Effect of Selector Coverage and Mobile Phase Composition on Enantiomeric Separations with Ristocetin A Chiral Stationary Phases

Three different amounts (i.e., selector coverages) of ristocetin A macrocyclic antibiotic have been covalently bonded to silica gel, packed in stainless steel columns, and evaluated as chiral stationary phases (CSPs) in high-performance liquid chromatography (HPLC). The three columns (i.e., CSPs) were examined and compared in the reversed-phase, polar-organic, and normal-phase modes. The retention behavior (k′), selectivity (α), resolution (RS), and number of theoretical plates (N) for all three columns are discussed and compared in each mobile phase mode. The coverage of the chiral selector had a profound effect on the chromatographic behavior of the more retained enantiomers. A special mobile phase composition (i.e., methanol plus very small amounts of acid and base modifiers) resulted in improved baseline separations, without greatly affecting the analysis times, for all chiral analytes evaluated. The column stabilities were excellent when switching between different mobile phase modes.