Carbon Assimilation. 
A characteristic difference between the two green pigments is to 
be found in the phase which appears on saponification with methyl 
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 bring about 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. 
