4 BULLETIN 1460, U. S. DEPARTMENT OF AGRICULTURE 
yellow oxide of mercury; 8 per cent, red oxide of mercury; and 18 
per cent, mercuric sulphate. The lowest quantity of mercury used 
was 0.5 gram and the highest was 1 gram. Copper sulphate in 
quantities varying from 0.05 gram to 2 grams was used by 47 per 
cent of the laboratories. The great majority used approximately 
0.2 gram. Three collaborators used metallic copper in the same 
ratio that copper sulphate was being used. 
The Kjeldahl x method or modifications thereof was used by 24 per 
cent of the laboratories. Forty per cent used the Gunning 2 method ; 
18 per cent, the method developed by the Kansas City Protein 
Referee Board, 3 and the remainder used methods peculiar to them- 
selves. The time of digesting the samples varied from 25 to 180 
minutes. Both electricity and gas were used. The average time 
consumed with the gas heaters was 75 minutes; with the electric 
heaters, 50 minutes. Although the differences are not great, a 
greater range and a higher average protein content were found by 
the laboratories using electricity than by those using gas. 
Three receiving acids were used — sulphuric, hydrochloric, and 
boric. Expressed as acid sufficient to hold 1 milligram of nitrogen, 
a sufficient quantity was used to hold from 14 to 122.5 milligrams. 
The strength of the hydrochloric and sulphuric acids varied from 
N/14 to N/2, whereas the usual 4 per cent solution of boric acid was 
used. When it is known that wheat will vary in protein content 
from 7 to approximately 20 per cent, it is evident that acid equivalent 
to 35 milligrams of nitrogen must be present to hold the nitrogen 
released from all types of samples, when a 1-gram sample is digested. 
These data show that few of the laboratories that submitted figures 
on this subject are using insufficient acid to hold these quantities of 
nitrogen. 
As so many variables entered into the final analysis, it was almost 
impossible to eliminate and study each factor separately to determine 
which were responsible for the differences reported in the collabora- 
tive studies. Investigations were therefore planned to uncover the 
causes of some of the differences. These investigations were carried 
forward in two sections, the first having to do with the methods of 
digesting the samples, and the second relating to the technic of dis- 
tilling. The outstanding points studied were: Analytical methods, 
time of digestion, quantity and nature of the catalytic agents, correct 
standardization of the receiving or titrating solutions, procuring and 
preparation of the sample for analysis, nature and volume of the 
receiving acids,, and care in distillation. The digesting and distilling 
apparatus used in these studies are shown in Figures 1 and 2. 
DIGESTION STUDIES 
In planning this work, recognition was given to the importance 
of time of digestion. It is evident that this will vary, depending 
upon the intensity of the heat applied. For these studies, therefore, 
three different intensities of heat were chosen. These are described 
1 Official and Tentative Methods of Analysis of the Association of Official Agricultural 
Chemists. Revised to July 1, 1924, p. 6, par. 17. 
2 Official and Tentative Methods of Analysis of the Association of Official Agricultural 
Chemists. Revised to July 1, 1924, p. 8, par. 20. 
3 Herman, R. S. a protein referee board. Southwestern Miller, vol. 3, No. 31, p. 35, 
1925. 
