40 Carbon Assimilation. 



instance, one should want to compare the amounts of chloropliyll 

 (a + b) in two different crude chlorophyll extracts, the simplest 

 thing to do is to separate the green from the yellow pigments by 

 saponification with alkali, and then to compare colorimetrically the 

 two chlorophyllin solutions so obtained. Further, by the saponifica- 

 tion of a solution of known chlorophyll content, which can be used 

 as a standard, it is possible to obtain an approximate value for the 

 chlorophyll content of the two extracts. 



if, however, one requires to find the ratio of the two chloro- 

 phyll components, their derivatives phytochlorin e and phytorhodin 

 g must be separated from one another and separate comparisons 

 made. 



i. Methods. 



We shall describe first a simple method for the quantitative 

 estimation of chlorophyll in a chlorophyll extract, and then the 

 more exact method used for the estimation of the four pigments in 

 fresh leaves. 



(a) Quantitative Estimation of Chlorophyll (a -\- b) in a 

 Chlorophyll Extract. 



Ten c.c. of an acetone, or alcohol, extract is diluted to 100 c.c. 

 with ether and 10 c.c. of this is poured into a separating funnel and 

 diluted with a further 40 c.c. of ether. 



An ether-diluted alcohol extract can be shaken at once with 4 

 to 5 c.c. methyl alcoholic solution of potash and the brown phase 

 will appear. If the solution contains acetone, however, this must 

 be completely removed from the solution before saponification, as 

 chlorophyllins are destroyed by acetone. In this case a little 

 methyl alcohol is added and the solution washed thoroughly with 

 water. 



After reappearance of the green colour, water is slowly added 

 during gentle rotation of the separating funnel and the aqueous 

 chlorophyllin solution is run into a 200 c.c. measuring flask. The 

 ether solution of the yellow pigment is washed with a little more 

 water to effect complete extraction of the chlorophyllins, and the 

 watery layer added to that in the flask. The whole is made up 

 with alcohol to 200 c.c. 



The chlorophyllin solution is then estimated in a colorimeter 

 by comparison with the standard solution. 



