28 Carbon Assimilation. 



alcoholic potassium hydroxide. This phase-test is best carried out 

 in ethereal solution. The methyl alcoholic solution is therefore 

 poured from the separating funnel into another and, as described in 

 Experiment 2 transferred to an ethereal solution. The petrol 

 ether solution and ether solution are then used for phase tests as 

 described in Experiment 4. 



The difference in the absorption spectra of the two chlorophylls 

 is not easily observed unless the solutions are very pure. 



Experiment 4. Saponification of the green pigments. Required: 

 5 c.c. of an ether solution containing the pigments. The petrol 

 ether and ether solutions containing chlorophylls a and b obtained 

 in Experiment 3 ; 10 c.c. methyl alcoholic potash. 



An ether solution of chlorophyll does not react with weak 

 alkali as being an ester it is without acid properties. If however, 

 strong alkalis are used, a brown colouration appears which changes 

 back later to green. 



Pour a little of the ether solution from Experiment 2 into a 

 test-tube and in a pipette take a little strong solution of potash in 

 methyl alcohol (obtained by dissolving 30 gms. potassium hydroxide 

 in 100 c.c. methyl alcohol). Place the lower end of the pipette at 

 the bottom of the test-tube and allow the potash to run in below 

 the chlorophyll solution. At the interface between the solutions 

 there appears immediately a brown coloured layer which diffuses 

 on shaking. In about ten minutes it changes back through an 

 olive green colour to pure green. The chlorophyll has been 

 saponified to the potassium salt of the acid chlorophyllin. This salt 

 is insoluble in ether, so if water is added to bringabout a separation 

 of the two layers, the green colour is no longer present in the 

 ethereal layer. 



The brown phase produced in this saponification of a mixture 

 of the two chlorophylls is the resultant of a yellow phase produced 

 by chlorophyll a and a brown-red phase produced by chlorophyll b. 

 The phase test should therefore also be carried out separately with 

 the petrol ether solution containing chlorophyll a and" the ether 

 solution containing chlorophyll b obtained in Experiment 3. 



It should be observed that if water is added directly the brown 

 phase appears, the greater part of the green pigment is soluble in 

 ether, and will again give the brown phase on treatment with alkali. 



Note Willstatter's theory of the lactam ring to explain the 

 appearance and disappearance of the brown phase. 



