350 PHYSIOLOGICAL CHEMISTRY 



Separation of Extractives from Muscles 



1. Creatine. Dissolve about 10 grams, of a commercial extract of meat in 

 200 c.c. of warm water (Test for Protein by Biuret and Coagulation Tests, see 

 Chapter V.) Precipitate the inorganic constituents by neutral lead acetate, 

 being careful not to add an excess of the reagent. Write the equations for the 

 reactions taking place here. Allow the precipitate to settle, then filter and remove 

 the excess of lead in the warm nitrate by hydrogen sulphide. Filter while the solu- 

 tion is yet warm, evaporate the clear filtrate to a syrup, and allow it to stand at 

 least 48 hours in a cool place. Crystals 6f creatine should form at this point. 

 Examine under the microscope (Fig. no, page 342). Treat the syrup with 200 

 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 in 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. no, page 342. 



(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 in Chapter XXII 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 

 remove 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 in the cold solution and separates on standing. After standing 

 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 in 

 Fig. 84, above. 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. 



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

 Physiological Constituents of the Urine. 

 2 Walpole: Jour. Physiol., 42, 301, 1911. 



