CHEMICAL BASIS OF THE ANIMAL BODY. 243 



stated the gall-stones of the ox or pig may consist of nearly half their 

 weight of bilirubin combined with calcium, they provide the best and 

 simplest source for the preparation of this substance 1 . The stones are 

 finely powdered, extracted with ether to remove any cholesterin, then 

 with water and treated with either strong acetic acid or dilute hydro- 

 chloric acid. By this means the bilirubin is set free from its calcium 

 compound and after being washed with water and alcohol is dissolved 

 in chloroform and finally separated by precipitation with alcohol as 

 already described. To obtain it quite pure the dissolving in chloroform 

 and precipitating by alcohol should be repeated several times. The 

 final product is amorphous. Crystals are most readily obtained by 

 slow evaporation of the first and hence slightly impure solution in 

 chloroform. 



When carnivorous bile is exposed to the air it turns more or less 

 rapidly green ; this is due to its oxidational conversion into biliverdin, 

 the normal pigment of herbivorous bile. A similar change is at once 

 produced by an oxidising agent such as nitric acid containing nitrous 

 acid, but in this case the change of colour does not stop short with 

 green but passes successively through blue, violet and red to a final 

 yellow. These later colours are due to products of the progressive 

 oxidation of the first formed biliverdin, but with the exception of the 

 final substance (choletelin), are as yet but imperfectly characterised. 

 The play of colours observed when either bilirubin or biliverdin is 

 oxidised, constitutes the well-known Gmelin's reaction 2 . This is ex- 

 tremely delicate and may be applied in either of the two following 

 ways. A few drops of the suspected solution are placed on a porcelain 

 slab and a drop of yellow fuming nitric acid is brought into contact 

 with it. A play of colours is observed at the junction of the fluids 

 if bile pigments are present. Or on the other hand some of the acid 

 may be poured into the bottom of a test-tube and the suspected fluid 

 carefully added so as not to mix with the acid but float on its surface. 

 If bile pigments are present coloured rings (layers) appear at the 

 junction of the two liquids, being yellow nearest the acid and 

 progressively red, violet, blue and green passing upwards. It is 

 stated that this test will detect as little as 1 part of bilirubin in 

 7000080000 parts of solvent. 



Other tests have been recommended but they are perhaps unnecessary in view of 

 the extreme delicacy of Gmelin's reaction when properly applied 3 . The certain 



1 The coloured residue from human gall-stones left after the extraction of 

 cholesterin (p. 133) may also be used for the preparation of bilirubin. 



2 Tiedemann u. Gmelin. Die Verdauung nach Versuchen, 1826, S. 80. 



3 See more particularly Capranica, Gaz. chim. Ital. Vol. xi. (1881), p. 430. 

 Moleschott's Untersuch. z. Naturlehre, Bd. xm. (1882), S. 190. Ehrlich. Centralb. 



