Carbon Assimilation. 
15 
It may be recalled that the yellow pigments in solution greedily 
absorb oxygen. Some observers, either unaware of this or 
assuming that the chlorophyll they used was free from yellow 
pigments without applying tests to prove it (Experiment 6), have 
mistakenly stated that chlorophyll greedily absorbs oxygen. 
In solution the two yellow pigments appear very similar. They 
can, however, be distinguished by means of their absorption spectra. 
(See section C of this chapter). 
Experiment 8. Phytochlorin and Phytorhodin. Required : 
5 c.c. ether solution containing both chlorophyll components 
(Experiment 2); 3 c.c. 30% potash solution in methyl alcohol; 
hydrochloric acid of various concentrations; separating funnel. 
Five c.c. of an ether solution containing both chlorophylls a 
and b are evaporated to dryness in a test-tube, and the residue 
treated with 3 c.c. of boiling, concentrated potash solution in methyl 
alcohol, and boiled gently for half a minute. A liquid with red 
fluorescence is produced, which consists of a solution of the 
potassium salts of isochlorophyllins. The solution is diluted with 
double its volume of water and concentrated hydrochloric acid is 
added until the solution is just acid. The liquid is then shaken 
with ether in a separating funnel; the dissociation products 
produced by the previous treatment go over to the ether solution 
which thus acquires an olive-brown colour. 
The ether solution is shaken twice, each time with 10 c.c. 4% 
hydrochloric acid, and the green-blue acid layer is separated and 
neutralised with ammonia and shaken with more ether, which then 
contains in solution phytochlorin e, the derivative of chlorophyll a 
The phytochlorin e gives to the ether an olive-green colour. 
The ether layer remaining in the funnel after the separation of the 
green-blue acid layer is now extracted with 10 c.c. 12% hydrochloric 
acid. The green acid solution so obtained is diluted with water 
and shaken with ether which then becomes coloured red and 
contains phytorhodin g, the derivative of chlorophyll b. 
It should be noted that saponification with hot alkali as in this 
experiment, produces changes in the chlorophyll compounds differ¬ 
ent from those produced by saponification in the cold. 
The potassium salts of the chlorophyllins which are produced 
by gentle saponification in the cold are not fluorescent, and under 
t,he action of acids pass over into the weakly basic phytochlorins f 
and g and phytorhodins k and i. 
