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. * We wish to report new types of compounds that give
characteristic color reactions with antimony trichloride.
These types comprise the five-membered monoheterocyclic
rings, thiophene, pyrrol, furfurane and their derivatives. d* Coward,K. H. , Dyer, D.J., MorVcn’,ff.AY"and*'GaddumVT “ Biochem, J. 251, 1102 (1931). 1. Heilbron, L.M. and Spring,H.S. Biochem,J,, pp. 24,133 (1930). 7
The formulas for these monoheteropentacycles are given as follows: C H H C C H H C\ / C H H C \/0HI Purfurane Pyrro1 The reagent we used was that of Carr and Price. Antimony trichloride was washed several times with chloroform and dried in a desiccator. A saturated solution of this produce was made by adding 30 grams to 100 c. c. of choloform (U. S. £.), allowing it to stand and decanting the clear liquid. The
chloroform contained 1 per cent alcohol. The compounds to be tested were me.de up to 20 per cent chloroform solutions or if insoluble into 20 per cent chloroform suspensions. To
the three drops of the chloroform solution or suspension were added 2 c. c. of the antimony trichloride solution. The characteristic color may not form immediately, but develops on standing. •
We have modified the above procedure by adding 0.5 c.c.
of acetic anhydride to the substance dissolved in chloroform
subsequent to admixture with the antimony trichloride reagent.
The acetic anhydride serves as a chromogenic stimulator. Por o ' the same purpose Whitby 1 and Rosenheinr have used formaldehyde. Brode and Magill 3 have employed acetic anhydride with their
antimony trichloride reagent in their experiments on fish
2. Seel, E . , Arch, exptl. Path, u Phaamiako'i. pp. 24, ,' 3, Moore, T, Lancet, 1, 499 (1929). 133 1. Whitby, ffi. S. , Biochem. J. , 17 , 5 (1923). 2. Rosenheim, 0., Biochem. J., 2 1 , 111 , 289 (1927). liver oil. As the reason for their use of the anhydride we may state that it serves to remove hydrochloric acid and water from the reaction mixture. To illustrate the chromogenic power of acetic anhydride, we will take skatol as an example. This compound gives with anti mony trichloride in the presence of acetic anhydride a
light purple color developing in ten minutes to a very deep royal purple. Skatol with, antimony trichloride alone gives a yellow color with a slight tinge of red only after twenty-four hours.
Besides acting in the capacity of a substance capable of stimulating color production, acetic anhydride acts as a solvent by its ability to form acetyl derivatives. Thus pyrrol fails to give color with antimony trichloride be cause it forms a white gelatinous precipitate. When, how- » ever, acetic anhydride is added before the antimony tri chloride solution, precipitation is prevented and a charac teristic color is permitted to form. The mixture without acetic anhydride we have designated as Reaction Mixture A and the one with acetic anhydride as Reaction Mixture B.
We have always found that mixtures with acetic anhydride gave a clearer and more distinct color than mixtures with out the anhydride.
" a. Erode, W. A. and Magi'llM.AV,~'J • Biol. Chen;., 92,87 (1921). 9.
PYKROL Compounds of the pyrrol type yield an intense color with Reaction Mixture B, The only exceptions a.re proline,
which is a tetrahydrol pyrrol with a carhoxyl group, and
nicotine which is composed of a pyridine nucleus and a re
duced pyrrol, and nicotine salicylate* Both nicotine and
its salicylate form precipitates in the reaction mixtures
with or without acetic anhydride. Hemoglobin, hematin and the bile pigments, bilirubin
and biliverdin, each contain four pyrrol rings. Hemoglobin
gives no reaction. It is insoluble in chloroform, Hematin,
bilirubin, and biliverdin dissolve in chloroform and yield
a characteristic color with antimony trichloride in the presence of acetic anhydride. Since the chloroform solu
tions of these three pigments are very deep in color, it is
imperative to make very dilute solutions in order to recog
nize the particular color obtained as a result of the in
teraction with antimony trichloride, but with acetic anhy dride and the antimony compound, a yellow color with a
tinge of green develops after prolonged standing in jx
solution of the plant pigment so dilute as to produce an almost colorless liquid.
We have tested a number of heterocycles which do not ■belong to the five-membered monoheterocyclic series. Among them may be mentioned histidine, a pentacycle with two nitrogens in the ring, and the monoheterocyclic six-membered
compounds, pyridine, quinoline, quinolinic acide, quin- aldine, quinaldinic acid and cincophene. These substances
give no reactions. Acridine, however, which is diben- zopyridine, displays with antimony trichloride green
fluorescene; but with the same reagent and acetic anhy
dride, the fluorescence is somewhajs more intense,
THIOPHEKE. Thiophene is the only sulphur-containing heterocycle
we have studied. With the antimony trichloride reagent the color develops but very slowly, finally yielding a
brownish red. With the reagent modified by the addition of acetic anhydride the color reaction is more rapid and * more intense and a purplish blue color develops changing
to light blue liquid and a dark blue precipitate. The color of the liquid and of the solid remains unchanged for many weeks,
FURAUE ( FUEPURA1IE ) We have extended our experiments with the antimony
trichloride reagents to include fürfurane and its de
rivatives. We have found compounds of the furane or fur-
furane type to react very strongly, very often with the formation of a "blue color. Furfurane itself yields a green color with, antimony trichloride, changing to green ish "blue and finally to a blue liquid with a dark brown precipitate. With the modified reagent employing acetic anhydride a deep purple wine color is obtained changing to purple and finally to blue, no precipitate is evident.
The color reaction for each particular furfurane deriva tive is to be found in Table II. With many of the com pounds that gave a greenish or bluish liquid a purplish or bluish precipitate formed after prolonged standing.
The intensity of the color and the variety of the color depends on the presence of the particular substi tuent in the furane ring. As a rule the presence of the aldehyde group strengthens the color and its permanence.
Derivatives of furfurane aldehyde (furfural) such as the » oxime and the condensation products, such as furoin, furil, furfural acetone, furylacrolein, furyiacrolein oxirne, fur fural acetophenone, and furfural diacetate react even more promptly and more intensely than the mother substance.
The presence of the hydroxyl group slows up,the re action and reduces its intensity. The presence of a car boxyl group attached to the heterocycle, be it pyrrol or furfurane, inhibits the reaction altogether. Prolin, which is pyrrolidene carboxylic acid, gives no reaction. Furoic acid does not react; neither do any of its derivatives which involve substitution in the carboxyl group. Furoyl chloride, furoamide and furonitrile and esters of furoic acid are therefore unresponsive. The fact that oxidation of the aldehyde prevents the reaction coupled with the finds that vitamin A when subject to oxidation is no longer potent biologically and no longer gives the characteristic color reaction leads to the belief that vitamin A is either an aldehyde or an alcohol. Cady and Luck1 working from another angle came to the conclusion that vitamin A is probably an aldehyde, while Karrer, Morf and Schopp,^
consider vitamin A to be an alcohol for which the empirical formula C20H30O has been suggested. When, however, the carboxyl group is on the side chain and not directly attached to the ring, as in the case of
furacrylic acid, the'reaction, although positive, is not intense and develops but very slowly. Furylacrylchloride
and furylacrylamide behave like furacrylic acid. Trypto
phane is B-benzopyrrol of B-indol ^-amino propionic acid.
The carbonyl group is on the side chain. The reaction is
weak, yielding with antimony trichloride and acetic anhy dride an' atypical color, deep yellow or yellow brown. This
»typical yellow color has also been reported by Heilbron
and Spring^ for o( and B-isoergo sterol and for dihydroergo-
!. DadyToTH. and LuckTj.N."," Bi'^l” “c¥enrr7^87'’,' T l Y T l ^ T . ' * 2 » Karrer,P,, Morf,R. and Schopp.K., Helv. Chim. Acta,14, 1036 (1931). 3. Heilbron,L.M. and Spring,F.S., Biochem. J. , 94, 133 (1930) 13.
sterol* Hemoglobin, bilirubin, biliverdin and chlorophyl react very feebly. These structurally complex compounds possess carboxyl groups.
Degree of unsaturation is held responsible for the antimony trichloride color reaction with unsaturated com pounds, acc6 rding to Euler and Hellstrom. 1 We hays found that complete saturation of the furfuryl radical does not abolish color formation, Tetrahydrofurfuryl alcohol yield a deep royal purple with antimony trichloride a,nd a deep greenish blue liquid and precipitate with the same reagent in the presence of acetic anhydride, Tetrahydrofurfuryl acetate, butyrate and lactate also yield characteristic color reactions. Proline is a saturated furfurane to which is attached a carboxyl group. It is negative in its behavior towards antimony trichloride because of the in terference of the carbo3^yl group. Strophantin gives with reaction mixture A a colorless liquid with flaky gray white solid particles which grad ually turn greenish blue. This result is interesting in view of the fact that Jacobs and Gustus2 Jacobs and Elder- 3 i field indicate the presence in strophantidin and in iso- s^ropphantidin two six-membered cyclic rings and one five- membered heterocyclic ring, which is a furfurane ring. 1. Von Eulcir, h T and Hellstrom,H. , Svensk.Kem.Tids. 41.11 f 1QPQ1 iaco?s‘V and Gustus’ Biol. Choi. ,74 , 811 (1927)®)# 3. Jacobs, W.A. and Elderfiend, R.C., J. Biol. Chem. , 97 ,727 (1932). 14
The six-membered mono heterocyclic compounds containing an oxygen atom, xanthone and xanthydro1 , do not react with the antimony trichloride reagent.
THE AHTEiOHY TRICHLORIDE REACT I Oil WITH OILS OTHER THAU LIVER OILS.
Harden and Ro'bison-1' maintain that the purple color obtained by the addition of sulphuric acid to cod liver oil bears a resemblance to Heuberg and Rauchwerger*s test^ for cholesterol in which the reagent is -methyl furfur- aldehyde obtained from rh&mnose by means of concentrated sulphuric acid. Harden and Robison postulated the pres ence of a furfurane derivative in fish oil, since they were able to obtain a similar color reaction by the interaction of sulphuric acid with furfural and cholesterol dissolved in petroleum or in chloroform. They were, however, unable to isolate from coal fish oil by distillation with team » 3 or under reduced pressure or by other methods either fur fural or some compound which could replace it in the re action system, cholesterol-furfural-sulphuric acid.
The presence or absence of furane derivatives in oils needs, nevertheless, further investigation. Shear3 re ported a color reaction with cod liver oil obtained by the use of a reagent consisting of 15 volumes of aniline to 1 volume of concentrated hydrochloric acid. This reagent T.*lEr^li7A™^OobiToT^Wo^cliemrJ™lT,*"lT5TTi9T3")7 S. Heuberg,C., and Bauchwerger,D., Chem.Centr,, 2, 1434 (1904). 3. Shear,M.G. »Proc.Soc.Biol, Med., 23, 546 (1925-26), 15.
yields a characteristic color reaction with furfurane and
its derivatives, with sterols, with ionone, and with caro
tene1. In this connection it is of great interest to men
tion that Tocher^ isolated from sesamin oil a crystal-line, unsaponifiahle compound, and named it 3esamin. Bertram, van der Steur and Waterman3 recently demonstrated a furfurane ring in its molecule, and ascribed to it the formula following:
0
We have tested 3esame oil with the antimony trichloride reagents. The oil reacted very strongly with antimony
trichloride with the development of a golden yellow color whicn rapidly transformed to a cherry red or wine red color.
In the presence of acetic anhydride sesame oil finally de- » veloped a clear deep purplish color.
Chaulmoogra oil is another oil that presents great
interest because it contains two compounds, chaulmoogric acid and bydnocarpic acid, the structures of which indicate -the presence of a pentacyclic ring;.4 This ring, however, is isocyclic instead of heterocyclic. Such pentacyclic rings are to be found in cholesterol3 in ergo sterol3 and in the 1. Tocher. Pharm. Jour. Trans., 638 (1890-91)“ 2. Tocher. Pharm. Jour. Trans., 700 (1892), 3. Tocher. Chem. Ayg. , 17, (1893). 4. Power, P. B. and Gornall, J.121 Chem. Soc. 85,838,851 (1904). 5. Rosehein, 0., Biochem J. , 47, 23 (1929). 6. Heilbron, L. M. and Spring, P.S. , Biochem,J. , 23,133 (1930). 16
"bile acids, 1 Chaulmoogrie acid has the following structural fo rmula: V C(CHrj)i2 C°°H
h 2 c
Chaulmoogrie acid
Hydnocarpic acid has ten CH>> groups in the side chain instead of twelve.
We have examined two samples of chaulmoogra oils, one from Eli Lily Co. and the other from Sargent and Company,
The first sample gave a slight lemon colored turbid mixture with antimony trichloride. With acetic anhydride and the antimony reagent, a slight lemon tinted hut clear liquid was obtained which turned to light amber. The other sample
gave an olive color, which changed to yellow. With antimony
trichloride in the presence of acetic anhydride, a brownish * olive color was obtained, changing to brown with a greenish tint and finally to reddish brown.
Since the five-membered cyclic ring occurs- in chaul-
moogric acid or in hydnocarpic acid present in the oil, we, -deemed it advisable to test these acids. We were, able to
obtain only a sample of chaulmoogrie acid from Eastman Kodak
Company and marked "practical." With antimony trichloride
we obtained a lemon yellow, cloudy color which finally turned
to olive. With the reagent and acetic anhydride, we obtained
1. Wieland, ~h 7 f T . Angew. Chem., 42 , 421 (1929 ) ." " " 17.
a clear lemon yellow liquid, changing to "brownish yellow and finally to brown. A sample of the ethyl chaulmoograte
(practical) obtained from Eastman Kodak Co. gave with antimony trichloride a yellow olive color changing to yellow. The same reagent in the presence of acetic an hydride gave a yellowish olive color turning to greenisn olive and finally to purplish red. Chaulmestrol, which
is the trade name for a mixture of ethyl esters prepared
from chaulmoogra oil by the Winthrop Chemical Company,
gave with antimony trichloride a brown color changing to
a mahogany color, while in the presence of acetic anhy dride and antimony trichloride, the reaction mixture as
sumes a brown color with a greenish tint and changes to
dark greenish blue and finally to purple. Since neither
the chaulmoogric acid, the ethyl chaulmoograte nor the
chaulmestrol we have ¿used were chemically pure we do not
know whether the colors obtained were due to the com
pounds mentioned or to impurities therein.
We hatfe also tested olive oil, cotton seed oil, and
wheat germ oil. Precipitates formed in all the reaction
mixtures containing antimony trichloride, but not in
those containing the antimony salt with acetic anhydride.
Uorris and Church-*- also examined 34 varieties of essen
tial oils with the antimony trichloride in chloroform
solution. They reported that three did not react at all.
T, ITorris,~"E. R. and Church, A. E. , JBiol. Chem, ~ Ppi 87 , 139 (1930). 18
Others gave various shades of yellow, brown and red. Oil
of wormwood gave a green color, ethereal oil gave a purple
color, and cedar wood oil gave an intense blue, which showed
an absorption band with a maximum at 580 p. p . They attribute the reac^_. ity of the essential oils to the presence of un
saturated compfeunds, We, however, hold the opinion that the
terpenes present in essential oils are responsible. We have
tested a number of compounds of the terpene series with anti-
mony trichloride and have found many of them reactive,'1'
Wheat germ oil developed a cloudy bluish green with anti
mony trichloride and a clear bluish green coloration in the re- action mixture containing- acetic anhydride. Wiliimott and Wokes2 reported a positive test «ton antimony trichloride reacted with decolorized yellow maize oil. Croxford and Stout and Schuette3 also obtained a positive toot with rye germ oil.
A typical color reactions are not to be regarded as in dicative of lack of vitamin A. Morton and Heilbron4 observed that nonsaponifiable material from Danish butter does not yield the typical blue color but rather a bluish green. Emmett, Bird, Hielsen, and Cannon6 made a similar observation for halibut livlr OH, Which is rnueh richer In vitamin A than cod liver oil,
Furthermore, the typical colcraction obtained with antimony
^ " BUbJe0t *» 4^ c r e n c e because of the presence si ~ r ion-— Schuette | W. and 19.
inhibiting agents. Horris and Church1 reported that such in hibiting agents are represented by oleic acid and by unsatur ated oils. The typical color may be subject to modificat ion because of the presence of other substances that may re act with antimony trichloride. Emmerie, van Eekelen and Wolff2 without being aware of the fact that five-membered heterocyclic compounds reacted with antimony trichloride, reported that the addition of furane, methyl furane, pyrrol, indol or skatol to fish liver oil before treatment with the Carr and Price reagent resulted in a purple color instead of the expected blue.
In order to determine the effect of oxidation on the color reaction, we heated a sample of wheat germ oil at
100» C for one hour while passing oxygen through it. At tne ena of that time the reaction was still positive and even stronger. We expected to find the reaction negative in view of the destructive action of oxygen on vitamin A. Hopkins3 , Drummond and Coward4 and Zilva5 reported loss of vitamin A potency at high i* Aileron, I.M. , Biochem. J. , 24, 870 (1950). 5. Emmett,A.D., Bird. O.D. , ITielaen.C. and Cannon,J.H. , J. In d . Eng. Chem., 24, 1073 (1932). 1. Horris, E.R. and Church,A.E., J. Biol. Chem.,85,477 (1920) 2. Emmerie,A.,van Eekelen,M. and Wolff ,L.K. tfatire Sept. 19, ) 3. Hopkins,P.C-. , Biochem. J. , 14, 725 (1930). '1931 4. grumond.J.c. and Coward, K.H. , Biochem. J., 14,743 (1920). o. ¿ H v « , S.S., Biochem. J, , 14, 740 (1920). 20.
temperatures in the presence of oxygen or an oxidizing agent.
We must, however, take into consideration the work of
Sherman, Quinn, Bay and Miller4, and the more recent work of Cady and Luck*5 who reported greater stability of vitamin A in plant than in animal substances, and of the 3till more recent work of Bann^, who finds that vitamin A may resist
the action of oxygen, the solvent and the impurities therein acting as inhibitors.
Increase in the intensity of the antimony trichloride reaction has also been noted by Hawk4 after exposing cod liver oil to air and sunlight for a total of seventy-nine
hours. This finding does not harmonize with that of
Zilva°, who maintains that ultraviolet rays destroy the
potency of vitamin A through the stimulus these rays
impart to the process of oxidation. Brummond® could not » confirm Hawk*s observations.
Other investigators, however, present evidence that the blue reaction can be intensified under certain con-
ditions. Mittlemann autoclaved at 120° C cod liver oil
sealed in air-evaculated tin cans and observed that the
liberated oil gave no blue color with antimony trichlor
ide immediately on opening after cooling. The same oils
. Shermah,H.C Q u i n n ,B.J. Bay,33,L. • and Miller,E.H., " “ 1 J. Biol. Chem., 18, 293 (1928). 2. Cady, 0.H. and Luck, J.N. , Bio 1.Chem, , 87 , 743 (1930). 3. Bann, W.J., Biochem, J., 26, 151 (1932). 4. Hawk, P.B., Science, 69, 200 (1929). 5. Zilva, S.S. , Biochem. J. 13, 164 (1919). 6 . Brummond, J.C., -7. Soc. Chem. Ind. , 49, 258T (1930). . Mittlemann, cited from Bezssonoff ,1?., Bull. Soc. Chem. Biol., 11 , 1146 (1929). ’ examined ten days latex gave a strongly positive test.
Lovern, Creed and Morton'1' obtained fresh, oils from auto claved and steamed cod livers which at first gave a read ing of 4.0 to 6.4 Carr-Price units, hut which after a storage period of three days under nitrogen yielded cor responding values of 8.0 and 7.2 blue units. They also reported the observations that cod liver oil exposed to air at 70° C or oxygenated at the same temperature underwent a considerable augmentation in its capacity to give the blue color with antimony trichloride.
It is probably that the intensification of the color reaction is due to another chromogenic substance present in the oil. In this connection it is interesting to note that Heilbron, C-illam and Morton2 maintain that liver oils contain two different substances each capable » of reacting with antimony trichloride. One substance yields with the antimony salt a band at 6C6 iu p and the other at 572 ju ju. The compound represented by the 606 u u band becomes on oxidation more reactive with the antimony
trichloride reagent, since treatment of the liver oil
with ozonized oxygen, hydrogen peroxide or benzoyl per
oxide previous to the addition of the antimony trichlor
ide solution induces a great increase in the intensity of
the band at 606 ju ju. but no increase in the band at 572 ju ju.
Lovern, Creed and Morton report that liver oils on standing
1, Lovern, J. A., Creed, R. "h , and Morton, R. A. Biochem. » 25, 1352 (1931). . Heilbron, I. M. , C-illam, A. E. , and Morton, R. A, , Biochem, 2 J. , 25, 1341 (1931). 22
or heated in air or oxygen "became modified bo that they react more intensely with antimony trichloride and at the same time show enhanced absorption in the visible spectrum at 606 yu p, "but no appreciable change in the absorption at 572 Mu or at 328 u p in the ultra, violet
spectrum.
THE CHROMOGEMIC SUBSTANCES OE LIVER OILS. The more recent investigations on the behavior of
liver oils towards antimony trichloride throw interest
ing light on the existence of more than one chromogenic
substance. The chromogenic power measured by color for mation with antimony trichloride is smaller than would
be expected from the values obtained with unsaponifiable
extracts. Smith and Hazley, 1 Smith2 , Coward, Dyer, 3 Morton, and Gaddum argue from this fact that for the
occurrence of an inhibitor in the unsaponifiable portion.
That some inhibitor is present is noted from the work of Morris and Church4 who find that oleic acid and un saturated oils depress the formation of blue color. Coward, Dyer, and Morton5 have recently come to ‘the con clusion that the intensity of absorption at 328 p p of
the oil itself gives the best measure of the vitamin A content of liver oil. Chevalier6 has reported, however, 1, Smith,E~.l V and* Hailey, V. Bïochenf, J, , 24, 1492, '('lÇSiy, 2, Smith,E.L. and J. Biol. Chem., 90, 597 (1931). 3, Coward, K. H. Dyer ,B.J", , Morton,R.A. and Gaddu, Biochem, J". 251, 1102 (1931). 4, Morris,E.L. and Church,A.E., J. Biol.Chem. 85, 467 (1930) 5, Coward,E.H.Dyer,E.J. and Morton,R.A., Biochem, J. 26, 159.3, (1932). ? Physiological Congress, heme, f e n r 10.^2.. 23.
that the intensity of absorption at 328 ju ju gives good agreement with the biologic value only in oils having a low free acid value. The inhibitor from our standpoint may as well be a compound that reacts with antimony tri
chloride to give a color differing from the typical color
so as to interfere with the accuracy of colorimetric or tintomeric observations.
Liver oils on treatment with antimony trichloride
exhibit complex and qualitatively variable absorption spectra, according to Marton, Heilbron and Thompson1 and Heilbron, Gillam and Morton2, Most workers in this
field are agreed upon the findings that two maxima in
the spectral absorption curves can be recorded. Ac cording to Gillam and Morton3 the blue color obtained with antimony trichloride yields two apparently inde
pendent absorption bands, one in the yellow green (722 ju y ) and the other in the orange (606 ja p.). In concentrates
the bands are displaced to 583 p. u and to 620 u u respec tively.
The intensity of these bands vary. Heilbron, Gillam 4 and Morton state that in oils characterized by a predom
inance of the 572 ju y. band over the 606 u ju band, a great
increase in the intensity of the latter can be secured by
treatment of the oils present to interaction by antimony
1541° (^231)"* * heilbron, I.M. aiid n 7 B i o c S , ’ J 21), 2* Heilbron, I.M,, Gillam,A.E., and Morton,E.A. Biochem. J. , 25, 1341 (1931). * 3, Gillam,A.E. and Morton,E.A., Biochem, J, 25, 1346 (1931), 24.
trichloride with ozonized oxygen, hydrogen peroxide of "benzoyl peroxide. Oils which initially show an excess of the 572 p p chromogen over the 606 p p chxomogan also undergo slow spon taneous oxidation with a resulting increase in the 606 u u band. The marked change in the 606 p p band does not take place at the expense of the 572 p p band, which, however, does not undergo an increase. We have found an intensification of the antimony trichloride reaction with wheat germ oil sub jected to heat and oxidation; and H a w k ’*' and others have re ported a similar finding for cod liver oil exposed to air and sunlight for many hours. In view of the destructive effect of ultra violet and of oxygen and ozone upon vitamin A, we are led to believe that the persistence of the blue color ob tained with antimony trichloride after oxidation or exposure to light and air is an indication of the presence in oils of a chromogenic substance other than the fat-soluble vitamin A .
* Evidence o^ the presence of two chromogenic substances bas been presented by other investigators. Emmerie, van Eekelen and Wolff2 add to vitamin A preparations small quan tities of such monoheterocyclic compounds as furane, methyl fur ore, pyrrol, indol and skatol, and subsequently treat the oil mixture with antimony trichloride. As a result of the addit ion of such compound they find that the 610 p p band is no long seen, while the 572 p p band still remained. The physio logical activity cf the vitamin A mixture is still retained 1. Hawk, P.B., Science, 69, ( ). 2 Emmerie, A. »Van Eekelen,M.200 and 1929Wolff, l .E. . 19,(1931) Hature, Sept. after the addition of an heterocyclic compound and continues to show the same hand, 328 ju ju as the original vitamin A pre paration previous to its admixture with the heterocycle. Morton1 studied the effects of 7-methyl indol on the color reaction induced with antimony trichloride. He ob served that a trace of this compound inhibits the formation of a blue color and that it masks the more intense 717 ju ja bana more readily than the 583 p. ju band. When, however, he adds 7-methyl indole before the antimony trichloride reagent in the ratio of one part to four parts of concentrate, the two bands appear with roughly equal intensity. The addit ion of still larger amounts of 7-ethyl indole induces a
Slow inhibition of 583 p p band, but the inhibition is al ways of lesser intensity than that of the 617 fa p. band. 4 * It ts evident from the recent work quoted at length that we are dealing in liver oils with two substances ca** able of reacting with antimony trichloride. These two substances exhibit two independent bands, it is not con- ceivable that one chromogenic substance should yield the two bands in view of the fact that carotene, according to koore2 , Duller, Morton and Drummond3 and Brode and Magill4 1* Morton, R.A., Biochem. J . , 26*,"" *Ï197 ( 19 52 ) . 2, Moore, T., Lancet, 2, 280 (1929). 3, Duller, W . , Morton,R.A . and Drummond,J,c., Ind., 48, 316T (1932). Soc. Chem. 4, Brode, W.A. and Magill , M.A., J, Biol. Ehern•> 92,87 (1921) 26
reacting with antimony trichloride, exhibits only one band at 590 ju ju, although Karrer, Euler and Euler^- re port two or even three bands with some carotinoid pig ments.
Ender^ reported that the influence of antimony trichloride and water upon halibut liver oil concentrates
à,lso serves to point out the existence of two chromogens. One chromogen, possibly identical with vitamin A is des troyed by treatment with antimony trichloride and water.
The other chromogen reacts with antimony trichloride forming a blue product from which the chromogen may be
regenerated by the addition of water. He also observes
that the destruction of vitamin A in concentrates subse
quent to treatment with antimony trichloride abolishes
the band at 622 p p, while the absorption bb&nd at 590 p p remains exceedingly pronounced. » These two chromogenic compounds present in liver oils
show variation in quantity as indicated by the absence of
one band or by the presence of two bands of different in
tensities* One of these substances seems to be briefly
influence by oxigenation, since an increase in the in
tensity of one of the bands (619 ju jU) takes place on spon
taneous oxidation, treatment with ozonized oxygen, hydrogen peroxide, or benzoyl peroxide. The same substances seem also to be influenced by certain heterocyclic compounds, as
1. Karrer,P., von Euler,B. and von Euler H.',‘^"r*chiV kemo--- Mineral Zeol. 103, Mo. , pp (1928). 2. Ender, E. A., Biochem. 2 J. , 626, 1197 (1932). indicated by the fact that in their presence the 610 |u ia t > band disappears, while the other remains. Prom the facts cited it is evident that liver oils rich in vitamin A contain substances chromogenic with fcespect to antimony trichloride. One of these substances is vitamin A, while the other is a yet unidentified sub stance. We have reported in this paper color reactions with antimony trichloride and various plant oils, although the reactions may not be the typical ones obtained in
liver oils. This finding leads us to the belief that
substances chromogenic with respect to antimony trichlor
ide are common components of oils and that the presence of these chromogenic substances do not necessarily indi cate vitamin A potency.
RELATION OP VITAMIN A TO SUBSTANCES REACTING WITH ANTIMONY TRICHLORIDE. Antimony trichloride gives characteristic reactions with oils containing vitamin A, with many sterols and their derivatives, with the carotinoid pigments, and with
compounds containing five—membered monoheterocyclic rings. It would be of interest to determine whether sterols,
carotinoid pigments, and compounds containing five-membered monoheterocyclic groupings have the ability to develop vitamin A potency. Eerppola1 prepared from cod liver oil a cholesterol fraction, differing from ordinary cholesterol
1. Eerppola, W. , Skand, Arch. Physiol., e o T T i i * ( 1 9 3 0 1 7 and acts as a chromogen towards antimony trichloride,
which cures xerophthalmia and restores activity to tissues of vitamin A depleted rats. Seel1 and Seel and Dannmeyer2 oxidized cholesterol with "benzoyl peroxide, according to the method of Lif-
schutz, and obtained a product presumably oxycholesterol,
resembling vitamin A in giving ultraviolet absorption
bands at 327 ju jx and 293 ju p, in yielding the typical
color reaction with antimony trichloride, and in curing
xerophthalmia. The minimum dose of the oxidized chole sterol required to cure xerophthalmia was 0 .1 milligram, whereas a natural vitamin preparation from shark liver
oil proved active in 0.0001 milligram doses. Seel ex
pressed the view that vitamin A is a very highly labile partial oxidation product of cholesterol.
That carotene possesses vitamin A potency has been definitely established by a number of workers. Both u!*u -carotene5 possess biologic potency as well as and B -dihydrocarotene.4 Chlorophyl carries in its mole cule four groupings of the five-membered heterocycle, pyr rol, Burgi5 hasdemonstrated in rats receiving no vitamin A that growth was started by the administration of chloro-
1, Seel, H., Arch, exptl. Path. u Pharmakol., 159, 92 (Ï ÎT. 2, Seel, H. and Dannmeyer, p., Strahlentherapie, 39,499 93 ( ) 3, Von Euler, B., von Euler,H. and Hellstrom,K., Biochem. 1931 Zeit., 203,370 (1928). 4, von Euler, B . , von Euler, H. 12, 278 (1S29). and Karrer,P., Helv. Chim. Act 5, ' Burgi, E . , Klin. V/ochschr.,10, 13 1 3 (1913), phyl and its derivatives phacophytin and chlorophyllin.
The pigments were free from carotin. In this connection we may state that Abbot-*- has reported that crystalline chlorophyl does not cure xerophthalmia. Burgi2 has also published work indicating that rhoding, obtained from chlorophyl b, also stimulates growth in the absence of vitamin A in the diet.
CONCLUSIONS. Antimony trichloride in chloroform solution gives characteristic color reactions with the five-membered * monoheterocyclic compounds, pyrrol, thiophene and fur- furane, and with more complex compounds containing pyr rol or furfurane configurations.
The color reaction is intensified and often modified by the addition of acetic anhydride-. The anhydride cat alyzes the chromogenic properties of the heterocycles.
It serves to remove water and free hydrochloric acid from the reaction mixture. It also aids in holding the heterocyclic compounds in solution.
Derivatives of the monoheterocyclic compounds also yield characteristic colors, the substituents in the mole cule modifying the intensity and rapidity of the reaction.
The greater the degree of oxidation represented by the derivative, the less the response. The aldehyde reacts
1* p • oy^ °* C* ’ Pla* Asr* Expt* St a. Ann. Reptl, 19297' * 2. Burgi, E . , Klin. Wochschr, 9, 789 (1930), more vigorously than the corresponding alcohol. Acids
fail to react when the carboxyl group is attached directly
to the heterocycle, and react hut feehly when the carboxyl
group is attached to a side chain. The acyl chloride, the
amivu-, and the nitrile behave like the corresponding acid obtained on hydrolysis*
The color reaction obtained with antimony trichloride in chloroform solution is not specific for vitamin A,
since positive tests have been obtained with sterols,
with pigments other than carotene, and with such five-
membered monoheterocyclic compounds as thiophene, furfur&ne
and many of its derivatives. The color reaction may, how
ever, prove to be indicative of the presence of a specific
grouping or configuration in the vitamin A molecule.
The fact that a gr
the sterols and Carotinoids react with antimony trichloride
lends support to the idea that fish liver oils contain be
sides vitamin A another compound possessing ehromogenic properties.
The ehromogenic compound not possessing vitamin A potency gives, when oxygenated or when- treated with oxi
dizing agents, a more intense antimony trichloride reaction.
Compounas other than vitamin A possessing ehromogenic properties with reference to antimony trichloride are in all probability common componehts of animal as well as vegetable oils* At present only the biologic method of examination is the most reliable for the detection and quantitative determination of vitamin A; and if attempting to essay this vitamin chemically the associateci. chromogenic sub~ stance must be first isolated. 32 Table 1
XNTIMONY TRICHLORIDE REACTIONS WITH COMPOUNDS CONTAINING EIVE- KEMBERED MONOHETEROCYCLIC RINGS. Thiophene and Pyrrol
R eaction Type and Name o f Compound M ixture______R esult Heterocycles with Sulphur Atom.
1. Thiophene...... A. Colorless at first, then developing pinkish purple hut finally changing to light blue and dark blue precipitate.
B. Colorless, finally chang ing after a few days to light straw and then to brownish red. Heterocycles with Imino Groujp.
2, Pyrrol...... A, Gelatinous gray white precipitate.
B, Bright red or cherry red which increased in intensity , on standing.
n-Metnyl pyrrol.,., A. Chalky white precipitate on the addition of acids; bottom of tube contains lemon colored precipitate.
B. Lemon color changing to light ye-llow orange then to dark brick red. ,
4. n-Etbyl pyrrol..... A. White ohalhy turbulence; bottom of tube yielding gradually brown viscid precipitate; on standing the liquid becomes color less and the precipitate red brown.
B * Lemon yellow changing to orange and gradually turn ing deep orange or red. n-Buty 1 pyrro 1...... A. White chal] B. Brown yellow color, chang ing to yellow orange; gradually darkening and finally becoming deep cherry or brick red. n-Phenyl pyrrol...... ,A. Lemon yellow turbidity and finally a red brown viscous liquid separates at bottom.. B. Lemon color, changing to deep orange or blood orange. IfrO-Tolyl pyrrol...... A, Lemon tinted turbity, gradually changing to a clear lemon colored liquid and a dark brown precipitate. H-m-Tolyl pyrrol...... Clear liquid with a deposit of brick red substance at bottom of tube.. B. Brick tint immediately changing to lemon color while pouring in the antimony trichloride solution, changing to light yellow orange, orange to red brick, and fin ally to a wine color. Pro line...... A. Ho reaction; slight (pyrrolidine car- turbidity. bo:xylic acid) B. Ho reaction. Indole-3-n-prcpicnic A. acid Does not go into sol ution; straw colored (benzopyrrol 3-n- liquid, propicnic acid) B. Reddish brown color, changing to deep wine color and finally to brownish red. Skatole A. Yellow with tinge of brown, giving after a few days cherry red or carmine red. B. Light purple developing in ten minutes to a very deep royal purple at first. Tryptophane a. Ho change on long standing, deep carmine red dense liquid separates at bottom. B. Deep lemon yellow brown color. Carbasole A. Does not go into solution reaction mixture; on long standing the suspended particles are bluish or greenish blue. -*->• Light reddish brown color, changing’ to wine color and finally to purple. B ilirubin A. Deep orange colored liquid from dilute solutions that are colored yellow. On standing colorless liquid and red brown precipitate. After a few days the pre- cipitate turns to bluish green and the solution assumes a bluish tinge. B. Light orange liquid* On standing becomes a yellow crown. After a few days »0 lution becomes green. Bilifaerdin A., Greenish precipitate. B. On long standing dilute reaction mixtures become dark green. 35. 16. Hemoglobin.... . Insoluble in the reagents (contains four used; no reaction. pyrrol rings) B. Insoluble in the reagents used; no reaction. 17. Hematin...... On standing brown precipitate (contains four and clear colorless liquid. pyrrols) B. On standing gradually becomes dark brownish red. 18. Chlorophyl...... Ho reaction. (contains four pyrrol rings) B. On long standing: green solution turns yellow with a greenish tinge. 19. nicotine and nicotine.A. White precipitate formed salicylate (contains one B. White precipitate formed, pyrrol ring) 20* Strophantin...... ,A, Colorless liquid and flaky translucent solid turning to a creamy color and gradually getting darker "brown. After a few'days a greenish blue on saides and bottom of tube. B. Colorless at first; but gradually developes a brown ish amber and finally an olive green color. Table II AITTIMOHY TRICHLORIDE REACTION WITH VARIOUS OILS Reaction Name of Oil Mixture Re suit 1. Almond oil, sweet...... A. Straw color changing to pink to deep golden yellow, brown color developing over night with a yellowish precipitate. B. Almost colorless, with a faint straw color, changing to pink deep brown with a purple tinge developing overnight. 2* Castor oil,.,.,...... A, Cloudy, straw color with pink wine, yellow preci pitate overnight and golden yellow color simi lar to that of bilirubin. B. Almost colorless liquid with a pinkish tint; dark brown with a tint of pink developing overnight, 3. Cottonseed oil A. Brownish gold, developing into a deep orange and finally cherry red. -o. Straw color, developing into brown with a purple tinge and finally a purplish wine. 4. Linseed oil A. Cloudy gold to cloudy wine, a brownish wine color with grayish precipi tate developing overnight. B. Amber, grayish amber, pinkish brown; brownish wine developing overnight. A. Straw color developing into a cloudy light orange; a gray precipitate develop ing overnight and a light brown color. 37. B. Light amber, developing overnight a light tan and finally changing to a pinkish brown. 6, S3same oil,A, Golden yellow changing very soon to cherry red, to wine; mahogany brown developing overnight to a chocolate-colored pre cipitate. 3, Light amber, pinkish yellow, light brown, purplish brown, devel oping overnight to a purplish broxTO color. 7, Wheat-Germ oil,,,,,,,A. Bluish green; overnight cloudy brown. B. Bluish green; overnight cloudy brown. 8, Chaulmoogra oil,.,,,.A. Slight lemon-colored turbid mixture. B. Slightly lemon-colored liquid, turning to light amber. 9, Chaulmoogra oil,*.... A, Olive color, changing (Sargent and Co.) to yellow. 3. Lemonish olive color changing to biown with a greenish tint and finally turning reddish brown. 10, Chaulmoogric acid,.,,A, Lemon yellow cloudy turbidity finally turning a reddish brown. B. Clear Lemon yellow liquid, changing to brownish yellow and finally to brown. 38. 11. Ethyl Chaulmoograte....A, Yellowish olive color turning yellow. -i. Yellowish, olive color turning to greenish olive and finally to purplish red. 12 Chaulrnestrols Brown color changing to a mahogany tint. B. Brown color with greenish tint, changing to dark greenish olive and finally to purple. 39. Tattle III ANTIMONY TRICHLORIDE REACTIONS WITH COMPOUNDS CONTAINING P I Y E - M B E R E D MO ITO HE TERO CYCLIC RINGS. Eurfure,ne Reaction Type and Name of Compound Mixture _ Result Heterocycles with Oxygen Atom. 1, Eurfurane (Eurane) 'A, Green, greenish "blue, "blue and dark Drown pre cipitate ; liquid "becomes yellowish green overnight. B, Deep purplish wine color which changes in 20 minutes to purple, five minutes later to a purplish blue, and finally to blue. 2. Eurfurai alcohol A, Dirty lemon precipitate, purplish liquid changing to blue and finally to purple. B, Dark brown red changing to purplish, greenish blue, and blue. 3, Eurfuryl acetate ■ A., Brown liquid and dark brown precipitate with oily drops at bottom of tubes; azure blue precipitate on bottom, straw colored liquid above; on standing overnight, deep purple brown precipitate on sides of tube, finally be coming deep royal purple, B. Instantaneous dark blue color. 4. T e t rahydrofurfury1 alcohol .A. Deep greenish blue suspension, 3. Oil standing deep royal purple, 5. Tetrahydrofurfury acetate 1 -A. Light lemon rapidly changing to orange brown and more slowly to purplish brown, to reddish brown. 40 B. Light lemoni to darker lemon, green, amber, brownish green dark green. 6. Te trahydro fur fury1.., Straw, golden brown, green, butyrate purple with light brown tinge, light purple, darker purple. B, Lemon reddish lemon, pink red, dark reddish purple. 7. Tetrabydrofurfury1... Yellow straw, golden brown; lactate red brown overnight. B. Yellow straw, golden brown; brown overnight. 8. Furfural...... Greenish brown, blue, purplish blue. B. Greenish brown, blue, purplish blue. 9. Faro in...... r Light brown precipitate; liquid green. B. Clear green liquid. 10. Furil...... Tt ujx u vviij. oxj. ytilQy/ aHu &G.X1C&X 0 • precipitate, yellowish green liquid. B. Brownish yellow changing to yellowish green. 11. Furfury1 oxime...t., White precipitate, on standing thick brownish liquid forms on bottom. B. Lemon, cherry red, wine, reddish purple deep purple. 12. Furfural acetone,,,, Bark orange, turning deep red with brown tinge. BS Green, becoming darker on standing. 41 1-3« Furfural acrolein,... .A, Amber color and somewhat turbid; overnight yellowish green liquid and greenish brown on sides of tube, B. Lemon green, wine, dark red, blue specks on sides of tube, bluish green on standing blue liquid and blue precipitate. 14. Furfuryl acrolein oxime. A. Lemon green solution with yellowish brown precipitate on standing. B. Green yellow, changing to green, to red brown, to a very dark green, finally to purple. 15* Furfural acetophenone. • * A « Lemon changing to brown. B. Lemon color, finally developing into deep lemon green or olive green. 16. Furfural diacetate.... On standing light bluish purple. B. Lemon, reddish lemon, pink * red, dark reddish purple. 1?. Bydrofuramide.. . f,,,, . A Greenish muddy liquid, and Jlue green precipitate on bottom and sides of tube 3. Reddish brown, brown with greenish blue tinge, be coming deep bluish green on standing, , 18 - :l Furonitrile,, ,, furoamide Bo reaction turoyl chloride, __ furoic acid U J. wco C o X 0 il* 42 22 - 27 Methyl, ethyl, propyl,.,». .A, No reaction "butyl, amyl and. iso amyl esters of furoic B. No reaction acid 2 8 , Furfuracrylie acid...... ■•*A. Yellow liquid with slight greenish precipitate on standing, B. On standing very light lemon with "brown tinge. 2/, Furylacroly1 chloride..T,,, * ?n standing overnight brownish green liquid and greenish blue pre- dip it ate. B * On standing very light lemon with brown tinge, 30, Fury lacro lyl amide.... f ..A. Colorless liquid, Qlue precipitate on bottom and sides of tube. * B. On standing light >0 lemon, finally assuming a green tinge. i 43. BIBLIOGRAPHY Boo-kfe : McCollum and Simmond. The Hewer Knowledge of nutrition Fourth Edition, Macmillan Co* ’ McLester. nutrition and Diet in Health and Disease. Second Edition. W. B. Sounders Co. Mendel, l . 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