Unexpected effects of mobile phase solvents and additives on retention and resolution of N-acyl-D,L-leucine applying Cinchonane-based chiral ion exchangers

Chiral ion exchangers based on quinine (QN) and quinidine (QD), namely Chiralpak QN-AX and QD-AX as anionic and ZWIX(+) and ZWIX(-) as zwitterionic ion exchanger chiral stationary phases (CSPs) have been investigated with respect to their retention and chiral resolution characteristics. For the evaluation of the effects of the composition of the polar organic bulk solvents of the mobile phase (MP) and those of the organic acid and base additives acting as displacers necessary for a liquid chromatographic ion-exchange process, racemic N-(3,5-dinitrobenzoyl)leucine and other related analytes were applied. The main aim was to evaluate the impact of the MP variations on the observed, and thus the apparent enantioselectivity (αapp), and the retention factor. Significant differences were found using either polar protic methanol (MeOH) or polar non-protic acetonitrile (MeCN) solvents in combination with the acid and base additives as counter- and co-ions. It became clear, that the charged sites of both the chiral selectors of the CSPs and the analytes get specifically solvated, accompanied by the adsorption of all MP components on the CSP, thereby building a stagnant “stationary phase layer” with a composition different from the bulk MP. Via a systematic change of the MP composition, trends of resulting αapp and retention factors have been identified and discussed.