206 CAROTINOIDS AND RELATED PIGMENTS 



resulting soap is dissolved in three volumes of distilled water. After 

 cooling, this solution is shaken with an equal volume of pure ether 

 in a separatory funnel. The extraction is repeated with a fresh 

 volume of ether equal to one-half the volume of soap solution. The 

 soap should now be colorless. 4 The combined ether extracts are 

 now washed many times with an excess of water, carefully at first 

 to avoid emulsions, and more vigorously with subsequent washings. 

 When the wash water no longer reacts alkaline to phenolphthalein, 

 the ether solution is dried by shaking with neutral, fused CaCl 2 or 

 anhydrous Na 2 S0 4 for a few hours, decanted or filtered from the 

 inorganic drying agents and evaporated to dryness in a dry vacuum. 

 Little or no heat need be applied because of the rapid volatilization of 

 ether under diminished pressure. The residue consists of pigment 

 mixed with large quantities of cholesterol and traces of other unsaponi- 

 fiable matter. According to Steenbock (1921a) and others, the fat- 

 soluble vitamine in butter fat is present in this fraction. The choles- 

 terol can be removed by the digitonin method of Windaus (1909), 

 by dissolving the residue in warm 95 per cent alcohol and adding 

 an excess of a hot one per cent solution of digitonin in 90 per cent 

 alcohol. This procedure is not necessary, however, for the study of 

 the chemical and physical properties of the pigment. 



The examination of pigment isolated from animal fat in the above 

 manner must be made at once unless facilities are available for keep- 

 ing the pigment in an atmosphere of inert gas. Kohl (1902b) states 

 that crystalline carotin can be protected completely from oxidation, 

 even in the sunlight, if placed under glycerin. The writer has never 

 tried this method for crude preparations of pigment from animal 

 tissues, so is unable to vouch for its usefulness for pigments prepared 

 by the method just given. 



Blood serum. The blood serum of man and certain animals may 

 be relatively rich in carotin, giving it a golden yellow color. While 

 this material can not be expected to serve as a suitable source of 

 pigment in large quantities the pigment can be isolated in sufficient 

 amounts for chemical examination without great difficulty. Serum 

 or plasma free from erythrocytes must first be obtained. This may 

 be done either by allowing the blood to clot and permitting the serum 



* Many of the early workers who saponified their plant or animal extracts evaporated 

 the alcohol and extracted the dried soaps with the solvents, or carried out the extrac- 

 tions with soaps which had been salted out of aqueous solution with NaCl. In the 

 writer's experience these procedures are not advantageous when working with pure 

 animal fats which contain carotinoids. 



