4^ 
D3GU2Liri AITD HDT-NOlIE PLUS DZGUSLIN 
The Durham Test 
A test vfidely used in work on rot-.none-containing plants is that based 
on the color recction discov.ircd "by Darham in 1902, in which treatment of certai 
constituents of derris root \vith nitric acid followed "by aminonia v;?.s foujid to 
produce an evanescent "blue-^reen color. Ishikawa (50) in 1916 independently 
discovor^id this reaction, using sodiur. hydroxide as the "base. According to 
Tattersfit^^ld and Hoach ( 12^) and Giralettc (3B) , who descrited Durham's work, 
concentratsd nitric acid v/a? added to solid rotenone, or rosin, and this mixture 
was treated directly v.lth strong ajanonium hydroxide. In this form the test was 
qualitative only and was unsuitatle for delicate testing oecause of the violence 
of the nt^utralization. 
Payer a:id Hincrbein (92) in 1931 described a qualitative test similar to 
that of DLirhpjD. To 2 cc. of dilute acetic acid extract of the sar.ple, 2 drops 
of fuming nit ric acid were added; the mixture WaS diluted with 10 cc. of water 
and then made alkaline with sodiu;n hj^'droxide. The color produced passed quick- 
ly from green to brown. The green color produced with aaaonia was said to be 
more stable than that obtained with scditLm hydroxide. 
In 1933 Jon( s and Smith (75) modified the I>urham test in an attempt to 
render the blue color more pemanant and make the test more nearly quantitative. 
They used the following procedure: 
T& 1 CG. of an acetone solution of rotenone (or a plant extract) 
1 cc. of 1 -♦. 1 nitric acid is added, and the mixture i? allowed 
to stand for l/2 minute. It is then diluted with 8 to 9 cc of 
water and 1 cc. of strong amaoni\im hydroxide is added. A blue 
color is produced wMch is ainost 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. Ey preparing a series of solutions contairing different concentrations 
of bromothymol blue in a buffer of pH 7.2 and standardizing these against the 
colors produced by different amounts of rotenone, it v/as possible to use the 
test to make a rough estimate of the amo-'ont of rotenone present. Since approx- 
imately the same intensity of color was given by deguelin, whereas toxicarol 
gave alm.ost no color, it wgs believed that the test gave a rough value for the 
total rotenone plus deguelin content of plant extractives. Sodium and potassiion 
hydroxides, or sodi\:m and potassium carbonates, used in place of the ammonia, 
were also foiond to produce the blue color. Vhen the nitric acid was partially 
neutralized with sodiixm bicarbonate a:id the neutralization was completed with a 
stronger base. Thitt'dolor w^is sli:;^htly 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 xeph rooia. i.ctracts that gave the test were rated in 
three grades aependirg on the depth of color produced. 
Fischer and Hitcohe (2^) in 1935 made a study of the tests proposed by 
Peyer and Huncrbein (92) and by Jones and Smith (75) in a further attemot to 
render the reaction more nearly quantitative. The test, carried out in 
