6 PHYSIOLOGY OF NUTRITION 



chlorophyll by Pelletier and Caventou 1 — must be studied. Chlorophyll can be 

 extracted from leaves by alcohol, but the solution thus obtained also contains 

 several other pigments, as well as colorless substances, for the removal of which 

 various methods have been devised. 2 The method of Fremy involves the 

 precipitation of the alcoholic extract with barium hydroxide; the green precipi- 

 tate is collected upon a filter and treated with alcohol until the yellow pigments, 

 xanthophyll and carotin, are completely removed. The remaining precipitate 

 is then decomposed by potassium hydroxide, according to the method of 

 Timiriazev. 3 The green solution thus obtained is treated with ether, and dilute 

 acetic acid is gradually added, with shaking, to neutralize the potassium 

 hydroxide. As long as the reaction is alkaline the ether remains without 

 color, but as soon as the hydroxide is neutralized the lower layer becomes yellow, 

 since all the green pigment passes into solution in the ether above. The color 

 of the ether solution is emerald green, more intense than that of the alcoholic 

 solution; it is cherry red, however, in reflected light, while the yellow solution 

 shows no fluorescence. Timiriazev was the first to succeed in separating chlo- 

 royhyll from yellow pigments, out of the alcoholic chlorophyll extract. This 

 chlorophyll is not the normal pigment, however, for it has been changed by the 

 treatment employed in separating it. 



The method of Kraus 4 is based upon the relative solubilities of the pig- 

 ments in alcohol and benzine. If benzine is gradually added, with shaking, 

 to the green alcoholic extract diluted with water so as to be about an 85 -per 

 cent, solution of alcohol, two sharply distinct layers are finally formed, an 

 upper, green layer (benzine) and a lower, golden-yellow one (alcohol and 

 water). By renewed shaking of the former solution, with further additions of 

 alcohol, the green pigment can be practically freed from the yellow coloring 

 matter. 



The green pigments 6 form an amorphous mass, readily soluble in alcohol, 

 ether and naphtha. The solution is intensely fluorescent, appearing cherry 

 red by reflected light and green by transmitted light. The chemistry of chloro- 

 phyll has been largely worked out by Willstatter and his co-workers. Two 

 closely related pigments are always associated to form chlorophyll, these having 

 been termed chlorophyll a and chlorophyll b. 



1 [Pelletier, [Joseph], and Caventou, [J. B.], Sur la matiere verte des feuilles. Ann. chim. et phys. 

 11,9: 194-196. 1818.] f 



2 Willstatter, Richard, Chlorophyll und seine wichtigsten Abbauprodukte. Abderhalden s Handb. 

 biochem. Arbeitsmeth. 2: 671-716. Berlin, 1910. Willstatter, Richard, and Hug, Ernst, Isolierung des 

 Chlorophylls. Liebig's Ann. Chem. u. Pharm. 380: 177-21 1. 1911. 



s Timiriazev, K. A., Spectral analysis of chlorophyll. [Russian.] St. Petersburg, 1871.* [Haas and 

 Hill give methods for obtaining chlorophyll, and present a good discussion. See: Haas, Paul, and Hill, 

 T. G., An introduction to the chemistry of plant products. London, 192 1.] 



■> Kraus, Gregor, Zur Kenntnis der Chlorophyllfarbstoff e und ihrer Verwandten. Stuttgart, 1872. 



b Some modifications have been made in this discussion of chlorophyll, so that it does not 

 agree entirely with Palladin's presentation. . An attempt has been made to bring it more into 

 accord with Willstatter and Stall's monograph. (Willstatter, Richard, and Stall, Arthur, 

 Untersuchungen liber Chlorophyll, Methoden und Ergebnisse. Berlin, 1913-) For English 

 resumes of this work, see: West, Clarence J., A review of Willstatter's researches on chloro- 

 phyll. Biochem. bull. 3: 229-258. 1914. Willstatter, R., Chlorophyll. Jour. Amer. Chem. 

 Soc. 38: 323-345. 1915— Ed. 



