120 IV. BILE PIGMENTS 



4.2. Individual Biladienes-(a,c) 



4.2.1. Bilirubin. Bilirubin, CssHseOeX^, is best obtained from 

 ox gallstones, which consist to a large extent of calcium bilirubinate. 

 This method had already been used by Stiideler in 1864 {2607) and 

 has been further developed by Kuster (1598,1599,1603,1610) and 

 Fischer i776,826;861,p.634'). 



The isolation consists in the liberation of bilirubin from its salt by acid, 

 the removal of impurities by extractions with ether and alcohol, in which 

 bilirubin is almost insoluble, and the extraction of bilirubin with chloro- 

 form. This procedure has to be repeated many times, since layers of the 

 bihrubinate appear to be coated with layers of impurities. AVith the fancy 

 prices demanded for ox gallstones, methods using other starting materials 

 are welcome. Two methods liave been descril)ed for its isolation from pig 

 bile (.9.96) ; one gives a smaller yield but is less laborious and the product is 

 almost pure (1688). More recently preparations from bile have been 

 described {1730) and patented {2119,2120) which use chlorobenzene as 

 solvent for bilirubin. All of the bilirubin preparations on the market require 

 recrystallization and some are very impure. 



Bilirubin crystallizes from chloroform in typical rhomboid prisms 

 or plates, occasionally in finer leaflets. Its surface color varies from 

 light orange yellow to deep red-brown and is no indication of purity. 

 While it keeps well in the dry state and moderately well in chloroform 

 solutions protected from light, alkaline solutions are very unstable; 

 and alcoholic solutions, even those containing chloroform, tend to 

 become colloidal, particularly if the bilirubin is pure. Great care is 

 thus needed in making up standard solutions of bilirubin. Bilirubin 

 gradually blackens on heating and does not melt. It forms a red 

 compound with strong hydrochloric acid {1211), which is perhaps 

 the hydrochloride of the tautomeric form with hydroxypyrrolene 

 nuclei. 



4.2.2. Mesobilirubin. Mesobilirubin, C33H40O6N4, is obtained from 

 bilirubin by catalytic hydrogenation (820,859,868) or by treatment 

 with hydrazine hydrate in pyridine {86 Jf). It is more easily soluble 

 in chloroform and of somewhat yellower color than bilirubin. It 

 melts at 315° C. (coalescing at 360°), which distinguishes it from 

 bilirubin. Two safer ways of distinguishing the substances exist, 

 which are applicable to small amounts. The first involves conversion 

 to the biliverdin, and heating the later in concentrated sulfuric acid 

 (c/. Section 4.1.); the second involves treatment with iodine in the 

 presence of zinc acetate in alcohol, followed by spectroscopic exami- 



