160 TERPENOIDS AND STEROIDS 



Special methods of purification are applicable to various categories of compounds. 

 The low molecular weight terpenes are usually separated by simple distillation or steam 

 distillation. The most likely contaminants are volatile esters which may be removed by 

 saponification. In purification of the carotenoids saponification of contaminants is carried 

 out without heating in order to avoid degradations. The carotenoids may be separated ac- 

 cording to their solubilities. The so-called epiphasic ones go preferentially into the petro- 

 leum ether layer when shaken with a mixture of this with methanol. Hypophasic carote- 

 noids are those with two or more hydroxy groups. They are preferentially extracted into 

 the methanol layer. Some monohydroxy compounds are found in both layers. 



Formation of molecular complexes has found application in purifying some compounds 

 of this group. Thiourea forms adducts with many types of branched hydrocarbon chains, 

 depending on their molecular dimensions. Some cyclic compounds may also be accommo- 

 dated. The thiourea adducts are insoluble in alcohol and non-polar solvents. They may 

 be prepared by mixing alcoholic solutions of thiourea and the sample to precipitate the 

 adduct, or dry material may be triturated with thiourea and a small amount of methanol. 

 When the adduct has formed, uncombined lipids are extracted with benzene, ether, etc. 

 The thiourea adduct is then decomposed with hot water and the desired material extracted 

 into ether. For a general discussion of this technique see (56). Straight-chain aliphatic 

 compounds can be removed from the branched or cyclic terpenoids by converting the 

 former to urea complexes (57). 



Complex formation is very common between various members of the steroids. 

 This phenomenon causes difficulties in purification, but may also be put to practical use. 

 In particular, digitonin is frequently used to precipitate sterols from alcoholic solution 

 as insoluble digitonides. The reaction is specific for 3-/J-OH sterols (as almost all natu- 

 ral ones are). Free sterols are then regenerated by partitioning the complex between 

 mixtures of hot water and benzene or xylene whereupon the saponin goes to the aqueous 

 and sterol to the organic layer. Another method used for splitting the complex involves 

 boiling it with pyridine, cooling and adding ether to precipitate the saponin and leave 

 sterol in solution. Conversely, this same technique may be applied to purifying many 

 saponins by adding cholesterol to form an insoluble addition complex. 



Further purification is usually carried out by column chromatography as a general 

 technique although special methods may be available for individual examples (e. g. frac- 

 tional distillation for the low molecular weight terpenoids). Purification of saponins and 

 other glycosides is difficult and not commonly performed. These compounds are usually 

 first hydrolyzed to their aglycones by boiling for several hours with 1-4N HCl, the agly- 

 cones extracted with benzene and purified as such (58). Alumina is the most common 

 adsorbent used for chromatography of these compounds, with non-polar developing sol- 

 vents such as petroleum ether, benzene, etc. In most cases highly active alumina is un- 

 desirable since it may cause degradative reactions. It may be neutralized with acid and 

 a few per cent water added to lower its activity. Column chromatography of steroids is 

 discussed by Neher (59). Sterols can be converted to colored urethanes to aid visualiza- 

 tion of their separation on florisil columns. After separation, the original sterols are 

 quantitatively regenerated (60). Some carotenoids are too sensitive for chromatography 

 on even deactivated alumina and milder adsorbents such as magnesium oxide and sucrose 

 are recommended for them. Chromatographic purification of carotenoids is extensively 

 discussed by Strain (61). Other common adsorbents such as calcium carbonate and silica 

 gel have been used for some of the terpenoids, cardiac glycosides, etc. Tropolones are 

 isolated by procedures similar to those used for plant phenols, but in addition advantages 

 can be taken of their property of forming chelate complexes (62). 



