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the cold acid alkylation products of isobutane with butenes. Its boiling point, 115.7’ C., is less than 1” C. above that of another isomer present, 2,3,3-trimethylpentane. I n this distillation the temperature did not rise rapidly until 116” C. had been reached. Raman spectra studies ( 5 ) of these alkylates indentified several other isomers than those mentioned. They must have been present in such small amounts as to remain undetected in exhaustive fractional distillations. Such isomers could be attributed to “secondary” reactlons such as isomerization, leaving only those isomers which are found in substantial amounts to be explained by our “primary” reaction theory. The proposed breaking of a carbon-carbon bond in this mechanism should not be objectionable since just such a break is also essential in the isomerization of n-paraffins. Why and how the carbon-carbon bond should be so affected by certain catalysts, we are not prepared to postulate. However, we hope that this theory may lead t o some clarification of the catalyst’s behavior. The vital presence of an isopara& in these alkylation reactions would suggest bonding between the tertiary carbon, or its lone hydrogen atom, and the catalyst as the first step.
Thermodynamic Equilibria Consideration of the thermodynamic equilibria of isomeric paraffinic hydrocarbons which have recently become available (3, 8, 10, 11) shows the following interesting point: In any group of isomeric paraffins formed by low-temperature alkylation, the relative amounts of the isomers agree closely with those computed by thermodynamic equilibria (when those isomers are excluded which are not permitted by the mechanism). This is illustrated in Table I which employs the calculations of Thacker et al. (11) so as to include heptanes and octanes. Those of Rossini and Prosen (10) for hexanes are somewhat discrepant with our idea with respect to 2methylpentane and 2,3-dimethylbutane.
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The bulk of the products of alkylation consist of isomers with the expected number of carbon atoms. But there are usually by-products of higher and lower carbon content; and the isomers of these other groups are the same and are in approximately the same relative amounts as if they were the main products; i. e., the preferred isomers are isopentane, 2,3-dimethylbutane, 2,3- and a,.l-dimethylpentane, isooctane, and 2,2,5-trimethylhexane. The butanes and pentanes, which cannot be main products of alkylation, are included in the table because they can be by-products and because they are of special interest for isomerization reactions. The calculated percentages of isobutane and isopentane are obviously a little too low, in view of experimental results. It is interesting that in spite of favorable thermodynamics, neopentane is not a factor in isomerization any more than are the other “disqualified” paraffins in alkylation.
Literature Cited Birch, S. F., and Dunstan, A. F., Trans. Faraday SOC.,35, 1013 (1939). Birch, S. F., et al., IND.ENG.CHEAI.,31, 887,1081 (1939). Ewell, R. H., Ibid., 32, 778 (1940). Grosse, A. V.,and Ipatieff, V. N., Rochester Meeting, A. C. S., 1937. Grosse, A. V., Rosenbaum, E. J., and Jacobson, H. F., IND. ENQ.CHEM.,ANAL.ED., 12, 191 (1940). Ipatieff, V. N., and Grosse, A. V., IND.ENG. CHEM.,28, 461 (1936). Ipatieff, V. N., and Grosse, A. V., J . Am. Chem. SOC.,57, 1616 (1935). Knowlton, J. TV., and Rossini, F. D., J . Research Natl. Bur. Standards, 22,416 (1939). Montgomery, C. W., McAteer, J. H., and Franke, N. W., Baltimore Meeting, A. C. S., 1939. Rossini, F. D., and Prosen, E. J. R., J.Am. Chem. SOC..62,2250 (1940). Thacker, C. M., Folkins, H. O., and Miller, E. L., IXD.ENG. CHEX.,33, 588 (1941). PRESEXTED before t h e Division of Petroleum Chemistry a t t h e 102nd Meeting of t h e American Chemical S x i e t y , ltldntio C i t y N J.
Lipides Isolated from Alfalfa-Leaf Meal H. G. PETERINGz, P. W. RIORGAL, AND E. J. MILLER Michigan Agricultural Experiment Station, East Lansing, Mich.
S h DEVELOPMENT of the general program for the study of the industrial utilization of agricultural products, the authors recently described a method for the isolation and purification of carotene extracted from green plant tissue (18, 19). This method has been improved and developed t o the point where it may be of value in the production of bland and rich concentrates of provitamin B and other useful materials (15). From the first the authors recog-
A
1 Present address, Biological Laboratory, E. I. du Pont de Nemours & Company, Ino., New Brunswick, PI’. J.
nized that a number of other lipides were isolated and concentrated in this process. Further work on these constituents has shown that some of these lipides are exceedingly interesting and useful materials. Therefore, since a knowledge of the nature of some of the materials isolated in the process for carotene may have an important bearing on its economics and on its interest to industry, the authors wish to report their findings on the biological activity of the carotene as provitamin A, on the nature of the sterols and other lipide constituents, and on the preparation of certain valuable chlorophyll derivatives.
INDUSTRIAL A N D ENGINEERING CHEMISTRY
November, 1941
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Constituents of the Unsaponifiable Concentrate The barium hydroxide reaction, whereby chlorophyll is removed from the acetone extract of green leafy plant tissue (16),is a saponification process in which the fats, fatty acids, esters, and chlorophyll are saponified to form insoluble barium salts. These salts or soaps are found in the green barium sludge (16,18), and the unsaponifiable extractives remain in the liquor after the reaction with barium hydroxide. The lipoidal unsaponifiable extractives are concentrated along with the carotene, and therefore the authors now prefer to call the “carotene residue” (18) the “concentrate of unsaponifiable constituents” according to the more recent work (16). This change in nomenclature is suggested because of the fact that the material contains a large amount of unsaponifiable lipides or constituents other than carotene. This concentrate, labeled “product 111” by the authors ( I @ , is in fact a water-in-oil emulsion which conhins barium sulfate and is stabilized by a small amount of magnesium soaps formed in the process. Since analysis of the concentrate showed that carotene formed only a small part of the,lipoidal constituents, it seemed of considerable importance to determine the effect of the other material on the utilization of the carotene as provitamin A, and to find out the effect of these other constituents on the stability of the carotene (8,16).Furthermore, since the concentrate contains an appreciable amount of sterols, it seemed of interest to find out something about their nature. Bethke, Kennard, and Kik (6) and Bechdel, Landsburg, and Hi! (4) reported that green forage and artificially dried hay have no vitamin D activity. However the work ‘of Steenbock, Hart, Elvehjem, and Kletzien (23) indicates that probably some of the sterols which are isolated by the authors’ process can be activated with ultraviolet light to produce antirachitic activity for rats. Therefore the authors had bioassays for vitamin D made on samples of these materials after they had been irradiated with ultraviolet light.
Animal experiments have shown that the pcarotene contained in the concentrate of unsaponiflable constituents of alfalfa leaves is completely utilized as a source of vitamin A, and furthermore that the concentrate contains sterols which can be activated by ultraviolet light to produce rat antirachitic activity. From one ton of alfalfa-leaf meal a concentrate is obtained which contains about 340,000,000 I. U. of vitamin A (as @-carotene) and at least 12,000,000 t6 16,000,000 units of vitamin D. This would be approximately equivalent to 34 kg. of a fish liver oil concentrate containing 10,000 I. U. of A and 500 units of D per gram. The data reported here also indicate that the irradiation of the concentrate for the activation of the sterols does not cause appreciable destruction of the carotene. Several methods for preparing derivatives of chlorophyll as by-products of the process for the unsaponifiable constituents are suggested.
diluted with refmed cottonseed oil by the assayer, as was the reference cod liver oil, in order to bring the carotene and vitamin A concentrations of the preparations into the range which made i t easy to feed accurate doses. These final dilutions Bioassays with rats were carried out on several samples of were fed to each of the rats in two doses per week, each dose the unsaponifiable concentrate of lipide and other constituents containing the equivalent of 3 I. U. (international units) of prepared from different batches of alfalfa in order to compare vitamin A. The vitamin A activity of the carotene was the biological activity of the carotene of the concentrate disbased on the assumption that it was all &carotene and solved in refined cottonseed oil with the activity of a standard that the carotene analytical figures could be converted into preparation of U. S. P. reference cod liver oil. vitamin A units by the standard conversion value (0.6 The unsaponifiable lipides were taken up in refined cottonmicrogram of /3-carotene equals 1 I. U.). Four rats were seed oil (Wesson oil), and these oil preparations were further carried through for a 4-week period for each sample of carotene-in-oil and for the reference cod liver oil, the animals having been previously depleted of their vitamin A store. TABLE I. VITAMINA ACTIVITY OF CAROTENE IN CONCENTRATES Then the average gain in weight for the test period of each group of rats on carotene concentrates was compared with OF UNBAPONIFIABLE CONSTITUElNTS FROM ALFALFA the average gain in weight of the animals on the cod liver oil Cmotene ~. Concn. of in order to determine the approximate efficiency of the utiliUnsaponifiAv. Gain able ConEquivain Wt. Relazation of the carotene by the animals when fed at the level stituent in lents of of Rats tive of 6 units of provitamin A in Wesson oil per week. Wesson Oil, Vitamin A, for 4-Wk. UtiliSample No. Mg./G. I. U./G. Period, G . zation If we assume that the animals on the cod liver oil gained the 2-86-1“ 0.855 1426 40 0.95 maximum weight, then a figure based on the ratio of the gain 2-92-1 t 1,889 3143 38 0.91 in weight of the other rats on the carotene to the gain in weight 2-96-1 2.280 3797 44 1.05 U. S. P. reference of the rats on cod liver oil w illgive some idea of the percentage 1700 42 1.00 cod liver oil utilization of the carotene. The results of the bioassays and Prepared by taking the unsaponifiable concentrate directly into Wesson oil with heating, subsequently pooling the material thoroughly at 4 O C., and the figures on the utilization of the carotene are given in then removin by centrifugation the sludge formed. This sample corresponds to pro&ct I V of Morgal el al. (16). Table I. b Purified scoordinq to our prooedure. corresponds to produot VI of MorThe figures on the relative utilization of the carotene comgal el al. (16) after this product has been taken into Wesson oil. The oolor nnnr pared with the reference cod liver oil show that, within the 0 %;:fied according to our propedure and corresponds to product VI after it has been taken into Wesson oil. This conaentrate had good color, mild limits of experimental error, the carotene was completely odor and bland taste It represents the type of produot oonsistently obutilized by the animals as a source of vitamin A at the level t a d d by Morgal at ai. in then improved method. at which it was fed. Indirectly these data corroborate the
Provitamin A Activity of Carotene in Concentrates
...
5
mPIl ..I”
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contention of the authors that all of the carotene in their concentrate is p-carotene. Furthermore, it indicates that the other constituents of the unsaponifiable concentrate from alfalfa do not interfere with the utilization of the carotene.
Provitamin D Sterol Content I n order to determine to what extent the unsaponifiable concentrate from alfalfa could be activated by ultraviolet light to yield antirachitic activity, a number of bioassays were made with rats; all of these must be considered preliminary experiments and not limiting, since no effort was made to get optimum irradiation conditions, nor has the minimum level of antirachitic dosage of the irradiated material as yet been found The samples for the assays were prepared in the following manner: 10.00 grams of crude concentrate of unsaponifiable constituents (sample 2-97-2), which contained 5 per cent sterol, were dissolved in petroleum naphtha (Skellysolve B) with heating. The mixture was cooled, and the xanthophyll and aqueous layer which had separated were removed. The solution was dried over anhydrous sodium sulfate and made UKJto a volume of 1 liter (100 ml. of this solution contains 50 &g. of sterol). The solution was divided into four equal parts of 250 ml. each and treated as follows: (1) The first portion was not irradiated: (2) the second portion was irradiated for 10 minutes; (3) ihe third portion was irradiated for 30 minutes; and (4) the fourth portion was irradiated for 60 minutes. A Luxor quartz mercury-arc lamp was used for the irradiation. The arc was placed 10 inches above the surface of the solution. The solution was contained to a depth of about 1 cm. in a large open crystallizing dish. No special stirring of the solution was maintained, nor was any effort made to exclude air or any gaseous products of irradiation of the air. Each sample was analyzed for carotene after the irradiation had been completed. These analyses showed that the irradiation experiments had been carried out with only a small and inappreciable destruction of carotene. The destruction of carotene for sample 2 was 1 per cent; for sample 3, 3 per cent; and for sample 4, 10 per cent. Since no effort was made to exclude air and other agents which might contribute to the destruction of carotene during the irradiation, these data are both interesting and important, for it is likely that even the loss during the hour period of irradiation for sample 4 could be greatly reduced by using a closed vessel for the concentrate during the irradiation, or by having the carotene solution under an inert atmosphere. Portions of the above irradiated and nonirradiated samples were then incorporated in the feed of the test animals by evaporating a suitable portion of the Skellysolve solution on the feed. Initial exploratory tests were made with two rats on each of the following levels: 20, 50, 100, and 200 mg. of original concentrate. These levels are equivalent to 1, 2.5, 5.0, and 10.0 mg. of sterol per rat, respectively. This amount of material, then, was the only source of vitamin D for the animals for the 10-day test which followed the period of rachitic diet. These initial tests showed that there was no antirachitic It also activity in the original nonirradiated material. showed that irradiation had produced an appreciable amount of vitamin D in the concentrate (compare citation 2 3 ) . Since all of the rats on all of the levels of irradiated samples showed a great amount of calcification of the rachitic metaphyses, no figure on the potency of the material could be calculated. Therefore more assays were made in which smaller amounts of the concentrate were fed to the animals. I n these later experiments only sample 3 (the 30-minute irradiation product) was used. Each of five rats received diets containing I
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the following amounts of this sample as the source of vitamin D-namely, the equivalent of l, 0.75,0.5, and 0.25 mg. of sterol This amount of antirachitic agent sufficed in all cases to produce broad bands of calcification of the rachitic metaphyses, and the minimum protective level was not attained. Although these experiments are only preliminary and further work needs to be carried out on the production of antirachitic activity of the sterols from the unsaponifiable concentrate of alfalfa-leaf meal, the assays which have been made indicate that a t least 30,000 to 40,000 I. U. of vitamin D per gram of sterol can be produced by the irradiation conditions described for sample 3. This is based on the assumption that the lowest level fed up to the present time-i. e., 0.25 mg. of sterol-contains or is equivalent to 7.5-10 I. U. of vitamin D. This figure is based on the assayer's judgment of the extent of the calcification since the lowest level for the production of a line of calcification had not been reached. This means that from a ton of alfalfa-leaf meal a t least 12,000,000 to 16,000,000 I. U. of vitamin D can be produced in addition to about 340,000,000 I. U. of vitamin A, since 1 ton of alfalfaleaf meal of the quality used would yield 421 grams of sterol in the unsaponifiable concentrate.
Chlorophyll Derivatives Although chlorophyll and some of its derivatives have been used in medicine and industry in the past, the demand for them has not been great. Chlorophyll derivatives have been utilized for coloring matter in soaps, and for some time various chlorophyll preparations have been used in heart therapy by European and British physiciansz. However the literature does not reveal that the medical profession in this country has taken this up to any extent. The recent report of Gruskin (10 ) indicates that water-soluble and oil-soluble derivatives of chlorophyll and chlorophyll itself may have important places in the treatment of suppurative diseases. If this becomes established, there may be a demand for cheap derivatives of chlorophyll; this possibility led us to investigate methods for preparing compounds identical with and similar to those mentioned by Gruskin. The material from which any chlorophyll derivatives could be made using materials available from the authors' process is the green barium sludge. The saponified chlorophyll undoubtedly exists in this material as barium isochlorophyllin, and is associated with barium soaps and unreacted barium hydroxide octahydrate as well as filter aid. Since the preparation of chlorophyll derivatives comes as a secondary development in this process, the starting material is different from that used by Willstaetter and his students (25), and the methods to be described are considerably different than those employed by Willstaetter or by Schertz ($2). In order to prepare any derivatives of chlorophyll, two general methods lie open, depending on whether magnesium chlorophyllins and their derivatives, or phytochlorin e-phytorhodin g and its derivatives are desired. In the first case the green barium sludge which has been freed of acetone is treated with a solution of sodium carbonate; thus by metathetical reaction, barium carbonate and sodium magnesium isochlorophyllin are produced as well as sodium soaps. The sodium salts are water soluble and are removed from the barium carbonate and filter aid by filtration or centrifugation. The amount of sodium carbonate should naturally be slightly in excess of the amount necessary to react completely with the barium to form barium carbonate. It is preferable to keep the solution of isochlorophyllins as concentrated as possible. 9
Compare the advertisement of "Phyllosan" in The Lancet, Nov. 16, 1940.
November, 1941
I N D U S T R I A L & N b ENGINEEBIbTG C H E M I S T R Y
The isochlorophyllin solution is cooled, the pH is adjusted to 8.5-9.0, and sodium chloride is added until a saturated solution is formed. This causes the precipitation or saltingout of the sodium magnesium isochlorophyllin and sodium soap. The precipitate is collected and washed with a saturated solution of sodium chloride to remove sodium carbonate. This precipitate when dried and ground represents a crude preparation of sodium magnesium isochlorophyllin, which contains sodium chloride and sodium soaps. The purification of this material is difficult but can be accomplished by taking it up in water, salting-out the pigments from a concentrated solution, drying the precipitate, and extracting the dried material with methanol to free it of sodium chloride.
W A V E LENGTH ( M y )
TRANSMISSION OF ETHAFIGURE1. SPECTRAL e-PHYNOL SOLUTIONS OF ( A ) PHYTOCHLORIN TORHODIN g, ( B ) ZINC COMPLEX DERIVATIVE OF A , AND (C) COPPERCOMPLEX DERIVATIVE OF A
The methanol solution is then evaporated under reduced pressure with the addition of ethanol toward the end of the distillation, and a dry product is obtained according to the method of Willstaetter (26). The isochlorophyllin, either dry or wet with ethanol obtained as described above, is then extracted with ethanol to remove a portion of the soaps remaining. The dried residue represents a fairly pure product of sodium magnesium isochlorophyllin. While i t is rather difficult to obtain a pure product of the sodium magnesium isochlorophyllins from the barium sludge, on the other hand it is rather easy to obtain a pure preparation of phytochlorin e-phytorhodin g. The procedure recommended for this purpose follows: The barium sludge is suspended in about an equal volume of water, and hydrochloric or acetic acid is slowly added with stirring until just a slight amount in excess of that necessary for reaction with all of the barium has been added. This mixture is allowed to stand warm for several hours until the reaction is considered to be complete. This reaction not only replaces the barium in the barium isochlorophyllins with hydrogen but also replaces the magnesium atom in the tetrapyrrole nucleus with hydrogen.
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In addition, the barium soaps are changed into fatty acids. Care should be taken not to add a great excess of acid, especially hydrochloric, because this tends to form the acid salt of the porphyrins and thus to solubilize them. After the reaction mixture is cooled, the waxy material containing the pigments floats on top of the liquor and the filter aid settles to the bottom. The liquor contains barium salts and some pigment associated with the excess acid. The waxy material is a mixture of fatty acids and phytochlorin ephytorhodin g. The waxy material is removed and heated with water to wash it free of excess acid, cooled, and removed once more from the wash liquor. This may be done several times until the water is free of acid. This waxy material is dried and then exhaustively extracted with Skellysolve B to remove the fatty acids. Some small amount of pigment i s lost in this process owing to the intersolubility of the pigment, fatty acids, and solvent, for ordinarily pure phytochlorin e-phytorhodin g is not soluble in Skellysolve B. The black residue is freed of solvent and represents a purified preparation of phytochlorin e-phytorhodin g. This material is a dark powder with a violet metallic luster. The yield of this powder is 75 to 80 per cent of the amount calculated from the amount of isochlorophyllin in the sludge. The absorption spectrum of an ethanol solution of this powder is shown in curve A , Figure 1. This phytochlorin e-phytorhodin g is a good starting product for the preparation of the metal complex chlorophyllins. We have prepared copper, zinc, cobalt, and iron complex derivatives by methods similar to those described by Rothmund (21) and Taylor (94) for porphyrins in general. However for the preparation of the copper and zinc complexes a simpler procedure can be used-i. e., mixing slowly a hot solution of phytochlorin e-phytorhodin g in glacial acetic acid or ethanol with an excess of a concentrated aqueous solution of copper or zinc salt, boiling the mixture for several minutes, and then allowing the mixture to be warmed for several hours. The mixture is diluted with water, cooled, and filtered. The precipitate is thoroughly washed with slightly acidified water t o remove metal salts and then washed with water to remove acid. When dried and ground, this precipitate represents a fairly pure preparation of metal complex chlorophyllin derivative. The yields of these preparations have been about 80 per cent. The zinc complex, which may be considered to have the formula (CazHaoONdZn) (COOH)2-(C32H2802N4Zn) (COOH)2, has a yellow-green color in alcohol or ether solution, while the copper complex has a blue-green color in these solvents. The absorption spectra for these compounds in absolute ethanol are shown in curves B and C, respectively, of Figure 1. These curves indicate that the metal complexes were relatively free of the chlorin e-rhodin g pigment. Since these derivatives are carboxylic acids, they and their esters are oil soluble while their sodium salts are water soluble.
Discussion Ordinarily green leafy plant tissue is not thought of as a source of lipoidal material, for it does not contain more than about one per cent of its dry matter as fatty material. Although some work has been done on the nature of the fatty acids and sterols of leaves in connection with other problems ( I $ ) , the methods for isolating large quantities of these materials from leaves are not very good. However, with the development of a process suitable for isolating, separating, and concentrating various portions of the lipide fraction of green leaves, this abundant and widely distributed material becomes a good source of some of the most valuable lipides known to the chemist. Therefore the improved method of Morgal et al. (I&) should not only permit investigators to go
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ahead with detailed study of the mture of the lipoidal constituents of leaves of different plants, but it should serve as a process for the commercial preparation of such valuable products as rich concentrates of provitamin A (p-carotene), vitamin D, and chlorophyll derivatives. The provitamin A and vitamin D concentrates can be obtained in a state of purity which will permit their use in foodstuffs, The question of whether carotene is a good source of vitamin A activity has recently been subjected to some critical experiments; the weight of evidence in the literature seems to indicate that for human beings, dogs, rats, and chicks there is little differencein the utilization, unit for unit, of vitamin A activity from carotene or from fish liver oil concentrates if the supplements are accompanied by an adequate intake of fat (6, 7, 17, 18, 30). The utilization of both vitamin A and carotene depends on the presence of adequate fat in the diet and on the establishment of normal fat metabolism (1, 7). I n addition to the errors which entered into the interpretation of early work on this subject due to the unknown rela$ion of fat intake to utilization, there exists a t present the anomaly of the varying biological activity of preparations of pure vitamin A alcohol, pure vitamin A esters, and the saponified and unsaponified fish liver oil concentrates. Emmett and Bird (8) reported that saponified fish liver oil concentrates had a lower biological activity than did unsaponified concentrates of vitamin A, based on spectrographic analysis, and that the completely saponified concentrates had as little as half the activity of the unsaponified concentrates. Hickman (11) corroborated this work. It would indicate that vitamin A alcohol is less potent than vitamin A esters. However, Holmes and Corbett ( I S ) and Baxter and Robeson (3)reported that their pure crystalline preparations of vitamin A alcohol and vitamin A palmitate have similar potency when based on spectrographic data; i. e., the potencies are in the range expected from data on unsaponified fish liver oil concentrates. Although the investigation on the sterol fraction of the unsaponifiable concentrates of alfalfa reported here is only preliminary and exploratory in nature, it is evident that the sterols contained in these concentrates from alfalfa contain an appreciable quantity of provitamin D sterols. The nature of these sterols is not known, nor is it yet known whether any of the sterols can be activated t o form chick vitamin D ; nevertheless it appears that in part they are different from those isolated from alfalfa seed by King and Ball (14) and the aspinasterol isolated by Fernhola and Moore (9) from alfalfa leaves. It is probable that much of the activatable sterol is ergosterol, but no proof has been obtained. The saponification compound of barium and chlorophyll has been designated as isochlorophyllin on the basis of the strong fluorescence which the sodium salt shows in alcoholic or aqueous solution. This is the criterion given by Willstaetter (66)for distinguishing the chlorophyllins from the isochlorophyllins. The phytochlorin e-phytorhodin g product is designated as such on the basis of the process by which it is obtained; according to Willstaetter’s data, this should yield a mixture of phytochlorin e and phytorhodin g from a mixture of chlorophyll a and b in the starting material, Furthermore, absorption spectrum A , Figure 1, checks well with that which might be expected from such a mixture.
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Literature Cited Basu, J.,2. Vitaminj’omA, 6 , 106 (1937). Baumann, C. A., and Steenbock, H., J . Bid. Chem., 101, 661 (1933). Baxter, J. G., and Robeson, C. D., Science, 92, 203 (1940). Bechdel, S. I., Landsburg, K. G., and Hill, 0. J., Penna. Agr. Expt. Sta., Bull. 291 (1933). Bessey, 0. A., and Wolbach, S. B., “T’itamins”, Chap. 11, pp. 31-6, Chicago, Am. Med. Assoc., 1939. Bethke, R. M., Kennard, D . C., and Kik, M.C., J . Biol. Chern., 63, 377 (1926). Drummond, J. C., Ann. Rea. Biochem., 7, 338-40 (1938). Emmett, A. D., and Bird, 0. D., J . Bid. Chem., 119, xxxi (1937). Fernholz, E., and Moore, M. L., J . Am. Chem. Soc.. 61, 2467 (1939). Gruskin, B., Am. J . S u r g e r y , 59, 49 (1940). Hickman, K., J . Bid. Chem., 128,xlii (1938). Hilditch, T. P.,“Chemical Constitution of Natural Fats”, New Yurk, John Wiley 8: Sons, 1940. Holmes, H. N., and Corbett, R. E., J . Am. Chem. SOC.,59, 2042 (1937). King, L. C., and Ball, C. D., Ibid., 61, 2910 (1939). Morgal, P. W., Petering, H. G., and Miller, E. J., IND. ENG. CHEM.,33, 1298 (1841). McDonald, F. G., J . Bid. Chem., 103,455 (1933). Nelson, E. M., and Tolle, C. D., Ann. Rev. Biochem., 8 , 419-21 (1939). ENQ. Petering, H. G., Morgal, P. W., and Miller, E. J., IND. CHEM.,32, 1407 (1940). Petering, H. G., Wolman, W., and Hibbard, R. P., IND. ENQ. CHEW,ANAL.ED.,12, 148 (1940). Record, P. R., Bethke, R. M., and Wilder, 0. H. M., PouLtry Sci., 16, 25 (1937). Rothmund, P., J . Am. Chem. SOC.,58, 625 (1938). ENC.CHEM.,30, 1073 (1938). Schertz, F. M., IND. Steenbock, H., Hart, E. B., Elvehjem, C. A., and Kletzien, 8. W. F., J . Biol. Chem., 66, 428 (1925). Taylor, J. F., Ibid., 135, 569 (1940). Willstaetter, R., and Stoll, A. (tr. by Sohertz, F. M., and Mers, A. R.),“Investigations on Chlorophyll”. Lanoaster, Penna., Science Press, 1928. PUBLISIIED with t h e permission of t h e Direotor of t h e Experiment Station as Journal h r & k S o . 520 ( n , a , ) .
Acknowledgment This research was supported by the Horace €€.Rackham Research Endowment of the Michigan State College of Agriculture and Applied Science for studies on the industrial utilization of agricultural products. The authors wish to express their appreciation to C. A. Hoppert of the Chemistry Department of Michigan State College for carrying out the bioassays reported in this article.
A STAFFT~CHNICIAN AT GOODYEAR’S AKRONPLANT Is PULLHANDFUL OF PARTIALLY PROCESSED CHEIMIGUM FROM
ING A
THE
CONVEYOR TO EXAMINE ITSCOMPOSITIOX (See text on page 1342.)
