316 BILIRUBIN. [BOOK n. 



further a common constituent of gall-stones ; it occurs in the urine, 

 and stains the conjunctivae and skin, in cases of jaundice. In old 

 blood extravasations it occurs in microscopic crystals which were first 

 discovered by Virchow and by him called haematoidin. 



Methods of In the separation of bilirubin, from whatever source 



separation. t - g obtained, we rely on the remarkable solubility 

 of the free colouring matter in chloroform, whilst its compounds are 

 insoluble in that liquid. 



1. Separation from Gall-stones. The gall-stones are finely pow- 

 dered and then thoroughly extracted with ether which extracts the 

 cholesterin which these concretions contain. The residue is then 

 extracted with boiling water, and afterwards treated with dilute 

 hydrochloric acid. The latter decomposes the bilirubin-calcium, the 

 pure bilirubin remaining as an insoluble powder which is thoroughly 

 washed with water, dried and then boiled with chloroform. The 

 chloroformic solution of bilirubin is evaporated on the water bath 

 and the residue is extracted with absolute alcohol and ether. The 

 former solvent separates a certain quantity of a pigment, termed by 

 Stadeler, bilifuscin. The residue insoluble in alcohol and ether is 

 then again dissolved in chloroform, the solution, if necessary, filtered, 

 and evaporated until bilirubin commences to separate ; alcohol is 

 then added to the concentrated chloroformic solution, when an 

 amorphous orange-coloured precipitate of bilirubin, which resembles 

 sulphide of antimony, is obtained. These operations may be repeated. 

 Ultimately the purified bilirubin is once more dissolved in chloroform, 

 and the chloroformic solution allowed to evaporate spontaneously, 

 when crystals of bilirubin separate. 



2. From bile. (1) Bile, preferably of the dog, is acidulated with 

 acetic acid and shaken with chloroform, care being taken to exclude 

 air as completely as possible 1 . The chloroformic solution is separated^ 

 evaporated to dryness, the residue treated with absolute alcohol, and 

 the matter which is left undissolved by this solvent is dissolved in 

 chloroform. By the spontaneous evaporation of the chloroformic 

 solution, crystals of bilirubin may be obtained. The purification of 

 the bilirubin may, if the quantity of material be sufficient, be carried 

 out as directed in the case of bilirubin prepared from gall-stones. 



(2) The bile from which bilirubin is to be prepared is diluted 

 with water and precipitated with milk of lime. A solution of 

 carbonic acid is passed through the mixture and the bulky pre- 

 cipitate, having been collected and washed, is suspended in water, 

 decomposed by means of hydrochloric acid and shaken with chloro- 

 form, care being taken in these operations to avoid the access of air. 

 The chloroformic solution is evaporated to a very small bulk, pre- 



1 In all cases in which it is sought to separate bilirubin, so long as the solutions 

 contain free acids or alkalies the greatest care must be taken to avoid the access of air ; 

 in the presence of atmospheric oxygen the conversion of bilirubin into biliverdin at 

 once commences, in acid or alkaline solutions. . 



