EXTRACTION OF CHLOROPHYLL 227 



To illustrate this Tswett invented the chromatogram : a 

 solution of chlorophyll in petroleum ether on filtering through 

 a column of calcium carbonate parts with its pigments at 

 different rates, which thus occur in zones in the column, the 

 more strongly adsorbed ones occurring successively at higher 

 levels. 



EXTRACTION OF CHLOROPHYLL. 



The usual method of extracting chlorophyll from green 

 tissues consists in first steeping the fresh material in hot water 

 and then extracting the colouring matter by means of warm 

 alcohol. Willstatter, however, recommends the use of dried in 

 place of fresh material, and extracting by shaking with organic 

 solvents (ethyl or methyl alcohol, ether or acetone) in the 

 cold ; the liquid is then filtered off over a pump and the 

 residue washed with more solvent until the volume of the 

 filtrate is equal to the original volume of solvent employed. 

 In working with larger quantities the chlorophyll may be 

 extracted by percolation. 



The chief advantage in using dried material lies in the fact 

 of its relatively small bulk; 100 grams of stinging nettle 

 leaves, for example, weighing only 25 grams after drying. It 

 has been shown, moreover, that the operation of drying pro- 

 duces no change of any importance in the chlorophyll, since 

 the results obtained from dried material have been repeated 

 and confirmed on fresh material. 



Associated with chlorophyll are two yellow pigments. 

 Carotin and Xanthophyll, which together with some lecithin, 

 waxes, etc., always occur in alcoholic extracts of green leaves. 



The purification of the chlorophyll contained in such 

 alcoholic extracts is a matter of some difficulty since the 

 solubilities of the various substances are very similar. 



The method recommended by Willstatter * is a modification 

 of Kraus's process. Two litres of methyl alcohol extract from 

 I kg. of leaf meal are mixed with an equal volume of low- 

 boiling petroleum and 400 c.c. of water are added. The 

 greenish-yellow alcoholic layer is run off from below by means 

 of a separating funnel, and the petroleum is shaken up again 

 successively with solutions of 1700 c.c. methyl alcohol in 300 



* Willstatter : " Annalen," 1906, 350, 48. 

 15 * 



