DISTRIBUTION OF NITROGEN 105 



5 grams of protein, containing approximately 800 mgm. of 

 nitrogen, are heated with 20-25 parts of 3N hydrochloric acid on 

 a water-bath until there is complete or nearly complete solution, 

 and then in an autoclave at 15 f r i"5 hours which effects com- 

 plete hydrolysis. 



The solution is concentrated on the water-bath as much as pos- 

 sible, the residue dissolved in water, the solution filtered and made 

 up to 250 c.c. The nitrogen retained in the filter paper, usually a 

 negligible quantity, is estimated Total nitrogen of the solution is 

 determined in 5 c.c. and amide nitrogen in 20 c.c. by distilling in 

 vacuo with ^N baryta dissolved in methyl alcohol (saturated baryta 

 in methyl alcohol, if much ammonia be present). 



200 c.c. are treated with saturated sodium carbonate solution, until 

 the reaction of the solution is alkaline to turmeric paper, and evapor- 

 ated to dryness in vacuo. The residue is dissolved in 100 c.c. of 

 water and its reaction tested : if not alkaline, more sodium carbonate 

 is added and the evaporation repeated ; this must be continued until 

 the reaction is alkaline. The residue is dissolved in 50 c.c. of water 

 and neutralised with hydrochloric acid ; 27 c.c. of concentrated hydro- 

 chloric acid and 25 grams of phosphotungstic acid are added; the 

 solution is diluted to 300 c.c. and heated on a water-bath until the 

 phosphotungstates have dissolved. After standing for forty-eight 

 hours, the crystalline phosphotungstates are filtered off by suction on 

 a hardened filter paper in an ordinary funnel. This method of filtra- 

 tion is preferred to that adopted by Van Slyke. The precipitate is 

 washed ten times with 15-20 c.c. of 2*5 per cent, phosphotungstic 

 acid and 3-5 per cent, hydrochloric acid by stirring up with the wash 

 solution and each time draining off completely. The precipitate is 

 dissolved and the bases estimated as directed by Van Slyke. 



The precipitate of barium phosphotungstate is dried in the air, 

 and the nitrogen contained in it estimated. This is humin-nitrogen I., 

 which is thrown down at this stage. 



The filtrate is diluted to 1000 c.c., made distinctly red to phenol- 

 phthalein with alkali and barium chloride added to remove phospho- 

 tungstic acid ; the solution is kept distinctly alkaline and not too 

 small an excess of barium chloride is used. The barium phospho- 

 tungstate is filtered off and washed till free from chlorides. The 

 nitrogen in the precipitate is estimated and forms humin nitrogen II. 



The filtrate is neutralised, treated with an amount of hydrochloric 

 acid equivalent to the total nitrogen, and evaporated in vacuo until salt 



