T H K S I S Approved 3y V u c X î V Major Adviser 7 ßd Dean CONTRIBUTIONS TO THE; CHEMISTRY OR VITAMIN A. A Critical Study of the Antimony Trichloride Reaction. By EUNICE B. RICKMAN. A Thesis submitted to the Raculty of the Graduate School of the Creighton University, in Partial Fulfillment of the Requirements for the de- * gree of Master of Science. 0 M A H A 1933 C O IT TENTS I. Introduction Pp. 1 - 5. A. Biological experiments, showing the necessity of Vitamin A as found in milk. B. Kinds of fats, vitamin A bearing fat and "pure” fat. C. Effect of heat on lipins bearing vitamin A. D. Indispensabilitjr of the non-saponifiable portion of fat containing vitamin A. II. Color Reactions. _ Pp. 5-29. A. Historical developments leading to the antimony trichloride reaction, B. Eive-membered monoheterocyclic groups. 1. Pyrro1, * 2. Thiophene 3. Purane C. Oils other than liver oils. D. The chromogenic substances of liver oils. E. Relation of vitamin A tc substances reacting with antimony trichloride. III. Conclusions. Pp. 29 - 31* A. Characteristic color reactions of antimony trichloride with the five-membered monoheterocyclic compounds. B. The color reaction is intensified and modified Toy adding acetic anhydride. C. Substituent»© in the molecule modify the intensity and rapidity of the reaction. D. Colors obtained with antimony trichloride is not specific for vitamin A. E. Compounds other than vitamin A possessing chromo- genic properties might he responsible for the color obtained. IY. Tables. P p . 32 - 4 3 . A. Thiophene and Pyrrol. B. Oils, C. Purfurane. Y. Bibliography. Pp, 43 - 43. C01TTRIBUTIOITS TO Tffii CHEMISTRY OP VITAMIN A.. A Critical Study of the Antimony Trichloride Reaction* The chemical study of vitamin A dates "back to the work of Hopkins. As early as 1906, he had determined and reported that, "no animal can live upon a mixture of pure protein, fat, and carbohydrate and even when the necessary inorganic material is carefully supplied, the animal still cannot flourish. The animal body is adjusted to live either upon plant tissue or other animals and these contain countless substances other than the proteins, carbohydrates, and fats." In his experiments, using rat© as subjects, Hopkins found that the addition of small amounts of milk to diets otherwise composed of purified foodstuffs resulted in growth, and that this was due to an alcohol-soluble organic substance or substances in the milk. This alcohol-soluble organic substance is known today as vitamin A . 1 In 1909, Stepp carried out the first experiment which demonstrated the indispensability of some substances con- tained in lipoids extracted from, certain natural foods. He believed that the fat-like bodies: the lipins, including licithin, kephalin, cholesterol, cerebron, to be indespen- sable dietary components. Most food-stuffs of animal or T, The Vit'amiFs'.' She'iSan and Smith*/ * *Seconcf Edition/*~ American Dhemical Society Monograph Series. Pp. 18-19, 2. plant origin contain these lipins. He sought to discover what would happen if they were eliminated from an other­ wise adequate diet. He prepared a bread from milk and wheat flour, and another from milk and protomol (a protein- rich preparation from rice) and extracted them thoroughly with alcohol and ether. Mice were fed on these food mix­ tures, Those that were fed on the extracted food diet, died within thirty days; whereas those fed the whole food remained healthy, Stepp concluded that certain lipins are essential for the maintenance of health. To prove which of the individual lipins was indispensable for the main­ tenance of health, he restricted mice to the lipin-free diet plus the added one or more of the individual lipins which was prepared in a state of purity. The diet proved to be incomplete. He then made an extract of egg yolk by shaking with cold alcohol and divided the solution into two portions; one was heated with 95% alcohol for two days in a water bath; the other was heated only to evaporate the alcohol. Each preparation was added to the lipin-free diet and the two mixtures were fed to different mice. Those that were fed with the heated yolk extract died within thirty days; whereas those receiving the unheated preparation remained healthy. The indispensable substances were changed by pro­ longed heating so that they no longer rendered the food com­ plete for nutrition. The unheated egg yolk, however, which is very rich in what is now known as vitsimin A rendered the diet complete.~ In IS 13 McCollum and Davis "began feeding young an­ imals with purified foodstuffs such as purified casein, starch, lard, and salt mixture. These investigators no­ ticed that the animals failed to grow. In the same year Osborne and Mendel also worked on the problem independently and found that animals whose sole fat in the diet was la,rd, but good in all other respects, the young would cee,se grow­ ing; these and the older animals developed xerophthalmia. But when young animals restricted to such a diet would grow well and prevent the development of the characteristic di­ sease of the eye when butter fat, egg yolk or cod liver oil was added to the diet, but not when lard was the sole source of fat. From this they concluded that certain fats contain a dietetry essential not hitherto recognized. Lard is as much a fat as the other fats mentioned. The differences among the fats are largely due to the differences in quantity, but not in the quality of the three fatty acids: palmeic, stearic, and oleic. The differences even in quan­ tity is not as profound as to warrant the nutritional def­ iciency in the animal whose sole fat in its diet is lard; and the thriving of the same animal fed on a diet containing butterfat, egg fat, or cod liver oil. Therefore, these in- 1. Sherman, Chemistry cTTood‘arXWtrTtYorX*'Mac"-“" ~~~ millan Co. Fourth Edition. Pp. 348 and passim. vestigators attributed the loss of weight and general decline of the animals fed on lard not to the fat itself but something lacking in this fat. This something is accompanied in those fats that bear the designation of vitamin A. And to prove further that vitamin A is not a fat, that is an ester of a fatty acid«*. vitamin A bear­ ing mixtures like cod liver oil when saponified, vitamin A appears in the non-saponifiable portion of the reaction product; and as such can be removed. 1 This ursaponifiable portion, is believed by some investigators, gives the blue color reaction with antimony trichloride color reaction, COLOR REACTIONS In 1922, Drummond and Watson suggested that the strik­ ing purple color developed when liver oils are treated with sulphuric acid might be due to vitamin A or other re­ lated substance; since there is a relationship between the intensity of the color reaction of the oil and its vitamin A value. In 1925 Rosenheim and Drummond described a color test given by cod liver oil upon interaction with arsenic t trichloride. This they believe to be a specific test for vitamin A. They also found that dimethyl sulphate, tri­ chloracetic acid, acetyl chloride, and benzyl chloride give blue color reactions with substances containing T l ShenST and" Smith.the VitamTnsT'~ Second EcQtTon“ American Chemical Society Monograph Series, Pp. 238 and passum. 5. vitamin A 1 , Carr and Price"2 introduced some modifications in the procedure adopted by Rosehheim and Drummond and substituted antimony trichloride for the arsenic compound. The color obtained with these reagents was taken to in­ dicate the presence of vitamin A. The Carr-Price reaction has been employed in the colorimetric method for the quantitative determination of vitamin A zn cod liver oil. Several investigators, in­ troducing various modifications, have reported agreements between the biologic method for the estimation of this vitamin and the chemical method involving the production Ox colox with antimony trichloride and its subsequent measurement in the Lovibond tintometer. Other investi­ gators, however, could not find any close coi’relation be­ tween the chemical and the biological method. By employ- * ing the unsaponifiable residue instead of the oil itself, Andersen and nightingale3 , Dorris and Church4 , Smith and Hazley5 , and also Coward, Dyer, Morton and Gaddum6 , claim to have obtained reliable value for vitamin A content. Tne antimony trichloride color test is hardly to be considered specific for vitamin A. although it may serve to establish some chemical relation between this vitamin 1. Rosenheim, 0 . and I)ru3no!id7 j . c, , Biochem— t----To— 2 . Carr, p. and Price,E.A.,Biochem,J ,2 0 , * '' 4* 0hUrOhl/‘-E-• End teeeum.J. Biol.Bhem. 65, 5. Smith,l.L. and Hadley,V. , Biochem. J. , 24, 1492 (1931 ). 6, and the type of compounds giving positive reactions. Sterols also have been reported by Wokes7 , by Heilbron and Spring1 , O and by Seel^ to give characteristic color reactions with antimony trichloride. Among the sterols may be mentioned phytosterol, cholesterol, ergo sterol and their various derivatives. Heilbron and Spring maintain that sterols giving a postive reaction contain the 1;2 or /\ 1:13 ethenoid linkage. The absorption spectra of the various reaction mixtures have, not as yet been reported. Carotinoid pigments also give characteristic colors with antimony trichloride*^: Among those reported as re­ acting positive are carotene, di-iodocarotene, bixin, cap- santhin, (X-crocetin, dihydro, o(-crocetin, dihydro-isonor- bixin, fucoxanthinV lutein, lycopin and zeaxanthin, The belief has been ventured that the antimony trichloride be­ comes attached to one of the conjugated double bonds (pyiyene groupings) in the carotinoid molecule. The color reaction differs in intensity with the change in the polyene groupings and is in all probabilities influenced by other por­ tions of the molecule.