Enantiomeric separations by HPLC:temperature, mobile phase, flow rate and retention mechanism studies

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1993-03-05
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Virginia Tech
Abstract

The effects of changes in temperature, mobile phase composition, flow rate, and stationary phase upon the enantiomeric separation of several racemic mixtures are investigated. The changes in capacity factor (k'), selectivity (α), and efficiency (N) and enantiomeric resolution (R), are explored. Resolution is then shown to be controlled by the specific combination of chiral stationary phase (CSP), solute, mobile phase and temperature.

The key to optimizing chiral resolution lies in understanding the retention mechanism(s) for a given CSP. The proposed retention mechanisms for the two CSP used in the optimization studies are evaluated using chromatographic/thermodynamic data. Inferences are made which support the well-characterized "Pirkle"-type R-dinitrobenzoylphenylglycine retention mechanism, which depends solely on attractive-repulsive interactions to establish two diasteriomeric complexes having unequal internal energy, and therefore eluting at different times from the chromatographic system. For comparison, a more complicated CSP composed of a tris cellulose(3,5- dimethylphenylcarbamate) coated to a silica gel support is also examined. For this CSP the proposed mechanisms, which include both attractive-repulsive interactions and inclusion complex types, are evaluated according to the chromatographic optimization data, and compared to similar data for the single-mechanism "Pirkle" CSP.

In contrast to the above"macromolecular"-level inferences about retention mechanisms drawn from chromatographic data, a second study was initiated using a model CSP attached to a l000-Å gold surface, with Fourier-Transform Infrared Spectrometry in a Reflectance-Absorbance mode, to probe the specific molecular interactions which make possible the diasteriomeric complex. This in situ experiment, in contrast to previous ex situ, stationary phase work, is designed to show that hydrogen bonding is, as predicted, a principal force holding the complexes together, and that a measurable difference exists between the weaker R-trifluoroanthrylethanol (TFAE) and the stronger S-TFAE complexes due to their different stereo-geometry.

A further experiment to characterize the difference in mechanisms between the "Pirkle" and cellulose CSPs involves relating their chromatographic retention behavior to their structure, known as Qualitative Structure Retention Relationships (QSRR). Some structure-specific physical-organic chemistry parameters are determined using an EPA-developed computer program and correlations are made between retention on a given CSP and some of the parameters.

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