CHLOROPHYLL AND THE CHLOROPLASTS 339 



a. The Extraction and Separation of the Leaf Pigments. 



A method which has aided considerably in the separation of the dif- 

 ferent leaf pigments and has been of much value in demonstrating the 

 presence of the different pigments is that devised by Tswett.^ The method 

 is based upon the difference in adsorption affinities of the various pigments 

 by dry, powdered calcium carbonate or other adsorbents. A dry benzol, 

 I>etroleum ether or carbon disulfide extract of the leaf pigments is filtered 

 through a column of powdered calcium carbonate. The latter thus becomes 

 dififerentiated into zones of dififerent color, the various pigments being ad- 

 sorbed in dififerent portions of the column. There is thus formed a strati- 

 fied column called a chromatogram, each zone of which contains a dififerent 

 pigment. The various zones can be sharply separated and the pigments 

 contained therein can be extracted by means of different solvents. 



Tswett prepared the solutions of the leaf pigments in the following 

 manner. The leaves were ground with emery and a small quantity of 

 magnesitmi oxide or calcium carbonate and extracted with alcoholic petro- 

 leum ether (1 : 10). The alcohol was removed by washing and the solution 

 was dried. Less successful were the methods of extracting with petroleum 

 ether leaves which had previously been treated with boiling water or of 

 extracting the neutralized leaf material with alcohols, acetone, ether or 

 chloroform, distilling off these solvents and redissolving the pigment in 

 petroleum ether or carbon bisulfide. Tswett's chromatograms had the 

 following adsorption-zones: L (from top) colorless; IL yellow, contain- 

 ing xanthophyll (3; IIL dark olive green designated by Tswett as chloro- 

 phyllin |3; IV. dark blue green, designated as chlorophyllin a; V. yellow, 

 containing two xanthophyll pigments; VL colorless; VIL orange yel- 

 low, xanthophyll a. The yellow carotin was not adsorbed by the calcium 

 carbonate. In this manner Tswett attained a separation of the leaf pig- 

 ments and by extracting the various zones he was able to differentiate the 

 pigments spectroscopically. Sufficiently large quantities for exhaustive 

 chemical analysis could, however, not be obtained by this method. It re- 

 mained for Willstatter and his co-workers to devise methods for the isola- 

 tion of the different leaf pigments. 



Although the pigments in almost all leaves are the same, they cannot 

 be obtained from different species with equal ease and with the same de- 

 gree of purity. For obtaining preparations of the dififerent pigments it 

 is very essential that proper consideration be given the choice of the plant 

 material. Leaves which have relatively little chlorophyllase, an enzyme 

 which partially breaks down chlorophyll, are to be preferred. The nettle 

 has proved to be very satisfactory material for extraction and was used 

 extensively by Willstatter and some earlier workers. 



Most of the early workers extracted the leaf pigments from fresh 

 leaves usually by means of boiling alcohol. Willstatter recommends pre- 

 viously drying the leaves. This procedure has several advantages ; a much 



'Tswett, Ber. bot. Gcs., 24, 316, 384 (1906). 



