384 PHYSIOLOGICAL CHEMISTRY 



cent alcohol. After 15 minutes filter off the slight precipitate which forms. 

 Treat the final filtrate with 30-40 c.c. of 30 per cent zinc chloride. Stir and let 

 stand over night in a cool place. Pour off the supernatant liquid and collect the 

 creatinine zinc chloride on a Buchner funnel, wash once with water, then thor- 

 oughly with 50 per cent alcohol, finally with 95 per cent alcohol and dry. A 

 nearly white, light crystalline powder should be obtained. The yield should be 

 90-95 per cent of the original creatinine (usually about 1.5-1.8 grams of creatinine 

 zinc chloride per liter of urine). 



Recrystallize the creatinine -zinc chloride by treating 10 grams with 100 c.c. 

 of water and about 60 c.c. of normal sulphuric acid, heating the mixture until a 

 clear solution is obtained. Add about 4 grams of purified animal charcoal, con- 

 tinue boiling for about a minute, filter with suction through a small Buchner 

 funnel, pouring the filtrate back on the filter three or four times until it runs 

 through perfectly colorless. Wash residue with hot water and transfer the total 

 filtrate to a beaker and while hot treat with a little strong zinc chloride solution 

 (3 c.c.) and with about 7 grams of potassium acetate dissolved in a little water. 

 After ten minutes dilute with an equal volume of alcohol, and allow to stand in a 

 cold place for some hours. Filter off the crystalline product and examine under 

 microscope (see Fig. 124). To remove the small amount of potassium sulphate 

 which it contains stir up with twice its weight of water, filter, wash with a little 

 water and then with alcohol. The preparation should be snow white. Yield, 

 85-90 per cent. 



Place the finely powdered recrystallized creatinine zinc chloride in a dry 

 flask and treat with seven times its weight (by volume) of concentrated aqueous 

 ammonia. Warm slightly and agitate gently until a clear solution is obtained, 

 care being taken to drive off no more ammonia during the warming than is 

 necessary to obtain a clear solution. Stopper the flask, allow to cool, place in 

 the ice-box for an hour or more. Pure creatinine crystallizes out. It may be 

 recrystallized from boiling alcohol or concentrated ammonia, but this is usually 

 unnecessary. The product is perfectly pure and can be used as a standard in 

 the quantitative determination of creatine and creatinine. See chapters on 

 Quantitative Analysis of Urine and Blood. 



i. Preparation of Creatine. Creatine may be prepared from creatinine zinc 

 chloride by decomposition with calcium hydrate, the process being one of hydrol- 

 ysis (Benedict). 



One hundred grams of creatinine zinc chloride are treated with about 700 c.c. 

 of water in a large casserole and the mixture heated to boiling; 150 grams of pure 

 powdered calcium hydrate are then added, with stirring, and the mixture boiled 

 gently for 20 minutes (with occasional stirring) . The hot mixture is then filtered 

 with suction, the residue being washed with hot water. The nitrate is then treated 

 with hydrogen sulphide gas for a few minutes and poured through a folded filter 

 to remove the zinc. The filtrate is acidified by the addition of about 5 c.c. of glacial 

 acetic acid and boiled down rapidly to a volume of about 200 c.c. This solution 

 is allowed to stand over night, preferably in a cool place. The next day the crys- 

 tallized creatine is filtered off with suction, washed with a very little cold water, 

 and then thoroughly washed with alcohol and dried. 1 This product is then recrys- 

 tallized by dissolving in about seven times its weight of boiling water and allowing 



1 The filtrate obtained at this point should be diluted with alcohol and treated with zinc 

 chloride (50 c.c. of a 30 per cent solution) for recovery of the unconverted creatinine. 



