Distillation of Tocopherols from Sovbean Oil J
F. W. QUACKENBUSH, H. L. GOTTLIEB, AND HARRY STEENBOCK University of Wisconsin, Madison, Wis.
In a cyclic molecular still a-tocopherol distilled quantitatively with a distillation maximum of 147’ C. Under the same conditions, soybean tocopherols distilled from soybean oil with a slightly lower maximum. Distillates containing 14 t o 18 per cent tocopherols were readily obtainable. Ten t o fifty per cent of the “tocopherols” of soybean and other natural oils remained i n the undistilled residue. The tocopherols were determined by a simple and rapid method based on the reaction of Furter and Meyer. The method is sensitive to 50 micrograms of tocopherol. It is adaptable to distillates and natural oils, but may give high results with the latter.
I
N EXPERIMENTS involving the molecular distillation of vegetable oils, attention has usually been focused upon the glyceride fractions ( 7 , 8 ) . The results show that only a slight degree of fractionation has been achieved. More hopeful is the prospect of separating the more volatile constituents from the glycerides. Hickman ( 3 ) found that sterols could be obtained from natural oils by alternate distillation and fractional crystallization from the distillate. From the vegetable oils he obtained an antioxidant fraction and also indicated that his distillate contained tocopherols. I n the present discussion evidence is presented that the tocopherols can be distilled directly from natural oils a t relatively low temperatures, and that a distillate containing 14 per cent or more of tocopherols is readily obtainable from soybean oil.
Determination of Tocopherols The method employed for determining tocopherols in oils and distillates was based on the reaction of Furter and Meyer ( I ) in which the tocopherol is oxidized with nitric acid to an orange-red compound. In its original form this procedure was found to be tedious and lacking in sensitivity, especially when applied directly to oils. Experiments with different mixtures of solvents for carrying out the reaction revealed that a mixture of n-butanol, chloroform, and nitric acid in the ratio 7:2: 1 eliminated the most serious disadvantage of the Furter-Meyer procedure in that it gave a completely homogeneous solution when the reaction was carried out directly with an oil. The mixture also tended to increase the sensitivity of the method since it gave a slightly deeper colored solution with a given amount of tocopherol. For the analysis, a sample containing 0.05 to 1.0 mg. of
tocopherol was placed in a 50-cc. ground-neck flask with 2.0 cc. of chloroform and sufficient n-butanol to bring the total volume to 9.0 cc. To this mixture 1.0 cc. of nitric acid wab added; the flask was then fitted with an air condenser and heated on a steam bath for 10 minutes. After cooling, the volume of the reaction mixture was brought to exactly 10 cc. with butanol; the extinction was read on an Evelyn photoelectric colorimeter. Under our conditions with synthetic d,Z-a-tocopherol, k4@0= 2920. Figure 1 shows that the color produced was proportional to the amount of tocopherol present. The smallest amount which could be determined accurately was about 50 micrograms. Furter and Meyer stated that the minimum with their method was 300 micrograms. Table I gives the results of determinations made on several oils. The values for soybean oil ranged from 0.12 to 0.21 per cent, and for wheat germ oil from 0.31 to 0.38 per cent. Those for cottonseed and corn oil were slightly lower than for soybean oil, and for olive and hydrogenated coconut oil were less than 0.03 per cent. These results compared favorably with values reported in the literature. Cod liver oil, though known to be low in vitamin E, gave a small amount of color. The data in Table I1 suggest that the color produced with this oil may be attributable to its vitamin A and D content. Thile the orange-red color produced by nitric acid has been shown to be quite specific for 6-hydroxychromans (9),other natural compounds produce some color which, while not of the same quality, may cause trouble in the determination. Among various compounds tested, the most highly chromogenic were the common polyphenols. One milligram of pyrogallol gave 84 per cent as much L490 color as a-tocopherol; catechol, hydroquinone, and FIGURE1. CALIBH.4TIOh’ CURVEFOR SYXTHETIC d,l-aguaiacol gave much less. TOCOPHEROL Carotene gave a considerable amount. Vitamin A, calciferol, and ergosterol also gave a chromogenic reaction. Of greater significance in the present work is the fact that the mixed sterols from soybean oil did not give a color. Cholesterol also showed a negative result. Vitamin K1and its corresponding methylnaphthoquinone gave a light green color which did not absorb appreciably a t 490 m,u, hence caused no interference. The usefulness of the improved Furter-Meyer method was shown when the procedure was applied to distillates containing tocopherol.
Distillation and Analysis The distillations of tocopherols were performed with a cyclic niolecular still of our own construction. A residue oil 1276
October, 1941
INDUSTRIAL AND ENGINEERING CHEMISTRY
which served as a diluent for synthetic tocopherol was prepared by distilling the volatile constituents from corn oil a t 210" C. In a typical experiment 100 mg. of synthetic d,Z-atocopherol was mixed with 50 grams of the residue oil and the mixture allowed to distill. Fractions were removed a t 10' temperature intervals and analyzed for their tocopherol content. Distillation curves are shown in Figure 2. The distillation maximum of 140", which we reported previously (6), was obtained with a 40-minute cycle. We have since found that equally satisfactory curves can be obtained with a 20-minute cycle and have adopted the latter as standard. The distillation maximum for a-tocopherol under these conditions wm 147'. Calculations based a on the individual fractions showed that 98 per cent of the added TEMPERATURE, "C. t o c o p h e r o l was recovered in the distillFIGURE 2. DISTILLATION CURVES ates. To determine to FOR SYNTHETIC d,Ga-TOCOPHEROL what extentit was conA , 40-minute cycle: B , 20-minute taminated with residue cycle oil, the individual fractions were recombined, the solvent was removed in B vacuum, and the residue weighed. Corrections were made for the aliquots removed for analysis, and the value obtained was 108 milligrams. It was evident, therefore, that the added tocopherol had been separated almost quantitatively from the glyceride. It was to be expected that the natural tocopherols would distill from soybean oil in a manner similar to the synthetic tocopherol, and that a similar curve would result from analyses made on the distillates. The result of such a distillation is s h o l n in Figure 3. The two curves are nearly identical, the only substantial difference being that the maximum of the curve for the soybean oil distillate is a t 145' C. which is 2" lower than that of a-tocopherol. Hickman and Gray (2, 4) showed that, in general, the removal of one methylene group from a compound decreases its distillation maximum by 5" C. It is therefore to be expected that p-
1
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1277
and y-tocopherols will show maxima of 142" under the same conditions, and it is probable that this lower maximum of 145" is due to the presence of p- and y-tocopherols in the distillate. The close parallelism throughout the two curves and the near purity of color given by the samples of distillate were interpreted as evidence for the absence from the distillate of appreciable amounts of chromogens other than tocopherol. Simple phenolic compounds would, for example, cause a distortion of the curve a t the lower temperatures. Since such compounds were not collected either on the condenser or in the cold trap, _ .it is evident that they were not present in free form in the soybean oil. The weights and tocopherol content of individual fractions from the soybean oil . are shown in Table 111. The weights of fractions increased continuously with increasing temperatures, irrespective of the distillation maximum of the toco100 140 180 pherols. It is probTEMPERATURE, OC. able that the relativelv large fraction obtain& FIGURE 3. COMPARISON OF DIS a t 180" C. consisted TILLATION CURVESOF SOYBBAN partly of glycerides. TOCOPHEROLS (SOLIDLINE) AND T I C CY-T0 c 0 P H E R 0 L The highest concentra- 8 Y N T H E(BROKEN LINE) tion of tocopherols was reached in the 140' fraction which, according to the analysis, contained 18.4 per cent tocopherols.
TABLE 111. TYPICAL DATAOBTAINEDON DISTILLATES FROM 50 GRAMSOF SOYBBAN OIL Weight of Fraction No.
Temperature, C.
Summary 50 g. oil, 180° C., 1 hr.
Fraction, Mg.
481.8 407.0
Tocopherols Mg. %
44.38 67.0
9.2 14.0 4
CONT~NT OF OILS TABLE I. TOCOPHEROL Soybean I (arude) Soybean I (refined) Soybean I1 (crude)
0.212'% 0.175 0.152
Cottonseed Wesson) Corn (Mano$ Olive Coconut (hydrogenated) Cod liver
0.110% 0.119 0.026 0.003
Included in Table I11 are the corresponding data for a different sample of oil with which distillation was begun a t 0.310 0.026 180" C. and continued for 1 hour. The distillate contained 0.380 14.0 per cent tocopherols. These figures represent somewhat bether than a hundredfold concentration of tocopherols in a TABLE 11. CHROMOQENIC PROPERTIES OF VARIOUS COMPOUNDS single operation. They are based on the assumption that other tocols in the soybean oil give the same color intensity Tooopherol Equivalent of as the trimethyl tocol. 1.0 Mg. at Compound Color 400 mp, Mg. While the tocopherols in the distillate behaved much as a-Tocopherol Orange-red 1.00 synthetic a-tocopherol, an important discrepancy arose in the Bronze-orange 0.84 Orange % % :O I:1 balance sheet of the distillation. Of the 87 mg. of tocopherol 0.33 Guaiacol Orange 0.06 indicated by colorimetric test to be present in the original oil, Yellow-orange Hydroquinone 0.20 8-Carotene Yellow-orange 0.31 only about half had distilled a t 180' C. Table IV shows that Vitamin Ai Golden- ellow 0.28 raising the temperature to 210' or even to 240" C., a t which Calciferol Golden-grown 0.06 Ergosterol Red-oranee 0.08 point about one third of the glyceride was allowed to distill, Cholesterol None 0.00 Phytosterols (soybean) None 0.00 was not sufficient to cause distillation of more than a small Vitamin Ki Light green 0.01 fraction of the remaining chromogen. A study of other 2-Methylnaphthoquinone Light green 0.00 samples of soybean oil revealed the presence of residual
INDUSTRIAL AND ENGINEERING CHEMISTRY
1218
chromogen generally, but the amount was variable. Similar results were obtained with wheat germ oil and cottonseed oil. TABLD IV. DISTILLABILITY OF CHROMOGEN FROM OILS
Oil a-Tooopherol due) (corn oil resiSoybean (crude) Soybean refined) Soybean [refined) Cottonseed (Wesson) Wheat germ (crude)
Tocopherol in Oil before Distn., T Mg. ture, 102.1 107.0
Distillate
Residue
180 170 210 240
99.5 43.4 45.7 53.4
65:4
~ ~
~o
in Dist.
59:2
97.5 40.9 43.2 61.1
86.8 64.5
180 180
44.4 57.0
9:o
50.9 88.3
48.2
180
38.2
13.4
80.0
193.0
180 240
155.2 12.0
5i:4
80.3 6.2
.
0-
~~
-
~
~
~
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The authors are indebted to Lever Brothers Company for a grant in support of this work. The synthetic tocopherol was kindly furnished by Merck & Company.
Literature Cited Furter, M., and Meyer, R. E., Helv. Chim. Acta, 22, 240 (1939). Hickman, K. C. D., IND. ENG.CHmf., 29, 968 (1937). Ibid., 32, 1451 (1940). Hickman, K. C. D., and Gray, E. LeB., Ibid., 30, 796 (1938). (5) Olcott, H. S., J. B i d . Chem., 110, 695 (1935). (6) Quackenbush, F. W., Gottlieb, H. L., and Steenbook, H., abstracts of papers, Div. Agr. and Food Chem., A. C. S., S t . Louis, 1941. (7) Rawlings, H. W., Oil & Soap, 16,231 (1939). ( 8 ) Riemenschneider, R. W., Swift, C. E., and Sando, C. E., Ibid., 17, (1) (2) (3) (4)
145 (1940).
(9) Ungnade, HeE., and Smith, L.
I,,J . Org. Chem., 4, 397
(1939).
P R ~ ~ ~ N Tbefore E D
the Division of Agricultural and Food Chemistry a t the 1Olst Meeting of the American Chemical Sooiety, St. Louis, Mo. Published with the approval of t h e Director, Wisconsin Agricultural Experiment Station.
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tocopherols occur naturally in part as the esters. However, upon saponification of the undistillable residue, we have been able to recover only a small percentage of the chromogen in the unsaponifiable fraction. Its true nature must await further experimentation.
Acknowledgment ~ M g . Tooopherol ~ ~ in:
C.
We know very little concerning the nature of this residual chromogen. The color produced with nitric acid was not a pure but it Was not to exclude the presence of tocopherol in some quantity. It is possible that the color was due to tocopherol esters, and that they were sufficiently nonvolatile to resist distillation under the conditions used. Olcott (6) also expressed the view that
25
Vol. 33, No. 10
1
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OR saturated aqueous solutions of potassium and sodium sulfates and of potassium, sodium, and cesium chloride, Foote, Saxton, and Dixon' presented equations connecting the vapor pressure, p , in mm. of mercury, with the temperature, T, in degrees Kelvin, The equations for the sodium and potassium chloride solutions are, respectively, logp = logp =
--2890'7 T - 4.715 log T + 22.612
- 2995*5 _ _ - 6.680 log T + 0.001024 T f 27.569 T
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(2)
The equations for the other solutions are of the form, logp-
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(1)
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(3)
The line coordinate chart facilitates calculation of vapor pressures, once equations of the form of Equation 3 are d e termined for solutions of sodium and potassium chlorides. Values of A and B are as follow: Satd. Soln.
A
KzSOd
2332.5 2258.0 2306.0 2198.5 2696.6
KC1 NaCl CSCl NaiSOi
B 9.1881 8.8750 8.9850 8.5621 10.3630
The index line on the chart shows that the vapor pressure
of a saturated aqueous solution of sodium sulfate is 10 mm. of mercury at 15' C. 1 Foote. H. W., Saxton, B., and Dixon, J. K., J . Am, Chem. Soc., 54, 563 (1932).
