24 ISOLATION III 



closely related pigments on the chromatographic column. A classical example 

 of the efficiency of the method is the separation of y-carotene from the a- and 

 jS-components of crude carotene, although the proportion of y-carotene only 

 corresponds to about one part in a thousand. Even differences in steric con- 

 figuration of the pigments are sufficient for a quantitative separation, as was 

 first shown by the investigations of Winterstein and Stein^ on cis- and trans- 

 crocetin methyl esters. 



The strengths of adsorption of different carotenoids on a given adsorbent 

 bear a definite relationship to their chemical structures. Hydroxyl substituents 

 exert the largest influence in this respect. (Carotenoids containing carboxyl 

 groups are not considered here). Of two carotenoids otherwise possessing the 

 same structure, that containing a larger number of 



a) hydroxyl groups, 



b) carbonyl groups, 



c) esterified hydroxyl groups, or 



d) double bonds, 



is adsorbed more strongly. 



The effectiveness of functional groups on the strength of adsorption de- 

 creases in the sequence : hydroxyl group, carbonyl group, esterified hydroxyl 

 group, double bond. 



The following table provides a summary of the positions of some caro- 

 tenoids (those whose functional groups are known) on the chromatogram. At 

 the top of the table are the pigments which are most strongly adsorbed, at 

 the bottom those which are least strongly adsorbed. 



In practice, adsorption analysis is carried out by percolating the solution of the 

 carotenoids through a long column of suitable adsorbent. The amount of adsor- 

 bent used depends on the amount of pigments to be separated. The individual 

 zones are then "developed" by further washing with the same (or a different) 

 solvent. The solution of carotenoids is poured into a tube which is filled to 

 the extent of about ^/g with the adsorbent and connected to an evacuated 

 bottle. As soon as the solution has been almost completely adsorbed by the 

 column, the latter is washed through with an organic solvent (usually the same 

 as that employed to prepare the solutions) until an optimum separation of the 

 zones has been achieved. It is of great importance that the chromatogram 

 should never be allowed to dry, as this results in the destruction of the polyene 

 pigments by aerial oxidation, and in a shrinking of the upper part of the 

 column and a distortion of the zones. As soon as the individual pigments have 

 been separated as shown by the formation of colourless zones between the in- 

 dividual coloured layers, the "development" is stopped. The adsorption 

 column is extruded from the tube and the individual zones are mechanically 

 divided. They are immediately placed in prepared vessels already filled with 

 References p. 28. 



