CHROMATOGRAPHY 1 47 



sugar column, the column can be "developed" as follows. More petroleum 

 ether is added to the tube, a process also accompHshcd by allowing the 

 liquid to flow slowly down the side of the tube from a pipette. The 

 upper layer of the sugar has been stained green by the mixture of pig- 

 ments, and the addition of petroleum ether causes a yellow band to move 

 downwards through the sugar. The green band is left behind at the top 

 of the column. More petroleum ether can be added to push the yellow 

 band farther downward. When the yellow band of carotenes is fairly 

 well separated from the green band, we add more petroleum ether con- 

 taining about 1 per cent acetone or ethyl ether. Another colored band 

 will move out of the upper colored layer. As we gradually increase the 

 amount of acetone or ethyl ether in each addition of the solvent mixture, 

 various colored bands will move downward through the sugar one after 

 another. One of these bands will be the bluish-green chlorophyll a, 

 followed by the green (or slightly yellowish-green) chlorophyll h. Yel- 

 low bands of carotenols (xanthophylls) will appear in various orders, 

 depending on the particular forms present in the mixture and on the 

 exact composition of the solvent mixture used for developing the column. 



The different pigments may be recovered in either of two ways. They 

 may be allowed to move downward until they drip out the bottom of 

 the tube. Each pigment can be caught in a separate container. In this 

 case we are "washing" the pigments off the sugar by the addition of 

 solvent, a process commonly know^n as elution. It is usually faster, how- 

 ever, to allow the pigments to become well separated from each other 

 on the sugar, and then push the plug of sugar from the tube. This plug 

 of sugar can be sliced into the different-colored bands. Each slice is placed 

 in a separate container, and acetone or ethyl ether is added to liberate the 

 pigment from the sugar. We now have several different solutions, each 

 containing a different pigment. 



Pretty chromatograms are possible only on evenly packed columns. 

 The technique of column packing apparently must be learned through 

 several trials. We have found, however, that columns prepared as de- 

 scribed percolate more rapidly and evenly than columns in which sugar 

 is first packed in the dry state and then saturated with petroleum ether. 

 When petroleum ether is used as the developing solvent, it is advisable 

 to work in the cold because at room temperature bubbles of solvent vapor 

 often disrupt the column. The gradual increase in the amount of acetone 

 or ethyl ether added to the developing solvent requires some judgment. 

 If it is increased too rapidly, several pigments may be pushed downward 

 together. If it is increased too slowly, a chromatogram may take all day 



