62 
this residue was added 6 to 8 drops of concentrated sulfuric acid and a 
crystal of sodiun nitrite. The red-violet color developed in from l/2 to 
5 minutes. A similr-r 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 dehydrorotenone , dehydrodoguelin, and isodihydrodehydrorotenone. Toxicarol 
was said to give a red-brown color only. 
This color reaction was developed into a quantitative test by Fischer 
and Nitsche (2S) in 1935. The method was essentially as follows: 
One cubic centimeter of an acetone solution containing 0.2 
to 0,5 ng. of derris resin is placed in a 25-cc. volumetric 
flask and evaporated to dryness. Tbe 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 
sulfiiric acid are added. The flask is heated on the water 
bath for 8 minutes, cooled, and the solution is made to volume 
with concentrated s-iulfuric acid. Comparison is made in a 
colorimeter with the color developed from a standard solution 
01 rotenone. 
Pure rotenone gave a "permang.'=inate-like" violet, while derris resins gave 
a more reddish violet. Since the authors used an "absolute" colorimeter, 
it was not necesspry 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 a-pve equal color 
values. Zther extracts, however, gave erratic results. Too much nitrite 
Was foiond 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 
comoo->jnds obtained in the Takei ( ll8 ) 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 susioension of the material to be test'^d was treated 
vlth' the reag-nt. The method was designed prin^^rily for the rapid 
determination of total ether extract and was said to require about 20 
minut.jR. It was as follows: 
A 1-gm. sample 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. v;ith distilled water. This milky suspension is well 
sh-aken, and 0.2 cc. in a dry teat 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. Tae violet -color obtained was measured 
in a step photometer with a 5300 A. filter. The results were compared 
with determinations m-de on samples of known total~-extract content. The re- 
lation between extinction coefficient at 5300 A. and percentage of ether 
