Nonideal behavior of void volume markers in polar-organic chiral HPLC on polysaccharide-based chiral stationary phases.

Polysaccharide-based chiral stationary phases account for a majority of chiral HPLC separations. Their behavior in normal-phase and reversed-phase modes of separation has been studied extensively. However, much less is known about their behavior in polar-organic mode. In the course of our studies on preparative separation of various cyclic imides, lactams, and acetamides on Chiralcel OD, Chiralcel OJ, Chiralpak AD, and Chiralpak AS stationary phases we sought to determine the void volumes of these columns using commonly used markers, LiNO3, thiourea, and acetone. We were surprised to notice extensive column- and solvent-dependent deviations from ideal (unretained and unexcluded) behavior. These deviations likely result from multiple types of noncovalent interactions possible for these chiral stationary phases. Therefore, great care should be taken when choosing void volume markers for such separations. Our results indicate that with acetonitrile as a mobile phase, acetone is a suitable void volume marker for all four of the above stationary phases.