A novel column based on silica containing immobilized carbon nanotubes (CNTs) was developed and evaluated in terms of its binding efficiency and resolution. First, CNT functionalized with amino groups (CNT-NH2) were prepared via chemical modification of carboxylic groups introduced on the CNT surface. Secondly the covalent immobilization of CNT-NH2 was carried out by using glutardialdehyde activating agent on aminopropyl (AP)-silica surface. This CNT stationary phase was applied to the HPLC separation of two molecule series, i.e. polychlorinated biphenyl (PCB) isomers with different degrees of substitution in the ortho-position (non-ortho to tetra-ortho substituted) and terpenes (linalool, geraniol, thymol, α-terpineol). The retention behavior of these solute molecules was measured under isocratic conditions with different mobile phase compositions, ranging from 0.05–0.70 v/v of toluene in cyclohexane. The retention factors of the solute molecule do not depend linearly on the toluene fraction but follow a quadratic relationship. This CNT stationary phase was a very useful column for the separation of PCB congeners and terpenes. It was demonstrated that a planar conformation of the solute molecule enhanced the solute retention on this CNT stationary phase. As well, a quantitative structure relationship derived, demonstrated the significant input to retention was due to the structurally selective dipole-dipole and charge transfer interactions with the solutes. These results were compared with those obtained on the AP stationary phase. The proposed CNT stationary phase for the separation possess distinctive and interesting retentive properties, and chemometric analysis of retention data of appropriate designed series of test solutes appears to be a convenient, objective and quantitative method to prove a new phase specificity.