The Application of Counter-Current Distribution to the Separation of Phospholipins

Counter-current distribution between two immiscible solvent phases, as first developed by Craig (1944), offers advantages over both direct and partition chromatography for analytical and preparative work in the field of phospholipins (Lovern, 1949). The only recorded use of the method for such purposes appears to be in a series ofpapers from one laboratory and deals with vegetable phosphatides (Scholfield, Dutton, Tanner & Cowan, 1948; Scholfield, McGuire & Dutton, 1950; McGuire & Earle, 1951). These workers obtained varying degrees ofseparation of constituents by distribution between hexane and aqueous methanol or ethanol in a metal apparatus of essentially the original Craig pattem. Various developments in design of apparatus (Lochte & Meyer, 1950; Craig, Hausmann, Ahrens & Harfenist, 1951; Lathe & Ruthven, 1951) have considerably extended the scope ofthe method, and recently the similar 'cascade distribution' (Kies & Davis, 1951) offers an alternative technique. The author has studied the applicability of the counter-current technique to the separation of the constituents of the crude glycerophosphatide fraction of the lipids of ox brain, using light petroleum (b.p. 40-60O) and aqueous ethanol as solvents. Preliminary tests, both in a metal apparatus of the original Craig pattem and in a series of separating funnels, suggested that phospholipins are likely to give trouble with apparatus in which all equilibrations and transfers are performed by one operation. They appear to have a considerable effect on the solubility ofone solvent in the other. Thus, although the two phases are saturated with each other at the start of the experiment, the relative volumes of the two phases alter appreciably as fractionation proceeds along a series of vessels. This could lead to trouble with mixed phase transfers in an automatic apparatus. Moreover, at certain stages ofthe separation-usually rather late stages-it occasionally happens that the contents of one vessel will form a very stable emulsion whereas all the rest separate rapidly. This may occur only once or twice in the course of hundreds of transfers, and it can easily be dealt with in a procedure based on individual transfers, but would pass unnoticed in an automatic apparatus. For these reasons, in spite of the great expenditure of time required, the use of a series of conical flasks has been adopted. They permit the use of any desired volumes and are thus ideal for preparative work. Separating funnels are more easily manipulated, but involve the danger of contamination with stopcock grease. Beroza (1951) prefers bottles to funnels for the same reason.