88 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



40 to a volume of 2-3 c.c. ; the greater portion of the hydrochloric 

 acid is thus removed. 



2. About 300 c.c. of water is then placed in the flask and a cream 

 of magnesia, which has been freed from every trace of ammonia by 

 long boiling, is added until in slight, but distinct, excess. The mix- 

 ture is distilled in vacuo at 40 and the distillate collected in excess of 

 standard acid ; about half the liquid should be distilled. Titration of 

 the standard acid gives the amount of amide nitrogen. 



3. The remainder of the solution is filtered through a nitrogen-free 

 paper and the residue, thus collected, washed thoroughly with water. 

 The nitrogen in this precipitate is estimated by Kjeldahl's method 

 and is the " humin " nitrogen. 



4. The filtered solution is concentrated to 100 c.c. and cooled to 

 20 J 5 grams of sulphuric acid and then 30 c.c. of a solution con- 

 taining 20 grams of phosphotungstic acid and 5 grams of sulphuric 

 acid per 100 c.c. are added. 



5. The precipitate is filtered off after twenty-four hours and washed 

 with a solution containing 2 '5 grams of phosphotungstic acid and 5 

 grams of sulphuric acid per 100 c.c. The washing is effected by rins- 

 ing the precipitate from the filter into a beaker and returning to the 

 paper three successive times, each portion of the wash solution being 

 allowed to run out completely before the next is applied. About 200 

 c.c. of washings are generally obtained. 



6. The precipitate is transferred to a 600 c.c. Jena glass flask and 

 the nitrogen estimated in it by Kjeldahl's method, digesting it with 

 35 c.c. of concentrated sulphuric acid for seven or eight hours. Potas- 

 sium permanganate crystals may be added three or four times. If the 

 phosphotungstic acid precipitate be small, less sulphuric acid may be 

 used, but sufficient must be taken to prevent bumping. 



7. The remaining nitrogen, belonging to the mono-amino acids, 

 is found by subtracting the sum of the nitrogen found in the preced- 

 ing operations from the total nitrogen contained in the protein. 



The data given in the table on page 131 show that there are con- 

 siderable differences in the amounts of the various kinds of nitrogen 

 in proteins. 



