584 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



III. The Separation and Isolation of Purine Bases from Tissues 

 or Extracts of Tissues. (Jones.) 



The tissue is allowed to digest or dissolve itself by placing the minced 

 material in water and allowing the mixture to stand at 40 for some days in 

 the presence of chloroform to prevent putrefaction. Except connective tissue 

 the whole tissue gradually dissolves. This solution, or an aqueous extract, is 

 heated to boiling and made faintly alkaline with soda to dissolve any purines 

 which are insoluble or soluble with difficulty and then acidified with acetic 

 acid. Proteins which are present are coagulated, filtered off and washed. 



The filtrate is treated at its boiling-point with 10 per cent, copper 

 sulphate solution and saturated sodium bisulphite solution which are added 

 alternately so long as a white precipitate is formed and until yellow cuprous 

 oxide begins to come down. 



The precipitate of purine copper compounds is filtered off, washed, sus- 

 pended in boiling water and decomposed with sodium sulphide. It is diffi- 

 cult to determine the end point of the reaction as an emulsion is formed, but 

 a test drop placed against a drop of lead acetate on a filter paper will form 

 lead sulphide when sufficient sulphide has been added. Acetic acid is added 

 to the boiling solution until the copper sulphide aggregates, or if guanine be 

 present, sulphuric acid is used. The solution contains the mixture of purine 

 bases. 



The separation of the pu/ine bases is greatly faciliated if it be known 

 whether one, two or all are present. 



(a) If uric acid be present in greatest amount it is separated by adding 

 25 c.c. of concentrated hydrochloric acid and evaporating the solution to 25 c.c. 

 Nearly the whole of the uric acid separates out. 



(b) If uric acid be mixed with xanthine, these may be separated by dis- 

 solving in concentrated sulphuric acid (*i gm. in 2 c.c.) and diluting with 

 four volumes of water. The uric acid separates out on stirring. The 

 xanthine is obtained by again precipitating as copper compound and treating 

 as under (d). 



(c) Guanine and adenine are separated before xanthine and hypoxanthine. 

 The solution is made alkaline with ammonia and precipitated with a slight 

 excess of ammoniacal silver nitrate. The well-washed silver precipitate is sus- 

 pended in hot water and decomposed with hydrochloric acid. An emulsion 

 may be formed if guanine be absent and xanthine be present, but it can be 

 made to sediment by an excess of hydrochloric acid. 



The filtrate from the silver chloride is heated to boiling and treated with 

 ammonia. Guanine is precipitated and purified by preparing the chloride 

 (p. 295). Ammonia is removed from the filtrate by boiling, the solution is 

 acidified to methyl orange with hydrochloric acid and the adenine is precipi- 

 tated as picrate by adding sodium picrate (p. 296). 



The picric acid is removed by extracting the solution acidified with 

 sulphuric acid with ether, the solution is neutralised with soda, the purine 

 bases thrown out as before with copper sulphate and bisulphite and recovered. 



(d) The solution containing xanthine and hypoxanthine is acidified with 

 hydrochloric acid and carefully evaporated to dryness with constant stirring, 

 the residue moistened with water and again evaporated. The xanthine hydro- 

 chloride is thus decomposed ; on extracting the residue with water it is present 

 as free base and is insoluble, whilst hypoxanthine hydrochloride dissolves. 



The xanthine is dissolved in 1 5 parts of warm 3 '3 per cent, caustic soda 

 and the solution is poured into two-thirds of its volume of 32 per cent, nitric 

 acid from which nitrous acid has been removed by boiling. Xanthine nitrate 

 crystallises out on cooling. It is dissolved in dilute ammonia and the solution 

 evaporated. Pure xanthine crystallises out. 



The solution of hypoxanthine hydrochloride is diluted, precipitated with 

 copper sulphate and bisulphite and the precipitate decomposed with hydrogen 

 sulphide. The solution is evaporated to dryness and the residue crystallised 

 from 30 parts of pure 6 per cent, nitric acid. Hypoxanthine nitrate crystallises 

 out. 



