Simple Laboratory Experiments on Leaf Pigments. 29 



The phase test also applies to the chlorophyllides and phaso- 

 phytin and to the pliseophorbides. It is not given with aliomerised 

 chlorophyll. 



This allomerisation takes place in alcoholic solution particularly 

 when water-free (see Section B of this chapter). The chlorophyll- 

 ides are also very easily aliomerised and lose thereby their power 

 of crystallisation. Small quantities of water and of acids protect 

 the substances against allomerisation, while alkalis increase the 

 velocity of the reaction. 



Experiment 5. Aliomerised chlorophyll does not give the brown 

 phase test. 



Dissolve a little crude chlorophyll, obtained by evaporating an 

 ether solution, in absolute alcohol. Add a little alkali, and perform 

 the phase test from time to time till at last the brown phase no 

 longer appears. 



Experiment 6. Separation of the green and yellow pigments. 

 Required : 5 c.c. ether solution of pigments ; 2 c.c. 30% potassium 

 hydrate in methyl alcohol ; 5 c.c. ether. 



Shake 5 c.c. of the ether solution of the pigments with 2 c.c. 

 of the strong alkali. After the green colour has reappeared, slowly 

 add 10 c.c. 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. 



This test is employed in the examination of the purity of a 

 chlorophyll preparation. If all the yellow pigments have been 

 removed, the ethereal layer in this experiment should remain 

 colourless after saponification of the chlorophyll. 



Experiment 7. Separation of the two yellow pigments. 

 Required : The ethereal solution of yellow pigments from Experi- 

 ment 6; 10 c.c. petrol ether; 30-50 c.c. 90% methyl alcohol; 1 

 separating funnel. 



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. petrol ether and next mixed with 10 c.c. 

 90% methyl alcohol. The methyl alcoholic layer is removed and 

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

 methyl alcoholic layer 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. 



