Carbon Assimilation. 
293 
means of water from its methyl alcohol, whereon the pigment in it 
is precipitated. 
The third and fourth and fifth alcohol extracts are similarly 
treated. The content of component b is now considerably reduced. 
The sixth methyl alcohol extract is treated with 900 c.c. methyl 
alcohol, and each successive extract with 100 c.c. less, so that to 
the 14th extract only 100 c.c. methyl alcohol is added. 
These extracts are cleaned in pairs with 1 litre of petrol ether, 
for the second of each pair a further ^-litre of petrol ether is added. 
All extracts thus cleaned are added to the same ether solution 
which is increased by continual additions of ether, beginning with 
1 litre, and decreasing in amount to about ^-litre with the 10th 
extract. 1 
A 15th and 16th extraction with methyl alcohol is made in 
order to free chlorophyll a from the last traces of chlorophyll b. 
The chlorophyll b solution is now freed from methyl alcohol by 
washing with water, it is dried with sodium sulphate and evaporated 
to 500 c.c., and then to 30 or 40 c.c. in vacuo. 
The chlorophyll b is then precipitated by the addition of 
300 c.c. petrol ether of B.P. 30° to 50"C. and filtered on talc. The 
filtrate contains much chlorophyll a. It is purified by solution in 
ether and precipitation with petrol ether, which is repeated several 
times. It is finally filtered and dried in a vacuum desiccator. 
(8) Purification of Chlorophyll a. The petrol ether solution, 
from which the last traces of chlorophyll b have been removed as 
indicated in the preceding section, is further purified by shaking it 
three times with 2 litres of 90% methyl alcohol. 
The methyl alcohol is removed and the petrol ether solution 
of chlorophyll a is washed with water until the chlorophyll is 
precipitated in quantity. Talc is added to the extent of from 30 to 
100 grams, and the whole filtered on a layer of talc on a Buchner 
funnel. The petrol ether should then run off colourless. 
The talc is washed with petrol ether of low B.P. and sucked 
dry with the pump till all petrol ether smell has disappeared. It 
is then transferred to a bottle and shaken with as little ether as 
possible. On filtration on a small Buchner funnel the beautiful 
deep blue ether solution of chlorophyll a runs through. The 
chlorophyll and talc are completely freed fropi one another by 
further filtration. 
1 The large quantities of ether are necessary because the watery methyl 
alcohol dissolves much ether and the petrol ether which separates out on dilution 
makes it difficult to carry the chlorophyll over from methyl alcohol to ether. 
