The paper electrophoretic separation of phosphatides.

A number of chromatographic methods have been previously reported for the separation of phosphatides. Column chromatography has been used successfully by Thannhauser and Setz (l), Klenk (a), and Hanahan et al. (3). Paper chromatographic techniques for phosphatide analysis have been reported by Bevan et al. (4), Hecht and Mink (5), Hack (6)) and Lea et al. (7). Garvin (8) has reported a separation of phosphatides by filter paper electrophoresis. In the present paper, a method for the separation of phosphatides by electrophoresis on a paper strip is described. This method is a result of the successful use of non-aqueous solvents and solvent systems containing low concentrations of water. The use of such solvents was first described by Paul and Durrum in 1952 (9). Experimental-Lecithin was prepared by a combination of the methods of Pangborn (lo), Taurog et al. (II), and Lea et al. (7). Three samples of synthetic lecithin analogues were kindly supplied by Dr. E. Baer and Dr. J. D. MacLennan. These included dimyristoyllecithin, dioleoyllecithin, and glycol stearyl lecithin. Cephalin was prepared and fractionated by the method of Folch (12-14)) which provides for the separation of three phosphatides: phosphatidylethanolamine, phosphatidylserine, and diphosphoinositide. Analytical-With the exception of the diphosphoinositide, the purity of the sample was based on the ratio of nitrogen to phosphorus. Phosphorus was determined by the method of King (15) and nitrogen by the microDumas method. The results are shown in Table I. Apparatus-The apparatus used was an electrophoresis unit designed by Dr. Samuel Raymond of the E. C. Apparatus Company, and consists of three separate buffer chambers, each provided with baffle plates. Between the opposing buffer chambers there is situated a 12 inch long watercooled plate. The power supply provided a potential of 1000 volts and a current of 200 ma. Method-An electrophoretic determination was conducted in the following manner: the electrolyte system was added to the buffer compartments of the apparatus, and the Whatman No. 3MR!I paper strips were placed on a Teflon pad with the ends of the paper strips kept out of the solvent system.