58 METHODS FOR THE ANALYSIS OF URINE 



an hour. The residue is moistened with 1 c.c. of 50 per cent 

 acetic acid to bring the calcium hydroxide and carbonate into 

 solution, and is then washed into a 10 c.c. flask and filled up to 

 the mark. One can use the entire solution for determination of 

 the amino-nitrogen in the large amino-apparatus, or use 2 c.c. 

 portions for the micro-apparatus. 



The length of time which the nitrous acid solution should be 

 shaken in order to drive off all the amino-nitrogen depends 

 somewhat on the temperature. When the latter is 15 to 20 

 the time should be five to four minutes; for 20 to 25 it is three 

 minutes; for 25 to 30, two and a half to two minutes. It is 

 preferable that the solution should be shaken vigorously with a 

 motor and the time kept down to these limits, for the sake not 

 only of rapidity but of accuracy. 



Van Slyke's Method for Free Amino-Add Nitrogen 



To 25 c.c. of urine in a 50 c.c. flask add urease solution and 

 allow to stand for one and one-half times the interval which has 

 been found necessary to affect the maximum decomposition of 

 urea, as observed by titration of the ammonia. The last traces 

 of urea are decomposed. At the end of the digestion period 10 c.c. 

 of a 10 per cent suspension of calcium hydroxide are added, the 

 mixture shaken and made up to 50 c.c. Then filter, evaporate, 

 and complete the determination according to the method out- 

 lined under total amino-acid nitrogen, above. 



HIPPURIC ACID 



Method of Folin and Flanders 1 



Principle. The hippuric acid is hydrolyzed to benzoic acid 

 in alkaline solution and then the solution is boiled with strong 

 nitric acid to remove pigments and emulsifying substances. 

 The benzoic acid is extracted with chloroform and titrated with 

 sodium ethylate. 



Procedure. Measure 100 c.c. of urine intoa porcelain evaporat- 

 ing dish by means of a pipette. Add 10 c.c. of 5 per cent NaOH 

 and evaporate to dryness on the steam-bath. Transfer the resi- 



1 Folin and Flanders: Jour. Biol. Chem., 1912, 11, 257. 



