Ho w a r d A. Jo n e s, Insecticide Division, Bureau of Chemistry and Soils, Washington, D. C.
T
H E insecticide rotenone (CaHaOo) is known to occur in a number of fish- poisoning plant materials, such as the roots of various species of Deguelia (derris root) found in the M alay Peninsula and the E ast Indies, the roots of Lonchocarpus nicou (locally termed cubé root) of Peru, and the stems of the haiari vine, a species of Lonchocarpus native to the Guianas.
In 1923 Tattersfield and Roach (14) first proposed a chemi
cal 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 th a t rotenone contained methoxyl groups and it was felt th a t this would prove the genuineness of the extract.
These investigators found th a t 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 th a t proposed by Tattersfield and Roach, except th a t the determination of methoxyl content was omitted, was used by Georgi and Curtler (S, 9) 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 th a t the ether extracts of both derris and cubé root contain a number of compounds of varying toxicity.
I t has also been found th a t several of these—i. e., toxicarol (1), deguelin (2), and tephrosin (S'), 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 cubé 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, 5, 18), many manufacturers may wish to stand
ardize 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, a determination of the amount of rotenone in a root should prove of consider
able value.
Et h e r Ex t r a c t i o n Me t h o d
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 re
cently outlined by Roark (12). 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 pro
duce crystallization of the rotenone and in some cases no rotenone could be obtained from the ether extract, whereas,
as will be shown later, a con
siderable quantity actually was present. Other ether extracts deposited amorphous material, which interfered with the filtra
tion and gave erroneous results for rotenone.
Accordingly, a s e a r c h w as made for a solvent which would give a more selective separa
tion of rotenone. A material w-as 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 tetra
chloride, 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. I t is interesting to note that, although Tattersfield and Roach stated th a t “benzene, dry ether, and carbon tetrachloride...have a selective dis
solving action on the poisons,” they found difficulty in drying extracts made with carbon tetrachloride and consequently abandoned the use of this solvent.
Duplicate extractions wrere made on about tw enty sam
ples of derris root, ten samples of cubé root, and two sam
ples of haiari stem (the three sources of rotenone investi
gated 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.
Ca r b o n Te t r a c h l o r i d e Ex t r a c t i o n Me t h o d
Fifty grams of plant material ground to about 20 mesh are completely extracted in a large Soxhlet extraction ap
paratus 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). I t is suggested th a t the extraction be run overnight (about 17 hours), thus accomplishing a reduction in the actual working time con
sumed 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 rotenone- carbon 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 tetra
chloride in small portions. As much as possible of the ex
cess 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 Derris root and cubé root 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 extrac
tion method previously in use.
24 A N A L Y T I C A L E D I T I O N Vol. 5, No. 1 freed from carbon tetrachloride by evaporation and dried for one hour a t 105° C. This weight added to the weight of rotenone gives the total carbon tetrachloride extract (without solvent of crystallization).
Sh o r t e n e d Pr o c e d u r e
Reasonably complete crystallization of rotenone from the evaporated extract m ay be obtained by cooling the extract, whole method is thus reduced to 24 hours or one actual work
ing day. This shortened procedure will give sufficiently good results for many purposes, although the longer method
chloride extraction. Thus, the value for rotenone obtained from one sample of derris (No. 402)1 was lowered from 1.4 per cent to 0.9 per cent by drying the root in a vacuum oven a t 100° C. for 5 hours. Furthermore, the rotenone obtained by extraction of the dried material was tan in color, while th a t from corresponding extractions, made without previous drying, was white. Similar results were obtained on this same sample by the ether extraction method. I t lead to incomplete extraction, whereas finer grinding ap
peared unnecessary.
We ig h t o f Sa m p l e. In using the original ether extrac
tion method it was necessary in many cases to use 100 grams of sample in order to produce crystallization of the rotenone.
However, since the usual large-size Soxhlet apparatus con
veniently holds only 50 grams of ground root, it was de
sirable to use a sample of this smaller size. No difficulty has been experienced in obtaining crystallization from ex
tracts made by the carbon tetrachloride method of 50 grams per cent rotenone in 8-hour extractions by this method, gave no more rotenone on additional 7-hour extractions. How
ever, samples giving over 5 to 6 per cent rotenone in an 8- hour period should receive further extraction. For exam
ple, a sample of cubé root (No. 686-A) yielding 10.2 per cent rotenone in 8 hours, gave 1.0 per cent more upon an addi
tional 7-hour extraction.
Some carbon tetrachloride extractions were followed by short acetone extractions with the idea th a t any remaining rotenone would be extracted by this solvent, since acetone is a good solvent not only for rotenone, but also for the resinous material in which the rotenone may be bound. One sample of derris root (No. 522) extracted 10 hours with car
bon tetrachloride gave 6.7 per cent rotenone. The marc was extracted for 4 hours with acetone, the extract evaporated to dryness, taken up in hot carbon tetrachloride, filtered to remove insoluble material, and allowed to cool. No further rotenone separated. On the other hand, another sample (No. 594-B) gave about 6 per cent rotenone on a 10-hour carbon tetrachloride extraction, with 0.3 per cent additional impure rotenone on acetone extraction. A third sample of root (No. 956) gave about 6 per cent in 10 hours with carbon tetrachloride and an additional 1 per cent with ace
tone. A 17-hour (overnight) carbon tetrachloride extrac
tion of this last sample gave the full 7 per cent rotenone,
chloride was kept actively boiling throughout the extrac
tions. Under these conditions the temperature of the solvent surrounding the root remained a t about 70°.
Cr y s t a l l i z a t i o n a n d Fil t r a t i o n o f t h e Ro t e n o n e.
In all extractions made in this laboratory, the rotenone, when present in quantities larger than 0.3 per cent, has readily crystallized from the evaporated carbon tetrachloride ex
tract on cooling. In a few cases, when less than 1 per cent of rotenone was present, it was necessary to seed the cold extract with a small crystal of the solvate, and when this was done, the crystalline material separated quickly. Usually scratching the inside of the flask with a glass rod was sufficient to induce crystallization and, in most cases, even this was concentrated extract in a refrigerator overnight.
The separated rotenone should be washed with as small point in any quantitative extraction method. Something in the nature of a check result may be obtained by heating the separated solvate compound to drive off the carbon tetrachloride of crystallization and reweighing as pure ro
tenone. This may be accomplished by heating the separated crystalline material for one hour a t 105° C ., when the ma
terial usually “melts” to a glassy mass (due to the presence
January 15, 1933 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y 25 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 th at 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.
In all extractions made in this laboratory a small por
tion of the separated crystalline material was recrystallized, and microscopic examination and melting point determina
tions were made. At first, ethyl alcohol was used for this purpose, but it was found th a t in many instances alcohol gave odd-shaped lathlike crystals with erroneous melting points when the substance was actually known to be quite 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 cubé—i. e., toxicarol, deguelin, and tephrosin—if present in the separated crystal
line material, would be recognizable by their different crystal
lography properties, but their presence has not been de
tected. According to Clark’s work (1, 2, 8) on these ma
terials, their presence in the separated crystalline material would be unlikely, as they are ordinarily obtained by alkali
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 crystal
lization from the rotenone. 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.
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
none 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, how
ever, too much washing is not advisable because of the possi
bility of loss. I t is believed th at this method, when properly handled (including examination of the separated crystalline material by the methods outlined to make certain th a t it used, in order to reduce the proportion of solvent to rotenone in the evaporated extract. 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 in wiiich no rotenone could be detected, gave neither crystal
line 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 w'as 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 low'er volatility.
Ot h e r Po s s i b l e Ex t r a c t i o n M e t h o d s
In an attem pt to shorten the time of extraction, some experiments w'ere made in wiiich the root was extracted with solvents having a high solubility for rotenone, and the rote
none then crystallized from the dried extract by means of car
bon tetrachloride. Acetone proved suitable for this purpose.
26 A N A L Y T I C A L E D I T I O N Vol. 5, No. 1 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 crystal
lization from carbon tetrachloride. A cubé root sample (No. 686-G), giving 6.3 per cent rotenone by the carbon tetrachloride method, gave about the same value when ex
tracted for 4 hours with acetone and the rotenone crystallized from carbon tetrachloride. I t is evident th a t acetone ex
tracts 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 th at extracted by carbon tetrachloride. Con
sequently, 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 be
cause of this, amounted to several tenths of a per cent more than th a t obtained by the carbon tetrachloride extraction method. Tins 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 ben
zene [ a ] j ’ = —224° for a 5 per cent solution) it was thought th at 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 opti
cal rotation was measured in a saccharimeter. By use of data previously obtained (11) the amount of rotenone repre
sented 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 cubé 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 th a t 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. I t is thus evident th a t the optical rotation of the extract cannot be used as a measure of the amount of the rotenone present in the root.
Co n c l u s i o n s
The carbon tetrachloride extraction method outlined gives good results for the rotenone content of thoroughly air- dried derris roots, cubé roots, and haiari stems. The re
sults of numerous extractions indicate th a t 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 contain
ing 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, b ut its complete separation from such extracts is difficult.
Values based on the optical rotation of the extracts are in
correct.
A purely chemical method for the accurate determination of rotenone in plant materials is needed.
Li t e r a t u r e Ci t e d
(1) Clark, J . A m . Chem. Soc., 52, 2 4 6 1 -6 4 (1930).
(2) Clark, Ibid., 53, 3 1 3 -1 7 (1931).
(3) Clark, Ib id ., 53, 72 9 -3 2 (1931).
(4) D arley, J . Econ. E nlom ol., 24, 111-15 (1931).
(5) D avid son , Ib id ., 23, 8 6 8 -7 4 (1930).
(6) D avid son , Ib id ., 23, 8 7 7 -7 9 (1930).
(7) Fryer, S tenton, Tattersfield, and R oach, A n n . A p p l. Biol., 10, 1 8-34 (1923).
(S) G eorgi, M a la y a n Apr. J ., 17, 36 1 -6 3 (1929).
(9) Georgi and Curtler, Ib id ., 17, 3 2 6 -3 4 (1929).
(10) Jones, J . A m . Chem. Soc., 53, 2738—11 (1931).
(11) Jones and Sm ith, Ib id ., 52, 255 4 -6 2 (1930).
(12) R oark, Soap, 7, 97, 99, 101 (1931).
(13) Shepard and Cam pbell, J . Econ. Enlom ol., 25, 142-44' (1932).
(14) Tattersfield and R oach, A n n . A p p l. B iol., 10. 1-17 (1923).
R e c e iv e d August 30, 1932.