Enantiomer and Topoisomer Separation of Acidic Compounds on Anion-Exchanger Chiral Stationary Phases by HPLC and SFC

Nov 01, 2012
Volume 25, Issue 11

Quinine- and quinidine-derived anion-exchanger chiral stationary phases are versatile tools for enantiomer separation of acidic compounds in high performance liquid chromatography (HPLC). This article demonstrates their recognition ability in specific HPLC applications, involving enantiomer resolution and plasmid topoisomer separation. The extension of their applications from HPLC to supercritical fluid chromatography (SFC) was also investigated, with the aim of assessing the influence of a series of parameters and gaining insight into the general approaches for SFC method development and optimization.

Quinine (QN) and quinidine (QD) are alkaloids of the Cinchona family with anti-malarial properties that have a long tradition in stereoselective methods as auxiliaries (as a base for fractionated crystallization of chiral acids), as chiral catalysts and as chiral selectors for enantioselective separations. Based on the investigations of Lindner and his fellow workers, the chiral recognition ability of various derivatives has been explored recently for the resolution of acidic enantiomers. Most notably, it was found that a carbamoyl modification of the secondary hydroxyl group at C9 of the alkaloid significantly enhanced the enantiorecognition capabilities of the resulting chiral selector (1-3). The tert-butyl carbamates of QN and QD immobilized on spherical silica gel (Figure 1) turned out to be the most versatile compromise of structure variations. When used with weakly acidic mobile phases — usually pH 4–7 — they act as weak anion exchanger chiral stationary phases (CSPs) to provide specific enantioselectivity for acidic compounds.

The enantiomer recognition mechanism is based on ionic interaction between the protonated tertiary nitrogen of the quinuclidine moiety of the chiral selector and the anionic analytes. Such an ion pairing is accompanied by additional intermolecular interactions including hydrogen bonding, dipoledipole, p–p and hydrophobic, as well as steric interactions (2–4).

These chiral columns have been exhaustively investigated in HPLC with aqueous and non-aqueous polar organic mobile phases and show remarkable performance in enantiomer resolution of a wide variety of acidic compounds (3–10) and so investigation of these columns for enantiomer separation by SFC is a current area of research.

Figure 1: The chiral stationary phases.
In this article we demonstrate that the application of these columns can be extended to different practical applications in LC and SFC for enantiomer resolution of acidic compounds and for separation of certain specific compounds like topoisomers of circular plasmid DNA.