654 VI. CAROTENOIDS AND RELATED COMPOUNDS 



stances change in exactly the same way when one varies the adsorbing 

 phase and the solvent. 



A second application of the method is for establishing the identity or 

 non-identity of two substances. When a solution of the unknown is 

 mixed with a known substance, the formation of a single zone on subse- 

 quent chromatography indicates that the two products are the same. 

 Such a mixed chromatogram is of very great value in supplementing the 

 mixed melting point technic for establishing identity. In cases in which a 

 solution contains two components, the identity of one of the zones with a 

 knowTi substance can be verified by the addition of a large amount of the 

 known compound to the solution, followed by rechromatography. By 

 determining which of the zones is widened, one can establish which layer 

 corresponds to the known substance. 



Another important application of the chromatographic method is for 

 the concentration of a product occurring in a high degree of dilution. 

 Several hundred liters of such a solution can be put through a relatively 

 small column, and the filtrate can then be rejected. By a single procedure, 

 the desired substance is quantitatively removed without resorting to 

 evaporation, Avhich is time-consuming, and which may alter the structure 

 of labile substances. 



The chief importance of chromatography is for the separation of a mix- 

 ture into its components. Each fraction may then be identified, and in 

 most cases quantitatively estimated. At the scale of operations usually 

 carried out in the laboratory, sufficient material of a homogeneous nature 

 can be prepared from each zone of separation for microanalyses. The 

 quantity of the product obtained can be increased by the use of larger 

 batches of adsorbent. The procedures can be carried out practically 

 without loss in the separation or concentration of materials. This renders 

 such a method even more satisfactory. 



Still another application of this technic is in the purification of products. 

 If the substance desired is adsorbable on the chromatographic column, it 

 can be eluted from that portion of the column; if a clean-cut separation 

 cannot be made in a single operation, the eluate can be rechromatographed, 

 and this procedure repeated several times. On the other hand, when the 

 desired product is not adsorbable, it can frequently be freed from some or 

 all of the contaminants, if the latter are adsorbable on the column. 



b. Theoretical Basis for Chromatography. Tswett recognized the 

 theoretical principles involved in chromatography. The positions occu- 

 pied by a series of adsorbable components were explained as being related 

 to the adsorption affinities of the several substances in a common solvent. 

 The pigments tend to form layers in the descending order of their affinities, 

 i.e., those with the highest affuiity are at the top of the column. Colorless 

 materials follow the same laws of adsorption as do the colored products. 



