The popular textbook and literature model I(λ_x, λ_m) = K(λ_x, λ_m)(1 - 10^-A_x) or its variants for correlating the sample absorption and fluorescence often fails, even for the simplest samples where the fluorophore is the only light absorber. Reported is a first-principles model I(λ_x, λ_m) = K(λ_x, λ_m)A_x,f10^{-(A_x,sd_x+A_m_,sd_m)} for correlating the sample fluorescence measured with a conventional spectrofluorometer and its UV-vis absorbance quantified with a UV-vis spectrophotometer. This model can be simplified or expanded for a variety of fluorescence analysis. First, it enables curve-fitting fluorescence intensity as a function of the fluorophore or sample absorbance over a sample concentration range that is currently impossible with existing models. Second, it provides the theoretical foundation for an inner-filter-effect (IFE)-correction method developed earlier and explains mathematically the linearity between the IFE-corrected fluorescence and the fluorophore concentration or absorbance. Third, this model can be expanded for quantitative mechanistic studies of the fluorescence intensity variations triggered by stimuli treatments. One demonstrated example is quantifying the temperature effects on the wavelength-specific and total quantum yield of three different polyaromatic hydrocarbons and xanthene fluorophores. This was achieved by utilizing a total photoluminescence spectroscopy (TPL) based approach, where the fluorescence intensity was measured at all excitation and emission wavelengths. When combined with UV-vis measurements, this TPL method enables experimental quantification of temperature effects on fluorophore fluorescence intensity and quantum yield at any combination of excitation and detection wavelengths. We expect that this first-principles model will be broadly adopted for both student education and a variety of fluorescence applications where disentangling sample absorption and emission are critical for reliable data analysis.

Xin Cui

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