THE ORGANIC CONSTITUENTS OF THE URINE. 259 



/NH 



(1) N~C NH 2 + NH(CH 3 )CH 2 .COOH = NH : C< 



\N(CH 3 ).CH 2 .COOH 



Cyanamide. Methyl-glycocoll. Kreatin. 



/NH 2 /NH CO 



(2) NH=C< = NH:C< | -f H 2 O 



X N(CH,).CH 2 .COOH X N(CH 3 ).CH 2 



Kreatin. Kreatinin. 



Isolation. The isolation of kreatinin from the urine is based upon 

 the formation of the chlorozincate, which is almost insoluble in alco- 

 hol : 240 c.c. of urine, which should be free from albumin and sugar, 

 are rendered alkaline with milk of lime, and treated with a solution 

 of calcium chloride so long as a precipitate forms. Water is then 

 added to the 300 c.c. mark. After standing for a while the phos- 

 phates are filtered off. The precipitate is washed with a little water. 

 Filtrate and washings are rendered slightly acid with acetic acid, 

 and are then evaporated to a syrup. This is stirred while still 

 warm with about 25 to 30 c.c. of absolute alcohol, transferred to a 

 glass-stoppered flask, and diluted with absolute alcohol to 100 c.c. 

 After twenty-four hours the mixture is filtered. The nitrate is 

 treated with a small amount of sodium acetate in solution, and is 

 concentrated to about 50 c.c. To this is added 0.5 c.c of a concen- 

 trated alcoholic solution of zinc chloride, which is prepared by dis- 

 solving a small amount of the salt in 80 per cent, alcohol, and 

 diluting with 95 per cent, alcohol to a specific gravity of 1.2. The 

 mixture is then well stirred and set aside in a cool place for several 

 days. Crystals of the chlorozincate of kreatinin are thus obtained. 



To isolate the kreatinin from the chlorozincate, the latter is dis- 

 solved in a small amount of hot water and boiled for ten minutes 

 with well-washed plumbic hydrate. After filtering off the insoluble 

 oxide of zinc and the chloride of lead the filtrate is evaporated to 

 dry ness and extracted with cold absolute alcohol. This takes up 

 the kreatinin, while a small amount of kreatin formed during the 

 process of boiling remains. On evaporation the kreatinin is obtained 

 in crystalline form, and can be further purified by recrystallization 

 from water. 



Quantitative Estimation. FOLIN'S METHOD. This method is 

 based on Jaffe's reaction of kreatinin with alkaline picric acid solution. 

 The red colored solution produced in this reaction has in proper 

 concentration and when viewed by transmitted light exactly the 

 same shade as a potassium bichromate solution. Half-normal potas- 

 sium bichromate solution (containing 24.55 grammes per liter) is 

 therefore used as a standard for comparison. 



A high-grade colorimeter, by means of which the depths both of 

 the unknown solution and of the bichromate can be adjusted to 

 tenths of millimeters, is necessary for the comparison. 1 



The following solutions are also necessary : The half-normal po- 



1 The French instrument of Duboscq, which can be obtained through Eimer & Amend, is 

 admirably suit'ed for the purpose. 



