6o 4 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



mark 15 c.c. and ether to the mark 30 c.c. The vessel is stoppered and the 

 contents mixed by inverting twice. After standing the lower layer is separated 

 and replaced by 20 c.c. of water which is allowed to run down the sides. 

 After 5 minutes the water is withdrawn and a second washing with water 

 carried out in the same way. The ether is put into a basin of 30 c.c. capacity, 

 the funnel washed with ether and the ether and the washings evaporated. 



The residue is dissolved in 2 c.c. of chloroform, the solution placed in a 

 graduated test tube of 10 c.c. capacity and the basin washed out with about 

 3 c.c. of chloroform which is also transferred to the test tube. 



2 c.c. of acetic anhydride and 3 drops of sulphuric acid (66 per cent.) 

 are added. 



At the same time 5 c.c. of a solution of *o6 per cent, cholesterol in chloro- 

 form together with 3 drops of acetic anhydride and 3 drops of sulphuric acid 

 are placed in another graduated test tube. 



The green colorations of the solutions are compared in half an hour when 

 the maximal coloration is reached. 5 c.c. of each of the solutions are poured 

 into the cups of a dilution colorimeter and the deeper one is diluted with a 

 mixture of chloroform, acetic anhydride and sulphuric acid in the above pro- 

 portions. 



If n is the number of c.c. of the diluted solution and P is the cholesterol 

 content of 1000 c.c. of serum, 



P = 0*30 x gm. if the unknown is diluted. 



7"5o 

 P = gm. if the control is diluted. 



In the case of tissues 0-2- 1 gm. is placed in a 90 c.c. flask, 30 c.c. of 70 per 

 cent, alcohol containing i per cent, of sodium hydroxide are added, and the 

 mixture heated in a boiling water-bath till the tissue dissolves and the volume 

 is about 15 c.c. These 15 c.c. are put in the special separating funnel 

 (above) and the flask is washed out with 15 c.c. of ether. The rest of the 

 process is the same as with serum. 



If P is the amount of cholesterol in 1000 gm. of tissue, 



P = . gm. if the unknown is diluted, 

 P = -. gm. if the control is diluted, 



where / is the weight of the tissue taken. 



For larger quantities of serum or tissue Grigaut describes a gravimetric 

 method: 



20 c.c. of serum are placed in a 250 c.c. flask, 20 c.c. of 40 per cent, 

 sodium hydroxide are added and the mixture is heated in an autoclave at 1 10 

 for i hour. 



5-10 gm. of tissue are mixed with 40 c.c. of 40 per cent, sodium 

 hydroxide diluted with an equal volume of water and heated in an autoclave 

 at 110 for i hour. 



The solution is put into a separating funnel and shaken whilst still warm 

 (at 40-45) with 60 c.c. of ether. The lower layer is withdrawn, warmed to 

 40-45 and again shaken with 60 c.c. of ether. This extraction is carried 

 out 10 times. 



The ethereal solutions are evaporated, the residue is dissolved in 50 c.c. 

 f 95 P er cent, alcohol containing i c.c. of i per cent, alcoholic sodium 

 hydroxide and the solution is evaporated on a water-bath. The residue is 

 heated in a steam oven for half an hour and the dry residue is dissolved in 

 petroleum ether whilst the vessel is still warm. The impurities are insoluble 

 and are filtered off through asbestos and washed with petroleum ether. The 

 petrol solutions are evaporated in a weighed basin. Pure cholesterol separates ; 

 it is dried at 100, cooled and weighed. 



