Determination of retention factors of phospholipids in gradient HPLC

Summary The mechanism of separation of solutes under gradient elution conditions has been studied. Separation of a mixture of soybean phospholipids (phosphatidylethanolamine, phosphatidylinositol, and phosphatidylcholine) in the ternary mobile phase: methanol, hexane, isopropanol was considered as an example. An analytical expression in terms of the composition of the ternary mobile phase was obtained to calculate retention factors in two-step gradient modes. The following new polynomial equation was derived: logk=A+BF+CF ²+DG+EG ² where A, B, C, D, and E are empirical coefficients; F and G are volume fractions of isopropanol and methanol respectively in the mobile phase based on hexane. The coefficients for phospholipids were obtained under gradient conditions.