E o and
n, can be adjusted to fit the computed curves to the experimental data with respect to potential, once curves of the proper shape and scale have been obtained. For the curves in Figure 1 we have taken Eo = +l.OO volt us. S.C.E., but found that n must be taken as approximately 0.5 to obtain a proper spread in charge with respect to potential. We would have preferred to take n = 1 to reflect a stepless removal of protons from the solvent sheath on the electrode. Vermilea (8) has found that, in the oxidation of tantalum, the activation energy of the primary process is not a simple linear function of potential and that the preexponential factor is a function of potential. Use of these ideas in formulating different forms for Equations 1 and 2 would permit a better fit with a more reasonable value of n. Similar effects could be obtained by allowing a to vary with coverage. Such modifications would introduce additional arbitrariness not justified by available data. No allowance for double layer charging has been made in the computed curves, but this is a small error. It is not intended to suggest that the above parameters are the correct ones.
In particular, it appears that the formulation of surface activity is quite in error. It is suggested, however, that a two-step model involving a nearly reversible surface reaction, followed by an exponential transfer to underlying layers is capable of explaining much of the kinetics of electrochemical oxidation of platinum. It provides for the storage of large amounts of charge in the subsurface form and partially shielded from the influence of applied potential, hence the hysteresis in the reduction of such surfaces; and for the observation that the total charge does not determine the nature of the surface. In this connection, the above computations show large fractions of the total charge in the subsurface form, even a t low potentials and short times. It is of interest that equations of the form of Equation 3 have been used to treat the growth of very thin films by place exchange of metal and oxygen atoms (4). It should be noted that results similar to, but not identical with, those obtained experimentally above were obtained if charges were measured by cathodic stripping of repeatedly oxidized electrodes, rather than by direct oxidation of well-reduced electrodes,
LITERATURE CITED
(1) Booman, G. L., Morgan, E., Crittenden, A. L., J . Am. Chem. SOC.78, 5533 (1956). (2) Feldberg, S. W., Enke, C. G., Bricker, C. E., J. Electrochem. SOC.110, 826 (1963): (3) Laitinen, H. A., Enke, C. G., Ibid., 107, 773 (1960). (4) Lanyon, M. A. H., Trapnell, B. M. W., Proc. Roy. SOC.A227, 387 (1955). (5) Muller, E. W., “Structure and Properties of Thin Films,” C. A. Neugebauer, J. B. Newkirk, D. A. Vermilea, eds., p. 476, Wiley, New York, 1959. (6) Smith, J. G., Thesis, University of Washington, Seattle, 1960. (7) Tucker, C. W., J . A p p l . Phys. 35,1897 (1964). (8) Vermilea, D. A., J . Electrochem. SOC. 102, 655 (1955). (9) Warner, T. B., Schuldiner, S.,’ Zbid., 112, 853 (1965).
KARLH. POOL’ JAMES G. SMITH$ A. L. CRITTENDEN Department of Chemistry Universit of Washington Seattle, $ash. 98105 1 Present address, Department of Chemistry, WFhington State University, Pullman, Wash. 2 Present address, Pennsalt Chemicals Corporation, King of Prussia, Pa.
Quantitative Determination of Individual Tocopherols by Thin Layer Chro matog ra p hic Se pa ration a nd S pectrophotometry SIR: The natural antioxidants such as the tocopherols are important factors affecting the shelf life of vegetable oils. The fact that the various tocopherols differ considerably both in their biological activities and in their ability to protect these oils from oxidative degradation emphasizes the need for good analytical methods. The high molecular weight and chemical similarity of the various tocopherols have hindered their efficient separation. I n the past few years a number of workers have reported thin layer chromatographic systems for the separation of the tocopherols (1, 2, 7-18). Several analytical methods have been reported which utilize such separation. Seher (10) determined the tocopherols semiquantitatively from the area of the spot obtained on the thin layer plate. Dilley and Crane (8) determined the tocopherol content, after thin layer separation, from the absorbance difference obtained after a two-step procedure involving oxidation followed by reduction. Katsui, Ichimura, and Nishimoto (7) analyzed only standard a-tocopherol solutions using the Emmerie-Engel reagent. The method described here was developed for the analysis of tocopherols in peanut oil and involves saponification 1244 *
ANALYTICAL CHEMISTRY
of the oil sample and the separation of the tocopherols in the nonsaponifiable fraction by thin layer chromatography. The CY-, y-, and &tocopherols were removed from the thin layer plate and determined spectrophotometrically by the Tsen (13) modification of the Emmerie-Engel method for total tocopherols (3) using bathophenanthroline. This modification increases the sensitivity of the original EmmerieEngel procedure 2.5 fold. The method was applied to peanut oil samples and the precision of the method was evaluated. EXPERIMENTAL
Reagents.
All reagents were of analytical grade. Bathophenanthroline was purchased from the G. Frederick Smith Chemical Co.; a-tocopherol was obtained from Merck and Co., Inc.; p-, y-,and &tocopherol were obtained from Hoffmann-LaRoche, Inc. The bathophenanthroline reagent was 6.0 X lO-3M in ethanol and was stored in an amber bottle in the refrigerator. The ferric chloride solution was 1.0 x 10-3M in ethanol, and was prepared fresh each day and protected from light by an amber bottle. Orthophosphoric acid solution was 0.1M in ethanol; pyrogallol was 5% in ethanol. Potassium hydroxide solution was pre-
pared by dissolving 160 grams in 100 ml. of distilled water. All distilled water used was boiled to remove dissolved oxygen. Potassium ferricyanide and ferric chloride were 5% in distilled water. Mallinckrodt anhydrous ether was found preferable; others gave irregular results due to peroxides. Saponification. One gram of peanut oil, 4 ml. of 5% pyrogallol in absolute ethanol, and 20 ml. of additional ethanol in a three-neck, lOO-ml., round-bottom flask were brought to reflux under a NP atmosphere. One milliliter of KOH (160 grams/100 ml. distilled HzO)was quickly added, and refluxing continued for 1 hour. The reaction mixture was cooled by immersing the flask into an ice bath without removing it from the reflux condenser or Nz atmosphere. Boiled, cooled, distilled water (20 mi.) was then added while the mixture was still under Nz. The flask was then removed from the condenser and Nz atmosphere and quickly stoppered. After the reaction mixture had dissolved in water, it was extracted five times with fresh anhydrous ethyl ether. The ether extract was kept under NZ until the extraction was completed. The extract was then washed with boiled, cooled, distilled water, until free of base. The ether was evaporated, and the dry nonsaponifiables were dissolved in 5 ml. (VI) of ethanol. Four milliliters ( V z ) was removed to a 20-ml. round-
bottom flask and the ethanol evaporated from both portions of nonsaponifiables. Ten milliliters (V3) of ethanol was added to the 0.2 portion A (1 ml. VJ, which was then ready for the modified Emmerie-Engel procedure. One milliliter (V,) of benzene was added to the 0.8 portion B and known microliter amounts were placed in a band on a large thin layer, silica gel G plate for subsequent elution and recovery of tocopherols. Benzene was used as the solvent for placing nonsaponifiables on the thin layer plates because it evaporates much faster than ethanol. Chromatography. Four-tenths milliliter (V6) of the benzene solution (fraction B ) was spotted in a 15-cm. band across a large (8 X 8 inch X 1 mm.) silica gel plate, which had been activated in an oven at 100’ C. for 1 hour and cooled. This left space for a 15-p1. test spot of the same solution and a test spot of each standard tocopherol solution. The plate was then developed with chloroform to a distance of 16 cm. After removal and drying, the test spots were sprayed with 5% K&’e(CN)6 followed by 5% FqC& to determine tocopherol positions. The appropriate zones were removed with a 1-cm.-wide blade by holding the plate in a vertical position over a funnel placed in a 15-ml. centrifuge tube. Silica gel adhering to the funnel was rinsed into the tube with 10 ml. (V7) of ethanol for a- and y-tocopherols and 5 ml. (V,) of ethanol for &tocopherol. The contents were stirred well and then centrifuged for 3 to 5 minutes. The modified Emmerie-Engel determinations were made directly on 3 ml. of the individual supernatants.
Spectrophotometric Determination. The modified Emmerie-Engel color test used in determining the amount of tocopherol present consisted of adding 0.5 ml. of bathophenanthroline (6.0 x lO+M) in ethanol and 0.5 ml. of FeC13. 6 HzO (1.0 X 10-3M) in ethanol to 3 ml. (VE) of solution to be tested. A reaction time of 15 seconds was allowed for a- and y-tocopherols; 3 minutes were allowed for &tocopherol, total tocopherol, and blank determinations. The blank value was determined using 3 ml. of ethanol instead of the test solution. The reaction was then terminated by adding 0.5 ml. of H3POa (0.1M) in ethano l(4.5 ml. Vg). The absorbance values of the solutions were obtained a t a wavelength of 534 mp with absolute ethanol in the reference beam. The ethanol blank value was subtracted from the test solution value. The absorbance value obtained was inserted into a previously constructed calibration durve (Table I ) for the tocopherol being measured. Values for pg. of various tocopherols per gram of oil were then calculated using the weight of oil taken for analysis and the appropriate formula below.
Table 1. Calibration Standards a-Tocopherol 8-Tocopherol ~- 7-Tocopherol pg./ml. Absorbance ~g/ml. Absorbance pg./ml. Absorbance 1.41 2.82 4.22 5.63 7.07 8.47 9.87 11.3 12.7
0.139 0.284 0.430 0.575 0.737 0.890 1.05 1.18 1.32
1.33 2.67 4.00 5.34 6.67 8.00 9.34 10.7 12.0
0.145 0.292 0.450 0.592 0.740 0.900 1.04 1.16 1.30
0.80 1.60 2.40 3.20 4.00 4.80 5.60 6.40 7.20
0.074 0.163 0.255 0.337 0.422 0.503 0.592 0.700 0.760
Rf Values for Silica Gel G Thin Layer Chromatography Development time, 35 minutes; distance, 16-17 cm. Tocopherol ( R f ) Solvent ff B Y 8
Table II.
Benzene Chloroform Petroleum ether-ether ( 5 : l )
0.26-0.32 0.42-0.53 0.32-0.38
-
46.91
VI = (5 ml.) volume to which nonsaponifiables were diluted. Vz = (4 ml.) volume taken to give portion B. V3 = (10 ml.) volume to which portion A was diluted. V4 = (1 ml.) volume taken to give portion A . V5 = (I ml.) volume to which portion B was diluted. vg = (0.4 ml.) volume placed on chromatographic plate. V , = (5 ml. or 10 ml.) volume used to elute tocopherol from silica gel. VE = (3 ml.) volume of spectrophotometric test solution. Vg = (4.5 ml.) volume of spectrophotometric test solution and reagents. t = weight of tocopherol obtained from calibration curve. g = weight of oil taken for analysis. DISCUSSION
Saponification was necessary to remove excess lipids which caused streaking and poor separation of the tocopherols in the chromatographic step. Care should be taken during srtponification to rigorously protect the tocopherols from oxidation, especially when base is present. Only freshly opened peroxide-free ether should be used for the extraction. The division of the solution into two portions, A and B , in order that portion A may be used for analysis of total tocopherols, required very little additional effort and gave an important check on the determination of the individual tocopherols. The values for tots1 tocopherols usually ran slightly higher than the sum of the individual tocopherols, however, because of small amounts of
0.17-0.22 0.30-0.36 0.28-0.33
0.17-0.22 0.30-0.36 0.24-0.29
0.10-0.15 0.20-0.24 0.194.24
nontocopherol reducing substances which were still present a t this point. The values obtained for total tocopherols before chromatography were calculated as a-tocopherol and are therefore not completely valid since there is a small difference in the standard curves for each tocopherol. Using chloroform as a solvent in the chromatographic procedure failed to separate 0- and y-tocopherols (Table 11), but gave a good separation of Q-, y-, and &tocopherols. Peanut oils have been shown to be essentially devoid of btocopherol(4). The 15-pl. test spot and test spots of standard tocopherol solutions were used as a guide in removing the tocopherol bands for analysis. After development, the plate was masked with another glass plate and the test spots were sprayed with potassium ferricyanide solution followed by ferric chloride solution which reveal reducing materials as bright blue spots. Care must be taken not to contaminate the other parts of the plate which must be removed for analysis. Quantitative removal of tocopherols from the plate was not a problem becasne they tend to move toward the outer surface of the thin layer during evaporation of the chloroform. The Emmerie-Engel method is based on the quantitative reduction of ferric ions by tocopherol and the excess ferric ions are subject to photoreduction if the solutions are exposed to strong light. No difficulty is encountered if the work is done under dim artificial light. After addition of the orthophosphoric acid solution, danger of photoreduction is eliminated because the excess ferric ions complex with the phosphate. RESULTS
Table I11 shows the results obtained by application of the method to a sample of New Mexico Valencia peanut oil. VOL. 38, NO. 9, AUGUST 1966
1245
Table IV shows the results obtained on the same peanut oil containing added amounts of each tocopherol. An average percentage recovery was ob-
Table 111.
tained for each tocopherol from the values of Tables I11 and IV. Values for total tocopherol analysis, which involves saponification and application
Tocopherol Analyses on New Mexico Valencia Peanut Oil Tocopherols (pg./gram)
Total 400, 394 402, 402 395, 399 422, 402 404, 387 390, 387 387, 398
Av:
Y
ff
176 159 164 156 166 153 150 122 156
6
199, 186, 185 176, 181, 186 185, 188, 191 150, 184, 159 186, 194, 166 189, 188, 176
11.6, 15.4 12.0, 15.4 27.5, 15.8 15.6, 11.0 12.6, 15.7 11.0, 10.8 12.5, 12.5
182
14.2
of the modified Emmerie-Engel procedure directly to the nonsaponifiable material, are also reported. The average recovery of total tocopherol from saponification was 97,9y0 with a standard deviation of f 5 . 9 4 . Values for CY-, y-, and b-tocopherol analysis, which involves thin layer chromatographic separation, are also reported. The average recovery values are 93.2y0, 87.9%, and 75.1%, respectively, with standard deviations of i 6 . 8 5 , &4.97, and f6.79, respectively. The order of recovery as exhibited by our analyses agrees with the relative antioxidative effectiveness of each tocopherol which increases in the following order: CY-, 8-, y-, b-tocopherol (6). It is therefore reasonable to obtain greater losses due to oxidation with y- and &tocopherol.
Table IV. Tocopherol Analyses on New Mexico Valencia Peanut Oil with Added D L - a - ,
DL-7-,
and DL-&Tocopherols
Total tocoDherols After a-Tocopherol ?-Tocopherol &Tocopherol saponification Recovery Recovery Recovery Recovery Sample" (rg./gram) (70) ( fig./gram) (Yo) (fig.lgram) (%) (rg./gram) (%) 708 92.3 269 107 368 1 93.0 39.6 85.0 712 93.5 216 326 2 . . .b . . .b 27.7 79.4 731 99.1 263 101 369 3 93.5 39.2 83.7 97.7 249 4 726 345 87.7 81.6 35.8 72.3 250 733 99.7 368 88.7 5 93.0 34.0 66.4 98.2 104 260 749 367 92.5 6 36.5 74.7 b b 214 755 331 7 106 ... ... 33.1 63.3 261 737 362 101 99.2 8 90.0 38.9 82.7 254 362 105 92.4 751 9 36.5 90.0 74.7 246 734 353 85.6 10 100 85.0 36.2 73.6 664 358 86.9 87.8 11 88.0 ... ... 248 246 340 766 84.: 12 100 79.1, 60.0 76.5 716 226 86.8 323 13 ... ... 57.6 72.6 786 261 366 106 99.2 92.1 14 56.8 71.2 b 219 346 ... 751 15 82.1 96.4 . . .b 46.1 248 82.1 346 737 92.6 86.8 16 59.4 75.5 93.2 87.9 97.9 Av. 75.1 a 106 fig. a-tocopherol and 200 fig. T-tocopherol were added to all of the above samples. 30 pg. &tocopherol was added to samples 1-10. 60 pg. &tocopherol was added to samples 12-16. &Thesevalues were omitted from determination of the average because they were found to be low because of incomplete removal of the layer from the plate or storage of the sample before analysis.
Table V.
Averaged Corrected Values of Tocopherolsa Tocopherol (fig./gram)
Sample Spanish Type Dixie Spanish Argentine Starr Spantex Virginia Type Virginia Bunch 46-2 Georgia 119-20 Virgiiia 61R Florigiant NC 2 NC 5
Total after saponification
a
Y
6
Total after chromatography
400 f 10.5 391 f 11 406 f 12.5
350 f 10.5
130 f 7 99.8 f 8.1 113 9 84.0 f 7.2
235 f 9 . 5 238 f 17 229 f 13.5 204 f 11
22.1 f 2.0 8 . 4 f 2.8 11.7 f 2 . 0 8.3 f 1 . 7
387 f 18.5 346 f 27.9 354 f 24.5 296 f 19.9
377 f 11 399 f 12.5 446 f 12 413 f 10.5 472 f 12 411 f 12.5 540 f 14.5
149 137 178 177 175 137 197
208 f 14 197 f 13 213 f 14 216 f 12.5 223 f 11.5 195 f 13.5 288 f 15
10.9 f 2.0 8.3 f 1.9 13.5 f 1.8 13.2 f 1.8 8 . 4 f 1.7 8 . 9 f 2.0 12.4 f 2.9
368 f 26.0 342 f 23.9 405 f 25.3 406 f 23.3 406 f 22.7 341 f 24.5 497 27.9
25.0 f 2 . 0 18.6 f 2.2 10.9 f 1.8 13.8 f 2 . 0 14.1 f 2 . 1 6.9 f 1 . 7
561 f 23.0 468 f 26.7 365 f 21.3 424 f 22.5 405 f 26.6 357 f 21.2
*
f 10 f
f
f
9 9.5 9 9.5 9
f f f 10
Florida 393 Runner Type 230 f 9 306 f 12 Southeastern Runner 56-15 542 f 12.5 196 f 10.5 253 f 14 Early Runner 505 f 14 129 f 8 225 f 11.5 453 f 12 Dixie Runner 282 f 12.5 128 f 8 Georgia 186-28 488 f 12.5 200 f 14 191 f 10.5 Virginia Bunch 67 409 f 12.5 122 f 8 228 f 11.5 418 f 11.5 Florida 416 a The oil samples were obtained from sound mature seed grown a t Holland, Virginia, in 1964.
1246
0
ANALYTICAL CHEMISTRY
*
Table V shows the results obtained on 17 peanut oil samples. The values have been adjusted to 100% recovery; averages and the error allowed by one standard deviation are also shown. This was calculated in the following manner: Standard deviations were calculated for the values shown in Table 111. These deviations were applied to sample values shown in Table V because the experimental procedure was identical. The limit,s of one standard deviation for an average value obtained on a particular sample were calculated using standard methods ( 5 ) . The number of measurements for Table V varied from two to four.
( 9 ) Schmandke, H., J . Chromatog. 14, 123 (1964). (10) Seher, A., Mikrochim. Acta 1961,308. (11) Skinner, W. A., Parkhunt, R. M., Alaupovic, P., J. Chromatog. 13, 240 (1964). (12) Stowe, H. D., Arch. Biochem. Biophys. 103, 42 (1963). (13) Tsen, C. C., ANAL. CHEM.33, 849 (1961).
LITERATURE CITED
(1) . . Dillev. R. A.. Ana2. Biochem. 7 . 240 (1964): ‘ (2) Dilley, R. A., Crane, F. L., Ibid., 5 , 531 (1963). ( 3 ) Emmerie, A., Engel, C., Nature 142, 873 (1938). (4) Fore, S. P., et al., J. A m . Oil Chemists’ SOC.30, 298 (1953). (5) Freund, J. E., “Modern Elementary
PRISCILLA A. STURM R. M. PARKHURST W. A. SKINNER Department of Pharmaceutical Chemistry Stanford Research Institute Menlo Park, Calif. WORKsupported by U. S. Department of Agriculture, Oilseed and Industrial Crops Research Branch, Beltsville, Md., Contract No. 12-14-10&7745(34).
Statistics,” Prentice-Hall, Englewood Cliffs, N. J., 1952. (6) Griewahn, J., Daubert, B. F., J. Am. Oil Chemists’ SOC.25, 26 (1948). ( 7 ) Katsuki, G., Ichimura, Y., Nishimoto, Y.. Arch. Pract. Pharm. 23(4) . , 299
(1963). (8) Lambertsen, G., Myklestad, H., Braekkan, 0. R., J. Sci. Food Agr. 13, 617 (1962).
Gas Chromatographic Study of the Separation of Resin Acid Methyl Esters on a QF-1 Column SIR: In preparation for our work involving some of the resin acids isolated from natural sources, the use of gas chromatography as one criterion of homogeneity of the isolated acids seemed very attractive. The literature cites the use of various polar and nonpolar liquid phases in conjunction with a thermal detector cell to monitor the separation of the diterpene acids present in gum rosin (I,,!?, 5-9). To date no one column has been found to resolve satisfactorily the seven commonly known resin acids. It appeared desirable to utilize a lower operating oven temperature to reduce the extent of thermal isomerization of the resin acid methyl esters on the column. Also bypassing the thermal conductivity cell which operates generally 30’ to 50’ C. above the column temperature by using a flame ionization detector appeared advantageous. Furthermore, the increased sensitivity of this detector cell would aid in the detection of trace impurities in our isolated samples. The use of a 4% QF-1 column provides better separation of the resin acid esters than anything previously reported.
The column was conditioned before use for 24 hours at 210’ C. with a slow stream of nitrogen, which was employed as the carrier gas. Hydrogen was generated by an Aerograph hydrogen generator, Model 650. The flow rate, determined by a soap bubble flow meter, was 30 cc./minute hydrogen and 31 cc./minute nitrogen. Materials. Resin acid samples were isolated by established procedures from W/W gum rosin. The acids were analyzed as methyl esters and were prepared by diazomethane treatment of an ethereal solution of the acid, followed by removal of solvent under nitrogen. The methyl esters were injected as a carbon disulfide solution with a Hamilton 5-pl. syringe.
Determination of Relative Retention Times. The point of initial emergence of the solvent peak was taken as reference.
The retention
times were measured as the difference in time between the reference peak and the peak in question. Relative retention times were taken with reference to methyl neoabietate. RESULTS AND DISCUSSION
As can be seen from Figure 1 a complete analysis of the gum rosin can be accomplished in 30 minutes. Dehydroabietic, abietic, and neoabietic acids can be cleanly separated (as methyl esters) on this column. Of the remaining two resin acids in the abietic-type series, palustric and levopimaric acids can clearly be differentiated. Previous reports have mentioned the failure in resolving these two homoconjugated dienes with Versamid 900 ( I ) , butanediol succinate (@, and diethylene glycol succinate (6) substrates.
EXPERIMENTAL
Equipment. Gas-liquid chromatographic separation of the gum rosin was accomplished using an Aerograph A-600 B unit equipped with a flame ionization detector and a Brown Honeywell 1-mv. full-scale recorder. The column used, prepared by Wilkins Instrument and Research Corp., Walnut Creek, Calif., was a 10-foot X ‘/rinch aluminum column packed with 4% QF-1 fluorosilicone on 60/80 mesh Chromosorb W, acid-washed, and treated with dimethyldichlorosilane.
Pimaric
Isopimaric
Elliotinoic
VOL. 38, NO. 9, AUGUST 1966
1247
