Partition and adsorption in the separation of long chain lipids by normal phase high performance liquid chromatography

Martin's adaption of Raoult's Law has been shown to describe partition phenomena in normal phase HPLC. Thus, in the separation of fatty acid pentaflorobenzyl esters and of triacylglycerols in dichloromethane or acetonitrile in half-water-saturated hexane, the natural logarithm of the corrected retention volume is proportional to the number of double bonds that these solutes contain. By extending Martin's adaption of Raoult's Law with activity coefficients, evidence was obtained that deviations observed for the homologous series of saturated fatty acid pentafluorobenzyl esters from C₂ to C₅ could be accounted for by a diminution of adsorption by the inductive effect. For chain lengths longer than C₅, it appeared that there were additional field and inductive effects from the methyl end of the hydrocarbon chains. Thus, with these half-water-saturated solvent systems, the silica surface is probably covered with a bilayer of water which, in the dynamic situation in an HPLC column, attracts several layers of the polar organic component of the mobile phase by dispersive interactions. This stationary phase surface may, then, be considered to be immiscible with the predominantly hexane mobile phase due to these dispersive interactions and thus partition can occur between these two phases. Solutes that enter the stationary phase are then free to interact with the underlying aqueous phase by adsorption.