XXIII] FATS AND OILS 311 



CHOLESTEROL AND PHYTOSTEROL IN MIXTURES OF ANIMAL AND VEGETABLE FATS. 

 32 Alcohol Extraction Method^°. — Tentative. 



Introduce 200-300 grams of the melted fat into a fiat-bottomed liter flask. Close 

 the neck of the flask with a 3-holed stopper and insert through these holes: (1) a 

 reflux condenser; (2) a right-angled glass tube, one arm of which reaches to a point 

 6 mm. above the surface of the melted fat, the other being closed a short distance 

 from the flask by means of a short piece of rubber tubing and a pinch-cock; (3) a 

 glass tube bent so that one arm reaches down to the bottom of the flask and the 

 other serves as a delivery tube for a 700 cc. round-bottomed flask containing 500 cc. 

 of 95% alcohol by volume. 



Place the flasks, containing the melted fat and the alcohol, on a steam bath and 

 heat so that the alcohol vapor passes through the melted fat in the liter flask and 

 is condensed in the reflux condenser, finally collecting in a layer over the melted 

 fat. After all the alcohol has passed in this manner into the flask containing the 

 fat, disconnect the flask from which the alcohol has been distilled and attach a 

 tube to the short piece of rubber tubing attached to the right-angled glass tube 

 [see (2) above] and siphon the alcohol layer back into the alcohol distillation flask. 

 Reconnect as at first and again distil the alcohol as in the first operation. When 

 all the alcohol has been distilled, siphon it again into the distillation flask and 

 extract in the same manner for a third time. 



Discard the fat and retain the alcohol which now contains practically all of the 

 cholesterol and phytosterol originally present in the fat. Concentrate the alcoholic 

 solution to about 250 cc. and add 20 cc. of potassium hydroxid solution (1 to 1) 

 to the boiling liquid. Boil for 10 minutes to insure complete saponification of the 

 fat, cool to room temperature and pour into a large separatory funnel containing 

 500 cc. of warm ether. Shake to insure thorough mixing and add 500 cc. of water. 

 Rotate the funnel gently to avoid the formation of e.xtremely stubborn emulsions, 

 but mix the water thoroughly with the alcohol-ether-soap solution. A clear, sharp 

 separation takes place at once. Draw off the soap solution and wash the ether layer 

 with 300 cc. of water, avoiding shaking. Repeat the washing of the ether solution 

 with small quantities of water until all the soap is removed. Transfer the ether 

 layer to a flask and distil the ether until the volume of liquid remaining in the 

 flask measures about 25 cc. Transfer this residue to a tall 50 cc. beaker and 

 continue the evaporation until all the ether is driven off and the residue is per- 

 fectly dry. If desired, a tared beaker may be used and the weight of the unsaponi- 

 fiable matter determined at this point. 



Add 3-5 cc. of acetic anhydrid to the residue in the beaker, cover the beaker 

 with a watch glass and heat to boiling over a free flame. After boiling for a few 

 seconds, remove the beaker from the flame, cool and add 35 cc. of 60% alcohol by 

 volume. Mix the contents of the beaker thoroughly, filter off the alcoholic solution 

 and wash the precipitate with 60% alcohol. Dissolve the precipitate on the filter 

 with a stream of hot 80% alcohol by volume and wash the insoluble portion well 

 with 80% alcohol. Acetates of cholesterol and phytosterol are dissolved while the 

 greater i)ortion of the impurities present (including paraffin and paraffin oil if pres- 

 ent) remain behind on the filter. Cool the combined filtrate and washings to a 

 temperature of 10°-12°C. and allow to stand at that temperature for 2-3 hours. 

 During this time the acetates of cholesterol and phytosterol crystallize from the 

 solution. Collect the crystals upon a filter, wash with cold 80% alcohol and then 

 dissolve them in a minimum amount of hot absolute alcohol. Collect the alcoholic 

 solution of the acetates in a small, glass evaporating dish, add 2 or 3 drops of water 

 to the solution and heat if not perfectly clear. Allow the alcohol to evaporate 



