809 



the fluid sliglitly troubled. These traces of water are removed by 

 shaking with glowed sulphate of sodium. The latter is removed bv 

 filtration. The result is a very pure solution of the yellow pigment 

 in chloroform (solution A). It may be easily proved that this yellow 

 pigment is bilirubin. 



1. If the chloroform solution is shaken with diluted KOH or NaOH 

 the pigment passes into the latter, while the chloroform loses its 

 colour (solution B). 



2. If now some acid is added till the fluid reacts distinctly as an 

 acid, then the fluid at the top loses its colour, the pigment passing 

 into the chloroform at the bottom. 



3. If to the alkaline solution (see sub 1) HNO^ containing some 

 HNO3 is added, the result is the well-known colour-play of the reaction 

 of Gmelin. 



4. If a slight quantity of a diluted iodine-solution in alcohol 

 (1 : 100) is carefully poured on to the alkaline solution, a l^lue ring 

 is formed. 



5. If to the alkaline solution first an equal volume of alcohol is 

 added, and then i of the original volume of the diazo-raixture ot 

 Ehrlich, a red colour is the result. An addition of a few drops of 

 concenti-ated HCl changes the red colour into blue. 



All these reactions together, prove conclusively that the pigment 

 obtained in the above way is indeed bilirubin. 



Crystals of bilirubin can be easily obtained from the pure chloro- 

 form-solution (sol. A) in the following manner. The latter is poured 

 out into a watch-glass which is covered with another watch-glass 

 and placed in the ice-safe. Tiie chloroform evaporates slowly and 

 on the watch-glass the microscopically visible, pretty, yellow bilirubin- 

 crystals are left. When HNO.„ containing HNO^ is added, these 

 crystals present under the microscope the reaction of Gmelin, 



We can also dissolve the yellow crystals again in some solvent 

 (chloroform, dil. NaOH etc.) and carry out the above-mentioned 

 reaction with them. 



If one has no good vacuum-pump at one's disposal the method 

 can also be applied with the following modification suggested by 

 Dr. Snappkr. 



10 cm^ of bloodserum are precipitated with 20 cm" of acetone. 

 The albumen-precipitate is centrifugalized. To the pipetted upper- 

 fluid some drops of water are added; then this fluid is washed 

 carefully with aether a few times, to remove the fatty substances 

 as much as possible. These volumes of aether are removed with 

 the pipette every time. Then some drops of ice-vinegar and 1 cm" 



