216 CAROTINOIDS AND RELATED PIGMENTS 



discarded. The extracted material was pulverized and extracted at 

 once (i. e., within a few days) with 5 portions of 85 per cent alcohol. 

 The last four fractions were combined (the first being discarded) and 

 amounted to 25 liters for the 20 kgs. of algae. This solution was next 

 shaken with CaC0 3 to neutralize acids, and decanted from the set- 

 tled chalk. The solution was now diluted with water, using 3.4 

 volumes for each 10 volumes of extract. The chlorophyll which pre- 

 cipitated was allowed to settle out and the supernatant fluid, amount- 

 ing to 40 liters in all, used as the mother liquor for the isolation of the 

 fucoxanthin. This was accomplished as follows: 



Four-liter portions were treated with one liter of a mixture of 

 ether and petroleum ether (b. p., 30-50 C.), 3:1, and 1.5 liters of 

 water added. The ether layer which took up the pigment was then 

 washed very carefully with water (to avoid emulsions) in order to 

 free it from the acetone which was used to extract the pigments 

 from the alga?. The petroleum ether was then concentrated to 0.5 

 liter in vacuum, diluted with an equal volume of ether and shaken 

 with 70 per cent methyl alcohol saturated with petroleum ether. This 

 removed the fucoxanthin, together with some xanthophyll. The xan- 

 thophyll was removed by shaking the methyl alcohol with an equal 

 volume of a mixture of petroleum ether and ether (5:1). The fuco- 

 xanthin was then transferred to ether, the ether solution was concen- 

 trated to a thick syrup and about 1 liter of low boiling petroleum 

 ether added. The precipitate of crude pigment obtained amounted to 

 about 2 grams from each 4 liter portion of mother liquor, representing 

 about half the total pigment present. The crude pigment was puri- 

 fied by recrystallization from methyl alcohol, giving crystals con- 

 taining three molecules of methyl alcohol of crystallization, which 

 could be removed in vacuum. Solvent-free crystals were obtained 

 by precipitation from ether with low b. p. petroleum ether. 



Isolation of Rhodoxanthin 



This pigment, as explained in an earlier chapter, appears to be 

 a red xanthophyll. It was discovered by Monteverde (1893) in the 

 Russian pond-weed, Potamogeton natans, later by Tswett (1911) as 

 the cause of the winter red color of the arbor vita?, Thuja orientalis, 

 and a little later by Monteverde and Lubimenko (1913b) in the 

 arillus of the seed of the yew, Taxus baccata. The isolation of 

 crystals for macroscopic examination can be carried out as follows, 



