Carbon Assimilation. 
89 
done so rapidly that the boiling of the pyridine solution is not 
interrupted. The brown phase appears and gives place rapidly to 
olive-green colour. 
A vertical condenser is now fitted to the flask which is heated 
for two minutes on the steam bath. 5 c.c. water are added through 
the condenser and the boiling continued for another 1 to 1^ 
minutes. 
The flask is now cooled under the tap and its contents 
transferred to a 500 c.c. separating funnel with water and some 
ether. The liquid is then acidified with 20% hydrochloric acid, upon 
which the colour changes to a dull grey-green. 200 c.c. of ether are 
added and the funnel shaken strongly for some minutes. A little 
ammonia is added to the dull coloured, watery layer which is shaken 
up with small quantities of ether until the last is no longer 
coloured. 
The mother liquor is made alkaline with ammonia on account 
of the flocks which separate out, and then acidified again in order 
that the ether may remove only a small quantity of the derivatives. 
If the flocks are again washed with ammonia and little pigment 
thereby goes into solution, no phytorhodin has been destroyed in 
the saponification ; otherwise this has taken too long a time. 
Before fractionation of the two chlorophyll derivatives the 
accompanying substances must be removed. 
To achieve this the combined ether solutions are extracted 2 
or 3 times with 30 c.c. of 12% hydrochloric acid and then with 10 to 15 
c.c. of 20% acid till the acid layer finally separates in a colourless 
state. Further flocks form at the boundary layer, but they 
usually contain no green pigment. Besides the carotin, the ether 
also contains brown coloured substances. 
The combined acid extracts are transferred to a 500 c.c. 
separating funnel with 200 c.c. of ether. They are neutralised with 
concentrated ammonia during gentle rotation until the watery 
layer is dull blue-violet in colour. The well-stoppered separating 
funnel is cooled under the tap and at the same time shaken first 
gently and then strongly. The watery layer is then usually pale 
blue and is run into a second separating funnel. By this method 
of neutralisation the last trace of the derivatives is brought into the 
ether. 
The ethereal solution of derivatives is freed from methyl 
alcohol and pyridine by washing with 200 c.c. of water three times, 
with 1 to 2 c.c. of 3% hydrochloric acid added, as pure water would 
remove phytochlorin. 
