PROPERTIES OF THE CAROTENOIDS 655 



It is generall}^ agreed that the capacity of a given amount of adsorbent is 

 Hmited. If all the surfaces of the particles in a thin layer are surrounded 

 by superficial active molecules, they will immediately adsorb them and 

 become exhausted, so that no additional material can be taken up on that 

 portion of the column. However, if the adsorbable molecules are in excess, 

 only that portion of the pigment with the strongest affinity will be retained 

 and the pigments with a lower adsorption coefficient will pass further down 

 the column. When these latter molecules are no longer subject to compe- 

 tition on the part of the molecules of higher adsorption affinity, they be- 

 come firmly adsorbed, forming a second zone of concentration. This 

 phenomenon continues until all the adsorbable material is removed. 

 Those molecules with the lowest adsorbability will appear furthest down on 

 the column. 



It is not believed that, under the actual conditions of the experiment, all 

 the particles of the adsorbent immediately become exhausted by the mole- 

 cules of highest affinity. Some molecules of lower adsorbability will also 

 be retained. However, when new quantities of the solution are allowed to 

 percolate through the column, the molecules of highest activity will dis- 

 place those of lower activity from the uppermost adsorption band. The 

 latter molecules will then pass further down the column. This same 

 separation also occurs when the column is washed with large amounts of 

 the solvent ; this brings about the development of the chromatogram, with 

 the clean-cut separation of the various pigments into separate layers. 



The experimental evidence for this hypothesis has been beautifully 

 demonstrated by Zechmeister and v. Cholnoky.^^^ When zeaxanthin in a 

 1:4 benzene-petroleum ether solution is passed through a calcium chloride 

 column, it forms a broad yellow band at the top of the column. If, after 

 the development of this chromatogram, a capsanthin solution in a similar 

 solvent is introduced into the tube, the red ring of the latter carotenoid is 

 immediately adsorbed in the portion of the column occupied by the zea- 

 xanthin; the latter pigment, which has a lower adsorption affinity than 

 capsanthin, is displaced and forms a new yellow ring further do^vIl the 

 colunm. When the reverse test is carried out, that is, by pouring a zea- 

 xanthin solution into a column in which capsanthin has been chromato- 

 graphed, no displacement occurs — the zeaxanthin passes through the upper 

 red layer occupied by the capsanthin and forms a yellow band some dis- 

 tance below on the chromatographic column. 



c. Practical Considerations in Chromatography. The general proce- 

 dure for chromatography involves the passage of a dilute solution of the 

 substance to be chromatographed in a suitable solvent through a vertical 

 column of the adsorbent of sufficient length (at least 10 cm.) to allow dif- 

 ferences in adsorbability to be manifest. Carbon disulfide (b.p., 46°C.), 

 petroleum ether, and benzene (b.p., 80°C.) are the solvents most frequently 



