THE RILE. 161 



crystallized bile. In this form the biliary acids are conveniently 

 estimated as a whole. If then it is desired to determine the rela- 

 tive amount of the two principal acids present, it is only necessary 

 to estimate the sulphur in Platner's bile, from which the corre- 

 sponding amount of taurocholic acid can be calculated. 



To separate the two acids from each other, Platner's bile is dis- 

 solved in water and completely precipitated with a solution of neutral 

 acetate of lead. The corresponding lead salts are thus formed, and 

 can now be readily separated from each other, as the glycocholate is 

 thrown down, while the taurocholate remains in solution. In the 

 filtrate the latter is then precipitated with ammonia. To obtain the 

 free acid from the glycocholate, the precipitated lead salt is sus- 

 pended in water and evaporated to dryness in the presence of sodium 

 carbonate. The sodium salt is thus obtained and extracted with 

 alcohol. The alcohol is evaporated off, the residue dissolved in 

 water and treated with hydrochloric acid, when the glycocholic acid 

 will separate out. The taurocholic acid can be similarly obtained 

 from its lead salt by decomposing the solution with hydrogen sul- 

 phide. The resulting plumbic sulphide is filtered off, the filtrate 

 evaporated to dryness, the residue dissolved with a small amount 

 of alcohol, and then precipitated with an excess of ether, when the 

 free acid is thrown down. 



Tests for the Bile-acids. Pettenkofer's Test. On treating the 

 biliary acids in aqueous or alcoholic solution with a few drops of an 

 0.1 per cent, solution of furfurol and 1 or 2 c.c. of concentrated, 

 chemically pure sulphuric acid, a beautiful purple color develops. 

 As furfurol results when concentrated sulphuric acid is added to 

 a carbohydrate, the test may also be conducted by treating the solu- 

 tion of the biliary acids (1 or 2 c.c.) with a few drops of a 10 per 

 cent, solution of cane-sugar and then with sulphuric acid. In either 

 case, however, care should be had that the temperature during the 

 reaction does not exceed 70 C., as otherwise the resulting pigment 

 is destroyed. This may be obviated by substituting strong phos- 

 phoric acid for the sulphuric acid, and placing the solution in boiling 

 water. The resulting pigment shows two bands of absorption on 

 spectroscopic examination. One of these is situated at F ; the other 

 between D and E, near E. On diluting with alcohol a green fluo- 

 rescence is observed. In the presence of an excess the red pigment 

 disappears, but reappears upon the further addition of sulphuric acid. 

 On standing, the color gradually turns to a bluish violet. 



As Pettenkofer's reaction can also be obtained with other sub- 

 stances, such as the phenols, the higher alcohols, certain bases of the 

 aromatic series, the higher hydrocarbons, and acids of the fatty 

 series and the benzol series, it is always necessary to isolate previ- 

 ously the biliary acids, as Platner's bile, before drawing conclusions 

 from the reaction. Albumins, if present, must in every case first 

 be removed. For, as has been stated, many of these contain carbo- 

 hydrate groups, which on contact with concentrated sulphuric acid 

 11 



