49 
essentially the same. manner as already describt-d except that the diluted re- 
action mixture vcs' cooled .to 0*^ C, "before addition of the annoriia, w^s as • 
follovs: • 
To, prepare color standards, 50 ng. of malachite green is 
dissolved in 1 liter of water, a portion of the solution is. 
diluted' to douhle the volune 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 times 
the volume, so that the three solutions contain 4, 2, and 1 mg. 
per cubic centimeter of rotenone or extract. At th© time of 
analysis 1 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. 
Jor 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 vlth 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 yellov; color 
with nitric acid was v-ry'much retarded, and in these cases it was necessary 
to add a drop of f-uming nitric acid to initiate the first reaction. 
The colorimetric method of Jones and Smith (75) was regarded by 
Groudswaard and" Timmers (^ in 1937 as unsuitatle for the estimation of 
rotenone, because the intensity of the color produced depended on the temper- 
ature and because various tints interfered wl':h the estimation of the color. 
Pozz.i-Sscot -(98) in 1937 stated tliat bases other than ammonia produced 
the final blue color in the Durham reaction. Even organic bases such as 
triethanolamine produced a blue-green color. 
Sievers and coworkers (ij^ 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 v/ere 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 
samples. 
Th9 Durham reaction has been adapted to the qualitative testing of 
mineral-oil fly sprays for rotenone and deguelin (l25). Directions for the 
test were as follows: 
