INDUSTRIAL AND ENGINEERING CHEMISTRY
362
U.S.P. standard of reference oil a t multiple levels and eytimating the biological vitamin A potency from log-dose interpolation curves. ACKNOWLEDGMENT
The writers wish to express their appreciation to The Best Foods, Inc., for permission to publish this work; also to E. D. Seiter and G. Rowland of this laboratory and Miss C. Nott, formerly of this laboratory, for valuable technical assiqtance. LITERATURE CITED
Official Agr. Chem.. Official and Tentative Methods of Analysis,4th ed., p. 412, Section 22, 1935.
(1) .Issoc.
Vol. 16, No. 6
(2) Barthen, C. L., and Leonard, C. S.. J . A m . Pharm. Assoc.. 26, 515-24 (1939). (3) Dann, a'.J., and Evelyn, K. A., B i o c h e n ~J . , 32, 1008 (1938). (4) Denlarest, Beaumont, 2. Vitaminforach., 9, 20-1 (1939). ( 5 ) Drurnmond, J. C., and Morton, R. h.,Biochm. J . . 23, 786 (1929). (6) Edisbury, J. R., Analyst, 65, 484-93 (1940). (7) Federal 6, 2761 (1941). (8) Morton, R . A , , and Heilbron, I. M., Bioehem. J . , 22, 987 (1928). (9) Neal, R. H., Haurand, C. H., and Luokmann, F. H., TND. ENG. CHEM.,ANAL.ED., 13, 150 (1941). (10) Oser, B. L., Federation Proc., 1, 343 (1942). (11) Vahlteich, H. W., and Neal, R. H., Food Industries, 16, 90 (March, 1944). (12) Wilkie J. B., J . Assoc. Oficial Ag7. Chen., 20, 208-12 (19371.
Qualitative Differentiation of the Methylcarbinols and Methyl Ketones JONAS KAMLET, Chemical Research Division, Miles Laboratories, Inc., N e w York, N. Y. Compounds yielding positive Lieben iodoform reactions may be either methyl ketones or methylcarbinols. O n reaction with a reagent containing o-nitrobenzaldehyde in alkaline solution, only the methyl ketones will form indigo and may thus be distinguished readily from the methylcarbinols.
IN
T H E course of a recent inveitigatioii, a simple and rapid method was needed for the qualitative differentiation of methyl ketones and methylcarhinols. The valuable Lieben iodoform reaction ( 7 ) has long been used to detect the grouping CHZCO- when joined to a hydrogen atom, or to a carbon atom which does not carry highly activated hydrogen atoms, or groups capable of exerting excessively high steric hindrance. However, the corresponding methylcarhinols (CHZCHOH-) will also give the Lieben reaction, owing undoubtedly to their prior oxidation by the alkaline hypoiodite reagent to the methyl ketones. Thus, while methyl ketones and methylrarhinols are readily identifiable by the Lieben reaction, there is 110 simple means of differentiating the two groups. The von Baeyer-Drewsen synthesis of indigo (1, 6), a t one time practiced on a commercial scale, is based on a reaction which lends itself readily t o such a differential identification technique. o-Nitrobenzaldehyde was found by these workers to condense with acetone, acetaldehyde, or pyruvic acid in the presence of alkali, to form indigotin. Feigl, Zappert, and Vasquez ( 3 ) applied this reaction t o six additional methyl ketones (methyl ethyl ketone, methyl heptenone, acetophenone, acetylacetone, diacetyl, and ethyl acetoacetate) and suggested its applicability in detecting other methyl ketones. Tananescu and his co-workers (8, 9) have suggested that' the reaction probably proceeds as follows: The methyl ketone first condenses with the o-nitrobensaldehyde to form the corresponding 4-2-nitrophenyl-8-hydroxyethyl ketone ( I ) :
A40* 0-CHO
+
--t
U C H O H . CH2.CO. R (I)
-NO2 -CHOH
,
)C-COR
CO. CHi. CO. R
(111)
to form Compound 111, two moles of which split off R. COOH by hydrolysis and form one mole of indigotin:
As the result of the author's invedtigation, it was found that a simple qualitative differentiation of rnethylcarbinols and methyl'ketones could be based on the Lieben iodoform reaction in conjunction with the von Baeyer-Drewsen indigo reaction, both being specifically modified for use as an analytical technique. A negative iodoform reaction rules out both methylcarbinols and methyl ketones; a positive iodoform reaction and a negative indigo reaction indicate a methylcarbinol; a positive iodoform reaction and a positive indigo reaction indicate a methyl ketone. This differentiation is specific. When a technical sample of a methylcarbinol was found to give a faint positive indigo reaction, this could invariably be traced to the presence of methyl ketone as an impurity. Scrupulous purification of the methylcarbinol (e.g., via a characteristic crystalline derivative) resulted in a product which reacted entirely in the expected manner.
A-SO,
CHZCOR
Compound I undergoes an intramolecular oxidation-reduction to form the corresponding p-2-nitrosophenyl-B-ketoethyl ketone (11) :
2
Compound I1 then cyclizes in the presence of the alkali:
CHz. CO . R
Co, CH2.Co,
('I)
I O D O F O R M REACTION
The procedure used for the iodoform reaction was that deand Tullock (5). scribed by hson REAGENTS REQUIRED. Dioxane, 10% Sodium . hYdfo$de solution, and iodine reagent: 200 grams of potassium iodide and 100 grams of iodine dissolved in 800 cc. of distilled water. About 100 mg. of the compound being tested PROCE~CRE. are placed in a 150 x 16 mm. test tube, 5 cc. of dioxane are added, and the sample is dissolved with shaking. First 1 cc. of 10% sodium hydroxide solution and then the iodine reagent
ANALYTICAL EDITION
June, 1944
are added dropwise with shaking until a slight excess of iodine causes a definite dark color which does not disappear on standing. The test tube is now placed in a water bath maintained a t 60" C., and the dropwise addition of the iodine reagent is continued until the definite dark color persists as before; but the warming a t 60" C. should not last over 2 minutes. The excess of iodine is now removed with a few drops of 10% sodium hydroxide solution, and the test tube is filled with cold water, allowed to stand for 15 minutes, and filtered. The characteristic odor of iodoform is readily distinguishable. -4s a confirmation, the crystals which are collected on the filter paper are dried a t 100" C. for one hour and identified by their melting point. Iodoform melts a t 114-121" C. INDIGO REACTION
Since o-iiitrobenzaldehyde is not a t present obtainable from any domestic source, it may be prepared by nitrating benzaldehyde by the method described by Friedlander and Henriques (,$) and separating the o-nitrobenzaldehyde from the mixture of isomers thus obtitincd by the method described by Erhart ( 2 ) .
PREPARATIOX OF REAGEST. One hundred grams of finely powdered sodium nitrate are added in small portions to 1 liter of 66' BB. sulfuric acid, the temperature of the mixture being kept below 20" C. by external cooling. C.P. benzaldehyde (106 grams) is now added to this nitrating mixture in small portions, the temperature being kept below 30-35" C. After all t'he benzaldehyde has been added, the reaction mixture is cautiously poured into a mixture of 1.5 liters of water and 1.5 kg. of ice. The oily layer of mixed o- and nt-nitrobenzaldehyde is separated by decantation and mixed without heating with 500 cc. of 44% sodium bisulfite solution (sp. gr. 1.37). Six hundred cubic centimeters of water heated to 45" C. are now added t o dissolve the magma of rrystals and the resultant solution is chilled in a refrigerator a t 0" to .5' C. for 48 hours. The copious precipitate of m-nitrobenzaldchyde sodium bisulfite which forms is filtered off. To the filtrate is added a saturated sodium carbonate solution to strong alkaline reaction, and the mixture is cooled and extracted with three successive 500-cc. portions of ether. The ether extracts lire dried overnight over anhydrous calcium chloride, filtered, and the solvent evaporated off. The residual oil (20 to 25 grams) solidifies on cooling and consists of o-nitrobenzaldehyde mixed with smaller amounts of m-nitrobenzaldehyde. This product is sufficiently pure for u e in the technique described below. The o-nitrobenzaldehyde reagent is prepared by dissolving 5.0 grams of the crystals or the oily product in 100 cc. of 95% ethanol. This reagent solution should be prepared fresh from the undissolved compound a t least once a month, and stored in an amber-colored glass-stoppered bottle. PROCEDURE. About 100 mg. of the compound being tested are dissolved or suspended with vigorous agitation in 5.0 cc. of the o-nitrobenzaldehyde reagent, and 1.0 cc. of 1Oyo sodium hydroxide solution is added dropwise. An immediate darkening of the solution will occur (partly due to the formation of dismutatioii products of o-nitrobenzaldehyde). After 60 seconds, a few drops of the solution are placed on a piece of filter paper and allowed to be absorbed. The filter paper is then washed under a stream of tap water and examined. If the brown stain has washed away, the test is negative-Le., the sample was a methylcarbinol. A positive test-Le., a methyl ketone-is evidenced by a distinct and unmistakable deposition of indigoblue dyestuff within the fibers of the filter paper. The "spot" of indigo blue is usually rimmed by a characteristic blue-green ring, which cannot be removed, even by prolonged washing. By comparing the washable brown stains obtained with known methylcarbinols and the permanent blue dye obtained with known methyl ketones, the chemist can soon become highly proficient in distinguishing the two. This method will detect as little as 1 mg. of methyl ketone in a 100-mg. sample of methylcarbinol. I t has been tried with a number of methylcarbinols and methyl ketones. The results obtained may be summarized by classifying the compound,. a.: follo\vs: CLASSI. Compounds which give a positive indigo reaction but fail to give a positive iodoform reaction: KO compounds were found which fall in this class rategorically. Sterically hindered vompounds like pinacolone (1) give the indigo reaction more readily than they form iodoform, but these belong properly in ('lass 111.
363
CLASS11. Compounds which give a negative indigo reaction and a positive iodoform reaction: ethanol (2), isopropanol (3), methylethylcarbinol (4), methyl-n-propylcarbinol ( 5 ) , methylisopropylcarbinol (6), methyl-n-butylcarbinol (7), methylisobutylcarbinol (8), methyl-n-amylcarbinol (9), methylisoamylcarbinol (lo), methyl-n-hexylcarbinol (1l), methylisohexylcarbinol (12), butandiol - 2,3 (13), benzylmethylcarbinol (14), 1 - phenylpropandiol-2,3 (15), lactic acid (If?), methyl lactate (17), and ethyl lactate (18). Thi.5 class, therefore, comprises only the methylcarbinols--i.a., the series of compounds characterized by the grouping CHs .CHOH- joined to a hydrogen atom or to a carbon atom which does not carry groups that exert an excessively great steric hindrance. CLASS111. Compounds which give a positive indigo reaction arid a positive iodoform reaction: acetaldehyde (19), acetone (20), methyl ethyl ketone (2l), methyl n-propyl ketone (22), methyl isopropyl ketone (23), methyl n-butyl ketone (24), methyl isobutyl ketone (25), methyl n-amyl ketone (26), methyl isoamyl ketone (27), methyl n-hexyl ketone (28), methyl isohexyl ketone (29) acetoin (30), diacetyl (31), phenylacetone (32), 1-phenyl ropanol-1, one-2 (33), pyruvic acid (34), methyl pyruvate (35f ethyl pyruvate (36), methyl cyclohexylketone (37), benzyl acetone (38), acetophenone (39), methyl p-tolyl ketone (40), p-chloroacetophenone (41), p-bromoacetophenone (42), methyl p-anisyl ketone (43), 2,4-dimethoxyacetophenone (44), o-hydroxyacetophenone (45), m-hydroxyacetophenone (46), p-hydroxyacetophenone (47), 3-methoxy-4-hydroxyacetophenone (48), o-nitroacetophenone (49), o-aminoaceto henone (50), 2-aceto-1-naphthoxyacetic acid (51),mesityl oxide f52), benzalacetone (53), salicylalacetone (54), vanillalacetone ( 5 5 ) , p-hydroxybenzalacetone (56), furfuralacetone (57), acetylacetone (58), acetonylacetone (59), benzoylacetone (60), levulinic acid (61), methyl levulinate (62), ethyl levulinate (63), ethyl acetoacetate (64), a-acetyl-~-butyrolactone (65), pentanol-1-one-4 (66), 5diethylaminopentanone-2 (67), methylheptenone (68), pionone (69), n-ionone (70), methyl vinyl ketone (71). This class, therefore, comprises only the methyl ketones-Le., the series of compounds characterized by the grouping CHpCOjoined to a hydrogen atom or to a carbon atom which does not carry groups that exert an excessively great hindrance. Interfering compounds in the iodoform reaction are usually (u) primary amines which are oxidized to methyl ketones by the
alkalfhe hypoiodite reagent (such as a-aminoisobutyric arid, a-phenylethylamine, 2-aminoheptane, 2-aminohexane, isopropyl amine, etc.), ( b ) esters of ethanol and secondary alcohols which are hydrolyzed by the alkaline reagent to compounds of Class I1 (such as ethyl acetate, ethyl propionate, diethylphthalate, diethyladipate, sec-butyl acetate, sec-amyl acetate, isopropglacetate, etc.), and (c) oximes which are hydrolyzed to methyl ketones (such as acetoxime, acetophenone oxime, acetone oxime, methyl ethyl ket oxime). The only compounds which have been found to interiere in the indigo reaction are those which are readily hydrolyzed by the alkaline reagent t o compounds of Class 111. These are ( a ) halogen derivatives (such as ethylidene chloride, 2,2dibromopropane, 2,24ibromobutane, etc.), ( b ) acetals (such> as acetaldehyde alcoholate, acetal), ( c ) oximes (such as acetoxime, acetophenone oxime, acetone oxime, methyl ethyl ketoxime, etc.), and ( d ) biiulfite addition products (such as acetaldehyde, aodium bisulfite, acetone sodium bisulfite, etc.). LITERATURE CITED (1)
(2) (3)
(4) (5)
Baeyer, A. yon, and Drewsen, V. B., (to Badische Anilin u. Sodfabrik), German Patent 19,768 (Feb. 24, 1882); U. 5. Patents 257,812-34-5 (May 9,1882). Erhart, Carl, German Patent 116,124 (Sept. 10, 1899). Feigl, F., Zappert, R.,and Vasquez, S., Mikrochemie, 17, 169 (1935). Friedlander, P., and Henriques. R . , Ber., 14, 2801-5 (1881). Fuson. R . C., and Tullock, C. W., J. Am. Chem. Soc., 56, 1638-40 (1934).
(6) Heller, G., Lauth, H., and Buchwaldt,, A , , BeT., 55, 483-9 (1922). (7) Lieben, Adolph, Ann., Suppl. 7,218 (1870). (8) Tananescu, I., and Baciu, A . , Bull. 8oc. chim., V Series, 4, 1673-83 (1937). (9) Tananescu, I., and Georgescu, A4.,I b i d . , I V Series, 51, 23440. (1932).
