Procedures for the microestimation of nitrogenous phosphatide constituents.

This paper describes procedures for the separation and quantitative estimation in minute amounts of several nitrogenous substances normally encountered as constituents of phosphatides; viz., ethanolamine, choline, and serine. The work is based on techniques employing paper chromatography, previously applied to the investigation, in a qualitative fashion only, of tissue lipides (1) ; its principal features were reported in a brief communication (2) ; its application to a survey of the distribution of nitrogenous constituents in a series of phosphatide fractions of beef brain also is included in the present communication. The use of these separation procedures for the study of several nitrogenous compounds, analogous to the ones discussed here, is described in an accompanying paper (3). Other applications will be illustrated in another publication (4). Similar methods also have recently been applied to a study of the composition of vitamin Bn (5). The method rests on the following principles: (a) hydrolysis of the phosphatide and removal of the fatty acids; (b) separation of the bases by unidimensional partition chromatography on filter paper (6) ; (c) demonstration on chromatographic guide strips of the location of adsorption Bones containing primary amines by meads of ninhydrin; (d) quantitative colorimetry, also with the employment of ninhydrin, of corresponding eluates; (e) development of separate chromatograms for choline by its conversion to choline phosphomolybdate, followed by the reduction of the latter to molybdenum blue; (f) quantitative planimetry (7) of the choline spots thns produced. The preferred range for the separation and estimation of primary amines is 5 to 20 y of each component; that of choline is 25 to 75 y. Satisfactory chromatographic analyses have been performed, however, w&h 20 to 100 7