Anal. Chem. 1995, 67, 2195-2200
Analytical Applications of Cylindrical Carbon Fiber Microelectrodes. Simultaneous Voltammetric Determination of Phenolic Antioxidants in Food M. L. Agui, A. J. Reviejo, P. Y&iiezgedeiio, and J. M. Phgarr6n* Department of Analytical Chemistty, Faculty of Chemisfry, Complutense University of Madrid, 28040 Madrid, Spain
Cylindrical carbon fiber microelectrodes (CFMEs)(8 pm in diameter and 8 mm in length) have been used to determine the phenolic antioxidants tert-butylhydroxyanisole (BHA) and tert-butylhydroxytoluene (BHT) in acetonitrile medium. The electrochemicalbehavior of the CFMEs was tested using ferrocene in pure acetonitrile. Pretreatment of the microelectrodes was not necessary for the voltammetric analysis ofthe antioxidants BHA and BHT. "he influence of the supporting electrolyte concentration on i, and E, values obtained by differential pulse voltammetry (DW) has been evaluated. Detection limits close to 70pg L-lof BHA or BHTwere achieved. Squarewave voltammetry (SWV) of these antioxidants at the CFMEs showed current densities remarkably higher than those obtained at a conventional glassy carbon disk electrode. A background current various orders of magnitude lower was also observed at the CFME. An optimization of square-wave parameters was performed in order to achieve the best sensitivity and resolution of the net current responses. Linear calibration graphs were obtained in the concentration ranges (1.0-10.0) x and (1.0-10.0)x mol L-l, their slopes being signitican* higher than those obtained by DW. Voltammograms from mixtures of BHA and BHT showed two well-defined oxidation peaks with a difference between peak potentials of -300 mV, which allows the simultaneous determination of both antioxidants in this medium. Interferences fi-om other substances commonly present in commercial antioxidant mixtures were tested. The developed method by SWVwas applied to determine BHA and BHT in spiked samples of dehydrated potato flakes. Cylindrical microelectrodes have been shown to possess several advantageous properties that can be exploited in electroanalysis. Such microelectrodes can be easily fabricated from quality micrometer metallic wires or carbon fibers and are less sensitive to an imperfect seal between electrode and insulator than are disk microe1ectrodes.l Moreover, the current at cylindrical microelectrodes can be of much greater magnitude than that at disk microelectrodes. As the area of the cylinder depends not only on its radius but also on its length, the length of the microcylinder can be adjusted to obtain the desired magnitude of current without changing the diffusional characteristics.2 This (1) Golas, J.; Osteryoung J. Anal. Chim.Acta 1986,186,1. (2) Singleton, S. T.; O'Dea, J. J.; Osteryoung J. Anal. Chem. 1989,61,1211.
0003-2700/95/0367-2195$9.00/0 0 1995 American Chemical Society
area is usually large enough to yield currents which can be measured with conventional instrumentation? In spite of these properties, cylindrical microelectrodes have not been as extensively used as would be expected. Their analytical utility seems to be limited because they cannot be polished reproducibly4 and they can easily be damaged during work. Nevertheless, cylindrical microelectrodes have been used as sensitive detectors for liquid ~hromatography,~ flow injection analysis,G and capillary electrophore~is,~ as well as probe electrodes for in vivo measurements.* The theory of various electrochemical techniques at microcylinder electrodes has been established, and so, the theoretical basis of chronoamperometry? cyclic voltammetry,1° squarewave voltammetry (SWV)," and normal and differential pulse voltammetry (DFV)12 have been examined and verified with experimental data. Consequently, in the last few years, a better knowledge of the Muence of cylindrical diffusion on the voltammetric response has been achieved, which has renewed interest in the use of these microelectrodes for analytical purposes.13 Most applications of cylindrical microelectrodes are referred to readily available carbon fiber electrodes, whose properties and electroanalytical applications were reviewed by Edmonds.14 Various procedures to fabricate carbon fiber microcylinder electrodes (CFMEs) have been proposed,15 the most popular consisting of sealing the carbon fibers into glass capillaries. SWV at a CFME has been used to determine N-acetylpenicillaminethionitrite with a great improvement in sensitivity with respect to conventional glassy carbon electrode^.^ Miniaturized glucose sensors based on enzyme deposition on CFMEs have been also developed.'6 Finally, CFMEs have been used as substrates for mercury films,l and the preparation and some properties of these mercury coated (3)Nuwer, M.J.; Osteryoung, J. Anal. Chem. 1989,61,1954. (4)Bixler, J. W.; Bond, A M.; Lay, P.A; 'Thormann, W.; van der Bosch, P.; Fleischmann, M.; Pons, B. S. Anal. Chim. Acta 1986,187,67. (5)Baur, J. E.;Wightman, R M. J. Chromufogr. 1989,482,65. (6) Wang, J.; Li, R Anal. Chem. 1990,62,2414. (7) Lu,W.; Cassidy, R M. Anal. Chem. 1993,65,1649. (8)Shannon, C.;Frank, D. G.; Hubbard, A T. Annu. Reu. Phys. Chem. 1990, 42,393. (9)Aoki, K;Honda, K; Tokuda, K; Matsuda, H. J. Electroanal. Chem. 1985, 186,79. (10)Aoki, K;Honda, K; Tokuda, K; Matsuda, H. J. Electroanat. Chem. 1985, 186,267. (11) O'Dea, J.; Wojciechowski, M.; Osteryoung, J.; Aoki, K Anal. Chem. 1985, 57,954. (12) Murphy, M.M.; O'Dea, J. J.; Osteryoung, J. Anal. Chem. 1991, 63,2743. (13)Peng, W.; Wang, E. Anal. Chem. 1993,65,2719. (14)kdmonds, T.E.Anal. Chim. Acta 1985,175,1. (15)Golls, J.; Osteryoung, J. Anal. Chim. Acta 1986,181,211. (16)Wang, J.; Angnes, L Anal. Chem. 1992,64,456.
Analytical Chemistry, Vol. 67, No. 13, July 1, 1995 2195
microelectrodes have been inve~tigated.'~ On the other hand, antioxidants are a large group of chemicals widely used in the food and pharmaceutical indu~tries.'~In particular, phenolic antioxidants, such as tert-butylhydrowanisole (BHA) and tert-butylhydroxytoluene (BHT), are commonly added to food products to improve their stability and especially to prevent rancidity in products containing lipids or fats. Several methods, usually involving separation steps, have been proposed for the determination of phenolic anti0~idants.l~Regarding electroanalytical methods, in the last few years some applications have been described. Wang and Freihal*studied the preconcentration and DW of BHA at a carbon paste electrode. Liquid chr~matographyl~ with amperometric detection has been also used to detect some antioxidants in oil and food samples. Flow injection methods for the determination of BHA and BHT separately, and of BHA in the presence of BHT, have been described on the basis of their oxidation processes at a glassy carbon electrode.2O Micellar and emulsified media have been also employed to carry out voltammetric studies of the BHA oxidation process and to determine this antioxidant in chewing gum samples.z1 Finally, the catalytic voltammetric determination of BHAzzand B W at a nickel phthalocyanine-modifed carbon paste electrode has been reported. The determination of these phenolic antioxidants in food samples usually involves the extraction of the analytes with organic solvents. As it is well known, one of the most interesting analytical advantages of the use of microelectrodes is the possibility of performing measurements in highly resistive media. The aim of this work is to investigate the voltammetric behavior of the antioxidants BHA and BHT at a cylindrical carbon fiber microelectrode of relatively high length (8 nun) , by applying DW and SWV as electroanalytical techniques, in an acetonitrile medium. This organic solvent has been used to extract BHA and BHT from food sam~les.2~ Consequently,the voltammetric analysis has been canied out directly in the analyte extract, thus avoiding some steps of the analytical procedure, such as the solvent evaporation. The possibility of resolving mixtures of both compounds in this medium with no separation steps has also been proved, and finally, the method has been applied to the determination of these antioxidants in samples of dehydrated potato flakes. EXPERIMENTAL SECTION Apparatus. Experiments were performed on an EG&G PAR 273 potentiostat using the electrochemicalanalysis software. Twoor three-electrode configurations were employed. In the former, the reference and counter electrode connections were both attached to the Ag/AgCl (BAS MF 2063) reference electrode. A Metrohm 6.0804.010 glassy carbon electrode (3.0 mm diameter, A = 0.0707 cmz) or carbon fiber microelectrodes (8pm diameter, A = 0.0020 cmz) were used as working electrodes. The auxiliary electrode consisted of a platinum wire directly immersed in the (17) Hudson, B.J. F., Ed. Food Antioxidants;Elsevier: London, 1990,pp v-vii. (18)Wang, J.; Freiha, B. H. Anal. Chim. Acta 1983,154, 87. (19) Grosset, C.; Cantin, D.; Villet, A; Alary,J. Talanta 1990,37,301. (20) Ythiez-Sedefio, P.;Pingarrh, J. M.; Polo, L. M. Anal. Chim. Acta 1991, 252,153. (21)GonAez, A;Ruiz, A; Y~ez-Sedefio,P.; Pingarrh, J. M. Anal. Chim.Acta 1994,285, 63. (22) Ruiz, A; Calvo, M. P.; Pingarrh, J. M. Talanta 1994,41. 289. (23)Ruiz, A;Yhlez-Sedefio, P.; Pingarrh, J. M. Electroanalysis 1994,6, 475. (24) OfficialMethods ofAnalysis of the Association of officMl Analytical Chemists, 14th ed.; AOAC: Arlington, VA, 1984;Procedure 20012, p 373. 2196 Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
solution. A l@mL electrochemical cell (BAS UC-2), a P-Selecta Meditronic centrifuge, and a Griffin flask shaker were also used. Fabrication of Carbon Fiber Microelectrodes. Carbon fibers (Union Carbide Corp.) 8 pm in diameter were used to prepare the microelectrodes. The fibers were soaked, washed, and stored in acetone and dried thoroughly at room temperature. A single fiber was inserted into a 2WpL polypropylene pipet tip and sealed with epoxy resin. The pipet was backfilled with mercury, and a copper wire was used as the electrical contact. Microcylinders were cut with a scalpel to the desired length (8 mm). In a second procedure, the use of epoxy resin was suppressed, and the fiber was sealed by heating the pipet tip carefully with a red-hot steel wire. This latter procedure was demonstrated to be preferable since many electrodes can be fabricated in a very short time, and problems associated with loss of adhesion of the sealant after working with the electrode for a few h o d 5 are avoided. mol L-I solutions Reagenb and Solutions. Stock 1.0 x of BHA (Sigma) and BHT (Fluka) in pure acetonitrile (Panreac) and 1.0 x mol L-l solutions of ferrocene in acetonitrile were used. In all cases, dry acetonitrile (0.01% H20 maximum) was used as received. Tetraethylammonium perchlorate (TEA€') or tetrabutylammonium perchlorate (Fluka, 99%)were used as supporting electrolytes. All other chemicals were of analytical reagent grade. The water used was obtained from a Millipore Milli-Q system. Sample. Samples analyzed were commercial dry potato flakes (Maggi), containing sodium bisulfite (E223) and ascorbic acid (E300) as antioxidants. These samples were spiked with BHA and/or BHT at levels of 100 mg k g - I BHA and 120 mg kg-' BHT. Procedures. After use, the CFMEs were immersed in acetonitrile and stored by night in this solvent. Determination of BHA and/or BI-lTin Spiked Dry Potato Flakes. About 1g of sample previously crushed in a mortar and spiked with the appropriate volume of the BHA and/or BHT stock solution in acetonitrile was accurately weighed into a 3@mL centrifugue tube provided with a screw tap. Next, 10 mL of acetonitrile was added, and the mixture was mechanically shaken for 5 min. After centrifugation at 3500 rpm for 5 min, an aliquot of 5.0 mL of the supernatant extract was transferred to a l@mL volummetric flask. TBAP was then added to obtain a final concentration of supporting electrolyte of 1.0 x mol L-l, diluting to the mark with acetonitrile. The determination of BHA and/or BHT was carried out by SWV using the standard additions method, which involved the addition of (2.0-8.0) x mol L-l of each antioxidant. RESULTS AND DISCUSSION In order to test the electrochemical behavior of the fabricated CFMEs, ferrocene in pure acetonitrile was used as a model of a reversible electrochemical system. Cyclic voltammograms from 0.00 to f1.00 V were registered using a two-electrode configuramol L-' ferrocene solutions tion. Results obtained from 1.0 x containing 6.0 x mol L-l TEAP showed typical Sshaped curves, indicating that depletion of the electroactive species at the electrode does not occur. At potential scan rates between 10 and 60 mV s-l, the forward and reverse sweeps were practically superimposed, as a result of the reversibility of the couple F i r e 1). As expected, higher scan rates, from 80 to lo00 mV s-', gave rise to the distortion of the voltammograms, which became more peak-shaped as the scan rate increased.
1.5
r 0.0
: :1
10.0
20.0 TMP,
m,L-~,,,,*
Figure 3. Influence of the supporting electrolyte concentration in acetonitrile on (0),Fpand (0)ipfor DPV at a CFME 1.O x mol L-l BHT; A € = 50 mV.
Oll
015
0:s
017 E,V'
Figure I. Cyclic voltammograms at a CFME of 1.O x mol L-I ferrocene in acetonitrile containing 6.0 x loV4mol L-' TEAP at scan rates of (a) 20, (b) 100, and (c) 1000 mV s-l.
1
0.a
1.2
1-6
E,V
Figure 2. Differential pulse voltammograms at a CFME of (1) 1.0 x 10-4 mol L-l BHA and (2) 1.O X - ~ O -mol ~ L-' BHT in acetonitrile; 1.0 x mol L-I TBAP, A € = 50 mV.
The reproducibility of microelectrodes was tested by measuring the limiting current and half-wave potential from cyclic voltammograms of ferrocene under the same experimental conditions using a scan rate of 20 mV s-l. In this case, a simple electrochemicalpretreatment of the CFMEs, consisting of applying a constant potential of -0.9 V for 60 s before each scan, was necessary. Results obtained for five different CFMEs yielded limiting current and El/2 mean values of 3.8 f 0.4 pA and 0.573 & 0.013 V, respectively. These values demonstrated that there are no significative differences in the electrochemical behavior of the electrodes. Merential Pulse Voltammetry of BHA and BHT. Differential pulse voltammograms at the CFME were obtained from BHA or BHT solutions in acetonitrile. As Figure 2 shows, a welldefined peak was obtained in each case, the peak potentials being +1.24 and +1.54 V, respectively. In order to test the reproducibility of measurements performed with the same CFME, repetitive voltammograms from 1.0 x mol L-l BHA or BHT solutions were registered. A relative standard deviation value for ip measurements near 4% (n = 20) was obtained in both cases, indicating that a cleaning pretreatment of the microelectrode was not necessary for the electrochemical analysis of these antioxidants. These results constitute a great advantage of the use of
CFMEs with respect to conventional carbon electrodes, especially in the case of BHT, where its strong adsorption is well known to cause fouling of the electrode surface.'* The influence of the supporting electrolyte concentration on the BHA and BHT D W oxidation peaks was studied by measuring ipand Epfrom voltammograms recorded in acetonitrile solutions containingTBAP in the 1.0 x 10-4-3.0 x mol L-' concentration range. Similar results were obtained for both antioxidants. As an example, Figure 3 shows the results obtained for BHT. As can be observed, at lower TBAP concentrations, a rapid increase in ip together with a strong decrease in Epvalues occurs when the supporting electrolyte concentration increases and, consequently, the solution resistance is reduced. At a TBAP concentration of 1.0 x loT3mol L-I, both parameters tend to level off. Furthermore, peak width decreases monotonically with increasing TBAP concentration. A fall in the uncompensated cell resistance from 1.2 MQ down to 270 kQ has been reported on passing from pure acetonitrile to a 1mM tetrabutylammoniumbromide (lBAl3) solution in acetonitrile.25 Since the currents measured at the CFMEs are not very low, the ohmic potential drop (iRd becomes important at very low supporting electrolyte concentrations, which causes an increase of the measured peak potentials. The lower ipvalues observed at these very low TBAP concentrations can be attributed to the lower conductivity of such solutions. At higher TBAP concentrations, a small peak at Ep= $1.2 V appears. The ipof this peak slightly increases with increasing TBAP concentration, which suggests that, probably, the peak may be due to the presence of water in the electrolyte. As a compromise between the best sensitivity and the lower supporting electrolyte concentration, and thus, the lower water and other impurities content, a 1.0 x mol L-' TBAP concentrationwas chosen for subsequent studies. Calibration Graphs for BHA and BHT by DPV. The plots of ip versus BHA or BHT concentration were linear over the (r = 0.9997 in both cases) and (1.0ranges (1.0-10.0) x 10.0) x mol L-' (r = 0.997 and 0.9992 for BHA and BHT, respectively). The slopes of these calibration graphs were (2.76 f 0.06) x 103 and (2.39 & 0.05) x 103p A L mol-' for BHA and BHT in the upper concentration range, respectively, and (2.3 f 0.2) x 103and (1.79 f 0.06) x 103,uA L mol-' for BHA and BHT, respectively,in the lower range of linearity. These values indicate a good sensitivity in both cases. Differences observed between the slopes in both ranges arise from the way the peak heights are measured (by drawing a baseline from the beginning of the rising portion of the peak to the portion where the peak begins (25) Wang, J.; Wu, L-H.; Angnes, L. Anal. Chem. 1991,63,2993.
Analytical Chemistry, Vol. 67, No. 13, July 1, 1995 2197
1.o
0.6
1.4
1.8
E,V
Figure 4. (a) Forward (f) and reverse (I) and (b) net (n) squarewave voltammograms at a conventional glassy carbon electrode (3 mm diameter) -) and at a CFME (-) for 4.0 x mol L-I BHA in acetonitrile; 0.1 and 1.O x 10-3 mol L-’ TBAP when using a glassy carbon electrode and a CFME, respectively; f = 25 Hz, E,, = 100 mV, A€, = 10 mV. (-e
to flatten). Intercepts were 5 f 4 and -0.4 f 1nA for BHA, and -2 f 3 and -0.1 f 0.4 nA for BHT, respectively. Relative standard deviations of 3.7% for BHA and 2.0% for BHT were mol L-l calculated for a concentration level of 5.0 x antioxidant (n = 10). Limits of detection, 4.0 x mol L-I for BHA and 3.1 x mol L-I for BHT, were determined according with the %b/m criterion, where m is the slope of the lowest calibration graph, and st, is the standard deviation (n = 10) of the signals for 1.0 x mol L-I BHA or BHT. Limits of determiand 1.0 x 10-6 mol L-1 were calculated for nation of 1.3 x BHA and BHT, respectively, according to the 10 x standard deviation criterion. Detection limits obtained, close to 70 pg L-I of BHA or BHT, demonstrate the validity of the D W methods for the determination of both antioxidants at low concentration levels. Square-WaveVoltammetry of BHA and BHT. Nonplanar diffusion at microcylinder electrodes has been shown to give enhanced SWV signals.2 The effect of the cylindrical diffusion is to increase the current density and, consequently, the sensitivity of the measurements. Thus, current densities at CFMEs remarkably higher than those obtained at glassy carbon disk electrodes of larger are have been reported.3 In the case of BHA and BHT, our investigations confirmed a similar behavior. As an example, square-wave voltammograms at the CFME obtained for BHA solutions in acetonitrile are shown in Figure 4. Furthermore, voltammograms obtained at a conventional glassy carbon disk cm2) are also displayed. As can be electrode (A = 7 x observed, the net current squarewave voltammogram at the CFME shows the typical bell-shaped symmetrical peak, whereas a broad, poorly defined peak was obtained at the glassy carbon electrode. Current densities per unit concentration at the glassy carbon disk electrode were 1.09 pA/(uM cm2>for BHA and 1.31 pA/(uM cm9 for BHT, whereas these values at the CFME were 3.94 pA/(uM cm2) and 3.82 N ( u M cm2), respectively. A background current of various orders of magnitude lower was also 2198 Analytical Chemistry, Vol. 67,No. 13, July 1, 7995
observed at the CFME (note the different current scales for the glassy carbon electrode and the CFME) . These results agree with theoretical and experimental data which demonstrated that the shape of the net current square-wave voltammograms is unaffected by the cylindrical geometry? It has been also demonstrated that, for a reversible system, the net square-wave response is independent of the electrode geometry.2 Consequently, the shifts observed in peak potentials as well as the d~erencesin peak width for both types of electrodes must be attributed to the nonreversibility of the oxidation process and also to the different electrode material^.^ Regarding individual forward and reverse currents, they have been shown to be dependent on the extent and nature of nonplanar diffusion.2 This fact is also made clear for BHA and BHT. Thus, voltammograms registered at the glassy carbon disk electrode were typical of an irreversible electron transfer with no cathodic contribution to the reverse current. Poorly defined peaks in the forward voltammograms were obtained for both antioxidants. When these results were compared with those obtained at the CFME, noticeable differences in shape for the forward and reverse currents were observed. Now, equivalent curves, both waving in form, were obtained. This change may be attributed to the effect of the additional flux at the cylindrical fiber which does not exist in the planar diffusion to the disk ele~trode.~ An optimization of square-wave voltammetric parameters was performed in order to achieve the best sensitivity and resolution of the net current responses for BHA and BHT. The influence of the frequency was examined in the 5-300 Hz range using AE,= 10 mV and E, = 50 mV. The net current from 5 to 50 Hz was observed to increase rapidly for both antioxidants, and then level off to practically constant Aip values. Moreover, slight increases of the peak potential values were observed as the frequency increased, indicating the nonreversibility of the electrode process. Finally, both background current and noise also increased with increasing frequency. Taking into account the best signal-to-noise relationship, a frequency of 25 Hz was chosen for further studies. The intluence of the square-wave amplitude (Esw) on the voltammetric responses was investigated withii the range 5-200 mV. An increase in the net peak current and in the peak width at half-height with increasing E, was observed for both antioxidants. Thus, the resolution of the square-wave response (Aip/ W ~ / Zshowed ) a maximum for a square-wave amplitude of 100 mV, which was selected for analytical purposes. Finally, the variation of the step height (AE$in the 2-20 mV range had little effect on W~lzand produced a slight increase of the net current peak height. A step height of 10 mV was chosen as a reasonable value to give well-defined voltammograms. Linear calibration graphs (r values ranging from 0.998 to 0.9995) for BHA and BHT were obtained with f = 25 Hz, E, = 100 mV, and AEs = 10 mV within the same concentration ranges mentioned for DW. Slopes were (1.25 f 0.03) x lo4 and (7.3 f 0.2) x 103p A L mol-’ for BHA and BHT in the upper concentration range and (7.3 f 0.4) x 103and (9.6 f 0.3) x 103p A L mol-’ for BHA and BHT in the lower range of linearity. Intercept values were 0 f 10 and 4 f 2 nA for BHA and 10 f 10 and 2 f 0.2 nA for BHT, respectively. Relative standard deviations of 1.6 and 1.4% were calculated respectively for BHA or BHT concentration of mol L-1 (n = 10). Limits of detection of 4.0 x 5.0 x mol L-l and limits of determination of 1.3 x and 3.7 x
BHA
BHA
1
I
1.0
b
1.4
1.8 E,V
Figure 6. Net square-wave voltammograms at a CFME of the acetonitrile extract from a potato flakes sample spiked with both BHA and BHT: (- - -) spiked sample and (-) successive additions of 2.0 x mol L-l each of BHA and BHT; f = 25 Hz, LEsw= 100 mV, A€, = 10 mV.
!
0.6
1.o
!
1
1.4
1.8
, E,V
Figure 5. Net square-wave voltammograms at a CFME of BHA and BHT mixtures in acetonitrile. (a) Successive additions of 2.0 x 10-5 mol L-l BHA in the presence of 4.0 x mol L-' BHT; (b) mol L-I BHT in the presence of Successive additions of 2.0 x 10.0 x 1 0-5 mol L-I BHA; 1.O x 10-3 mol L-' TBAP; f = 25 Hz, E,, = 100 mV, A€, = 10 mV.
and 1.2 x mol L-' for BHA and BHT, respectively, were calculated according to the same criteria as in DW. As can be observed, the slopes of the calibration graphs were both significantly higher than those in DW. These results indicate an improvement in sensitivity when working with SWV. On the other hand, limits of detection were similar for both methods. Simultaneous Determination of BHA and BHT. Voltammograms obtained from mixtures of BHA and BHT in acetonitrile showed two well-defined peaks with both D W and SWV. The differences between peak potentials were -300 mV in each case, which seemed to be large enough to allow the simultaneous determination of both antioxidants in this medium. Recovery studies of each antioxidant in the presence of the other were performed by applying the standard additions method. The individual determination of BHA and BHT was accomplished in solutions containing BHA or BHT at a fixed concentration of 2.0 x mol L-I, in the presence of BHT or BHA, respectively, in the (2.0-10.0) x mol L-' concentration range. As an example, Figure 5 shows square-wave voltammograms obtained for the determination of BHA in the presence of 4.0 x mol L-' BHT (Figure 5a) and for the determination of BHT in the presence of 10.0 x mol L-' BHA (Figure 5b). A recovery of 96 f 4% was found for five determinations of BHA using both DPV and SWV techniques, the slopes of the standard additions plots being (2.2f 0.1) x 103and (1.2 f 0.1) x 104p A L mol-' for D W and SWV, respectively. Regarding BHT, recoveries of 102 & 3%by D W and 98 f 3%by SWV were obtained (n = 5), with slopes of the standard additions graphs of (1.76 f 0.05) x 103 and (9.9 f 0.1) x 103 p A L mol-', respectively. All confidence intervals were calculated for a signiicance level of 0.05. On the other hand, five different solutions containing 2.0 x low5mol L-' each of BHA and BHT were used to carry out the
simultaneous determination of both antioxidants by SWV and the standard additions method. Recoveries ranging between 98 and 100%were obtained in this case. Interferences. Different substances commonly present in commercial antioxidants mixtures (tert-butylhydroquinone VBHQ), citric acid, propyl gallate, and propylene glycol) were tested by DPV in order to check for interferences with respect to the BHA and BHT peaks. Various interferent-to-analyte ratios were studied for a BHA or BHT concentration of 2.0 x mol L-I. Under the same experimental conditions used for the determination of these antioxidants, voltammograms of solutions of propylene glycol did not show any analytical peak in the +0.8 to +2.0 V potential range. Moreover, the shape of the BHA or BHT oxidation peaks did not change appreciably in the presence of this compound, which indicates that it does not interfere. In fact, in the presence of a 1:100 BHA-to-propylene glycol or BHT-topropylene glycol ratio, the calculated concentrationsof the analytes using the respective calibration graphs yielded values within the two standard deviationsrange for 2.0 x mol L-' BHA or BHT (n = 10). Citric acid showed a poorly defined broad peak at Ep ~ 1 . V 4 which does not interfere in the determination of BHAup to a 1:lOO BHA-tocitric acid concentration ratio. Nevertheless, the shape of the BHT peak changes slightly in the presence of this compound, which leads to relative errors in the concentration higher than 10%from a 1:lO BHT-tocitric acid concentration ratio. Voltammograms from solutions of TBHQ showed a welldefined oxidation peak at $1.18 V. This Epvalue is very close to that of BHA (Ep= f1.25 V). Thus, an overall peak was obtained from mixtures of BHA and TBHQ, whose ip increased with increasing TBHQ concentration. Relative errors higher than 10% were obtained from a 501 BHA-to-TBHQ concentration ratio. On the other hand, voltammograms from BHT and TBHQ mixtures showed two well-defined peaks, with a separation between both peak potentials of about 360 mV. However, relative errors higher than 10%were obtained for the determination of BHT from a 1:2 BHT-to-TBHQ concentrationratio when the BHT calibration graph was used. In this case, the standard additions method must be used to avoid matrix effects which seem to be responsible for the aforementioned errors. Finally, voltammograms from propyl gallate solutions showed a well-defined oxidation peak at Ep= +1.30 V. Results similar to those mentioned above in the case of TBHQ were obtained for Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
2199
BHA and propyl gallate mixtures, which exhibited a broad overall peak. Relative errors higher than 10%were obtained for a 1:l or lower BHA-to-propylgallate concentration ratio. Regarding BHT, two well-defined peaks appeared in the voltammograms registered from the corresponding mixtures. No interference in the determination of BHT was found up to a 1:5 BHT-to-propyl gallate concentration ratio. Determination of BHA and BHT in Spiked Dehydrated Potato Flakes Samples. SWV, under the experimental conditions described in the Experimental Section, was used to determine BHA and BHT in spiked samples of dehydrated potato flakes. The described procedure was applied to different samples containing (a) BHA, @) BHT, and (c) BHA BHT. In all cases, the final concentration of each antioxidant in the analytical solution was 2.75 x mol L-I. Voltammograms obtained from these samples exhibited well-defined peaks corresponding to the oxidation of each analyte. As an example, those registered from a sample spiked with the mixture of both antioxidants are shown in Figure 6, as well as those for the corresponding successive additions used to carry out recovery studies. As can be observed, net current increased with increasing BHA and BHT concentration. Furthermore, SW voltammograms of blank solutions o b tained from unspiked samples did not show any peak in the potential range scanned. The results obtained from five samples of each type are as follow: BHA found, (2.68 f 0.05) x mol L-I, which corresponds to a recovery of 97 f 2% BHT found, mol L-’ (recovery of 98 f 2%);BHA BHT (2.69 f 0.04) x found, (2.67 f 0.05) x + (2.68 f 0.09) x mol L-’ (97
+
+
2200 Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
f 2 and 97 f 3%,respectively). The coniidence intervals were calculated for a significance level of 0.05, and the relative standard deviations were f1.5% (BHA), f1.2% (BHT), and f1.5 and f2.7% for BHA and BHT, respectively. CONCLUSION
The results obtained show fairly well that CFMEs can be used for the determination of food additives such as the antioxidants BHA and BHT in commercial samples. The advantageous properties of these microelectrodes allow this determination to be canied out directly in the antioxidants extract using acetonitrile as extraction solvent. Furthermore, the application of electroanalytical techniques such as DW or SWV permits the sensitive determination of the analytes, and the selectivity of the proposed method is rather good when mixtures of antioxidants are present. Moreover, the CFME seems to be a suitable indicator electrode in flowing systems for electrochemical detection of phenolic antioxidants. ACKNOWLEWMENT
Financial support from the Spanish C.I.C.Y.T. (Project ALI 92-0049) is gratefully acknowledged. Received for review November 28, 1994. Accepted March 21, 1995.@ AC941139T @Abstractpublished in Advance ACS Abstracts, May 1, 1995.
