Carbon Assimilation. 
291 
is then removed by adding 4 successive ^-litres of water, with 
gentle rotation of the liquid and running off the lower layer each 
time. (The first time 06 litre of acetone is removed and in 
successive removals 05, 04 and 0-2 litre). In the acetone thus 
removed are many of the impurities accompanying chlorophyll in 
the crude extract. 
From the solution remaining, the xanthophyll is first separated 
by shaking the solution with 3 successive additions of 2 litres of 
80% methyl alcohol. After each addition and shaking, the methyl 
alcoholic layer is removed, and if the last extract is still considerably 
yellow, one or two further additions of methyl alcohol are made. 
From these methyl alcohol extracts xanthophyll is prepared. 
From the petrol ether solution, which should now have a 
volume of 3-6 litres, the last traces of acetone and methyl alcohol 
are removed by washing with water four times, each time using 
2 litres of water. With the disappearance of the last parts of the 
acetone and methyl alcohol, the chlorophyll is precipitated as a 
suspension from the petrol ether, which thus loses its fluorescence. 
This suspension in petrol ether is shaken with some fused 
sodium sulphate and about 150 gms. of talc, and then filtered 
through a layer of talc on a Buchner funnel. From the filtrate 
carotin can be isolated as described later. 
The talc and chlorophyll on the Buchner funnel are washed 
with ordinary petrol ether until this runs off yellow in colour, and 
then the washing is completed with 300 c.c. petrol ether of B.P. 30° 
to 50 n C. The talc is then sucked completely dry with the pump, 
and the chlorophyll in it dissolved in pure ether. The ether 
solution of chlorophyll so obtained is filtered through fused sodium 
sulphate, concentrated to 100 c.c., filtered twice more, and evaporated 
to 25 c.c. 
From this solution the chlorophyll is precipitated by the slow 
addition of 800 c.c. of low B.P. petrol ether. The precipitate so 
obtained may be a blue black powder easily filtered, or it may be so 
fine that it has to be filtered on talc. 
The precipitate is again dissolved in ether, and the solution 
concentrated to 20 c.c. and dried in a dish in a desiccator. 
The pure chlorophyll so obtained consists of about 13 grams 
(i.e., 6’5 grams per kilo of dried leaves of mixed chlorophyll a and 
chlorophyll b) forming a thin shining steel blue crust. The yield is 
about 75% of the total chlorophyll content of the leaves. 
