November 15, 1942
ANALYTICAL EDITION
studied thoroughly (4, 7-10) and may be taken into account if necessary. The effect of varying the iron concentration was studied for two water samples containing relatively small and large amounts of magnesium, respectively, and the results are summarized in Table IV. Iron in natural waters rarely reaches concentrations which mould interfere seriously in the spectrophotometric procedure. Although the amount of sulfide in the mater samples was not determined, some of the ground waters \%-ereknown to contain considerable amounts, since they yielded colloidal sulfur on standing exposed to air. The resulting error is negligible if the sulfur is allorred to settle out.
Summary Various natural and treated waters and concentrated magnesium solutions may be analyzed rapidly and accurately for magnesium by a spectrophotometric adaptation of Kolthoff’s Titan yellow method. For most practical purposes the
897
interferences likely to be encountered do not appreciably affect the results. The procedure is recommended for control use.
Literature Cited (1) Am. Public Health Assoc., “Standard Methods for the Examination of Water and Sewage”, 8th ed., p. 79, Lancaster, Penna., Lancaster Press, 1936. (2) Becka, Jan, Biochem. Z.,233, 118 (1931). (3) Collins, W. D., Lamar, W. L., and Lohi, E. W., Geol. Survey
Water-Supply Paper 658 (1934). (4) Eilers, H., Chem. Weekblad, 24, 448 (1927). (5) Hirschfelder, A. D., and Serles, E. R., J. Bid. Chem., 104, 635 (1934). (6) Kolthoff, I. XI., Biochem. Z., 185, 344 (1927); Chem. Weekblad. 24, 254 (1927). (7) Mellan, I., “Organic Reagents in Inorganic Analysis”, pp, 19. 212, 447, Philadelphia, P. Blakiston’s Son Co., 1941. (8) Muller-Xeugliick, H. H., Gluckauf, 77, 34 (1941). (9) Schmidt, R., and Gad, G., Kleine Mitt. Jfitglied, VeT. Wasser-. Boden- u. Lufthyg., 13, 326 (1937); J . Am. Water W o r k s Assoc., 30, 173 (1938). (10) Urbach, C., and B a d , R., Mikrochemie, 14, 343 (1934).
Determination of Rotenone Improvements in the Gravimetric Method S. I. GERTLER ’ Bureau of Entomology and Plant Quarantine, L. S. Department of Agriculture, Washington, D. C.
F
OR the determination of rotenone in plant materials the
method adopted as official by the Association of Official Agricultural Chemists ( 1 ) is most widely used. The procedure involves room-temperature extraction with chloroform, crystallization as a solvate from carbon tetrachloride solution. and purification of the solvate b y allowing it to stand in alcohol. A variation developed by Jones (2), and until recently extensively used by the writer of this paper, depends upon the isolation of the rotenone as a dichloroacetic acid solvate. This variation has been found satisfactory in this laboratory, but has met with some objection from others because of the difficulty of obtaining the solvate free from interfering gummy material. I n order t o get a shorter and simpler procedure the official method has been varied with respect t o the mode of extraction and purification of the solvate.
Procedure for Powdered Roots Although parts of the official method are duplicated more or less, for the sake of continuity and ease of following the method through, the procedure will be completely outlined. Weigh a sample of finely powdered root of such size that the quantity of extract finally obtained and used for analysis wdl contain at least 1 gram of rotenone, This will not be possible in the case of roots of exceptionally low rotenone content, and the method is modified accordingly, as noted later. Transfer the powder to a 1-liter round-bottomed flask, preferably one having a standard glass joint. Add 10 grams of decolorizing carbon and then an accurately measured volume of chloroform a t some definite (near room) temperature in the ratioof 1Oml. toeach gram of root used. Connect the flask to an efficient reflux condenserthat is, one shown to be able to conserve all the chloroform-and boil the contents for 1 hour. If there is any question as to the efficiency of the condenser, the flask and contents should be weighed before heating and any loss in weight made up by the addition of fresh chloroform. As soon as the condenser has drained, remove the flask, immediately stopper it, and cool it first under the tap and then in the refrigerator for 30 minutes. Filter the contents rapidly through a fluted filter, keeping the funnel covered with a watch glass to avoid evaporation of the solvent. Bring the filtrate back to the original temperature by immersing the flask in warm water and shaking until the chosen tempera-
ture is reached. Measure the volume of the filtrate in a graduate to the nearest milliliter, and calculate the proportion of the sample that it represents. Transfer the chloroform extract to a round-bottomed flask, distill until about 25 ml. remain, and then transfer this remainder to a 125-m1. Erlenmeyer flask with a little chloroform and distill almobt to dryness; in this may most of the solvent is recovered. Remove the last traces of solvent by warming slightly under diminished pressure and evaporating twice more after adding two small portions of carbon tetrachloride. Dissolve the residue by warming under a reflux condenser with exactly 25 ml. of carbon tetrachloride, cool, and seed if necessary to induce crystallization. If a t this point crystallization does not take place readily or occurs only in small amount because of low rotenone content, add an accurately weighed quantit of pure rotenone to bring the amount up to at least 1 gram, andrkeep the flask at 0” C. overnight. Pour the contents of the flask quickly through a small frittedor sintered-glass funnel of medium porosity, allowing the flask to drain as completely as possible. (These glass funnels and also crucibles have been found very useful by the author and can be cleaned easily with acetone and used repeatedly.) Continue the suction for about 5 minutes after the solvent has drained off. Place the flask in which the solvate was crystallized, and which still contains some crystals adhering to the sides, under the funnel containing the filtered solvate. Dissolve the solvate by pouring small portions of acetone through the funnel, about 25 ml. usually being sufficient. Evaporate the acetone solution of the solvate to dryness on the steam bath, and then place it under gentle suction for a short time to remove all traces of acetone. Treat the residue from the acetone with 25 ml. of ethyl alcohol saturated with rotenone a t room temperature, and boil the contents, still in the flask originally used for crystallization of the solvate, gently under reflux until completely dissolved. If necessary, add more alcohol to effect complete solution. Allow the alcohol solution to come to room temperature. Usually crystallization begins readily, but it may be induced, if necessary, by adding a crystal of pure rotenone; it then usually proceeds very rapidly. Shake the flask vigorously for 2 full minutes, and allow to stand at room temperature for 30 minutes. Filter the rotenone crystals through a weighed sintered-glass or Gooch crucible, using about 10 to 15 ml. of alcohol saturated Kith rotenone at room temperature to transfer and wash them. After aspiration for 3 to 5 minutes dry to constant weight at 105” C., xhich takes about 30 minutes. Weigh the final product, which consists of purified rotenone, add 0.07 gram to correct for the solubility in 25 ml. of carbon tetrachloride, and subtract any amount of pure rotenone that may have been previously added. Multiply the net weight by 100 and
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Vol. 14, No. 11
completely as possible. Suction is continued for about 5 minutes. Usually a t this point a fairly light colored crystalline product is obtained. Since the crystals are not washed Total Extractives at all, a possible source of loss, especially in warm weather, is Complete Sample No. Root Boiling method extraction eliminated. The drying to constant weight for about an hour % 70 is also avoided, since weighing as crude solvate is unnecessary. Instead of decomposing the solvate by allowing it to stand from 4 hours to overnight a t room temperature, the same result was obtained by complete solution in hot alcohol saturated ,with rotenone and allowing to crystallize for about 30 OF ROTENONE IX ROOT SAMPLES minutes. A test sample of pure rotenone under these condiTABLE11. DETERMINATION tions gave practically complete recovery of the rotenone. (Proposed Method as Compared with the A. 0. A . C. Method) Thus a considerable saving of time is effected. Rotenone
TABLEI. COMPARISON O F BOILINGMETHODWITH COMPLETE EXTRACTIOK OF ROTENONE FROM DERRISAKD CUBE ROOTS
By proposed
SsmDle No.
Root
gravimetric method %
By A. 0 . A. C method
R
Derris 3.8 3.9s Derris 4.8 4.8" Derris 4.6 4.5 Cube 4.8 4.8 5.1 Cube 5.0 Cube 4.8 4.7 5.9 Derris 5.7 7 Derris 3.6 3 7 8 Derris 8.9 8 6 9 4.4 Derris 4.2 10 6 Analyzed by titrimetric variation instead of complete A. 0. A . C . method. 1 2 3 4 5 6
divide by the weight of sample represented by the aliquant taken, to obtain the percentage of pure rotenone.
Discussion of the Method
It is agreed that chloroform is one of the best and most convenient solvents for the extraction of the powdered root, and the official method recommends shaking a weighed quantity of powdered root and decolorizing carbon with a definite volume of chloroform for not less than 4 hours, preferably interrupting the process with an overnight rest, or else shaking continuously overnight. The author accomplishes the same result by refluxing for only 1 hour. To show that 1 hour of refluxing will give sufficient extraction of the rotenone, five root samples were extracted in two ways. First, they were completely extracted in a Butt-type extractor with chloroform, the solvent was distilled off, and the residue dried in an oven a t 105' C. overnight and weighed. Other portions of the same samples were then boiled with chloroform as in the proposed method, except that the charcoal was omitted, since it absorbs part of the nonrotenone resins and would cause low results for total extract. An aliquant was then taken, the solvent distilled off, and the residue dried and weighed as before. I t can be assumed that under these conditions the weight of total extractives would be proportional t o the weight of rotenone, and would give a measure of the completeness of extraction. Table I gives the results obtained. The results show that, for all practical purposes, one method of extraction is as good as the other. The boiling method, however, has some advantages over any other extraction method. It greatly reduces the extraction time, and it does not require a shaking machine or any special extraction apparatus. It also enables much larger samples (up to 200 grams with a reduction in proportion of solvent) to be conveniently handled in one container. B y using the entire filtrate instead of a fixed aliquant it is possible to recover all the rotenone and solvent present in one step, in the course of the method; hence, small portions of solution do not have to be accumulated for subsequent recovery. I n the proposed method the contents of the flask containing the rotenone solvate, after crystallization, are poured quickly through a small fritted- or sintered-glass funnel of medium porosity. No weighing is necessary, and the transfer is generally complete within a few seconds. The flask need not be put back into an ice bath, but i t should be allowed t o drain as
Experimental Results Ten samples of derris and cube roots were analyzed by this method. Eight of the samples were also analyzed by the regular A. 0. A. C. method, and the other two by the titrimetric variation. The results are listed in Table 11. These results indicate fairly good agreement between the method as proposed and both the official and titrimetric methods.
Adaptation t o Other Types of Samples Occasionally a root sample is encountered which contains an unusually large proportion of nonrotenone resins. I n this case the crystallization will not proceed correctly and there is no choice except to add enough pure rotenone to the extract to bring the ratio of rotenone to resin u p to about 1 to 5 . Otherwise the procedure is the same. Dusting ponders consisting of ground roots and an inert diluent such as talc, kaolin, etc., which is insoluble in the solvents used, may be treated in the same way except that the sample should be large enough (up to 200 grams) to give a minimum of 1 gram of rotenone, and the proportion of chloroform adjusted to the ratio of 5 ml. of 1 gram of sample. If the amount of material available is limited, a smaller sample can be used and pure rotenone added to make up the amount. Samples containing sulfur have to be treated somewhat differently. The boiling chloroform takes up a large quantity of sulfur, most of which crystallizes out and is filtered off with the marc. The whole solution may be taken down to dryness and the residual chloroform removed under vacuum. The residue is then digested with several portions of warm acetone. Each portion of the solution is then cooled and filtered into a 125-m1. Erlenmeyer flask. The acetone dissolves all rotenone, but only a small amount of sulfur, The acetone is then completely removed by evaporation, the residue dissolved in carbon tetrachloride in the usual way, and the normal procedure resumed. What would normally have been the final product consists of rotenone still contaminated with a little sulfur, and should be weighed. A saturated solution of sulfur in acetone is poured through the crucible until all the rotenone is dissolved out, and the crucible containing the small amount of sulfur is dried and weighed. The loss in weight represents the rotenone content.
Summary A n e a procedure for the gravimetric determination of rotenone has been developed from the official A. 0. A. C. method, shortened and simplified in several respects. Extraction time was shortened b y refluxing the sample with the solvent for a short time. This procedure also permits the use of samples u p to 200 grams as well as mixtures of powdered roots with sulfur and most inert ingredients. The purification of the crude rotenone was also modified and several unnecessary steps were omitted.
Literature Cited (1) Assoc. Official Agr. C h e m . , Official and T e n t a t i v e Methods of Analysis, 5th ed., pp. 64-6, 1940. (2) Jones, H. A , , IND. ENQ.CHEM.,ANAL.ED.,10, 684 (1938).
