CHAP. IV.] 



BILIRUBIN. 'GMELIN'S REACTION.' 



319 



' Gmeliu's 

 reaction.' 



Stadeler and Maly, or probably a multiple of it, is correct. The 

 various reactions are best explained by doubling Stadeler's formula. 



Action of When bilirubin is treated with pure dilute nitric 



nitric acid on acid (containing 20 per cent, of HN0 3 ) no change 

 occurs at ordinary temperatures. When the solution 

 is heated, however, dark-violet resinous flakes are formed 

 which as the temperature rises assume a light-brown 

 colour and ultimately dissolve, yielding a yellow-coloured liquid. 



Pure concentrated* nitric acid acts in the cold and a cherry-red 

 liquid is obtained which retains its colour for many days. Nitric 

 acid which has a slightly yellow colour and which contains nitrous 

 acid 1 (as the nitric acid of commerce does) gives rise in solutions 

 which contain bilirubin, to a remarkable play of colours already 

 referred to as ' Gmelin's reaction.' The reaction may be tried with 

 a dilute alkaline solution of bilirubin, with diluted bile, or with any 

 liquid, such as the urine of jaundice, which contains bilirubin. 



Various methods of exhibiting Gmelin's reaction may be adopted. 

 The most common is to pour some of the solution to be tested into 

 a test tube containing nitric acid, so that the two liquids are not 

 mixed. Near the line of junction the colour-reaction at once 

 commences to develope, and a succession of zones of colour appear, 

 the tints being, from above downwards, as follows: green, blue, 

 violet, red and reddish-yellow. These tints represent the successive 

 stages of the reaction, the first being the green and the last the 

 reddish-yellow, which is observed in the region where the oxidising 

 action is most intense, viz. in close proximity to the nitric acid. 



Instead of employing a test tube, a few drops of diluted bile, or 

 bilious urine may be poured upon a flat plate, so that a thin layer of 

 liquid is obtained. On now adding a drop or two of coloured nitric 

 acid, wherever the acid falls a series of concentric coloured rings 

 of beautiful colour is developed, the succession of tints being the 

 same as in the experiment previously described. 



The delicacy of 'Gmelin's reaction' is such that it permits of the 

 detection of bilirubin in solutions which contain only 1 part of the 

 colouring matter in from seventy- to eighty-thousand parts of water. It 

 must be remembered that in order to be sure of the presence of bilirubin the 

 whole series of tints must be observed, as lutein (the yellow crystalline 

 matter obtained from corpora lutea, from the yolk of egg, and which is 

 also present in the liquor sanguinis of some animals), when treated with 

 nitric acid, exhibits a green and also a blue tint very similar to those 

 developed in Gmelin's reaction. The spectroscopic characters of lutein 

 are, however, sufficiently distinctive to enable the observer to ascertain 

 whether this substance is present in a solution or not. 



Each tint in Gmelin's reaction corresponds apparently to a 

 definite chemical change, probably to a definite oxidation product, 



1 If the acid is too highly coloured (i.e. if the amount of nitrous acid and of nitrogen 

 peroxide be large) it exerts so energetic an action on the bilirubin that the successive 

 stages of Gmelin's reaction cannot be properly observed. 



