METHODS OF ISOLATION OF CAROTINOWS 205 



pigmentcd adipose tissue which one finds in certain breeds of dairy 

 cuttle. At the most butter fat contains little more than 0.005 per 

 cent carotin, and in many cases considerably less than this amount. 

 Ten to 20 kgs. of fat would therefore be required to secure 0.5 grams 

 of pigment, assuming that all of it could be recovered. The problem 

 is rendered still more difficult by the fact that the fat must be com- 

 pletely saponified before the pigment can be extracted; and, if it is 

 necessary to use a procedure in connection with the saponification 

 and extraction of pigment from large quantities of fat such as has 

 been found practicable for small quantities, the volumes of soap 

 solution and ether required for the operation would soon reach a 

 magnitude all out of proportion to the facilities of the best appointed 

 laboratories. To be specific, at least 120 liters of ether would be 

 necessary to secure the carotin from 10,000 grams of fat, and inas- 

 much as the yield could not be over a few tenths of a gram of crystal- 

 line product the mechanical difficulties involved would not justify 

 the attempt. 



It is readily possible, how r ever, to obtain sufficient carotin from 

 animal fat for a macroscopic study of the chemical and physical 

 properties of the pigment. Twenty to thirty grams of well colored 

 butter fat or rendered adipose tissue fat are ample for such a study. 

 The butter fat must not be artificially colored, the pure rendered 

 butter fat from Jersey or Guernsey cows on a fresh pasture-grass 

 diet being best suited for the experiment. The fat must first be 

 saponified; and, in this connection, an important precaution must 

 be taken, namely, to avoid the use of alcohol which has not been 

 completely purified from aldehydes which produce yellow to red col- 

 ored resins with alkali. 3 The resins thus formed follow the caro- 

 tinoids in their isolation and interfere greatly with the study of the 

 properties of the pigments. 



For the saponification of the fat, 2 cc. of colorless 20 per cent 

 alcoholic potash is added for each gram of fat and the mixture al- 

 lowed to boil for about one hour under a reflux condenser. The 



8 Ethyl alcohol is especially likely to contain such impurities. It can be purified best 

 by treatment with silver nitrate, in which about 2.0 grains of crystals are added to 4 

 liters of alcohol and allowed to stand, with shaking, for several days. 200 g. unslaked 

 lime are now added to precipitate the AgO, neutralize the acids and remove any excess 

 water. The lime can now be filtered off and the filtrate distilled. Usually one such 

 treatment will prepare an excellent 98 per cent alcohol which will show no coloration 

 on boiling in the presence of 20 per cent KOII. Should a color develop under these 

 conditions the purification must l>e repeated. 



Methyl alcohol can also be used for the saponification of the fat, but it, also, must 

 show no coloration when a 20 per cent KOII solution of the alcohol is boiled. 



