342 PHOTOSYNTHESIS 



thereby the color becomes brown, and in a few minutes the original chloro- 

 phyll color appears in the alkaline medivim. During this operation the 

 ether should remain colorless : if carotin or xanthophyll are present, these 

 pass into the ether on the addition of water. A quantitative determina- 

 tion of the phytol-content of the preparation can also be used as a test 

 of its purity. 



Willstatter and Stoll have concluded that the chlorophyll from differ- 

 ent plants does not differ in composition. This conclusion is based upon 

 the analyses of chlorophyll from a large number of different species, 

 including marine algae, fresh water plants and land plants from the 

 temperate zone and the tropics, representing many different families. 

 They concluded that most plants contain four pigments, viz., two yellow 

 pigments, carotin and xanthophyll and two green pigments, chlorophyll a 

 chlorophyll b. Thus far in this chapter the two latter pigments have 

 been treated together as "chlorophyll." That the latter is actually com- 

 posed of two pigments was already considered probable by Stokes * and, 

 as has already been mentioned, by Tswett who by means of his chromato- 

 grams was able to demonstrate the existence of two chlorophyll pigments. 

 These he designated as a- and (3-chlorophyllin which Willstatter and 

 Stoll called chlorophyll a and chlorophyll b. Marchlewski ^ used the terms 

 neo- and allochlorophyll for the two components. 



The separation of chlorophyll a and chlorophyll & is a rather tedious 

 process ; Willstatter and Stoll accomplished this by taking advantage of 

 the fact that cholorophyll a is more soluble in petroleum ether while 

 chlorophyll b is more soluble in methyl alcohol. Eight grams of chlorophyll 

 are dissolved in 150-200 cc. of ether, and the dark solution is filtered 

 into 4000 cc. of petroleum ether (0.64-0.66). In order to prevent the 

 precipitation of the chlorophyll 50-100 cc. of methyl alcohol are added. 

 The ether is removed by shaking with 2000 cc. of 80 per cent methyl 

 alcohol. 



The petroleum ether solution is now extracted with methyl alcohol in 

 order to remove the chlorophyll b component. For this purpose 85 per 

 cent methyl alcohol is saturated with petroleum ether and 0.01 gram 

 oxalic acid is added to each liter. About 14 extractions with 2000 cc. 

 each of 85 per cent methyl alcohol are required to extract the chloro- 

 phyll b; the chlorophyll a remains in the petroleum ether. The first 

 methyl alcohol extract, after separation, is brought to a concentration of 

 90 per cent by the addition of 1000 cc. of methyl alcohol, washed with 

 1000 cc. of petroleum ether, added to 2000 cc. of ether and then con- 

 siderable water is added to this. 



To the second methyl alcohol extraction a liter of methyl alcohol is 

 also added, washed with the petroleum ether used for washing in the first 

 extraction to which another 500 cc. has been added. This methyl alco- 



* Stokes, Proc. Roy. Soc, 13, 144 (1864). Sorby, ibid.. 21, 442 (1873). 

 'Jacobson and Marchlewski, Biochcm. Zcit., 39, 174 (1912); 40, 296 (1912). 



