BILE ACIDS 323 



Preparation of Cholic Acid, Choleic Acid and Deoxycholic Acid. 



The most recent methods of preparing these acids from bile are described 

 by Pregl and Buchtala, 1 by Schryver 2 and by Main 3 



SchryveSs Method. 



2*5 litres of bile are heated in an iron vessel under a reflux condenser for 30 

 hours with 170 gm. of sodium hydroxide dissolved in 300 c.c. of water. Two 

 volumes of water are added and the hot solution acidified with excess of 

 hydrochloric acid, the mixture being vigorously stirred after each small ad- 

 dition of acid. The crude acids separate out as a thick oil, which forms a 

 pasty mass on cooling ; a small quantity separates in a granular form. After 

 standing for 1 2 hours, the acids are filtered off, washed free from mineral acid 

 by kneading with water, dried on a water-bath and powdered. They are 

 dissolved in dilute ammonia, the solution is diluted so as not to contain more 

 than 5 per cent, of the salts and boiled for 10 minutes with animal charcoal 

 to remove some of the pigment. The acids are reprecipitated with hydro- 

 chloric acid, washed, dried in vacua over calcium chloride and soda lime, the 

 mass being removed from time to time to powder the surface lumps and to 

 expose the under layers. The mass is readily powdered as more water is 

 removed and is finally obtained in a granular form. It is added to boiling 

 acetone till the solvent is saturated, the solution is filtered and allowed to 

 cool. The crystals so formed are filtered off and washed with cold acetone. 

 The filtrate and washings on concentration yield further crops of crystals. A 

 syrupy mother liquor finally results from which only a few crystals separate on 

 prolonged standing. Over 80 per cent, of the acids having a light greenish 

 tinge are thus obtained. 



The separation of the three acids depends on the different behaviour 

 of their magnesium salts. The crude product is suspended in alcohol, the 

 alcohol warmed and caustic soda added from a burette till the solution is just 

 alkaline to phenolphthalein which is added to the liquid. The alcohol is 

 evaporated and the sodium salts dissolved in water so that they form a i per 

 cent, solution. The alkaline solution is filtered, dilute acetic acid added till 

 the red colour disappears and treated with J^ of its volume of 20 per cent, 

 magnesium chloride solution. On heating the clear solution on a water-bath, 

 a bulky crystalline precipitate gradually separates; sometimes it becomes 

 almost a paste. It is heated for i hour and allowed to cool. 



The precipitate of magnesium choleate and deoxycholate (with not more 

 than ^ of its total of cholic acid) is filtered off by suction and the mother 

 liquor removed as far as possible (below). The acids are regenerated from 

 the magnesium salts by rubbing in mortar with excess of dilute hydrochloric 

 acid ; they become granular on standing, are easily filtered off, washed free 

 from acid and dried in vacua. They are dissolved in alcohol with the addition 

 of caustic soda as before, the solution is neutralised, diluted so as to contain 

 about 2 per cent, of salts and treated with about \ of its volume of 20 per 

 cent, barium chloride solution. The precipitated barium salts become 

 granular and crystalline on standing for 16 hours. The liquid is syphoned 

 off on a Buchner filter and the solid added. It is washed by grinding in a 

 mortar several times with water. This insoluble barium salt consists of 

 barium choleate from which almost pure choleic acid is obtained by stirring 

 up in mortar with dilute hydrochloric acid. 



The filtrate and washings are acidified, the precipitate of deoxycholic and 



a Zeitschr. physiol. Chem., 1911, 74, 198. 2 J. Physiol., 1912, 44, 265. 



3 Biochem J., 1917, n, n. 



