178 CAROTINOIDS AND RELATED PIGMENTS 



ficulty. Willstatter and Escher announced their discovery of the 

 carotin nature of the corpus lutcum pigment in connection with their 

 studies on egg yolk xanthophyll. The details of the corpus luteum 

 work were later published by Escher (1913). 



The relatively small size of corpora lutea tissue naturally required 

 the collection of ovaries on a large scale. It was fortunately found 

 possible to preserve the ovaries for many months under 60 per cent 

 alcohol without impairment of the pigment (preservation in dilute 

 formalin prevented the isolation of crystals), thus making it feasible 

 to collect the material in large slaughter houses over an extended 

 period of time. After 146 kgs. (about 10,000 ovaries) has been col- 

 lected from cows and sheep, the tissue was hashed in 10 kg. lots, 

 further dehydrated with 95 per cent alcohol, and then shaken in the 

 cold with petroleum ether (b. p. 50-70 C.) for several hours. 

 This effected an almost complete extraction of pigment. This extract 

 (about 3 liters for each 10 kg. of ovaries) was then washed seven suc- 

 cessive times with one-sixth volume of 90 per cent methyl alcohol, 

 the alcohol removed by washing four times with one-third volume of 

 water, the extract freed from water by shaking with anhydrous sodium 

 sulfate, filtered and concentrated to a syrup in vacuum. On adding 

 six to ten volumes of absolute ethyl alcohol and cooling in an ice-salt 

 bath nearly all fatty impurities were precipitated. These were sepa- 

 rated quickly by means of a cloth filter, for in a short time crystalliza- 

 tion of the pigment began and continued for several hours, at ice-box 

 temperature. Some very large (1-2 mm. long), beautiful crystals 

 were obtained. Purification was secured by filtering and washing 

 with a mixture of equal parts petroleum ether and absolute alcohol. 

 Only 0.45 grams of pigment in all were secured in this way from the 

 146 kgs. of ovaries. 



The crude ovarian pigment proved to be remarkably pure as judged 

 from the microscopic examination and melting point of the crystals. 

 For the elementary analyses, however, the pigment was recrystal- 

 lized first from alcohol, again by addition of an excess of absolute 

 alcohol to a concentrated carbon disulfide solution, and finally from 

 pure petroleum ether (sp. g. 0.64-.66). The elementary composition 

 showed the pigment to be a hydrocarbon. The average of four closely 

 concordant analyses carried out for Escher by Fisher and Sonnenfeld 

 under Willstatter's direction, using Preyl's micro-method, showed 89.55 

 per cent carbon and 10.66 per cent hydrogen. This corresponds even 

 more closely to the calculated composition of the compound C 40 H 5G 



