CHLOROPHYLL AXD THE CHLOROPLASTS 367 



d. The Quantitative Estimation of Leaf Pigments. 



The early reahzation that the leaf pigments were an essential part of 

 the photosynthetic apparatus naturally led to efforts to determine whether 

 a quantitative relation exists between the chlorophyll-content of plants 

 and their photosynthetic activity. For such investigations quantitative 

 methods of determining the leaf pigments were necessary. Various 

 efforts were made to devise such methods; the principle of these was 

 usually the extraction of the pigment and the determination of the absorp- 

 tion thereby of light of a given intensity, thus enabling a comparison 

 to be made between two sets of leaves. The great advance in our 

 knowledge of the leaf pigments, contributed by the investigations of 

 Willstatter, made possible the development of methods for the estimation 

 of these substances in fresh plant material on a far more rational and 

 accurate basis than had hitherto been possible. Through the investiga- 

 tions of Willstatter it was made evident not only what the essentia! 

 leaf pigments are, but also how these can be extracted without under- 

 going alteration and how they can be separated from one another. Will- 

 statter's investigations thus constitute an exceedingly valuable contribution 

 to our knowledge of photosynthesis. At the same time, it should be 

 mentioned that the methods for estimating the quantities of leaf pigments, 

 devised by Willstatter and Stoll are by no means simple. The pigments 

 themselves are exceedingly labile, many operations are involved, numerous 

 special precautions must be observed, so that considerable experience is 

 necessary in order to obtain results which, at best, are not of the highest 

 degree of accuracy. 



\\'illstatter and StolP^ have given a detailed description of their 

 method of determining the leaf pigments. A rehearsal in moie general 

 terms of their statements would be of very little value. Before endeavor- 

 ing to use this method the original publications should be consulted and 

 a thorough knowledge of the chemical reactions involved in each step 

 must be gained. 



The principle of the method is given here very briefly. The pigments 

 are extracted from the fresh, macerated leaves with acetone in the cold 

 after a preliminary treatment with 40 and 35 per cent acetone. The 

 pigments are then transferred to ether and the acetone is removed com- 

 pletely by washing with water. One portion of the pure ether solution 

 is used for the determination of the two chlorophyll components, another 

 portion for the estimation of carotin and xanthophyll. The two chloro- 

 phyll components are determined by converting them into decomposition 

 products through hydrolysis with acid and alkali. Chlorophyll a thus 

 yields phytochlorin e and chlorophyll h, phytorhodin g : 



"WillstaUer and Stoll, "Untersuchungen iiber die Assimilation der Kohlensaure," 

 Berlin, 1918, p. 11. 



