In the Laboratory
Using Square Wave Voltammetry on Ultramicroelectrodes To Determine Synthetic Antioxidants in Vegetable Oils
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Claudio D. Ceballos Departamento de Ciencias Básicas y Departamento de Tecnología Química, Facultad de Ingeniería, Universidad Nacional de Río Cuarto, Río Cuarto, Córdoba, Argentina María A. Zón and Héctor Fernández* Departamento de Química, Facultad de Ciencias Exactas, Fisico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Agencia Postal No 3, (5800)–Río Cuarto, Córdoba, Argentina; *
[email protected] Voltammetric techniques based on new electronic and digital instrumentation aid in the study of electrochemical reactions and their electroanalytical applications (1–4). Particularly, square wave voltammetry (SWV) is a voltammetric technique that is currently being used in a wide variety of analytical chemistry fields (1, 5, 6). It is well known that SWV offers a number of advantages over linear sweep, normal, and differential pulse excitation signals (5, 7). Many articles can be found in the literature citing SWV applications in biochemistry (8), pharmaceutical products (9, 10), veterinary formulations (11), natural waters (12), trace quantity of metals (13, 14), organic phase biosensors (15), food chemistry (16), and so forth. The determination of natural and synthetic antioxidants in food has gained importance in the previous years (17, 18). We have studied the implementation of electrochemical techniques to determine synthetic antioxidants in edible vegetable oils since 1993 (19–21). As a result of these studies, we have recently proposed a methodology for the determination of tert-butyl hydroxyanisole (BHA) and tert-butylhydroxytoluene (BHT) based on SWV using ultramicroelectrodes (UME) (21). This methodology appears to be more convenient than conventional techniques. As far as we know, no education SWV experiments have been published, except for Osteryoung’s interesting report on pulse polarography (22). Commercial educational electrochemical instrumentation necessary to perform the SWV is available for an undergraduate laboratory (23, 24). In addition, a computer-controlled homemade portable potentiostat can be constructed for these purposes (25, 26). We propose an experiment that introduces SWV using UME as an electroanalytical technique to determine BHT added as a synthetic antioxidant to vegetable oils. This experiment is appropriate for an undergraduate course on chemical instrumentation. The experiment would also enable exploration of other concepts such as the extractive technique and the standard-addition method (27), topics that should have been taught in a previous course work. This experiment should take approximately 4–5 hours to complete, but could be divided into two sessions if necessary.
stability and prevent rancidity in lipids. It has been shown that the anodic oxidation mechanisms of phenolic antioxidants in acetonitrile (ACN) follow a sequence of steps (electron transfers and coupled homogeneous chemical reactions) similar to those proposed for the oxidation of phenolic compounds (Scheme I) (28–30). Voltammetric experiments on antioxidants show characteristic anodic peaks under acidic conditions, which can
OH
OH C(CH3)3
OCH3
(H3C)3C
C(CH3)3
CH3
Figure 1. Chemical structures of antioxidants used in food: (left) tert-butylhydroxianisole (BHA) and (right) tert-butylhydroxytoluene (BHT).
Preliminary Concepts BHA and BHT are compounds frequently used as antioxidants in food industry (Figure 1). Antioxidants improve
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Scheme I. Mechanism of anodic oxidation of simple substituted phenols (26), where R is an organic substituent.
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In the Laboratory
= 1.25 V, respectively. Both voltammetric peaks are in a useful potential window. The difference in peak potentials (∆Ep) for the discharge of each antioxidant enables independent determination of BHA and BHT. As BHT is the most common antioxidant chosen for edible oils, BHT determination is the only one proposed in this experimental work. The experiment can also be employed for BHA determination if desired. Equipment, Chemicals, and Instruments A potentiostat with electrochemical software is the primary equipment necessary to accomplish this experimental work. A Faraday cage is recommended for the electrochemical cell to minimize the electrical noise. Working, counter, and reference electrodes are also necessary to perform the experiment. They can be obtained from analytical instrument companies but they could be also home made. Use of the published (33–35) UME construction direction is a useful alternative. Hazards Figure 2. The current as a function of applied potential for a typical SWV experiment.
be used to perform the antioxidant analytical determination. Figure 2 is a typical voltammogram, that is, the current, I, as a function of the potential, E, obtained in a SWV experiment. These measurements give a peak output, rather than a wavelike response. A peak current, Ip, and a peak potential, Ep, are the characteristic parameters of this electrochemical signal. A given antioxidant, i, can be identified by its peak potential, Ep,i, by assuming that Ep,i ≈ E ⬚f,i, where E ⬚f,i the formal potential of the i antioxidant. In addition, its quantification can be performed by using the peak current intensity, Ip,i, by considering that Ip,i is proportional to the antioxidant bulk concentration, Ci (5 –7, 27). Besides the inherent advantages of UME electrochemistry (31), the use of SWV combined with UME for BHT and BHA determination in edible oils has proved to be useful to obtain reproducible results and to improve both speed and sensitivity when compared with other voltammetric methods (21). Square wave voltammograms recorded for mixtures of BHA (at a given fixed concentration) and different concentrations of BHT in 0.1 M H2SO4兾ACN are shown in Figure 3. In this figure, voltammetric scans are presented from 0.4 V to 1.5 V with the anodic oxidation of BHA and BHT at the potential regions indicated. A small peak, T, appears at about 0.72 V (Figure 3). This peak corresponds to the oxidation of α-, γ-, and δ-tocopherols (natural antioxidants) also extracted by ACN from oil (17, 21). These compounds are usually found in vegetable oils (32). Antioxidant formal potentials can be obtained from the corresponding peak potentials, that is, E ⬚f,BHA ≈ Ep,BHA = 0.92 V and E ⬚f,BHT ≈ Ep,BHT
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Acetonitrile (ACN) is a solvent that can irritate the eyes, nose, throat, skin, and lungs. It is a flammable liquid and a fire hazard. Poisonous gases are produced in fire. ACN is particularly incompatible with strong oxidizers. It reacts with them causing fire and explosion hazard (36). Sulfuric acid can be corrosive to all tissues, including the skin, eyes, nose, mucus membranes, and respiratory and gastrointestinal tracks. Since eyes are particularly sensitive, blindness can result from contact (37). Therefore, manipulation of all the experimental laboratory material must be done wearing solvent-resistant gloves and splash-proof chemical goggles. The experimental work should be performed in a well-ventilated laboratory, equipped with extinguishers, and away from heat. Other reagents used in this experimental work do not present significant hazards. Results and Discussion The most attractive aspect of UME (from a student’s perspective) is the extremely small electrode size (2). Prior to the experiment, students should perform some simple UME cyclic voltammetry (CV) experiments specifically designed to help them understand the properties of UME (2). In this way, the current–potential responses obtained from a cyclic voltammogram can be discussed and analyzed. Then, the advantages of application of SWV using UME will be easily understood. Initially, students obtain SWV data for the solution prepared from an extract. SWV voltammograms are recorded from 0.4 V to 1.5 V versus SCE. The presence of tocopherols does not alter the determination of BHT, but when the simultaneous determination of BHA is desired, the voltammetric peak should be corrected by a blank in some cases (see Ep,BHA value in Figure 3). On the other hand, because the BHT discharge is close to the onset of the supporting electrolyte–solvent system discharge, it may be necessary to
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In the Laboratory
Figure 3. Square wave voltammograms for BHA and BHT in 0.1 M H2SO4兾ACN: T represent the tocopherols, CBHA = 7.90 ppm; CBHT = 10.36 ppm (1). Successive additions of BHT: (2) CBHT = 15.0, (3) 20.65, (4) 30.87, (5) 41.03, (6) 51.12, and (7) 61.14 ppm.
subtract residual anodic currents (Figure 3). If that were the case, a straight line drawn between both minima of the peak or even a given polynomial calculated I–E pseudo-blank curve could be used for subtraction from each voltammogram. In most cases a straight line is adequate enough. This correction is simple to accomplish through the software incorporated in most of the commercial potentiostats or other commercial software packages. This laboratory experiment combines an extraction procedure and the application of the standard-addition method with electrochemical measurements. SWV voltammograms should be obtained for at least six additions of standard BHT. Then, the students plot the measured Ip (after correction if it is necessary) versus the corresponding standard BHT concentration (CBHT). A typical plot for student data is shown in Figure 4. A straight line is obtained (correlation coefficient, r = 0.9991). Using the intercept (1.14 ± 0.05 nA) and slope (0.082 ± 0.002 nA ppm᎑1) data values (errors are standard deviations of the intercept and the slope, respectively) students calculate the value of the BHT concentration in the oil sample from the conventional standard-addition method, CBHT = intercept兾slope. The intersection of the extrapolated straight line with abscissa at intercept equal to zero gives a direct estimation of BHT concentration, CBHT, as shown in Figure 4 (this option has a greater error than that calculated from CBHT = intercept兾slope). A BHT concentration of 13.9 ± 0.9 ppm was determined (the original sample was 15.0 ppm). Error in CBHT is calculated through error propagation. The recovery percentage (RP) was 92.7%, which is very close to the value reported in literature obtained by using the same analytical method, that is, 91.33% (21). However, if the recovery percentage is not known beforehand, the corresponding experimental approach using standard additions in the initial sample could be also a good and instructive alternative to proceed for the proposed exercise. Students are asked to explain the strong correlation between Ip and BHT concentration as well as to explore the
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Figure 4. Ip versus CBHT plot from data of the standard-addition method.
concepts of recovery percentage when a real matrix sample is analyzed. Students are also asked to check the experimental results with the theoretical dependence between Ip and reagent concentration predicted by SWV theory (see the Supplemental MaterialW). This experiment clearly shows how SWV can be used with UME as an electroanalytical technique. Acknowledgments We would like to thank to the Departamento de Química, Facultad de Ciencias Exactas, Físico-Químicas y Naturales and Departamentos de Ciencias Básicas y de Tecnología Química, Facultad de Ingeniería to allow this work be performed. We acknowledged financial support from SECyT (UNRC), Agencia Córdoba Ciencia, and CONICET. We are indebted to Lilia Fernández for language assistance. W
Supplemental Material
Notes for the instructor, including questions for the student and answers, are available in this issue of JCE Online. Literature Cited 1. Kissinger, P. T.; Heineman, W. R. Laboratory Techniques in Electroanalytical Chemistry; 2nd ed.; Dekker: New York, 1996. 2. Stanton, C.; Ray, D.; Elie, T. J. Chem. Educ. 1994, 71, 602. 3. Heffner, J. E.; Raber, J C. ; Moe, O. A., Jr.; Wigal, C. T. J. Chem. Educ. 1998, 75, 365. 4. Queiroz, S. L.; de Araujo, M. P.; Batista, A. A.; MacFarlane, K. S.; James, B. R. J. Chem. Educ. 2001, 78, 89. 5. Osteryoung J. G.; O’Dea J. Electroanalytical Chemistry. In Square Wave Voltammetry; Bard A. J., Ed.; Marcel Dekker: New York, 1987; p 209. 6. Osteryoung, J. In Microelectrodes: Theory and Applications; Montenegro, M. I., Queirós, M. A., Daschbach, J. E., Eds.; NATO ASI Series; Kluwer Academic Publisher: Dordrecht, The Netherlands, 1990; pp 139–175.
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In the Laboratory 7. Oldham K. B.; Myland, J. C. Fundamentals and Electrochemical Science; Academic Press: San. Diego, CA, 1994. 8. Radi, A.; El-Sherif, Z. Talanta 2002, 58, 319. 9. Economou, A.; Fielden, P. R. Electroanalysis 1995, 7, 447. 10. Berzas, J. J.; Rodríguez, J.; Castañeda, G. Electroanalysis 1999, 11, 268. 11. Berzas, J. J.; Rodríguez, J.; Lemus, J. M.; Castañeda, G. Electroanalysis 1995, 7, 1156. 12. Barra, C. M.; Correia dos Santos, M. M. Electroanalysis 2001, 13, 1098. 13. Wojciechowski, M.; Go, W.; Osteryoung, J. Anal. Chem. 1985, 57, 155. 14. Economou, A.; Fielden, P. R. Analyst 1993, 118, 47. 15. Iwuoha, E. I.; Smyth, M. R. Biosensors and Bioelectronics 2003, 18, 237. 16. Bradshaw, M. P.; Prenzler, P. D.; Scollary, G. R. Electroanalysis 2002, 14, 546. 17. Clough, A. E. J. Am. Oil Chem. Soc. 1992, 69, 456. 18. Aguí, M. L.; Reviejo, A. J.; Yañez-Sedeño, P.; Pingarrón, J. M. Anal. Chem. 1995, 67, 2195. 19. Ceballos, C.; Fernández, H. J. Braz. Chem. Soc. 1995, 6, 1. 20. Ceballos, C.; Fernández, H. Food Res. Intern. 2000, 33, 357. 21. Ceballos, C.; Fernández, H. J. Am. Oil Chem. Soc. 2000, 77, 731. 22. Osteryoung, J. J. Chem. Educ. 1983, 60, 296. 23. Princeton Applied Research Home Page. http:// www.princetonappliedresearch.com. (accessed May 2006). 24. Bioanalytical System, Inc. http://www.bioanalytical.com/ (accessed May 2006). 25. Zorzán, F. A.; Romero, M. R.; Zón, M. A.; Fernández, H. Desarrollo de una Aplicación para Voltametría de onda Cuadrada
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Empleando un Micropotenciostato; XIV Congreso de la Sociedad Iberoamericana de Electroquímica (SIBAE) y XV Congreso de la Sociedad Mexicana de Electroquímica; Oaxaca, México; 07 to 12/05/00. Economou, A.; Bolis, D.; Efstathiou, C. E.; Volikakis, G. J. Anal. Chim. Acta 2002, 467, 179. Skoog, D. A.; Leary, J. J. Análisis Instrumental; McGraw Hill: Madrid, 1994. Hammerich, O.; Svensmark, B. In Organic Electrochemical: An Introduction and a Guide, 3rd ed.; Lund, H., Baizer, M. M., Eds.; Marcel Dekker: New York, 1990; p 615. Evans, D. H.; Jimenez, P. J.; Kelly, J. J. Electroanal. Chem. 1984, 163, 145. Ceballos, C. Ph.D. Thesis. UNRC: Río Cuarto, Argentina, 1996. Williams, D. E. In Microelectrodes: Theory and Applications; Montenegro, M. I., et al., Eds.; NATO ASI Series; Kluwer Academic Publisher: Dordrecht, The Netherlands, 1990; p 415. Valenzuela, B. A.; Nieto, S. Grasas y Aceites 1996, 3, 186. Wightman, R. M.; Wipf, D. O. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1989; p 267. Zón, M. A.; Moressi, M. B.; Sereno, L. E.; Fernández, H. Bol. Soc. Chil. Quim. 1994, 39, 139. Reprints could be sent by authors upon request. Wightman, R. A. Science 1988, 240, 415. Niosh: Acetonitrile. http://www.cdc.gov/niosh/npg/ npgd0006.html (accessed May 2006). Australian Government Safety Hazards. http:// www.nohsc.gov.au/OHSInformation/Databases/Archived/ pamdetails.asp?pgmid=2082 (accessed May 2006).
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