THE EHRLICH DIAZO REACTION 335 



Reagent 



( Sulphanilic acid, 1 



Solution I. . . i Hydrochloric acid, 50 



( Distilled water, ad. 1000 



CI 1 ^- TT \ Sodium uitrite. 0.5 



Solution II. . . ] Distilled water, ad. 100 



To fifty parts of solution I. add one part of solution II. and 

 shake. To a few c.c. of this mixture, and an equal quantity of urine, 

 add a quantity of ammonia equal to about one-eighth of the com- 

 bined volume of the mixed urine and solution, letting it run down 

 the side of the test tube. 



At the point of contact of the ammonia and the mixture, colored 

 rings of various tints form, ranging from light yellow, through dark 

 yellow, orange, and brown, to eosin or garnet, depending upon the 

 urine. The formation of a red zone is an indispensable part of the 

 true Ehrlich diazo reaction. It is also essential that, on shaking, the 

 foam take on a pink color. This color varies considerably in its 

 intensity, depending upon the strength of the reaction, from the 

 palest rose to the deepest pink, but must not be any other color, such 

 as salmon, orange, etc. 



A third part of the reaction which Ehrlich considered important, 

 consists in the separation of a greenish-black or violet-black precipi- 

 tate, which forms a layer on the surface of the light-colored sediment 

 when the tube has been allowed to stand for twenty-four hours. 

 The changes which take place in this reaction may be represented 

 graphically as follows: 



(1) NaNo,+Hcl=HNO,-fNaCl. 



Sulphanilic acid. Diazobenzolsulphonic acid. 



(2) C,H,NH2HS0,+HN0,=C,H,N,HS0,-hH0. 



The resulting fluid contains small quantities of diazobenzolsul- 

 phonic acid, to which the reaction is due, together with some excess of 

 sulphanilic and hydrochloric acids which are indifferent or beneficial- 

 The diazobenzolsulphonic acid unites with certain aromatic bodies in 

 the urine to form analine dyes. The solutions should be kept in 

 dark bottles. If the mixture of solutions I. and II. is not used 



