January 15, 1933
INDUSTRIAL AND ENGINEERING
LITERATURE CITED
(6) Sando, IND. EKG.CHEM.,Anal. Ed., 3, 65 (1931). ( 7 ) Snell, Ibid., Anal. Ed., 2, 287 (1930).
Conte,Ibid., Anal Ed., 2,200 (1930).
(3) Dennis, (4) Fisher,
IND. ENQ.CHEM.,12, 366 (1920).
“Laboratory Manual of Organic Chemistry,” 1920.
75
( 5 ) Mulliken, “Identification of Pure Organic Compounds,” Vol. 1, p. 218, Wiley, 1911.
(1) Bell, IND.ENG.CHEM.,15,375 (1923).
(2)
CHEMISTRY
p. 58,
Wiley,
RECEIVED July 20, 1932.
A Color Test for Rotenone HOWARD A. JONES AND CHARLES M. SMITH,Insecticide Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture, Washington, D. C .
T
H E increasing use of rotenone as an insecticide renders methods of testing for this material of great importance. In a review of the literature it was found that Durham (1) in 1902 had discovered a peculiar color reaction of rotenone which showed promise of being a good qualitative test for the material. When he treated rotenone, either the crystals or powder, with a drop of concentrated nitric acid on a glazed porcelain plate, it became red. The addition of a few drops of strong ammonium hydroxide gave a deep,.greenish blue color, which quickly faded to yellow. I n this form the reaction is unsuitable for delicate testing because of the violence of the neutralization and the extremely rapid disappearance of the blue color. I n the course of the present study it was found that when an acetone solution of rotenone is treated with 1 to 1 nitric acid it gives a bright red color which on addition of ammonium hydroxide changes to the characteristic blue color. This procedure makes the test more delicate and more suitable for general use. Other alkaline reagents, such as solutions of sodium and potassium hydroxides and sodium and potassium carbonates, give the same color. Such weakly alkaline materials as sodium bicarbonate and borax do not produce it. All attempts to render the blue color permanent have thus far failed. However, it was found that dilution with water after the nitric acid reaction rendered the blue color developed with ammonium hydroxide somewhat more lasting. Neutralization of the acid with sodium bicarbonate followed by the addition of the ammonia gave an even more lasting blue color. Even this, however, faded in the course of 2 or 3 minutes. The method finally adopted as most suitable for a qualitative test consists of the following steps: One cubic centimeter of an acetone solution of rotenone is treated with 1 cc. of 1 t o 1 nitric acid, and the mixture allowed t o stand for 0.5 minute. It is then diluted with 8 to 9 cc. of water and 1 cc. of strong ammonium hydroxide added. A blue color is produced which is almost identical with that given by bromothymol blue indicator at a pH of 7.2. As little as 0.1 mg. of rotenone can be detected by this method. Several pure materials related to rotenone were tested by the method just outlined, to see if they would interfere. One milligram of material per cubic centimeter of acetone solution was used. Deguelin gave a test apparently identical with that given by rotenone. Tephrosin gave no blue color, and toxicarol only a very faint blue as compared with a deep blue for rotenone a t this concentration. Dihydrorotenone, isorotenone, and rotenone hydrochloride also gave about the same depth of blue color as rotenone. Dehydrorotenone and rotenonone, oxidation products of rotenone, gave no blue color. Acetone extracts of pyrethrum flowers and tobacco did not give the test.
Tephrosin gave a faint blue color when concentrated nitric acid was allowed to act on the dry material, the remainder of the procedure being as above. Tested in this way toxicarol gave a deep purple color. Naturally it was hoped that this color test might prove adaptable to a quantitative colorimetric method for the determination of rotenone. The principal difficulties were the marked dependence of the depth of color on the temperature and time of reaction, and the rapid fading of the blue color. By controlling the reaction conditions it was not difficult to reproduce about the same intensity of blue color. At first standard rotenone solutions were run simultaneously with an unknown, but this proved entirely unsatisfactory because of inability to manipulate the addition of reagents to the several tubes a t the same time. A standard series of color solutions containing different concentrations of bromothymol blue in a buffer of pH 7.2 was then prepared. These solutions were standardized against the colors produced by different amounts of rotenone. By using the test as previously outlined, or better, by neutralizing the acid witla sodium bicarbonate solution, the color obtained was sufficiently stable to allow of a hasty comparison with these standard colors. I n this way the test may be used to give a rough estimate of the amount of rotenone present. However, it will have no value as an exact colorimetric method unless some means is found of rendering the blue color much more lasting. The exact nature of the chemical reactions upon which the test depends is not known a t present. The nitric acid produces a t least two compounds, of which one, red in color, is extractable from sodium bicarbonate solution by means of ethyl acetate. It is this one which, while stable in acid or nearly neutral solution, becomes blue and unstable in alkaline media. It contains nitrogen in some form. This color test has been used in the Insecticide Division for surveying plants as sources of rotenone, acetone extracts of the plants being made and tested as outlined above. As stated before, deguelin, a natural constituent of some plants which are sources of rotenone, also gives the test, but deguelin is also of value insecticidally, so that any plant material which gives a definite color test is worthy of study for its insecticidal properties. A direct test on plant material may also be made by placing several drops of concentrated nitric acid on the sample, allowing it t o react for 0.5 to 1 minute, and then adding several drops of ammonium hydroxide. Roots containing as little as 1 per cent rotenone gave an evanescent blue color in this way. It is suggested that this form of the test might be used by field expeditions and plant explorers. The test has also proved useful in determining the completeness of extraction during recovery of rotenone from derris and cub6 roots; in detecting rotenone in spray resi-
ANALYTICAL EDITION
76
Vol. 5 , No. 1
dues; and in following the course of decomposition of thin deposits of rotenone exposed to light and air, an adaptation rendered possible by the fact that the test is not given by the products of such decomposition. The testing of proprietary insecticidal preparations is an obvious use for this method. I n the case of dry powdered preparations the test may be made on an acetone extract.
Liquid preparations and extracts including oily samples usually respond to direct treatment with nitric acid.
‘Use of Kohlrausch Sugar Flasks in Determinations of Biochemical Oxygen Demand
LITERATURE CITED
IVANC. HALL
(1)
LITERATURE CITED Durham, H. E., quoted by Gimlette, J. D., “Malay Poisons and
Charm Cures,” 2nd ed., p. 221, J. and A. Churchill, London, 1923.
RBCEIYED
(1) Hall, I. C., J . Infectious Diseases, 29, 317 (1921). (2) Hall, I. C., University of California, Publications in Pathology, 2, 147 (1915). (3)
Pomeroy, J. L., and Stone, R. V.,
OHLRAUSCH sugar flasks were first advocated for putrescibility tests in 1928 by Pomeroy and Stone thus applying the principle of the constricted tube with marble seal devised by Hall (1, 2) in 1915. These flasks are equally suitable for biochemical oxygen demand tests and are much more convenient than the reagent bottles. It is difficult to replace stoppers in reagent bottles without trapping air bubbles, and more care is reauired with the reagent bottles in d i s p o s i n g safely of the excess of liquid f o r c e d o u t upon the addition of reagents, although these objections do not apply to the special flasks devised by Theriault (4, 6). With the Kohlrausch flasks air bubbles are also easily excluded; the excess of liquid is caught above the m a r b l e s e a l a n d c a n be e a s i l y dumped in the sink when the flask is inverted, with the finger holding the marble in place to mix its contents. The author has worked with flasks of 200.6 cc. calibrated capacity, holding about 225 cc. to the marble seal. The graduation is an advantage in me a s u r i n g samples for titration. During i n c u b a t i o n evaporation is minimized by means FIGURE1. KOHLRAUSCH SUGAR FLASK of an inverted beaker, as shown in WITH MARBLESEAL Figure 1. The flask has one disadAND BEAKER vantage, that its shape slightly delays thorough mixing as compared with bottles, and necessitates specialattentionin securing thorough diffusion of reagents. If Kohlrausch flasks should receive favor for these tests, an improved form with a short neck would overcome this one objection. In the writer’s experience the results of comparative determinations of biochemical oxygen demand on single samples showed less variation with flasks than with bottles, but further comparative studies are needed and particularly by those accustomed to using bottles.
Am. J . Pub. Health, 18, 1172
(1928). (4) (5)
Theriault, E. J., Pub. Health Bull. 173, 185 (1927). Theriault, E. J., Suppl. Pub. Health Repts., 90 (1931).
RDCDIVDD August 29, 1932. Presented before the Division of Water, Sewage, and Sanitation Chemistry at the 84th Meeting of the $merican Chemical Society, Denver, Colo., August 22 to 26, 1932.
Department of Bacteriology and Public Health, University of Colorado School of Medicine, Denver, Colo.
K (a),
November 2 5 , 1932.
A Continuous Extractor ARMANDJ. QUICK Cornell University Medical College, New York, N. Y.
T
H E usefulness of a continuous extractor, especially in the analysis of biological fluids such as urine and blood filtrates, is being gradually recognized, A form of extractor which the author has found highly satisfactory is given in the accompanying sketch. It consists of an outer jacket, J (length, 450 mm.; internal diameter, 32 nim.); an extraction tube, T (length, 280 mm.; internal diameter, 15 mm.), having a 5-mm. opening 45 mm. from the top of the tube; a funnel tube, F (length 340 mm.; external diameter 7 mm.), with a bulb a t the lower end containing four small openings; a condenser, C; and a 125-cc. Erlenmeyer flask. The extraction tube is suspended by means of a silk thread or a thin wire. This tube is designed for a sample of 20 cc. or less. A battery of six to eight extractors can easily be set up. At the completion of the extraction, the flask is removed, fitted with an ordinary distilling head, and the solvent removed by distillation. RECBIVED August 2, 1932.
INCREASED SHIPMENTS OF U. S. BIOLOGICAL PREPARATIONS TO PHILIPPINES. Modification by the Philippine Health Service of its regulations affecting the period of potency of biological preparations is expected to react to the advantage of American exporters of such products, according to the Commerce Department. Changes in the Philippine regulations bring them into line with the regulations of the United States Hygienic Laboratory. Formerly, European manufacturers could comply with the Philippine regulations because it usually meant only shortening their datings while American manufacturers, prohibited from lengthening their datings, found it difficult to compete.
