44 Carbon Assimilation. 



subsequent separation of the watery methyl alcohol layer. The 

 methyl alcohol is completely removed by further washings with 

 water. The xanthophyll solution is then passed through a filter 

 into a 100 c.c. measuring flask. 



The solution is then cleared with a few drops of absolute alcohol 

 and ether added up to the 100 c.c. mark. 



The petrol ether solution of carotin is similarly washed, cleared 

 and made up to 100 c.c. 



4. The Standard Solutions. The Chlorophyll Components. 

 The standard solution of the chlorophyll components are prepared 

 by the saponification of a mixture of the methyl phseophorbides as 

 follows : 



0'0369 grams IVIethyl phagophorbide a (half hydrate) 



(12 X 10-'' gm.-mols. per litre) ; 



0-0124 grams Methyl phaeophorbide b (water-free) 



(4 X 10—' gm.-mols. per litre). 



The mixture is dissolved in 2 c.c. of pyridine and saponified with 35% 

 methyl alcoholic potash in the manner described above, under (2), 

 only as pure substances are used it is unnecessary to go through 

 the treatment for purification with 12 and 20% acid. This gives 

 about 500 c.c. phytochlorin e in ether saturated 3% acid, and of 

 phytorhodin g in 12% acid. 



These solutions can be kept for about a week, but after a 

 longer time the colours change somewhat. In such a case the 

 comparisons are better if the experimental solution is allowed to 

 stand for a day. 



The Yellow Pigments. The carotin solution is made up with 

 petrol ether and the xanthophyll with ether, as follows : 



00134 grams carotin in 500 c.c. petrol ether containing a little 

 ether (5 x 10—° gm.-mols. per litre). 



0-0142 grams xanthophyll in 500 c.c. ether (5 x 10~° gm.-mols. 

 per litre). 



The carotin solution can be kept in a well stoppered bottle in 

 the dark for at least three weeks. The xanthophyll solution must 

 be made fresh every day as it bleaches quickly, perhaps on account 

 of impurities in the ether. 



Instead of using solutions of the actual pigment it is possible to 

 employ a solution of potassium dichromate as a standard solution. 

 Willstatter gives the following thicknesses of aqueous potassium 



