122 1'RACTICAL ORGANIC AND BIO-CHEMISTRY 



(6) Hurt ley's 7'es/. The reaction involved in this test is the conversion of 

 aceto-acetic acid into isonitroso-acetone by nitrous acid. This compound 

 forms salts ; the ferrous salt has a purple colour : 



CH 3 . CO . CH 2 . COOH - CH. { . CO . CH : NOH -> (CH, . CO . CH : NO),Fe 

 Aceto-acetic acid. Isonitroso-acetone. Ferrous salt. 



2-5 c.c. of concentrated hydrochloric acid and i c.c. of a i per cent, solu- 

 tion of sodium nitrite are added to 10 c.c. of the solution, or of urine, and 

 allowed to stand for 2 minutes. 15 c.c. of strong ammonia and 5 c.c. of 

 a 10 per cent, solution of ferrous sulphate or of ferrous chloride (2 gm. iron 

 in TOO c.c.) are added; the mixture is shaken and poured into a 50 c.c. 

 Nessler glass where it is allowed to stand undisturbed. It is inadvisable to 

 filter. A violet or purple colour is slowly produced at a speed depend- 

 ing on the amount of aceto-acetic ac id present. The colour deepens for several 

 hours after its first appearance. 



The reaction appears at a dilution of i in 50,000. Ethyl aceto-acetate 

 gives a blue colour when more nitrite than prescribed above is used ; acetone 

 does not react. 



The reaction is very useful for gauging the amount of aceto-acetic acid in 

 a solution or in urine. 



By diluting a i per cent, solution of sodium aceto-acetate prepared as de- 

 scribed on p. 119 so that it contains "025, '02, '15 and -01 gm. of aceto- 

 acetic acid in 100 c.c. and applying the test to these four solutions at the same 

 time as it is applied to the unknown, and allowing them to stand 4 or 5 

 hours the initially turbid liquids become clear and the unknown can be placed 

 in position amongst the standards. Solutions, or samples of urine, containing 

 more than the standard amounts must be diluted 10 or 20 times, and the test 

 repeated. 



Estimation. 



The estimation of aceto-acetic acid is based upon its decomposition into 

 acetone and carbon dioxide by heating with dilute acid. The acetone is 

 separated by distillation and determined by converting it into iodoform with 

 excess of -iN iodine solution and caustic soda, the excess of iodine being found 

 by titration with *iN sodium thiosulphate solution : 



CH 3 . CO . CH a . COOH = CH 3 . CO . CH 3 + CO 2 



I 2 + aNaOH = NaOI + tfal + H 2 O 

 / CH 3 . CO . CH 3 + 3 NaOI = CH 3 . CO . CI 3 + 3 NaOH 

 I CR, . CO . CI 3 ' + NaOH = CHI, + CH., .' COONa 



I 2 + 2Na 2 S 2 O 3 = 2NaI + Na.^S ,(),.. 



The estimation of aceto-acetic acid in ur'ne and tissues is described on 

 P- 593- 



