30 Carbon Assimilation. 



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 pijjments 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 isochlorophylUns. 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 

 the action of acids pass over into the weakly basic phytochlorins f 

 and g and phytorhodins k and i. 



