ZnO Nanoparticle Ionic Liquids Carbon Paste ...

ZnO Nanoparticle Ionic Liquids Carbon Paste Electrode as a Voltammetric Sensor for Determination of Sudan I in the Presence of Vitamin B6 in Food Samples Jahan Bakhsh Raoof, Nader Teymoori & Mohammad A. Khalilzadeh

Food Analytical Methods ISSN 1936-9751 Volume 8 Number 4 Food Anal. Methods (2015) 8:885-892 DOI 10.1007/s12161-014-9962-z

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Author's personal copy Food Anal. Methods (2015) 8:885–892 DOI 10.1007/s12161-014-9962-z

ZnO Nanoparticle Ionic Liquids Carbon Paste Electrode as a Voltammetric Sensor for Determination of Sudan I in the Presence of Vitamin B6 in Food Samples Jahan Bakhsh Raoof & Nader Teymoori & Mohammad A. Khalilzadeh

Received: 6 June 2014 / Accepted: 5 August 2014 / Published online: 20 August 2014 # Springer Science+Business Media New York 2014

Abstract Room-temperature ionic liquid n-hexyl-3methylimidazolium hexafluoro phosphate as a binder and ZnO nanoparticle (ZnO/NPs) as a sensor were used to construct a new ZnO/NPs carbon ionic liquid paste electrode (ZnO/NPs/IL/CPE), which exhibited enhanced electrochemical behavior as compared with the traditional carbon paste electrode with paraffin for electrooxidation of Sudan I. This modified electrode exhibited a potent and persistent electron mediating behavior followed by well separated oxidation peaks of Sudan I and vitamin B6. The peaks current of square wave voltammograms (SWV) of Sudan I and vitamin B6 increased linearly with their concentration in the ranges of 0.01–400 μM Sudan I and 0.5–800 μM vitamin B6. The detection limits for Sudan I and vitamin B6 were 0.008– 0.2 μM, respectively. The modified electrode has been successfully applied for the assay of Sudan I and vitamin B6 in food samples. Keywords Vitamin B6 . Sudan I . Ionic liquid . Modified electrode . Food analysis

Introduction Sudan is an organic compound, typically classified as an azo dye (http://en.wikipedia.org/wiki/Sudan_I, 4 Jun, 2014). J. B. Raoof (*) : N. Teymoori Electroanalytical Chemistry Research Laboratory, Department of Analytical Chemistry, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran e-mail: [email protected] M. A. Khalilzadeh (*) Department of Phytochemistry, Qaemshahr Branch, Islamic Azad University, Qaemshahr, Iran e-mail: [email protected]

Sudan I has also been adopted for coloring various foodstuffs, especially curry powder and chili powder, although the use of Sudan I in foods is now banned in many countries because Sudan I, III, and IV have been classified as category 3 carcinogens by the International Agency for Research on Cancer (Refat et al. 2008). While Sudan I is strictly forbidden to be added to food products, it is still found in foodstuffs such as, poultry feed, paprika, ketchup, sausage, pie, etc., as additive due to its low cost, bright color, and stability (Karimi-Maleh et al. 2014a, b). Therefore, it is significant to develop a selective, simple, and high sensitivity method for detection and determination of Sudan I in food samples. A number of methods have been proposed for the determination of Sudan I including high-performance liquid chromatography (HPLC) (Long et al. 2011; Tateo and Bononi 2004), high-performance liquid chromatography–mass spectrometry (HPLC-MS) (Calbiani et al. 2004; Zhang et al. 2006), gas chromatography–mass spectrometry (GC-MS) (He et al. 2007), capillary electrophoresis (Mejia et al. 2007), and electrochemical methods (Lin et al. 2008; Ma et al. 2013; Elyasi et al. 2013). Among these methods, electrochemical sensors have attracted wide attention due to their convenience, fastness, high sensitivity, selectivity, and reproducibility (Yola et al. 2013, 2014a, b; Yola and Atar 2014; Sanghavi and Srivastava 2010, 2011; Sanghavi et al. 2013a, b; Gupta et al. 2000, 2002; Sanghavi and Srivastava 2013). Vitamin B6 is also called pyridoxine. It is involved in the process of making serotonin and norepinephrine, which are chemicals that transmit signals in the brain. It is also involved in the formation of myelin, a protein layer that forms around nerve cells. vitamin B6 deficiency in adults may cause health problems affecting the nerves, skin, mucous membranes, and circulatory system. In children, the central nervous system is also affected. Vitamin B6 has been studied for the treatment of many conditions, including anemia (low amounts of healthy

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red blood cells), vitamin B6 deficiency, certain seizures in newborns, and side effects of the drug cycloserine (http:// www.mayoclinic.org/drugs-supplements/vitamin-b6/ background/hrb-20058788; 4 Jun, 2014). Therefore, it is necessary to suggest a fast and simple method for its determination in biological and pharmaceutical samples. The science of nanomaterials has created great excitement and expectation in the recent years (Moradi et al. 2013; Sanghavi et al. 2013a, b, 2014a, b; Yola et al. 2014a, b; Karimi-Maleh et al. 2013, 2014a, b). Among various nanomaterials, metal oxides have the most attention with applications such as electrochemical sensor (Sanati et al. 2014; Sadeghi et al. 2013; Roodbari Shahmiri et al. 2013) and starting materials for preparing advanced structural ceramics (Reddy et al. 2010). On the other hand, metal oxide nanoparticles of a variety of shapes, sizes, and compositions are changing nowadays the electro-active materials measurement. In continuation of our studies on the preparation of chemically modified electrodes (Ojani et al. 2014; Raoof et al. 2008, 2011, 2012, 2013), a ZnO nanoparticle (ZnO/NPs) carbon ionic liquid paste electrode (ZnO/NPs/IL/CPE) was prepared and used as a high-sensitive voltammetric sensor for determination of Sudan I in food samples. We have also evaluated the analytical performance of the modified electrode for quantification of Sudan I in the presence of vitamin B6 in some food samples.

Food Anal. Methods (2015) 8:885–892

heated solution under high-speed stirring. The beaker was sealed at this condition for 2 h. The precipitated ZnO/NPs were cleaned with deionized water and ethanol then calcined at 250 °C for 2 h. Preparation of the Sensor ZnO/NPs/IL/CPE was prepared by mixing 0.2 g of n-hexyl-3methylimidazolium hexafluoro phosphate, 0.8 g of the liquid paraffin, 0.2 g of ZnO/NPs, and 0.8 g of graphite powder. Then, the mixture was mixed well for 45 min until a uniformly wetted paste was obtained. A portion of the paste was filled firmly into one glass tube as described above to prepare ZnO/ NPs/IL/CPE. When necessary, a new surface was obtained by pushing an excess of the paste out of the tube and polishing it on a weighing paper. Preparation of Real Samples To prepare the samples, 2.5 g sample was weighed exactly and 100.0 mL ethanol was added. After 30-min ultrasonication, the mixture was filtrated with a 0.25-μm organic filter membrane and the liquid phase was collected in a 100.0-mL volumetric flask. Then, a proper amount of the sample solution was transferred to the cell and detected by square wave voltammograms (SWV) under the optimal conditions.

Experimental Results and Discussion Chemicals X-Ray Diffraction of ZnO Nanoparticles All chemicals were of analytical reagent grade and were purchased from Merck (Darmstadt, Germany) or Fluka unless otherwise stated. Double-distilled water was used throughout for all experiments. Phosphate buffer solution (PBS) with different pH values was used. Sudan I stock solution, 1.0×10−3 μM, was prepared by dissolving 0.028 g of the reagent in a 100-mL volumetric flask (ethanol/water (1:1) solution). Vitamin B6 stock solution, 1.0×10−2 mol l−1, was prepared by dissolving 0.169 g of the reagent in a 100-mL volumetric flask.

The x-ray diffraction (XRD) patterns of the ZnO/NPs showed diffraction peaks being absorbed at 2θ values (Fig. 1a). The

Synthesis of ZnO/NPs To prepare ZnO/NPs, in a typical experiment, a 0.25 M aqueous solution of zinc nitrate (Zn (NO3)2·4H2O) and 0.5 M aqueous solution of sodium hydroxide (NaOH) were prepared in distilled water. Then, the beaker containing NaOH solution was heated at the temperature of about 55 °C. The Zn (NO3)2 solution was added drop wise (slowly for 2.0 h) to the above-

Fig. 1 XRD patterns of as-synthesized ZnO/NPs

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prominent peaks were used to calculate the grain size via the Scherrer equation, expressed as follows: D ¼ Kλ=ðβ cosθÞ

ð1Þ

Where λ is the wavelength (λ=1.542 Å) (CuKα), β is the full width at half maximum (FWHM) of the line, and θ is the diffraction angle. The grain size of the ZnO nanostructure was 21 nm, and the peaks were observed at the (100), (002), (101), (102), (110), (103), (200), (112), (201), (004), and (202) planes. These peaks correspond to ZnO/NPs. Electrochemical Investigation Sudan I, as a phenol derivative, can be oxidized at positive potential. It depends on the electrode type and solution pH. We anticipated that the oxidation of Sudan I would be pH dependent. In order to ascertain this, the voltammetric response of Sudan I at the surface of ZnO/NPs/IL/CPE was obtained in solutions with varying pH (Fig. 2 inset). Results show that the peak potential of the redox couple was pH dependent with a slope of −67.0 mV/pH unit at 25 °C. This was found to be equal to the anticipated Nernstian value for a one-electron, one-proton electrochemical reaction. It can be seen that the maximum value of the peak current appeared at pH 8.0, so this value was selected throughout the experiments (Fig. 2). Figure 3 (inset) shows the current density derived from the cyclic voltammograms responses of 150 μM Sudan I (pH 8.0) at the surface of different electrodes and with a scan rate of 50 mV s−1. The direct electrochemistry of Sudan I on the modified electrode was investigated by cyclic voltammetry. ZnO/NPs/IL/CPE exhibited significant oxidation peak current of 9.45 μA around 640 mV (Fig. 3, curve a). In contrast, low

Fig. 2 Current-pH curve for electrooxidation of 100.0 μM Sudan I at ZnO/NPs/IL/CPE with a scan rate of 50 mV s−1. Inset influence of pH on cyclic voltammograms of Sudan I at a surface of the modified electrode, (pH 6, 7, 8, and 9, respectively)

redox activity peak was observed at ZnO/NPs/CPE (Fig. 3, curve c) and at unmodified CPE (Fig. 3 curve d) over the same potential range. The Sudan I oxidation peak potential at ZnO/ NPs/CPE and at CPE observed was around 670 and 710 mV in contrast to the Ag/AgCl/KClsat reference electrode and have an oxidation peak current of 5.9 and 3.7 μA, respectively. In addition, at the surface of bare IL/CPE, the oxidation peak appeared at 650 mVand the peak current achieved was 6.9 μA (Fig. 3, curve b). This indicated that the presence of ILs in CPE could enhance the peak currents and decrease the oxidation potential (decreasing the overpotential). A substantial negative shift of the current starting from the oxidation potential for Sudan I and dramatic increase of current of Sudan I indicated the catalytic ability of ZnO/NPs/IL/CPE to Sudan I oxidation. The results indicated that the presence of ZnO/NPs on ZnO/NPs/IL/CPE surface had a great improvement on the electrochemical response, which was partly due to the excellent characteristics of ZnO/NPs such as good electrical conductivity, high chemical stability, and high surface area. The suitable electronic properties of ZnO/NPs, together with the ionic liquid, gave the ability to promote charge transfer reactions and good anti-fouling properties, especially when mixed with a higher conductive compound such as ILs when used as an electrode. The active surface areas of the working electrodes are estimated according to the slope of the IP vs. ν1/2 plot for a known concentration of K4Fe(CN)6, based on the RandlesSevcik equation and from the slope of the Ipa–ν1/2 relation, the microscopic areas were calculated. They were 0.25, 0.18, 0.14, and 0.09 cm2 for ZnO/NPs/IL/CPE, IL/CPE, ZnO/ NPs/CPE, and CPE, respectively. The results show that the presence of ZnO/NPs and IL, together, causes the increase of the active surface. According to scientific investigations, nanomaterials have high surface area and can increase current



Fig. 3 Cyclic voltammograms of a ZnO/NPs/IL/CPE, b IL/CPE, c ZnO/NPs/CPE, and d CPE in the presence of 150 μM Sudan I at pH 8.0, respectively. Inset the current density derived from cyclic voltammograms responses of 150 μM Sudan I. Conditions: 0.1 mol L−1 PBS (pH 8.0); scan rate of 50 mV s−1

density and sensitivity in modified electrodes. On the other hand, to summarize contrary to the electrodes modified by unsupported organic phase, the electrochemical generation of charge in IL deposit may generate the transfer of its component across liquid|liquid interface. These points can increase the active surface area for modified electrode. The effect of potential scan rate on the peak current of Sudan I was also studied. It can be seen that Sudan I (Fig. 4 inset) is completely irreversible in nature. From the inset in Fig. 4, it can be seen that the oxidation peak shifted to a more positive value for Sudan I with increasing scan rates along with a concurrent increase in current. The cyclic voltammetric results indicated that the anodic peak currents (Ip) of Sudan I increase linearly with the square root of the scan rate (ν1/2) in the range from 20 to 250 mVs−1. This finding implied that the

Fig. 4 Plot of Ipa vs. ν1/2 for the oxidation of 100 μM Sudan I at ZnO/NPs/IL/CPE. Inset shows cyclic voltammograms of Sudan I at ZnO/NPs/IL/CPE at different scan rates of a 20, b 50, c 100, d 150, and e 250 mV s−1 in 0.1 M phosphate buffer, pH 8.0

oxidation of Sudan I is diffusion controlled on the ZnO/NPs/ IL/CPE (Tavana et al. 2012; Ensafi et al. 2011a, b). To obtain information about the rate-determining step, the Tafel plot was drawn, as derived from points in the Tafel region of the cyclic voltammogram (Fig. 5). The slope of the Tafel plot was equal to 2.3 RT/n(1−α)F, which came up to 0.267 V decade−1 for scan rate 20 mV s−1. Therefore, we obtained the mean value of α equal to 0.78 (for n=1). The electrooxidation of Sudan I by a ZnO/NPs/IL/CPE was also studied by chronoamperometry (Fig. 6). Chronoamperometric measurements of different concentrations of Sudan I at this sensor were accomplished by setting t h e w o r k i n g e l e c t r o d e p o t e n t i a l a t 7 0 0 m V. I n chronoamperometric studies, we determined the diffusion coefficient (D) of Sudan I. The experimental plots of I vs.

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Fig. 5 Tafel plot for ZnO/NPs/ IL/CPE in 0.1 M PBS (pH 8.0) with a scan rate of 20 mV s−1 in the presence of 120 μM Sudan I

t−1/2 were employed with the best fits for different concentrations of Sudan I. The slopes of the resulting straight lines were then plotted vs. Sudan I concentrations. By using the Cottrell equation (Bard and Faulkner 2001): I ¼ nFAD1=2 C b π−1=2 t −1=2

ð2Þ

The diffusion coefficient for Sudan I was calculated as 5.13×10−5 cm2 s−1. The stability and reproducibility of any sensor are two important parameters. Our experiments showed that after ZnO/NPs/IL/CPE was stored for 4 weeks at 4 °C, only a small decrease of peak current sensitivity with a relative standard deviation (RSD) of 1.5 % (for 15.0 μM Sudan I) was

Fig. 6 a Chronoamperograms obtained at ZnO/NPs/IL/CPE in the presence of a 200; b 300 and c 400 μM Sudan I in the buffer solution (pH 8.0). b Cottrell’s plot for the data from the chronoamperograms

observed. This showed good stability of the modified electrode. Furthermore, the reproducibility of the determination was performed with nine successive scans in the solution containing 15.0 μM Sudan I. The RSD values were found to be 2.4 % for the analyte, indicating good reproducibility of the modified electrode. The electrode can be immersed in an aqueous media for 2.5 h with stable response. After that, the background current began to increase, which may be due to the partly leakage of ionic liquid from the electrode and the roughness of the electrode surface was increases gradually. Calibration Plot and Limit of Detection Since square wave voltammetry has a much higher current sensitivity than cyclic voltammetry, therefore, it was used for the determination of Sudan I. The plot of peak current vs. Sudan I concentration consisted of two linear segments with slopes of 0.5799 and 0.0557 μA/μM in the concentration ranges of 0.01–4.53.0 μM and 4.53–400.0 μM, respectively. The decrease in sensitivity (slope) of the second linear segment is likely due to kinetic limitation. The difference in the slopes for the calibration curves is due to the different activity of the electrode surface with low and high concentration of the analyte. In the lower Sudan I concentration, due to a high number of active sites (in relation to the total number of the analyte molecules), the slope of the first calibration curve is high. While in the higher Sudan I concentration, due to decreasing active sites (in relation to the total number of analyte molecules, mainly at the surface of the electrode), sensitivity dramatically decreased and therefore, the second calibration slope decreased too. On the other hand, the sensitivity for vitamin B6 in the range of 0.5–800 μM is 0.0302 μA/ μM. The detection limits were determined at 0.008 μM for



Fig. 7 The plots of the electrocatalytic peak current as a function of Sudan I concentration. Inset A SWVs of ZnO/NPs/IL/ CPE in 0.1 M PBS (pH 8.0) containing different concentrations of Sudan I– vitamin B6 in micrometers. a–f 0.38+5.1; 1.13+10.0; 2.03+15.0; 3.2+22.0; 3.9+28, and 4.5+30.0, respectively. Inset B SWVs of ZnO/NPs/IL/CPE in 0.1 M PBS (pH 8.0) containing 4.5+30.0 μM of Sudan I–vitamin B6, respectively

Sudan I and 0.2 μM for vitamin B6 according to the definition of YLOD =YB +3σ (Skoog et al. 1998).

Simultaneous Determination of Sudan I and Vitamin B6 The main object of this study was to detect Sudan I and vitamin B6 simultaneously using ZnO/NPs/IL/CPE. This was performed by simultaneously changing the concentrations of ZnO/NPs/IL/CPE, and recording the SWVs. The voltammetric results showed well defined anodic peaks at potentials of 640 and 800 mV, corresponding to the oxidation of Sudan I and vitamin B6, respectively. This is indicating that simultaneous determination of these compounds is feasible at ZnO/NPs/IL/CPE as shown in Fig. 7 inset a. On the other hand, at an unmodified electrode these compound signals cannot separate very well (Fig. 7 inset b). Also, the sensitivity of the modified electrode towards the oxidation of Sudan I in the presence of vitamin B6 was found to be 0.5446 μA/μM

(Fig. 7). This is very close to the value obtained in the absence of vitamin B6 (0.5799 μA/μM) indicating that the oxidation processes of these compounds at the ZnO/NPs/IL/CPE are independent and therefore, simultaneous determination of their mixtures is possible without significant interferences.

Interference Studies The influence of various substances as potentially interfering compounds with the determination of Sudan I was studied under the optimum conditions with 10.0 μM Sudan I at pH 8.0. The potential interfering substances were chosen from the group of substances commonly found with Sudan I in food samples. The tolerance limit was defined as the maximum concentration of the interfering substance that caused an error of less than ±3 % for the determination of Sudan I. After the experiments, we found that neither 950-fold of methionine, alanine, phenylalanine, valine, tryptophan, glycine, valine,

Table 1 Determination of Sudan I in food samples using propose sensor Sample

Added (μM)

Expected (μmol L−1)

Founded (μM)

Published method (μM)

Fex

Ftab

tex

ttab(95%)

Chili sauce

– 5.0 – 10.0 – 0.5 – 20.0

– 5.0 – 10.0 – 0.5 – 20.0