V O L U M E 23, NO. 2, F E B R U A R Y 1 9 5 1 Jlacerate a tablet containing amithiozone with pure synthetic inethanol in a mortar with the pestle. Repeat three times, and each time decant through a filter into a volumetric flask. In the case of essentially pure powder dissolve in a corresponding volume of methanol, omitting maceration. Use enough methanol to have an approximate concentration of not more than 0.5 mg. per ml. Make a secondary dilution in methanol, so that a final dilution of 3 * 1 micrograms per ml. results. Using the same methanol as li blank, determine the extinction a t 20' C. a t 3280 A,, using 1-cni cells and a Beckman DU or similar instrument. In Table I1 two sets of ten replicate results are listed: ailallsee of an essentially pure preparation and of a single commercial lot of tablets. The latter rrsults are corrected for tablet weight. diinilar analyses were pcarfornied on e-xperimenttlland commercial products of a. number of manufacturers, of both foreign and domestic nianufacture. S o evidence of interference was found The precision is comparable to other spectrophotometric methods. The extraction etep, required for tablet analysis, reduces pre-
335, c4sion somewhat. The technique is recommended for the routiiit. analysis of amithiozone products. ACKNOW LEDGMERT
The authors are indebted to Bruno Puetzer for suggesting this problem and for discussions, for Kurt Ladenburg for advice and encouragement, and to Schenley Laboratories, Inc., for permission to publish this note. LITERATURE CITED (1)
Doniagk, G., Behnisch, R., Mietzsch, F., and Schmidt, 11.
.Vaturwissenschuften, 33, 315 (1946): Angew. Chem.. 60, 11 3 (1948); Am. Reti. Tuberculosis, 61, 1 (1950). ( 2 ) SDinks. A. Brit. J. PharmucoZ.. 4. 254 (1949). (3) Wollenberg, O.,private communication; Am. Rea. Tuberculosis 61, 5 (1950). RECEIVED July 1, 1950.
Modification of Sanchez Color Test for Nicotine Application to Nicotine, Nornicotine, and Anabasine LOUIS FEINSTEIN AND EDWARD T. MCCABE Bureuu of Entomology and Plant Quarantine, ti. S. Department of Agriculture, Beltsuille, M d . S a search of the literature on tobacco alkaloids, it was noted
1 that Sanchez (S) gave a color test for nicotine using vanillin
and strong hydrochloric acid, the color varying from a rose red to a deep cherry red, according to the nicotine concentration. Sanchez's test is not directly a test for nicotine and this note presents the authors' experience with this test when it is applied only t o nicotine, nornicotine, and anabasine (4). These three alkaloids are perhaps not the only ones which can be made to give colors in this test, but Sanchez's test, when correctly applied, can be used to provide additional information in a study of these three alkaloids. Anabasine is an isomer of nicotine, both being closely related physiologically. Both alkaloids are liquids, forming picrates of melting points 212-213' and 223-224" C., respectively, for anabasine and nicotine. Sirupy 85% orthophosphoric acid has been used because of convenience in place of strong hydrochloric acid in the Sanchez test. In experiments with vanillin-phosphoric acid, carefully purified samples of nicotine and nornicotine failed to give the Sanchez test, but anabasine (extracted out of Nicotiana glauca)gave a faint brown-violet color. Further experiments showed that the following compounds (all having a six-member ring containing nitrogen) failed to give a red color with vanillin and phosphoric acid: ttnabasine, piperidine, y-dipyridine, 3-aniinopyridinc, benzoyl piperidine, 3-broniopyridine, and acridine. Any change in color appearing is toward a yellow or brown-violet. Pyrrol, a compound with a simple five-member ring containing nitrogen, gives an immediate red color in dilute concentration, while carbazole, where the five-member ring containing nitrogen is bound a t each carbon as part of benzene rings, gives the red-color reaction s l o ~ d y(after being stirred 20 to 70 minutes). Pyrrolidine gives no color change x i t h vanillin and phosphoric acid. Myosmine crystals change to red when vanillin-phosphoric acid solution is dropped upon them. Xicotyrine gives an immediate deep red color T-iith vanillin and phosphoric acid solution. The authors then dehydrogenated nicotine and nornicotine, using platinum-black prepared by the method of Linstead and Thomas ( I ) , and found that the dehydrogenated samples gave red color solutions with vanillin and phosphoric acid. Anabrtsine does not give this red color before or after a similar treatment The procedure for dehydrogenating and reacting with vanillin and phosphoric acid is as follow:
A small quantity of the alkaloid is mixed with a quantity of the platinum-black and heated in a vessel to 170" C. for 1.5 hours, taking care to prevent the alkaloid from evaporating off. After the sample is cooled, 5 ml. of distilled water are added and the whole is filtered. To 3 ml. of the filtrate are added 12 ml. of the vanillin-phosphoric acid solution (0.5 gram of vanillin in 100 ml. of 85% orthophosphoric arid a t room temperature) and the color change is noted.
A somewhat *differenttest is that using Meltzer's reagent, which is made up of carbon disulfide, ethyl alcohol, and dilute copper sulfate solution. RazvadovskiI (2) used this reagent t o distinguish nicotine from anabasine. Xicotine gives a water-white solution or slight turbidity with Meltzer's reagent. Anabasine gives a brown-black solution. This test will also show the presence of nornicotine, which reacts like anabasine, giving a brown-black solution. Meltzer's reagent can b e w e d t o show the absence of nornicotine and anabasine in nicotine. The presence of nornicotine, anabasine, or both in nicotine will cause a blackening of the reagent solution. To use the reagent, 5 ml. of carbon disulfide are mixed with 95 ml. of ethyl alcohol and 20 ml. of this mixture are added to 0.05 ml. of alkaloid. After a thorough mixing, 2 drops of 20% copper sulfate solution are added and the mixture is shaken. ACKNOWLEDGMENT
The authors wish to thank Abner Eisner, Eastern Regional Research Laboratory, Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U.S. Department of Agriculture, Philadelphia, Pa., for samples of pyrrolidinr and myosmine. LITERATURE CITED
( 1 ) Idinstead, R. E'., and Thomas, S. L. S., J . Chem. Soc.. 1940. 1126. (2) Razvadovski:, V.A,, Farmatsiya, 1939, No. 12, 12-15. (3) Sanchez, J. A., Semanamid. (Buenos Aires), 28, 61-4 (1923). ( 4 ) Smith, C. R.. J . Econ. Entomol., 24, 1108 (1931). RECEIVED June 22. 1950. Presented before the Division of Biolopcal CHEMICAL SOCIETY. Chemistry at the 117th Meeting of the AYBRICAN Philadelphia, Pa. Report of a study made under the Research and Market ing Act of 1946.
