652 VI. CAROTENOIDS AND RELATED COMPOUNDS 



{5) Adsorption Phenomena (Chromatography) 



The carotenoids show marked variations in their abihty to be adsorbed 

 on a chromatographic cohimn. This variation is exhibited not only by 

 carotenoids having gross differences in composition, but even by the stereo- 

 isomers in which the change in structure is confined to the replacement of a 

 single trans bond by a cis bond. This procedure thus presents an excellent 

 and simple means, not only for the differentiation of the components of a 

 stereoisomeric mixture, but also for the separation and preparation of pure 

 carotenoids. No other chemical or physical procedures offer as much 

 opportunity for the purification of the carotenoids as does chromatography. 

 It is safe to say that little would be known about stereoisomerism of these 

 polyenes were it not for this especially useful method. 



The use of the chromatographic column in the separation of plant pig- 

 ments is generally attributed to the botanist Tswett,^"^"^^^ whose first 

 report was published in 1906. However, Weil and Williams^^^" have re- 

 cently called attention to the work of Day^^^* who, in 1897, employed a 

 column of powdered limestone for the fractionation of crude petroleum. 

 Furthermore. Engler and Albrecht^^^'' reported the use of a liquid chromato- 

 gram as early as 1901. However, it must be admitted that the practical 

 application of the method can be credited to Tswett. When petroleum 

 ether, benzene, or carbon disulfide extracts were poured through columns 

 of calcium carbonate, inulin, or sucrose, the most strongly adsorbed 

 pigments were held fast at the top of^the column, while the weakly ad- 

 sorbed components were carried for varying distances do\vn the column. 

 When the column was washed with pure solvent, definite zones appeared 

 where colored pigments were concentrated; these were separated from 

 each other by interspersed white zones. This record of a mixture is spoken 

 of as its chromatogram. Tswett prepared the components chromatographed 

 individually in this manner by cutting out each zone from the column 

 which had been pressed out of the glass tube, and by elution of the adsorbed 

 pigment with an appropriate solvent. 



Little use was made of the Tswett column in the study of carotenoids for 

 the next 25 years, until its application by the Karrer and Kuhn schools. 

 In 1931 Kuhn and Lederer,"*^ as well as Kuhn, Winterstein, and Lederer,^^^ 

 successfully introduced chromatography into the preparation of carote- 

 noids. By the application of this method, these workers were able to demon- 

 strate that a carotene preparation which was believed for 100 years to be a 



5»9 M. Tswett, Ber. deut. botan. Ges., 24, 316-323 (1906). 

 "0 M. Tswett, Ber. deut. botan. Ges., 24, 384-393 (1906). 



611 M. Tswett, Die Chromophylle in der Pflanzen- and Tierwelt (in Russian), Warsaw 

 1910. Cited by L. Zechmeister, Die Carotinoide, Springer, Berlin, 1934, p. 94 ff. 

 5ua H. Weil and T. I. Williams, Nature, 166, 1000-1001 (1950). 

 5116 D. T. Day, Proc. Amer. Phil. Soc., 36, 112-115 (1897). 

 611-= C. Engler and E. Albrecht, Z. angew. Chetn., 1901, 889-893. 



