A long alkyl group like C30 (triacontyl group) has been known tobe more suitable than a conventional C18 phase for separation of hydrophobic structurally related isomers such as vitamin E or vitamin K1. In this study, separation factor of beta-tocopherol and gamma-tocopherol which were structurally related isomers was evaluated to vary both a pore diameter of the superficially porous silica and a ligand density of the C30 group. Regarding a pore diameter, 12 nm showed the largest separation factor of beta and gamma-tocopherol among 10nm, 12 nm and 16 nm. Regarding a ligand density, the higher a ligand density, the larger a separation factor of beta and gamma-tocopherol. However, when a ligand density was too high, much high hydrophobicity caused peak tailing and a drop of theoretical plate. The most suitable ligand density existed for the highest resolution. Finally separation of cis and trans-vitamin K1 was compared and the same result as separation of beta and gamma-tocopherol was obtained.