368 PHYSIOLOGICAL CHEMISTRY 



c.c. of 88 per cent alcohol, stir well with a glass rod to bring all soluble material 

 into solution, and then filter. The purine bases have been dissolved and are in 

 the filtrate, whereas the creatine crystals were insoluble hi the 88 per cent alcohol 

 and, remain on the filter paper. Wash the crystals with 88 per cent alcohol, 

 then remove them and bring them into solution in a little hot water. Decolorize 

 the solution by annual charcoal and concentrate it to a small volume. Allow 

 the solution to cool and note the separation of colorless crystals of creatine. 1 

 Make the following tests on the crystals : 



(a) Microscopical Examination. Examine some crystals under the micro- 

 scope and compare the form with those reproduced in Fig. 115, page 360. 



(b) Transformation of Creatine into Creatinine. Dissolve the crystals in 

 about 30 c.c. of hot water. To one-half of the solution in a flask add an equal 

 volume of normal hydrochloric acid and heat on a boiling water-bath for five 

 hours with reflux condenser. The creatine has been changed into creatinine. 

 Apply tests for creatinine as given hi Chapter XXIII to the original solution as 

 well as to the acidified solution. 



Diacetyl Reaction. To 5 c.c. of a dilute creatine solution add an equal vol- 

 ume of saturated sodium carbonate solution and a few drops of a solution of 

 diacetyl. A pink color should develop. This test has been made the basis of a 

 method for the quantitative determination of creatine. 2 



2. Hypoxanthine. Evaporate the alcoholic filtrate from the creatine to re- 

 move the alcohol. Make the solution ammoniacal and add ammoniacal silver 

 nitrate until precipitation ceases. The precipitate consists principally of hypo- 

 xanthine silver and xanthine silver. Collect these silver salts on a filter paper 

 and wash them with water. Place the precipitate and paper in an evaporating 

 dish and boil for one minute with nitric acid having a specific gravity of i.i. 

 Filter while hot through a double paper, wash with the same strength of nitric 

 acid and allow the solution to cool. By this treatment with nitric acid hypo- 

 xanthine silver nitrate and xanthine silver nitrate have been formed. The 

 former is insoluble hi the cold solution and separates on standing. After stand- 

 ing several hours filter off the hypoxanthine silver nitrate and wash with water until 

 the wash water is only slightly acid in reaction. Examine the crystals of hypo- 

 xanthine silver nitrate under the microscope and compare them with those hi 

 Fig. 117. Now wash the crystals from the paper into a beaker with a little 

 water and warm the liquid. Remove the silver by hydrogen sulphide and filter. 

 By this means hypoxanthine nitrate has been formed and is present in the filtrate. 

 (For crystalline form of hypoxanthine nitrate see Fig. 40, p. 135.) Concentrate 

 on a water-bath to drive off hydrogen sulphide, and render the solution slightly 

 alkaline with ammonia. Warm for a time, to remove the free ammonia, filter, 

 concentrate the filtrate to a small volume and allow it to stand in a cool place. 

 Hypoxanthine should crystallize hi small colorless needles. Examine the 

 crystals under the microscope. 



3. Xanthine. To the filtrate from the above experiment containing the xan- 

 thine silver nitrate add ammonia hi excess. (The crystalline form of xan- 

 thine silver nitrate is shown in Fig. 118.) A brownish-red precipitate of 

 xanthine silver forms. Filter off precipitate, suspend in water and treat with 



1 For an improved method of preparing pure creatine from creatinine see chapter on 

 Physiological Constituents of the Urine. 

 * Walpole: Jour. PhysioL, 42, 301, 1911. 



