250 CAROTINOIDS AND RELATED PIGMENTS 



Arnaud made an interesting study of the seasonal variations in the 

 carotin content of green leaves, using the stinging nettle and chest- 

 nut leaves as the source of his material. He found that the maximum 

 carotin content (on the dry basis) occurred at the time of the flower- 

 ing of the plants, and that it diminished regularly with the growth 

 of the leaves. Thus, in the case of the nettle, a carotin content of 

 172 mgs. per 100 grams of dried leaves was noted on May 2, but this 

 had decreased to about 100 mg. by the middle of July. In a com- 

 parison between etiolated and green leaves of the same plant, Arnaud 

 found that the carotin content, on the dry matter basis, increased 

 about 5 times during the formation of the chlorophyll. 



Kohl (1902i) used Arnaud's method for determining the carotin 

 content of the leaves of a few plants, but secured somewhat lower re- 

 sults. His values for spinach and stinging nettle leaves were about 

 half those reported by Arnaud, and for grass about 70 per cent of 

 Arnaud's value. These low results may have been due, however, to 

 the fact that Kohl apparently ignored Arnaud's precaution and dried 

 his material at 100 C. 



Monteverde and Lubimenko (1913a) have devised a spectro-colori- 

 metric method for the quantitative estimation of carotin, as well as 

 xanthophylls and chlorophyll, in green leaves. The writer has not 

 been able to secure a clear translation of this method which has been 

 published only in Russian. In general, however, the method appears 

 to be based on the extraction of all the pigments from fresh leaves, 

 0.1 gram quantities, by grinding in a mortar with alcohol. Measured 

 quantities of the extract are then treated with strong Ba(OHj 2 solu- 

 tion to throw down all the pigments. After standing for some hours, 

 the precipitate is filtered off and extracted completely with abso- 

 lute alcohol, which is said to take out only the carotinoids. These 

 are fractionated by the Kraus method between 80 per cent alcohol 

 and petroleum ether, and these fractions compared in the spectro- 

 colorimeter with standard 0.001 per cent carotin and xanthophyll so- 

 lutions. By keeping all extracts in definite volumes the data can be 

 calculated back to the quantity of pigments in the plant tissues exam- 

 ined. The feature of the spectro-colorimetric method is the compari- 

 son of the solutions on the basis of the depth of unknown solution 

 required to give an absorption spectra of equal intensity as the stand- 

 ard. The authors found that the first faint appearance of absorp- 

 tion bands for the standard solutions gave a more sensitive compari- 



