Simulation of Signal-to-Noise Ratio and Detection Limit of Fluorescence Spectroscopy

Signal-to-Noise Ratio and Detection Limit of Fluorescence Spectroscopy

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Computes the theoretical signal-to-noise ratio for a fluorescence specrometer with a continuum source (e.g. Xenon arc lamp) modeled as a blackbody, dispersive emission and excitation monochromators, and a photomultiplier detector, given the molar absorptivity and fluorescence quantum yield of the analyte at the excitation wavelength. Note that this simulation predicts only a theoretical maximum signal-to-noise ratio limited by detector noise and by the photon noise of the analyte's fluorescence emission. In practice, the measurement of small fluorescence intensities is likely to be limited by photon noise from spectral interferences, background fluorescence from the solvent, or the contamination on the sample cuvette. Even if the systematic error caused by background fluorescence interference is eliminated or compensated (for example by using synchronous scanning, wavelength modulation, derivative spectroscopy, or advanced chemometric techniques such as rank annihilation), the photon noise from the background remains and sets an ultimate limit to signal-to-noise ratio performance.

Assumptions: Room-temperature prompt fluorescence of a stable fluorophor in aqueous solution with right-angle geometry in a standard cuvette, measured with a dispersive spectrofluorometer; no background fluorescence from the solvent; scattering is resolved from the fluorescence; self-absorption is ignored in this version; sample cell is larger than entrance slit height and width; fluorescence emission is isotropic and completely fills the emission monochromator solid angle. Spectral response of the detector is much wider that the spectral bandpass of the monochromator.

Note: The initial values for molar absorptivity (5600) and quantum efficiency (0.53) are for quinine sulfate in aqueous solution excited at 350 nm.

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(c) 2008, Prof. Tom O'Haver , Professor Emeritus, The University of Maryland at College Park. Comments, suggestions and questions should be directed to Prof. O'Haver at toh@umd.edu. Number of unique visits since May 17, 2008: