Various chiral stationary phases were immobilized on supports using suitable alkyl spacers. Aminopropylsilica, which is aminopropylsilylated silica gel, is a typical spacer-carrying support for immobilization. Silanol and aminopropyl groups left unreacted on the support surface disturb the separation of enantiomers through a non-chiral inteaction. The following three types of chiral stationary phases were prepared using different preparation methods and the effect of the total structure on the chiral separation was studied: (1) a chiral group was immobilized on aminopropylsilica; (2) a chiral group connected with a spacer was immobilized on silica gel; and (3) a chiral group connected with a spacer was immobilized on dimethylchlorosilane-treated silica gel. It was shown for (1) that when the amount of chiral groups immobilized was lower, free silanol groups and unreacted amino groups would preferentially interact with sample molecules. When the amount immobilized was increased, its interaction with samples became effective, and also aminopropyl groups left unreacted were thought to enhance the interaction. Too many chiral groups, however, resulted in lower separatility. In case (2), a similar tendency to that with (1) but lower separabilities were obtained. A more effective performance was found with (3), with lower immobilization. For the preparation of an effective chiral stationary phase, the amount of chiral groups on the support and the structural and topographical conditions of the support material should be assessed.