Forty-seven saturated synthetic diacyl PA, PC, PE, PG, and PS and five unsaturated diacyl phospholipids (PL) underwent normal- and reversed-phase (RP) HPLC with isocratic isopropanol/hexane/water (5∶4∶1) and methanol/chloroform/acetonitrile/water (79.5∶9∶8∶3.5) mobile phases, respectively. For normal-phase HPLC, capacity factors (k′ _i ) decrease with chain length (n) of the two identical PL FA residues, whereas the opposite occurs with RP (C₁₈)-HPLC. Plots of In k′ _i vs. n for individual PL classes are in general curved, violating the linear free-energy relationship. For PL of the same n but with different head groups, k′ _i with normal-phase HPLC varies as PEn≥16, when the order is PEk′ _i values is PGn≥16, when it is PA≈PGk′ _i values of PL with unsaturated FA of n=18 are ordered as PEk′ _i .