.20M B-R buffer solution at various scan rates, are drowned (Fig. 3). Thismethod can estimate an approximation of the surface coverage of the electrode byadopting the method applied by Sharp (1979).

According to this method, the peak current is related to the surface concen-tration of mediator,C, by the following equation:

Ip ¼ n2F2An=4RT ð2Þ

where n represents the number of electrons involved in reaction, A is the surface area(3.14� 10�2 cm2) of the MWCNT=CPE, U (mol cm�2) is the surface coverage ofmodifier, and other symbols have their usual meanings. From the slope of anodicpeak currents vs. scan rate (Fig. 3B) the calculated surface coverage of MWCNT=CPE, U, is 8.23� 10�6mol cm�2 for n¼ 1.

The charge transfer coefficient, a, and apparent charge transfer rate constant,ks, of a surface-confined redox couple can be calculated from cyclic voltammetricmeasurements and using the variation of anodic peak potentials with logarithm ofscan rate, according to the procedure of Laviron (Bard and Faulkner 2001). InsetB of Fig. 3 demonstrates the variations of peak potentials (Ep) as a function of thelogarithm of the potential scan rate. According to this study, when scan rates werehigher than 50.00mV s�1, Ep values were proportional to the logarithm of the scanrate (Fig. 3C). Under these conditions transfer coefficient (a) and electron transfer

Figure 2. The Nyquist plot of the bare CPE and MWCNT=CPE in 0.20M B-R buffer solution (pH 6.00),

containing 5.00mM [Fe(CN)6]3�=4�.


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rate constant (ks,s�1) between probes andMWCNT=CPE can be obtained from slope

of such plot and the following equation, respectively.

Logks ¼ alog 1� að Þ þ 1� að Þloga� log RT=nFnð Þ � a 1� að ÞnaFDEp=2:3RT ð3Þ

where n¼ 1, DEp¼Epa - Epc, n (Vs�1) is the scan rate, and all other symbols havetheir conventional meanings. From the values of DEp corresponding to different scanrates, an average value of ks and a were to be 11.68 s�1 and 0.49, respectively.

Linear Sweep Voltammetry Measurements in Oxidation of SM

Figure 4 A is shown the linear sweep voltammograms of solutions containingdifferent concentrations of SM with a sweep rate of 100mV s�1. The rising part ofthese voltammograms is known as the Tafel region, which is affected by the electrontransfer kinetics between SM and modified electrode. If deprotonation of SM is asufficiently fast step, with assuming the number of electrons involved in the rate deter-mining step of na¼ 1, charge transfer coefficient (a) and ionic exchange current den-sity (j�) can be obtained by average of the slopes and intercepts of the Tafelplot,respectively (Bard and Faulkner 2001). The average Tafel slopes of 0.2805V per

Figure 3. CV of MWCNT=CPE in Fe2þ=Fe3þ probes solution in 0.20M B-R buffer (pH¼ 6.00), at

various scan rates, a to r correspond to 20, 30, 40,50, 60, 70, 80, 90, 100,110,120, 200, 300, 400, 500,

600, 700,and 800mV s�1 scan rates, respectively. (A) Variations of Ip vs. scan rates, (B) variation of Ep

vs. logarithm of the scan rate, and (C) magnification of the same plot for high scan rates. (Figure

available in color online.)


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decade obtained in this case a charge transfer coefficient of a¼ 0.79, assuming na¼ 1.Also, j� was found to be 2.78� 10�12 mA cm�2 by average of Tafelintercepts for SM(Fig. 4B).

Effect of Scan Rate in CV Behavior of SM

The electrochemical behavior of SM on the MWCNT=CPE was studied by CV.Voltammograms obtained at the MWCNT=CPE presented an irreversible chemicalbehavior for SM (Fig. 5A). Furthermore, the relationship between peak current andscan rate can obtain useful information regarding electrochemical mechanism. As aresult, the effect of scan rate on the peak current of SM was investigated by CV(Fig. 5A). For totally irreversible diffusion controlled process according to Eq.4(Antoniadou, Jannakoudakis, and Theodoridou 1989):

Ip ¼ 3:01� 105n½ð1� aÞna�1=2AD1=2Cn1=2 ð4Þ

where n, a, na, A, D, C, and n are the number of electrons involved in the thoroughoxidation process, transfer coefficient, the number of electrons involving in the ratedetermining step, surface area, diffusion coefficient, concentration of reagent, andscan rate, respectively.

According to this study, for a solution containing 50.00 mM of SM in B-Rbuffer (pH¼ 6.00), a linear relationship was observed between the peak current

Figure 4. (A) Linear sweep voltammograms of MWCNT=CPE in B-R buffer (pH¼ 6.00) containing

23.00, 31.00, 38.00, and 46.00mM SM at a sweep rate of 100mV s�1; and (B): Tafel plot derived from

linear sweep voltammograms. (Figure available in color online.)


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and square root of scan rate in the range of 20–180mVs�1 (Fig. 5B). This resultindicates that oxidation peak current of SM is a diffusion-controlled process onthe modified electrode.

Chronocoulometric Measurements of SM

The catalytic oxidation of SM at the surface of MWCNT=CPE was investi-gated by chronocoulometry. Chronocoulograms are obtained for solutions contain-ing various concentrations of SM ranging from 8.00 to 31.00 mM in B-R buffer(pH¼ 6.00) at MWCNT=CPE with a potential step of 1000mV (Fig. 6A). TheseChronocoulograms were used for calculating the diffusion coefficient (D) of SMat the surface of modified electrode.

A plot of Q vs.t1=2 for different concentrations of SM is shown in Fig. 6 B. Sub-sequently, slopes of the resultant straight lines were plotted vs.SM concentration(Fig. 6C). According to the integrated Cottrell equation (Eq. 5), the averagediffusion coefficient of SM is obtained 4.91� 10�5 cm 2s�1by using the slop ofFig. 6C (Bard and Faulkner 2001).

Q ¼ 2nFAD1=2Cp�1=2t1=2 ð5Þ

where D (cm2s�1) is the diffusion coefficient and C (mol cm�3) is the bulkconcentration.

Figure 5. Cyclic voltammograms of MWCNT=CPE in a B-R buffer solution (pH¼ 6.00) for 50.00mMSM, using various scan rates: 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, and

180mVs�1 (from inner to outer). (A) Peak current vs. E (mV), (B) Intensity, I (mA) vs. t1=2. (Figureavailable in color online.)


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Effect of pH on the Oxidation Behavior of SM

For investigation of the pH effect on the SM oxidation signal at the surface ofMWCNT=CPE, differential pulse voltammetry technique was studied. Therefore, asolution containing 50.00 mM of SM in 0.20M B-R buffer was used. Differentialpulse voltammograms of this solution were measurement in pH values ranging from2.00 to 10.00 in optimal conditions, that is, a scan rate of 139mV s�1, a step potentialof 3.5mV, and modulation amplitude of 37.1mV (Fig. 7A). In accordance with thesestudies, the maximum anodic current for SM was obtained at a pH of 6.00 (Fig. 7 B),which was in agreement with the optimum value obtained from experimental designdata. Furthermore, in the pH range of 2.00–10.00, there is a linear relationshipbetween pH and potential (Fig. 7 C) as expressed by the Nernst equation [Eq. (6)]:

Ep ¼ Eþð0:0591=nÞlog Oxð Þa= Rð Þbh i

� ð0:0591m=nÞpH ð6Þ

Epa Vvs:Ag=AgClð Þ ¼ 1:029� 0:0315pH;R2 ¼ 0:9972

where Epa represents oxidation peak potential (V vs. Ag=AgCl); n and m representthe number of electrons and protons involved in reaction respectively; and a andb represent the reagents coefficients in the reaction equation. The dependence of

Figure 6. (A) Chronocoulograms obtained at MWCNT=CPE in 0.20M B-R buffer solution (pH¼ 6.00)

for various concentrations of SM, with a potential step at 1000mV. a to d correspond to 8.00, 16.00,

23.00,and 31.00mM of SM;(B) Plots of Q vs. t1=2 (s1=2) obtained from chronocoulograms a-d; and (C)

A plot of the slope of the straight lines vs. the concentration of SM. (Figure available in color online.)


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Epa on the pH with a slope of �0.0315V pH�1indicates that the electrochemicalreaction involves proton transfer. This value is next to 0.0296V pH�1 (at 25.0�C),which was predicted for a two electron electrode reaction. This indicated that theproportion of the electron and proton involved in the reaction is 2:1. The oxidationprocess of SM probably starts with an electron oxidation to form a cation radical atthe nitrogen. Subsequently, a rapid loss of a second electron and proton occurred toobtain an iminium ion to which the water is subsequently added (Arvand, Ansari,and Heydari 2011;Bragaet al. 2010). Furthermore with increasing pH between2.00 to 10.00 Epa is shifted to less positive amounts. The decrease of over potentialwith increasing pH shows the catalytic effect of MWCNT=CPE on the anodicoxidation of SM. Also, from the intercept of Fig. 8C curve, the standard formalpotential of SM was obtained to be 1.029V.

Quantification of SM by DPV Investigations

As DPV has a better resolution and much higher current sensitivity than cyclicvoltammetry, it was applied to obtain the lower limit of detection of SM. Furthermore,a limiting factor in the analytical determination is the charging current contribution tothe background current. Fortunately this problem is negligible in DPV mode. Thus,under optimum conditions established in the Response Surface Study: CCRD

Figure 7. (A) DPV of a solution of 50.00mM SM at various buffered pHs, a to i correspond to pH values

of 2.00, 3.00, 4.00, 5.00, 6.00, 7.00, 8.00, 9.00, and 10.00, respectively;(B) Intensity, I (lA) vs. pH; and (C)

electrochemical potential, Epa (V) vs. pH. DPV conditions: scan rate, 139.00mV=s; step potential,

3.50mV; modulation amplitude, 37.10mV. (Figure available in color online.)


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section, DPV was used to estimate the limit of detection of SM. Figure 8A is shown asdifferential pulse voltammograms obtained for the oxidation of various concentra-tions of SM at the surface of MWCNT=CPE in 0.20M B-R buffer solutions(pH¼ 6.00) in optimized experimental conditions. As shown in Fig. 8 B, peak heightis linearly related to the SM concentration over the range 0.10 to 131.94mMand can bedescribed as following linear regression equation:

Ip mAð Þ ¼ 0:0213C mMð Þ � 0:013;R2 ¼ 0:9980

From the analysis of the data, we estimate that the limit of detection (3r) ofSM is 14.31 nM at the surface of MWCNT=CPE.

Figure 8. (A) DPV of MWCNT=CPE in a 0.20M B-R buffer solution (pH¼ 6.00), containing different

concentrations of SM. The letters a to p correspond to: 0.10, 0.50, 1.32, 2.13, 3.98, 5.96, 7.94, 15.75,

23.44, 31.01, 49.40, 67.10, 84.20, 100.70, 116.08,and 131.94mM of SM; (B) Plot of the electrocatalytic

peak current as a function of SM concentration. (Figure available in color online.)

Table 1. Influence of some foreign substances for 50.00mM SM

Foreign substances Tolerance level (mM)

Naþ, Kþ, NH4þ 10000

Cl�, CO32� 10000

L-phenyl Alanine 10000

Glycine 10000


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Interferences

The influences of some foreign substances on the determination of 50.00 mMSMat the surface of MWCNT=CPE were investigated. The proposed method showedgood selectivity for SM determination without the interferences from common coex-isting compounds. According this study the concentrations of Naþ, Kþ, NH4

þ, Cl-,CO3

2-, L-phenyl alanine, and glycine (200 times content) have not significantlyinfluence on the current response of SM (signal changes below 5%). The results areshown in Table 1.

Real Sample Analysis

The practical analytical application of the method was further established bythe selective measurement to detect the SM content in human blood plasma samples.The standard addition method was used for achieving this purpose. Prior to theiranalysis, the samples were diluted 100 times with B-R buffer solution (pH¼ 6.00).The efficiency for the determination of real samples at the modified electrode is dem-onstrating the accuracy of the proposed method (Table 2).

Stability and Repeatability of MWCNT/CPE

Stability and repeatability are two important properties of electrodes. Tochecking of stability of the proposed modified electrode DPV technique was used.Therefore successive DPV determination a solution containing 50.00 mM SM inB-R buffer (pH¼ 6.00) in the optimal conditions was carried out for a two weekperiod. Decreasing in peak current on 94.07% of initially current after this periodsuggests excellent stability of the modified electrode.

Also, the repeatability of the modified electrode was investigated by DPVmeasurements offive separately prepared MWCNT=CPEs. In this study, a solutioncontaining 50.00 mM SM in B-R buffer (pH¼ 6.00) for DPV determinations inoptimized conditions was used. The relative standard deviations (RSD) of the datawere obtained between 1.07 to 3.18%. These results indicate that repeatability of thesurface of the modified electrode is satisfactory.

CONCLUSIONS

A method of differential pulse voltammetry coupled with CCRD wasdeveloped in this paper for determination of sulfamethizole at the surface of

Table 2. Determination of SM in human blood serum samples

Sample Added (mM) found (mM)a Recovery (%)

Human Blood Serum _ _ _

4.00 3.78 94.59

6.00 6.21 103.52

aResult based on three replicate determinations per samples.


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MWCNT=CPE. CCRD permitted to optimum simultaneously all of experi-mental effective variables on voltammetric determination of SM. The resultsshow that the oxidation of SM is dependent on the pH, and the peak currentof SM is increased by 2.67 times at the surface of modified electrode rather thanbare CPE. Overall, fabrication of this modified electrode is simple with as table,repeatable, renewal surface, and rapid response. Electrochemical studies of SMat the surface of MWCNT=CPE were carried out using CV and LSV. Also, thediffusion coefficient of SM was determined by chronocoulometry technique. Thedetection limit for SM (3r) was found to be 14.30 nM by DPV. The results indi-cated that MWCNT=CPE is highly sensitive, with great potential for electro-chemical sensor applications. The proposed method could be applied to theanalysis of SM in real samples. To our knowledge, no previous literature hasdemonstrated the use of MWCNT=CPE for the determination of SM in purifiedand real samples.

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determination of trace amounts of sulfamethizole using a multi-walled carbon nanotube modified...

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