144 CHROMATOGRAPHY 



but a discussion of these principles is better delayed until after a de- 

 scription of the method. 



The technique of chromatographic separation seems to be an art, and 

 a certain amount of "feel" is developed after some practice. This feature 

 is to be kept in mind in going through the following discussions. 



Separation of plant pigments 



Chloroplasts of higher plants contain two chlorophylls, a and h, as 

 well as two types of yellow pigments, the carotenes and the carot- 

 enols (also called xanthophylls). The chemical properties of all these 

 pigments are similar, and they have similar solubilities in organic solvents 

 such as acetone, petroleum ether, and ethyl ether. Classical chemical 

 methods for separating these pigments are difficult, but chromatography 

 is relatively easy. 



Preparatory Steps: A sample of leaves (2 to 3 g) of a higher plant such 

 as spinach is ground up with a mortar and pestle with a small quantity 

 (a pinch) of CaCOa (to neutralize the acids of the leaf cells) and about 

 40 to 50 ml of acetone. The dark green acetone solution is filtered 

 through coarse filter paper or cloth to remove cellular debris. More acetone 

 may be added to the filter to extract more of the pigments. A separatory 

 funnel is set up and about 10 to 25 ml of petroleum ether (a mixture of 

 hydrocarbons, mostly pentanes and hexanes) is placed in the bottom. 

 About 10 to 15 ml of the acetone solution is added and mixed by gentle 

 swirling. If the mixture separates into an upper green layer and a lower, 

 faintly yellow layer, the lower layer is drawn off and discarded. If there 

 is no such separation, water is added a few drops at a time with gentle 

 swirling after each addition. When enough water has been added, the 

 separation into a petroleum ether layer and an acetone-water layer will 

 occur. (Petroleum ether and acetone are soluble in each other; acetone 

 and water are soluble in each other; petroleum ether and water are 

 almost completely insoluble in each other.) After the lower yellowish 

 layer, containing chiefly carotenols, is drawn off, more green acetone solu- 

 tion can be added, and the same steps can be repeated. 



After four or five additions of acetone solution, it is well to add water 

 to the petroleum ether solution (by now dark green) to remove excess 

 acetone. Care must be taken at this point not to agitate the water- 

 petroleum ether mixture, or an undesirable emulsion may form. Addition 



