Biochemical Studies on Carotenoide : Part II. Isolation of Green Leaf Xanthophylls by Esterification Method and Determination of Hydorxyl Groups by Gas Liquid Chromatography

As the chromatographic behavior of the fatty acid esters of green leaf xanthophylls was different to that of free xanthophylls, the fractionation and the isolation of these xanthophylls were carried out with application of this properties by silicagel column chromatogray, followed by determination of hydoroxyl groups in these xanthophylls using the quantitative analysis of fatty acid to be bound in GLC method. By these chromatographic method six fractions of green leaf xanthophylls were able to be obtained as esters in which at least two were considered to isolate. When the hydroxyl gloups of these fractions were subsequently estimated by absolutely quantitative determination of fatty acid with GLC, only one xanthophyll had four hydroxyl groups and others two. These conclusions partially agreed with the results of hydroxyl numbers of the known xanthophylls, that is, VI-1 fraction which was considered to correspond to the lutein with one hydroxyl group had two hydroxyl groups, while VII-3 corresponded to auroxanthin with two hydroxyl groups had the same number of the groups. It was deduced that the difference between the estimated and the known hydroxyl numbers probably accounts for both the oxidation of the carotenoids during the chromatographic procedure and the subsequent production of epoxide groups. Therefore, the usefulness of these methods for fractionation of green leaf xanthophylls and for decision of hydroxyl numbers is now not able to be judged. But, up to date, the such above method has not been used and then is considered to have methodological originality.