Rotenone Determination by Colorimetric Methods H. D. ROGERS
AND J.
A . CALAMARI, Laboratory, Medical Section, New York General Depot, Brooklyn, N. Y.
concentrated nitric acid to 100 ml. of concentrated hydrochloric acid). Agitate for about 30 seconds. A bluish green to blue color appears when rotenone is present. The color usually apears in from 30 seconds to 2 minutes and deepens on standing. f n the presence of the yellow coloring matter of pyrethrum flowers and of derris extract, the color is green and turns upon standing to a bluish green and finally to blue.
and Smith (Z), realizing the necessity for a sensitive
J O Ncolorimetric ES test for rotenone, modified the Durham qualitative reaction in an attempt to render the blue color more lasting. The modification, although a sensitive qualitative test, failed to make the color permanent enough for quantitative work. Gross and Smith (1) discovered a red color reaction which they developed into an accurate colorimetric test, but the test was limited to rotenone in acetone solution. Because of the restriction to acetone solution, difficulties were encountered in applying this test to liquid insecticides having refined kerosene bases. The Jones-SmithDurham test was found useful for detecting rotenone in some liquid insecticides but it failed in several cases when rotenone was known to be present. One substance which interfered with this test was the oil of sassafras. In the search for a more universal test, the authors found that rotenone in the presence of hydrochloric acid and certain phenols developed color reactions in certain organic solvents. These reactions varied from violet-red to deep blue in color, depending upon the solvent and phenol selected. The organic solvents used were chloroform, ethylene dichloride, carbon tetrachloride, ether, alcohol, and acetone. Phenol and some of the homologs of phenol, such as guaiacol and thymol, also reacted similarly in the solvents named. Small amounts of hydrogen peroxide, nitric acid, and light exerted a marked influence in accelerating the reaction. Isorotenone gave a blue color. Acetylrotenone gave no characteristic color within an hour, but upon standing for several hours a blueviolet color developed. The advantages of the tests developed over the older methods are its increased sensitivity, making possible an accurate quantitative test satisfactory within the limits stated, and the fact that substances usually found in insecticides do not interfere with the reaction. Some samples of commercial rotenone gave results approximately 15 per cent higher than the known rotenone content. Gross and Smith (1) obtained similar results with their test, which they attributed to the presence of deguelin or some unidentified compounds. The difference in color observed in the detection of rotenone and certain rotenone derivatives may be used as a future basis for investigation in differentiating such substances, as well as serving as a n identity test upon comparison with known standards. A qualitative and two quantitative tests were developed which were not interfered with b y substances usually found in proprietary liquid insecticides containing extracts of pyrethrum flowers, aliphatic thiocyanates (Lethane), or oil of sassafras. The following qualitative test is sensitive to 0.01 mg. of rotenone per ml.: Dilute 1 to 2 ml. of the insecticide or solution containing rotenone to 5 ml. with chloroform. Add 5 ml. of a chloroform solution of thymol (10 grams of thymol to 100 ml. of chloroform). Add 3 ml. of a nitric acid-hydrochloric acid mixture (0.2 ml. of
Two quantitative tests were developed. KO.1 can be applied to almost colorless liquid insecticides, such as those which contain rotenone and aliphatic thiocyanates (Lethane) . If the insecticide base is a refined kerosene, an amount of refined kerosene equal to the amount of liquid insecticides used in the test must be added to the standards. TEST1. To 10 ml. of a chloroform solution containing from about 0.05 to 2.5 mg. of rotenone per ml. in a glass-stoppered cylinder, add 10 ml. of a chloroform solution of thymol (10 grams of thymol to 100 ml. of chloroform) and 2 ml. of a reagent made by adding 2.5 ml. of 3 per cent hydrogen peroxide to 100 ml. of concentrated hydrochloric acid. Agitate for 1minute, loosen the glass stopper, and expose the cylinder to the intense radiation of a quartz mercury vapor lamp having a minimum output of 1000 microwatts per square centimeter in the field of exposure. (The use of bright sunlight produced similar results in approximately 3 hours.) A greenish blue color begins to appear in the chloroform layer in about 15 minutes. At the end of 30 minutes, compare with standards containing known quantities of pure rotenone prepared at the same time in the same manner. When the rotenone sample is not in solution in a hydrocarbon base, concentrated hydrochloric acid may be used in place of the reagent described. TEST2. To 5 ml. of an acetone solution containing from 0.1 to 2.0 mg. of rotenone per ml. in a glass-stoppered cylinder, add 5 ml. of an acetone solution of thymol (10 grams of thymol to 100 ml. of acetone), 0.1 ml. of 3 per cent hydrogen peroxide, and 5 ml. of concentrated hydrochloric acid. A reddish violet color appears within 30 seconds. After 1minute place the glass container in a water bath at about 20' C., and at the end of 20 minutes compare with rotenone standards, similarly prepared, at the same time. When the liquid insecticide contains pyrethrum and rotenone (not derris extract) in a refined kerosene base, proceed as follows: Prepare an extract of pyrethrum with refined kerosene. 1.8 kg., 4 pounds, of pyrethrum to 3.785 liters, 1 gallon, of re ned kerosene and allow to stand a t least 2 days.) Filter and preserve.
LAdd
Match the color of the insecticide, approximately, with the extract by diluting with refined kerosene. Prepare a chloroform solution of rotenone of suitable concentration. Use the diluted pyrethrum extract and rotenone solution in preparin the standards using the same amount of extract in the standarfs as insecticide used in the test. Proceed as in quantitative test No. 1.
Literature Cited (1) Gross, C. R., and Smith, C. M., J. Assoc. Oficial Agr. Chom., 17, 336 (1934). (2) Jones, H. A., and Smith, C. M., IND.ENC). CAEM.,Anal. Ed. 5, 75 (1933). R ~ C E I Y EDecember D 17, 1935.
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