On the Function of Chlorophyll. 



3 



1 — 3 minutes in boiling water. As much water as possible was then squeezed 

 from them with a strong screw press. Sufficient absolute alcohol was added 

 to moisten them, and within a few minutes all liquid was squeezed out in the 

 press. The pale green liquid was thrown away. The residue was just 

 covered with cold alcohol and digested in a closed glass cylinder in dark- 

 ness in a cool chamber for at least one day. All the liquid was then 

 poured off and squeezed out. One half of the volume of petrol ether was 

 added, and then sufficient water to bring about separation. After standing 

 in a large separating funnel the lower liquid was run off, then the scum 

 between, and then the petrol ether. This was evaporated to dryness by a 

 partial vacuum and gentle warming, redissolved in a minimum quantity 

 of cold absolute alcohol, and again separated as before. The petrol-ether 

 extract was allowed to stand in contact with water for some days. If 

 any red carotin separated out it was again purified.* 



This method is more satisfactory than when dried leaf meal is used, since 

 the preliminary treatment removes a large amount of the waxy and other 

 extractives that would otherwise be dissolved along with the chlorophyll. 

 The separation is based upon the fact that carotin is only sparingly soluble 

 in cold alcohol, that xanthophyll is soluble in water and in any mixture of 

 alcohol and water, whereas carotin and chlorophyll are thrown out of an 

 alcoholic solution by the addition of water and are readily soluble in petrol 

 ether. Although xanthophyll is soluble in water, contact with the latter 

 will not remove it from a petrol-ether solution of a waxy solid like chloro- 

 phyll unless sufficient alcohol is present, but pure solid xanthophyll dissolves 

 slowly in water forming a solution which filters as a clear yellow liquid. 

 If any carotin, wax, or chlorophyll is present, the xanthophyll dissolves in 

 water with difficulty or not at all, owing to the lack of proper contact. 



To obtain pure xanthophyll, the brownish-yellow alcoholic liquid was 

 enriched witli alcohol, shaken up again with petrol ether, and separation 

 produced by the addition of_ water. After long standing in darkness the 

 liquid beneath becomes perfectly clear and, after filtering, it may be 

 evaporated to dryness by gentle warming and a partial vacuum in darkness. 

 The residue was extracted with cold absolute alcohol, filtered again, 

 •evaporated to dryness, dissolved in petrol ether, and filtered. This gave a 

 dark liquid, brownish yellow when shaken up, yellow when dilute, reddish 

 brown by transmitted light when concentrated, but free from chlorophyll and 

 with no red fluorescence. 



Pure carotin can be obtained by treating the prepared grass material with 



* A very strong .solution of chlorophyll in petrol ether will always throw out some 

 of its carotin in contact with water if any is present. 



B 2 



