6 CHOLINE 



On the basis of a careful study of the reineckate method, as originally 

 modified by Jacobi et al." and Engel,^- Glick^^ has proposed the following 

 procedure for the isolation of choline from natural materials: 



A weighed sample, containing the equivalent of 2 to 5 mg. of choline 

 chloride, is placed in an alundum thimble of medium porosity (80 mm. long 

 and 22 mm. in diameter) for extraction in a Soxhlet apparatus fitted with 

 a 125-ml. boiling flask. About 100 ml. of methanol is used as the solvent, 

 and the extraction is allowed to proceed for 24 hours. With some finely 

 divided materials such as flour, the tendency to form a hard cake makes it 

 desirable to mix the sample intimately with No. 2 pulverized pumice to 

 facilitate the extraction. The boiling flask containing the methanol extract 

 is placed on a steam bath and, when only a few milliliters of solvent remains, 

 30 ml. of a saturated solution of barium hydroxide is added and the heating 

 is continued for 90 minutes. After the mixture is cooled, a drop of 1 % al- 

 coholic thymolphthalein is added to the hydrolyzate and glacial acetic is 



TABLE I 



Recovery of Added Choline by the Reineckate Procedurei^ 



Choline chloride, mg./ml. 



introduced until the blue color is just discharged by one drop. The liquid 

 is then filtered by suction through a sintered glass filter tube of medium 

 porosity (15 to 30 ml. capacity) into a 125-ml. suction flask. The boiling 

 flask is rinsed with small portions of distilled water, and the rinsings are 

 used to wash the filter, a total of about 15 ml. of water being used. To the 

 combined filtrate and washings is added 6 ml. of a 2 % solution of reineckate 

 salt in methanol, and the flask is placed in a refrigerator at about 5° for 2 

 hours. The choline reineckate precipitate is filtered with suction into a 

 30-ml. sintered glass filter tube of medium porosity. The dried precipitate 

 is washed three times with 2.5-ml. portions of w-propanol and again dried 

 by means of the suction. 



The above procedure avoids the precipitation of betaine reineckate which 

 is insoluble in acid solutions but soluble in slightly alkaline solutions. How- 

 ever, it has been noted^^ that dimethylaminoethanol appears to be carried 

 down in part in the choline reineckate precipitate when a solution contain- 

 ing the two bases is treated with reineckate at a slightly alkaline pH. 



" T. H. Jukes, A. C. Dornbush, and J. J. Oleson, Federation Proc. 4, 157 (1945). 



