312 METHODS OF ANALYSIS [Chap. 



spontaneously, the contents of the dish being stirred occasionally to mix the deposit 

 of crj-stals, which form upon the edges, with the main body of the liquid. As soon 

 as a good deposit of crystals has formed, collect them upon a hardened filter, wash 

 twice with cold 90% alcohol and dry by suction, drying finally at 100°C. for 30 min- 

 utes, and determine the melting point in the apparatus shown in 1 1 , Fig. 12, using 

 sulphuric acid in the outer beaker and glycerin in the inner tube. 



The melting point of the first crop of crystals usually gives definite information 

 as to the presence or absence of phytosterol but the conclusion indicated should be 

 confirmed by recrystallizing the crystals from absolute alcohol and again determin- 

 ing the melting point. If the crystals are pure cholesteryl acetate, the melting point 

 of the second crop should agree closely with that of the first. If phytosteryl acetate 

 is present, however, a higher melting point will be noted, as phytosteryl acetate is 

 less soluble in alcohol than cholesteryl acetate. The melting point of cholesteryl 

 acetate is 114°C., that of phytosteryl acetate 125°-137°C. 



33 Digitonin Method. (Marcusson and Schilling^^) — Tentative. 



Shake vigorously 50 grams of the oil or fat for 15 minutes in a separatory funnel 

 with 20 cc. of a 1% solution of digitonin in 95% alcohol by volume. Allow the 

 mixtm-e to stand for a time until the emulsion separates. The lower or fat layer 

 should be quite clear while the alcohol layer contains a bulky, flocculent precipitate. 

 Draw off as much as possible of the fat, avoiding any loss of the precipitate. Add 

 100 cc. of ether to the alcohol layer and filter the mixture. Wash the precipitate 

 with ether until free from fat; after drying in the air, transfer it to a tall 50 cc. 

 beaker, add 2-3 cc. of acetic anhydrid and cover the beaker with a watch glass. 

 Then boil slowly over a low flame for 30 minutes. After cooling, add 30-35 cc. of 

 60% alcohol by volume and mix the contents of the beaker thoroughly. Filter 

 off the alcohol solution and wash the precipitate with 60% alcohol, then dissolve it 

 on the filter with a stream of hot 80% alcohol by volume from a wash bottle and 

 set aside the filtrate in a cool place (10°C. or below). After the acetates have 

 crystallized out of this solution filter them off, recrj'stallize from absolute alcohol, 

 dry and determine the melting point of each crop of crystals, as directed under 32. 



34 UNSAPONIFIABLE RESIDDEi^.-TENTATIVE. 



Saponify 5 grams of the oil or fat with alcoholic potassium hydroxid solution and 

 remove the alcohol by evaporation. Wash into a separatory funnel with 70-100 cc. 

 of water and extract with 50-60 cc. of ether. If the 2 liquids do not separate, add a 

 few cc. of alcohol. Separate the water solution and wash the ether with water con- 

 taining a few drops of sodium hydroxid solution. Again extract the soap solution 

 and washings with ether and evaporate the combined extracts to dryness. In most 

 cases it is advisable to add a little alcoholic potassium hydroxid solution to the 

 residue and heat in order to saponify any traces of fats left unsaponified and extract 

 again with ether. Transfer to a weighed dish and dry as quickly as possible in a 

 water oven. 



Many of the hydrocarbon oils are volatile at 100°C., so that the drying should 

 not be carried any further than necessary. With resin oil, paraffin wax and the 

 denser mineral oils there is little danger of loss at 100°C. 



On account of the solubility of soap in ether and petrolemn ether it is well to 

 wash the residue with warm water containing a little phenolphthalein. If the 

 reaction is alkaline, soap is present and the residue must be further purified. 



