Carbon Assimilation. 
87 
pigment-content of various extracts and preparations. Thus, if for 
instance, one should want to compare the amounts of chlorophyll 
(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 -f 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. 
