Fluorescence Concentration Calculation Formula


Quantitative fluorescence measurements are used to determine amounts or concentrations of analytes in unknown samples. These quantities may be determined in absolute units, such as moles or moles per L, or in relative units, such as the ratio of the concentrations of two fluorescent analytes contained in a single unknown solution. These determinations use the following proportionality relating fluorescence signal (S) at a given pair of excitation and emission wavelengths (λex, λem) to fluorescent analyte concentration (c):


  • /0 = intensity of the excitation beam
  • Ω = fraction of the fluorescence collected by the detection system
  • Rd = responsivity of the detection system
  • α = absorption coefficient
  • Φ = fluorescence quantum yield
  • c = concentration of the fluorescent analyte

This linear proportionality with concentration applies to optically dilute samples (e.g., solutions with an absorbance of less than 0.05 at a path length of 1 cm).

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Reference: USP 〈853〉

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