METHODS OF ISOLATION OF CAROTINOIDS 213 



in this way the residue (94 kgs. in all) was divided into portions of 

 2.8 kgs. and each portion shaken twice with fresh two liter quan- 

 tities of acetone in a shaking machine for one hour, the acetone being 

 sucked off each time through a sand filter. Practically all the color 

 was extracted by this means. 



All the acetone extracts were now combined and amounted to 200 

 liters. About 2.25 liters of oil settled out on standing. Although 

 highly colored it was discarded. The next problem was to remove the 

 phosphatides and cholesterol. The phosphatides were removed by 

 mixing each 6 liter portion of acetone extract with 0.5 liter of petro- 

 leum ether (sp. g., 0.64-0.66), and adding three volumes of water 

 carefully, to avoid an emulsion. The lower watery acetone layer was 

 drawn off after standing a day and the dark brown, thick oily syrup 

 rinsed out with petroleum ether. Twenty liters, in all, of this oily 

 material were obtained. Large clumps of almost colorless phospha- 

 tides, amounting to nearly 2 kgs. were thrown down by adding 2 

 volumes of acetone to the petroleum ether solution of this syrup. The 

 pigmented acetone-petroleum ether solution was decanted, filtered 

 through linen, and freed from acetone again by washing with water, 

 first by decantation and finally by direct addition of water, allowing 

 about one hour between each addition. The reddish-brown petroleum 

 ether solution was now filtered through fused Na 2 SO 4 and the filtrate 

 concentrated to 2 liters at 30-35 C., in vacuum, i. e., until the syrup 

 set to a crystalline mass of cholesterol. This was filtered off and the 

 deep colored filtrate diluted with 4 liters of petroleum ether (b. p. 

 30-50 C.). On standing in the ice box for a few days most of the 

 pigment crystallized out as a bright red blanket of very fine needles. 

 The yield of crude pigment amounted to 4 grams. The purification 

 of the pigment was described in Chapter VI. It is of interest that 

 the method of isolation used by Willstatter and Escher shows that 

 a portion, at least, of the egg yolk carotinoids are not present dis- 

 solved in fat. It was not found necessary to resort to a saponification 

 of the extracts in order to isolate crystals of pigment. 



The separation of sufficient egg yolk pigment for macroscopic ex- 

 amination can be effected in a satisfactory manner from a single well- 

 colored egg yolk. For this purpose the following procedure gives 

 very satisfactory results. The raw yolk is thrown into 100 cc. of 

 acetone, and, after heating to boiling, filtered to remove the coagu- 

 lated, colorless proteins. The filtrate is evaporated and the residue 

 saponified with 50 cc. of 20 per cent methyl alcoholic potash solution 



