2 HYPOPHASIC AND EPIPHASIC CAROTENOIDS 21 



from peroxide), carbon disulphide, chloroform (free from hydrochloric acid), 

 ethanol, methanol and acetone. Extraction at room temperature is carried out, 

 by allowing the material to stand with the solvents in wide-necked bottles or 

 percolators in a carbon dioxide or nitrogen atmosphere. If large amounts of 

 material have to be extracted, large metal extractors can be used to advantage 

 and also make it possible to work at higher temperatures. The extracts must 

 be concentrated in vacuum as quickly as possible, and the concentrates thus 

 obtained must be kept sealed up under vacuum in large ampoules until they 

 are worked up. 



2. SEPARATION INTO HYPOPHASIC AND EPIPHASIC CAROTENOIDS 



As a result of the pioneering work of Willstatter and Stoll^, it is usual 

 to divide the extracted carotenoids into two groups by partition between two 

 immiscible solvents. Carotenoids with two or more hydroxyl groups are thus 

 obtained as hypophasic pigments and those without hydroxyl groups as epi- 

 phasic pigments. Mono-hydroxy compounds such as cryptoxanthin and rubi- 

 xanthin occupy an intermediate position and are found in the epiphase as well 

 as in the hypophase. The following table provides a summary of the epiphasic 

 and hypophasic carotenoids (table 4, p. 22). 



As most of the hydroxylated carotenoids occur in nature in the form of 

 esters as the so-called "pigment-waxes", it is necessary to saponify before 

 partition between methanol and petroleum ether. This can be done by means 

 of about 12 % methanolic potassium hydroxide at room temperature. The 

 carotenoid mixture to be saponified is dissolved in petroleum ether and a 

 requisite quantity of alkali is added. If the resulting mixture is homogenous it 

 is simply allowed to stand for about 20 hours. If two phases are formed, the 

 mixture must be shaken mechanically. In either case, the space above the 

 solution should be filled with an inert gas (e.g. hydrogen or nitrogen) in order 

 to prevent aerial oxidation. 



In some cases a solution of sodium methoxide in methanol is preferred to 

 methanolic potassium hydroxide. Saponification can also be carried out at 

 more elevated temperatures (about 60-70° C.) ; in this case an alkali concentra- 

 tion of about 5% is usually sufficient. 



After saponification is complete, petroleum ether is added, followed by 

 sufficient water to result in a separation into two phases. The upper phase 

 contains mainly the epiphasic carotenoids and the lower phase mainly the 

 hypophasic carotenoids. The petroleum ether phase is then repeatedly ex- 

 tracted with methanol, and the methanol layer is repeatedly extracted with 

 petroleum ether, and the appropriate extracts are combined. The solution of 

 epiphasic pigments is washed with water, dried over sodium sulphate, concen- 

 References p. 28. 



