554 Non-Protein Nitrogen of Wheat Flour 
determination of ‘‘total non-protein nitrogen”’ in flour extracts by 
the above method is to pipette 100 cc. of flour extract into a 
200 cc. sugar flask, add 25 cc. of 0.1N NaOH followed by 27 
ec. of 0.1 N CuSQ,, shake vigorously several times until a water- 
clear supernatant liquid remains after the precipitate settles, 
make to the mark, filter, and determine nitrogen by the Kjeldahl 
method in 100 or 150 cc. of the filtrate. 
The effectiveness of the method was then examined by study- 
ing the nature of the nitrogen compounds left in the filtrate from 
the copper-protein precipitation. In. this work, two flours, A 
and B, were used. Flour A was the Ceretana patent flour men- 
tioned previously, while B was a flour milled from some slightly 
frosted Karkov wheat grown in Montana. 1 liter portions of 
filtered flour extract were placed in 2 liter flasks, and 400 cc. 
portions of 0.1 Nn NaOH were added, followed by 400 cc. of 
0.1 Nn CuSO,. Then small portions (of about 10 cc. each) of 0.1 N 
CuSO. were added, the whole being well shaken and allowed 
to settle after each addition, until an absolutely clear, colorless 
supernatant liquid remained after the precipitate settled. Jt 
appears to be absolutely necessary that the 0.1 N alkale be kept in 
paraffin-lined containers, or else be made up fresh for each occasion, 
as it was found that alkali containing dissolved glass would neither 
cause a sharp separation nor give a clear, colorless filtrate. — Fi- 
nally, distilled water was added up to the 2 liter mark, and the 
filtration performed on a large paper filter. The filtration pro- 
ceeds rapidly and easily when the precipitation is made in the 
manner described. The filtrate was then slightly acidified with 
acetic acid and concentrated under diminished pressure to one- 
twentieth of its volume. The concentrated solutions were slightly 
viscous on account of dissolved carbohydrate material, and gave 
a slight test for copper with ferrocyanide, which they did not give 
before concentration. They also showed the slightest trace of 
color due to copper salts. These concentrated solutions are re- 
ferred to in the remainder of this paper as Solution X. These 
solutions gave no perceptible biuret reaction of any kind, but did 
give a very faint Millon’s reaction, and also a slight Adamkiewicz’ 
reaction. The latter two reactions are indicative of the presence 
of the amino-acids, tyrosine and tryptophane, but do not neces- 
sarily show that they are in protein combination. Solution X 
