Development of a bimodal polarimetric response model for improved quantitation of enantiomeric mixtures under conditions of poor chromatographic resolution.

The combination of a chiral-selective separation mode with polarimetric detection was investigated in terms of its ability to quantitate enantiomeric mixtures of the dansylated derivatives of phenylalanine, threonine, and valine under conditions of poor chromatographic resolution. Using the difference between two Gaussian functions to model the bimodal response obtained using polarimetric detection and incomplete chiral resolution, correlation coefficients as high as 0.999 were obtained when actual enantiomeric fractions were plotted against observed enantiomeric fractions calculated from the fitting procedure. The methodology was evaluated at different levels of chromatographic resolution, and as a function of the sign and magnitude of the specific rotation of the model compounds. Limits of quantitation calculated at different levels of enantiomeric excess demonstrate the potential of the method to quantitate enantiomeric mixtures even under conditions of poor chromatographic resolution. Minimum measurable quantities (MMQ) at, or below the 1.0% enantiomeric excess (ee) level were obtained for the three amino acid derivatives tested under various conditions of chromatographic resolution when the polarimetric data were fit to the bimodal response model. Even under the lowest resolution conditions tested, the MMQ was determined to be at the 5% ee level.

Determination of enantiomeric excess using a chiral selective separation mode and polarimetric detection.

The quantitative capabilities of a system that combines a chiral selective separation mode with polarimetric detection (CSS/PD) were investigated in terms of the ability to quantitate enantiomeric mixtures even under conditions of poor chromatographic resolution. The laser-based polarimetric detection system can provide a rotational sensitivity on the order of 10 mudeg corresponding to a minimum measurable quantity of 10 ng for a compound with a specific rotation of 100 deg (g/ml)(-1) dm(-1). For the chromatographic studies, peak height and peak area were measured as analytical descriptors of the bimodal response function of this polarimetric detector. Analyses of the dansylated enantiomers of the amino acids phenylalanine, threonine, and valine have shown that chromatographic resolution and specific rotation are important factors affecting the shape of the polarimetric response curve. With CSS/PD, enantiomeric materials can be quantitated at both low and high enantiomeric excess (ee) even at resolutions of 0.3-0.4.