6 Journal of Agricultural Research voi. xii, no. i 



whereas the 15-hour digestion was sufficient to bring about complete 

 hydrolysis and the 24- and 48-hour experiments were overdigested to 

 the extent that nitrogen was lost. 



The insoluble carbonaceous residues which were filtered from the 

 hydrolyzed solutions were dried at 100° C. and the total nitrogen de- 

 termined in each. 



The insoluble residue from experiment 2, or the 12-hour starch-casein 

 digestion, contained 1.30 per cent of nitrogen. That from the 15-hour 

 digestion contained 0.83 per cent of nitrogen. That from the 24-hour 

 digestion contained 0.80 per cent of nitrogen and that from the 48-hour 

 digestion contained 0.855 per cent of nitrogen. The results show that 

 a 15-hour digestion removed as much nitrogen from the insoluble residue 

 as the 24- and 48-hour digestions. 



Two determinations of total nitrogen on a sample of the dry starch 

 showed an average nitrogen content of 0.05 per cent. The small amount 

 of nitrogen contained in the starch and the comparatively greater amount 

 found in the insoluble residues indicate that some nitrogen compound 

 was absorbed by the latter. 



Seven gm. of the dry-carbon residue were weighed out and transferred 

 to a Claisen flask, 60 c. c. of a 10 per cent calcium-hydrate suspension 

 added, together with 250 c. c. of distilled water. The apparatus was 

 connected up as in an ammonia determination and distilled under 

 reduced pressure at from 40° to 45° C. for 30 minutes. Nine-tenths 

 c. c. of N/io hydrochloric acid was neutralized by the ammonia evolved, 

 which shows that the insoluble-carbon residue contained only a trace 

 of ammonia nitrogen. The insoluble-carbon and calcium-hydrate 

 precipitate remaining in the Claisen flask was filtered and washed 

 thoroughly, the filtrate made acid and concentrated under reduced 

 pressure to about 50 c. c. The concentrate was transferred to a Kjeldahl 

 flask and the total nitrogen determined in the usual way. The filtrate 

 contained 0.0032 gm. of nitrogen or 5.3 per cent of the total nitrogen 

 contained in the insoluble residue. The ammonia nitrogen was 2.1 

 per cent of the total nitrogen in the carbon residue. It is therefore 

 evident that a very small percentage of the total nitrogen contained in 

 the insoluble residue is affected by distilling with calcium-hydrate sus- 

 pension, which indicates that the nitrogen remaining in the insoluble- 

 carbon residue after digestion and washing is in what may be considered 

 an inert form and should not be included in the humin group. 



CONCLUSIONS 



From the data contained in this paper the following conclusions may 

 be drawn: 



(i) The Van Slyke method for protein analysis, when applied to 

 mixtures of casein and starch in the proportion of i to 5, and hydrolyzed 



