T2 i'L.,' lO^1,-'
April 1942, -563
A REVIEW OF METHODS FOR THE
CHEMICGAL'ANALYSIS OF ROTiNONEOi-BEARING PLANTS
By Howard A. Jones, Division of Insecticide Investigations
Introduction - - - - - - - - - - - - 2
Sampling - - - - - - - - - - - - - - 2
Moisture - - - - - - - - - - - - - - 5
Total extract - - - - - - - --- - - - - 7
Rotenone ----------- --- - - -- ----- - 14
Degurlin and rctenonu plus -egue3in - - - - - - - 48
Toxicarol and other alkali-sol-uble su-.bstances - - - - 59
Yiscellanoous oolorimnetric determinations - - - - - 61
Ot"ier determinations - - - - - --- - - ----66
Literature cited - - - - - - - - - - - -71
. u I.*
An attempt has been made to collect all references to methods used
for the chemical analysis of rotenone-btaring plants, particularly of
derris and c:'e roots. Th! compilation has been confined to articles
describing methods or critically copa.rinr results obtpirn.ed by different
methods. ;,eferenc3s jgiring only results of analysis or reviews ccntzining
no ori-'in-rl contributions or critical observations h niv rot been incs.'ed.
Likewise methods of utiliinr the results of chemical anl-rsis, directly
or through various calculations, to obtain a measure of insecticidal effec-
tiveness are-not treated here. This Froblom is itself sufficiently dis-
tinct and important to require sep rate treatment.
The methods discuss-?d here have been ,rouped rrin.qrilr according
to the substances which tV-o- determine. This classification, althou0:h
difficult because of uncertainty ss to .us't what substances are determined
bz, marLy of th9 methods, was believed to be the most useful. At the end
of the sections on methods for detsrmir.in: the more ir.ortant constituents,
a brief statement has been ma.e of the oreeer.t status' cf. methods for the
purpose. In the last two sections are -rc ,;, d certain methcds that give
more or less empirical values, which it is difficult or imoossible to
interpret in terrs of actual substances presei.t in the sample.
In this section on!l- those fe-iw articles are reviewed which jive
sroecific details on the surplin- of eerris or cut-. rcot for an-lysis.
Georgi said Teik (Z4) in 1933 su,-ge ted a trocea-I-e to be followed
in selecting a s m ele for analysis from a shipment of derris root.
In 1j3 these authors (35) elaborated the method somewhat, so that,
for the preparation of fresh roots, the procedure was as follows:
"Fresh roots are sun-dried until they can be broken
without exudation of plant-juices. The semi-'ried roots are
then cut into pieces var-lnr from 1/4 to 1/2 inch long and the
chor.ppe material is f-.rther sun-Sried until of constant wei-ht.
The m-*' "'al is then q'i. -tered for an--lysis until 'oproximntely
1/4 lb. remains. One hun:d r.-d..; s are weij'ed, then rerrund in
a drtg mill. The root is first passeJ thro' a e is. rnt':-tor
having snces l/38 inch wide, the snrc-i; -.in. 4w::.'?tly
1/4 inch apart. Th-" materi1. is -ro-ur- e second time, x',.- dis-
inte,- tor beinr r pl"-ee( by a 1 m. sieve. The v:--il r-ount
of wo;.y matter ren: inin is c)rri;:d "e ')r.itely I2, a !--n<_ I ill
and incorn'oratsd with th"e ain ,"-.'le. T o 'rir.. a "'re,.ntative
eam.pl-! of the 'ro.r:'4 root for anr.:'.sis, the various fr-'.Ctions are
shaken to-ether in a jar prcviler with c clo. .-13y-fittirln lid, and
tho required aniounit of material is wei-,hed before sagrer.otion of
the fine particles has token ploce."
In sampling small shipments it was propr.sed that !1 pound 'of roots "be
withdrawn at random.from a bale of 200 .pounds. In the case of a large
shipment it was suggested that -10 percent -of the bales be .s-'S'. 'ed and 5
percent by weight of each of these tales be removed. The roots were to
be chopped into short lengths and quartered until 1 around remained.
Cahn and Boam (13) in 1955, in an article on the determination
of rotenone, made the following statement in regard to sampling:
"Derris root should pass entirely through a 50-mesh sieve
before it is sampled. Root, as ordinarily ground, frequently con-
tains a proportion of coarse, spicular material, through which
the powder (often e:rtrenely fine) tends to filter, thus causing;
an undue amount of coarse pnrticles to be present in' the top
layers. If the amount of spicular material is consi er.-le,
the whole batch should be sieved-through a 50-mcsh sieve, and
the samples taken fro: propArtionate counts of the fine arid
Cahn (12) again in 1936 stressed the necessity of proper sampling.
He found thn.t, of 10 bales anly7.ed out of a cons; fuie-t of 40 bales
from the sarne estate, the total extract rar.-ed from 15 to 25 percent.
The only satisfactory method of saaplin.7 derris root was said to 1- to
grind it first, mix it thorou.,ly, ar-6 then ar.l-le their mi:ed fine powder.
G-orgi (3l)I in 1937 described a method used in snmaling kiln-dried
root during the process of tailn".. Vhen half the quantity required for
a bale had been loaded into the press, about Z pounds of the root was
taken at random from the heap and. set aside as a sample from that b:le.
When 20 sampl3s, say 40 ,ourds, had accumulated, the bulk sample of root
was mixe-3d, spread out in a thin liyer on the floor, an' quartered until
about 1 pound remained. This arooixrt was cut into 1-inch len-ths and
quartered until atout 3 ounces remair.ed. This quantity was further cut
into 1/4-inch lengths and ground in the laboratory mill for analysis.
In one case 25 samples covering 497 bales were annlys-ed.
In 1937 Levallois (82) stated that the sampling of rotenone-
bearing roots was very delicate, as the rotenone0content of a sample
depended on the abundance of long fibers. Progressive grinding of roots
gave fractions with rotenone contents as follows: triable parts and
short fibers 6, medium parts 4-to 4.5., and. long fibers 3 to 4 percent.
This author did not take samples of derris or cube at rardom, but
classed root in three or four categories according to size aiLd sampled
accordingly. From each lot ha took an aliquot part of 1/10 to 1/20 and
chopped up these parts. From each pa.rt h. took a new fraction. 7--,eese
fractions made up the samples for analysis.
Chevalier and Chevalier (:,0) recor.mendid that, since large and small
roots differ in rotencne and total-extract contest, samples tpken for
analysis should, have about the same proportion of large and small roots as
the whole lot.
Krukoff and Smith (80) in 1937 reported that, if a lot of crude
Lonchocarpus roots is a mixture of different species, no sample will
accurately represent the entire lot. In sampling large, unbaled lots of
a single species, fairly accurate results were obtained by making a care-
ful ocular estimate of the weight of small, medium, and large roots in
a given lot and taking a corresponding sample. For smaller lots, such
as roots from a single plant, the different sizes were sorted out, weighed,
and the sample was taken accordingly. This method would be impractical
for sampling baled shipments as they arrive in this country. In the pre-
paration of samples of fresh roots for analysis more uniform results were
obtained by drying them to about 12 percent moisture before reducing them
to slices or shavings.
Guillaume and Hervg (53) in 1939 emphasized the importance of obtain-
ing a representative sample in the analysis of plants containing rotenone.
They suggested that a sample of at least I kg. of root be taken for grinding.
The ground material should be mixed and sifted and then mixed again at the
moment of sampling.
The Puerto Rico Experiment Station (128) recently reported that the
already difficult problem of sampling baled derris root was further com-
plicated by the variation in rotenone and total-extract content of roots
of the same diameter found in worK at this station. It was suggested that
a representative sample of the baled product could be obtained by using a
small tube with a rotary cutter at the sampling end. Such a tool would
make it possible to take a fairly large number of small samples and thus
remove from the bale the minimum amount of root material required for a
Information on sampling was recently furnished by four large import-
ers of derris and cube root: McCormick and Company, S. B. Penick and
Company, John Powell and Company, Inc., and Derris, Incorporated (private
communications). These firms agreed on the matter of sampling about 10
percent of the shipment, although S. B. Penick and Company stated that
frequently half, or even all, the bales in a shipment were sampled. John
Powell and Company stated that where possible they preferred to grind at
least one-tenth of the shipment. Two sources described the methods employed
in removing samples of whole root. Derris, Incorporated, stated that 10
percent of the bales were opened and small bundles of root picked out at
random from various sections of each bale. This material was ground, mixed,
and a 1-pound sample drawn for testing. McCormick and Company removed from
each of the bales chosen three small bundles of root, and from each bundle
chopped sections about 4 inches long from the fine ends, the large ends,
and the middle. These sections were used to make a composite sample for
The sampling of derris and cube roots for analysis is very important.
The proper sampling of whole root is extremely difficult, and it is doubted
if any method is entirely satisfactory. In addition to differences in
rotenone and total-extract content of different roots in a shipment due to
differences in plant source, it has also been shown that fine and coarse
- 5 -
roots of both derris and. cube vary in their content of rotenone and total
extract (32, 33, 63, 64), Hence, in .:mplnr.g: A whole-root shipment care
must be taken not only to draw'fromr different parts of the bale or case,
but also to obtain a sample having approximately the same proportion of
fine and coarse roots as does the whole shipment, This proceed, e was
recommended by Chevalier and Chevalier (20) and by Krukoff and T-.th (80).
The method cited above as used by one American importer is an attempt to
The proper sa.Tplix of powdered root is no less important, although
less difficult. Cshn and Boam (13),and other workers have pointed out
that marked.segregation of fine and coarse particles occurs on standing.
In addition to Leva.llois (82) and others, Kcolhaas and MeijerI have
shown large differences in the rotenone and total-extract content of fine
and coarse powder from a single sample. They found in a sample prepared
by them that the material passing an 80-mesh sieve analyzed 9.8 percent
of rotenone and 19.6 percent of total ether extract, while the coarse
material, which did not pass the sieve, had 1.3 percent of rotenone and
4.0 percent of ether extract. This variation indicates the importance of
correct sampling of powdered root, particularly material that has been
subjected to shipment *
In this section are discussed only procedures for the determination
of moisture. Methods of drying the sample before extraction, which are not
actual moisture determinations, are discussed in connection with the various
rotenone and total-extract methods.
Tattersfield and Roach (122) in 1923, in determining moisture in
samples of Derris elliptica, dried the materials to constant weight at the
temperature of boiling dichloroethylene (550-58 C.) in a partial vacuum
over phosphorus pentoxide. Elevated temperature was avoided, as the powdered
root was said to decompose on prolonged heating.
A similar method was used by Georgi and Curtler (32) in 1929 in
analyzing derris roots.
Spoon (115) in 1931 determined moisture in derris by heating 5 grams
of the ground root at I00-102 C.
Geergi and Teik (34) stated in 1933 that moisture determinations
might be made either by drying the finely ground material to constant weight
in a steam oven at 100 C. or by distilling with xylene. In the former
method a 5-gm. sample was used,, while in the distillation method 40 to 50
gm. was employed. The distillation method was preferred, as it was said to
eliminate oxidation during drying.
- - - --- - - -
1 Report of the analysis of a sample of derris root by various laboratories.
19 pp., typewritten. Jan.. 19,, 1937.
- 6 -
ipttersfield and Martin (120). in their work on evaluation of roten-
cne-containing plants in 1935, reported- determinations of moisture by these
two methods. The xylene-distillation method was u"ased"in Mala.ya, while the
saine samples were analyzed for moisture in 3ngljnd.!.by drying at 1009 C..
Hiieher-results- were-obtained in M'alaya, but the -authors attributed the dif-
ference to greater absorption of atmospheric- moisture there., -'
Again in 1936 CGeorgi and Teik (75) stateA. that rcisture was best
determined by the xylene-distillation met'.c., but could also be determined
by drying to constant weight at 10' C.
-oolhaas and :.ei.jer2 in l97'in a report on-'the analysis of a sample
.of derris root by-various laboratories througha-,.t the world, gave the fol-
lowing results for moist'--e as 6etermrined by the several laboratories:
.. ... ... .- Moisture
SLaboratory . ethod ?. ; Parcent
A--United States Department of .Ariculturle
(Jones) .... ...
B--"enpartmrent of Agriculture, traits Set- X
tiements and Federatad I'alv.- States (Georgi
C--Commercial "useumn of the Colonial X
Institute, Amsterdam (Rowaan). ..
D--The Cooper, Tec'-nical Bureau, London
(Cahn and Boam)
Tied at- 13-1040 C.. 6.1
-for 2 hours ... -
.-lene distillation -. 1.3
)ried at. 100 0
E--Rothamsted Experimental- Station (Tat- Dried at 1010 C.
*tersfield and Martin *
F--Sell,. Putt, and Rus'yj, New.York (Seil)
. ... 9.9
G--Caeser and Lorentz, Halle-Saale, Germany Over calcium chloride
S. -, ... .. for 2 days. ,
H--Diethelm, Ltd., Si,-)pore
I--Lao,oratcv-y for Chemical Research, iuiten-
zorg, Java (Koolhaas and Meijer)
Dried at 85 C.
ried at 10,5 -,.
2 See footnote 1,
They concluded that moisture determ.I ationr sioild be mide by drying a 3--m.
sample at 105 0. t'o'constonrit weight. The xylene or heptane method gave
higher values, but the, higher temperature..to which the material was exposed
was thought to lead' to decomlositiorn, and the method was not recommended.
Jones and Graiam (71) in 1938, in the analysis of a large number of
derris, cube, and timbo roots, determined moisture by drying 2-gm. samples
at 106 C. for 2 hours. Two days' additional drying of some of the-samples
caused insignificant additional loss, in weight.
Results for moisture content of derris root by heating in the oven
and by toluene distillation were compared in a report of collaborative
work by the Imperial Institute and. the.Rothamsted Exoerimental Station (59).
Values bythe oven method were only slightly lower than those by the dis-
tillat tion meth6d..
Mei.jer and Roolhaas (89). in 1940 determined .the moisture content of
powdered derris root by drying a 2-. .to 3-gm. smole to constant weight at
1050 C. .. "
Of some interest in connection with the determination of the moisture
content of derris powder is the hygrroscopicity of the material. leeijer (87)
in 1938 studied this question by keepin,7 samples at different relative
humidities at approximately room temr.p3ratur3 .and neasurinrj the increase in
weight. After standing at 75 percent relative humidity roots originally'
having 6 to 8 percent of moisture were fouxid 1to co.ta,.in 11 to 12 percent of
moisture, while at 95 percent relative humidity these roots had about 24
percent of moisture. He concluded that powdered derris root was "freely
ILt may be concluded from the references given that the most suitable
method for determining moisture is the drying of a 2- to 5-gm. sample at
about 1050 0. to constant.weight. This should require only a few hours in
many cases, but overnight drying before the first weighing should be a good
Ihe determination.of the total-extract content-of derris and cube
roots, although a comparatively simple procedure and one that has been made
from the time of the earliest work on these materials,, is nevertheless
subject to considerable variation.
Tattersfield and Eoach (122) in 1923, in the course of an investi-
gation of Deri'is' elliptica, pointed out that, the following factors are to '
be considered in the determination of total extract: -.
(1) The- extraction solvent must be selective.
(2) The root must be ground to an almost impalpable powder.
(3) Extraction must take place at fairly low temperatures;
otherwise soarinplv soluble comDounds are formed.
(4) The extract undergoes chemical change on drying.
(5) The type of extraction flask may modify results.
(6) The extract is freed from the last traces of solvent with
They proposed the extraction of the root in a Soxhiet apparatus, using a
flat-bottomed flask, with ether (dried over anhydrous calcium chloride and
sodium), and drying the extract as rapidly as possible to constant weight
of 100 C.
A similar method was used by Georgi ard Curtler (32) in 1929. They
also pointed out that slight decomposition took place; on drying the extract,
and the results were slightly low.
In the 1929 report of the Colonial Institute of Amsterdam (77) it
was stated that when a large sample of root had been ground once to a
coarse condition, a portion of this sample ground again to a finer state,
and some of the latter ground a tnird time to a very fine powder, the
amount of ether extract was found to be higher as the root was more finely
Dodwell and Company (private communication) in 1930 outlined the
method in use in the trade for determining total extract of derris root.
Ten grams of powdered root was dried in a vacuum over sulfuric acid and
extracted exhaustively in a Scxhlet apparatus with dry ether, free from
alcohol. The powder was then reground in a mortar and extracted for an
additional 4 hours. The extract was dried for about 1/2 hour in a water
oven. Later Dodwell and Company. (private communication) quoted a revised
method for total extract in which the root was dried at 100 C. to constant
weight and then extracted with dry, alcohol-free ether until exhausted.
No second treatment was used. The extract was dried to constant weight at
Total ether extract was determined by Spoon (115) in 1931 by extract-
ing 5 gm. in a Soxhlet with absolute ether for 15 hours and drying the
extractives at l0.0-l0-2 0.
Koolhass's (78) ether extraction method for rotenone included the
determination of total extract. The filtrate and washings from the rotenone
separation were evaporated, heated at 80 C. in a vacuum for 1/2 hour, dried
in a desiccator, and weighed. Their weight was added to that of the crude
The method for rotenone proposed by Jones (62) in 1933 and involving
extraction of the root with carbon tetrachloride included a procedure for
total extract, by which the filtrate from the rotenone separation, upon
evaporation, was dried 1 hour at 105 C. and its weight added to that of
the separated rotenone.
Georgi and Telk (34) in 1933 compared other solvents with ether
for the determination of total extract- of derris root. Petroleum ether
was unsatisfactory, since its solvent action was so low. Acetone'gave'"t
higher value than ether. Slightly higher figures, were obtained with
chloroform, and slightly lower results with carbon tetrachloride than with
ether. Preliminary drying of the root did not change the amount of ether
In a comparison of. chemical composition and toxicity to insects of
rotenone-bearing plants Jones, Campbell, and Sullivan (69) in 1935 deter-
mined total carbon tetrachloride extractives of several samples of derris
and cube by the method cf Jones (62). Acetone and benzene e.:tractives
were pre-pared by Soxhiet extraction of 10- to 20-onr. r3znples for 7 to
8 hours. In general the amount of material extrEctea with acetone was
higher than that with carbon tetrachloride, while that with benzene was
Tattersfield and "'artin (120) in 1935 ave results of the determina-
tion of total ether extract on several samples of derris. In the extrac-
tion of 5-gm. samples with ordinary ether slightly higher results were ob-
tained in Malaya than in EnE-lan.d. The results that Vere obtained with
sodium-dried ether in England were slightly lower.than those obtained by
the use of ordinary ether.
In proposing their trichloroethylene extraction method for rotenone,
Cahn and Boam (13) in 1935 stated that total extract could be determined
by evaporation of the trichloroethylene solution to dryness and heating to
constant weight in an oven at 100 C.
In 1936 Georgi and Teik (35) gave directions, for the determination
of ether extract of derris root. Five grams of the' finely powdered root
was extracted with ether in a Soxhlet for 16 hours. No appreciable addi-
tional extract was recovered when the marc was reground with sand and again
extracted with ether. The ethereal solution was,-filtered if necessary to
remove tiaces of .suspended matter, the solvent distilled off, and the
residue dried to constant weight in a steam oven. Constant weight was
usually reached in 6 hours. .
Meijer (85) in 1936 proposed a colorimetric method for the approxi-
mate determination of the total extract of derris root. This method is
described in the section on Viscellaneous Colorimetric Determinations.
'.orsley (132) in 1937 determined total ether extract by extraction
of a 10-am. sample in a Soxhlet for 24 hours an.d subsequent drying of the
extract to constar.t weight at 100 C.
Guillaume and Proeschel (54) in 1937 determined total ether and ace-
tone extract of derris and other plants by treatment of 5-gn. samples in
a specially designed continuous percolator. Only 150 cc. of solvent was
required, and the extraction was said to be complete in 12 hours. 3x-
tractions with chloroform and carbon tetrachloride were made in a contin-
uous percolator of different desi2-n. All extracts were filtered, con-
centrated to about 20 cc., placed in the refrigerator until the solvent
S- 10 -'
evaporated, and then dried at 500- C. for 12 hours and weighed. Values for
acetone extracts were highest, those for ether and chloroform lower and
about equal to each other, and those for carbon tetrachloride generally
Koolhaas and :.:eijer in 1937 reported on the analysis of a sample
of derris root by several laboratories in various parts of the world.
Some of the methods and results for. total extract were briefly as follows.
A--United States Department of
B--Deoartment of Agriculture,
Straits Settlements and Fed-
erated Malar States (Georri)
C--Commercial Museum of the
Colonial Institute, Amsterdam
D--The Cooper Technical Bureau,
London (Cahn and Boam)
E--Rothamnsted Experimental Station
(Tattersfield and Martin)
F--Seil, Putt, and Rusby, New York
25 gm. benzene, 2 periods
totaling 24 hours
5 gm. ether, 18 hours
5 gm. ether, 48 hours
10 gm. ether, 3 periods
totaling 96 hours
5 gm. Analar ether, 3 periods
totaling 30 hours
5 gm. anhydrous ether, 2
periods, totaling 16 hours
5 gm. ether, until exhausted
G--Caeser and Lorentz, Ealle-Saale, 5 gm. ether
H--Diethelm,. Ltd., Singapore
I--Laboratory for Chemical Re-
search, Buitenhorg, Java
(Koolhaas and Meijer)
25 gmn. ether
50 gm. ether, 2 periods
totaling 48 hours
3 See footnote 1.
- 11 -
The authors also reported their own results on 81 samples of derris.
Results using ether were in close agreement with those using benzene, but
thetlme required for exhaustive extraction with ether was 2 das and 3
nights, whereas benzene required only 36 hours.
In discussing the determination of total extract Meijer (86) in
1937 recommended slow percolation of derris powder with ether in a Soxhiet.
This was accomplished by placing the powder in the extraction tube of the
apparatus without using a thimble. He also stated that drying of the
extract, after removal of most of the solvent, was better accomplished at
400 C. 'under reduced pressure than'by the''usual drying at; 100 C.
Jones and Graham (71) in 1938 determined total benzene extract by
Soxhiet extraction of 5-gm. samples.
The determination of total-extract content was studied by Jones 'and
Sullivan (76), in 1938, in an endeavor to select the solvent and method
that would most,readily extract all the toxic Substances. from derris and
cube roots together with.: the least' amount of nontoxic material.,, The total-
extract content of several samples wag determined by several procedures
with various solvents., Successive extracts of some of the marcs with
acetone, methyl. .alcohol,.and water were tested against mosquito larvae.
In 7-hour Soxhlet extractions of 5-gn. samples the percentages of material
extracted by benzene, carbon tetrachloride, and ether were generally lower
than by .chloroform,, ethylene !dichloride, trichloroethylene, and ethyl
acetate, which gave values, of about equal magnitude. Acetone generally
extracted more material and; methyl alcohol considerably, more than did the
other solvents. When samples were extracted with ether or benzene for an
extended length of time, the amount of material removed agreed with that
extracted by chloroform.- Acetone extracts of marc from extraction with
chloroform were in general nontoxic. Methyl alcohol and water extracts
following this were completely nontoxic. Acetone extracts of marcs from
benzene and ether .extractions were toxic. Thus, of the solvents tested,
chloroform appeared to bea-the most satisfactory .from the standpoint of
selective extraction of the, toxic material. Results by the multiple-
extraction procedure and by the aliquot procedure with chloroform at room
temperature were in, agreement with 'those by 5oxhlet extraction. Because
of its convenience, particularly when rotenone was to,.be determined by
the same method, the aliquot procedure was suggested as the most suit-
able for determination of total-extract content.
Rowaan and Van Duuren (108) in 1938 suggested chloroform extraction
at room temperature for the determination of total-extract content of
derris and cube root.
keijer (87) in 1938 showed the effect of drying the sample at
elevated temperatures on the :mourit of ether extract obtainable. Thus, a
sample of powdered derris root which before being heated contained 23.6
percent of ether extract gave 19.1 percent after being heated at 600 C. for
2 hours and only 14.4 percent'after being heated at 90..0. for 2 hours.
He concluded that heating above 50 C.. before analysis should be avoided.
.~ ~ s shul be avoided'. "'-
Braak (9) inj1939 reportedon the analysis of a samOle' of 'dBrris
root &anal;ized bytheLaboratory ifo Chemical Research`In Java; by Seil,
Putt, and Rusby in New York; and by Salamon and Seaber In London. The
three laboratories obtained' almost Identical values' for' the eti'- -
In collaborative work carried out between' the 'Imperial Inst'itutate
and Rothamsted Experimental Station (5) 5the' ether;'-xtract contents of
three samples of derris root were determined. Values obtained 'at the
former laboratory, where a 16-hour extraction was: made., were abouit- per-
cent higherr than those obtained at' the latter station, where two' `-hour '
periods were used. By the latter scheme little additional extract was
obtained' in the second 8-hour period except in the case of'h igh-rotanone
root, which gave 1.4 percent additional material. 1l values ranged from
25 to, 30 percent of total extract..
iieijer and Koolhaas (89) in 1940 described th. method used in'' .
their laboratory for determining roterone by.- extraction of a 50-gm.
sample~for '65 hours with ether. The determination of total-ether extract'
was made in conjunction with,: this as follows:
"The ether is distilled off (from the mother liquor '
from rotenone separation) on a water bath'and the last
traces are removed in a vacuum in a water bath nbt exceeding
40 C. The contents of the flask are blown upi to a voluminous
mass, and placed.in a. desiccator over lime for 2 days, after
which time constant weight has beer, reached,'
"Tne difference in weight between the flask with resin
and the empty. flask gives the amount of resin. To this the
amount of crude ro'tenone is added', giving the ether extract
in 50 %. of the sample..."
These authors stated that the total-extract content was about the same
when benzene, chloroform, or ether was uses; hence the extra determination
of ether extract was unnecessary- when chloc.i'oorm or benzene had been used
as extraction solvent.
A scheme of automatic hot percolation was described by .Martin (83)
in 1940. The apparatus consisted of a glass liner fitted into a wider
glass Lube connected with a 250-ml. flask and a condenser. The powdered
root was s:.:,-orted in t he liner on a pad of cotton wool, and rapid extrac-
tior. at th ceiling point of the solvent was said to be effected. The
percentages of resin extracted by this method with several solvents from
25-'m. portions of a sample of Derris elliptica were determined. 4ith most
solvents extraction was reasonably complete in 2 hours. Solvent efficien-
cies of a 'similar order to those given by Jones and Sullivan (76) were
found. jthyl acetate,et-2l'ene dichloride, and chloroform were equally
effective in extracting the toxic principles with a minimum of extraneous
matter. The method described was said to' have the advantages over the
percolation method of 'Iorsley (130) of being automatic and requiring
little more than 150 ml. of solvent.
Graham (47), in his 1938 report to the Association of' Official'
Agricultural. Chemists, gave results .of' collaborative analyses of one
sample of derrip and, one of cube root.. -Results: for total ether extract
were in fair agreement and ranged from 13.7 to 14.5 percent for the derrie
and from 21.1 to 23.3 percent for the cube sample. Details of the method
were ,no.t.given..,-,n his. 1939 reportAto the same Association Graham (49)
recommended that the method for total ether extract used in the 1938 work
be adopted, as official. In a discussion*(48) of insecticide analysis
before the 1939 meeting of the National Association of Insecticide and
Disinfectant T'anufacturers,. he described. the method briefly. The method
(2) was given in detail in the Qfficial and Tentative Methods of Analy:sis
of the, Association of fOffieial Agricultural Chemists. It is as follows:
... extractt 5, g. of finely, powdered root in a'Soxhlet or
,,other efficient extraction apparatus with ethyl ether f'or'.
48 hours. After extraction, concentrate extract and filter
off an% ,insoluble material that may be present. "Receive
filtrate in a tared.,beaker, evaporate off ether on steam
bath, and dry n ,oven at 1050. 0. to constant weight." *
Details of a modification ocf this method to be used on derris and cube
powder in the presence of sulfur have been furnished by the A&ricultural
Marketing Service (124). The total ether extract obtained'essentially as
in the official method is:weighed and then treated as follows:
Add 25 cc. .of acetone (which'has previously been sturated
with. sulfur at room temperature), disintegrate the residue
with a.astirring rod, and stir ,until the plant resins are in
solution; filter,. under, suction, through a' weighed 'Gooch crucible
fi.ttedwith.a disk of filter paper, ri1h'se ouit the 'sulfur frdm
.the beaker, and wash with acetone' (s tura.te'; with sulfur)..
Allow the. crucible to remain, under sutftion for 5 minutes, and
then place in an oven at 105- C.' for'15 minutes. Cool and weigh.
The' weight of this sulfur residue, saltracted from the weight of
the residue in the original beaker, represents the ether-soluble
plant extract material in the sarnple.
Aymonr' commercial firms haydlinr derris and cube root who recently
furnished information pon,'metho.s of:analysis (private communications ,
McCormick and. Company', S. B. Penick and Com'pan:,, and John Powell and
Company ..useadprocedures for total,'extract essentially similar to the
official method. S. B. Penick.and.' Compan.r also 'used total chloroform
extract and found this determination more convenient to handle. Derris,
Inc., used the. ether extract ,'eeterrrmination'only'as a check and ordinarily
determined .total extract in. con.4 unction wit'a analysis for rotenone. The
method used by.this. firm. *for .eterT inning 'rotenone, described in the
section on Rotenone,., involved .extraction, of the"'sample wit!i acetone and
crystallization of the rotenone from carbon tetrachloride. The mother
liquor from the rotenone separation was then treated as follows:
The nonrotenone portion (from a 25- to, 50-gm. sample), in
carbon tetrachloride- is' concentrated to a, small volume, and
the small amount of carbon tetrachlboride remaining is removed
- 14 -
by distillation with about 25 cc. of isopropyl alcohol. Usually
thee 85-cc. additions of isdpropyli alcohol are made before the
resins are finally brought't6 constant weight by heating the
tared distillation flask containing them in a vacuum oven'at
800 C. .
Results by this method were said to check with those by ether extraction.
Undoubtedl- the most generally used method for determining total
extract of derris and cube roots is extraction with ether. The principal
0isadvai-ta:e in the uise of this solvent is the great length of time re-
quired to obtain complete extraction. As a matter of fact any solvent
that gi-s comDarable values may be used for this purpose. Meijer and
Koolhnas (9), Jones and Sullivan (76), and others have pointed out that
that. the amount of total extract obtained with chloroform agrees very
well with that obtained with ether, anl the time required for complete
extraction is a matter of a few riours instead of days. The use of chloro-
form for determining total extract would therefore seem to be much pre-
ferred to the use of ether. Determinations of total extract in the
laboratories of this Bureau are made with chloroform.
Unfortunately, the use of decolorizing carbon in the chloroform
extraction method for rotenone, alth-iough. a distinct advantage from the
standpoint of rotenone determination, prevents the use of another aliquot
for total extract as su.-:ested by Jones and Sullivan (76). Graham (46)
has shown that some of the extract is absorbed by the carbon. However,
it should be possible to devise a scheme in which the sample is extracted
with chloroform without carbon and filtered at in the older rotenone
method (72), a suitable aliquot taken for total extract, the remainder
treated with carbon and filtered, and the proper aliquot taken for rote-
none determination. A study should be made, however, to ascertain whether
or not this scheme would involve loss of rotenone due to adsorption by
-:ethods designed to determine only rotenone are included in this
section Many other methods, particularly colorimetric procedures, have
been proposed for roteiione, cut when applied to whole derris and cube
roots or extracts the-' also Oeterrine additional materials. These methods
are discussed in ot'ier sections of tnis review.
Geoffroy (2) in 1895 was the first investigator to isolate rote-
none in a Pure condition. Although not quantitative, the method of separa-
tion is of interest. It involved prolonged extraction of the plant mater-
ial (cube) with petroleumn ether and crystallization of the crude product
The solubility of rotenone in various solvents (73, 74) is of interest
in connection with its determination.
Subsequent early investigators (Nagal (90), Ishik4wa (60), and
Takei (117)) generally employed ether for the separation of rotenone.
Tattersfield and Roach (122) used aldohol.. The same authors quote Luriam
as stating that the best method. of'6btaining pure rotenone was extraction
with petroleum ether followed'byrecrystallization from alcohol, the
method employed earlier by Seoffroy. .
Roark (100) in 1930 was the first to propose a quantitative method
for determining rotenone. This m-thod, based on the separation used at
that time in the United States Department of Agriculture, consisted
essentially in extracting 100 grams of root with ether and crystallizing
tie rotenone from the extract concentrated to 25 cc. The filtered
crystals were washed with ether, dried, and.weighed.
Brown and Skinner (10), as editors of wiley's Principles and Prac-
tice of Agricultural Analysis, quoted Roark's method. For the determina-
tion of rotenone in commercial derris preparations they suggested extrac-
tion with ether and precipitation of te rotenone, along with some resin,
from the concentrated extract by the addition of petroleum ether.
Spoon (115), in the Oeterrination of rotenone in 12 samples of
derris root, utilized crystallization :from ether by Roark's method.
In 1931 Jones (61) suggested the use of carbon tetrachloride ,to
replace ether in the an-lytical extraction of rotenone from. derris and
cube roots. 'Ether often gave extracts from which it was difficult to
separate rotenone. OCarbon tetrachloride extracts gave a quicker and more
selective separation Of the rotenone. In several cases in which no
rotenone could be separated from the ether extract, carbon tetrachloride
extracts separated rdtenone readily. The rotenone separated from solu-
tions in carbon tetrachloride as a solvate containing 1 mole of the sol-
vent to 1 mole of rotenone.
Blackie (7):in 1932 described an apparatus with groundglass joints
and mercury seals, whichwas especially designed for the determination
of the rotenone content of a Fijian plant byr extraction. with ether. In
the same -year'llackie (8) published results obtained for the rotenonr.e con-
tent of Derris uliginosa using this apparatus and extraction : for 36 '.ours
Georgi and Teik (33) in 1932 described a modification of Roark' s
ether extraction method. They dried a 100-g-. sample in a water-jacketed
vacuum drying oven at approximately 75 C. and then extracted with ether.
The extract was concentrated to 40 cc. and crystallized in the cold cabinet
In the same. year -1oolhaas (7_8) discussed the methods for the esti-
mation of rotenone then in uae. The ether extraction methods in particular
were said tc. be subject to error from such sources as insufficient fine-
ness of root, too large a sample so that on evaporation of the extract the
other substances present exerted an appreciable solvent effect, difficulty
in washing the rotenone free of impurities, and difficulties encountered
in the filtration of viscous ether solutions. He proposed an ether extrac-
tion method for derris root designed to overcome these objections.
- 16 -
As a measure of the purity of the crude rotenone the melting point was
determined and, by means of a curve plotted from the melting .points of
mixtures of various percentages of residual extract and pure rotenone, the
amount of pure rotenone in the crude crystalline material was obtained.
This method with few modifications has been used in the Dutch Mast Indies
since that time. The modified method now in use is described later in this
section in connection with the more recent article by Heijer and Koolhaas
In 1933 Jones (62) described a method of assaying derris and cube
for rotenone using" carbon tetrachloride for extraction and crystallization.
The plant material was extracted in a Soxhiet with the solvent, and the
extract concentrated and set aside to crystallize. The rotenone separated
as its carbon tetrachloride solvate, containing equimolecular proportions
of rotenone and solvent. The extract was cooled in ice, and then the
crystals were filtered through a G6och crucible and washed with ice-cold
carbon tetrachloride. The crystalline material was dried to constant
weight at room temperature and weighed. For more rapid but less accurate
results the extract was cr-stallized inr an ice bath for a few hours and
the solvate, after separation, dried in an air draft. This early method
has been superseded by the method of Jones and Graham (72),from which has
been evolved the official method of the Association of Official Agricultur-
al Chemists (2), which is given in detail later in this section.
If the sample contained over 5 percent of moisture, it was suggested
that it be air-dried at not much over room temperature before extraction.
Lower results for rotenone were obtained when samples of derris root were
dried at 100 C. in a vacuum for 5 hours. Overnight (17 hours) extraction
was recommended for most samples, as it was found that some samples giving
6 to 10 percent of rotenrone in 8 to 10 hours gave results about 1 percent
higher when extracted for 15 to 17 hours. As a means of checking the
purity of the separated rotenone solvate,heating to drive off the carbon
tetrachloride of crystallization and weighing the resulting rotenone were
suggested. A determination of the optical rotation of the separated
material was stated to give an indication of purity, while the chlorine
and methoxyl contents were also suggested as of possible value for this
purpose. The separated crystalline material was examined qualitatively
to make certain that it was rotenone. This was done by microscopic examin-
ation and melting-point determination of the material recrystallized from
Several samples that gave no rotenone bv the earlier ether extraction
and crystallization procedure gave as much as 2 percent by the carbon tetra-
chloride method. The latter method was believed to give correct results
with roots containing over 0.5 percent of rotenone but with roots contain-
ing 0.3 per cent of rotenone or less it was inaccurate. For such material
it was suggested that large samples be used. A scheme involving extraction
with acetone and crystallization of the extract from carbon tetrachloride
was tried. Although acetone .-ave a more rapid extraction of tre rotenone,
much of the acetone extract was insoluble in carbon tetrachloride. The
additional manipulation necessary resulted in a less pure product.
Also in 1933 Georgi and Telk (34) studied the extraction and. crystal-
lization of rotenone .aid suggested. method !hvolvinr. the use of carbon
tetrachloride. They suggested r:odifications to. the ether extraction meth-
ods of both IRoark (100) and Koolhaas (78.) but: considered that evwn w-ith
the modifications these methods did not give satisfactory results,. One
change introduced in'P-oark's metnod involved drying thi root in a vacuum
at 75 C. for 6 hours. They stated. that the drying did not, change the
amount of extractives obtained, but believed that the presence of moisture
interfered with the crystallization of the rotenbne. Thel method suggested
by these authors for estimating rotenone 'usini carbon tetrachloride is
briefly as follows:;
Fifty ,rams of the finely-ground root is treated for 72
hours (three active 8-hour periods) in a Soxhiet with, carbon
tetrac'iloride. The extract is concentrated until the solution
begins to thicken, 'and is allowed to cool and Peeda.d if
necessary. ,The flak is allw to stand in the cold. cabinet
for 24 hours. The crude solvate is filtered on a tared Gooch
crucible and washeA with 10 tp 15 cc. of ice-cold carbon
tetrachloride. After standing in the air for 24 hours it
is weighed. The f'Itrate from. the firet crystallization
is concentrated, cooled, and ee d. It is allowed to stand
in the cold cabinet overnight i. d eny additional crystalline
material. treated a's with the first crop. The total crystalline
V -material is.-treated with boilin.: alcohol, the solution cooled,
and the separated rotenone dried,at 10o0 C. and weighed. A
correction, is made for tlhe solubility of rote..cne in alcohol.
The purity of..the recrystallized. product is checked by melting-
These investigators tried acetcnp sn'. chloroform for extraction followed
by crystallization from carbon tetr-.c-loride. .he se paratioij,,of carbon
tetrachloride-insoluble0.resinous material in the acetone extracts inter-
fered with the separation- of the rotenone, ane, the use of t-iis .solvent was
not recommended. Chloroform proved satisfactory and e'trscted the .rotenone
in less time than did carbon tetrachloride, but ant,' saving of.time over
direct treatment,with carbon tetrachloride seemed doubtful. The rotenone
values obtained by the chloroform and carbon t.trachloride methods were in
close agreement. The. values ,by the modified ethe.r methods'were in r3ncral
lower and wer2 irreu-lar.
C-eorgi (30) in 1933 stated that carbon tetrachlcride had been
substituted for ether in the estimation of rotenone.
Spo6n and Rowaan (116) in'i933 described the method used by them
for determir.in,- rotenone inr derris root. This was essentially the method
of Roark with some modifications, such as lor.,er crystallization and deter-
mination of the meltingpoint of the separated rotenone. The latter step
was designed, hot as a means of calculating the purity of the material as
in the method of Zoolhaas, but as a chec- on its identity and approximate
A procedure pv'onosed by TaeKei, Miyajima, and Ono (118) in 1933 was
a close approach to a trul4 chemical method for rotenone. Since the method
was designed to determine both rotenone and deguelin, it will be discussed
only briefly here and described in more detail in the section on Deguelin
and Rotenone Plus Deguelin. The root sample was extracted with ether and
after crystallization of a first crop of rotenone from the extract the
evaporated mother liquor was oxidized so that there were formed dehydro-
rotenone, from the remaining rotenone, and dehnydroAeguelin. The mixture
of insoluble dehydro compounds was separated and weighed. It was then
subjected to catalytic hydrogenation under suitable conditions, when the
dehydrorotenone was converted to the alkali-soluble isodihydrodehydro-
rotenone while the dehydrodeguelin remained unchanged, thus in effect
giving separate values for rotenone and deguelin. Some derris-root
samples gave surprisingly large amounts of additional rotenone by this
method. In one extreme case only 0.56 percent of rotenone was obtained
by crystallization, while an additional 4.74 percent was obtained by the
chemical treatment of the mother liquor. On the other hand. in a sample
giving 6.38 percent of rotenone bi, crystallization only 0.52 percent of
additional rotenone was obtained. Values on some other samples ranged
between these extremes. One sample giving no rotenone by ether crstalliz-
ation gave 0.97 percent by the chemical method. Later work by Tatters-
field and Martin (120), however, has shown that the ether crystallization
used by Takei and coworkers was incomplete.
In the same year I)anckwortt and Budde (24), in an investigation
of methods for the evaluation of derris root, proposed the separation of
rotenone by extraction with criloroform followed by crystallization from
ether. The powdered root was covered with 10 times its weight of chloro-
form, and the mixture allowed to stand for 24 hours with frequent agitation.
An aliquot of the chloroform extract equivalent to six-tenths of the
original sample was filtered, the chloroform removed, and the residue
crystallized from ether.
In 1934 Danckwortt, Budde, and Baumgarten (25) reviewed various
crystallization methods for the determination of rotenone in derris and
described other means of evaluation. The ether-extraction method of
Koolhaas (78) was said to be too long. They considered the Jones (62)
carbon tetrachloride method to be good only for rotenone contents above
4 percent. The method of Takei, Miyajima, and Ono (118) was said to be
unobjectionable theoretically but ver" troublesome and time-consuming.
The method of Danckwortt and Budde (24) was also mentioned.
Gstirner (51) in 1934 compared results by the polarimetric method
with those by the ether-extraction and crystallization method. The much
lower results by the latter method were explained as largely due to part
of the rotenone remaining: in toe mother liquor.
Cahn and Boam (13) in 1935 made an extensive study of the determina-
tion of rotenone in ,1erris root and resin. irhen derris resins were dis-
solved in carbon tetrachloride tc separate thie crystalline solvate, the
solubility was said to be increased to an unusual extent by the presence
of the resin. -A maximum -ield of rotenone was obtained when 2 cc. of
carbon tetrachloride (saturated with rotenone) was used to dissolve each
gram of resin. Room temperature was adopted for crystallization and fil-
tration, as little difference was found between results obtained at 00 and
- 19 -
at 180 C. Excessive wqshinT with carbon tetrachloride saturated with
rotenone led to marked decrease in yield, with no increase in purity.
In the determination of rotenone in derris roots 8 to -17 hours' extrac-
tion with carbon tetrachloride was not sufficient to remove all the
resin or the rotenone. The authors preferred to use trichloroethylene,
which usually gave complete extraction of rotenone in 8 to 12 hour.
The rotenone was determined by evaporating the extraction solvent and
crystallizing from carbon tetrachloride saturated with rotenone.
When pure rotenone-carbon tetrachlori'?e solvate was stirre,1 with
5 parts of alcohol sat-urated with rotenone, a quantitative yield of pure
rotenonrie was obtained. Tests with mixtures of rotenone and "de-rotenon-
ized" resin showed that'a quantitative yield of the added rotenone was
obtained from such a'mixture when kept for 3 hours. It was later found
advisable to keep such a mixture overnight. The alcohol recover-, of the
separated solvate from derris-root samples was said to range usually
from 83.5 to 89 percent and rarely from 80 to 90 percent. Tests of purity
by methoxyl content, chlorine content., and optical rotation gave values
ranging from 89 to 95 percent on samples on which the alcohol-recovery
values ranged from 84.5 to 89 percent. rhe question as to whether the
purity of the solvate should be taken into consideration wien a rotenone
content was stated was considered by these investigators. !he yield of
roternone-was sai'd to be lower than the amount actually present, and the
amount of rotenone left in the mother liquor might be greater or less than
the 10 to 15 percent impurity in the solvate. The value given was there-
fore only a minimum. figure obtained by determining the yield and the
purity of the solvate.
When an appreciable amount of pure rotenone was added to resins
containing no rotenone by the usual procedure, an excess of rotenone over
that added was obtained. The excess ranged from 7 to 15 percent of the
resin in the samples tested. Resins of this kind were called "Sumatra-
type" b- the authors, an? the rotenone obtained from them was termed
"hidden" rotenone. The highest yields of hidden rotenone were obtained
when 4 gm. of resin and 1 gm. of rotenone were dissolved in 10 cc. of
carbon tetrachloride saturated with rotenone and the mixture was kept
overnight and then filtered as in the usual procedure. Six samples of
"normal" resins, containing appreciable proportions of rotenone, gave no
significant amount of excess rotenone when treated in this way. However,
a sample of root that had given a rotenone content of only 6.2 percent
of the resin (1.5 percent of the root) as determined by the ordinary
method gave 17 percent of rotenone in tae resin when treated for hidden
These authors stated that derris rcot should be dried, prior to
extraction, in a vacuum desiccator to not more than 5 percent of moisture.
The presence of much moisture was said to retard the rate of extraction
and also to cause the development of acidity in chlorinated solvents.
Drying at 100 C. caused decomposition and low results for rotenone. The
procedure proposed by these authors was as follows:
- 20 -
A quantity of root sufficient to give 5 to 10 gm. of
extract is extracted with trichloroethylene for 8 hours in a
Soxhiet apparatus. The solvent is changed anc the extraction
continued for 4 hours 'longer. If the second solution is more
than pale yellow, the extraction is repeated with fresh solvent
for an additional 4 hours. The combined extract is evaporated
until th, extract becomes thick. A gentle current of air is then
blown into the flask while the flask is rotated over a naked
flame until the odor 'of solvent is replaced by the odor of derris
resin. The flask-is'weif-hed to determine the approximate weight
of resin, and 2 cc. of warm carbon tetrachloride, saturated with
roten.one, is added for each gram of resin and the resin dissolved
rapidly. The solution is cooled, seeded if necessary, and kept
overnight. It is filtered br suction through a tared Gooch
crucible containing a disk of filter prper, and the solvate is
washed with carbon tetrachloride saturated with rotenone until
the filtrate is nearly colorless. The crystalline material is
dried to constant weight in air below 50 C. The purity of the
solvate is tested by the alcohol-recovery method already described.
A method for deterz,ininr hidden rotenone in resins poor in rotenone in-
volved the procedure already described for this purpose. The authors con-
cluded that the usual carbon tetrachlorice method cave low results if the
rotenone content of t-e resin was below about 17 percent, was seriously
in error if it was below 10 percent, and failed completely for resins of
very low rotenone content.
Tne determination of rotenone byr extraction with carbon tetrachloride
was described by Pozzi-Escot (9_3)in 19'5. If the determination was made
at roorm temperature, the sample was extracted for 18 to 22 hours. Hot
extraction, which was recommended by the author, required only 6 to 8 hours.
The rotenone was crystallized as the carbon tetrachloride solvate, separa-
ted, and weighed. With certain products it was founra preferable to extract
with acetone or ether. After the solvent had been evaporated, the residue
was extracted with hot carbon tetrachloride and the crystallization con-
ducted as before.
Later in this year the same investigator (94) suggested that extrac-
tion with acetone, alcohol, ether, or ethyl acetate considerably shortened
th time required for t'is operation. The use of a "Shumnaawa"l instead of
a Soxhiet extractor was also said to pern'it more rapid extraction. The
extract, freed of solvent, was treated with hot carbon tetrachloride, and
the rotenone crystallized from this solvent.
.owran (104) in 1935 described the method for determining rotenone
in derris arid cube roots used at the Colonial Institute of Amsterdam. In
condensed form it was as follows:
5 Svi'lntly the apparatus of Kumagawa and Suto (81), which is also men-
tioned by Guillaume and Aerve (53).
- 21 -
To 50 gm. of the plant material 250 cc. of chloroform is
added in a 500-cc. beaker, which is covered with a watch
glass and allowed to stand 6 hours, with occasional stir-
ring. The chloroform solution is then filtered, and the
mass on the filter is washed with 50 cc. of chloroform. The
filter with the rnmp.ss on it is replaced in the same beaker,
and 100 cc. of chloroform is added. .The mass is stirred again
and allowed to stand overnight. Then the chloroform is filtered
through a new filter and the mass washed with another 100 cc.
of chloroform in two 50-cc. portions. The chloroform is eva-
porated completely from the combined filtrates, the last traces
being removed by stirring the flask carefully while holding
it over a naked flame and blowing a current of carbon dioxide
through it. The extract freed from chloroform is dissolved
in 20 cc. of carbon tetrachloride by boiling under a reflux
condenser for several minutes. Upon cooling rctenone separates
as rotenone-carb6n tetrachloride solvate. When crystallization
is retarded, the solution is seeded with a small quantity of
solvate. The mass is kept overnight in an ice chest to ensure
complete crystallization. The separated solvate is collected
by suction in a tared (fritted) glass crucible, washed with
about 15 cc. of carbon titrachloride (saturated with rotenone)
in small portions, and dried overrnight to constant weight (in
air at room temperature).
In another article in the sare year Rowaan (105) compared results
obtained on authentic derris samples by a modification of Roark's method
(100) and by the polarimetric method of Danckwortt and his coworkers (25).
He recommended the extraction methods of Roark or of Jones (62) or modifi-
cations of them as the most workable for the determination of rotenone.
In the course of a study by Tattersfield and Fartin (120) in 1935
of the evaluation of derris root, determinations of rotenone were made on
the. same samples both in Malaya, the source of the material, and at
Rothamsted, England. In Malaya rotenone was determined by the method of
Georgi aid Teik (34). At Rothamsted a 50-gm. sample was extracted with
ether and the extract was freed of solvent and dissolved in carbon tetra-
chloride; the crude solvate obtained was recrystallized from alcohol.
Rotenone was also determined by the trichloroethylene method of Cahn and
Boam (13). In one sample an, extraction was also .made with chloroform.
The values obtained for rotenone by thise various methods were in general
in good agreement. The rotenone content of the samples ranged from about
2 to about 10 percent. In working with the method of Takei, Viyajima,
and Ono (118) the writers foune that in some samples much less rotenone
coule be crystallized out from ether by the preliminary crystallization
recom"-ended by Takei and his coworkers than was separated by the Jones (62)
method from carbon tetrachloride.
In further work on the evaluation of rotenone-containing plants,
Martin and Tattersfield (84) in 1936 studied the effect of removing the
toxicarol upon th-ie separation of rotenone from carbon tetrachloride solu-
tions of Sumatra-type and Derris ,.xlaccends resins. It was found that,
with a Sumatra-type root -from whici no rot'enone seppr-ted by t.e noriral
procedure, after most of the toxicsrol had been removed by alkali tre.t-
ment of the ether extract the rotenond separated reae.ily wien the residual
resin was taken up in .carbon t-trachloride'. Two Sumatra-type roots from'
which no rotenone coula be obtained by the usual methoi- without t removal
of toxicarol), gave .about .2 percent of crude rote-ione by Cahn and Boaml'
(13) proced'he for "Ithidden" rotenone (adding exces-s rotenone). Purifi-
cation of the crude solvate resulted in the greatly reduced values of
0.54 percent and 0.67 percent. Aliquots of resin from. the same tworoots
were dissolved in ether,:extractedi with 5-percent pota.ssium hydroxi..e, '
and the ether was recovered from the alkali-insoluble portion. 'The
residual resin was dissolved in carbon tetrachloride and the solvate
crystallized in the usua-l way. Values of 0.47 and 0.89 percent, respec-
tively, of crude roterncner were obtained 1.- this method, and these results
were reduced to 0.40 and 0.66 percent, respectively, by purification.
It thus appeared that in Sumatra-tyrpe roots the presence .of lar'P amounts
of resinous material rich in toxicarol prevented the sepr)rption of the
rotencrne present, or treatment with potash removed some otl.her inhibitor
of crystal nation. Further-.ore, tlhe solvate, which i.-:rated readily
after triiatmcnt with alk. li, was obtained in as r*ncunrt as-rnoin- closely
with the fl-rqe obtained for the purified rotenone the ncrnal method.
With 1*erris mp!lcc sis root, although rotenone separpnted without the
use of t-e -idden-rotenone tec---iaue, the product was ver-.- i--:re. Here
again tle *.':-aili treatment -ave a v-slue for crude roterone agreeing
closely vit.i that obtained for par.fied rotenone by th- u.--al method.
The authors adiritted the possibility that the al':ali c'x-i..z3 some loss by
induce': o-afion of the rotenone, but the-.- .u.Lested th,.t, an alkali
pretrcatnet such as the.:... described, if suit"--bly contrc-].Ied, mi
tne crude solvate byr trit-ration with alcohol these investigators preferred
to filter at 0 C., using alcohol saturated with rotenone at this tempera-
Beach (3) in 1936 proposed a servicesbla method, for determinin.-
rotenon- b'?se on extraction with chloroform, evaporation of an aliquot
of the -'i.-ed e:.i--rovct., and cry'Astallization of the rotenone from carbon
tetrachlorie0. -.e method was essentially as follows:
Shake 30 gm. of root with 330 cc. of chloroform in
a sto) -erer 500-cc. fl-sk at root temTperatura fcr 2 or 3
hour. Let stand overnight and then shake 1 hour more.
Chill flask and filter contents rapidly inte a suitable flask,
observing: prec-':tior.n to prevent loss from evaporation, Ad-
just the temrnerature of the filtr.te to that of. the oririnn1
chloroform and transfer a 200-cc. aliquot to a 500-cc. flisk.
Remove the chloroform by v..=,xn distillation, and treat with
two-successive portions of carbon t-trachloride, evaporating'
under vacuum each time to remove traces of chloroform,.
Dissolve th3 e;etract in 15 to 20 cc. of carbon tetrtchloride
saturated with rotenone solvate P-t refrigerator temperature.
Induce crvstillizatin of the extract ix an ice bath and
allow to crystallize overnight in the refrigerator. Cool the
extract in an ice bath, filter through a Gooch crucible as in
- 23 -
other methods, and wash with ci-bor. tetrachloride saturated.
with robeLonc so3vats at room te-nor-utre. Leave the
crucible on the vacuum for i5 to 20 'niniites and then weigh.
Warm (niot over 50 C.), again apply vacuum, and reweigh.
Repeat this process until constant weight is attained.
Fine grindlr-i of the sa-ple was said to be necessary in this method; the
root need be Fround only to 20 mesh. It was stated thet the methoC gave
*complete extraction of.roots of high rotenone content.
In 1936 lRobinson (10) proposed a method for estimating rotenone
in haiaris from British Guiana, which involved extraction with carbon
tetrachiloride in a Soxhiet for 12 to 24 hours, cr-rstallizaticon of the
concentrated and filtered extract for about 22 hours in a freezing chamber,
and filtratii of the extract after chilling to -100 C. The crystals were
washed with carbon tetrachlorideat -10 C., dried in a desiccator over-
night, and weighed.
Georgi and Teik (35) in 1935 urged the adoption of a stanr.dard method
for the estimation of roteriona .ncd .gave det".1Is cf the method tentatively
adopted in the Department of Al_-'icu]ture of t Straits Settlements and
Federated Malay States. The method was bsp'e on orne that they nad described
earlier (34), the chief difference bgin. that recrycta]lization of the
crude complex from boiling alcohol was replaced b-r trituration with cold
alcohol. Other modifications were as follows: (1) -r,- 2.0 to 50 gm. of
root was used, depending on the rotenone content, Fo that not more than
4 gm. of solvate would. be obtained. (2) rie o._r action time ws reduced
to 16 hours by using two b-hour periods and r-movini the sample and lightly
grinding and mirin.- between these periods. The mtniod was.s said to be satis-
factory with all species of. Derris in which thn! rctenone content exceeds 15
percent of the extract. With z. i-laccensis, -intea typ (Sumatra ty,-pe of
Cahn and Boam), in which the rotenone content is about 2 n6rcent, it was
necessary to add sufficient rotenone to raise the proportion of that sub-
stance to about 30 percent of the total extract to induce crystallization.
It was suggested that tne final figure -iAht be low because of rotenone
remaining in the mother liquor and sore passing into solution when the
solvate was triturated. with alcohol. The possibility of uzinge the weight
of the crude solvate and applying an appropriate correction factor was
studied, but the purity of the solvate varied too-widely, both in roots
of the same and different species. Conrequ.ently-tr- authors did not
recommend the weight of the solvate as a star.sard On which to base the
Buckley (11.) in 1936, in a' stuc$y of the constituents of derris root,
mentioned, that long eating of extracts to expel revia-al solvent ,was to
be avoided, as some chsng. occr-red which rendered subsequent crystalliza-
tion of rotenone incomplete.
In this -eaSr elso Worsley (130) rpme a study of the determination of
rotenone in derris root and the bark of VWnd-ilea subercsa. Fe described
an apparatus for extracting the root by percolation with hot ethyl acetate,
wnicnh comprised a. percolator with a writer jacket for maintaining an elevated
temperature and a receiver immersed in cold water. With this apparatus
- 24 -
a 10-t3r. sample of high rotenone content could. be completely extracted
with 200 cc. of hot ethyl acetate in about 45 minutes. Roots of lower
content, of which larger samples were used, required more solvent and
longer time but seldom over 2 1/2 hours. Worsley found it advisable to
add sufficient rotenone before crystallization from carbon tetrachloride
to bring the ratio of rotenone to total extract up to 40 percent. The
purity of the carbon tetrachloride solvate prepared by the author's method
was stated to range from 92.5 to 97 percent and averaged 94.7 percent,`
as .urlged by the alcohol recovery of Cahn and Boam. The rotenone obtained
nfter the alcohol treatment was found by measurements of its optical rota-
tion to range in purity from 98 to 99.9 percent, with an average of 99.2
percent. On the basis of the latter value the average purity of the crude
solvate became 94 nercent. Worsley proposed using" this value in calculating
the pure-rotenone content of a.8, sarnole, unless particular accuracy was re-
ouired. He also suggested the. use of optical rotation to determine the
purity of the solvate, stating that the carbon tetrachloride presenrt had
no effect on the rotation. Results by this method were slightly higher
than by alcohol treatment. Tne amount of rotenone left unicrystallized in
the resin was not .-reat, as shown by cooling and by adding further large
amounts of rotenone and crystalli"in-. Addition of 5 percent of charcoal
to Derris root before extraction gave a lighter colored extract and a
slight but definite increase in the purity of th- solvate. Similar results
were obtained when 10 percent of charcoal was r.dd3d to f"undulea bark before
extraction. The following is a conder.sed description of the method pro-
posed by Worsley:
Sufficient air-dried, rou.iud material is taken to give about
1 gm. of rotenon-?, and 5 percent by weight of decolorizing charcoal
is added for Derris or 10 percent for Mundulea. The mixture is run
into the percolation tube, which is placed in the constant-tempera-
ture bath, maintained at a few degrees below the bo'llng point
of ethyl acetate, and the lower end is fitted to a filter flask.
Suction is applied by means of a water pump. Tne calculated amount
of ethyl acetate is tnen heated almost to boiling mid is poured
into the top of the percolation tube. lhe amount required is
approximately 20 cc. o'er gram when 10 co &"0 gm. of material are
used, 15 cc. per r'fm for 25 to 40 gr:. 500 cc. for 50 gm., and
800 cc. for 100 -a. of material. As soon as the solvent appears.
at the bottom of the tube, suction is adjusted so. that the 'rate
of percolation is about 2 drops per second; whexi slightly more
tnan hialf the solvent his come through, it is incrs'-sed to about
4 drcTr7 a second; and finally, when pr2ctica-lly all the solvent
is through, full suction is applied.
The extract is filtered into a distillin.- flas- eand prac-
tically all thn ethyl acetate distilled off. The resins are
transferred to a small, weighed weaker, and the ethyl -cetate
is e,,oved on the water rath. The beaker is weighed to determiine
the amount of resins. Sufficient ourifieO rotenone (40-mesh) is
then added to brin. th rotenor.one- content in the resins up to at
least 40 percent; in -u:- cns9 at least 1.0 gm. is added. It
is stirred into the heated resine on a water bath, 2 cc. of
- 25 -
carbon tetrachlerije saturated with rotenone for every gram of
resins plus rotenone is added, and the mixture is warmed until
solution is complete. The beaker is set aside until morning '
in a desiccator containing a dish of carbon tetrachloride;
seeding is unnecessary. The crystals are filtered through a
Gooch crucible on a disk of filter paper. They are washed
with solvent (carbon tetrachloride-rotenone) until no further
color is removed and dried for about 6 hours at about 40 .
The weight obtained time@ 0.719 gives the amount of crude
The purity is most accurately determined by triturating
the rotenone-carbon tetrachloride complex with absolute alcohol
saturated with rote.none, 5 cc.. for every grj, of complex, and
leaving overnight in a eesiccator containing a dish of alcohol.
The rotenone is collected in a Gooch crucible, washed with 30
to 40 cc. of the solvent, and dried at 100 for 6 hours. From
the weight of rotenone thus obtained the amount originally
added is deducted; the difference is the P.ncunt in the sample.
The purity of this rotenone may be determined by weighing out
between 0.48 and 0.50 an., dissolving in a stoppered vessel in
10.00 cc. of pure benzene, and determining the angle of rota-
tion in a 200-mm. tube. From a previously prensred curve, or
from the formula C = (alpha 1.428)/4.066, the concentration of
pure rotenone is obtained and the purity thus determined. The .
purity may be take, as being 93.2 percent, and this means that
for rotenone contents above 6 percent a correction of -0.1 percent
is made, but for contents below 6 percent no correction is necessary.
A less accurate result for pure rotenone can be obtained by
determining the angle of rotation of the rotenone-carbon tetrach-
1oride complex by weighing out between 6.8 and 7.2 gm. and dissolv-
ing in 10.00 cc. of pure benzene. As before, the concentration
of pure rotenone is determined and the percentage purity of rotenone
in the 'conplex calculated; from this figure 2.6 is d'educted and the
difference used to calculate pure rotenone in the sample.
An even more rapid method is to assume tae purity of the com-
plex to be 94 percent and to calculate oure rotenone on this basis.
Except for a few unusual derris samples results of sufficient
accuracy for routine estimations are obtained.
In 1936 many commercial analysts of derris and cube roots were using
a method for roteio;..e developed by H. A. Sell.6 This method has not been
published, and several variations have been brought to the attentiQn of
the reviewer. In this method 50 gm. of root was extracted with carbon
tetrachloride in a Soxhlet for 5 to 7 hours, or longer if necessary. Before
extraction 0.34 gmn. of rotenone-carbon tetrachloride solvate was added to
the extraction flask, and after extraction the extract was concentrated to
40 cc. Crystallization and filtration of the solvate were carried out as
6 Details of this method were obtained in 1936 in private communications
from !cCormick and Co. and John Powell Pnd Co,
- 26 -
in other methods. The crystals were washed with a cold saturated solution
of rotenone in carbon tetrachloride and allowed to air-dry to constant
weight. The value for rotenone was calculated from this weight of crude
Rowaan (106.) in 1936 again warned against the use of Danckwortt's
(25) polarimetric method for the determination of rotenone and stated that
the most reliable procedure was some modification of the extraction-c-rystal-
The appearance of microscopic crystals of rotenone was described
by Pozzi-3scot (96) in 1936 as an aid in the identification of this mater-
ial in analytical 'work.
In 1936 government agencies of the Dutch East Indies prepared a
sample of derris root and sent subsamples for analysis to nine labora-
tories in various parts of the world. In 1937 Koolhaas and !eijer7"re-
ported the results of this Investigation. Values for rotenone on a mois-
ture-free basis were as follows:
A--United States Department of Agriculture (Jones)
B--Department of Agriculture, Straits Settlements
and Federated l1alay States (Georgi)
C--Commercial Museum of the Colonial Institute,
D--The Cooper Technical Bureau. London (Cahn and Boam)
E--Rothamsted Experimenrtal Station (Tattersfield and
F--Seil, Putt. and Rusby, New York (Seil)
G--Caeser and Lorentz, Halle-Saale, Germany
H--Diethelm, Ltd., Singapore
I--Laboratory for Chemical Research, Buitenzorg,
Java (Koolhaas and 1'eiJer)
8. 4 (pure)
- 12.3 (crude)
7 See footnote 1.
- 27 -
Various methods of extraction were used, but in all except two of the
laboratories the roterione was separated as the carbon tetrachloride solvate.
Laboratory A used both Soxhlet extraction and maceration with refluxing;
the latter method, using benzene, was preferred; the crude solvate was
purified by an alcohol-recovery test. In laboratory B the sample was mixed
with sand and extracted with carbon tetrachloride for two 8-hour periods;
an alcohol-recovery test was included. The method used by laboratory C
involved msceration with chloroform for two extended periods; no test of
the -urity of the solvate was made. Laboratory D made comparative Soxh-et
extractions with trichloroethylene, chloroform, and ether; ether extrac-
tion gave markedly lower values, and the first method was preferred; alcohol
recovery was used for purification. Ether extraction of the ssmpli mixed
with sand in a Soxhiet for 24 hours was used by laboratory E; the usual
alcohol-recovery test was employed. Laboratory F extracted the sample for
6 hours in a Soxhlet with carbon tetrachloride and used no final purifica-
tion. In laboratory G an extraction method using chloroform in a Soxhlet
was compared with the optical rotation of a benzene extract of the sample;
both methods gave results much higher than those obtained by other labora-
tories. Ether extraction in a Soxhlet for 6 hours, followed by crystal-
lization directly from tne ether, with no purity tests, was the method
used by laboratory H. The laboratory of the authors of the report (labora-
tory I) used Soxhlet extrction with ether for 48 hours, followed by
crystallization from the ether and measurement of the purity by determi-
nation of the melting point.
Koolhaas and Meijer criticized the results on the basis that many
of the laboratories did not obtain complete extraction of the root. Ben-
zene, chloroform, and ether, the last-named as used in their laboratory (I),
were stated to be suitable extraction solvents, but longer extraction than
was used by most of the laboratories was said to be necessary. Use of melt-
ing-point determination in judging the purity of the separated rotenone
gave higher values than alcohol recovery, and these authors preferred this
method. They also pointed out that the optical rotation of the whole
extract is of no value in determining the rotenone content. They recom-
mended that for analysis derris root should be ground so that at least 75
percent passes an 80-mesh sieve, and that the moisture content should not
be over 12 percent.
Georgi and Teik (36) in 1937 stated that rotenone may be lost at
two stages of its determination. Thus, some may not crystallize from the
carbon tetrachloride but may remain in the mother liquor, while some may
pass into solution when the complex is triturated with alcohol. Determina-
tions of the optical rotations of alcoholic filtrates after r-co"ery of
rotenone indicated that the loss in triturating the complex witn alcohol
amounted to approximately 6 percent, calculated on the weight of rotenone
recovered. It was not found possible to devise a method for estimating
the amount ir. the carbon tetrachloride mother liquor, but the low optical
rotation of the residual bodies in this liquor pointed to a smaller degree
of retention than with the alcohol liquor. The total loss was estimated
as possibly 10 percent.
Begtrup (4) pointed out in 1937? that extr'-ction of derris or- cube
root with a low boiling solvent, such as ether in a Soxhiet apparatus,
is incomplete. On the other hand, he stated that if a solvent of higher
boiling point is used the prolonged raeatin- genjrelly destroys the extrac-
tive substances and thus renders crystallization of the rotenone difficult
or impossible. To overcc-.e taese difficulties h' reco'rended :xtraction
with toluene at room tem.erature. The procedure su:.eeted by Pegtrup was
Thirty grams ofl00-mesh material is packed in an ordinary
funnel, thoroughly cmoistened with toluene, and washed six times
with 20 cc. of toluene (ea.ca extract is per-itted to drair off
ccMpl3tely). Tne combined. extract is diluted to 150 cc., and a
50-cc. aliquot is taken. The toluane is distilled off on an
oil bath maintained at about 130 0. ."he residue is dissol,ed in
7-8 cc. of carbon teaccrice turatsd with rotenone at 100 0;
and trars.ferr-d to a weijb!:T.- bottle. Washii.zs bring the total ,
volume to 12-15 cc. Cryr .allizv.tion is allo--:.d to proceed over-
night at 10 C. T'in. cryot-.ls r.-c filtered, washed, and dried,.
as in other methods.
Chevalier and Chevalier (20) in 1937 dec.-r-Lbed a method. involving
extraction with chloroform ind cr:--s*L-ltization from carcon tetrachloride,
similar in all essential details to oreiois methods. rotenone was
purified by recrystallization from warm alcohol.
Ripert (99) used dichloroethylene for the -xtraction of rotenone.
The determination of rotenone in samples of Derris and F'undulea
was described by Guillaure and Froesihel (54) in 1937. They used a
modification of the method of --oolhaas (7 7. xtr-r-tion of the 5-ga.
sample with ether in a continu-ious perrolptor was ca2ried out as described
in the section on Total ytra.ct. The evaporated -xtrect w.s then treated
Add 15 cc. of cold. ether to the extract in a tared crystal-
lizing dish (A), cov-er, and let stand in the refrigerator for
about 24 hours. The extract dissolves and the crystals of
rotenone deposit. >ecant the solvent into a second tared
crystalli-!in; dish (B). itapidly wash the crystals with a
little cold ether. vacant the -.rash liquid into the crystal-
lizing dish (3). Keep this dish in refrigerator for 24 hours.
Dry cr:-tallizing dish (A) in oven at 50 C. for 1 hour and
weigh. If crystals form in dish (B), decant the liquid, wash
as before, dry dish and contents at 50 C., weigh "nd add to
weight of rotenone in dish (. ).
These investigators attermpted to check the sensitivity of the cry.tal-
lization method. They added to the powdered. root of a species of
Lebeckia that contained no roterone b- qualitative tests progressively
increasing amounts of rotenonr.e in the form of c-rlon tetrachloride sol-
vate and analyzed the mixtures by the methods indicated. As shown in the
following tabulation, very .cod recovery of the rotencne was obtained:
- 28 -
2 9 -
SR otenone added per 5 gn. lRotenone found
:' (as pure rotenone) (by etheri'method) ':
,'I .071 .0683
S.. .,. ,035.5 .0348
They stated that when less thanQ O..0355 gm. of rotenone was'emoloyed
value's weretoQ low. Determinations,.by chloroform extraction (5-&g.
.sample): andacrystallization, from carbon tetrachloride on some of these
prepared samples,as well as on samples of Derris and lundulea, gave
results in good agreement with those by. ether. .
Levallois (82) in 1937 said that ethyl acetate and chloroform were
tne two best extraction solvents for use in the determination of rotenone.
He emphasized that all trace of extraction solvent must be eliminated
under .vacuum before crystallizing from carbon tetrachloride. Levallois
stated. that.the tendency was to abandon the cOlorimetric methods in favor
Sof the. gravimetric methods for rotenone. '
.- Crystallization methods for rotenone were said by Schonberg (110)
to be open to the .objection .that they were, not applicable to very small
amounts of rotenone, .that the results were not in accord with the in-
secticidal activity, and.that they were very laborious. 't was stated
that-the carbon tetrachior id- complex contained variable proportions of
impurities and that its content of pure rotenone could not be ddedulced.
Colorimetric methods were .recommended.
Worsley (13)! in 1937..stated that prior to the publication of the
work, of Cahn and 3oem.(l3) he had found that addition of pure rotenone
to extracts of Mundulea suberosa bark resulted in a considerably greater
net yield of rotenone. He obtained higher yields of rotenone by adding
decolorizing charcoal to the powdered bark before extraction with ether.
: It was necessary, however, to increase the time of extraction and better
extraction solvents were tried. Percolation with hot ethyl acetate gave
the most satisfactory results. Details of this method have been described
R owaan (107). in 1937 recommended the use of, chloroform at room
temperature for the extraction of rotenone. he stated that drying at
60 .0, for 1 hour brought the rotenone-carbon tetrachloride solvate to
constant weight. :Rowaan tentatively recommended determination of the
purity .by alcohol, recovery, although he said that this process needed im-
.,,.XIeijer (86'). in. 1.937., before a meeting of the Buitenzorg (Java)
Experiment Statios staff, .discussed the evaluation of derris and reviewed
methods for..the determination of rotenone. Treatment of the other liquor
from rotenone separation by chromatographic adsorption, by formation of
the hydrazine of rotenone, and by other methods was said to show as much
- 30 -
additional rotenone as 10 percent of the remaining extract. Of three
methods of determining purity alcohol recovery was said to give the lowest
values, the melting-point method the highest, and the polarization method
intermediate values. Because of ease of handling the lest two were pre-
A method for rotenone usinz ethyl acetate for extraction was sug-
gested by Pozzi-'scot (97) in 1937. Extraction waB accomplished by
boiling under reflux. The extract, concentrated to a sirup, was treated
with sufficient activated charcoal to make a dry powder and then extracted
with a known volume of carbon tetrachloride already saturated with rotenone.
This extract was diluted with carbon tetrachloride if necessary. The
extract was made to the original volume of the carbon tetrachloride solu-
tion and set aside to crystallize for 24 hous at the temperature at which
the solvent was saturated with rotenone. The crystals were filtered and
weighed in the usual way.
Seaber (111) in 1937 reported analyses of derris, barbasco, and
timbo for rotenone content using carbon tetrachloride, chloiroform,
trichloroethyleneiand ethyl acetate as extraction solvents. In'the case
of carbon tetrachloriie the sample.was extracted at least 16 hours, and
during crystallization the extract was kept at room temperature for'2 days
and then in ice at least 3 hours before filtering. The chloroform extrac-
tion method was iessenrtially that of Beach (3). The error introduced by
change in volume due to: solution of the extract was found to be small and
was shown to be almost exactly compensated for by the effect of evaporation
during filtering. Instead of. using a mechanical snaker in this method it
was found that equally good results were obtained by allowing the sample
to stand in chloroform overnight, sha dng occasionally by hand the next
day, allowing to stand over another night, and filtering the next day.
Extraction in a Soxhlet with chloroform was tried, but the eolvate obtained
was less pure. The trichloroethylene method of Cahn and Boam (13) and the
hot ethyl acetate percolation of Worsley (13Q.)'were, also tried. Purity
of the solvate :was calculated by polarization in benzene, which gave re-
sults a little-higher than those by the alcohol-recovery method.
In almost all cases the chloroform method gave'higher results than
die' carbon tetrqchloride, and in some cases the differences were large.
The question as to whether these differences were due to failure of the
carbon tetrachloride to extract the rotenone or to decomposition was
studied. In one typical extraction 82 percent of the rotenone was obtained
in the first 8 hours' extraction with carbon tetrachloride. 'However, with
this solvent a limit seemed to be reached before all the rotenone was
extracted out of the root. Decomposition of rotenone in boiling carbon
tetrachloride was found to be a factor in lower, results by this solvent.
Boiling rotenone in this solvent for 16 hours gave a loss equivalent to
about 0.2 percent on a 5-percent rotenone root, and for 72 hours a. loss
equivalent to about 0.5 perce-nt on a similar root. It was stated that
carbon tetrachloride cannot be .relied upon "to extract rotenone completely.
Extraction by the ethyl acetate method gave .result? in agreement with those
by the room-temperature chloroform method but ,the solvate obtained was less
pure.< Seaber recommended as the best method for commercial purposes extrac-
tion with cold chloroform, crystallization irom carbon tetrachloride, and
determination of purity by polarization, the result to be reported in terms
of pure rotenone.
- 31 -
Seablr also investigated the possibility bf making'use&'of the
maximum of the ultraviolet absorption cutirve to estimate the'percentage of
rotenone In the crude solvate. E extreme dilution was necessary', but never-
theless results- close to those by polarization were obtained in som? cases,
although in others the values were too high. The method could not be used
as a routine process for purity but right be useful in identifying an4d
roughly estimating rotenone present in small amounts in mixtures. An
attempt to. estimate rotenone by this method in whole extracts'of the root'
was not successful because of the effect of impurities.
Jones (66) in 1937 studied the crystallization cf the rotenrone-
carbon tetrachloride solvate from extracts and proposed a modified pro-
cedure for this step irn the rotenone determination. The proposed crystal-
lization procedure was as follows:
The solvent-free extract from a 25-pn. sample of root is
dissolved in 25 cc. of carbon tetrachloride, cooled in an ice
bath, and seeded with crystals of rotenone-carbon tetrachloride
solvate. If only a small amount.of crystalline material sepa-
rates, an accurately weighed. amount of rotenone is added, so
that at least 1 gm. of pure rotenone is present. This extract
and awash solution having 0.27 =n. of rntenone for 100 cc. of
carbon tetrachloride are maintained, at 0 C'. in an ice.bath
overnight. The crystals are then filtered in a t?red Gooch
crucible, washed with 6-10 cc. of ice-cold wash solution, and
dried to constant weight at 40 C. One gram is treated with 10
cc. of alcohol saturated witn rotenone at room temperature and
set aside at this temperature for-4 hours. TInis material is
filtered through a tared Goocn crucible, washed with about
5 cc. of alcohol saturated with rotenone at 'the same tempera-
ture, and dried, to constant weight at 105. Corrections are
made of 0.07 gm. for the roterione dissolved by the 25 cc. of
carbon tetrachloride used and also for any rotenone added.
,The purity of the solvate was found to deoend principally upon the Dropor-
tion of rotenone to total extract and upon the proportion of solvent used
in crystallization.. tAs the so-called "pure" rotenone obtained by the
alcohol treatment was' not entirely pure, and as its purity depended on
the purity of the solvate from which it was prepared, it was desirable to
obtain the solvate in as nearly pure a form as possible. For this reason
the..author preferred to crystallize the solvate from a larger proportion
of solvent than that used- by Cahn and. Boam (13). 1.ore rapid conversion
of the solvate to rotenone was obtained when 10 cc. of alcohol per gra i of
solvate was' used rather than the 5 cc. proposed by Cahn and Boam.
A determination of the precision of replicate results on one sample
of derris of about 4-percent rotenone content showed a standard' deviation
of 1 0.05 percent. A'study was made of the accuracy of the oropos3d
crystallization procedure when ap-rlied to extracts of both derris and
cube roots with various proportions of rotenone to total extract. t-e
method used assumed that thie *.onrotenone portionn of the extract exerts only
a retarding effect on the cr,-stal]ization end has little or no actual sol-
vent effect on the rotenone, an assum-otion tnat was indic-ated by all the
- 32 -
writer's work i, to is.; point. On this assumiption extracts of approxi-
mately known rotenone contents were prepared'from large samples' of roots
of 4-percent roter.one content or'over. The extracts were subjected to a
preliminary crystallization in the usual way,' and the solvate was removed
by suction filtration. The filtrate was then made t6 'a definite volume,
and aliquots were taken of such a size as to be equivalent to 25 n. of
root samples. The amount of rote non remaining in- each aliquot 'WPs
calculated from known solubility figures. To the driei aliquots amounts
of pure rotenone varying from 0.2 t~o 2.0 gm. were added.each was treated
with 25 cc. of carbon t'trachloride, and crystallization was carried out
by the proposed method. The actual weight of pure rotenone obtained was
used witnout correcting for solubility in the cartoon tetrachloride.
Hence from the knoun solubility of rotenone in carbonh tetrachloride at
0 C., a loss of 0.07 gin. was to be exyoected. : The values for rotenone
present (amount calculated from solubility plus amount added) were plotted
against the amount lost in 'crystallization (aznmou'nt present minus amount
actually recovered). In extracts with norwr proportions of extractives
other than rotenone, the loss of rotenone in crys6alliation wS great
when only small amounts -.re o-res3nt, but decreased with increasing amounts
of rotenone until at 0.06" to 1.0 gn. it becqae practically constant at
approximately the loss to be expected from the solvent effect of 25 cc.
of carbon tetrachloride. Similar fezv..lts wcre obtained'with extracts
of the same roots prepared to have both abnorinamaly n.igh ard abnormally low
proportions of nonrotrnone resins. .",nen extracts with nhormn. proportions
of rotenone were allo'.;ed to crstallize for 48 hours, complete crystal-'
lization to a value approximately equnl to the solubility was obtained when
only about 0.6 gm. of rotenone was present.
An extract of a Siiatra-tyoe derris root was subjected' to the same
preliminary crystallization except that sufficient pure rotenone was added
to assure quantitative crystallization. The filtrate was treated In'the
same way as described for those of the 4-percent roots'. When about 1 gm.
of rotenone was present, the loss on crystallization w&aS constant and
approximately equal to the solubility loss; consequently these resins
are similar to ordinary ?esins in their effect on rotenone crystallization.
The author therefore believed that the "hidden" rotenone described by
Cahn and Boam (13) was a result of the retarded crystallization obtained
with any extract having a low proportion of rotenone to nonrotenone
resins; the addition of rotenone merely hastened the crystallization.
The accuracy of the Cahn and Boam method, in which 1 g. of extract is
dissolved in only 2 cc. of carbon tetrachloride, was briefly studied and
appeared to be equal to that of the author's method.
It was concluded that Rccurate results by the proposed crystalli"a-
tion method were obtained only w-ier the rotenone present was equivalent
to 4 percent of tne root, or when sufficient rotenone was added to bring
the amount present during crystallization above this value. For the'
extracts studied the method- rave values which, in view of the precision,
were not significantly different from the actual rotenone contents.
Because of tne widely varyinr composition of different sam-nples of Oerris
and cube root, no general estimate was m".de of th-n accuracy.
In 1938 Jones and 'GrFah.t, w('71_ et, d,.afho.1s .for .t.he q..iartitative
extraction of rotenone fo". d-3rris -,nd cubq rc'.ts. T:ie follo'wing general
methods were compared:
(1) Soxhlet extraction u'n-.nF car-to.r, tetrachloride.
(2) Boiling-rrultip e xxtractior, in.which the sample was
refluxped witt ,'-e roLert on t.e :;te A "ath AE,, t'hen filtered
by suction ar.d the marc vs.s trestet twice with fresh solvent
in the s'.ime %a-'. lT:enene, carbon tetr.chloride, chlorofor:,
ethylene dichlorid.e, trichloropt-lene, ethyl acetate, and
the benzene-lcohol a7eotropic ,:i.:;'tu'e *-'ere tested as solvents
by this method." .. ;
(3) Boilirng-aliquot method, in.which tV-e, sample was
treated with a weighed -ourit of solvent, refilxe on othe
stOran bath, cooled, .olvett Pd ei to.:TJrlace that lest, the
extract filtered, and. an: a.ilquot taken. Only, Ien7ene was .
tried in this ;.ethod6.'
(4) Rocm temperature-wultiple e.trpection method, similar
to method (2) but carried cut at roo.i temper:.tre. Only
chloroform was tried. '
(5) Room temperaturm-
ethyl acetate were tested.
After rernova? of the solvent, cr r.'tallization of the rotenone was
carried out by the iretor proposed by Jones (66). -np marcs from multiple-
extraction methods were tested for rotenone by a qualitative color test.
Tests were rLade-3 o*a large number of finely pnwd1rei samples of
derris, cube, and timbo roots and on one sa-nrlo of 0 -phirosia virginiana
root. Maxce from the bern.sne-boiling-mniultiple extraction method and the
chloroform-rocmn temF3ratu-re-multi le extraction method showed practically
complete extraction of the rotnone. RssultF for rotenone by these
methods were in agreement with t-.ope by the chloroform-room t-"np'3rature-
aliquot methcd. Tne latter method was 9rePfrred hb, the authors because
of its convenieii.ce. Various O)-eses of the chloroforki-room temperature-
aliquot method were t Ien studied. It was ftoune thP.t the time of shaking
during extraction might be:re.i'ceA to 2 hours witho-it seriously affecting
the results, but to ensure complete ext-acticn overnight shaking was
advised. Fineness of th" s-.mole ,as an important factor in obtaining
complete extraction. Results on coarr'ly, .roundd camnple'- were in some
cases 1 percent lower than' on tha srie, rogte reground to a finer size.
It was stated that, to give sati3factcry extrnction by th? aliquoting
procedure, coarse samples should be gro'-rid so that at least 95 percent
passed a 60-mesh sieve. Samples con'ts.ining a hi0h ratio of rotenone to
total extract were found to be more difficult to extract than those with
lower percentages of rotenone. Whea the ratio of rotencne to total
extract was about 40 percent or over, particularly in the case of der-is
roots, it was necessary to employ the chloroform-room temperature-multiple
extraction method to obtain satisfactory extraction. Cube roots in general
were more readily extracted of their rotenone content thanr were derris roots.
Preliminary drying was unnecessary, since the moisture content of erris
and cube roots as received in the United States was found not to be suf-
ficiently great to interfere wit'i their analysis. In addition the results
for rotenone and the purity of the solvate were lower when the root was
dried either at 100 0. or at 50 under vacuum before analysis. In the
chloroform-room temperature-aliquot method replicate results on a sample
by a single investigator in general agreed within about 5 percent. The
average difference between the results by the two authors was only about
As a result of this work Jones and Graham (?2) in 1938 proposed a
complete extraction and crystallization method for rotenone based'on the
room temperature-aliquot extraction using chloroform and the crystalliza-
tion as proposed by Jones. The method with some modification has recently
been adopted by the Association of Official Agricultural Chemists (49) and
is described in detail later in this section. For roots of abnormally
high ratio of rotenone to total extract, or in any case of doubt as to the
completeness of extraction, the alternative room-temperature extraction
with successive lots of chloroform was suggested.-
Cahn, Phipers, and Boam (17) in 1938 discussed methods for deter-
mining the various constituents of derris extract. They did not agree
with Jones (66) that the nonrotenone resins exert no solvent effect on the
rotenone. They cited as experimental evidence one derris extract which
gave 39 percent of rotenone by the usual method including correction for
purity, but gave 42 percent of pure rotenone when a first crop was crystal-
lized and the mother liquor was allowed to be adsorbed on charcoal and
rotenone recovered from the numerous fractions obtained. Hence the authors
believed that the rotenone content calculated from the crude solvate was,
by a compensation of errors, closer to the correct value than results
based on pure rotenone. They tested the effect of the removal of the
alkali-soluble material as suggested by 1'artin and 'attersfleld (84),
and found that in a series of Sumatra-type extracts substantially the
same results were obtained by this method as by the hidden roterone tech-
nique of Cahn and Boam (1_3) in which excess rotenone is added. They also
di scassed results by the Goodhue modification of the Gross and Smith color
test (40), which determines primarily rotenon? plus ?eguelin (see section.
on De'-uelin and Rotenone plus Deguelin). They found that with the numerous
samples of derris extract studied (except, Sumatra-type extracts) the fol-,
lowin.; relation held: Goodhue value = percent rotenone + 22 , 3, wharae the
values are expressed as percentage of the extract and the rotenone value
is that for crude rotenone. They stated that this relation had been used
successfully in the inverse sense to determine approximate rotenCne con-
tents from Goodhue values, and was especially valuable for this purpose
when only small amounts of material were available.
Rowaan and Van Duuren (108) in 1938 recommended room-temperature
extraction with chloroform and removal of an aliquot for the determination
of rotenone in Derris and Lonchocarpus roots.
Seaber (112) in-1938 pubiishef results of analyses of derrls, timbo,
barbasco, and cube roots by thp room temp6rature-chloroform-aliquot extrac-
tion method of Beach'(3) and the short-time (6 hours) carbon tetrachloride
Soxhiet extraction of Sail (see p. 25). Thie first method almost invariably
gave higher results for derris and generally lower results for timbo and
barbasco, while results for cube were only a little higher by the first
method. It was suggostac'. that derris roots be amsayed by the room tempera-
ture-chlor6form method, while other roots be an-.-z-d by both methods and
the higher result taken. As in previous work, Seaber determined the purity
of the solvate by polarization.
The following method for the isolation of small quantities of
rotenone from oleaginous seeds was described by Guichard (52) in 1938:
The -am-le is extracted in the cold with ether or petrolerjn
ether. After evaporation of the solvent the oily liquid is
extracted with 50-percent acetic acid in a separatory funnel
until the last lot gives no color reaction for rotenone. This
acid solution is extracted with ether and t.e1 ether evaporated.
The residue is dissolved in about 10 cc. of 50-pe:ccent acetic
acid, and the solution is filt:,'Ed. and dried under vacuin until
all the acetic acid is removed. This :"sidue is dissolved in
several cubic centimeters of ether and placed in a partly covered
weighing bottle in the refrigerator for 2 weeks to crystallize.
If crystallization Cdoes not occur in this tine, tho solution is
evaporated and the residue, dissolved in 4 to 5 cc. of 50-percent
acetic acid, is filtered and driecd as before. This residue is
now dissolved in 2 to 3 cc. of anhydrous ether end left in the
refrigerator as before for 2 weeks. Minen crystallization has
occurred the ether solution is decanted, the crystals are
dissolved in cold anhydrous ether, and this solution is replaced
for crystallization as before. By such successive crystalliza-
tions as this pure rotenone is obtained.
The determination' of rotenone was discussed by Koolhaas and MLeijer
(79) in 1938. Not only was some rotenone left uncrystallized but varying
amounts remained in the mother liquor. As an extreme case, that of a root
with a total-extract content of 25 percent and an actual rotenone content
of 3 percent, rotenone amounting. to 2.2 percent might be left in the
mother liquor. In a root of this type by the usual method only about 60
percent of the true rotenone content would be obtained.
Methods for determining rotenone were discussed at length by
Bertaud-Rossi (6) in 1938. The method of Jones (62) was said to give
satisfactory results, but several changes designed to give more rapid
results were introduced. To obtain ready crystallization of the rotenone
from carbon tetrachloride, the extract should be dry. Rather than dry
the sample itself and thus cause decomposition of the rotenchdne, this in-
vestigator dried the carbon tetrachloride extract over anhydrous sodium
sulfate. The time of crystallization varied with the proportion of rotenone
to resin; crystallization took place rapidly in extracts rich in resin. A
device for enclosing the filtering crucible in an ice bath to maintain the
STATE PLAkW BOARD
- 36 -
filtration at 0 C. was described andillustrated. In case a rapid result
was desired, instead of waiting for the carbon tetrachloride solvate to
come to constant weight, it could be dissolved in ether, the solution
evaporated, and the process repeated. This scheme sufficed to remove the
carbon tetrachloride completely, and the dried product was weighed as
'Ieijer (87) reported in 1958 that preliminary drying of the derris
root sample at 60 and 80 C. caused a marked lowering in the rotenone
results. Thus, a sample originally analyzing 10.6 percent"of rotenone
gave the following results after drying:
Thmoerature Time Rotenone
0 C. Hours Percent
40 2 10.3
60 2 8.1
80 0.5 6.2
80 2 5.2
The Imperial Institute (58) reported in 1938 on work done by the
Department of Agriculture of the Fe orated ;'alay States. Soxhlet extrac-
tion with carbon tetrach'oride h-.L been ab-indoned, and extraction with cold
chloroform had been adopted as a routine method for rotenone. Results by
this method fell only a little short of those by extraction with hot
chloroform and were in agreement with results obtained with hot ethylene
dichloride and ethyl acetate.
Graham (44), in his 1937 report to the Association of Official Agri-
cultural Chemists, described work on tha determination of rotenone done
in collaboration with Jones, and recommended that the chloroform extrac-
tion method of Jones and Graham (72) be adopted by the Association as a
A titrimetric step in the procedure for determining rotenone, mak-
ing use of the rotenone-dichloroacetic acid solvate, was described by
Jones (67) in 1938. With dichloroacetic acid rotenone was found to form
a solvate containing an equimolecular proportion of the two constituents.
Formation of this solvate "vas adapted to determining the purity of the
crude carbon tetrachloride solvate obtained in the usual gravimetric
crystallization method. The method v'as briefly as follows:
The carbon tetrachloride solvate obtained in the usual way
is dissolved in acetone, end the solution is evaporated to remove
carbon tetrachloride. The residue is dissolved by warming in 10 cc.
of 60-percent dichloroacetic acid. Thne solution is cooled in an
ice bath, 10 cc. of water is aAled slowly, !nd crystals of the
dichloroacetic acid. solvate are edded for sliding. After the solu-
tion his been 2 or 3 minutes in thq ice bath, wqter is add3d 10
to 15 drops at a time, with cooling in the ice bath between addi-
tions, until about 25 cc. has been added. Then 25 cc. more of
water is added dropwise and finally, after further cooling, 50 cc.
of water is added more rapidly. After some- additional cooling the
crystals are filtered on a Gooch and washed with about 250 cc. of
- 37 -
water. The crystals are th?n dissolved in chloroform, about :O cc.
,of water is added, and the.nixture titrated with 0.1 1 .?l,_:l. us-r.
phenolphthalein as in?-cator.
With proper cere in the addition of w-3ter during: the precinitatiorn,
the-acid solvate separated in a crystalline form from wh-.ich excess acid
was readily washed. In tests on sneci lly prpnreO. carbon tetr-ichloride
solvates, the method gave values for purity ,bcut 2 perc?-t i'-i her t'."?.
those by the older alcoh.ol-recovery test. Results onr. samples of po,.'dered
root were in good P.greement with those by the older 7ravimrtric proc-z'Jur3.
The procedure effected a saving in time over the rivlrr._',tric method and.
neutr!? imDurities, such as sulfur, did not interfere. Attempts to pre-
cipitate the acid solve.t% directly from whole derris and cube extraIcts
in an effort to shorten the proc ,uye? still further met with failure.
In his 1938 report to thi Association of Official Agricultural
Chemists, Graham (47) gave the results of a collbcrs.tiTe s-L'y comT*arinl;
the crystallis.tion method of Jones and Graham (72) with the titration
method (67). The former method gave good results on both .5*rris and cube,
but several collaborators had difficulty with the latter procedure.
At the 193B meeting of the Eational Association of Insecticide and
Disinfectant manufacturerss Graham (45) reviewed methods of P-nrlysis for
rotenone. Extraction of the swa-rple with carbon tetrachloride in a Soxhiet,
although widely used, was said to have several dipdvantages, amon- which
were difficulty in obtaining complete extraction an- decorr':osition of the
solvent due to moisture present, with consequent d-icomrozition of the
rotenone. Chloroform extraction at room temperature was recommended.
It was stated that a modification of the crystallization method nhad. been
developed for the detcmrninition of rotenone in the presence of sulfur.
The method was not described, but it was said thit the rotenone and con-
taminating sulfur w.aere weighed and a correction was then f:.-'J for the aslount
of sulfur present. The titration method was also said to be applicable
to such mixtures.
Details of thi nethod' used in the pre,?rnce of sulfur have been
furnished by the Agricultural .:ark.ting Se-vic. (126). ]xtractior. was
carried out in the usual way and the ..'..c."ted extract was then treated
*"Add 30 cc. of acetone to the residue, iarm to dissolve
the rotenone, cool in running tap winter for 15 minutes, filter
through a disc of filter paper in a Gooch crucible and wash 2
or 3 times with 10 cc. portions of acetone. r jsfr the
acetone solution to a 125 cc. rlernmeyer flask F'nJ ev'port3
almost to dry-ness on a steam bath in a, current of air. Then
completely remove th? remainder of the solvent under reduced
The remainder of the procedure was thae same as that usually employed,
beginning with the addition -of c'.rbon tetrachloride.
In 1939 the Imperial Institute (59) reported the results of work
carried out in collaboration wi':h the R;otn'mt3id xerim-ntal Station.
Throunout the irve-ti',.tion three rtnles o' dr-s were used, one each
of D. mnlacce-.-is (ino (P.uLatra) 4pe), D. 1. c-'.nis var. sarawakensis,
and D. li* ca, C A-?i (iigh rcterone). C.- methlud used by the Imperial
Institute t1) involved r,'colticn of "he roct with co.d ethyl acetate,
crystallization front cairbonr t -ch.oice, u prific'tion of the sol-
ve.te by tritaration, if n- ces b.-ry twice, with alcohol. In the Rothamsted
method (II) the root w.s jxt:-ec;ed in .n automatic percolator with boiling
ethyl acetate, chloroform, or ethylene dichloride. The dried extract was
dissolved in ether and this solution. e.'trrcted with dilute alkali to
remove toticarol and other alkali-soluble mn-eriala. The alkali-insoluble
portion war slljected to -rystalliat-ur: from carbon tetrrchloride, and
the rotenone was c',culat'-d from the yoight of sclvate. In method I it
wps found necessary to ad' roteione to oit:Kir separation from the Kinta
type root. The general concordance by method II w'-s CoO(.
As a :result of this work method I was rodtfipd so that extraction
was made by hot percolation, pj.- rotenone *"'s acded if ncecss.ry, and
a second crystiLlii- tion of, th3 solvate frc-, carL:-. tetrachloride was
introduced. 'ethod II was modified by the ure of an ether-benvene mixture
(25 percent benzene) instead of ether rs solvent durin- 2.Ikali extraction
of the resin. In method I othy'L acetate, eth'--lene -icY.1orle, and chloro-
form were tried; in method II only chloroform was 'il-d. Purity of the
complex was Ceterrmlred in both v cr t.lietior from bolute
alcohol. The results by method 1 ndcated that the three sol-nts tried
were of about equal value a3 extr:ct-.., even for Lhe hrgh-rotenone root.
It was also found th2t a.-Kition of ro,.e-rione to aid crystll'7tion in the
case of the high-rotenon' root ef'fcted no Sr,prov-'-.-nt in yield. When
method II, involving remroval of vakal-" lubi tri, s it
was unnecess-ry to -dd rotironr. to produce 1c3-.let1? cr;.":.liz .tion even,
in the case of the Kinta type root. In -encral, results bj both methods
at both laboratories were in fair agree.-ent, although values for the
Kinta-type rooe showed consideracle variation.
Because the purity tost was unsatisfactory, further work was done
on schemes for determininfx purity. In mnethcd I the first crystallization
was m-iCe at two coiicelitr:.tions, namely, from volume of carbon tetra-
chlori.i (a) numerically 10 tits the wAi7ht of resin, ani a voli.me (b)
10 times the w'ight of resin plus adiJ rotcr-ne. In ecch cas3 the com-
plex was recrysil lized fro.: crbcn tetrachlori.de, and the pu-ity of the
second complex w;as detbrL."';d botih by rjcryr'-tallirat'on .from alcohol and
by optical rot.tion. In method II thi first ccmDex'. was crystallized in
the usual w-y r-d recrf'ztalli7-.-" from carbor. tetrachlori',1, and1 the purity
of this com.nlex was 2.etsr'-."ed I ,- optical. rotation. Values b:, method II
were in ,ooJ agreed, .t. In mn-thod I fi-re was no ap-nreciable difference
in the purit:- of second colvatcs obtainod froi solvates cr-sinnaly crys-
tallized at dilutions (a) and (b). In fnra, th, purit:y of the second
complex s fet.'i:nine' by rotation '.ren withi the value o`'.ained by
alcohol recr.stsli2-zation when the detarmiinations w-e mode in either
laboratory. In method I, 'iewivr, when dilution (a) was used, there
were serious discreoarncies between the values obtained by the two labor-
atories, For this reason additional work was Oone at the Imperial
- 33 -
- 39 -
Institute by.a worker from each lp.boratnry, using both general methods and
the several purification schemes. There was Cood agretmeiLt between values
obtained by, the two workers. Agreement betweenumethods I and II. was also
generally good. .It was pointed out that earlier discrepancies might have
been due. to the fict that at one laboratory the crbon tetrachloride -used
for crystallization was prepared by dissolving the theoretical weight nf
rotenone in the solvent while at the other an excess of crystals was kept
in the solvent in the refrigerator. The former method'was now agreed to
bn the bettor.
Some analyses were made by the two general methods and using titra-
tion to determine purity (c7). In method I abnormally high results were
obtained on thn Klnta-typs root, but in method II, after removal of alkali-
eoluble, material, thio root gave values that agreed with previous figures
by other methods of measuring purity. The titration method appeared to be
applicable only to solvstes of a certain degree of purity.
In 1939 Clhn and,' ,. m (16) confirmed the finding of Jones that the
dichloroacetic acid and alcohol-recovery procedures gave substantially
the spmin results. The relation between rotenone content and values by
the Good-hue (40) color test was studied further.- There was found to be an
approxi-mnto rolatibn between the values by this test and the pure-rotenone
content, but the correl31tion was more exact wnen crude-rotenone content
wan considered.. An abnormal2y small difference between Goodhue value and
rctenone content w.as said. 'to indicatee the presence of Sumatra-type root-
6 %3 . I ( .,
In 1939 Graham .(4 3) reported that, in the an.'lysis of cube powders
by the chloroform-extraction method, higher percentages of rotenone were
obtained., and the rotenone-carbon tetrachlcride solvate crystallized mnre
readily nid was -purer when decolorizin carbon was used in the extraction
flask. When 10 n.; (orit-AY) w's mixed with 30 gm. of cube powder and
extracted in the usu-.l way, the values for rotenone were from 0.5 to 0.8
percent higher than when no carbon was used, The use of carbon with the
derris tested caused no significant difference in the results for rotenone.
Braak (9) in 1939 reviewed the results of analysis of the sample
of derris root submitted by the Dutch l.ast Indies authorities to various
laboratories throughout the world and "which previously had been reported
by Koolhaas and l!eijer (see p. 26). Braak also gave the results obtained
on another sample of derris prepar"'-d in the Laboratory for Chemical Re-
search at Buithnzorg, Java.. and'submitted to the laboratories of Seil,
Putt, and .Rusby in New York and of Salamon and Seaber in London. The
sample, with approximately 8.5 percent .of rotenone and 22 percent of
total extract, was an.alyzed-by" the methods of Sell (see p. 25) using
longer extraction time, Jones"ind Graham' (72), Seaber (111), and Koolhaas
(78). The Laboratory for Chemical Research and Seill Putt, and Rusby,-
when applying the method of Jones and Graham, obtained practically iden-
tical results both for pure and crude r"6ten6ne. 'The Laboratory for
Chemical Research found that the values for crude rotenone by the Jrnes
and Graham method were almost the sarne as.thcse for pure rotenone by the
method of Koolhaae. The method of Seaber gave very different results when
applied in the Dutch 'East Indies and in the commercial laboratory in London.
The method of Seill gave results. considerably lower than by the other
methods. However, another sample prepared by the Dutch laboratory and
analyzed by Seil, Putt, and Rusby gave slightly higher results by the Sell
method than by the Jones and Graham method,' Although Braak believed the
method of Koolhaas to give figures closer to the actual rotenone content,
he stated that it would be most desirable for the method of Jones and Graham
to be applied universally, since it satisfied all reasonable demands for
reproducibility in various laboratories and the chance of its general accept-
ance seemed better than for any other method.
Begu6 (5) ip 1939 stated that the technique of Worsley was one of the
best gravimetric procedures for rotenone determination, and he advised its
use for derris root.
Numerous methods for evaluatin- rotenone-bearing plants were criti-
cally reviewed by Guillaume and He'rvg (5S) in 1939. All crystallization
methods were said to be impractical for determining small quantities of
rotenone, as crystallization is not effected. Increasing the size of the
sample was said to diminish the sernsitivity of the method. Such methods
were said to be inapplicable to leaves and fruits. Colorimetric methods
or a determination of mr.ethoxyl groups was recommended.
Georgi and Talk (37) in 1939 stated that the findings of Seaber (iii)
on decomposition of rotenone. after prolonged boiling with carbon tetra-
chlorid* had been confirmed in their laboratory. Consequently they had dis-
continued the use of this solvent an..- substituted rocm-temperature extrac-
tion with chloroform. Extraction eni cr-stallization were carried on as in
the methods of Beach (3) nd. Jones and C-rahamn (72), except that the extract
was allowed to crystallize for 2 days. One gram of pure rotenone was added
to extracts of roots containing 6 percent or less of rotenone. After sepa-
ration of the first crop of crude carbon tetrachloride solvate, the volume
of the mother liquor was reduced to 15 cc, an3 placed in the refrigerator
for 1 day to obtain a second crop of crystals. They were added to the
first crop and the purity of the whole was determined by the alcohol-
recovery method of Cahn and Boam (13).
Graham (49) in his 1939 report to the Association of Official Agri-
cultural Chemists, described the results of collaborative analyses of five
samples of derris, timbo, and barbasco by the crystallization method (72)
and by the titration m-thoe. (67). Decolorizing carbon was used in the
extraction flask as a result of the findings of Graham (46) on this point.
Results were in fairly close agreement except on one sample of derris root
of low rotenone content, which gave poor results by both methods. It was
recommended that the crystallization method, adopted as "tentative" in
1937, be amended to include the use of decolorizing carbon and be adopted
as "official, first action."
Graham (48) in 1940 reviewed the results of the collaborative analyses
just described., He pointed out that the addition of decolorizing carbon re-
sulted in higher values for cube.samples, and stressed the necessity of
using the multiple-extraction procedure with chloroform for samples with a
ratio of rotenone to total extract greater than 40 percent,
- 41 -
In 1940 ,e.ijer and i1,oolhaas (89) described the method in use at
the Laboratory for* Cheulical research, Buitenzorg, Java, for determining
rotenone in clderris root. This is a modification of the original method of
Kooihaas (78), and since it is one of the more important methods now in use
it is presented in some detail here:
extraction Fifty gr'ims of powder (at least 75 percent
must.oass an 80 mesh sieve) are percolated with ether in a
Soxhiet, without a thimble but with cotton wool at the bottom,
for 65 hours. The h3at used for boiling the ether comes from
a 60-watt electric bulb.
"Distillino off the Solvent. The ether is distilled off
in a 100 cc. centrifuge tube on a water bath; the tube is
filled with the ether solution by means of a dropping funnel.
Tha rotenone which may have separated during the extraction is
transferred quantitatively to the tube, and the flask is rinsed
out with ether. a few times. The final solution in the tube
must be 25 cc. T'he tube is then tightly closed with a cork.
"Crystallization of Rotenone. Th3 tube with the ether
extract is kept at room temperature for 1 day and then in a
refrigerator for 2 days. The mother liquor is poured into a
50 cc. Srlenmeyer flask and the remaining crude rotenone is broken
up with a spatula, with the addition of 10 to 15 cc. of ether.
The tube and the Zrlenmeyer flask are closed with a cork and
placed in the refrigerator for another day.
"Determination of Rotenone. The rotenone is centrifuged
for 3 to 5 minutes at 3500 revolutions per minute and the
supernatant liquor is added to the mother liquor in the
Erlenmeyer flask. The centrifuge tube with the crude rotenone
is dried for 10 minutes in a water bath at 70 C., and after
a slow current of air has been passed into the tube, it is
dried in vacuo on- a boiling; water bath for 15 minutes. After
cooling in a desiccator the tube is weighed. *'* The purity of the
crude rotenone is determined by the melting point, using an
empirical table in which the correlation between the purity
and melting point is given. If the melting point is lower
than 1400 C., the mass in.!the centrifuge tube is treated with
another 10 cc. of .ether, centrifuged, and dried, and the
melting point is a-ain determined & The authors determine' tha
optical rotation, from which the purity of rotenone can also
be determined. A correction is made for the amount of rotenone
dissolved in the ether of':the mother liquor and the ether used
for washing. For each cubic centimeter of ether used 4.2 mg.
of rotenone are added to the amount of'pure rotenone. If ioten-
one has separated from the-rother liquor to which the wash ether
has been added, after standing in the refrigerator for another
night, it is centrifuged off and added to the crude rotenone .
I'-... . . '' . ** * *
- 42 -
These authors attempted .to determine by two schemes the amount of
rotenone left in the resin after crxrstallization. The first involved
extraction of the nonrotenone-resins with petroleum ether and cyclohexane
and crystallization-from carbon tetrpchloride of the rotenone in the
residue, while the second method was based on adsorption on activated
fuller's earth and washing out with benzene, the evaporated residue
being crystallized from carbon tetrachloride. ,From a number of samples
of resin a range of from 4.5 to 17.0 percent (with two exceptions), with
an average of about 10 percent, of the amount of the original rotenone was
Results of the analyses of 40 samples of ,-erris root by the authors'
method and by that of Jones and Grahpm (72) were compared.. The.results
for pure rot:-.-one by the authors' method were in close agreement with those
for crule rotenone by the latter method.. It was fo'-nd that finer grinding
in -en':,.1 resulted in hi c1er values for both rotenone Pnd total ether
extract. Meijer and Koolh..as believed that samples with a ratio of rotenone
to total extract higher than 40 percent which were said to be common in
the Dutch 7ast Indies, cou-ale. be handled satisfactorily either by finer
grindin- (75 percent through a 200-nesh sieve) or by extraction several
times with chloroform. They stated that a satisfactory uniform method for
rotenone mi.ht be based on the Jones and Graham method, provided the values
for crude rotenone were used and this method was adapted to samples with a
high ratio of rotenone to total extract. The authors compared results for
purity of the carbon tetrachloride solvate by polarizat.on, alcohol recovery,
and the titration method of Jones (57). The results wer-e generally in good
agreement; alcohol recovery in general gave the lowest values, polarization
highest, and titration between these two. The degree of purity when deter-
mined by the melting point was said to be generally higher than when deter-
mined either by polarization or alcohol recovery. As they experienced
difficulty with the titration method when 20 percent or more impurity was
present and as the polarization method was even less time-consuming, the
authors preferred, the latter method. The figures previously given by Mleijer
(87) to show the effect on the rotenone content of drying the sample were
repeated. Heating the sample above 50 C. before analysis was strongly
In further work on the evaluation of rotenon.-containing plants,
i'artin (83) in 1940 described the procedure for determining rotenone in
derris, which involved some modification of previous methods. Sufficient
root to give about 5 Ln. of resin was extracted, by hot percolation with
ethyl acetate for 3 hours in the apparatus already described under methods
for Total Extract. The resin, freed of solvent, was dissolved in 100 ml.
of a mixture of benzene and. ether (25 percent by volume of benzene) and
extracted rapidly with 50 ml. of 2-percent potassium hydroxide and then
with two lots of 5-percent potassium hydroxide. Water was then immediately
added to the benzene-ether solution to dilute an, residual alkali. The
combined alkaline extract was washed with ben7ene-ether and the wash added
to the main solution. The combined benzene-ether solution was then washed
three times with water and dried with anhryrous sodium sulfate, and the
solvent was removed. The resin was dissolved in carbon tetrachloride and
the rotenone solvate crystallized in the usual way. Determination of the
- 43 -
purity of the complex was carrioi out byv a poiarimetric method. The optical
rotation of a 4-percent solution of the solvate in ben-enf was -etermined,
and the percentage of rotenone then calculnt-d from tho rotation of pure
carbon tetraciiorlde solvate in benzene. It ias stated that im-purities
present were likely to show a specific rot-,1tion ai:;jj-c: rmtely one-fourth
that of rotenone -. error involved sa-d to 1- smals but mi ht be
allowed for if it was cons-'.Ceied necessary. Values obt31ne.d bc this method,
in general, ar-reed with tVlose cbtiL.ed by the dichloro-.cetic acid titration
The Imperial Institute (57), n a survey of insecticides from plant
materials in 1940, briefly discussed the ev-uatio-i of eerris and cube.
It was stated that results for rotenone deTe-ded on the solvent, the number
of extractions, tha temn.e-'s.ture &nd lei-'th of tire of ertraction, as well
as on the fin.ress of grinding an. moisture content of the sjrozle. The
need of a standardized method vac, stras_3d.
In the Official Tan.T:.tajtive i.thocs of Analy-sis of th3 Association
of Official Agricul'-Aral CLemists (2) the follow:n.-: method for determining
rotenoiie, that recoi-mended by Grhain (9), is given as official, first
"Weigh 30 g. (if. sar.rkle contains more th-'-n 7z, rotenone
use a quantity that will eiva 1.0 1.5 g. of rotenone in the
200 ml. aliquot) of finely powdered root ane 10 g. of decolor-
izing carbon into 500 ml. glass-stoppered "rlezm-eyer flask.
Add 300 rl. of chloro-form repszuroA at definilt, rocm temperature;
place flask on s-.ki:.r. .-chine an. f-nsten stopper securely.
Agitate vigorn" ?ly for not less than four hours, preferably
interrupting sp-:in_ with overni-'-t re't (or Uasksy be shaken
continuously overni.- t). -.6,ove flsk from machine end allow
to cool in refriF;rator for at least an hour. Filter mixture
rapidly into suitable flask, usinz fluted paper without suction
and keepin funnel covered with watch glass to avoid loss from
evaporation. Stopper flask ane adjust temperature of filtrate to
that of original chorofcrm.
"Transfer exactly 200 ml. of this solution to 500 ir.l. Pyrex
irlenmeyer flask and distil until only about 25 ml. remains in
flask. Transfer extract to 2`5 m1. _riennenme.er flask, using
carbon tetrachloride to rinse out the 500 ml. flask. Tvcqporate
al&m.Ot to dryress on steaa bath in current of air. I-ern remove
remainder of solvent uzder reduced pressure, heating cautiously
on steam b.ath when n;-c3ssary to hasten evaporation (suction may be
applied cirectl-- to flask). Dissolve eyt-.-act in 15 ml. of hot
carbon tetrachloride and agaiin, in a similar manner, remove all
solvent. Repeat with another 10 15 ml. portion of hot carbon
tetrachloride. (Tnis treatment removes all chloroform from the
resins. The chloroform extract is usually completely soluble in
carbon tetrachloride. If small quantities of insoluble material
are present, t'-e purification procedure described later will
eliminate them. However, if large quantity of insoluble residue
should remain when -extract is dissolved in first portion of
carbon tetrachloride, it should be filtered off and thoroughly
- 44 -
washed with further portions of hot solvent, after which the
filtered solution plus washings should be treated as directed
above for removal of chloroform.)
"Add exactly 25 ml. of carbon tetrachloride and heat gently
to completely dissolve extract. Cool flask in ice bath for several
minutes and seed with a few crystals of rotenone-carbon tetra-.
chloride solvite if necessary. Stopoer flask and swirl until crystal-
lization is apparent. If at this stage cnly, a small quantity of
crystalline material separates, add an accurately weigheO quantity
of pure rotenone estimated to be sufficient to assure that finnl
result, expressed as -sure rotenone, is at le9st 1 g. Then warm to
effect complete solution, and agein induce crystallization. At same
time prepare a saturated solution of rotenonr in carbon tetra-
cdhbAde for washing. Place flasks containing extract and washin-r
solution in ice bath capable of maintaining temperature of 0 0.
and allow to remain overnight.
"After 17-18 *-.ours in ice bath, rapidly filter extract
throvLh weighed Gooch crucible fitted with disc of filter paper,
removing flask from ice ba.th only lonc enough to povu e'rh frac-
tion of extract into crucible. .inse residue of crystalline
material from flask and wash under suction with sufficient of ice-
cold saturated solution (usually 10 12 ml.) to remove excess
mother liquor. Allow crucible to remain under suction for about
5 minutes anc, then dry to constant weight at 40 C. (about an hour).
lhe weight obtained is 'crude rotenone-carbon tetrachloride solvate'.
"break up contents of crucible with spatula, mix thorou,7nly,
and weigh 1 g7. into 50 ml. Erlenmeyer flask. Add 10 ml. of alcohol
that has previously been saturated with rotenone at room tempera-
tuxre, swirl flask a few minutes, stop-,r tighti, aid set
aside at least four hours, preferably overnir-ht, ,.t the same
temperature. ?ilter on weighed Gooch crucible fitted with disc
of filter uaper. Rinse crystals from flask and wash under suction
with solution of ethyl alcohol satura+-I with rotenone at tempera-
ture of recr, stsilization (ca. 10 ml ;ill usually be required).
Allow crucible to r:m-:'in uner suction 3 to 5 minutes and then dry at
105 C. to constant wi.--ht, which should be effected in 1 hour.
"'ultiply weight expressed in ramr, by weight of the 'crude
roteno:--:e-carbon tetrachloride solvate' and to project add 0.07 g.
which re1iesents correction for rotencne held in solution in the
25 ml. of carbon tetrachloride useO in crystallization. If any pure
rotenone '-as been nd.ed, subtract its weight from .lue obtained..
This -ives the weight of *pre rotenone contained in the aliquot of
the extract, representir-n 20 i-rcms of the sample.
"Alternate extraction prociTdL'-e. If sample is one in which
ratio of rotenone to total extract is greater than 40.-, use quantity
sufficient to contain 1.0 1.5 .:. of rotenone' and successively
extract four times with CvCl3, usinc- 200 ml. each for second to
fourth L-tractions. Filter after each extraction and return marc
to flask for extraction with fresh solvent. Finally combine
extracts, evaporate almost to dryness, and: proceed as directed
above, beginning at point where aliquot has been evaporated
almost to dryness."
The same commercial sources that supplied information on the deter-
mination of total extract also discussed the methods used by them for the
determination.of rotenone (private communications). Two of them, !cCormick
and Company and John Powell and Company, employed the official A. 0. A. C.
method. The laboratory of S. B. Penick and Company, regularly used the
official method but also occasionally employed a modif'ration for compara-
ti-vr purpo-s. This method, which depended on measurL.E, the carbon tetra-
chlorie of crystallization and assumed that rotenone is the only solvated
material present, was as'follows:
Place the crucible containing, the crude solvate (dried to
constiit weight) in a 100-cc. beaker and weigh. Add 5 cc. of
alcohol (or icetone). to the material in the-nruwide and keep in
a mrodWCr.-ely warm place, where evaporation w-sl prl-e'c b]owly,
until all the solvent has evaporated. Repa. thi. 'p ...-ce
and then dry the beaker and contents at 100 C. to cons;:.nt weight.
This procedure removes the carbon tetrachloride of crystallization,
whi-ch was combined in equimolecular ratio with the rotenone in the
solvate. The difference in weight divided by 0.281 and multiplied
by 0.719 gives the weight of rotenone.
Another firm, Derris, Incorporated, preferred to use acetone as
the extraction solvent, claiming that this solvent extracted the resins
and rotenone in less time than any other solvent tried. The method was
briefly as follows:
'rom 40 to 50 gm. of the coarsely :ground or 25 to 50 gm. of
finely powdered root is extracted in a Soxhiet with acetone.
(If powdered root is 'jsed, it is mixed with an equal volume of
fine sand or sodium chloride.) At the end of 3 hours the extraction
flask is replaced by another containing fresh acetone and the extrac-
tion'continued for about 20 minutes, or until a.negative color test
(Durham) shows that th'e marc is depleted: of rotenone. The combined
acetone extracts 'are' then concentrated to about 10 cc., when two or
three auditions and evaporations of 25 cc. each of carbon tetra-
chloride ar'e made to remove the acetone. .
'One hundred cubic centimeters of carbon tetrachlbride and about
3 gm. of Celite are added to the residue and the whole is heated
to boiling and filtered by suction. The residue is washed with two
or three lets of about 25 cc. each of hot carbon tetrachloride.
The carbon tetrachloride solution is concentrated to about 25 cc.
and transferred to a 50-cc. graduated cylinder with ground-glass
stopper. The volume .is adjusted to. ,40 cc. with carbon tetrachloride
and the cylinder placed in 'the refrigerator to remain overnight.
The filtration, washing, and determination of purity are carried out
approximately as in the official A. 0., A. C. method.,
- 46 -
Results by this method were said to be in agreement with those by the various
published procedures, including the official A. 0 C. method. .
It is generally agreed that derris and cube root should be finely
#rounra fanl air-dried before extraction. Several investigators (71, 87)
have shown that drying at elevated temperature is detrimental to the
* determination of rotenone. .
One of the best solvents for extracting rotenone for analytical
.piarposes is chloroform. Extraction at room temperature seems to be pre-
ferred by most workers. This method is rapid, yet it extracts less extra-
neous material and involves less' decomposition than the use of other sol-
vents or higher temperatures. For most roots the aliquot procedure appears
to be entirely suitable, but for roots with a high proportion of rotenone,
as pointed out by Jones and Graham (71) and Meijer and Koolhaas (89), a
more exhaustive extraction must be resorted to. For this reason the alter-
native multiple-extraction procedure is included in the official A. 0. A. 0.
Crystallization from carbon tetrachloride as carried out by most of
the present methods may be considered to be reasonably complete. It is
possible, as indicated by the work of Cahn, Phipers, and Boam (17), and
of 'eijer and Xoolhaas (89), that in some few samples, particularly those
with a very low ratio of rotenone to total extract, a small proportion of
the rotenone rermq.ins unaccounted for in the mother liquor. While the
reviewer (66) believes this to be'primarily a result, of greatly retarded
rote of crystallization in certain types of extracts, other investigators
(17) state that it is due to an actual solvent effect of other constituents.
WThichever may be the case, anythin- that can be done to increase the
relative proportion of rotenone will aid in producing more nearly complete
crystgllization in the time allowed. The addition of pure rotenone (13,
66), the treatment with 6ecolorizing carbon (130, 46), and the removal
of alkali-soluble material (84) all tend to accomplish this result.
In the. reviewer's opinion, when these schemes or combinations of them
are employed, samples in which rotenone remains unaccounted for in the
resin will be encountered only rarely, and even then results will not be
greatly in error. If derris samples with a very low proportion of rotenone
to total extract are to be encountered-'egularly on the market, alkali
extraction ma: become a necessary step. Such roots as these usually
coiLtain a large proportion of toxicarol ahd other phenols, and their
removal undoubtedly improves the crystallization of the rotenone.
8 Recently a sample of derris root encountered in the laboratories of this
Bureau was not completely extracted even by this method and it was neces-
sary to resort to hot extraction. This sample contained about 10 percent
of rotenone and 24 percent of total extract.
- 47 -
?.ost investigators now feel that pure rather than crude
rotenone should be reported. With regard to the oes' method of
determining purity there is soie diflerrence of cp'.nior.. The
alcohol-recovery method (13, 63) is the most*u',ed, although it
generally gives lightly lower values than other methods. The
titration scheme (67) 'may give more accurate v.lu)3s, but difficulty
has been experienced in apolying it to low-rotori'onie roots. Polar-
ization (111, 89, 13S0) is used but probably ive.s values thL.t are
slightly too high. Accord inr to Meijer and Koolhaas (89) the
purity determined by meltin.; point is ger:erlly higher than by
polarization. In any of these methods more ne-.rlr the correct value
will be obtained the purer thes ori:in-al solvato. Since the three
methods already discussed for treating tlie extract before crystal-
lization .-n-amely,addition of rotencne, use of carbon, and alkali
extraction-in general result i a purer solv.te thy are to
be recomqerned fxau this sta"!&&p6int as well as from that of
improving the comnleter.esp of crysta!llization. Thus -t appears
from work reported by the Imperial Inztitute (59) that alkali
extraction might obviate muc'- of the ifficty encc-L't=red by
some workers in the use of the titiation method with low-rotenone
Without doubt the procedure for the deter.mtnat.on of rotenone
that involves room-temperature ext-raction within chloroform and
crystallization from carbon tetrachloride, as exeimplifi3d by the
official A. 0. A, 0. method (2), is now the most widely used.
DGU3-IIN AND MT-70:7 PLUS D-GUELIN
The Z)rham Test
A test widely used in work on rotenoue-containing plants is that based
on the color reaction discovrcd by Durham in 1902, in which treatment of certal
constituents of derris root, with nitric acid followed by ammonia was found to
produce an evanescent blue-graer color. Ishikawa (60) in 1916 independently
discovered this rection, using sodium hydroxide as the base. According to
Tattersfield4 nd Roach (2) uand. Gimlette (38), who described Durhin's work,
concentrated nitric acid -.':i a'dad to solid rotenone, or rosin, and this mixture
was treated directly :ith strong =moniuam hydroxide. In this form the test was
qualitative only and was unsuitable for delicate testing because of the violence
of the neutralization.
Peyer and Ehun-rbein (92) in 1931 described -A qualitative test similar to
that of Durham. To 2 cc. of dilute acetic acid extract of the sample, 2 drops
of fuming ni ric acid were added; the mixture WaS diluted with 10 cc. of water
and then made alkaline with sodium hjydroxiie. The color prcluced passed quick-
ly from green to brown. The gren color produced with ammonia was said to be
more stable than that obtained with sodium hydroxide.
In 1933 Jon(s and S;nith (7.) modified the Durham test in an attempt to
render the blue color more prl: sn.rant and make the test more nearly quantitative,
They used the following procedure:
T 1 cc. of an acetone solution of rotenone (or a plant extract)
1 cc. of 1 + 1 nitric acid is added, -nd the mixture is allowed
to stand for 1/2 minute. It is then diluted with 8 to 9 cc. of
water and 1 cc. of strong am4nonium hydroxide is aided. A blue
color is produced which is alMost identical with that given by
bromothymol blue indicator at a pH of 7.2.
It was said that as little as 0.1 mg. of rotenone could be detected by this
method. By preparing a series of solutions contqiring different concentrations
of bromothyn-.ol blue in a buffer of pH 7.2 and standardizing these against the
colors produced by different amounts of rotenone, it was possible to use the
test to make a rough estimate of the amount of rotenone present. Since approx-
imately the same intensity of color was given by deg-elin, whereas toxicarol
gave almost no color, it wes believed that the test gnve a rough value for the
total rotenone plus deguelin content of plant extractives. Sodium and potassiun
hydroxides, or sodium and potassium carbornates, used in place of the ammonia,
were also fourd to produce the blue color. When the nitric acid was partially
neutralized with sodi'.= bicarbonate and the neutralization was completed with a
stronger base. Thisvolor was slightly more permanent.
The first reported semiquantitative application of this test was made by
Jones, Campbell, and Sullivan (70) in 1935, in working with a series of samples
of several species of e-or-.A.ia. -.:tracts thnt rave the test were rated in
three grades de endir- on tie depth of color produced.
Fischer and illtsahe (2) in 1935 mncde a stud-- of the tests proposed by
Peyer and Hu5ncrbein (92) and by Jones and Smith (75) in a further attemot to
render the reaction more nearly quantitative. The test, carried out in
essentially the same.manner as already described except that the diluted re-
action mixture w.s* cooled to 0 0. before addition of the amonis, 's 25
To prepare color standards, 50 mg. of malachite green is
dissolved in 1 liter of water, a portion of the solution is, .
diluted'to double the volume and the procedure is repeated un-
til six solutions of successively greater dilution have been '
prepared. In general, in the analysis of rotenone and rotenone-
rich extracts a 0.4-percent solution in acetone is needed. One
part is -diluted to double the volume and another to four t times
the volume, so that the three solutions contain 4, 2, and 1 mg.
per cubic centimeter of rotenone or extract. At the time of
analysis i cc. of acetone solution is mixed with 2.cc. of dilute
nitric acid (sp. gr. 1.2), allowed to stand 2 minutes,diluted with
7 cc. of ice-cold water, and.cooled to 0 Two cubic centimeters
of 2S.percent ammonia is then added rapidly, and the liquid is quickly
mixed and compared visually with the standards. estimations must
be made not later than 3 to 4 seconds after addition of the ammonia.
Orange-yellow color filters may be used in making the comparison.
For a repetition of the measurements another series of concentrations
of the unknown sample should be chosen. Values obtained may be
plotted graphically with the standards if desired. As the end value
of an analysis the middle value from three observations at different
concentrations may be used. It should not differ more than about
10 percent from the end value of a second analysis.
In some cases extraction with benzene followed by evaporation and solution
in acetone removed impurities that interfered with the measurement of the .
color. In' the presence of many substances the appearance of the yellow color
with hitric acid was vry' much retarded, and in these cases it was necessary
to add a drop of fuming nitric acid to initiate the first reaction.
The colorimetric method of Jones and Smith (75) was regarded by
Goudswaard and Timmers (43) in 1937 as unsuitable for the estimation of
rotenone, because the intensity of the color produced depended on the temper-
ature and because various tints interfered with the estimation of the color.
Pozz.i-Escot-(98) in 1937 stated that bases other then amonia produced
the final blue color in the Durham reaction. Even organic bases such as
triethanolamine produced a blue-green color.
Sievers and coworkers (lj1) in 1938 extended the semiquantitative.
use of the Jones' and Smith' (7) variation of the Durham reaction .to several
hundred samples of Tephrosia virginiana. Extracts were rated in five dif-
ferent grades depending on the degree of color, and two investigators work-"
ing independently gave substantially the same rating to the majority of
The Durham reaction has been adapted to'the qualitative testing of ..
mineral-oil fly sprays for rotenone and deguelin (j25). Directions for the
test were as follows:
"Transfer 5 cc. of the material to a large test tube, add
2 cc. of concentrated nitric acid and shake for 20 to 30 seconds,
dilute immediately with 20 cc. of wvrater. Close the tube and
shake to disperse the oil in the aqueous solution; allow the oil
to veparatk, which should not require more than 30 to 40 seconds,
and add 1 or 2 cc. of amnonia poured do.,,mn the siie of -the tube.
The presence of rotenone or derris extract is indicated by the
formation of a fugitive blue color."
Quantities of rotenone as small as 2.5 mg. in 5 cc. of a mineral oil-pyrethrum
extract were said to have been detected. by this method.
Guillaume and Herve (53) in 1939. described a modification of the Durham
test which rendered' the color slightly more perioaent rnd permitted a roughly
quantitative estimation., This scheme involved r.-ding only a few drops of nitric
acid to the acetone solution of rotCnone or extract, covering with a layer of
toluene and allowing to str-ind in vacuo at -8 tc-.10 for '1 hour. The blue
color then obtained by aidin- a few drops of F-ionia persisted unchanged for
about 1 minute. Colors prod'iced simultaneously from the sample and from stand-
ard solutions of rotenone were com-a.red :r'ocld in test tub s.' In this form
the method was said to be sufficiently precise end sensitive for many purposes.
The Gross and Smith Test
A more useful color reaction than that of Darh-m from the quantitative
standooiat was discov-red in 1934 by Gross and Smith (50). It involved treat-
ment of an acetone extract of the sarnole with alcoholic alkali followed by
nitric acid containing nitrite. A rather permanent red color was given by
both rotenone and degaelin, and also by dinhdrorotenone, Briefly the proce6.
dare was as follows:
To 2 cc. of an acetone .solution or extract containing 0.05
to 0.30 mg. of Trotenone oer cubic centimeter, add 2 co. of 10'
percent alcoholic potassium hydoxide solution, and allow to
stand at 20 C. for 2 minutes. Then add 6 cc. of a nitric acid-
sodium nitrite mixture containrin- 1 voiunmo of concentrated nitric
acid to 1 volume of aqueous sodium nitrite having 0.25 gm* of
sodium nitrite per liter. Mix, cool to 20 C*, and allow to stand
at this temperature for 15 minutes. Compare visually with stand.-
ards containing pure rotenone prepared at the scme time.
When applied to samples containing only rotenone, the results agreed with those
by the gravimetric methods. Applied to derris and cube samples, the results
were 50 to 100 percent higher than for rotonone alone, owing to the presence of
deguelin. The method was applied to rotenone spray residues 'on fruits and
In using the Gross and Smith test for total rotenone and deguelin in
several samples of derris root, Tattersfleld and Martin (120) employed two
methods of extraction. In one method the samples were extracted with acetone
and aliquots of this extract were diluted to the proper concentration for the
test. In the other procedure the samples were extracted with ether and the
dried extract was dissolved in acetone and treated as in the first method.
Colors developed from extracts by the second method were more readily matched
than those obtained from the first method.
Ambrose and Haag () in 1936 shortened this test for use in detecting
rotenone and deguelin in the excreta of animals fed rotenone and derris. The
material was extracted with ether and the evaporated extract taken up in acetone.
To this solution was added one-third its volume of freshly prepared 10-percent
alcoholic potassium hydroxide, and the resulting mixture warmed. A wine-red
color developed to a maximum in about 15 minutes. The test was said to be.
sensitive to 1 part of rotenone in 35,000.
Goodhue (4) in 1936 described a more serviceable modification of the
Gross and Smith test, which has been widely used in subsequent work. Sulfuric
acid was substituted for nitric, the concentration of the alcoholic potassium
hydroxide was reduced, and the nitrite necessary to produce the color was added
in the alkali instead of in the acid. The method was as follows:
Piepare the following reagents: (a) mix 1 volume of sulfuric
acid (sp. gr. 1.84, free from nitrous acid) with 3 volumes of water.
(b) Dissolve 1 gram of sodium nitrite in 10 cc. of water and dilute to 1
liter with 95-percent alcohol. (c) Dissolve 40 grams of potassium
hydroxide in 100 cc. of water. (d) MKx 1 rolume of'1eagent (c) and 7
volumes of reagent (b). Prepare this solution fresh daily.
Proceed as follows: Prepare an acetone extract of the sample containing
from 0.005 to 0.25 mg. of rotenone per cubic centimeter and pipette 2
cc. into a dry test tube. Add 2 cc. of reagent (d) and place the
tube in a water bath at about 250 C. for 5 minutes. Add 5 cc. of
reagent (a), stopper, shake, and place the tube back in the water
bath. The color reaches a maximum after about 15 minutes, 6nd then
remains unchanged for 2 hours. Determine the amount of rotenone by
comparing the color with standards "prepared at the same time from
known quantities of rotenone.
The .turbidity or brown color which sometimes developed during the analysis of
orade plant extracts was removed upon extraction of the final mixture with a
small portion of ether. The red color due to rotenone and deguelin was not
removed. In this work comparisons were made with Lovibond color slides in a
roulette comparator. The small amount of blue which accompanied the red was
filtered out by a dichromate filter for easier matching. -By this modified
procedure the sensitivity of 'the original test was increased 20 times. The
specificity for rotenone and deguelin remained the same. Deguelin was said to
give the same amount of color-as did rotenone.
This method was criticized by Goudswaard and Timmers (4 in 1937 on the
same grounds as the Jones and Smith (75) test-namely, dependence of color in-
tensity on the temperature and interference of various tints with the estimation
of the color. On the other hand, the method was said by Schonberg (= to
present several advantages over the crystallization method.. -Among these were
the fact that it would determine very small quantities and *that it was rapid
and practical. Purtnermore, the oxidation of rotenone to dehydrorotenone could
be followed by this colorimetric method but not'-Ty crystallization methods.
C.^, Phipers, and Boam (7.) in 1938, in an extensive study of the com-
position of derris root, used this colorimetric method, making color comparisons
in a Dabosccn colorimeter. They proposed to call the result obtained the "Good-
hue value"; this was the percentage of rotenone that the material would contain
if rot.nor.nu ,-r the only ingredient giving a color in the test. They found
these values to bear a definite relation to the rotenone content of derris
extract. For ordinary extracts the relation was as follows: Goodhue value =
percent rotenone 4 22 1 3. This relation did not hold for, Sumatra-type extracts,
which usually had Goodhue values of 10 to 15. As mentioned in the section on
Rotenone, this relation was used by CahaPhipers, and Boam to determine approxi-
mate rotenone contents by the color method. According to these, writers,,.ot.enone
and. deguelLn r ,were the only substances (known at that time) in derris
extract which gave this color test. They found that the color given by deguelin
was only fouir-fifths the intensity of that given.by rotenone. Consequently, they
stated. that the "excess'" value alove the actual rotenone content must be multi-
plied b.1 ..5 to indicate the deguelin content of an extract'.. Using this value,
they cornclriled the deguelin content of derris extracts to be 27 t 4 percent,
exc-pot Sumatra-type extracts, which -contained 9 to 15 percent of degaelin.
Again in 1939 Cahn and 'Toam (6) discussed the determination of approxi-
mate roten-one content by means of the Goodhue value. This scheme has been
mentioned in the section on Rotenone.
In a study of colorimetric procedures in 1939.Jones.(68) used this method
and made color comparisons in a neiitral wedge photometer using a filter with
its optical center at 0.56 micron. This means of measuring the color had been
in use for come time by Goodhue and by Oassil in the laboratories of this
Begue' (J) in 1939 recommended thi-s method for the analysis of products
containing less than 1 percent of active principles."
Guillaume and Here (53) in 1939 described and discussed Goodhue's
modification of this method. They suggested that for this test a 1-gm. sample
be extracted with 100 cc. of acetone in a Soxhlet or a Kumagava( l) apparatus
for 5 to 8 hours. They also found that complete exhaustion of 1 gm. of powder
was obt~i.id by agitating with 100 cc. of acetone inBa:'flask.for 2 hours. They
found it unnecessary to prepare standard solution of rotenone each time the
test -as made. Instead, they made a 0.05-percent solution in acetone and kept
it in the dark, in ice (or at laboratory temperature) for use in a large number
of deterniii-tions. The procedure used for the development of the color was
essentially. th'e same as that already described. Although this method gave
results on freshly prepared powders agreeing with those of the blue color test
and the methoxyl method, it gave lower results on old powders than the other
Goodhue and Haller (w) in 1940 used this method for the estinption of
dihydrorotenone in the hydrog2nation products of rotnair.e. Although this
derivative of rotenozor hrs rot bePn foand in 6Frrrias f-.d cl'ue roots, the modified
procedure is equally rppAccblc to the .etnrminatio-. of rotenone ad d;i.:lin
in extracts of these pla-i-s. The prjcceure was prscticRlly identical vith
Goodhue's earlier modification (4-) of the test except that 6 cc'(instpead of 5
Cc) of dilute sulfuric acV. was uzed following the addition of'a.k:lAi 'i- the
solution was then maintain-ed in a cold-wat'-r bath at 15 to 20 C. for about 5
minutes (instead of at 25 for a ?.on.?er period). However, the n.s^rei,,nt of
the color intensity was ir.nroved. Tilis pc-tion of the procEdu.re as as follows:
"The use of a photorieter has been found to be the most eccurrte
method of comparing the sta-.-.,rd.s a.d the unknowns. The colrs can
be devetloued direcUly in selected test tribes wiiich fit the ;hotometer,
or they can be developed in sny test tube ond poured ir.to a rnecia.)
cell. A Brice photometer whichh gives pe.:centage trans.-isLsion ac". direct
rpsadings has been found satifactory. Glass lilters (Corr.'-:, ,5 mm.,
No. 430, dark shade blx.e-groer) were used. T'-, blanr. is ta'en as 109
percent transmission, tnd the photometer is thr .efore d-justed to ive
a reading of 100 with the ce'_1 count inir: the bl-r': in plece. The
readi.,gs for the strnd.ards and u n.'ciow is wrhich sr, ncxt obtained are
therefore in percent t-_ensrrision.
"*4'the color for rotenone has b:.en found by 'ssil (unpublisheJ)
to follow Beer's la?*`*1 A plot is therefore made on3 s =rleorith ic
paper with transmission as o:.'iinate (logaritihmnic) and concentration as
-'abscissa (arithmetic), and a straight line is drr.vn frCm the point of
zero concentration and 100 percert transmission to the point determined
by the concentration and the transmission of the standard. The trans-
mission of the uniriown having been determined, its concentration can be
read from the curve and the percentage calculated."
Cassil (19) in 1941 used the red color test for the determination of
derris-dust reziduaes on cabbegas. The residues were extract-ed by washing the
leaves with chloroform, the extract evaporated, and the rs-idue dissolved in
hot acetone. The acetone solution was chilled to remove w:jxymaterial, and the
filtered solution was used for the color test, The colors i-ere comopred with
those from extra-cts of known quantities of the derris3, root actually usrd in the
original dusts. Comparisons of color intensity were made in a photoelectric
T-kei, Miyajima, and Ono (__8)in 1933 developed a gravimretric m-'thod for
determining rotenone arideguclir. This method, which has been briefly discussed
under Potenone, is ba.cd on the fact that rotenone and deguelin are readily
oxidized under alkaline conditions to rotenolones and deguelinols, respectively,
which are quantitatively dehydrated by alcoholic suluric acid to the highly in-
soluble deh-drorot-enone a-nd dehyarodeguelin. The mixture of d.hydro compounds
gives a value for total rotenone plus deguelin. A value for degelin r.lone
was obtained by subjecting the dehydro comonoounds to aporop-.Lite catlytic
hydrogenation, vhich con-rerted the dehydrorotenone to the alkali-soluble
isoci l:_rod. yc-dorotenone (dehydrorotenonic acid) while the dehya.rodegu-elin
re-ained unc3"-o72ed. In the original method much of the rotenone was first
crystallized out. The method is briefly as follows:
The rotr.one is first cryistalliz'd fran. an ether extract in
the usual i-sr. Five grams of th ?vcr'-e mot- r liquor is
dissolved in 150. cc. of alco'-.el '-rl 3 n. of 5-percent alcoholic
sodium h.ydroxide is .-. .. C.-men is passed in at the rpte of
150 cc. per minute for 1/2 hour. If air is used instead of oxgan,
it is run through for 1 114 to 2 hours. The reaction product is
acidified v/ith 15 s. of 50--ercent alcoholic sulfur c acid, and
12- cc. of alcohol is distilled off on the water bath during 1/2
hour. The residue is rafld another hour. It is nixsd with
50) cc. of water and shrea': n v.-'-.rrousrly with ?CO cc. of ether in a
separatory funnel. The dhv,'dro %eriv -tivcs are suspended in the
ethr !i7er, other materials dissolve in either the water or the
ether layer. The er.tire mixture is filtered by suction, and the
crystals are washed with 10 co. of rethyl! alcohol. The yellow
needle crystals are dried at 1000 ari weighed. This value gives
the weight of any rotenone not seoar-.ted in the origir-l ether
crystallization plus the deguelin. A small quantity of crystals
may also be obtained from thC filtrate. Five-tcnths gram of tV-e
mixtvrc of dehydro compounds and 0.5 gmn. of Ta1nisiLpn-b-iriumn sulfate
cat-l-,.'t are placed in 100 cc. of a.lchol and mixed with 3 c. of
3-p,'-rc. .nt sodium h:.fro:ide. The mixture is shaven in an atmoanph'-re
of }t drogen for 2 hours, and then filtered. The redidue consists
of dehydrodsuelin nnd c:italist. It is extractrd with acetone, and
evaporation of the ext zit livess the dch7-c.rodeTuel'n. Most of the
alcohi-l is distilled from the filtrete, which is then acidified
with sulfuric acid and extracted with ether. Evaporation of the
ether extract gives the isodi-..irodehydrororcnonre.
Danc*:.ortt, Budde, and ?e' nrgrten (U) in 34 stated that the foregoing
method igrht be theoretically unobj,-ction-ble but it was very trouobl'soir.e and
time-consamnirng ,nd requirpd a lar *e amount of p..-.?araitus.
Fischer 'nd 'iteche (?8) stated that the method was too bothersome and
time-con:m;lr.n- in its complete c.x:-cition. They found it useful, howev r, for
determining only the rum of r>te:vn.e ann. de-uelin. essentially the directions
of Taicai ct -,* were used, b,'.t in 'shiA the sea-:r.ted dehydro comounds the
methyl alcoh,:l wqh was follow.'d with a small .,-,nt of ether. Frequently a
second or third methyl alcohol or ether rinse was necessary when the filtrate
from the first was dark colored. In the case of plant materials inclined
to resinify on oxidation, the substance was extracted with ben-ene and the
evaporated benzene extract used for the oxidation. The disturbing materials
were insoluble in benzene.
In 1935 Tattersfield end Martin (.20) found that pure roten.one, when
put through the first portion of the Teicei process, gave a yield of only
about 80 percent of dehyirorotenone. The renairider could be recovered from
the solvents used for the final sena-ration of dehydro co-.pourds. Conseqfuent-
ly they modified this separation. After dehydration with alcoholic sulfuric
acid the residue was cooled for some time in ice, the cry-stals filtered
by suction and washed successively with a little ice-co]d. ether, 100 cc. of
distilled water, end a few cc. of ice-cole. methyl alcohol. The crystals
were dried at 100 0. and weighed. Thc filtrate was then separated by add-
ing 400 cc. of distilled water and 1-00 cc. of ether. If a further yield of
crystals was obtained, they were filtered, washed with a little ice-cold
methyl alcohol, and the wcight added to that of the first lot. Tatttersfield
and Martin in some ceaecs used the whole eth 3r extrct of derris without
preliminary separation of the rotenone, end. In other cn. e the mother liquor
from the separation of the carbon- tct -chloride complex, which they dissolved
in the alcoholic mother liquor from the purification of the rotenone solvate.
Higher results were obtained in' general by the mrdcified. method, either
with or without removal of the rotenone, than by the original 'fZkei method
made on the whole ether extract.
In the following year the samne investigators (f4) attempted the
quantitative preparation of "degaelin concentrates"' of several samples of
derris root, following the qualitative scheme uscd in the earlier work of
Haller and La~orge (55). Twenty grams of the roots was extracted with
petroleum ether, with rapid refli-xing, for 55 hours. The evaporated extracts
were dissolved in ether, and the rotenone was allowed to crystallize for
2 days in the ice chest and then seEra:ted. The ether filtrates were then
extracted with dilute potash, washed with water, dried ovT'r sodium sulfate,
concentrated to a small volume, and placed in the ice chest for 5 de.ys. Any
further rotenone was separated. The mother liquor from this separation was
termed the "deguelin concentrate'." It probably contained most of the deguelin
but may have contained other materials also. The values were somewhat higher
than those obtained for dehydro compounds by the modified Takei method (120)
on the alkallnsoluble portions of the oririina?.l ether extracts. In this
work also the term rotenonet plus du.elin" was applied to a value calculated
from the methoxyl content of the alkali-insoluble portion of the extract -nd
based on the methoxyl content of rotenone and deguelin of 15.74 percent.
In using Takcits dehydro method 'lorsley (132) in 1937 found the amount
of wash solvents recommended by Tatt.-rafiold and martin n (.20) to b, in-
adequate. Accordingly he adopted th:c following proc,'dur'', The cooled
residue from the dehydration process was filtered throljgh a Gooch crucible
and washed with 30 to 40 cc. of ether cooled to -10 C., the crystals were
then pressed down and 100 cc. of water cooled to 2 or 3 C. was poired
through; finally methyl alcohol cooled to -100 C. was poured through as
long as any color was removed. The residue was dried and weighed. In some
cases the crystals were dark colored and resins appeared to be present.
Hence the dried crystals were stirred with about 10 cc. of ether cooled
to -10 0., filtered off, washed with 10 cc. more ether, then with 5 cc.
of methyl alcohol cooled to -100 C. and finally dried and weighed again.
The second series of washings gave parer crystals, and no further yield
could ce obtained from the filtrate.
Cahn, Phipers, and Boam (17) in 1938, in briefly discussing the
method of Takei and. coworkers, stated that they had found the reactions
involved not to be quantitative for pure rotenone anid. deguaelin, and that
tozicarol bcheied partly like deguelin and artificially swelled the re-
sulting 1egelin contents.
Jones (68) in 1939, in using this method., removed the alkali-
soluble material prior to the oxidation to avoid interference from toxicarol.
This plan had also been used in the work of Martin and Tattersfield (84-),
In 1939 Goodhue and. Haller (4A) develoT.ed a method for determining
deguelin in derris and cube ba3.i on its racemization and. the separation of
the inactive form as the carbon ttrachloride solvate. The method was as
A 50-m sample is extracted with chloroform in a Soxhlet
for 7 hours. The chloroform is removed and the extract dis-
solved in about 75 cc. of ether. This solution is extracted
with two 15-cc. portions of 5- rz'nt potassium hydroxide
saturated with sodium chloride. These portions are extracted
with ether, and the combined ether la.er is washed once with
1+ 10 hydrochloric acid and once with a saturated sodium chloride
solution. The alkali soluble extract is discarded.
The ether is removed, the resin dissolved in 40 cc. of carbon
tetrqchloride, and the rotenone solvate crystallized and separated
in the uoual way. The carbon tetrachloride is remov'd front the
filtrate end the residue is dissolved in 10 to 15 cc. of methanol.
Vh,'nile warm. this solution is placed in a 25-cc. Erlermeyer flask
and 10 drops of 40-percent pot-iassium hydroxide aided. The contents
are swirled and the flask is filled w.th warm methanol. A one-
hole stopper carrying a funnel made from a dralnm-out test tube is
ir-..7diptely inserted so that no air bubbles remain in the flask
nnd some of the colorless liquid is forced up into the funnel.
More methanol is ptured in the funrnel to allow for contraction on
cooling arnd for evaporation. The solution is kept at about 45 0.
for an hour to prevent separation of resin before it is racemized.
.If ieielin is present, crystals soon separate, but rrco izr-tion
is usually not complete until the matoriil has stood overnight.
The flask is then cooled it 0 C. for 1 ho-ur. The methanol
is then decanted through P small filter and the residue allowed
to drain as co-.pletely as possible. For purification the
de.'elin cryptals are dissolved in a little chloroform, nnd the
chloroform is roelt-ced by evapor.tin- to a thick solution twice
with carbon tetrachloride. Fi i-lly, the deguelin is crystal-
li", fromi 5 or 10 cc. of crbor. t-tr chloride. It is usually
necessE.ry to s9-d the solution with the carbon tetrachloride
solvate of deguelin at 0 0. and let it stand overnight for
complete crystallization. The crystals are then filtered on
a tared Gooch crucible, washed with cold carbon tetrachloride
saturated with deguelin,. air-dried at room temp-ratuLre for 4
hours, and wei hd as the l;1 deguelin-carbon tetrachloride
solvate. .. "
The amount of deguelin in this itpure solvate is deter-
mined by the red color test (40). It is assume d that deguelin
alone is responsible for the color, and the fact that racemic
d-guelin gives only 80 percent of the color given by rotenone
is taken into consideration when rotenone is used as the standard
of comparison. The effect of solubility in the solvents used
is compensated for by adding 0.08 percent when 5 cc. of carbon
tetrachloride is us.?d for crystallization and 0.11 percent when
10 cc. is used.
The accuracy of the method was checked from several angles and it appeared
that no gresit error was Introduced. The possibility of decomposition of
deguelin during the- racemization was studied by using a concentrated sample
of active deguelin orenq3red by hi ':-vacuum distillation. Upon racemizdtioa
of this'aatetial, 83 per6ent of Inactive dfguelin was obtained. Hence not
more than 17 percent was believed to be destroyed in the racemization and
probably much less, as the active dagielin was not pure. 'The precision
was said to be equal to that of the rotenone analysis (72)). Results for
deguelin by this method were markedly lower than those by either the red
color test (40) or the dehydro method (120). For example, on 13 samples of
derris and cube values by racemization ranged from less than one-tenth to
about four-tenths of those by the red color test. It was pointed out that
some of this difference might be'due to compounds other than deguelin that
giv? the color test or dehydro derivatives.
As in previous work on the evaluation of rotenone-containing plants,
Martin (83) in 1940 made quantitative determinations of the'amounts of;
"deguelin concentrate" in several samples of derris. The method was similar
to that employed in the earlier investigation (84)wlth certain'modifications.
The roots were extracted by percolation with ethyl acetpte for 3 hours in-
stead of by prolonged extraction with petroleum ether in a Soxhiet. Alkali
extraction of the resins in ether solution was carried out before crystal'-
lization of the rotenone. In the c-se of roots rich in rotenone a mixture
of ether and benzene was used as solvent for the extract during the alkali
extraction. This scheme, which prevented crystallization of the rotenone
during the extraction, has already been mentioned in connection with the
determination of rotenone (59). After separation of the rotenone as the
carbon tetrechloride solvate, the residual resin was designated the "deguelin
concentrated It was also stated that an assessment of the "deguelin con-
centrate" was obtained simply by subtracting from the percentage of "neutral
resin" (alkali-insoluole material) the percentage of purified rotenone.
-' ... .. Discussion .
The Durham color reaction (75) has become established as a rapid
*qualitative test for rotenone and related compounds. Likewise the red dolor
test originated by Gross and Smith (50), particularly in the Goodhue modi-
fication (40), is widely used in the quantitative evaluation or material
containing these substances. Since both reactions probably depend essentially
on the same structural grouping, the substances present in derris and cube
which will"give one of these color reactions will probably giveboth. The
dehjdro reaction has not been used so widely as the color tests. Both the
dehydro' reaction and the color tests have been suggested as means of evaluat-
ing the t'exicity of rotenone-containing plants.
At one time it was thought that rotenone and deguelin were the only
substances -resent in derriS and cube which were determined by these reactions,
but there is now increasing evidence that this is not strictly true. Thus,
although Faller and LaForge (55), 1orsley (132), Tattersfield and Martin
(120), and other investigators obtained comparable results on.many samples
by the 'debydro method and by the red color test, on many other samples it
has been found that the methods do not agree. For example, on a series of
derris roots Jones (60) obtained results that were substantially lower by
the dehydro method than by the red color method., while results for cube roots
were comppr3ble. It thus appears that in many samples substances are present
which do not give both reactions, or at least not to the same degree.
Furthermore, new compounds have been found in derris which both give the red
color test and form dehydro derivatives (Ll,56, 881' The amounts present of
these other compounds have been stated to be small. The recent work of
Goodhue and Haller (41), however, presents the possibility that large amounts
of substances other than rotenone and &eguelin may be determined by both the
red color test and the dehydro reaction. On the other hand, it is possible
that the deguaelin may not all be present in such a form as to be determined
by the racemization method. The earlier work of Haller and LaForge (55) had
already indicated that not all the deguelin was present in a single form.
Only further work can give a clear understanding into the cause of the con-
siderable discrepancies between this and the earlier methods. In the mean-
time results by any of these methods should be considered as essentially
TOXICAROL AID ,OT'r-RH ALKAL!-SOLULE SU3STATNCES
In 1935 Jones, Canre-bell, and Sullivan (659) determined the amount of
alkali-soluble material in several samples of derris and cube roots follow-
ing the method used by HEa]lir aind' LaF'org. (55) in preparative work. This
involved extraction of an eti-er soluhion of the ertr:_ct with 5-percent
aqueous potassium hydroxide until no more material was dissolved. The
alkaline layer was acidified-'and extracted with ether, and the extract was
evaporated and. weighed.
Martin and Tattersfield (e_4) in 19356 used essenti-lly the same method
for separating alkali-soluble' and aYicwli-insoluble fractions of derris root.
In one test the separation ws made on an extract from which the rotenone
had been crystallized; in the other the whole extract was used. In the
latter case 5-gm. samples of root were extracted with ether and the ether solu-
malaccensis extracts precipitates forced in the alkaline extract. The
alkaline extract of D3. elliptica resin showed no precipitate and very little
alkali-soluble material. The :Esi'ns from the precipitates and alkali-soluble
fractions were recovered by acidification with dilute h-r-.rochloric acid
and solution in ether. The ether extracts were washed, dried with anh,-droua
sodium sulfate, evaporated, and dried to constant weight. Yethoxyl deter-
minations were rade and the contents of "active princiipls," based upon the
methoxyl content of zcxicarol, were calculated, In one series of tests
saturated barium hydroxide -was su.sti-tued for potash. TLe. amount of
alkali-insoluble material agreed with that obtained by the use cf potash,
but the berium hydro:ide extractior. precipitated a lF-rgr-, proportion of the
alkali-soluble material than did potash. The fractionat3on with 1quLous potash
was repeated using benzene infistead of ether as solvent for the Ixtract, but
only a relatively small percentage of the resin was extracted by the alkali
froth the benzene extracts of the three types of derris roob examined.
Potash did not effect complete separation of the potessiur. salt of toxi-
carol from a benzene solution of Stiratra-type or Derris m.accensis resins.
Cahn, Phipers, and Boam (17) inr. 1938 described a color test for the
determination of toxicarol based on the deep green color obtained with
"One drop (0.06 cc.) of a 5 percent aqueous solution of commercial
*ferric chloride (hbydered) is added to.10' ce. of'ihol'containing
an ainount of Derris extract approximately equivalent to 1 mg. of
toxicarol; the color developed is matched in a Duboscq colorimeter
against that from a standard solution of exactly 1 mg. of toxicarol
in 10 cc.,of alcohol. The approximate amount of extract to be
taken may-be det-rmined by rough visual matching, using solutions
containing 5 mg. of toxicarol or its equivalent in 10 cc. of
alcohol.,.. The color is permanent for quite long periods."
It was found that absolute or 96-percent alcohol could be used. Pure ferric
chloride offered no advantage over the commercial grade. Ferrous sulfate
gave no color, while ferric alum gave only a slight color. The size of the
drop was determined by the dropping pipette arbitrarily chosen; a similar
drop of 2.5-percent ferric chloride solution gave & slightly weaker color,
but 1 and 2 drops of the 5-percent solution gave identical colors. withini n
limits, therefore, the amount of ferric chloride appeared to be immaterial
provided sufficient was used. The alcoholic solution could contain at
least 3 percent of acetone, benzene, or ahloroformn without interfering with
the results. Extracts of Sumatra-tyoe roots gave'a deep green ferric chloride
color and were easily matched against pure 'toxicarol. However, as the
rotenone content of an extract increased the ferric chloride color became
more and more brown, and with rotenone-rich extracts matching in the Dhaboscq
colorimeter was vpry uncertain. This difficulty was overcome by employing
secondary standards. Thus it was found that extracts containing less than
20 percent of rotenone could be matched against Sumatra-type extracts. One
particular Sumatra-type extract was accordingly taken as a subsidiary stand-
ard. For extracts containing more than 20 percent of rotenone even this
was not satisfactory. Ethereal solutions of such extracts were shaken ',,th
5-percent aqueous potassium hydroxide and the phenols recovered from the
alkaline layer and separated solid salts (if any) by acidification and
extraction with ether. The ferric chloride value was then determined on
this alkali-soluble part only.
The authors proposed the term "ferric chloride value" to denote the
percentage of toxicarol that the sample would contain if the intensity of
color developed was due entirely to toxicarol. Although sumatrol gave a
brown color with ferric chloride, this color was believed to contain a
green component which contributed to the total ferric chloride color.
Consequently, the ferric chloride values of derris extracts were inter-
preted as giving the sum of the percentage of toxicarol and sumatrol. Hiow-
ever, the yield of sumatrol isolated from derris was said to be always
much less than the yield of toxicarol. Other substances giving a green
color night also be present in derris, but they had not been isolated up
to this time.
Rowaan and Van Duuren (108) in 1938 described a quantitative method
for the determination of toxicarol based on the earlier qualitative
separation of this substance by Clark (21). It was as follows:
The carbon tetrachloride filtrate from the rotenone
determination is freed of solvent and the residue dissolved,
by warming, in 100 cc. of 95-percent ethyl alcohol. After
the addition of 10 cc. of lN. sodium hydroxide the solution
is boiled. On cooling-, the separated sodium salt of toxicarol
is filtered, washed with alco'iol, dried at 100o- 105 C., and
weighed. The weight multiplied by 0.95 gives the weight
The ferric chloride color test of Cahn anrid his coworkers (17) was used
by Jones (68) in 1939 in the examination of a series of derris and cube
roots. Determinations were made by the method already described including
the use of secondary standards where necessary. Difficulty .was encountered
in matching the colors from the cube samples, and the use of.the alkali-
soluble fractions of the extracts did not improve the color matching. The
amount of alkali-soluble material was determined by the method previously
Martin (83) in 1940 made determinations of alkali-soluble material,
or of "toxicarol fraction," on several samples of derris. The method was
similar to that used in previous work (84) except that extraction of the
ether solution was made first with one lot of 2-percent and then with
two lots of 5-percent potassium hydroxide. As mentioned under the sections
on Rotenone and Deguelin, a mixture of ether with 25 percent of benzene
was employed as the solvent for extracts of high-rotenone roots. It was
also stated that the percentage of "toxicarol fraction" removed by the
potash could be determined,from the difference in the percentages of original
and "neutral" (alkali-insoluble) resins.
Both the determination of alkali-soluble material and the ferric
chloride color test must be considered to give only empirical values. It
is probable that in Sumatra-type derris roots both these methods give
reasonably close approximations of the toxicarol plus. sumatrol content. In
other samples of derris and particularly in cube samples large amounts of
interfering substances are undoubtedly present. Since the ferric chloride
test was proposed an additional phenol, malaccol, has been isolated from
derris (88). Rowaan and Van Duuren (108) state that toxicarol has not
yet been isolated from Lonchocarpus, (cube),. and attempts to isolte this
compound from cube root in the laboratories of this Bureau have been un-
successful. Nevertheless, the samples of cube examined by Jones (68) gave
ferric chloride values equivalent to 1.5 to 2.8 percent of toxicarol,
presumably due to other phenolic bodies, and contained 2.8 to 5.0 percent
of 'alkali-soluble material.
The method of Rowaan and Van Dauren (108), based on the separation of
inactive toxicarol, should be subject to less error from interfering sub-
stances. whoever, the accuracy of the scheme May be questioned when only
small amounts of toxicarol are present.
MISCELIANEOUS COLORIMETRIC IETEB41NATIONS
Sulfuric Acid-Nitrite Color Reaction
In 1899 Sillevoldt (114) found that his "derrid,".which undoubtedly
contained a high percentage of rotenone, gave a brown-violet color with
concentrated sulfuric acid. Danckwortt, Budde, and Baumgarten (25) in 1934
found that sulfuric acid followed by a small amount of sodium nitrite gave
an intense red-violet color with rotenone. This finding suggests that
Sillevoldt's acid contained a trace of nitrite. '.'hen the test was applied
to derris root, a 0.5-gm. sample was shaken with 5 cc. of chloroform and
1 to 2 drops of the filtered extract were evaporated on a wateh glass. To
this residue was added 6 to 8 drops of concentrated sulfuric acid and a
crystal of sodium nitrite. The red-violet color developed in from 1/2 to
5 minutes. A similar result was obtained when the test was carried out in
the same way but with water instead of chloroform. The color was also given
by dehVyrorotenone, dehydrodeguelin, and isodihydrodehydrorotenone. Toxicarol
was said to give a red-brown color only.
This color reaction was developed into a quantitative test by Fischer
and 3Titsche (28) in 1935. The method was essentially as follows:
One cubic centimeter of an acetone solution containing 0.2
to 0,5 m,. of derris resin is placed in a 25-cc. volumetric
flask and evaporated to dryness. The residue is cooled and
dissolved in 2 cc. of concentrated sulfuric acid, and 5 drops
of a 1-percent solution of sodium nitrite in concentrated
sulfuric acid are added. The flask is heated on the water
bath for 8 minutes, cooled, and the solution is made to volume
with concentrated sulfuric acid. Comparison is made in a
colorimeter with the color developed from a standard solution
Pure rotenone Pave a "perman.qnate-like" violet, while derris resins gave
a more reddish violet. Since the authors used an "absolute" colorimeter
it was not necessary to repeat the rotenone standard. A red filter was
found to be suitable in the color measurement, although a green filter was
sometimes used. Acetone, benzene, and chloroform extracts ave equal color
values. Eth'ner extracts, however, gave erratic results. Too much nitrite
was found to destroy the color. Toxicarol gave a color reaction equally as
strong as did rotenone but of a more reddish violet. The mixture of dehydro
com-)ounds obtained 'in the Takei (118) method gave a color exactly like
that of rotenone in tone and intensity.
The sulfuric acid-nitrite test was modified by Meijer (85) in 1936.
In this form an aqueous suspension of the material to be tested was treated
with' the reagpnt. The method was cdesignp.d primarily for the rapid
determi. etion of total ether extract and was said to require about 20
minutes. It was as follows:
A 1-gn. snrnmole of derris .root is extracted by shaking
in a test tube with 10 cc. of acetone for 5 minutes. One
cubic centimeter of the filtered extr-ct is diluted to 25
cc. with distilled water. This milky suspension is well
salcen, and 0.2 cc. in a dry test tube is treated with 5
cc. of a solution of sodium nitrite in concentrated sulfuric
acid containing 10 mg. of sodium nitrite per 100 cc.
The addition of the sulfuric acid to the aqueous suspension caused sufficient
heat to produce the maximum color. The violet-color obtained was measured
in a step photometer with a 5300 A. filter. The results were compared
with determi:iations mr-de on samples of known total-extract content. The re-
lation between extinction coefficient at 5300 A. and percentage of ether
extract followed Beer's law.. It was found that the.color could also be
compared visually with permanent color standards prepared from cobalt
chloride. The standards were made by mixing in various proportions a 10-
percent solution of the cobalt salt in 96-percent alcohol, a 10-percent
solution of the cobalt salt in water, and 96-percent alcohol. When kept
inlsealed tubes these color standards were stable for a long period. The
color reaction required a strongly acid medium. Seventy percent trichloro-
acetic acid instead of sulfuric also gave the color but with a more reddish
shade. Acetic, oxalic, and tartaric acids gave no color. iHydrochloric
acid gave only a faint pink color. Since the variety of derris known in
the Dutch East Indies as "Toeba woeloeng" had previously been shown to
have a definite ratio' of rotenone to total extract, it was 'suggested that
this method would give approximate values for rotenone in this type of root.
Goudswaard and Timmers (43) in 1937 stated that this reaction could
be usi.d in the inverse sense for the detection of nitrates and nitrites
in sulfuric acid. Sulfuric acid that conformed to the Dutch pharmacopoeia
contained sufficient nitrites to give a color with rotenone. In the usual
form of the test the color developed was proportional to the rotenone
present and was stable for 24 hours. The test was said to be unsuitable
for the estimation of rotenone in derris root because the reaction was not
specific for rotenone.
Cahn, Phipers, and Boam (17) in 1938 found that the Meijer color
test was given by many substances other than derris extract; consequently,
the method was applicable only in the absence of interfering substances and
when the genuineness of the root was certain. The color was given with
equal intensity by rotenone, deguelin, toxicarol, and sumatrol, as well as
by many of their' derivatives. All derris extracts tested ,wre found to
give the color with about 90 percent of the intensity given by rotenone.
A modification of the Meijer test was used in this work (private communication).
In 1939 Jones (68) used the sulfuric acid-nitrite test in the form
described by Meijer, and made color comparisons with a rotenone standard in
a Daboscq type of colorimeter without a filter. Values for the derris
samples averaged about 90 percent of the extract, but those for the cube
samples were lower. The test was said to give a rough estimate of the
total materials of the rotenone type.
The Rogers and Calamari Test
Rogers and Calamari (102) found in 1936 that in the presence of
hydrochloric acid and certain phenols rotenone developed colors ranging
from blue to violet-red depending upon the -solvent and the phenol, used.
Such organic solvents as chloroform, ethylene dichloride, carbon
tetrachloride, ether, alcohol, and acetone were used. Phenol, -guaaiacol,
and thymol reacted similarly in these solvents. Snmall amounts of hydrogen
peroxide and nitric acid and light exerted a marked influence in accelerating
the reaction. The color was also given by certain derivatives of rotenone.
Both qualitative and quantitative tests based on this color reaction were
developed. Substances usually found in proprietary liquid insecticides,
such as pyrethrumn extract, aliphatic thiocyanates, and oil of sassafras,
were said not to interfere with the tests. In the qualitative test a
chloroform solution of the sample was treated with a chloroform solution
of thymol. A mixture.,of 0.2 part of concentrated.nitridacid:and 100 parts
of hydrochloric a&c.d was then added and -the solution shaken. AL blue-green
to blue color appeared in from 30 seconds to 2 minutes when rotenone-was
present. The following quantitative test was designed for use with colorless
liquid insecticides: ...
To 10 ml. of a chloroform solution containing from about
0.05 to 2.5 mg. of rotenone per milliliter in a glass-stoppered
cylinder, add 10 ml. of a chloroform solution of thymol (10 gm.
of thymol to 100 ml. of. chloroform) and.*-2 ml. of a reagent made
by adding 2.5 ml. of 3-percent hydrogen peroxide to 100 ml. of '
concentrated hydrochloric acid. (When the sample is not in
solution in a hydrocarbon base, concentrated hydrochloric' acid
may be used in place of this reagent.) Agitate for 1 minute,
loosen the glass stoijer, and expose the' cylinder to the intense
radiation of a quartz-mercury vapor lamp (minimum output 1000
microwatts per square cubic centimeter in the field of exposure).
A greenish-blue color apoers in the chloroform layer in about
15 minutes. (Exrposure to bright s'nlight produces similar results
in about 3 hours.) At the end of 30 minutes compare with stand-
ards containing known quantities of pure rotenone prepared
simultaneously in the same way. If the insecticide base is refined
kerosene, use an equal amount of refined kerceene in preparing the
A more rapid method making use of an acetone solution was as follows:
To 5 ml. of an acetone solution containing 0.1 to 2.0 mg. of
rotenone per milliliter add 5 ml. of an ceptone solution of
thjmol (10 gm. of thymol to 100 ml. of acetone), 0.1 ml. of 3-
;percent hydrogen peroxide, and 5 ml. of concentrated hydrochloric
acid. A reddish-violet color a ooears within 30 seconds. After
1 minute place the cylinder in a water bath at about 200 C., and
at the end of 20 minutes compare with rotenone standards similarly
prepared at the same time.
If the liquid extract contained pyrethrum extract, it was recommended that
a standard containing pyrethrum extract be prepared to match the original
color of the liquid insecticide.
Cahn and Boam (14) in the same year reported failure to obtain the
Rogers and Calamari qualitative test with the rapidity and intensity
stated by the authors of the test. These factors were found to be markedly
affect d by the nitrous acid content of the nitric acid. With a mixture of
fresh nitric and hydrochloric acids the rotenone color was produced only
slowly. ..then nitrite was added to the mixture, or when the acid mixture
was allowed to stand before use, the rotenone color developed rapidly. It
was concluded that the reaction depended more upon the presence or
production of nitrous acid than upon the presence of nitric acid.
In their reply Rogers and Calamari (103) stated that it was essential
to use concentrated hydrochloric acid containing not less than 35 percent
of hydrogen chloride and pointed out that Cahnlm and Boam used an acid contain-
ing only about 30 percent of hydrogecn chloride. They found that when the
stronger hVdrochloric acid was used the nitrogen peroxide content of the
nitric acid was unimportant. The intensity and speed of the color reaction
depended on the concentration of the acid used. They believed that the acid
reag- nt depended on the formation of nitrosyl chloride for its action and
that the reagent should stand at least 3 minutes before use.
Cahn and Boam (15) in 1937 reported that when they used 36.45 percent
hydrochloric acid (sp. gr. 1.18) the'dr results were in substantial agreement
with those of Rogers and Oalairari. They found, however, that even then the
rate of development of color and its final intensity increased greatly as
the acid mixture was allowed to stand before use. They emphasized that
the sensitivity of the reaction depended .-reatly on the exact conditions
According to DeOng (27) the Rogers and Calamari method proved un-
satisfactory for the quantitative det- rminntion of rotenone in fly sprays.
In 1939 Jones (68) modified the Pogers and Calamari test for the
analysis of srvernl samnoles of derris and cube roots. The color produced
in the acetone test was not proportional to the rotenone present. In
chloroform solution the proportionality between color and concentration
held. Feat as well as light accelerated the color formation, but the
latter was adopted. The solutions were exposed in glass cylinders to
daylight (not direct sunlight) for 24 hours. Perchloric acid was found
to effect a more rapid development of the color than hydrogen peroxide.
At a rotenon- concentration of 0.12 mg. per cubic centimeter, 2 drops of
60-percent prchloric acid Pve a moderately intense, pure-blue color In
24 hours. Even in this form results were err-tic and duplicate standards
varied as much as 10 percent. De.fuelin was found to give a color intensity
about 125 percent of that given by rotenone, whereas toxicarol gave only
about on.e-Llf the rotenone color. When rotenone was used as a standard
values for the derris roos ranged from 60 to 120 percent, and those for
cube and timbo from 140 to 160 percent of the total extractives.
The method of Rogers and Calamari was recently used as a qualitative
test for rotenone in miscellaneous insecticides (127). The characteristic
blue color did not develop vwhen too much rotenone was present; therefore,
in case of failure to obtain the test a repeat should be made with a
Other Color Reactions
Geoffrey (29) in 1-95 was the first to study the color reactions of
rotenone with several reagents. The most characteristic involved treatment
with bromine, followed by application of concentrated sulfuric acid to the
residue. A violet color was produced.
Dennis (26), in a patent on cube issued in 1927, described a color
reaction for testing the material using sulfuric and nitric acids followed
by pots *ium hydroxide. Jones (68) in 1939 found that this test gave
identical color reactions with roots of derris, cube, and Tephrosia
Schmitt (109) in 1930 stated that rotenone may be detected in ground
root by treatment with nitric acid, whereupon the affected portions turned
PozZi--scot (95) in 1936 stated that rotenone dissolved in concentrated
sulfuric acid with the formation of a red-cerise or rose-cerise color. When
the acid contained mercuric oxide in solution, as in Denige's reagent, the
color reaction with rotenone was from 100 to 1,000 times more sensitive and
was not subject to interference from as many other substances as when acid
alone was used. On being warmed with this reagent, the rotenone dissolved
giving an intense orange-yellow color. The intensity of color was observed
to be proportional to the amount of rotenone.
In later work Pozzi-3scot (98) discussed the color reactions given by
sulfuric acid, sulfuric and nitric acids, and Denige's reagent with sub-
stances that might interfere with the rotenone reaction, and described methods
for differentiating between the reaction of rotenone and reactions of other
In 1937 Tapia Freses (119) reported that rotenone gave a red-violet
color with a solution containing Q.1 gmn. of vanadium pentoxide in 10 cc.
of sulfuric acid.
Danckwortt and Budde (?4) in 1933 stated that polarimetric determinat-
ions might ultimately be used for the evaluation of derris root.
In the same year Jones (62) briefly studied the possibility of using
the high optical rotation of rot-enone (74) (in benzene ralpha320= P24o for
a 5-percent solution) as a means for its determination. The optical
rotator powers of derris and cube extracts were det-rmined in several
solvents, and a hypothetical rotenone content was calculated from the values
for pure rotenone. In most cases the results were very much higher than
those obtained by crystallization. Since one derris extract was dextrorotat-
ory, it was concluded that optical rotation cannot be used as a measure of
the amount of rotenone present in the root.
A polarimetric method proposed by Danckwortt, Budde, and Baumgarten
(25) in 1934 was claimed to be superior to the crystallization methods, at
least as a measure of the toxicity of derris. Directions were as follows:
3 gm. of finely pulverized sample is digested with 30 cc.
of benzene at room temperature for ?4 hours. The optical
rotation of the filtered benzene solution is determined in a
lOC-mm. tube. The rotenone content is obtained by the follow-
Percent rotenone alpha X 1,000
In 10 out of 11 samples of derris root the method gove values higher
than those by crystallization. The method was also adapted to the deter-
mination of rotenone in aqueous extracts of derris, 'nd this use of the
procedure was described in detail.
Gstirner (51) in 194 emp)ojed the method of D.-:'nckwortt and coworkers
in the anal, sis of 19 s.-uir.les and fractions of samples of derris root.
In all cases except One r:,,'.lts w-re hi-ht-r th-n those by crystallization,
in most CPSLS srtcily;r hi,-.-r. This diffe`Lnce was attributed to the
rotenone remaining in the.rosin from the crystallization.
Danckworttts method was also usrd by 7tscher and ritsche (28) in 1935
in a study of chemical methods for the evaluation of cormercial derris
preparations. These Inves'tgators found, that extraction of the sample for
this purpose-was comn-lete in 3 to 4 hours. In certain cases it was
necessary to have more dcrris resih present then specified by Lanck'wortt
Rowapn (105) in 1935 report,ci that in ar.sn.l.'ses of samples of derris
root the rotenone content was usually n!gh r by the polarirrmetric method
of Dnc'wortt rnd his cowo rkers than by cryst.Alization, but that in two
samples the values by crystallization were about double thaose by the optical-
rotation method. Ro-ann also reported the isolation of a dextrorotatory
preparation of toxicarol from derris. Because of these circumstances
analysts were warned against the use of the polarimetric method.
In reply Dflnc*.wortt (23) pointed out that the polpr>metric method
was.a means of determining the effective value of derris and not of deter-
mining rotenone. He also reported the use of the method in a stidy of the
relative stability of industrial derris extracts over a period of time.
The extracts wcre dried with sEnd, the residue was shaken with benzene for
20 minutes, and the optical rotation of the solution was JRtc'rm'.ned as
previously described. Stror.nly colored solutions were shaken with animal
charcoal. Too much charcoal was avoided, since it caused s decrease in
Bowaan (106) in 1936 pain warned against the use of the polari:-ietric
method for the determination of rotenone in derris rnd stated that the
most reliable method was soma. modification of the extraction-crystallization
Tattersfield and Martin (121) in 1936 studied the optical activity of
benzene solutions of resins nnd their fractions derived from sanples of
Derris elliptica, D. mralaccensis, and Sumatra-type derris root. A fraction
of Sumatra-type resin obtained by means of methyl alcohol was dextrorotatory
in methyl alcohol but lev-:rotatory in benzene.
A study of the optical rotatory power of acetone and benzene ex-.racts'
of 16 samples of derris and 10 samples of cube root was teporte-d by Jones
(65) in 1936. Calculations of h;,-pothetical rotenone contents were b-scd on
the known rotations of pure rotenone in acetone -nd benzene, as was done
in earlier work (62). All extracts were levorotatory in benzene, but
acetone extracts of certain derris roots with little or no rotenone by
crystallization were dextrorotatory. There was no agreement between the
value obtained by crystallization and the values obtained from the optical
rotatory powers of either acetone or benzene extracts. A further calculation
based on optical rotatory power which used the rotations of both acetone and
benzene extracts was made by Jones. It was based on the assum-ption that the
optical rotatory power of all the rotenonelike com.-ou'.ids -oresent was .greater
in benzene than in acetone to the same degree as was that of rotenone, and
that the rotation of the oth r optically active material was the same ih both
solvents. All values by this method were lharkedly higher than values obtainedb
crystallization. It was concluded that the optical rotatory power could not
be recommended as a means of evaluation of derris and cube root.
One of the laboratories participating in the collaborative analysis of
derris reported by Koolhans and Mi"ijer (see p. B) in 1937 used the -)olari-
metric method of Danckwortt. The use of this method for the determination
of rotenone was criticized b-,, the authors of the report because of the general
lack of knowledge of the significance of optical activity and of the propor-
4 of othcr constituents present.
Worsley (132) in 1937 studied the use of optical rotatory.pow',r as a
means of assessing the toxicity of derris root. The dried extract from
hot percolation of the root with ct'thyl tastet e Was dissolved in b';nzene and
the concentration of the solution. adjust, d so that the angle of rotation
in a 200-mm. tube was about 82 degrees. Concentrations between 25 and 200
gm of original sample in 25 cc. of benzene were involved. Direct extraction
with benzene in a Schlet gave the sane results. From a curve for the optical
rotation of pure rotenone a value was read off corresponding to the angle
obtained as described in ;'orsley's method for rotenone.detcrmin-tion (130).
Because in most samples this value agreed rather closely with that for
dehydro compounds, Worsley used the term "optical dhydro compounds.11 In a
subsequent article Worsley (133) proposed the term "optical constituents "
instead of "optical dehydro compounds" for the value described in the previous
work. He also proposed the use of the reciprocal of this value for toxicity
Guillaume and Proeschel (54) in 1937 stated that the polarimetric
method was used in Gernmany, but that investigators in other countries found
it gnve too high r sults.
PRowqn and Van Daurern. (1068) in 1938 confirmed in general the observations
of Jones (65) on the optical activity of derris and cube extracts, particular-
ly with regard to the dextrorotation of derris extracts that gave no rotenone
Thung (123) in 1939 stated that in the analysis of derris powder a 2-
gn. sample was extracted by shaking repeatedly with 53 cc. of benzene during
24 hours. The optical rotation of the filtered extract was determined in a
200-mm. tube (045 V. 1 percent rotenone).
Dtd.rminatlon of Methoxyl Content
Tatte.rsfield and Roach (122) in 1923 s'ttud that 'the genuineness 6of
derris :ext"rcts might be crnfirn.d by the dattrrnin-'tion of their methoxyl
cCnt *rt. DctPrmnirationE. w'-ro mrdo on the dr'ed eth--r.extrectives" byPerkin's
rmo')i-'ic.tion (`l1). of the Zuisl eth-d. T!-,e methoxyl co.ntent of different
s'r..lus of.e7-tract ranr d between 13.5 ?nd 14.7 percent.
fl"'anc*'ortt &e-d Zu'de .(?) in 1933 deterin-:'d the meThoXyl content"of
benzr.e'cxtracts of deri,'is root.
In l--4 Denc.i'.ortt.,. Bud'.L, P nd Bai, nrt-n (25) described their method
for the detr-rin.Ation of the mctnovl contn.t of derris c.trqct. The benzene
extract '.'as trr 2feTEr.d directlyy to the fl.?sk of a Zeisel metho-xyl determine-.
tii app-r-tus rr.- evK T o,-nt&d to dr:,n-ss therein. The usual Zcisel method was
used. O:-.e gran of silver iodice corrFs-no'-did to O.c394 Pram of "active
substance," c]C..l ptd. d as roteonone. This .detrrminr?-ion was said to have only
an orienting v,-.ie ,
Carpb01l, Sullivs'i, Pnd Jcnes .1..) in 1 31 determined the methoxy1l
contents of derris and cube roots by tto method of 1Vi(o0cc. and Schwappach
as modified by Clerkc (e1). dcte detct4r ittios w-'re r.nde on acetone extractlves,
which had boen tr.?td with bc-',Ene to r^.:-ve tr-c-s of acetone. In 1935
these invest gatas (69) usd the sa-ie n<.thc o.n benzene e:t:-:-.cts of derris
and cube and' calc'-aL td. the results to ,,t fiall having ths ane reth1xyl
content as rotenone. These vn\'Z: %.-,7re said to be significant, as all the
known naturally occv-rri.-1"- corp.,--'.s of the rotenone ri-nup had approximately
the same methoxyl ccntenb as rotenone.
Metloxyl determinations .i-ere ride by Tattersfield and M'.-rtin (1.20) in
1935 on sanr-les of three s:;.cies of Derris. VTlur:s were det :-pined on dried
ether extracts by the method of Cipri: [27. The results were closely
correlated with the weiits of the ether extract.
Martin and T.ttersfield (P4) in 1936 fc-ind the methoxyl content of the
ether extracts of three rmples of derris root in good "re-iment with that
of the benzene extraecs. They also made methoxyl determinations on various
fractions of the extracts.
Guillaume and Eervd (53) in 1939 used a modification of the Zeisel
method in determining the methoxyl content of derris, cube, an'l other rotenone-
containing mt.Irr-'s. _r? meot,-l iodide oramed was run into 0oIT silver
nitrate a:,-d the excess of silver nitrate was titrated vith 0.1 fsodi-um
thiosuilfte. The value obtained was calculated to material of the sane
methoxyl conte-nt as rotenone. The results, in the 11 samples tested, were
in good agrecrent with those by the modified Durham blue color test used by
Danckwortt and BIdd3 (2._) in 1953 stated that attempts to measure the
absorption of iodine or bromine by the double bond of rotenone as a method
of determination 1.,d to practical difficulties. Again in 1934 they reported
(25) that, since the halogen co.sumption was dependent on factors other than
the quantity of rotenone, a reproducible method could not be attained.
Wihittaker and Glicknan (129) in 1934 described a method for the
determination of rotenone which was an adaptation of the method of Gnadinger
and Co0rl (39) for the pyrethrins. It involved reduction of an alkaline copper i
solution, a modified Folin's solution, by standard solutions of dextrose and
of rotenone and by the unknown solution of rotenone. The reagents and pro-
cedure were almost identical with those described by Gnadinger and Corl.
A standard rotenone solution in 95-percent ethyl alcohol was used. Results
for standard roterone solutions ranged from 9.60 mg. of rotenone equivalent
in reducing action to 1.25 mg. of dextrose to 21.09 mag. of rotenone equiva-
lent to 4.19 mg. of dextrose. From these results an equation was obtained
for calculating the amount of rotenone present from the equivalent amount of
dextrose. In a sac6nd series of tests the results agreed well with those
calculated from this equation. The precision of themethod was said to be
usually about 2 parts per thousand.. Traces of chloroform and carbon
tetrachloride interfered with the determination.
This procedure was successfully used for the determination of rotenone
in alcoholic solutions containing antioxidants. When the method was applied
to the analysis of derris root, the results were much higher than those
obtained by the crystallization method. For example, two samples which gave
about 6 percent of rotenone by crystallization gave 11.5 an 3A5 praent by the
reduction method. A sample with no rotenone gave 15.5 percent by the present
- 71 -
LITY-RA TU1RE CIT3D
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1936. Toxicological study of derris. Indus. and Fngin. Chnm,
2. ASSOCIATION OF OFFICIAL AGEICU.TUTjL ChElI STS.
1940. Official and tentative method; of analysis of the Associa-
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Ed. 5, 757 pp., illus.
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1936. Rotenono determination- observations on the dotermina.-
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1937. Rotenonbc twumnelsc .i Dorris-og Cuber)ddor. Dansk.
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1939. Lo do-age chimiquae des poudres rot6nonecs. Ann. Agron.
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8. - .
192. Derris uliLinosa. Agr. Jour. (Fiji) 5(1); 34-35.
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S1939. Evaluation of dorris and allied insecticides. The
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10. B;:OJ1PE, C. A., and SKI-iTEP-- W. W.
1931. Wiley's Principles -nd practice of agricultural analysis.
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1936. The toxic constituents of dorris root. Soc. Chem. Ind.
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1936. Derris versus wirbloe. Farmer and Stock-Breeder and Agr.
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