High-Efficiency Chiral Analysis by Supercritical Fluid Chromatography Coupled with Circular Dichroism Spectroscopy | POSTER

Introduction

In the research and development of chiral pharmaceuticals and functional organic molecules, the preparation of enantiomerically pure compounds and their stereochemical characterization are essential. High-performance liquid chromatography (HPLC) using chiral stationary phases, which offers high resolution, reproducibility, and versatility, is widely used as a key analytical technique for the separation of enantiomers.[1] However, efficient screening is required to rapidly determine the optimal mobile phase and chiral stationary phase for various chiral compounds.

In this regard, supercritical fluid chromatography (SFC), which provides higher throughput and resolution than HPLC, is particularly effective because it can reduce the analysis time required for method screening.[2,3] After identifying the optimal separation conditions (mobile phase and column) using analytical-scale SFC, the method is scaled up to preparative SFC, and each eluted peak is collected and concentrated to obtain the enantiomers. The enantiomers obtained by preparative SFC are subsequently subjected to structural analysis using techniques such as circular dichroism (CD) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and X-ray diffraction (XRD). As mentioned above, the isolation and structural analysis of enantiomers are generally performed offline. However, performing these procedures offline is time-consuming and laborious and generally requires larger sample amounts; therefore, the development of hyphenated techniques that combine separation and structural analysis online is expected.

In this study, a hyphenated system was established by online coupling SFC with electronic circular dichroism (ECD) spectroscopy. ECD spectroscopy is particularly advantageous for chiral analysis compared with other structural characterization techniques, because it provides stereochemical information in solution without the need for crystallization and requires only a small amount of sample. Furthermore, its optical detection principle facilitates online coupling with chromatographic techniques. This system enables the real-time acquisition of high-quality CD spectra simultaneously with chromatograms, allowing the chiral information of each peak to be directly evaluated without fractionation. As a result, sample consumption and analysis time canbe significantly reduced, enabling faster and more efficient structural analysis in the study of chiral compounds. In this presentation, examples of the application of this system to several chiral compounds (flavanone, warfarin, and bromuconazole) will be presented. Flavanone and warfarin each exists as two enantiomers, whereas bromuconazole exists four stereoisomers because it contains two independent chiral centers.

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