II. CHEMISTRY AND INDUSTRIAL PREPARATION 5 



are capable of existing in more than one stereochemical form. Changes of 

 this type bring about changes not only in adsorption affinity and absorp- 

 tion spectra but also in biological activity. Table I shows some of the well- 

 known naturally occurring pro\itamin A carotenoids together with their 

 important sources, physical properties, and relative physiological activity. 



2. Isolation of Provitamin A Carotenoids 

 a. General Procedure 



Well-dried and well-ground materials of either plant or animal origin 

 are extracted in large vessels, percolators, or Soxhlet apparatus by any of 

 the well-known solvents such as low-boiling saturated hydrocarbons, ben- 

 zene, ether (peroxide-free), chloroform, trichloroethylene, ethanol, or ace- 

 tone. Extraction is carried out at room temperature in an atmosphere of 

 carbon dioxide or nitrogen. The extracts are then concentrated under a 

 reduced pressure and the concentrates saponified at 60 to 70° with a 5 to 

 10% methanolic potassium or sodiimi hydroxide. Water is then added, 

 and the non-saponifiable portion extracted thoroughly with low-boiling 

 petroleum ether. The petroleum ether extract is then partitioned with an 

 equal volume of methanol. Carotenoids which go into the methanolic layer 

 are known as hypophasic and usually contain groups such as hydroxyl, 

 carbonyl, or oxirane, and those which remain in the hydrocarbon layer are 

 known as epiphasic and are usually hydrocarbons such as a-, ^-, 7-, or 5- 

 carotenes. 



The epiphasic carotenoids have somewhat small differences in molecular 

 structure and are therefore difficult to purify by the well-known classical 

 methods. They show, however, strong selective adsorption on various solid 

 adsorbents such as alumina, AI2O3, calcium hydroxide, Ca(0H)2, calcium 

 carbonate, CaCOs, and zinc carbonate, ZnCOs, and can therefore be sepa- 

 rated in the pure state by the chromatographic adsorption method first 

 introduced by Tswett.^ (See also general references on chromatography.^ ■ ^) 



8 M. Tswett, Ber. deut. botan. Ges. 24, 316, 384 (1906); Chromophylls in Plant and 



Animal World. Warsaw, 1910. 

 ' General references on chromatography: 



(a) L. Zechmeister and L. Cholnoky, Chromatographic Adsorption (trans, from 

 the 2nd ed. by A. L. Bacharach and F. A. Robinson). John Wiley and Sons, New- 

 York, 1941. 



(b) H. H. Strain, Chromatographic Adsorption Analysis. Interscience Publishers, 

 New York, 1942. 



(c) T. I. Williams, Chromatographic Analjsis. Blackie and Son, London, 1947. 



(d) E. Lederer, Chromatographic Analysis. Hermann, Paris, 1949. 



(e) L. Zechmeister, Progress in Chromatography 1938-1947. Chapman and Hall, 

 London, 1950. 



(f) H. G. Cassidy, Chromatographic Analysis. Interscience Publishers, New York, 

 1951. 



