Improving signal to background ratio for on-the-fly fluorescence lifetime detection in capillary electrophoresis.
On-the-fly fluorescence lifetime detection (OFLD) in capillary electrophoresis (CE) was previously demonstrated using a commercial multiharmonic Fourier 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 signal-to-background ratio (S/B) of the dynamic measurement. Strategies included using various combinations of optical filters including a holographic filter and longpass or bandpass filters, tilting the capillary relative to the incident laser beam, employing a confocal design and adding an iris to remove 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-fly detection of fluorescein in CE were achieved with most modifications.
Li
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Department of Chemistry, Duke University, Durham, NC 27708-0346, USA.
McGown
L B
LB
D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D012815:Signal Processing, Computer-Assisted; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*