[CONTRIBUTION FROM THE SCHOOL OF CHEMISTRY OF TEE UNIVERSITY OF MINNESOTA]
THE CHEMISTRY OF VITAMIN E. XV AN EXTENSION OF THE ANALYTICAL METHOD OF FURTER AND MEYER' HERBERT E. UNGNADE
AND
LEE I. SMITH
Received April 84, 1939
In a recent paper, Furter and Meyer2 have described an elegant analytical method for the determination of tocopherols which in simplicity and exactness leaves little to be desired. The method consists essentially in treating an alcoholic solution of the substance with nitric acid under definite conditions, whereby a red color is produced. This red color is then measured quantitatively by means of a photometer or colorimeter, and from the extinction coefficient at the maximum wavelength (4670@, the amount of tocopherol can be calculated, since the solution obeys Beer's Law. The whole procedure requires only about 30 minutes, and the accuracy of the method, as checked against the potentiometric method of Karrer and his collaborators3 is well within 3 per cent., with a sensitivity of 0.2 mg. of tocopherol in 4 cc. of solution. Furter and Meyer found that the method was highly specific in that the red color was given by tocopherols (a-and S-) alone among the substances investigated. These substances were hydroquinones, phytol, and phytyl bromide (possible impurities in synthetic tocopherols) ;phenols and phenolic acids, sterols, tyrosine, j3-carotin, and ascorbic acid (possible contaminants in tocopherols from natural sources); glucose, starch, lactose (possible diluents present in tablets containing tocopherols) ; and various oils (possible vehicles for liquid preparations containing tocopherols). Of the oils, only wheat-germ oil gave a red color; all the other substances gave a yellow color or else no color a t all. Acetyltocopherol produced a t first a yellow color, and gave the red color only as it was gradually hydrolyzed. The method, however, does not distinguish between CY- and &tocopherols, for these two substances give the same values. Since a- and 8-tocopherols differ markedly in biological action, the method cannot be used generally as a substitute for the bio-assay of tocopherols from unknown substances. We were interested in extending the method to some simple analogs of Paper XIV: J. ORG. CHEM.,4, 389 (1939). FURTER AND MEYER,Helv. Chim. Acta, 22, 240 (1939). a KARRER et al., ibid., 21, 939, 1161 (1938). 397 1
2
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the tocopherols and compounds related to them, to determine whether or not the reaction was limited to the tocopherols specifically, or was characteristic rather of the ring system present in the tocopherols. We have found the latter to be true; the method appears to be specific for 6-hydroxy chromans and 3-hydroxy coumarans. Our results are given in the following table, which shows only the value of E a t the maximum wavelength. The detailed curves for five of these substances which showed a red color are given in Figures 1 and 2. Figure 1 shows the curves obtained from known 6-hydroxy chromans, including the tocopherols, and includes for comparison, the curve which Furter and Meyer obtained with synthetic RESULTS OF
THE
TABLE FURTERAND hxEYER REACTION UPON
VARIOUS SUBSTANCES
BUBBTANCl
a-Tocopherol (impure).. 7-Tocopherol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2,2,5,7,8-Pentamethyl-6-hydroxychroman ........... 2-Ethyl-5,7,8-trimethyl-6-hydroxychroman*. ................ Reduced condensation product of geraniol and cumohydroquinone'. ....................... 2,4,6,7-Tetramethyl-5-hydroxycou 2,4,6,7-Tetramethyl-3-ethyl-5-hyd ............. 2,3,4,6,7-Pentamethy1-5-hydroxyc 2,4,6,7-Tetramethylcoumaran. .............................. 2,2,5,7-Tetramethylchroman.. ..
COLOB
Red Red Red Red
773 588 850
Red 684 Red Red 650 Red Yellow-pin€ 113 Yellow Yellow Yellow Yellow 2,4,6,7-Tetramethyl Yellow 2-Methylcoumaran.. 3-Carbethoxyd,7,8. . . . . . . . . . . Yellow Colorless Coumarin.. . . . . 4-(2,5-dimethoxy-3,4,6-trimethylphenyl)butanone-2.. . . . . . . . . Colorless
* Structure not fully determined. dl-a-tocopherol. Figure 2 shows the curves obtained from known coumarans. Our a-tocopherol was a synthetic product, distilled only once and not particularly pure. This no doubt accounts for the rather low value of E which we obtained. The y-tocopherol was a sample isolated from corn oil by Dr. 0. H. Emerson.$ While this specimen of y-tocdpherol showed a maximum a t 4670w, identical with those of a- and 8-tocopherols, the value of E for our y-tocopherol was somewhat low. Our work does not enable us to say whether this is characteristic of y-tocopherol, or is due to 1W e wish to thank Dr.Emerson for the specimens of 7-tocopherol used in them experiments.
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CHEMISTRY OF VITAMIN E
lack of purity in the sample. A sample of y-tocopherol from cottonseed oil also showed a maximum a t 4670& and thus all three of the tocopherols, a-,8-, and y-, show this same maximum. It is surprising how closely the curve for 2 ,2 ,5 ,7 ,8-pentamethyl-6hydroxychroman follows the curve of Furter and Meyer for a-tocopherolin fact, the agreement between the two curves is better than the experimental error of the method. This suggests at once that the method may be applied to 6-hydroxy chromans in general and leads one to suspect strongly that this chroman and a-tocopherol, when subjected to the action - am
IINGaTilOUS
FIGURE 1 @ 2,2,5,7,8-Pentamethy1-6-hydroxychroman @ a-Tocopherol (Furter) 0 a-Tocopherol,once distilled (impure) 0 7-Tocopherol (impure?)
FIGURE2 2,3,4,6,7-PentamethyI-5-hydroxycoumaran 0 2,4,6,7-Tetrsmethyl-5-hydroxycoumaran @ 2,4,6,7-Tetramethylcoumaran Q
of nitric acid under the conditions used in the method give very similar compounds indeed. A red color was obtained from both 2 ,4 ,6 ,7-tetramethyl-5-hydroxy- and 2,3 ,4 ,6 ,7-pentamethyl-5-hydroxycoumarans. This red color, however, contains a great deal more yellow than that produced by the corresponding 6hydroxy chromans. This difference is clearly ap arent from the curves shown in Figures 1 and 2. The maximum at 4670 is still present, but E is reduced to 650-700 while a new maximum at 4400A (E 750-800) has appeared. Thus the colorimetric method is still applicable to these compounds, but by determining the values of E at 467011. and 4400A it appears possible to distinguish between isomeric coumarans and chromans.
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HERBERT E. UNGNADE AND LEE I. SMITH
Coumarans and chromans without the hydroxyl group para to the bridge oxygen atom give a yellow color which may have a tinge of pink in it, as is the case with 2,4,6,7-tetramethylcoumaran.The value of E at 467d is however, extremely low. Moreover, the high specificity of the reaction, which Furter and Meyer have pointed out in other connections, is also apparent from the fact that 2,4,6,7-tetramethyl-6-hydroxycoumaron gives a yellow color with no trace of red. Our results lead us to the conclusion that the method of Furter and Meyer is not only the best so far developed for rapid determination of tocopherols, but that it is also a very promising method indeed for distinguishing between chromans and coumarans hydroxylated para to the bridge oxygen. Much more work will of course be necessary in order to determine the limits of the method; Dr. Furter has such studies already under way. We are very grateful to Dr. Furter, who most generously invited us to carry out and to publish our work upon the new method. EXPERIMENTAL
The apparatus used was a Bausch and Lomb spectrometer equipped with a Martens type photometer. The determinations were carried out as follows. The substance (1-5 mg.) was weighed into a 25-cc. volumetric flask and dissolved in exactly 5 cc. of absolute ethanol. To this solution was added with shaking exactly 1 cc. of concentrated nitric acid (from a burette). A small piece of porous tile was added and a cooling “finger” was placed loosely in the neck of the flask. The solution was heated t o boiling on the steam bath and then refluxed for exactly 3 minutes, after which i t was removed and allowed to cool for 15 minutes on the laboratory table. The solution was then placed in the photometer cell and covered with a slide. The compensating liquid was a mixture of absolute ethanol (83.5 vol. %) and nitric acid (16.5 vol. %). The readings were taken usually within an hour or two after the solutions were prepared, but little change occurs during the first 24 hours. The values for the extinction coefficient were calculated using the formula, tan 61 log tan 8 E=-, cx1 where c = concentration in moles per liter, and I = length of the cell in centimeters. SUMMARY
1. The colorimetric method of Furter and Meyer for determination of tocopherols has been extended to several simple chromans and coumarans. The method appears to be specific not only for all of the tocopherols, but also for &hydroxy chromans generally. By means of this procedure, it appears possible to distinguish clearly between 6-hydroxy chromans and &hydroxy coumarans.
