The separation mechanism of solutes under gradient conditions has been studied. The separation of a mixture of soybean phospholipids (phosphatidylethanolamine, phosphatidyl-inositol, and phosphatidylcholine) in the ternary mobile phase of methanol, hexane, and isopropanol was investigated. An analytical expression was obtained to calculate the retention volume under a two step-gradient mode. The following new polynomial equation was also presented, log k''=A+B×F+C×F²+D×G+E×G² where A, B, C, D, and E are empirical coeffieients and F and G are volume percentage of isopropanol and methanol in the mobile phase based on hexane, respectively. The resulting equations of the three phospholipids were used to describe the effect of the composition of the ternary mobile phase on the retention factor under step-gradient conditions.