6 PHYSIOLOGY OF NUTRITION 



chlorophyll by Pelletier and Caventoui— 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 devjsed.^ The method of Fremy invdves the 

 precipitation of the alcohohc 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. ^ 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 alcohohc 

 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- 

 rophyll 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^ 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 8s-per 

 cent, solution of alcohol, two sharply distinct layers aire 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'" 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.l, Sur la matidre verte des feuilles. Ann. chim. et phys. 

 //, 9: 194-196. 1818.I 



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 : 177-211. 1911. 



"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, 1913.I 



' Kraus, Gregor, Zur Kenntnis der Chlorophyllfarbstoffe und ihrer Verwandten. Stuttgart, 1872. 



' 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 StoU's monograph. (Willstatter, liichard, and StoU, Arthur, 

 Untersuchungen uber Chlorophyll, IVIethoden und Ergebnisse. Berlin, 1913,) For English 

 r6sum6s of this work, see: West, Clarence J., A review of Willstatter's researches on chloro- 

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

 800.38:323-345. 1915.— £(/. 



