Rapid Voltammetric Method for the Estimation of Tocopherols and Antioxidants in Oils and Fats Harold D. McBridel and Dennis H. Evans2 Department of Chemistry, Uniuersity of Wisconsin, Madison, Wis. 53706 Linear sweep voltammetry has been applied to the measurement of the tocopherol content of vegetable oils. Separate anodic voltammetric peaks are obtained for CY-,7- and &tocopherol. The p-tocopherol peak is superimposed on that of ?-tocopherol. Under the same conditions, butylated hydroxyanisole (BHA) may be determined in vegetable oil at concentrations exceeding 0.001%. However, 6-tocopherol interferes. Propyl gallate (PG) may be determined in lard at concentrations greater than about 0.001%. The procedures are quite rapid. The entire operation from sample weighing to acquisition of the voltammogram requires only about ten minutes.
PHENOLIC ANTIOXIDANTS are added to many food products to enhance their stability. In addition, certain foods, particularly vegetable oils, contain significant quantities of natural phenolic materials, the most prominent of which are the various tocopherols (vitamin E group). In this paper, an electrochemical method for the rapid analysis of fats and oils for tocopherols as well as synthetic antioxidants present as food additives will be described. Procedures for the analysis of tocopherols (1) and the phenolic antioxidants used as food additives ( 2 ) have been reviewed. Almost every procedure requires considerable sample preparation such as saponification and extraction before chromatographic separation and quantitation. Existing electrochemical methods (3-5) for tocopherol analysis involve oxidation of the tocopherol extract with Ce(1V). The resulting tocopherylquinones give polarographic reduction waves whose heights permit calculation of the tocopherol content of the original sample. An amperometric titration of tocopherol extracts with Ce(1V) has also been reported (6). The electrochemical behavior of pure solutions of phenolic antioxidants has been reported (7, 8) but only rarely have methods been developed for analysis of real samples (Y, 10). In the present investigation, voltammetry with linearly varying potential using a stationary, planar glassy carbon electrode is applied to the problem. The tocopherols and certain synthetic phenolic food additives can be determined
2
Present address, Nebraska Wesleyan University, Lincoln, Neb. Author to whom requests for reprints should be directed.
(1) R. H. Bunnell, Lipids, 6,245 (1971). (2) J. Jonas, J . Pharm. Belg., 21, 3 (1966). (3) E. Knobloch, Abh. Deut. Akad. Wiss. Berlin, KI. Chem., Geol. Bioi., 1964 (I), 12. (4) A. Niederstebruch and I. Hinsch, Fette, Seifen, Anstrichm., 69, 559 (1967). (5) K. Wisser, W. Heimann, and C. Fritsche, Fresenius' Z . A n d . Chem., 230, 189 (1967). (6) M. Cospito, G. Raspi, and L. Lucarini, Anal. Chim. Acta, 47, 388 (1969). (7) E. Barendrecht, ibid., 24, 498 (1961). (8) M. A. Solinov, E. A. Agaeva, and N. L. Babaeva, Reakrs. Sposobnost Org. Soedin., 4, 80 (1967); Chem. Abstr., 68, 118791r (1968). (9) V. F. Gaylor, A. L. Conrad, and J. H. Landerl, ANAL.CHEM., 29, 224, 228 (1957). (IO) C. Franzke, F. Kretzschmann, and K. Beinig, Fette, Seifen, Anstrickm., 70, 472 (1968).
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from a single voltammogram with a total analysis time of about ten minutes. EXPERIMENTAL
Butylated hydroxyanisole (BHA) was obtained from Eastman while butylated hydroxytoluene (BHT) and propyl gallate (PG) were supplied by Aldrich Chemical Company. The d-a-tocopherol was obtained from General Biochemicals, Chagrin Falls, Ohio. All other chemicals were reagent grade. The origin of the samples was as follows: Corn oil A, Mazola Corn Oil; corn oil B, Magnus, Mabee and Reynard Company; soy bean oil A, Soya Food Products Company, Los Alamitos, Calif.; soybean oil B, Crisco Oil, Procter and Gamble; cottonseed oil, Welch, Holme and Clark Company, New York; peanut oil, Norganic Foods, Los Angeles; olive oil, Hain Pure Food Company, Los Angeles; lard, Oscar Mayer and Company, Madison, Wis. An inexpensive voltammetric instrument was constructed for this study. As the design involves no novel features, it will not be presented here but a circuit diagram is available on request. Voltammograms were recorded using a HewlettPackard Model 2D-2A X-Y recorder. The three-electrode voltammetric cell was equivalent to the Type 1 Polarographic Cell available from Princeton Applied Research Corporation. An aqueous saturated calomel electrode (SCE) was used as reference electrode and a platinum wire served as the counter electrode. All experiments were performed at 25 "C. The working electrode was constructed from 3-mm glassy carbon rod (Grade GC-20, International Carbon Company, New York). The rod was sealed in the end of 5-mrn glass tubing with epoxy cement. A few millimeters were removed from the end in a 90" cut using a glass cutting wheel. The end of the glass tube-carbon rod assembly was polished with emery paper until it was quite smooth. Then using a rotary polisher and polishing alumina (Fisher), the surface of the glassy carbon was brought to a mirror finish. In order to obtain reproducible results, a standard pretreatment procedure was applied before each voltammogram. The previous sample solution was rinsed from the electrode and the surface of the carbon electrode was buffed for about five seconds using the wheel and 1 ,u polishing alumina. This was followed by a n ethanol rinse, a rinse with the next sample solution, and insertion into the cell. The solution was stirred gently for a few seconds, then allowed to become quiescent for at least 30 seconds before recording the voltarnmogram. Mixtures of ethanol and benzene proved most useful as solvent. Though numerous electrolytes ranging from potassium hydroxide to nitric acid were investigated, dilute sulfuric acid seemed most generally useful. Most analyses were performed using 0.12.44 sulfuric acid in 2 : l ethanolbenzene (v/v) as solvent. The sample solutions were prepared by dissolving a carefully weighed oil or lard sample in the appropriate solvent mixture. RESULTS AND DISCUSSION
Tocopherols. Typical voltammograms for five oils are presented in Figure 1. Waves with peak potentials of 0.57 and 0.67 V cs. SCE are evident in all of the current-potential
/cj)/
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1
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I
I.o
6
0.8
p+a a 0.6
0.4
E VS SCE, VOLTS
0.2
Figure 1. Voltammograms of various vegetable oil samples Each solution contains 9.0 g oil per 100 ml. Solvent: 0.12M sulfuric acid in 2 : 1 ethanol-benzene. Scan rate: 0.040 V/sec. Soybean oil A and corn oil B were used for these voltammograms curves and an additional wave with a peak potential of 0.74 V is present in the voltammogram of the soybean oil sample. The peak potential of authentic a-tocopherol is 0.57 V in this medium. Hence, the first wave may be attributed to the oxidation of a-tocopherol. The structures of the various tocopherols are given below:
Tocol a-tocopherol = @-tocopherol= y-tocopherol = &tocopherol =
5,7,8-trimethyltocol 5,8-dimethyltocol 7,8-dimethyltocol 8-methyltocol
Cospito, Raspi, and Lucarini (6) studied the oxidation of the various tocopherols at bright platinum anodes using 0.5M sulfuric acid in 75 ethanol as solvent. They reported that p- and y-tocopherol are oxidized at a potential 85 mV more positive than that of a-tocopherol, whereas &tocopherol is oxidized 170 mV positive of a-tocopherol. Assuming that these separations in potential also pertain to the electrode and solvent used in the present study, we may assign the waves in Figure 1 with a peak potential of 0.67 V to the oxidation of the p- and y-tocopherol present in the oils. The peak at 0.74 V should be due to &tocopherol oxidation. (The substance in the corn oil sample causing the peak at 0.84 V has not been identified.) Determination of the concentrations of the various tocopherols was accomplished by means of a standard addition procedure as shown in Figure 2. The peak height of the wave at 0.57 V increases linearly with the concentration of added a-tocopherol. For the particular conditions employed, each
I
l
l
I
I
0.8 0.6 E VS. SCE, VOLTS
I
1
0.4
Figure 2. Additional of a-tocopherol to corn oil sample A . 10.0 g of corn oil B per 100 ml of solution. Solvent: O.12M sulfuric acid in 2 : 1 ethanolbenzene. B. Solution A plus 4.8 X lO-;M 01tocopherol. C. Solution A plus 9.6 X 10-5M atocopherol. Scan rate: 0.040 V/sec for all voltammograms. Dashed curve: residual current base line (see text). Dotted curve: extension of first peak; i,: peak current for a-tocopherol; ip: peak current for p- ?-tocopherol
+
microampere of peak current corresponds to 3.8 X lO-&M a-tocopherol in the sample solution or 0.23 mg of a-tocopherol per gram of original oil. Evaluation of the peak current for a-tocopherol oxidation in the corn oil sample requires estimation of a base line from which to measure the peak height. The normal procedure of obtaining a residual current base line from a voltammogram of the supporting electrolyte is inadequate in this case because all of the oil samples contain an oxidizable substance or substances which cause a base-line current somewhat larger than that obtained with the pure supporting electrolyte. Thus, it was necessary to construct a somewhat arbitrary base line as shown in Figure 2. This base line was used for the evaluation of peak currents in voltammograms of all the oil samples. Evaluation of the peak current of the wave at 0.67 V requires an accurate means of calculating the current due to the first wave ( E p = 0.57 V) at the potential of the second peak. Fortunately, this is readily accomplished by examining the shapes of voltammograms of a-tocopherol obtained in the supporting electrolyte in the absence of oil. The current 100 mV positive of the peak was 36 of the peak current. Thus, the contribution of the first wave at the potential of the second peak was eliminated by subtracting 36 of the first peak current from the total current observed at the second peak. This procedure is illustrated for curve A in Figure 2 where the corrected second peak current is i2. In this way, a peak current for the oxidation of p- and y-tocopherols may be obtained. It is assumed that the sensitivity is identical for all of the tocopherols. This requires that they have nearly identical diffusion coefficients, a reasonable assumption in view of their very similar structures. The voltammograms for the various oils were analyzed as described above and the results are summarized in Table I. The total tocopherol concentrations as well as the relative abundance of the individual tocopherols are seen to fall in the range generally found for these oils. None of the sub-
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
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Table I. Determination of Tocopherols in Vegetable Oil
Tocopherol concentration, mg/g Oil ff P+r 6 Total This work: CornA 0.26 . . . .a 0.87 1.13 CornB 0.26 . . . .a 0.92 1.18 Soybean A 0.07 0.24 0.78 1.09 Soybean B 0.06 0.26 0.61 0.93 Cottonseed 0.56 . . . .a 0.38 0.94 Peanut 0.23 .... 0.31 0.54 Olive 0.24 . . . .a 0.24 Literature values: Corn 0.020.430.460.44
Soybean 0.15 Cottonseed 0.47 Peanut 0.080.23
1.19 0.70 0.34 0.200.31
0.33 0.010.02
Olive Concentration too low to be evaluated.
1.64 1.18 0.81 0.350.54 0.24.3
Refer-
ence
(12) (12) (13) (14)
I .o
0.8
0.6
0.4
E VS. SCE, VOLTS
Figure 3. Addition of peanut oil sample
BHA
to
A . 10.0 g of peanut oil per 100 ml of solution. Solvent: 0.12M sulfuric acid in 2 : 1 ethanol-benzene. E, C, D , and E. Solution A plus 1.0 X 10-5M, 3.0 X
stances studied contains appreciable amounts of @-tocopherol so the second peak is undoubtedly a measure of the y-tocopherol present in the oils. However, as the present method cannot distinguish the two, the results are labeled as the sum of 0-and y-tocopherol. The electrochemical behavior of the structurally related tocotrienols is not known so they may be contributing to the current at one or more of the voltammetric waves. Usually these are only minor constituents of the oils investigated in the present work (11). The products of the electrochemical oxidation of the various tocopherols have not been identified in this work though the tocopherylquinones are probably the final products as has been shown to be the case for a-tocopherol oxidation (15). The reproducibility of the method is quite good. The standard deviation in peak currents leads to an uncertainty of 10.02 mg of tocopherol per gram of oil. The accuracy of the determination of the amount of a-tocopherol added to an oil sample is excellent. For example, 0.22 mg a-tocopherol per gram of oil was added to corn oil B. The amount found by voltammetric analysis was 0.21 mg/gram. The accuracy of the determination of the natural tocopherol concentrations is difficult to evaluate due to the arbitrary character of the base line (cf. Figure 2). The uncertainty is greatest for the most positive peak and least for the a-tocopherol peak where a rather reliable base line may be obtained by extrapolating the curve just prior t o the peak. As a rough estimate of the maximum error to be expected due to improper evaluation of the base line, one may take the difference between the base line selected in Figure 2 and the base line obtained with solvent and supporting electrolyte alone. At the potential of the most positive peak, the difference is 0.7 pA which corresponds to 0.16 mg of tocopherol per gram of oil. Synthetic Antioxidants. Of the various synthetic antioxidants commonly added to foods, three were investigated in this study. These were: butylated hydroxytoluene, BHT (11) C. K. Chow, H. H. Draper, and A. S. Csallany, Anal. Biochem., 32, 81 (1969). (12) J. Green, “Vitamin E,” Atti del terzo congress0 internazionale, Venezia, Edizioni Valdonega Verona, Italy, 1956. (13) P. A. Sturm, R. M. Parkhurst, and W. A. Skinner, ANAL. CHEM.,38, 1244 (1966). (14) J. M. M. Moreno, Fette, Seifen, Anstrichm., 66, 903 (1964). (15) M. F. Marcus and M. D. Hawley, Biochim. Biophys. Acta, 201, l(1970). ANALYTICAL CHEMISTRY, VOL. 45,
-6 BHA IN PEANUT OIL
(11)
Q
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1 0 - 5 ~ , 5.0 x 10-5~4,7.0 x 1 0 - 5 ~ BHA, respectively. Scan rate: 0.040
Visec (2,6-di-tert-butyl-p-cresol), butylated hydroxyanisole, B H A (a mixture of 2- and 3-tert-butyl-4-methoxyphenol), and propyl gallate, PG (n-propyl ester of 3,4,5-trihydroxybenzoic acid). The peak potential for the oxidation of BHT in 0.12M sulfuric acid in ethanol-benzene is 1.03 V US. SCE. This is sufficiently close to the rapid increase in current observed at 1.1 V with the oil samples (cf. Figure 1) that it is not practical to determine BHT in these oils unless a very large amount is present. On the other hand, BHA is considerably more easily oxidized than BHT. Its peak potential is 0.78 V, coinciding approximately with that of d-tocopherol. Hence, BHA may be determined in any of the oils which do not contain appreciable amounts of d-tocopherol. This is illustrated in Figure 3 where various quantities of BHA have been added to the peanut oil sample solution. The peak height at 0.78 volt is linearly dependent on BHA concentration. The method possesses ample sensitivity for monitoring BHA content of vegetable oils. The legal tolerance in the United States is 0.01% BHA (16). Curve E in Figure 3 corresponds t o 0.013 BHA in the peanut oil. The peak potential of PG is very close to that of the @- and y-tocopherols. Hence, determination of PG in vegetable oils would not be practical under the conditions used in this study. However, animal fats contain very little tocopherol and PG may be determined easily in such samples. Voltammograms of a lard solution t o which various amounts of PG have been added are presented in Figure 4. Again the peak currents are directly related to PG concentration though a plot of peak current US. PG concentration is slightly nonlinear at high concentrations. (The small peak at 0.75 V is attributed to 0.006 BHA present in the commercial lard sample used.) A precision of approximately 1 2 % was observed for PG peak currents in a series of experiments like those in Figure 4. The base line for a pure lard sample is rather smooth from 0 to 1.2 V. This suggests that both BHA (E, = 0.78 V) and (16) Code of Federal Regulations, Title 21, 121.101(d)(2); ibid., Title 9, 318.7(~)(4).
I
PG
IN LARD
1
Figure 4. Addition of PG to lard sample A. 3.60 g lard per 100 ml solution. Solvent: 0.12M sulfuric acid in 1 : 1 ethanol-benzene. B, C , D, and E. Solution A plus 1.0 X lO-jM, 3.0 X lO-jM, 5.0 X 10-jM and 7.0 X lO+M PG, respectively. Scan rate: 0.040 Vlsec
BHT (E, = 1.03 V) may also be determined. In fact, preliminary experiments indicate that mixtures of PG, BHA, and BHT in lard may be determined from a single voltammogram. The principal advantage of the proposed method is the rapidity of analysis. The entire operation from sample weighing to the acquisition of the voltammogram requires only about ten minutes. The speed of analysis is aided by
the fact that no prior treatment or separation procedures need be applied to the sample. Furthermore, as dissolved oxygen does not interfere with the voltammetry, no solution deaeration is required. A disadvantage is that the electrode pretreatment procedure must be applied before every voltammogram in order to ensure reproducibility. In addition, the method is of only moderate sensitivity. Typical concentrations of naturally occurring tocopherols or synthetic antioxidants are readily determined but traces (sub ppm) cannot be detected. It may be possible to improve the sensitivity by utilizing a pulse voltammetric method. No attempt was made in this work to minimize sample size. For example, the tocopherol analyses were performed with 10 grams of oil made up to 100 ml. Sample solution volumes may readily be reduced 100-fold by redesigning the electrochemical cell for smaller volumes. The method should find application whenever a rapid analysis of tocopherols or synthetic antioxidants is required. For example, it could be used to monitor tocopherol content at various stages of the refining process. A survey of oil samples for antioxidant content could readily be accomplished or the method could be applied to monitor changes in antioxidant content during manufacture or storage. With modification in sample preparation, the method may be applicable to the determination of antioxidants in other food products such as cereals or processed meat products. RECEIVED for review July 20, 1972. Accepted October 13, 1972. This research was supported by the National Science Foundation through Grant No. GP-19579.
Determination of Nitrogen Oxides in Ambient Air Using a Coated-Wire Nitrate Ion Selective Electrode Barbara M. Kneebone and Henry Freiser University of Arizona, Tucson, Ariz. 85721 A method has been developed for the determination of NO, in ambient air utilizing a coated-wire nitrate ion selective electrode. Air is collected in gas-washing bottles containing 2% H202,the solution is treated with M n 0 2to destroy the remaining peroxide, and the nitrate concentration is measured potentiometrically. The optimum conditions for preparation and use of the electrode were determined. The effect of interfering anions was studied. The method can be used in the presence of at least a 40-fold excess of SO2 or SOs. The results obtained with this method compared well (1-4% relative error) with accepted spectrophotometric methods.
DETERMINATION OF NO, in the atmosphere by a simple potentiometric method would be a highly advantageous alternative to the presently available spectrophotometric methods. The phenoldisulfonic acid ( I ) (PDS) and the xylenol (2) methods are both based on the formation of nitric acid and (1) American Society for Testing and Materials, Philadelphia, Pa., Method D 1608-58T. (2) M. B. Jacobs, “The Chemical Analysis of Air Pollutants,” Interscience, New York, N. Y . , 1960.
its subsequent reaction with the reagent to form a colored product. These methods involve time-consuming sample handling with the attendant dangers of contamination and loss. The PDS procedure takes about three hours to complete, while the xylenol method takes about an hour. Recently the Orion Research nitrate electrode was applied to NO, determination by DiMartini (3). The analytical technique he employed involves gas phase oxidation of N O or NOs by ozone, followed by absorption and hydrolysis of N205. Quantitative determination of the nitrogen oxides as nitrate ion is then performed with the electrode. According to the data reported, a serious problem exists with the efficiency for 5.7 to 0.062 ppm NO,. Driscoll et al. ( 4 ) also used the Orion electrode t o determine NO, in combustion effluents by modifying the phenoldisulfonic acid method absorbing solution and measuring the nitrate produced. This method was shown to work well in the case of NO, in combustion effluents where concentra(3) R. DiMartini, ANAL.CHEM., 42, 1102 (1970). (4) J. N. Driscoll et al., J . Air Pollut. Confr. Ass., 22, 119 (1972). ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
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