An Iodoform Microtest for the Higher Alcohols and Ketones FRAXK H. ST0DOL.I Columbia University, New York, N. Y .
I
then covered with a thin layer of powdered potassium hydroxide. This mixture is made slightly moist by the addition of a small drop of water. Tube B is then heated on an asbestos-covered hot plate until bubbles appear. Without removing the tube from the hot plate, several drops of solution from tube A are at once dropped on the resorcinol-potassium hydroxide mixture. One minute of heating should give only a pale green color. The material under investigation is tested as follows: Because some compounds discolor on warming with alkali or give a red color with heated potassium hydroxide-resorcinol, it is necessary to carry out a blank on 1 mg. of the compound. The material is placed in a small test tube and warmed at 60' for several minutes with 5 drops of solution I. A slight discoloration does not interfere with the test; a deep color shows the compound to be unsuitable for investigation. Several drops of this solution on addition to heated potassium hydroxide-resorcinol should cause no change in the pale green color of the potassium hydroxide-resorcinol. The compounds used in this study met these requirements. TESTOX KETOKES.Five drops of solution I are pipetted into tube A, containing 1 mg. of the ketone to be tested. The tube is held in a water bath until the compound is in solution, and solution I1 is added dropwise until the brown color appears. The tube is placed in a water bath a t 60" for 3 to 4 minutes, solution I1 being added from time to time t o restore the brown color. Sufficient powdered potassium hydroxide is finally added to discharge the brown color when the tube is rotated on its side. If considerable iodoform is present, a yellow color may remain. In tube B are placed a few milligrams of resorcinol, which is covered by a thin layer of powdered otassium hydroxide. A small drop of water is added to make tRe mixture slightly moist. Tube B is then placed on an asbestos-covered hot plate and when bubbles begin to appear, several drops of the solution in tube A are added. If the compound tested is a methyl ketone, a deep red color is observed in less than 15 seconds. TESTON ALCOHOLS.Eight drops of solution I are pipetted into tube A containing 3 mg. of the alcohol to be tested, The solution is warmed until the compound has dissolved and an excess of solution I1 is then added. The tube is kept in a water bath at 60" for 3 minutes. By this time, the brown color will have faded, so more of solution I1 is added to restore it. The solution is heated for another 2 minutes at 60" C. One drop of solution I is added and then an excess of solution 11. After another 3 minutes of heating at 60" C excess powdered potassium hydroxide is added. This solution% added dropwise to moistened potassium hydroxide-resorcinol on the hot plate, If the alcohol is capable of being oxidized to a methyl ketone, a deep red color results in a very short time.
N THE study of the lipides of the tubercle bacillus, higher alcohols such as d-eicosanol-2 (1, 7) and phthiocerol (9) (C3J37403) have been among the many compounds isolated. T o prove the presence or absence of the -CH(OH)CH3 grouping in these alcohols, it was hoped that the widely used iodoform test might be applied. Unfortunately, the methods heretofore described for carrying out this test were not applicable to these compounds. Scarcity of material or insolubility in water prevented the use of procedures based on the isolation of iodoform for a melting point determination or microscopic study (2-4). These difficulties have now been circumvented by employing the sensitive color test described b y Lustgarten ( 5 ) , in which iodoform gives a deep red color when heated with resorcinol and potassium hydroxide. As a solvent for the higher alcohols and ketones, methanol. has been found satisfactory. By this method it is possible to detect 1 mg. of eicosanone-2 or 3 mg. of the corresponding alcohol. The fact that the alcohols can be used directly in this test is very helpful in those cases where the amount of material is insufficient to allow oxidation to the ketone. The use of the alcohol is also of advantage in work with compounds such as phthiocerol, which cannot be converted to ketones without disrupting the molecule. The test was positive with 1-mg. samples of p-phenylacetophenone, methyl octadecyl ketone (eicosanone-2), methyl pentadecyl ketone, o-benzoxyphenyl acetone, methyl$naphthyl ketone, and benzoyl acetone and with 3-mg. samples of d-eicosanol-2 and 5,5-dimethyl-2,4-hexanediol. It was negative with citric acid, hydantoin, mannitol, benzophenone, biphenylene oxide, tricarballylic acid, benzyl sulfonamide, and acetyldurene.
Apparatus The formation of iodoform is carried out in a Pyrex test tube, A, 2.5 cm. in length. This tube is made by fire-polishing the open end (7 mm. in inside diameter) until this dimension is reduced to 4 to 5 mm. The reaction between the iodoform and resorcinol-potassium hydroxide takes place in a Pyrex test tube, B, 4 cm. long and 7 mm. in inside diameter. A medicine dropper having an opening of 1.5 mm. is used to transfer the contents of tube A to tube B. If the opening is smaller than this, some difficulty may be encountered from solidification of the solution during transfer.
Limitations of the Method
-
Some halogen compounds [CC13CH(OH)2, CC1,CHOH. NHCHO, CC13COOH, and CeH2. Cl,OH] have been observed b y Ware (11) to give red colors when heated with resorcinol and OH
Reagents SOLUTIOK I. Three grams of potassium hydroxide are dissolved in 2 cc. of water and 18.7 cc. (15 grams) of pure methanol are added. This solution is stored in a glass-stoppered flask equipped with a medicine dropper. SOLUTION 11. Pure methanol is saturated with iodine with gentle warming. It is kept in the same manner as the alcoholic potassium hydroxide solution.
potassium hydroxide. I n this laboratory CCl,. CH-COOH was found to behave similarly; hence care must be taken in the interpretation of results with halogen compounds. The sterically hindered ketone, acetyldurene, was found to give a negative test. This is to be expected on the basis of the behavior of acetylmesitylene observed b y Fuson and Tullock (4). For other anomalous haloform reactions, see Suknevich and Chilingaryan (IO), Slotta and Neisser (8), and Paggi (6).
The Color Test BLAKKTEST. The reagents are tested as follows: Five drops of solution I are pipetted into tube A, and to this solution solution I1 is added dropwise until a brown color persists. The tube is held for 5 minutes in a water bath a t 60" C. and then cooled to room tem erature. Sufficient powdered potassium hydroxide is added to Jscharge the brown color. This is accomplished by rotating the tube on its side, to make sure that the entire inner surface is moistened. In tube B are placed a few milligrams of resorcinol, which is
Literature Cited (1) Anderson, R. J., Crowder, J. A., Newman, M. S., and Stodola, F. H., J . Biol. Chem., 113, 637 (1936). (2) Foulke, D. G., and Schneider, Frank, IND.ENG.CHEM.,ANAL. E D . , 12, 554-6 (1940).
72
January 15, 1943
ANALYTICAL EDITION
(3) Fuson, R. C., and Bull, B. A., Chem. Revs., 15, 275-309 (1934). (4) Fuson, R. C., and Tullock, C. W., J . Am. Chem. SOC.,56, 163840 (1934). (5) Lustgarten, S., hfonatsh., 3, 717 (1882). (6) Paggi, Raoul, Gazz. chim. ital., 70, 328-42 (1940). (7) Pangborn, M. C., and Anderson, R. J., J . Am. Chem. SOC.,58, 10 (1936).
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(8) Slotta, K. H., and Neisser, Klaus, Ber., 71B, 1611-16, 1984-6 (1938). (9) Stodola, F. H., and Anderson, R. J., J . Biol. Chem., 114, 467 (1936). (10) Suknevich, I. F., and Chilingaryan, A. A., Be?., 68B, 1210-16 (1935); 69B,1537-42 (1936). ( l i ) Ware, A. H., Chemist and Druggist, 123, 282 (1935).
Detection of Zirconium with 5-Chlorobromamine
Acid JOHN H. YOE AND LYLE G . OVERHOLSER, University of Virginia, Charlottesville, Va.
B
R O M A M I K E acid is the name used b y dye chemists for the sodium salt of l-amino-4-bromo-2-anthraquinonesulfonic acid. A number of substituted bromamine acids react with the tri- and tetravalent cations in aqueous medium, giving red precipitates. In acid solution, the reactivity is limited to only a few ions, of which the reaction with zirconium is the most sensitive. The chloro derivativesnamely, 5-chloro-, 6-chloro-, and 7-chlorobromamine acidwere found to be the most selective of the substituted bromamine acids studied. Although these three compounds behave similarly, the 5-chloro derivative is recommended because of its slightly more selective reactions. The chlorobromamine acids give heavy bright red precipitates with cadmium, copper, cobalt, nickel, palladium, and zinc in ammoniacal medium. Although the attempted application of 5-chlorobromamine acid as a quantitative precipitant for zirconium was unsuccessful, the reagent may be used for the detection of zirconium.
reagent. The reaction rate is greatest a t low concentrations of nitric acid and decreases markedly with increasing acid concentration-for instance, using a zirconium concentration of 10 p. p. m. and 0.05 ml. of 0.3 M nitric acid, the precipitate forms within a minute; with 2 M acid, 2 to 3 minutes are required. The 2 M acid is recommended, despite the slower reaction rate, to avoid interference by a number of ions.
T.4BLE
I. LIMITIKG CONCENTRATION Limiting Concentration Mg./ml.
Ion
Na Ba++ Ca++ Sr++ K + Cd++: CO++’C u + v , M g + + , M n + t , N & + Ni++ pbt+’Zn++ Rarekarths*’,Fe+++,G a + + + , H g + +I. n + ’ + , S c + + + , Y + + + +
--
20 10
5 5
1
ce+-++
0.03
Be++ A ] + + + C y + + + ,T h + + + + T , ifit+
0.1
Po,--*-, sod-* Tsivalent ions.
0.26 1
Reagents ~ - C H L O R O B R O XACID. ~ ~ N E The compound was obtained from E. I. du Pont de Nemours & Co., Inc. A 50 per cent acetone-water solution containing 2 mg. per ml. of the reagent was used. ZIRCOSIUM.A stock solution containing 1 mg. of zirconium per ml. and 0.2 ilf with respect to nitric acid was prepared from zirconium nitrate and n-as diluted with water to the desired concentration. NITRICACID. A 2 31 solution was employed.
Procedure Transfer 0.05 ml. of the test solution to a depression in a white tile spot plate, and add 0.05 ml. of nitric acid and finally 0.03 ml. of the 5-chlorobromamine acid solution. Shake the spot plate continuously for several minutes before making the observations. A blank prepared in the same manner, except that distilled water is added in place of the test, solution, should be used for comparison. Occasionally, the blank may have a small amount of a pale red colored precipitate, which floats on the surface of the liquid and has the appearance of a slight scum. This should not be confused with the precipitate formed in the presence of zirconium, which is more granular and darker colored and settles to the bottom of the solution.
Characteristics of Reaction The reaction rate is dependent upon agitation of the solution, the concentration of zirconium and of the reagent, and the type and concentration of acid present. Continuous shaking hastens the formation of the precipitate. A t the higher Concentrations of zirconium the reaction rate is faster than a t the lower. T h e use of a higher concentration of the reagent hastens the precipitation but may cause interference. The reaction rate is about the same in nitric or hydrochloric acid but the latter tends to cause increased interference by the
Interference I n testing for the interference of the various ions, the procedure previously given was followed using a zirconium solution containing 10 p. p. m. Solutions of the ions alone, and mixtures of zirconium and the respective ions were used. Suitable blanks were used in all cases. Interference was noted if the ions gave a precipitate in the absence of zirconium and if precipitation was prevented or the character of the precipitate altered in the mixtures of the ions and zirconium. The limiting concentrations given in Table I are for a zirconium concentration of 10 p. p. m . A t higher zirconium concentrations they are somewhat greater. Phosphate and sulfate prevent the formation of the precipitate. Fluoride must be absent. I n the case of aluminum, beryllium, chromium, ferric, thorium, and titanium ions, mixtures consisting of zirconium and the respective ions were precipitated as the hydroxides, the precipitates were Utered off and dissolved in nitric acid, and the interference was determined under conditions comparable t o the preceding interference studies. The results were comparable to those listed in Table I.
Sensitivity I n the absence of interfering ions, the minimum concentration of zirconium that may be detected is 2 p. p. m. About 5 minutes of continuous shaking are required and a blank must be used. The reaction rate is slow at 5 p. p. m . A zirconium concentration of not less than 10 p. p. m . (0.5 microgram in 0.05 ml.) is recommended.
