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Voltammetric determination of caffeine by using gold nanoparticle-glassy carbon paste composite electrode
Measurement 106 (2017) 26–30
Contents lists available at ScienceDirect
Measurement journal homepage: www.elsevier.com/locate/measurement
Voltammetric determination of caffeine by using gold nanoparticle-glassy carbon paste composite electrode Tug˘ba Ören, Ülkü Anık ⇑ Mug˘la Sıtkı Koçman University, Faculty of Science, Chemistry Department, 48000 Kötekli/Mug˘la, Turkey
a r t i c l e
i n f o
Article history: Received 30 March 2016 Received in revised form 2 September 2016 Accepted 18 April 2017 Available online 20 April 2017 Keywords: Caffeine AuNP Composite electrode Glassy carbon paste electrode
a b s t r a c t In the present study, simple and sensitive voltammetric determination of caffeine was carried out by using a gold nanoparticle-glassy carbon paste composite electrode (AuNP-GCPE) in 3.2 mM H2SO4 (pH 2.5). The effects of supporting electrolyte, pH and AuNP amount on caffeine peak current were investigated. After the optimization of experimental parameters, analytical characteristics of AuNP-GCPE were examined and two linear ranges were obtained between 25–150 lM and 200–1000 lM with limit of detection values of 0.96 lM and 4.90 lM, respectively. Relative standard deviation for 50 lM caffeine was also calculated as 7.52% (n = 3). In order to evaluate the analytical performance of AuNP-GCPE, caffeine contents of various brands of cola samples were measured and compared with the results obtained by UV–Vis spectrometer. The obtained results by using the proposed composite electrode are in accordance with the caffeine contents that manufacturers claimed compared to UV–Vis. This composite electrode which utilizes the catalytic effect of AuNP with the simple preparation of GCPE, offers a fast and sensitive determination of caffeine in beverages as a practical application. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Caffeine, a methylxanthine derivative, is a natural alkaloid which is the main component of daily consumed beverages and food like coffee, tea, coke, energetic drinks and chocolate [1,2]. Besides, caffeine is also used in several pharmaceutical formulations due to its psychoactive effects such as stimulation of central nervous system, diuresis and gastric acid secretion [1,3]. On the other hand, excessive intake of caffeine may cause mutagenic effects in DNA repair. This situation also affects cyclic AMP phosphodiesterase activity and causes cardiovascular problems, kidney diseases and complications in pregnancy [2,4,5]. Furthermore, caffeine intake in doses of 150–200 mg kg 1 of body weight is considered to be fatal [2]. Thus, the development of a fast and sensitive method for caffeine determination is vital for testing food quality and for clinical applications. Numerous analytical methods have been developed for the caffeine determination up to now. Most of these methods are based on spectroscopic [6–8] and hyphenated chromatographic techniques [9–11] in which remarkable results were obtained in terms of sensitivity. However, these methods are complicated, laborious and time consuming which need sample pre-treatment procedures
⇑ Corresponding author. E-mail address: [email protected] (Ü. Anık). http://dx.doi.org/10.1016/j.measurement.2017.04.031 0263-2241/Ó 2017 Elsevier Ltd. All rights reserved.
and expensive bulky equipments [2]. As an alternative choice, electrochemical methods have attracted a considerable attention by providing fast and sensitive caffeine detection in a wide variety of samples with simple and low cost instrumentation [2,4,5,12– 18]. Moreover, in most cases there is no need to use sophisticated sample pre-treatment procedures prior to analysis. However, electrochemical methods suffer from more positive oxidation potential of caffeine which leads to large background currents in blank solution and strongly adsorbed oxidation products on the electrode surface [13]. In order to overcome these limitations, carbon based composite electrodes may provide an opportunity for the electrochemical determination of caffeine by combining the superior properties of the materials used in the electrode fabrication. Also, since these electrodes are practical in terms of renewal and preparation procedures, they can solve problems. Besides these, composite electrodes may be adapted to various electrode configurations with great flexibility in size and shape of the material [19]. Glassy carbon paste electrode (GCPE) is a kind of composite carbon electrode which can be easily prepared just by mixing proper amount of glassy carbon microparticles and oil binder. GCPE can be defined as a favourable candidate among carbon based electrodes due to its higher electrochemical reactivity, wider potential window and low background current as well as its easy preparation at low cost. Also, since it is easy to renew the surfaces of these electrodes, fresh
T. Ören, Ü. Anık / Measurement 106 (2017) 26–30
surfaces are obtained prior to each measurement which make their usages advantageous in the case of strongly adsorbed oxidation products. Since the components used in electrode fabrication are homogeneously dispersed and compressed in the bulk of the structure, it is possible to obtain reproducible results with every new surface [19–21]. To the best of our knowledge, although there are several research papers based on the usage of carbon based electrode itself and also as the component of the composite electrode fabrication for caffeine detection, GCPE has not been used for this purpose [22–26]. To date, several researches dealing with GCPE have been performed and nanomaterials and biomolecules were used to improve the electronic and catalytic properties of these composite electrodes [19,21,27]. Among these materials, owing to its higher conductivity, larger surface area and applicability in biosensing platforms, gold nanoparticles (AuNP) became very popular and were also utilized for the electrode modification in voltammetric determination of caffeine [28,29]. By taking into consideration of these unique properties of GCPE and AuNP, these mentioned features may be combined to bring new insights into the composite electrode fabrication especially for caffeine detection. Thus, in the present study, it was aimed to fabricate a composite electrode by combining the properties of AuNP and GCPE for the rapid and sensitive voltammetric determination of caffeine for the first time. After the optimization of the experimental conditions and the examination of analytical characteristics, AuNPGCPE was applied to the determination of caffeine amount in various cola beverage samples and the results were compared with UV–Vis spectroscopy.
27
on a weighing paper and rinsed with double distilled water prior to measurement. 2.4. Procedure The oxidation peak of caffeine was obtained by scanning potential from 0.8 V to 1.6 V in differential pulse mode with a 0.005 V of step potential, 0.025 V of modulation amplitude and scan rate of 10 mV/s using 3.2 mM H2SO4 (pH 2.5) as the supporting electrolyte. Prior to each measurement, AuNP-GCPE surface was renewed and the reference electrode was rinsed with double distilled water. Pt wire was sonicated for 3 min in 0.1 M H2SO4 to remove strongly adsorbed oxidation products of caffeine. 2.5. Sample preparation Different branded cola samples were purchased from a local supermarket and stored at room temperature until sample preparation. Cola samples were degassed by ultrasonication for 10 min and diluted in the ratio of 1:10 (v/v) with supporting electrolyte containing a known amount of caffeine standard solution [4]. 3. Results and discussion The optimization studies including pH effect and AuNP amount were conducted in the first place. For optimum pH study, plain GCPE was used. Then, optimization of AuNP amount was found. After that, analytical characteristics and sample application were made.
2. Experimental
3.1. Influence of pH
2.1. Apparatus
The influence of pH on caffeine oxidation peak current was evaluated in presence of 50 lM caffeine by varying pH from 1.0 to 6.0. Within this scope, pH dependence of caffeine peak current was examined using GCPE in pH ranges 1.0 to 3.0 with H2SO4 solution, 3.0 to 5.0 with acetate buffer and at pH 6.0 with phosphate buffer, respectively. As shown in Fig 1, the highest peak current was obtained for pH 2.5 in case of using 3.2 mM H2SO4 at GCPE. Since the highest peak current at GCPE was obtained in pH 2.5, further experiments using AuNP-GCPE were also conducted at the same pH.
Differential pulse voltammetry (DPV) measurements were carried out with AUTOLAB PGSTAT 12 electrochemical measurement system (ECO CHEMIE Instruments B.V., The Netherlands) equipped with NOVA 1.10 software. The experiments were conducted in a 10 mL voltammetric cell using conventional three-electrode configuration consisted of GCPE or AuNP-GCPE as the working electrode, Ag/AgCl and platinum wire as the reference and the counter electrodes, respectively. Absorbance measurements were performed on a T60 model UV–Vis spectrometer (PG Instruments, England). 2.2. Reagents and materials All reagents were used as received without further purification. Caffeine was purchased from Merck. Glassy carbon spherical powder (2–12 mm, 99.95% metals basis), mineral oil and gold colloid (0.75 A520 units/mL; 10 nm 0.001% as HAuCl4) were obtained from Sigma-Aldrich for the preparation of GCPE and AuNP-GCPE. 3.2 mM H2SO4 (pH 2.5) was used as the supporting electrolyte. pH of the supporting electrolyte was adjusted with 0.1 M KOH (Riedel-de Haën). All solutions were prepared with double distilled water.
3.2. Effect of AuNP amount The amount of AuNP was varied as 0.5, 1, 2, 5 and 10 lL to examine the relationship between AuNP amount and peak current of 50 lM caffeine. As depicted in Fig 2, peak current increased up
2.3. Preparation of AuNP-GCPE AuNP-GCPE was prepared in the composition of 80:20 (w/w%) glassy carbon paste powder/mineral oil with 1 lL of gold colloid by hand-mixing. The required amount of the homogeneous paste was filled into the electrode cavity of a Delrin tube where electrical contact was provided via a copper wire. The surface was smoothed
Fig. 1. The influence of pH on voltammetric response of 50 lM caffeine using GCPE.
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T. Ören, Ü. Anık / Measurement 106 (2017) 26–30
Fig. 2. The effect of AuNP amount on peak current for 50 lM caffeine in 3.2 mM H2SO4 (pH 2.5).
to 1 lL and then a sharp decrease on peak current was observed. The current decrease with increasing AuNP amount may be explained with the phenomena which was previously reported by Liu et al. [30] and Çubukçu et al. [31]. According to these studies, increasing AuNP amount causes the resistance and capacitance increment due to double layer formation. Thus, as the ratio of carbon sensing sites in the paste decreases, peak current also decreases [30,31]. As a result of this finding, 1 lL was used in further experiments.
Fig. 3. DPV responses of (A) supporting electrolyte, (B) plain GCPE and (C) AuNPGCPE in presence of 50 lM caffeine in 3.2 mM H2SO4 (pH 2.5). Step potential: 0.005 V, modulation amplitude: 0.025 V and scan rate: 10 mV/s.
3.3. DPV responses of caffeine at GCPE and AuNP-GCPE Within this perspective, DPV responses of 50 lM caffeine in 3.2 mM H2SO4 (pH 2.5) were recorded where GCPE or AuNP-
Fig. 4. DPV responses of caffeine in concentration range of (A) 25–150 lM and (B) 200–1000 lM at AuNP-GCPE. The linear relationship between oxidation peak current and concentration of caffeine at Au-GCPE in the ranges of 25–150 lM and 200–1000 lM (C) and in the range of 50–1000 lM when used GCPE (D).
T. Ören, Ü. Anık / Measurement 106 (2017) 26–30 Table 1 The effect of interfering species on DPV response of caffeine at AuNP-GCPE. Recovery of DPV response of caffeine (%) Interferent/caffeine concentration Interfering species
1:1
5:1
10:1
Glucose Fructose Ascorbic acid Hypoxanthine Glycine Citric acid
97.2 96.6 92.2 105.4 102.8 101.2
103.9 106.9 104.9 94.8 91.9 90.3
104.8 103.6 108.0 104.8 101.0 95.7
Table 2 Determination of caffeine contents in spiked cola beverage samples by using AuNPGCPE and UV–Vis. Sample
Cola beverage 1 Cola beverage 2 (sugar-free) Cola beverage 3 (sugar-free)
Average caffeine content ± SD(g/L) (n = 3) AuNP-GCPE
UV–Vis
Declared by manufacturer
0.141 ± 0.029 0.155 ± 0.039 0.133 ± 0.028
0.205 ± 0.007 0.234 ± 0.004 0.207 ± 0.003
Max. 0.150 Max. 0.150 0.120
29
hypoxanthine and glycine, DPV responses of 50 lM caffeine in presence of these interfering species were recorded. According to Table 1, the results were obtained in acceptable limits without significant changes in peak current. 3.6. Sample application The applicability of AuNP-GCPE was evaluated to determine the caffeine content in various cola beverage samples. Subsequent to sample preparation which is described earlier according to the reported procedure [4] required aliquots of caffeine standard solution spiked samples were analyzed and presented as average of three measurements ± standard deviation (SD) in Table 2. In order to examine the accuracy of the method, the same sample preparation procedure was used for UV–Vis measurements which were carried out at 272 nm [6]. As can be seen in Table 2, the results obtained by AuNP-GCPE are in accordance with the caffeine contents declared by manufacturer whereas UV–Vis results are quite different. These results indicated that a sample pre-treatment procedure should be conducted to eliminate the interferences in sample matrix for UV–Vis measurements prior to analysis while the dilution of the sample with supporting electrolyte suggests a practical way for the proposed method by using AuNP-GCPE.
4. Conclusions GCPE was served as the working electrode to examine the effect of AuNP. As demonstrated in Fig. 3, a peak at 1.38 V with 1.029 lA oxidation current was obtained at plain GCPE whereas a peak at 1.36 V with 2.721 lA oxidation current was observed at AuNPGCPE. As can be seen from these voltammograms, introduction of AuNP into GCPE structure increases the current value and also slightly decreases the peak potential indicating the feasibility of AuNP in voltammetric caffeine detection. This situation may be attributed to the increase of electron transfer rate due to electrocatalytic effect of AuNP providing a promotion in the electrochemical oxidation process of caffeine on the electrode surface [28,29].
Combination of AuNP with GCPE provides sensitive results to caffeine detection. Also the nature of composite electrode brings practicality to the preparation and renewal of electrode surface which eliminates problems like adsorption of oxidation products on the electrode surface. On the other hand, developed sensor showed closer caffeine values to manufacturer declarations when compared to UV–VIS spectrophotometer. As a result, it can be concluded that practical and effective electrochemical sensor for caffeine detection was obtained. References
3.4. Analytical characteristics The analytical characteristics of GCPE and AuNP-GCPE were investigated after the optimization of experimental parameters. In case of using AuNP-GCPE, two linear ranges were obtained between 25–150 lM and 200–1000 lM caffeine with equations of y = 0.0439x 0.5442 (R2 = 0.99) and y = 0.0086x + 5.57 (R2 = 0.98) (Fig 4). Limit of detection (LOD) was defined as 3s/m, where s is the standard deviation of blank (n = 7) and m is the slope of the calibration graph and calculated for both linear ranges as 0.96 lM for the range of 25–150 lM and 4.90 lM for the range of 200–1000 lM. For comparison, calibration graph was also plotted between 50 and 1000 lM using plain GCPE and the equation of the calibration graph was found as y = 0.0174x + 0.7757 (R2 = 0.99) (Fig 4D). Besides, LOD was also calculated for GCPE as 2.49 lM. It can be obviously seen that more sensitive results were obtained for lower caffeine concentrations using Au-GCPE compared to plain GCPE. In order to evaluate the repeatability, relative standard deviation was calculated for 50 lM caffeine (n = 3) as 7.52% in case of serving Au-GCPE as the working electrode. 3.5. Interference study In order to test the selectivity of AuNP-GCPE towards possible interferents such as glucose, fructose, ascorbic acid, citric acid,
[1] L. Švorc, P. Tomcˇík, J. Svítková, M. Rievaj, D. Bustin, Voltammetric determination of caffeine in beverage samples on bare boron-doped diamond electrode, Food Chem. 135 (2012) 1198–1204. [2] W.Y.H. Khoo, M. Pumera, A. Bonanni, Graphene platforms for the detection of caffeine in real samples, Anal. Chim. Acta 804 (2013) 92–97. [3] M.A. Rostagno, N. Manchón, M. D’Arrigo, E. Guillamón, A. Villares, A. GarcíaLafuente, A. Ramos, J.A. Martínez, Fast and simultaneous determination of phenolic compounds and caffeine in teas, mate, instant coffee, soft drink and energetic drink by high-performance liquid chromatography using a fusedcore column, Anal. Chim. Acta 685 (2011) 204–211. [4] K. Tyszczuk-Rotko, I. Be˛czkowska, Nafion covered lead film electrode for the voltammetric determination of caffeine in beverage samples and pharmaceutical formulations, Food Chem. 172 (2015) 24–29. [5] W.D.J.R. Santos, M. Santhiago, I.V.P. Yoshida, L.T. Kubota, Electrochemical sensor based on imprinted sol–gel and nanomaterial for determination of caffeine, Sens. Actuators B 166–167 (2012) 739–745. [6] A. Belay, K. Ture, M. Redi, A. Asfaw, Measurement of caffeine in coffee beans with UV/vis spectrometer, Food Chem. 108 (2008) 310–315. [7] X. Zhang, W. Li, B. Yin, W. Chen, D.P. Kelly, X. Wang, K. Zheng, Y. Du, Improvement of near infrared spectroscopic (NIRS) analysis of caffeine in roasted Arabica coffee by variable selection method of stability competitive adaptive reweighted sampling (SCARS), Spectrochim. Acta Part A 114 (2013) 350–356. [8] L. Franzen, J. Anderski, M. Windbergs, Quantitative detection of caffeine in human skin by confocal Raman spectroscopy – a systematic in vitro validation study, Eur. J. Pharm. Biopharm. 95 (2015) 110–116. [9] S.S. Verenitch, C.J. Lowe, A. Mazumder, Determination of acidic drugs and caffeine in municipal wastewaters and receiving waters by gas chromatography–ion trap tandem mass spectrometry, J. Chromatogr. A 1116 (2006) 193–203. [10] H. Li, C. Zhang, J. Wang, Y. Jiang, J.P. Fawcett, J. Gu, Simultaneous quantitation of paracetamol, caffeine, pseudoephedrine, chlorpheniramine and cloperastine in human plasma by liquid chromatography–tandem mass spectrometry, J. Pharm. Biomed. Anal. 51 (2010) 716–722.
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[11] G.M. Hadad, R.A.A. Salam, R.M. Soliman, M.K. Mesbah, Rapid and simultaneous determination of antioxidant markers and caffeine in commercial teas and dietary supplements by HPLC-DAD, Talanta 101 (2012) 38–44. [12] R.N. Goyal, S. Bishnoi, B. Agrawal, Electrochemical sensor for the simultaneous determination of caffeine and aspirin in human urine samples, J. Electroanal. Chem. 655 (2011) 97–102. [13] Y. Gao, H. Wang, L. Guo, Simultaneous determination of theophylline and caffeine by large mesoporous carbon/Nafion modified electrode, J. Electroanal. Chem. 706 (2013) 7–12. [14] J.-M. Zen, Y.-S. Ting, Y. Shih, Voltammetric determination of caffeine in beverages using a chemically modified electrode, Analyst 123 (1998) 1145– 1147. [15] B.C. Lourenção, R.A. Medeiros, R.C. Rocha-Filho, L.H. Mazo, O. Fatibello-Filho, Simultaneous voltammetric determination of paracetamol and caffeine in pharmaceutical formulations using a boron-doped diamond electrode, Talanta 78 (2009) 748–752. [16] J.-M. Zen, Y.-S. Ting, Simultaneous determination of caffeine and acetaminophen in drug formulations by square-wave voltammetry using a chemically modified electrode, Anal. Chim. Acta 342 (1997) 175–180. [17] B.C. Lourenção, R.A. Medeiros, R.C. Rocha-Filho, O. Fatibello-Filho, Simultaneous differential pulse voltammetric determination of ascorbic acid and caffeine in pharmaceutical formulations using a boron-doped diamond electrode, Electroanalysis 22 (2010) 1717–1723. [18] S.Y. Ly, Y.S. Jung, M.H. Kim, I. Kwon Han, W.W. Jung, H.S. Kim, Determination of caffeine using a simple graphite-pencil electrode with square-wave anodic stripping voltammetry, Microchim. Acta 146 (2004) 207–213. [19] U. Anik, M. Çubukçu, Y. Yavuz, Nanomaterial-based composite biosensor for glucose detection in alcoholic beverages, Artif. Cells Nanomed. Biotechnol. 41 (2013) 8–12. [20] J. Wang, Ü. Anik Kirgöz, J.-W. Mo, J. Lu, A.N. Kawde, A. Muck, Glassy carbon paste electrodes, Electrochem. Commun. 3 (2001) 203–208. [21] S. Çevik, Ü. Anik, Banana tissue-nanoparticle/nanotube based glassy carbon paste electrode biosensors for catechol detection, Sens. Lett. 8 (2010) 667–671.
[22] M. Aklilu, M. Tessema, M. Redi-Abshiro, Indirect voltammetric determination of caffeine content in coffee using 1,4-benzoquinone modified carbon paste electrode, Talanta 76 (2008) 742–746. [23] B.J. Sanghavi, A.K. Srivastava, Simultaneous voltammetric determination of acetaminophen, aspirin and caffeine using an in situ surfactant-modified multiwalled carbon nanotube paste electrode, Electrochim. Acta 55 (2010) 8638–8648. [24] T. Alizadeh, M.R. Ganjali, M. Zare, P. Norouzi, Development of a voltammetric sensor based on a molecularly imprinted polymer (MIP) for caffeine measurement, Electrochim. Acta 55 (2010) 1568–1574. [25] G.A.M. Mersal, Experimental and computational studies on the electrochemical oxidation of caffeine at pseudo carbon paste electrode and its voltammetric determination in different real samples, Food Anal. Methods 5 (2012) 520–529. [26] Z.M. Khoshhesab, Simultaneous electrochemical determination of acetaminophen, caffeine and ascorbic acid using a new electrochemical sensor based on CuO–graphene nanocomposite, RSC Adv. 5 (2015) 95140–95148. [27] N.G. Khare, R.A. Dar, A.K. Srivastava, Determination of carbendazim by adsorptive stripping differential pulse voltammetry employing glassy carbon paste electrode modified with graphene and amberlite XAD 2 resin, Electroanalysis 27 (2015) 1915–1924. [28] B. Rezaei, M.K. Boroujeni, A.A. Ensafi, Caffeine electrochemical sensor using imprinted film as recognition element based on polypyrrole, sol-gel and gold nanoparticles hybrid nanocomposite modified pencil graphite electrode, Biosens. Bioelectron. 60 (2014) 77–83. [29] G. Yang, F. Zhao, B. Zeng, Facile fabrication of a novel anisotropic gold nanoparticle–chitosan–ionic liquid/graphene modified electrode for the determination of theophylline and caffeine, Talanta 127 (2014) 116–122. [30] S. Liu, J. Yu, H. Ju, Renewable phenol biosensor based on a tyrosinase-colloidal gold modified carbon paste electrode, J. Electroanal. Chem. 540 (2003) 61–67. [31] M. Çubukçu, S. Timur, Ü. Anik, Examination of performance of glassy carbon paste electrode modified with gold nanoparticle and xanthine oxidase for xanthine and hypoxanthine detection, Talanta 74 (2007) 434–439.
Contents lists available at ScienceDirect
Measurement journal homepage: www.elsevier.com/locate/measurement
Voltammetric determination of caffeine by using gold nanoparticle-glassy carbon paste composite electrode Tug˘ba Ören, Ülkü Anık ⇑ Mug˘la Sıtkı Koçman University, Faculty of Science, Chemistry Department, 48000 Kötekli/Mug˘la, Turkey
a r t i c l e
i n f o
Article history: Received 30 March 2016 Received in revised form 2 September 2016 Accepted 18 April 2017 Available online 20 April 2017 Keywords: Caffeine AuNP Composite electrode Glassy carbon paste electrode
a b s t r a c t In the present study, simple and sensitive voltammetric determination of caffeine was carried out by using a gold nanoparticle-glassy carbon paste composite electrode (AuNP-GCPE) in 3.2 mM H2SO4 (pH 2.5). The effects of supporting electrolyte, pH and AuNP amount on caffeine peak current were investigated. After the optimization of experimental parameters, analytical characteristics of AuNP-GCPE were examined and two linear ranges were obtained between 25–150 lM and 200–1000 lM with limit of detection values of 0.96 lM and 4.90 lM, respectively. Relative standard deviation for 50 lM caffeine was also calculated as 7.52% (n = 3). In order to evaluate the analytical performance of AuNP-GCPE, caffeine contents of various brands of cola samples were measured and compared with the results obtained by UV–Vis spectrometer. The obtained results by using the proposed composite electrode are in accordance with the caffeine contents that manufacturers claimed compared to UV–Vis. This composite electrode which utilizes the catalytic effect of AuNP with the simple preparation of GCPE, offers a fast and sensitive determination of caffeine in beverages as a practical application. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Caffeine, a methylxanthine derivative, is a natural alkaloid which is the main component of daily consumed beverages and food like coffee, tea, coke, energetic drinks and chocolate [1,2]. Besides, caffeine is also used in several pharmaceutical formulations due to its psychoactive effects such as stimulation of central nervous system, diuresis and gastric acid secretion [1,3]. On the other hand, excessive intake of caffeine may cause mutagenic effects in DNA repair. This situation also affects cyclic AMP phosphodiesterase activity and causes cardiovascular problems, kidney diseases and complications in pregnancy [2,4,5]. Furthermore, caffeine intake in doses of 150–200 mg kg 1 of body weight is considered to be fatal [2]. Thus, the development of a fast and sensitive method for caffeine determination is vital for testing food quality and for clinical applications. Numerous analytical methods have been developed for the caffeine determination up to now. Most of these methods are based on spectroscopic [6–8] and hyphenated chromatographic techniques [9–11] in which remarkable results were obtained in terms of sensitivity. However, these methods are complicated, laborious and time consuming which need sample pre-treatment procedures
⇑ Corresponding author. E-mail address: [email protected] (Ü. Anık). http://dx.doi.org/10.1016/j.measurement.2017.04.031 0263-2241/Ó 2017 Elsevier Ltd. All rights reserved.
and expensive bulky equipments [2]. As an alternative choice, electrochemical methods have attracted a considerable attention by providing fast and sensitive caffeine detection in a wide variety of samples with simple and low cost instrumentation [2,4,5,12– 18]. Moreover, in most cases there is no need to use sophisticated sample pre-treatment procedures prior to analysis. However, electrochemical methods suffer from more positive oxidation potential of caffeine which leads to large background currents in blank solution and strongly adsorbed oxidation products on the electrode surface [13]. In order to overcome these limitations, carbon based composite electrodes may provide an opportunity for the electrochemical determination of caffeine by combining the superior properties of the materials used in the electrode fabrication. Also, since these electrodes are practical in terms of renewal and preparation procedures, they can solve problems. Besides these, composite electrodes may be adapted to various electrode configurations with great flexibility in size and shape of the material [19]. Glassy carbon paste electrode (GCPE) is a kind of composite carbon electrode which can be easily prepared just by mixing proper amount of glassy carbon microparticles and oil binder. GCPE can be defined as a favourable candidate among carbon based electrodes due to its higher electrochemical reactivity, wider potential window and low background current as well as its easy preparation at low cost. Also, since it is easy to renew the surfaces of these electrodes, fresh
T. Ören, Ü. Anık / Measurement 106 (2017) 26–30
surfaces are obtained prior to each measurement which make their usages advantageous in the case of strongly adsorbed oxidation products. Since the components used in electrode fabrication are homogeneously dispersed and compressed in the bulk of the structure, it is possible to obtain reproducible results with every new surface [19–21]. To the best of our knowledge, although there are several research papers based on the usage of carbon based electrode itself and also as the component of the composite electrode fabrication for caffeine detection, GCPE has not been used for this purpose [22–26]. To date, several researches dealing with GCPE have been performed and nanomaterials and biomolecules were used to improve the electronic and catalytic properties of these composite electrodes [19,21,27]. Among these materials, owing to its higher conductivity, larger surface area and applicability in biosensing platforms, gold nanoparticles (AuNP) became very popular and were also utilized for the electrode modification in voltammetric determination of caffeine [28,29]. By taking into consideration of these unique properties of GCPE and AuNP, these mentioned features may be combined to bring new insights into the composite electrode fabrication especially for caffeine detection. Thus, in the present study, it was aimed to fabricate a composite electrode by combining the properties of AuNP and GCPE for the rapid and sensitive voltammetric determination of caffeine for the first time. After the optimization of the experimental conditions and the examination of analytical characteristics, AuNPGCPE was applied to the determination of caffeine amount in various cola beverage samples and the results were compared with UV–Vis spectroscopy.
27
on a weighing paper and rinsed with double distilled water prior to measurement. 2.4. Procedure The oxidation peak of caffeine was obtained by scanning potential from 0.8 V to 1.6 V in differential pulse mode with a 0.005 V of step potential, 0.025 V of modulation amplitude and scan rate of 10 mV/s using 3.2 mM H2SO4 (pH 2.5) as the supporting electrolyte. Prior to each measurement, AuNP-GCPE surface was renewed and the reference electrode was rinsed with double distilled water. Pt wire was sonicated for 3 min in 0.1 M H2SO4 to remove strongly adsorbed oxidation products of caffeine. 2.5. Sample preparation Different branded cola samples were purchased from a local supermarket and stored at room temperature until sample preparation. Cola samples were degassed by ultrasonication for 10 min and diluted in the ratio of 1:10 (v/v) with supporting electrolyte containing a known amount of caffeine standard solution [4]. 3. Results and discussion The optimization studies including pH effect and AuNP amount were conducted in the first place. For optimum pH study, plain GCPE was used. Then, optimization of AuNP amount was found. After that, analytical characteristics and sample application were made.
2. Experimental
3.1. Influence of pH
2.1. Apparatus
The influence of pH on caffeine oxidation peak current was evaluated in presence of 50 lM caffeine by varying pH from 1.0 to 6.0. Within this scope, pH dependence of caffeine peak current was examined using GCPE in pH ranges 1.0 to 3.0 with H2SO4 solution, 3.0 to 5.0 with acetate buffer and at pH 6.0 with phosphate buffer, respectively. As shown in Fig 1, the highest peak current was obtained for pH 2.5 in case of using 3.2 mM H2SO4 at GCPE. Since the highest peak current at GCPE was obtained in pH 2.5, further experiments using AuNP-GCPE were also conducted at the same pH.
Differential pulse voltammetry (DPV) measurements were carried out with AUTOLAB PGSTAT 12 electrochemical measurement system (ECO CHEMIE Instruments B.V., The Netherlands) equipped with NOVA 1.10 software. The experiments were conducted in a 10 mL voltammetric cell using conventional three-electrode configuration consisted of GCPE or AuNP-GCPE as the working electrode, Ag/AgCl and platinum wire as the reference and the counter electrodes, respectively. Absorbance measurements were performed on a T60 model UV–Vis spectrometer (PG Instruments, England). 2.2. Reagents and materials All reagents were used as received without further purification. Caffeine was purchased from Merck. Glassy carbon spherical powder (2–12 mm, 99.95% metals basis), mineral oil and gold colloid (0.75 A520 units/mL; 10 nm 0.001% as HAuCl4) were obtained from Sigma-Aldrich for the preparation of GCPE and AuNP-GCPE. 3.2 mM H2SO4 (pH 2.5) was used as the supporting electrolyte. pH of the supporting electrolyte was adjusted with 0.1 M KOH (Riedel-de Haën). All solutions were prepared with double distilled water.
3.2. Effect of AuNP amount The amount of AuNP was varied as 0.5, 1, 2, 5 and 10 lL to examine the relationship between AuNP amount and peak current of 50 lM caffeine. As depicted in Fig 2, peak current increased up
2.3. Preparation of AuNP-GCPE AuNP-GCPE was prepared in the composition of 80:20 (w/w%) glassy carbon paste powder/mineral oil with 1 lL of gold colloid by hand-mixing. The required amount of the homogeneous paste was filled into the electrode cavity of a Delrin tube where electrical contact was provided via a copper wire. The surface was smoothed
Fig. 1. The influence of pH on voltammetric response of 50 lM caffeine using GCPE.
28
T. Ören, Ü. Anık / Measurement 106 (2017) 26–30
Fig. 2. The effect of AuNP amount on peak current for 50 lM caffeine in 3.2 mM H2SO4 (pH 2.5).
to 1 lL and then a sharp decrease on peak current was observed. The current decrease with increasing AuNP amount may be explained with the phenomena which was previously reported by Liu et al. [30] and Çubukçu et al. [31]. According to these studies, increasing AuNP amount causes the resistance and capacitance increment due to double layer formation. Thus, as the ratio of carbon sensing sites in the paste decreases, peak current also decreases [30,31]. As a result of this finding, 1 lL was used in further experiments.
Fig. 3. DPV responses of (A) supporting electrolyte, (B) plain GCPE and (C) AuNPGCPE in presence of 50 lM caffeine in 3.2 mM H2SO4 (pH 2.5). Step potential: 0.005 V, modulation amplitude: 0.025 V and scan rate: 10 mV/s.
3.3. DPV responses of caffeine at GCPE and AuNP-GCPE Within this perspective, DPV responses of 50 lM caffeine in 3.2 mM H2SO4 (pH 2.5) were recorded where GCPE or AuNP-
Fig. 4. DPV responses of caffeine in concentration range of (A) 25–150 lM and (B) 200–1000 lM at AuNP-GCPE. The linear relationship between oxidation peak current and concentration of caffeine at Au-GCPE in the ranges of 25–150 lM and 200–1000 lM (C) and in the range of 50–1000 lM when used GCPE (D).
T. Ören, Ü. Anık / Measurement 106 (2017) 26–30 Table 1 The effect of interfering species on DPV response of caffeine at AuNP-GCPE. Recovery of DPV response of caffeine (%) Interferent/caffeine concentration Interfering species
1:1
5:1
10:1
Glucose Fructose Ascorbic acid Hypoxanthine Glycine Citric acid
97.2 96.6 92.2 105.4 102.8 101.2
103.9 106.9 104.9 94.8 91.9 90.3
104.8 103.6 108.0 104.8 101.0 95.7
Table 2 Determination of caffeine contents in spiked cola beverage samples by using AuNPGCPE and UV–Vis. Sample
Cola beverage 1 Cola beverage 2 (sugar-free) Cola beverage 3 (sugar-free)
Average caffeine content ± SD(g/L) (n = 3) AuNP-GCPE
UV–Vis
Declared by manufacturer
0.141 ± 0.029 0.155 ± 0.039 0.133 ± 0.028
0.205 ± 0.007 0.234 ± 0.004 0.207 ± 0.003
Max. 0.150 Max. 0.150 0.120
29
hypoxanthine and glycine, DPV responses of 50 lM caffeine in presence of these interfering species were recorded. According to Table 1, the results were obtained in acceptable limits without significant changes in peak current. 3.6. Sample application The applicability of AuNP-GCPE was evaluated to determine the caffeine content in various cola beverage samples. Subsequent to sample preparation which is described earlier according to the reported procedure [4] required aliquots of caffeine standard solution spiked samples were analyzed and presented as average of three measurements ± standard deviation (SD) in Table 2. In order to examine the accuracy of the method, the same sample preparation procedure was used for UV–Vis measurements which were carried out at 272 nm [6]. As can be seen in Table 2, the results obtained by AuNP-GCPE are in accordance with the caffeine contents declared by manufacturer whereas UV–Vis results are quite different. These results indicated that a sample pre-treatment procedure should be conducted to eliminate the interferences in sample matrix for UV–Vis measurements prior to analysis while the dilution of the sample with supporting electrolyte suggests a practical way for the proposed method by using AuNP-GCPE.
4. Conclusions GCPE was served as the working electrode to examine the effect of AuNP. As demonstrated in Fig. 3, a peak at 1.38 V with 1.029 lA oxidation current was obtained at plain GCPE whereas a peak at 1.36 V with 2.721 lA oxidation current was observed at AuNPGCPE. As can be seen from these voltammograms, introduction of AuNP into GCPE structure increases the current value and also slightly decreases the peak potential indicating the feasibility of AuNP in voltammetric caffeine detection. This situation may be attributed to the increase of electron transfer rate due to electrocatalytic effect of AuNP providing a promotion in the electrochemical oxidation process of caffeine on the electrode surface [28,29].
Combination of AuNP with GCPE provides sensitive results to caffeine detection. Also the nature of composite electrode brings practicality to the preparation and renewal of electrode surface which eliminates problems like adsorption of oxidation products on the electrode surface. On the other hand, developed sensor showed closer caffeine values to manufacturer declarations when compared to UV–VIS spectrophotometer. As a result, it can be concluded that practical and effective electrochemical sensor for caffeine detection was obtained. References
3.4. Analytical characteristics The analytical characteristics of GCPE and AuNP-GCPE were investigated after the optimization of experimental parameters. In case of using AuNP-GCPE, two linear ranges were obtained between 25–150 lM and 200–1000 lM caffeine with equations of y = 0.0439x 0.5442 (R2 = 0.99) and y = 0.0086x + 5.57 (R2 = 0.98) (Fig 4). Limit of detection (LOD) was defined as 3s/m, where s is the standard deviation of blank (n = 7) and m is the slope of the calibration graph and calculated for both linear ranges as 0.96 lM for the range of 25–150 lM and 4.90 lM for the range of 200–1000 lM. For comparison, calibration graph was also plotted between 50 and 1000 lM using plain GCPE and the equation of the calibration graph was found as y = 0.0174x + 0.7757 (R2 = 0.99) (Fig 4D). Besides, LOD was also calculated for GCPE as 2.49 lM. It can be obviously seen that more sensitive results were obtained for lower caffeine concentrations using Au-GCPE compared to plain GCPE. In order to evaluate the repeatability, relative standard deviation was calculated for 50 lM caffeine (n = 3) as 7.52% in case of serving Au-GCPE as the working electrode. 3.5. Interference study In order to test the selectivity of AuNP-GCPE towards possible interferents such as glucose, fructose, ascorbic acid, citric acid,
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