New Instrumentation for Use in Excitation-Emission Fluorescence-Polarization Measurements

Abstract

A fluorescence excitation-emission matrix (EEM) is an excellent way to determine a specific substance in a multicomponent sample. In the present investigation, a novel, rapid-scanning spectrofluorometer has been developed for measuring an EEM; the instrument employs a 0.35-meter monochromator for choosing the excitation wavelength and a continuously variable interference filter for selecting the emission wavelength. With the system, a 51 × 93 excitation-emission matrix can be collected in 3.2 min, and detection limits of 500 ng/L are obtained for fluorescein dye in ethanol. Fluorescence polarization measurements in the plane parallel and perpendicular to the polarized excitation light adds another dimension to the instrument for characterizing a sample. The instrument is controlled by an IBM XT computer although data analysis is performed on a Macintosh computer with Spyglass software. A series of two-dimensional contour plots and multidimensional excitation-emission-polarization plots can be rapidly generated.