QUANTITATIVE ESTIMATION OF CAROTINOIDS 257 



with an equal volume of a mixture of five parts petroleum ether and 

 one part ether. Some fucoxantliin is lost in this extract but it is re- 

 covered by concentrating the extract to 250 cc. in vacuum, adding an 

 equal volume of ether and extracting twice with 500 cc. of 70 per 

 cent alcohol, which has been saturated with petroleum ether. The 

 new alcohol extract is added to the first main extract. The fuco- 

 xantliin is finally transferred to ether, which is freed from methyl 

 alcohol by washing with water, and made up to a volume of 250 cc. in 

 a graduated flask. 



The solution is now compared in a colorimeter with either a stand- 

 and fucoxantliin solution (using a 5 x 10" 5 molar, or 0.0304 per cent 

 solution) or the 0.2 per cent KoCr 2 7 standard which is used for esti- 

 mating carotin and xanthophylls. The standard is set at 50 mm. of 

 standard fucoxantliin or 85 mm. of the dichromate solution, which is its 

 equivalent, and the depth of the unknown solution which is required 

 to match the color determined. If the height of the unknown is h f the 

 content of fucoxantliin in 1 kg. of fresh alga? as calculated from the 



1 50 



40 gram sample will be 25 x 0.00608 x x -r-^if the standard fucoxan- 



2 li f 



thin solution is used, or a similar result if the dichromate has been 

 used, since the latter will be set at the equivalent of 50 mm. of stand- 

 ard fucoxantliin. 



Using this method Willstatter and Page determined the fucoxan- 

 tliin content of Fucus, Dictyota and Laminaria to be, respectively, 

 169 mgs., 250 mgs., and 81 mgs. per kg. of fresh algae. 



Application to Other Biological Materials 



It seems obvious that the colorimetric methods of analysis for caro- 

 tin, xanthophylls and fucoxanthin as worked out by Willstatter and 

 his co-workers should be applicable to any biological material con- 

 taining these pigments if a suitable method can be devised for free- 

 ing the pigment from the tissues involved. It would seem that the Will- 

 statter technic can be applied without modification to plant tissues, 

 including flowers, fruits and leaves, with the exception of the fruits 

 containing lycopin, for which no quantitative method has yet been 

 devised. Moreover, the writer is not aware of any methods for separ- 

 ating carotin from lycopin so that even a quantitative estimation of 



