Improving signal to background ratio for on-the-fly fluorescence lifetime detection in capillary electrophoresis

On-the-fly fluorescence lifetime detection (OFLD) in capillary electrophore sis (CE) was previously demonstrated using a commercial multiharmonic Fouri er transform (MHF) spectrofluorometer interfaced to a commercial CE system. This paper discusses optimization of the interface design for minimization of background fluorescence and scattered light, thereby maximizing the sig nal-to-background ratio (S/B) of the dynamic measurement. Strategies includ ed using various combinations of optical filters including a holographic fi lter and longpass or bandpass filters, tilting the capillary relative to th e incident laser beam, employing a confocal design and adding an iris to re move out-of-focus light, using a microscope objective in the emission beam to increase the collection efficiency, and using square instead of ciruclar capillary columns. Significant improvements in S/B for on-column, on-the-f ly detection of fluorescein in CE were achieved with most modifications.