- 16 - 
As a measure of the purity of the crude rotenone the melting point was 
deterr.ined 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 ".as been used in the Butch 3ast Indies 
since that time. The modified metiod now in use is (described later in this 
section in connection with the more recent article by Mei.jer and Koolhaas 
(89). 
In 1933 Jones (62) described a method of assaying derris and cube 
for rotenone usin? carbon tetrachloride for extraction and crystallization. 
The plant material was extracted in a Soxhlet 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 throug-^. a Gooch crucibla and washed with ice-cold 
carbon tetrachloride. The crvstalline material was dried to constant 
weight at room te-'iperature and weighed. For more rapid but less accurate 
results the extract was cr-"-8talli2ed in 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has been superseded by the method of Jones and Graham (72), from which has 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 Agricultxir- 
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 saTiples of derris root were 
dried at 100° C. in a vacuum for 5 aours. Overnight (17 nours) extraction 
was recommended for most samples, as it was found that some samples giving 
6 to 10 percent of rotenone 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, neating to drive off the carbon 
tetrachloride of crystalli7ation and weighing the resulting rotenone were 
suggested. A deterrination 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 deternination of the material recrystallized from 
amyl acetate. 
Several samples that gave no rotenone by 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 ma.terial 
it was suggested that large samples be used, a scheme involvinr extraction 
with acetoiie and crystallization of the extract from carbon tetrachloride 
was tried. Although acetone ^:ave a more rapid extraction of tae rotenone, 
much of the acetone extract was insoluble in carbon tetrachloride. The 
additional manipulation necessar?' resulted in a less pure product. 
