42 Carbon Assimilation. 



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. 5c.c. water are added through 

 the condenser and the boilinj* 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 

 or3 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, 

 withl to 2 c.c. of 3% hydrochloric acid added, as pure water would 

 remove phytochlorin. 



