HYDROXY-, KETO- AND DIBASIC ACIDS 121 



(4) Arnold's Test. This test depends upon the formation of a colouring 

 matter irom aceto-acetic acid and diazotised para-amino-acetophenone : 



CH 3 . CO . C 6 H 4 NH 2 . HC1 + HNO 2 = CH 3 . CO . C 6 H 4 . N : N . Cl + 2 H 2 O 



/CO . CH 3 

 CO . C 6 H 4 . N : NCI + CH 3 . CO . CH 2 . COOH = CH 3 . CO . C 6 H 4 . N : N . CH<; 



\COOH 



Two solutions are required : 



(a) i gm. of para-amino-acetophenone dissolved in a little water with 

 the aid of 2 c.c. of concentrated hydrochloric acid and made up to 100 c.c. 



(l>) i gm. of sodium nitrite dissolved in water and made up to 100 c.c. 



2 volumes of (a) and i volume of (b) are mixed, an equal volume of the 

 solution is added to the mixture and i or 2 drops of strong ammonia. A 

 large excess of concentrated hydrochloric acid (15 c.c.) is added to a portion 

 of the above (2 c.c.). A fine purple colour is produced. 



In testing urine, Arnold recommends filtering it through animal charcoal 

 before applying the test. The delicacy is increased and more urine can be 

 added to the mixture of (a) and (b) than if unfiltered urine be used. 



The sensitiveness of the test is increased according to Lipliawsky if, after 

 adding the excess of hydrochloric acid, 3 c.c. of chloroform and 2-4 drops 

 of ferric chloride be added and the mixture carefully shaken so as to avoid an 

 emulsion. The purple colour is taken up by the chloroform forming a very 

 stable solution. 



The delicacy of the test is i in 40,000. The reaction is also given by 

 ethyl aceto-acetate, but not by other substances, and can therefore be directly 

 applied to the urine of patients who have taken salicylic acid preparations, 

 antipyrine, etc. 



(5) Rieglers Absorption of Iodine Test This test depends upon the forma- 

 tion of iodo-aceto-acetic acid, a colourless substance ; 



CH 3 . CO . CH 2 . COOH + I 2 = CH 3 . CO . CHI . COOH + HI. 



The conditions necessary for this test are that the solution must be acid 

 and that iodine must be present. 



10 c.c. of the solution, or of urine, -are acidified with 5 drops of 30 per 

 cent, acetic acid and 5 drops of iodine solution are added. This mixture 

 is shaken with 2 or 3 c.c. of chloroform. No colour appears if aceto-acetic 

 acid be present. 



The amount of aceto-acetic acid present in the solution can be gauged 

 from the amount of iodine solution which is required to be added so as to 

 form an excess over that required to combine with the aceto-acetic acid and 

 so as to colour the chloroform violet. 



Ondrejovich has modified the test as follows : 



5 c.c. of the solution, or urine, are acidified with 5 drops of 50 per cent, 

 acetic acid and i drop of i in 2000 solution of methylene blue added. The 

 liquid should be distinctly blue. The liquid turns red on adding cautiously 

 2 to 4 drops of tincture of iodine or iodine solution, and the red colour 

 turns blue or green at a speed depending on the amount of aceto-acetic 

 acid present. The green colour is restored if the solution has been made too 

 acid. 



Indican also absorbs iodine, but its amount in urine is so small that it 

 may be neglected. No other substance seems to show this reaction. 



