Effect of FA chain length on normal- and reversed-phase HPLC of phospholipids

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 (C-18)-HPLC. Plots of ln 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 PE<PG<PA<PS<PC, except when ngreater than or equal to16, when the order is PE<PS≈PA≈PG<PC. For RP-HPLC, the order of k'(i) values is PG<PA≈PS<PCapproximate toPE until ngreater than or equal to16,when it is PAapproximate toPG<PS<<PC≈PE. With normal-phase HPLC, k'(i) values of PL with unsaturated FA of n=18 are ordered as PE<PA<PC. Increasing degrees of unsaturation lead to increasing k'(i).