316 URINE. [CH. XII. 



389. Rothera's test for acetone and aceto-acetic acid. To 



10 cc. of the urine add an excess of solid ammonium sulphate, so 

 that the urine is completely saturated. Then add two or three 

 drops of a freshly prepared 5 per cent, solution of sodium nitro- 

 prusside and 2 or 3 cc. of concentrated ammonia. Mix and allow to 

 stand undisturbed for at least thirty minutes. A characteristic 

 permanganate colouration, that may only develop above the layer 

 of undissolved crystals, indicates the presence of acetone or aceto- 

 acetic acid. 



NOTES. i. The test is much more sensitive for aceto-acetic acid than 

 for acetone. The reaction with acetone can just be detected in a dilution of 

 i in 20,000, whilst aceto-acetic acid shows it in a i in 400,000 dilution. 



2. The intensity of the colour and the rate at which it develops vary 

 with the concentration. Kennaway* suggests the following scale : (i) quick- 

 strong ; (2) slow-strong ; (3) quick-weak ; (4) slow- weak. A " quick-strong " 

 reaction shows a deep permanganate colour within a few seconds, and indicates 

 the presence of at least 0-25 per cent, aceto-acetic acid. A " slow-weak " 

 reaction may show no pink for some minutes, and the amount present is 

 probably less than 0-0005 P er cent. 



3. Though aceto-acetic acid gives a vivid reaction, aceto-acetic ester 

 actually inhibits the reaction when the free acid or acetone are present. 



4. For the sake of experience it is advisable to practise the tests on 

 urines containing varying amounts of acetone and aceto-acetic acid. The 

 latter is prepared from the ester by the following method of Hurtley. 



Into a beaker weigh out 13 grams, (i-io molecule) of aceto-acetic ester. 

 Add 100 cc. of N. soda (i-io molecule) diluted with about 300 cc. of distilled 

 water. Transfer to a 500 cc. measuring flask and make the volume up to 

 500 cc. with distilled water. Allow the solution to stand for at least 24 hours 

 at room temperature. The ester is completely hydrolysed to sodium aceto- 

 acetate. 



CH 3 .CO.CH 2 .COOC2H 6 + NaOH = CH 3 .CO.CH 2 .COONa + C 2 H 5 .OH. 



The solution obtained corresponds to a 2 per cent, solution of aceto- 

 acetic acid. 



390. Gerhardt's test for aceto-acetic acid. A. To 5 cc. of 

 the urine in a test-tube add ferric chloride solution, drop by drop, 

 till no further precipitate of ferric phosphate is formed. Filter. 

 To the nitrate add some more ferric chloride. A Bordeaux-red 

 colour indicates aceto-acetic acid. 



NOTE. A similar colour is given by a large number of substances, such as 

 salicylic acid, and the bodies excreted after the administration of aspirin, 

 antipyrin, thallin, etc. The majority of these substances are not destroyed by 

 boiling, whereas aceto-acetic acid is converted into acetone. The reaction is 

 not obvious when less than 0-07 per cent, of aceto-acetic acid is present. 



* Guy's Hospital Reports, Ixvii., p. 161. 



