PROPERTIES AND METHOD* or I Dl-XTIl'K '.\Tlo\ 227 



The small end is left with only a small opening, Mo 2 mm. in diame- 

 ter. A plug of cotton is placed in the small end, pn>.-rd dmvn tightly 

 and the tube is then filled with the dry CaCO ;t , which is poured in 

 a little at a time and packed in as tightly and evenly as possible 

 with a glass rod or wooden stick. The success of the chromatograph 

 depends upon the evenness with which the adsorbent is packed into 

 the tube. The tube is filled within 2 or 3 cm. of the top, and a final 

 plug of cotton placed upon it. The tube is now set up through a 

 rubber stopper fitted into a small filter flask, gentle suction applied 

 and a stream of pure solvent (either petroleum ether or carbon disul- 

 fide, depending on the solvent selected for the pigment solution) 

 passed through the column until the adsorbent is moistened with 

 it. The suction is stopped and the upper cotton plug removed. Suf- 

 ficient pigment solution is now poured into the tube to color about 

 1 cm. of the adsorbent. When this has passed into the column with 

 the aid of gentle suction, the tube is filled with solvent and suction 

 continued. The upper part of the tube is kept filled with pure solvent 

 in order to establish a stream of the solvent through the adsorbing 

 column. The layer of pigment will now pass through slowly and will 

 differentiate itself into zones of relative adsorption, that of greatest- 

 adsorption affinity being at the top, and that of the least at the bottom. 

 Inasmuch as all the chlorophylls and carotinoids form dissociable com- 

 pounds with CaC0 3 the stream of pure solvent will slowly wash them 

 through the column as differently colored zones. If the column has 

 been packed perfectly evenly with the adsorbent the zones will be 

 true rings, otherwise they will be irregular. Perfectly true adsorp- 

 tion rings are difficult to secure. Pigments obtained in the various 

 zones by this method are not pure, as Tswett has pointed out, but 

 can be purified by repeating the analysis on the pigment obtained 

 in any desired zone. 



A chromatographic analysis applied in the above manner to a pe- 

 troleum ether or carbon disulfide solution of carotin and the four 

 xanthophylls recognized by Tswett should show the following result. 

 Assuming that carbon disulfide has been used and the differentiation 

 has been continued with a stream of solvent until the least adsorbed 

 pigment has reached the bottom of the column the lowest zone will be 

 rose colored due to carotin; above this, probably separated by a 

 more or less colorless region, will be a wide orange-yellow zone, due to 

 xanthophyll a, which apparently comprises the major part of the 

 xanthophylls; still higher in the column and separated from the 



