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Photometric determination of fatty acid ester groups in phospholipides.

M. Rapport,N. Alonzo

Published 1955 in Journal of Biological Chemistry

ABSTRACT

In order to facilitate a study of chromatographic separation of phospholipides currently in progress in this laboratory, a simple, rapid, calorimetric analysis was required which would be applicable to quantities of less than 1 mg. of material. For this purpose, we examined the reaction of hydroxylaminolysis of the carboxyl ester linkages, followed by formation of colored ferric hydroxamic acid complexes, according to the modification of Kornberg and Pricer (1). This method was not satisfactory, since large variations were encountered in the color yields obtained with various naturally occurring phospholipides, the color was not stable, and the effects produced by changes in the quantity of color-producing reagent were considerable. Goddu, LeBlanc, and Wright (2) carried out an extensive study of the factors affecting the reaction. They found that color stability and maximal color yield were obtained with a final ferric ion concentration greater than 0.002 M and a final hydrogen ion concentration less than 0.6 M. Instead of the complex reagent of Hill (3), commercially available ferric perchlorate has been found to be satisfactory (2, 4). We have studied the combination of these modifications with those of Kornberg and Pricer to determine ester groupings in phospholipides, both natural and synthetic, and have found the revised method to be completely satisfactory for characterizing small quantities of material with respect to convenience, sensitivity, and reproducibility.

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