Micromelting- Point Determination with the Thiele Tube EUGENE W. BLANK,Allentown, Pa.
A
HOT stage, though a requisite in microwork, is not always available for melting-point determinations. In its place an ordinary Thiele tube or one of the various modifications of the tube (1, 2, 3) may be substituted. If an electrically heated tube, as devised by Sando (6),is available, it can be utilized. Otherwise the tube is heated in the usual manner by means of a small flame. Thaapparatus is shown in detail in F i g u r e 1. T w o glass rods are fused to the side walls of the tube c o n t a i n i n g t h e sample and thermometer to support it in the Thiele tube. To observe the melting point, optically, a microscope-preferably a reading m i c r o s c o p e , as used in optical experimentation-is turned to a h o r i z o n t a l p o s i t i o n and the stage r e m o v e d to make room for the Thiele tube. T h e s a m p l e is placed between two small q.q uares cut from a microscope cover glass with a d i a m o n d point, or, in the absence of a diamond point, broken by hand. One 18 by 18 mm. cover glass will yield six s m a l l s q u a r e s sufficient for three me1t i n g-po i n t d e t e r m i n a t i o n s . The glass slips, with the sample between, are held in position by a small clip made of thin platinum wire.
CALIBRATION To calibrate the instrument several materials of corrected melting point are taken and their melting point in the apparatus observed. A calibration curve is drawn for subsequent work. Table I presents the results of several determinations, from which a graph may be constructed. The differences between the observed and corrected melting points are small at low temperatures because of the small stem exposure in the case of the corrected melting point. At higher temperatures the protecting action of the tube surrounding the thermometer becomes apparent, the corrections being smaller than when the thermometer is directly immersed in the Thiele tube. Since the calibration curve is specific for each individual instrument and empirically dependent upon the dimensions, depth of immersion, etc., the apparatus has been designed t o take care of these effects automatically. The projecting lugs of the tube permit it to be inserted to the same depth at each observation; the thermometer should always be inserted till it touches the bottom of the tube. The size of the aperture in the wall of the tube produces a discernible effect on the melting point: a very small hole gives a low melting point, and a very large hole gives a higher melting point on the same material. The only variable factor is the height of the bath liquid. This should be regulated by keeping the bath liquid level with an etched ring on the Thiele tube. Sulfuric acid will require no attention for long periods of time; water, particularly at high temperatures, will require leveling more frequently. The instrument requires no external support for the thermometer, the lip of the Thiele tube being ground flat to insure the stability of the tube and thermometer. TABLEI. CALIBRATION DATAAND TYPICAL DETERMINATIOWS OF MELTING POINT
PROCEDURE
*
The sample, c on t a i n ed between the sections of cover glass, is placed in the empty tube and supported v e r t i c a l l y b y means of the therFIGURE 1. DETAIL mometer which is subsequently inserted. The thermometer is secured OF APPARATUS in the tube by means of a small roll of a s b e s t o s p a p e r . The tube is now inserted in the Thiele tube. The sample should be well illuminated. Temperature readings and the progress of melting of the material under test can be simultaneously observed by means of an Abbe drawing camera. A 32-mm. objective possesses a sufficiently long working distance to focus on the sample and a t the same time avoid undue heating of the objective due to the proximity of the hot Thiele tube. The customary precautions essential to a determination should be observed (4). The temperature of the bath liquid, in particular, should be very gfadually increased. The usual bath liquids may be used (6). Snell (7) has proposed the use of phosphoric acid Water is preferable to sulfuric acid for substances melting a t less than 100" C. To correct the observed melting point, recourse is had to a calibration curve based on the observed and corrected melting points of several materials.
MATERIAL
OBSERVED MELTING CORRECTED MELTING POINT CORRECMELTINQ POINT MICROTION CORRECTED POINT IN T K I ~ L E FROM MELTING STANDARDLITERATUBE GRAPH POINT METHOD TURE
c. Thymol Naphthalene Benzoic acid Urea. Anthraoene Phenolphthalein Silver nitrate Salicylic acid
51.0 80.2 121.0 130.9 211.6 253.6 205.1 157.5
0
(7.
0.3 0.6 1.3 1.5 4.0 6.4 3.8 2.0
O
c.
51.3 80.8 122.3 132.4 215.6 260.0 208.9 159.5
0
c.
51.3 80.8 122.3 132.4 '215.6 260.0
c. 61.5 80.8 122.5 132.5 216.0 261.1 209.0 150.8
ADVANTAGES OF INSTRUMENT The preparation of capillary tubes, usually regarded in the light of a task, is entirely eliminated. The operation of placing a minute quantity of sample between two glasses is infinitely easier than filling a melting-point capillary. A small amount of material spreads over a larger area and its melting point is much sharper between two glasses than when examined in a capillary. This instrument is superior to a hot stage for rapid work, since the inner tube and thermometer can be quickly transferred to a cold Thiele tube for subsequent determinations, whereas a hot stage is so thoroughly insulated as to retain its heat for a long period of time. 74
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-
