An examination of the effects of water on normal phase enantioseparations

Abstract

Superficially porous silica bonded with macrocyclic glycopeptides can separate enantiomers in various chromatographic formats, including normal phase liquid chromatography (NPLC). The conventional wisdom in NPLC is to avoid intentionally adding water in the eluents. Herein we examine the effects of small quantities of water as an additive on chiral separations in NPLC with the n-hexane-ethanol system. A phase diagram (n-hexane-ethanol-water) is used to analyze the physicochemical properties of the mobile phase. The relative polarity change of solvents upon adding water was determined by using bathochromic shifts of dissolved Nile Red dye. The effectiveness of chiral NPLC with water traces is demonstrated for various pharmaceutically relevant enantiomeric compounds. It is postulated that water molecules weaken stationary phase-solute interactions, resulting in lower retention times for both enantiomers in addition to significantly higher efficiencies. Gibbs free energy changes provided an understanding of the different enantioselectivity shifts caused by water addition. Some interesting kinetic effects also were observed. Classical van Deemter curves are not observed on macrocyclic glycopeptide stationary phases due to slow mass transfer kinetics and thermal effects at high flow rates. The most significant advantage of adding water in NPLC is reducing mass transfer kinetics and altering the mass overloading properties which is highly beneficial on macrocyclic glycopeptide phases. By overloading a 10 × 0.46-cm column with up to 0.6 mg alprenolol, it was found that the relative adsorption isotherm of the first eluting enantiomer was switched from Langmuir to anti-Langmuir type by water addition. The peak shape tuning effect demonstrated the strong influence of water on specific interaction sites of the chiral stationary phases. Water addition effects were most beneficial for enantiomeric and preparative separations in NP mode.

Keywords: Adsorption isotherm; Efficiency; Enantiomer separation; Macrocyclic glycopeptide; Normal phase; Selectivity; Water additive.