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ing for about 1 5 t o 2 0 min., a precipttate is formed, this is filtered off and the filtrate nearly neutralized with sodium hydroxide and tested for sulfates. Commercial hydrogen p2roxide contains sulfates and naturally cannot be used for t h a t test. I n the a b s a c e of sulfate in the original sample, and subszquent formation of sulfate by the action of the chlorine, a good indication is had of the presence of sulfonic acid.guaiacol. I n the presence of sulfate in the original, determination of sulfates before and after have t o be resorted to in order t o note increase due t o sulfonic acid. ( b ) Some of the filtrate is made alkaline and t o it is added drop by drop a c o l b solution of diazotized p-nitro aniline. (The diazo salt is prepared by dissolving 140 mg. p-nitro aniline in 8 cc. H z O and I or 2 cc. concentrated HC1, cooling and adding 7 5 mg. sodium nitrite dissolved in a few cc. of HzO.) I n the presence of guaiacol sulfonate the solution will be colored deep red. If substances are present which obscure the color, the solution is made acid with HC1, extracted in a separatory funnel with amyl alcohol, t h e lower aqueous layzr is tapped off, and the alcohol layer re-extracted with NaOH solution, when t h e azo dye will cblor the aqueous layer deep red. On acidification, t h e solution changes very sharply t o yellow, the aao dye behaving like a n indicator in t h a t respect. If guaiacol or other phenols are present in t h e original solution, they should be removed beforehand by steam distillation or extraction with organic solvent as t h e case may require. The guaiacol sulfonic acid will remain behind and can subsequently be tested for as indicated above. (c) I n the absence of much organic material or possible removal of the same the following procedure may be used. To a concentrated solution of t h e sample ( a few cc.) in a hard glass test tube are added about 5 cc. syrupy phosphoric acid containing a little NaC1, and the mixture is boiled or preferably distilled over. Distillate, which may be less than I cc., may then be tested for the presence of guaiacol and pyrocatechol, as both are generally formed. A few drops of a very dilute ferric chloride solution will give with the distillate (in the absence of much HCI) a green coloration which changes t o yellowish and on addition of ammonia changes t o violet-blue. The neutral, or better, ammoniacal solution causes marked reduction of silver nitrate. QUAKTITATIVE DETERMINATION-(Q) I n the absence of much contaminating matsrial, a known amount of the sample, which should not contain much more than 2 0 0 mg. of potassium guaiacol sulfonate, is diluted somewhat with water in a 1 5 0 t o 2 0 0 cc. Erlenmeyer flask and I O t o 2 0 cc. concentrated HC1 are added and then a few cc. liquid bromine. The solution is boiled gently and bromine added several times. It is then evaporated down t o a small volume on the steam bath, using air blast, I O cc. concentrated " 0 3 added, and some more bromine and boiled. This is done to convert the last traces of sulfonic acid guaiacol. The process is repeated twice and the whole evaporated t o
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dryness on the steam bath. It is then diluted with water and sulfates determined in the usual manner. ( b ) I n the przsence of much organic material, which is most often the case, a weighed quantity of the sample in a 1 5 0 cc. Erlenmeyer flask is treated repreferably fuming peatedly with concentrated " 0 3 , "03, heating gently a t first until nearly all of the organic material has been oxidized. The samz process is then repeated, using bromine and concentrated "03, several times. The whole solution is then evaporated t o dryness on the steam bath and the sulfates determined as above.
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Factor for conversion of Bas04 to SO, = 0.3428; factor for conversion of Bas04 t o potassium guaiacol sulfonate 1.0376. BUREAUOF CHEMISTRY OF AGRICULTURE DEPARTMENT WASHINGTON, D . C.
THE OCCURRENCE OF CAROTIN IN OILS AND VEGETABLES By AUGUSTUSH. GILL Received April 5, 1918
I n a previous paper' i t was shown t h a t the peculiar bluish reaction of palm oil was due t o carotin, and t h a t palm oil could not be detected in oleomargarine by this test, because the animal fats also contained carotin. A t t h a t time, the subject was being further studied, as t o what other fats and oils might contain it. As the carotin is undoubtedly dissolved out from the seeds by the oils they contain, and as it is present in them in extremely small amounts, it was deemed best t o extract it from the seeds, rather t h a n from the oils 8 themselves. The substances investigated were: ( a ) Seeds: Yellow corn, flax, mustard, black sesame, rape, and white sunflower. ( b ) Yellow colored vegetables or products: Carrots, squash, turnip, orange peel, safflower, cottonseed meal, turmeric, and neat's-foot and linseed oils. The procedure used in isolating carotin from various oils and vegetables consisted in extracting the finely divided dried vegetable with carbon bisulfide a t a return flow condenser, .evaporating off the solvent, and saponifying the residue or oil with alcoholic sodium or potassium hydroxide, leaving a slight excess of alkali; t o ensure the absence of free oil, the alcohol was evaporated, keeping the temperature below 70' C. as much as possible. and the residual soap was then dissolved in water. The solution was shaken out with carbon bisulfide, which, in the presence of carotin, assumed a yellow or orange color, depending upon the amount of carbon bisulfide used. The carbon bisulfide was evaporated off, and the residue again treated with a n excess of sodium hydroxide t o ensure the complete removal of, any oil which might possibly have escaped saponification. The resulting soap solution was extracted with carbon bisulfide, as before, with similar results. Those vegetables t h a t were free from oils, such as carrots, were extractea directly with carbon bisulfide, after first drying the finely divided sample a t a low temperature. 1
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The carbon bisulfide solution, after t h e extraction from sunflower seeds, was colorless, showing a t once the absence of carotin. Turnip gave a pink solution, safflower, neat's-foot oil, and cottonseed meal a yellow one, while turmeric gave a yellow solution with a greenish fluorescence. All the other solutions were blood-orange in color. T o prove the presence of carotin in these different substances, advantage was taken of various chemical and physical properties which carotin exhibits, being the same tests as employed by Palmer and Ecklesl in their work. These properties of carotin are: ( I ) It absorbs bromine. ( 2 ) I t is not extracted from its petroleum ether solution by 80 t o 90 per cent alcohol. (3) It gives a deep red or blood-orange color in carbon bisulfide solution. (4) It is not adsorbed b y precipitated chalk. ( 5 ) I t gives characteristic absorption bands when tested in the spectroscope. (6) I t gives a blue color with the Crampton-Simons test. Test 6 was carried out by adding the carbon bisulfide solution of the carotin t o cottonseed oil-proved free from carotin-evaporating the solvent and applying the Crampton-Simons test to the oily solution in the usual way. The results of the tests, except the spectral analysis, are tabulated in Table I ; the analysis follows in Tables I1 and 111. TABLEI-SHOWINGTHE TESTSFOR CAROTININ EXTRACTS EROM VARIOUS SUBSTANCES
(6) CramD(4) ton: (2) Alcohol (3) CaC03 Simons SEEDOK (1) ExtracColor of Ad- Color VEGETABLE Bromine tion CS2 S o h . sorpn. Test Blood-Orange Large Blue Yellow Corn.. ... Absorbs None Blood-Orange None Blue Flaxseed.. ...... Absorbs None Blood-Orange Large Blue Mustard.. Absorbs None Blood-Orange None Blue Black Sesame.. Absorbs None Rapeseed'.. Absorbs Extract6:d Blood-Orange Some None .... . . . . . . Colorless White Sunflower. None Blood-Orange &me Blue Squash., ........ Absorbs None Pink Some Brown Turnip'.. . . . . . . . Absorbs Trace Blood-Orange Large Blue Orange Peel. . . . . Absorbs None Xone Yellow Safflower'. . . . . . . Absorbs Yellow . . . . None Cottonseed Meal' Turmeric'. . . . . . . . . . . . Yellow Large None . . . . . . Yellow Neat's-foot Oil.. . ? Blood-Orange Node Blue Linseed Oil.. Absorbs None 1 Shows no evidence of carotin.
...... .. .....
....
....
....
....
....
Relative Amts. of Carotin Small Small Small Small None None Some iVone Small hTone None None Trace Trace
The fifth test, the spectroscopic test, is probably the most reliable test for carotin. It depends upon t h e fact t h a t carotin cuts off the violet end of the spectrum sharply, as if a card had been placed between the instrument a n d the test tube containing the solution. The instrument used in this investigation w,as a Kruss single-prism spectroscope and the solutions were tested according t o the method of Formanek.2 Carotin from carrots was used as a standard. The carbon bisulfide solution from this material was run through CaC03 and then extracted with 80 t o 90 per cent alcohol in order t o remove other pigments, although if xanthophylls were present they probably would not interfere with t h e readings, as the bands of this substance are shifted furt'her toward the blue end of the spectrum from the corresponding bands of carotin. 1
J . B i d . Chem., 17 (1914), 190-249. Nachweis kunstlicher organischer Farbstoffe,
* Spectral-analytischer
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Phytosterol which might have been extracted along with carotin gives no absorption spectrum.' The spectroscope was equipped with a n arbitrary scale. By setting this scale at a constant point, before taking a n y measurements, i t was possible t o standardize the absorption bands from carotin so t h a t the bands of a n unknown pigment could be compared with them. The solvents used were carbon bisulfide and ethyl alcohol. The colors of all unknown solutions were brought to the same tint as the solution b y means of the Lovibond tintometer. This was the only means available of standardizing the strength of the solutions, which is an important factor. This was not so pronounced in the case of the carbon bisulfide solutions, being mostly a question of clearness of reading, but in the case of the alcoholic solutions a marked effect was noted. The more the solutions were diluted the greater was the shifting of the absorption bands toward the blue end of the spectrum. S o , in order to get comparative results, it was necessary t o have all solutions as nearly as possible a t the same concentration. Palmer and Eckles, as well as other investigators, noted and read three absorption bands for carotin. I n this investigation, a n attempt was made t o read only the extremity of one band, the end of the band toward the red, between the E and F lines. This line was particularly clear and distinct. The readings obtained are given in the following table: TABLE 11-CAROTIN ABSORPTIONSPECTRA-CSa SOLVENT 14.02 13.64 Rape Seed.. Carrots.. Palm Oil.. 13.64 Turnipl.. 13.09, 14.89, 16 10 13.46 Grass . . . . . . 13.88 Corn (ydlow). 15.29 Squash 13.65 Safflower'. 13.87 Turmeric' 15.10 Flax Seed.. 13.84 Orange Peel.. 13.51 Linseed Oil.. 13.66 Sesame Oil. ... 13 -60 Mustard Seed.. 1 Shows no evidence of carotin.
..
.............. ............. .......... ................ ............ .......... .........
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...
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..
Neat's-foot oil and cottonseed meal were too weak t o give readings, although both were colored yellow. Neat's-foot oil responded t o none of the other tests, the color being so dilute. The amount of cottonseed meal obtainable was so small t h a t the failure t o obtain a reading of this substance cannot be taken seriously. Turnip was interesting i n the fact t h a t it gave three distinct absorption bands, none of which corresponded t o the carotin reading. Black sesame seed gave considerable trouble as the carbon bisulfide solution dissolved resinous material which, interfered with t h e readings. By evaporating off the carbon bisulfide and treating with alcohol the resins precipitated, and on filtering i t was possible t o get readings both i n carbon bisulfide and alcohol. Safflower, rape seed, and turmeric gave readings considerably further toward the blue end of the spectrum t h a n carotin. All readings given in the tables are mean readings of a series of nine observations. TABLE111-CAROTIN ABSORPTIONSPECTRA-C2HbOH SOLVENT Carrots.. . . . . . . . . . . . Palm Corn Squas . Flax Seed., ......... Orange Peel. . . . . . . . .
15.38 15.38 15.32 15.38 15.46 15.36
Sesame Seed. . . . Rape,S Turnip Grass. Mustard Seed., .
15.24 15.99 14.80 15.47 15.49
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Neat's-foot oil, cottonseed meal, and linseed oil were too weak t o give readings, although a reading could be obtained with the carbon bisulfide solution 1
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from linseed oil where the absorption was more distinct. No reading was obtained with safflower, as on the addition of alcohol t o the coloring matter, a white precipitate formed and the solution which remained after filtering was too weak t o read. It is quite evident from this, its behavior towards bromine and its high reading in carbon bisulfide solution, t h a t the coloring matter in this substance is not carotin. Rape seed and turnip gave high and low readings, respectively, which is not surprising in view of their behavior in the previous tests.
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important in determining the length of time necessary for complete removal of the ammonia, as other experimenters have shown1 and as has been our experience. The titration method is preferred wherever possible, but in case a large number of samples a r e . t o be run in a short time, the colorimetric method can be substituted. I n the latter case the amounts of a m monia t o be determined are so small t h a t great care must be exercised t o keep the apparatus free from ammonium salts.
S U M MARY
From this work i t would seem t h a t carotin is contained in corn, squash, orange peel, flaxseed, mustard seed, and black sesame seed. Palmer and Eckles showed its presence in butter fat and beef tallow, Gill in palm oil, and it has long been known t o be in carrots and grass. I t does not seem t o be present in rape seed, white sunflower, turnip, safflower, cottonseed, or turmeric. I n conclusion the writer wishes here to acknowledge his indebtedness t o Messrs. James F. Maguire, Jr., and In-shing Wan, by whom the experimental work was .performed. MASSACHUSETTS INSTITUTE O F TECHNOLbGY CAMBRIDGE, MASS.
DETERMINATION OF LOOSELY BOUND NITROGEN A S AMMONIA IN EGGSL By N. HENDRICKSON AND G. C. SWAN Received February 18, 1918
The chemical methods for the detection of incipient decomposition in foods must be selected in accordance with the character of the substance under examination. As is well known, ammonia is one of the decomposition products of proteins, and the determination of loosely bound nitrogen as ammonia has proved t o be one of t h e best chemical methods in general laboratory use for the grading of eggs.2sS The principle is t h a t of F ~ l i n namely, ,~ of aerating an alkaline fluid until all the loosely bound nitrogen is driven off as ammonia. This is caught in a known amount of standard acid for titration, or merely in a n excess of acid for a colorimetric determination. The size of sample and the time in which i t must be run are the determining factors in the selection of the method. The apparatus used for this purpose has been changed from time t o time as improvements were devised until it is now most satisfactory and may be of interest t o those who have t o deal with the determination of loosely bound nitrogen in biological material. Of the two optional methods of aeration (suction or blowing), the latter is preferable, for i t is easier t o keep the conditions of aeration constant, and this is Published by permission of the Secretary of Agriculture. M. E. Pennington and A. D. Greenlee, “An Application of the Folin Method t o the Determination of the Ammoniacal Nitrogen in Meat,” J . Am. Chem. SOL.,32 (1911), 561. a H . W. Houghton and F. C. Weber, “Methods Adapted for t h e Determination of Decomposition in Eggs and in Other Protein Food Products,” Biochem. Bull., 1914, 447. 4 Z. phys. Chem., 37 (1902), 161. I
2
FIG.I-APPARATUS BOR TITRATION METHOD A-Pipefrom air pump. B-Wash bottle containing 35 per cent sulfuric acid. C-Pipe t o which aeration cylinders are connected. D-Aeration cylinder (14S/a in. high X l’/z in. inside diameter) containing sample. The glass tube for aeration extends t o within 1/z in. of t h e bottom apd is open a t the end. E-Trap. F-Flask in which ammonia is caught, containing 10 cc. of N/50 sulfuric acid plus 2 drops of 0.2 per cent methyl red (dissolved in alcohol), and 75 cc. of water. G-Dispersion tube made according to method of Folin and Farmer [ J . B i d . Chem., 11 (1912), 4931 to insure complete absorption. NOTE-It has been found by test t h a t the ammonia is always completely absorbed in the one flask b y this method. H-Water gauge for keeping air pressure constant and thus ensuring the passage of an equal volume of air through the cylinders in a given time. DIRECTIONS F O R T I T R A T I O N METHOD
Mix samples well (preferably with one of the electric mixers in common use a t soda fountains) a n d weigh out 2 5 g. Pour the bulk of the egg into t h e aeration cylinder D and transfer the remainder b y means of four 2 5 cc. portions of distilled water, stirring each time with a rubber-tipped glass rod t o remove the egg adhering t o the sides of t h e weighipg vessel. Add 7 5 cc. of alcohol, mix well, and let stand 1 5 min. Now add about one gram of.sodium fluoride, 2 cc. of 50 per cent potassium carbonate and I cc. of kerosene. Connect the apparatus, blow air through until no more ammonia comes over, and titrate solu1 P. A. Kober and S. S. Graves, “Quantitative Ammonia Distillation by Aeration, for Kjeldahl, Urea, and Other Nitrogen Estimations,” J . A m . Chem. Soc., 36 (1913), 1594.
