IV] CARBON ASSIMILATION 29 



being poured gently down the side of the funnel in order to avoid the formation of 

 emulsions. In the course of a few minutes, tlie ether layer separates out and now 

 contains the pigments. The lower layer, which is slightly green, is run oflf. The 

 addition of distilled water and subsequent removal of the lower layer is repeated 

 about four times, in order completely to remove the acetone from the ether solution. 

 If the ether solution should have become at all emulsified, it can be cleared by 

 shaking with anhydrous sodium sulphate and filtering. 



The whole process should be rei)eated with another 2 gms. of leaf-powder and the 

 pigment transferred to ether, since a sohition in this solvent is recpiired for later 

 experiments. 



Expt. 19. Demonstration of the presence of chlorophylls a and h. Of the petrol 

 ether solution from the last experiment, 10 c.c. are shaken with 10 c.c. of 92 % methyl 

 alcohol. Two layers are formed of which the petrol ether layer contains chlorophyll 

 a, and the methyl alcohol layer chlorophyll b. The solution of chlorophyll a is blue- 

 green, while that of chlorophyll b is a purer green, but the colour difference between 

 them is diminished owing to the presence of the yellow pigments, of which carotin 

 is in the petrol ether, and xanthophyll in the methyl alcohol. 



As will be explained later, the green pigments of chlorophyll can be 

 saponified by alkalis and are then insoluble in ethereal solution. This 

 method can be adopted to separate the green from the yellow pigments, 

 xanthophyll and carotin. 



Expt. 20. Separation of green and yellow pigments. Shake 5 c.c. of an ether 

 solution of the pigments (Expt. 18) with 2 c.c. of 30 "/q caustic potash in methyl 

 alcohol (obtained by dissolving 30 gms. of potassium hydroxide in 100 c.c. of methyl 

 alcohol 1). After the green colour has reappeared, slowly add 10 c.c. of water and 

 then add a little more ether. On shaking the test-tube, two layers are produced, of 

 which the lower watery-alkaline one contains the sapiniified green pigments, while 

 the carotin and xanthophyll are contained in the upper ethereal layer. 



Expt. 21. Separation of the two yellow pigments. The ether layer obtained in 

 the last experiment is washed with water in a separating funnel, and evaporated 

 down to 1 c.c. It is then diluted with 10 c.c. of petrol ether and next mixed with 

 10 c.c. 90 0/o methyl alcohol. The methyl alcoholic layer is removed and the petrol 

 ether layer is again treated with methyl alcohol and the methyl alcohol again 

 removed. This process is repeated until the methyl alcohol is no longer coloured. 

 The methyl alcohol contains the xanthophyll, the petrol ether the carotin. 



Further accounts of the yellow pigments are given on p. 39. 



The best known reactions of chlorophyll are those which take place 

 with acids and alkalies respectively. 



Chlorophyll is a neutral substance, and, on treatment with alkalies, 

 it forms salts of acids, the latter being known as chlorophyllins. These 

 salts are soluble in water forming green solutions which are not however 



1 The methyl alcohol must be very pure, otherwise the alcoholic potash solution will 

 become brown and discoloured. 



