HPLC / fluorescence detected circular dichroism detection by three-dimensional fluorescence detector

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An HP LC detection method based on fluorescence- detected circular dichroism (FDCD) is described. The proposed detector consists of a polarizing prism and a retardation plate fitted on the excitation light side of a conventional threedimensional fluorescence detector and uses a modulated beam in the wavelength axial direction. The FDCD wave is superimposed on the excitation spectrum measured at a fixed emission wavelength. When estrone with a large anisotropy factor, Δε/ ε, was used as a probe analyte, the difference between the two excitation spectra obtained by reversing the direction of the optical axis of the retardation plate showed a wave-like curve whose phase is consistent with that of the plate. W e suggest that FDCD can be measured by using the optical system of threedimensional fluorescence detector, which obtains a spectrum by the grating with high rotational speed. However, in the detection of an enantiomer with a low anisotropy factor, such as catechin, the amplitude of the artifact wave was comparable with that of the FDCD wave and obstructed the FDCD measurement. This wave was thought to be induced by a lack of uniformity of the rotation of the grating. As both the artifact and FDCD waves varied sinusoidally with wavelength, the FDCD wave with a short period was successfully extracted by using the differential properties of a trigonometric function.

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