Development, optimization, and application of polarimetric methods for chiral-selective analysis.

摘要

The unique response characteristics of laser-based polarimetric detectors have made them an important HPLC detection method for the analysis of optically active molecules. Over the last decade, as the relationship between optical activity and biological activity has become clear, interest in the use of polarimetric detection has steadily increased. This interest is fueled by the unique capabilities of this detection mode, as well the ability of polarimetric detection to provide information not available from other detection approaches. This dissertation details the development of several new methodologies, which use the unique capabilities of laser-based polarimetric detection to address problems critical to progress in pharmaceutical and biotechnology science.; The combination of laser-based polarimetric detection and UV/Vis absorbance spectroscopy is shown to be advantageous for the determination of enantiomeric purity in real-time, large-scale chiral separation systems. This methodology could prove to be a valuable tool for directing and optimizing enantio-selective preparative scale separations that exhibit a moderate to high degree of chromatographic peak overlap. The determination of enantiomeric excess (ee) at the extremes of the enantiomeric excess scale (99%) is also demonstrated. To date, no other chiral-selective detection method has been shown to be able to quantitate at such a high degree of precision and accuracy at enantiomeric excess levels greater than 99.0%.; The use of high precision specific rotation measurements, via HPLC coupled to a laser-based polarimeter, for the identification of five structurally similar tetracycline analogs is shown to be effective and specific. Separation and quantitation of these closely related structural analogs provides a great challenge to the analyst due to the very subtle changes in molecular structure that differentiate each analog. Thus, the ability to simultaneously separate, quantitate, and identify structural analogs could be of great interest to biomedical and biological scientists. Finally, the direct monitoring of two biologically significant chemical reactions: interconversion of β-D-glucose and the enzymatic conversion of β-D-glucose via glucose oxidase are demonstrated. The ability to monitor a reaction's progress and subsequently calculate kinetic parameters is a unique property, especially when considering that many biologically important reactions involve amino acids and sugars, which in general, do not exhibit a significant UV absorbing chromophore.