Assay of Plant Material for Its Rotenone Content An Extraction Method HOWARD A. JONES,Insecticide Division, Bureau of Chemistry and Soils, Washington, D. C.
T
HE insecticide rotenone ( C Z S H Z ~isN known to
Derris root and cub; roof are at present the principal Sources of the insecticide rotenone. The chemical evaluation of these roots and of other Plant materials containing rotenone is a subject of increasing importance. A method of extraction using carbon tetrachloride has been devised which gives a more selective and ready separation of the rotenone than the ether extraction method Previously cn use.
occur in a number offishpoisoning plant materials, such as the roots of various species of Deguelia (derrisroot) foundinthe Malay Peninsula and the East Indies, the roots of Lonchocarpus nicou (locally of Peru, and termed the stems ofroot) the haiari vine, a species of Lonchocarpus native to the Guianas. I n 1923 Tattersfield and Roach (14) first proposed a chemical method for evaluating derris root. This consisted, briefly, in exhaustively extracting the finely-powdered, dried root in a Soxhlet apparatus with anhydrous ether, this extract being dried to constant weight a t 100" C. The methoxyl content of the extract was then determined because it was known that rotenone contained methoxyl groups and it was felt that this would prove the genuineness of the extract. These investigators found that in three samples of Deguelia elliptica the insecticidal value of the root (7') bore a direct relationship to the amount of extract. A method for ether extraction similar to that proposed by Tattersfield and Roach, except that the determination of methoxyl content was omitted, was used by Georgi and Curtler (8, 0 ) in a study of Deguelia elliptica and Deguelia malaccensis, and has been used by a number of subsequent investigators and by commercial firms. However, it is now known that the ether extracts of both derris and cub6 root contain a number of compounds of varying toxicity. It has also been found that several of these-i. e., toxicarol ( I ) , deguelin (g), and tephrosin (3), which have a lower toxicity to insects than rotenone (6)-have almost the same methoxyl content as rotenone. Although the total extracts of derris and cub6 roots, which contain a number of materials of insecticidal value, may be widely used commercially, there will be many applications for preparations containing only rotenone. Furthermore, since rotenone is a definite compound of known toxicity to insects (4, 6, IS), many manufacturers may wish to standardize their preparations on the basis of the content of this material, rather than on the basis of an extract of unknown composition and toxicity. Consequently, 8 determination of the amount of rotenone in a root should prove of considerable value.
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as will be shown later, a considerable quantity actually was present. Other ether extracts deposited amorphous material, which interfered with the filtration and gave erroneous results for rotenone. Accordingly, a S e a r c h w a s made for a solvent which would a selective sel)aration of rotenone. A material was desired which was a good solvent for rotenone a t the temperature of the extraction, but which would not extract too large a proportion of the resinous constituents of the root, and from which the rotenone would separate readily and completely on cooling. Carbon tetrachloride, in which the solubility of rotenone a t 20" C. is 0.4 per cent (11) and a t 65" C. is about 5 per cent, was found to meet these requirements. It is interesting to note that, although Tattersfield and Roach stated that "benzene, dry ether, and carbon tetrachloride have a selective dissolving action on the poisons," they found difficulty in drying extracts made with carbon tetrachloride and consequently abandoned the use of this solvent. Duplicate extractions were made on about twenty samples of derris root, ten samples of cub6 root, and two samples of haiari stem (the three sources of rotenone investigated by this laboratory to date), comparing the results obtained by carbon tetrachloride with those obtained by ether. (The results of these and other extractions will be contained in a subsequent article.) As a result of this series of extractions, the following method for the determination of rotenone by carbon tetrachloride extraction was developed.
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CARBONTETRACHLORIDE EXTRACTION METHOD Fifty grams of plant material ground to about 20 mesh are completely extracted in a large Soxhlet extraction apparatus with carbon tetrachloride. The material should be extracted for 8 to 10 hours (longer for samples giving more than 5 per cent rotenone in this period). It is suggested that the extraction be run overnight (about 17 hours), thus accomplishing a reduction in the actual working time consumed by the method. The extract is concentrated to 50 to 25 cc. in a small beaker flask and set aside for 18 to 24 hours to allow the rotenone to crystallize. The rotenone separates from the concentrated extract as needle-like crystals containing one molecule of solvent of crystallization (10). Just before filtering, the extract is cooled for 10 to 15 minutes in ice to assure complete crystallization. The rotenonecarbon tetrachloride solvate is filtered by suction through a tared Gooch crucible containing a disk of hardened filter paper and washed with 10 to 20 cc. of ice-cold carbon tetrachloride in small portions. As much as possible of the excess solvent is removed from the precipitate by suction and then the crystalline material is dried in the crucible to constant weight in air a t room temperature (an overnight drying is
ETHEREXTRACTION METHOD
A procedure designed primarily for determining the amount of rotenone in the ether extract of these roots has been in use by the Insecticide Division for some time, and was recently outlined by Roark ( l a ) . Based on the methods adopted by earlier investigators, this method was found to be not entirely satisfactory for all samples of root. For instance, in many cases it was found very difficult to produce crystallization of the rotenone and in some cases no rotenone could be obtained from the ether extract, whereas, 23
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ANALYTICAL EDITION
sufficient). The solvate is quite stable, and the loss of solvent of crystallization occurs so slowly as to be insignificant in the time mentioned. It is then weighed as the solvate, and this weight multiplied by the factor 0.719 (the ratio of C23HzzOs to C Z ~ H ~ ~ O ~ Cgives C I , ) the weight of rotenone. If the amount of total extract is desired, the filtrate is freed from carbon tetrachloride by evaporation and dried for one hour at 105" C. This weight added to the weight of rotenone gives the total carbon tetrachloride extract (without solvent of crystallization).
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traction is sufficient for samples of low rotenone content, Thus, a sample of derris root (No. 549) giving 1.9 per cent rotenone, and another derris sample (No. 535) giving 4.5 per cent rotenone in 8-hour extractions by this method, gave no more rotenone on additional 7-hour extractions. However, samples giving over 5 to 6 per cent rotenone in an 8hour period should receive further extraction. For example, a sample of cub6 root (No. 686-A) yielding 10.2 per cent rotenone in 8 hours, gave 1.0 per cent more upon an additional 7-hour extraction. Some carbon tetrachloride extractions were followed by SHORTENED PROCEDURE short acetone extractions with the idea that any remaining Reasonably complete crystallization of rotenone from the rotenone would be extracted by this solvent, since acetone evaporated extract may be obtained by cooling the extract, is a good solvent not only for rotenone, but also for the seeding with a small crystal of the solvate compound, and resinous material in which the rotenone may be bound. One allowing to stand in ice, with occasional stirring, for only sample of derris root (No. 522) extracted 10 hours with car2 to 3 hours. The separated solvate crystals may be freed bon tetrachloride gave 6.7 per cent rotenone. The marc of excess solvent by drying in an air draft for 3 to 4 hours. was extracted for 4 hours with acetone, the extract evaporated By using the overnight extraction, the time required for the to dryness, taken up in hot carbon tetrachloride, filtered to whole method is thus reduced to 24 hours or one actual work- remove insoluble material, and allowed to cool. No further ing day. This shortened procedure will give sufficiently rotenone separated. On the other hand, another sample good results for many purposes, although the longer method (No. 594-B) gave about 6 per cent rotenone on a 10-hour carbon tetrachloride extraction, with 0.3 per cent additional is recommended for more accurate values. impure rotenone on acetone extraction. A third sample DISCUSSION OF TEE CARBON TETRACHLORIDE of root (No. 956) gave about 6 per cent in 10 hours with METHOD carbon tetrachloride and an additional 1 per cent with acetone. A 17-hour (overnight) carbon tetrachloride extracCONDITION OF SAMPLE. This method was designed for samples of roots as received without additional drying. tion of this last sample gave the full 7 per cent rotenone, Such roots are usually thoroughly air-dried by the time they however, showing that the rotenone is completely extracted reach this country. The moisture content of our samples from such a sample by carbon tetrachloride if sufficient time of derris root, as determined by vacuum drying a t 100" C., is allowed. It is for these reasons that an overnight extracaveraged about 3 per cent, while that of cub6 root samples tion is recommended. The temperature of the solvent is probably a factor in the was slightly higher, no doubt because of the shorter shipping distance and the larger size of the roots. This content rate of extraction. In these experiments the carbon tetraof moisture in no way interferes with the determination of chloride was kept actively boiling throughout the extracrotenone by the method described above If, however, tions. Under these conditions the temperature of the solvent the sample contains over 5 per cent moisture, as was the surrounding the root remained a t about 70". CRYSTALLIZATION AND FILTRATION OF THE ROTENONE. case with one cub6 root and one haiaxi stem sample reI n all extractions made in this laboratory, the rotenone, ceived, the material should be air-dried at not much over when present in quantities larger than 0.3 per cent, has readily room temperature before extraction. Oven-drying of samples of derris root before extraction crystallized from the evaporated carbon tetrachloride exwas found to interfere with the results of the carbon tetra- tract on cooling. In a few cases, when less than 1 per cent chloride extraction. Thus, the value for rotenone obtained of rotenone was present, it was necessary to seed the cold from one sample of derris (No. 402)' was lowered from extract with a small crystal of the solvate, and when this 1.4 per cent to 0.9 per cent by drying the root in a vacuum was done, the crystalline material separated quickly. Usually oven a t 100" C. for 5 hours. Furthermore, the rotenone scratching the inside of the flask with a glass rod was sufficient obtained by extraction of the dried material was tan in color, to induce crystallization and, in most cases, even this was while that from corresponding extractions, made without unnecessary. If the extract evaporates to a gummy conprevious drying, was white. Similar results were obtained sistency, crystallization may often be produced by the addion this same sample by the ether extraction method. It tion of a small amount of cold carbon tetrachloride. The had been previously noted that samples of root dried a t procedure of cooling the extract in ice for 10 to 15 minutes 100" C. for several hours gave much darker ether extracts before filtering was adopted to insure more nearly complete separation of the rotenone. A good plan is to place the than those extracted as received. A fineness of about 20 mesh was chosen as the most adapt- concentrated extract in a refrigerator overnight. The separated rotenone should be washed with as small able to this method. It was felt that coarser grinding would lead to incomplete extraction, whereas finer grinding ap- an amount of cold solvent as possible. Although the solubility of rotenone in carbon tetrachloride at 0" C. is very peared unnecessary. WEIGHTOF SAMPLE. I n using the original ether extrac- low, an appreciable loss may occur if the rotenone is washed tion method it was necessary in many cases to use 100 grams too extensively. In our experiments the solvate was washed of sample in order to produce Crystallization of the rotenone. until the filtrate was colorless. PURITYOF THE SEPARATED ROTENONE.An examinaHowever, since the usual large-size Soxhlet apparatus conveniently holds only 50 grams of ground root, it was de- tion of the purity of the separated rotenone is an important sirable to use a sample of this smaller size. No difficulty point in any quantitative extraction method. Something has been experienced in obtaining crystallization from ex- in the nature of a check result may be obtained by heating tracts made by the carbon tetrachloride method of 50 grams the separated solvate compound to drive off the carbon of samples actually containing over 0.3 per cent rotenone. tetrachloride of crystallization and reweighing as pure roTIMEOF EXTRACTION. It was found that an 8-hour ex- tenone. This may be accomplished by heating the separated crystalline material for one hour a t 105" C., when the ma1 These numbers refer to Insecticide Division samples and are given for terial usually "melts" to a glassy mass (due to the presence convenience in comparing with subsequent extraction results.
January 15, 1933
INDUSTRIAL AND ENGINEERING CHEMISTRY
of the solvent of crystallization). Check results were obtained in this way on 6 samples of derris and 2 samples of cub& A determination of the optical rotation of the separated rotenone is of value as an indication of its purity. This should be made on the material from which the carbon tetrachloride of crystallization has been removed, as the presence of this solvent may cause incorrect values. This determination should be made in benzene because of the high rotation of rotenone in this solvent. If it is assumed that the impurities are optically inactive, the apparent purity of the rotenone may be calculated from the optical rotation (11). However, many of the impurities likely to be present may possess optical activity, and hence the result of such a calculation will be only approximate. I n all extractions made in this laboratory a small portion of the separated crystalline material was recrystallized, and microscopic examination and melting point determinations were made. At first, ethyl alcohol was used for this purpose, but it was found that in many instances alcohol gave odd-shaped lathlike crystals with erroneous melting points when the substance was actually known to be quite pure rotenone. This may be due to the formation of an alcohol solvate as suggested by Tattersfield and Roach ( I C ) , although the author was unable to obtain such a solvate from pure rotenone (10). Amyl acetate was finally found to be a most suitable solvent for this purpose. It consistently gave the characteristic hexagonal plates of rotenone. The melting points of the recrystallized materials obtained in this way ranged from 155" to 163" C. The melting point of this material mixed with rotenone of known purity may also be obtained. The three other crystalline substances thus far isolated from either derris or cub6-i. e., toxicarol, deguelin, and tephrosin-if present in the separated crystalline material, would be recognizable by their different crystallographic properties, but their presence has not been detected. According to Clark's work (1, b, 3) on these materials, their presence in the separated crystalline material would be unlikely, as they are ordinarily obtained by alkali treatment of the mother liquors from the rotenone separation. It is possible that a determination of thk chlorine content of the crystalline solvate would give some indication of the purity of the rotenone. The determination of the methoxyl content of the separated rotenone should also be of value. In the extracDRYINGTYE NONCRYSTALLINE EXTRACT. tions made in this laboratory, the carbon tetrachloride was removed from the filtrate after the rotenone separation by heating on the steam bath in a draft of air until no odor of the solvent was detected and then drying the residue a t 105" C. for one hour. It was found that a one-half hour drying was not sufficient. These filtrates were dried in shallow aluminum dishes. The difficulty encountered by Tattersfield and Roach (14) in drying carbon tetrachloride extracts was no doubt due to difficulty in removing the carbon tetrachloride of crystallization from the rotenone. ACCURACY OF METHOD. The accuracy of an extraction method of this type depends primarily on the thoroughness of the extraction and the completeness of the crystallization from the extract, The solubility of rotenone in carbon tetrachloride a t 20" C. is 0.6 gram per 100 cc. of solution, so that the 25 cc. of extract in this method might hold 150 mg. of rotenone in solution. In a 50-gram sample this would amount to 0.3 per cent of the original root. The resinous materials present might be expected to increase this solubility slightly. On the other hand, the fact that the extract is cooled in ice should markedly decrease error from this source.
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In order to determine the approximate accuracy of the carbon tetrachloride extraction method, mixtures were made of different amounts of pure rotenone with a derris root (No. 401) which was known to contain no rotenone. This root had given from 0.2 to 0.3 per cent separated material from ether extractions, but on careful examination this material was found to contain no rotenone. Carbon tetrachloride extractions of the same root gave less than 0.1 per cent separated material, which again was found to contain no rotenone. Mixtures of this root with pure rotenone, containing 0.3, 0.5, 2.0, and 5.0 per cent rotenone, were prepared and extracted by the method in question for 10 hours. No crystalline material could be separated from the 0.3 per cent mixture; the 0.5 per cent mixture gave exactly 0.5 per cent, the 2.0 per cent gave 2.3 per cent in two determinations, and the 5.0 per cent gave 5.2 per cent in two determinations. It would appear from these results that the method is of no value for samples containing 0.3 per cent, or less, rotenone. On the other hand, when sufficient rotenone is present to produce crystallization (over 0.3 per cent), the method gives correct or slightly high results. The latter effect is no doubt due to incomplete washing when large quantities of rotenone are separated. As already noted, however, too much washing is not advisable because of the possibility of loss. It is believed that this method, when properly handled (including examination of the separated crystalline material by the methods outlined to make certain that it is entirely rotenone), and used on samples containing over 0.3 per cent rotenone, will give results which differ from the true values by not more than $0.3 per cent. Probably no extraction method will give more accurate results than this. A chemical method is undoubtedly needed to obtain exact results on the rotenone content. It is suggested that when no rotenone can be crystallized from an extract by this method, a larger sample should be used, in order to reduce the proportion of solvent to rotenone in the evaporated extract. SUPERIORITY OF METHODOVER ETHER EXTRACTION, In general, carbon tetrachloride was found to give a much more selective separation of the rotenone than is possible with ether. Four samples of derris root (Nos. 408, 524, 537, and 739), which gave no rotenone by ether extraction, even when the concentrated extract was seeded with a few crystals of rotenone, readily yielded crystalline rotenone when the carbon tetrachloride method was used, the rotenone amounting in each of the four cases to about 2 per cent of the root. About 1 per cent rotenone was obtained by carbon tetrachloride extraction of a sample of haiari stem (No. 627), which gave no rotenone by ether extraction. On the other hand, a sample of derris root (No. 412) whose ether extract yielded 2 to 3 per cent of amorphous material, in which no rotenone could be detected, gave neither crystalline nor amorphous material from a carbon tetrachloride extract. The ether extract of another sample of derris root (No. 548) deposited about 2 per cent of mixed amorphous and crystalline material which on examination was found to contain only a small proportion of rotenone, whereas only a fraction of 1 per cent of crystalline rotenone separated from the carbon tetrachloride extract. Carbon tetrachloride will also be found superior to ether for extractions under tropical conditions because of its lower volatility. OTHERPOSSIBLE EXTRACTION METHODS I n an attempt to shorten the time of extraction, some experiments were made in which the root was extracted with solvents having a high solubility for rotenone, and the rotenone then crystallized from the dried extract by means of carbon tetrachloride. Acetone proved suitable for this purpose.
ANALYTICAL EDITION
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A derris sample (No. 594-B), yielding 6 per cent rotenone by the carbon tetrachloride method, also gave 6 per cent when extracted for 4 hours with acetone, followed by crystallization from carbon tetrachloride. A cub6 root sample (No. 686-G), giving 6.3 per cent rotenone by the carbon tetrachloride method, gave about the same value when extracted for 4 hours with acetone and the rotenone crystallized from carbon tetrachloride. It is evident that acetone extracts the rotenone in a much shorter time than carbon tetrachloride, probably because it is a better solvent for the resinous material in which the rotenone may be incorporated. However, the total material extracted by acetone amounts to more than that extracted by carbon tetrachloride, Consequently, in extractions such as the two just cited, a very considerable proportion of the dried acetone extract was insoluble in hot carbon tetrachloride. This necessitated filtration and numerous washings of the dried extract with hot carbon tetrachloride. The rotenone obtained in several tests made by this method was less pure, and, probably because of this, amounted to several tenths of a per cent more than that obtained by the carbon tetrachloride extraction method. This modified method thus has certain objections, although it may be useful when a rapid but approximate estimate of the rotenone content is desired. Because of the high optical rotation of rotenone (in benzene [or]'%" = -224" for a 5 per cent solution) it was thought that a method for its determination might be based on this phenomenon. Extractions were made with such solvents as acetone, benzene, chloroform, and ethylene dichloride. The extracts were made to a definite volume, and the optical rotation was measured in a saccharimeter. By use of data previously obtained (11) the amount of rotenone represented by the rotation was calculated. The values obtained in this way for rotenone in two samples of derris root (Nos. 402 and 407) and one sample of cub6 root (No. 584) were about twice those obtained by crystallization from ether or carbon tetrachloride. The optical rotation of an extract of one derris root (No. 412) indicated a content of over 5 per cent rotenone and that of another (No. 406) over 3 per cent, whereas neither of these samples gave any rotenone by crystallization from carbon tetrachloride extracts. The extract of one derris sample (No. 401) was dextrorotatory. Such erroneous values as the above may be expected, since there are numerous variable constituents of these extracts whose optical rotations are not known. It is thus evident that the optical rotation of the extract cannot be used as a measure of the amount of the rotenone present in the root. COXCLUSIONS The carbon tetrachloride extraction method outlined gives good results for the rotenone content of thoroughly airdried derris roots, cub6 roots, and haiari stems. The results of numerous extractions indicate that this method is superior to a similar method using ether. The method gives correct or slightly high results for roots containing over 0.5 per cent rotenone. For roots containing 0.3 per cent rotenone or less the method is without value unless larger samples are used. Acetone gives a more rapid extraction of the rotenone, but its complete separation from such extracts is difficult. Values based on the optical rotation of the extracts are incorrect. A purely chemical method for the accurate determination of rotenone in plant materials is needed. LITERATURE CITED (1) Clark, J . Am. C h m . Soc., 52, 2461-64 (1930). (2) Clark, Ibid., 53, 313-17 (1931).
(3) (4) (5) (6) (7)
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Clark, Ibid., 53, 729-32 (1931). Darley, J . Econ. Entomol., 24, 111-15 (1931). Davidson, Ibid., 23, 868-74 (1930). Davidson, Ibid., 23, 877-79 (1930). Fryer, Stenton, Tattersfield, and Roach, Ann. Appl. Biol., 10,
18-34 (1923). (8) Georgi, Malayan Agr. J., 17, 361-63 (1929). (9) Georgi and Curtler, Ibid., 17, 326-34 (1929). (10) Jones, J. Am. Chem. Soc., 53, 273-1 (1931). (11) Jones and Smith, Ibid., 52, 2554-02 (1930). (12) Roark, Soap, 7, 97, 99, 101 (1931). (13) Shepard and Campbell, J. Econ. Entomol., 25, 1 4 2 4 4 (1932). (14) Tattersfield and Roach, Ann. A p p l . B i d , 10 1-17 (1923). RECEIVED August 30, 1932.
New Reagent for Deterrnination of Zinc ARMAND J. QUICK Cornel1 University Medical College, New York, N. Y.
Z
INC is readily precipitated from an acid or a neutral solution by means of borneolglycuronic acid. Since this acid does not form insoluble salts with the other common metals, except cadmium, it offers an easy method not only for detecting zinc, but also for separating it from a mixture of other cations. Zinc in as low a concentration as 0.03 per cent will give a characteristic crystalline precipitate when treated with a 5 per cent aqueous solution of borneolglycuronic acid. Zinc borneolglycuronate is a white glistening salt containing 2 molecules of water of crystallization. Andusis. Calculated for (C16H2601)2Zn.2H?O: glycuronic acid, 51.1. Found: 50.8, 51.5. The development of a simple and satisfactory quantitative method for zinc based on the precipitation of zinc borneolglycuronate is feasible and promising. The precipitate can be weighed directly, or it can be hydrolyzed by boiling with 1.0 N hydrochloric acid for 15 minutes. I n this procedure, borneolglycuronic acid is split, and the glycuronic acid thus liberated can be determined by any of the common sugar methods, of which the Shaffer-Hartmann (2) is especially satisfactory From the determined glycuronic acid, zinc can be calculated. The reagent, borneolglycuronic acid, is best prepared by the method described by the writer (1). For the convenience of the reader, the essential details of the method are again outlined: Five grams of pulverized borneol are fed to a dog. The drug can be mixed directly with the food. The urine is collected for 24 hours, acidified, and treated with excess lead acetate to remove pigments. The precipitate is filtered off, and the filtrate heated to boiling. An excess of zinc acetate solution is added, and the precipitated zinc borneolglycuronate filtered off and washed with hot water until all coloring matter has been extracted. To prepare borneolglycuronic acid, 100 grams of the zinc salt are dissolved in 140 cc. of hot 3.5 N sulfuric acid. The solution is rapidly cooled in ice and allowed to stand for several hours. The crystalline borneolglycuronic acid is filtered off and washed with cold water. One recrystallization from hot water is necessary to obtain a pure product. LITERATURE CITED (1) Quick, A. J., J . Biol. Chem., 74, 331 (1927). (2) Shaffer. P., and Hartmann, A., Ibid., 45, 365 (1920-21). RECEIVED August 2, 1932.
