D 



30 CHLOROPHYLL [ch. 



quantity of distilled water is added, this being poured gently down the side of the 

 unnel in order to avoid the formation of emulsions. In the course of a few minutes, 

 the ether layer separates out and now contains the pigments. The lower layer, which 

 is slightly green, is run off. 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 repeated with another 2 gms. of leaf-powder and the 

 pigment transferred to ether^ since a solution in this solvent is required for later 

 experiments. 

 ^ i/ Expt. 20. 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 7o methyl 

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

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

 green, while that of chlorophyll 6 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. Keep the two extracts 

 for Expt. 24. 



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

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

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

 xanthophyll and carotin. 



y Expt. 21. Separation of green and yellow pigments. Shake 5 c.c. of an ether 

 solution of the pigments (Expt. 19) with 2 c.c. of 30 ^Iq caustic potash in methyl 

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

 alcohol'}. 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 saponified green pigments, while 

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



Expt. 22. 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 o/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. 40. 



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. 



