Page 39 - Instrumental Analysis - Pharm D Clinical- 07-PA403

Spectrofluorimetry

Fluorescence and Quantum Efficiency

• Not all absorbing molecules give fluorescence.
• Fluorescence occurs when the molecules have structures that decrease the

rate of non-radiative relaxation to the point that there is chance for
fluorescence to occur.
• The efficiency of fluorescence is measured by the Quantum Yield or
Quantum Efficiency (Φ). The quantum yield is the ratio of the number of
molecules that fluoresce to the total number of excited molecules, or the
ratio of photons emitted to photons absorbed.

Φ=

• It ranges from 0 to 1. Highly fluorescent molecules, such as fluorescein,
have Φ values that approach 1. Molecules that do not fluoresce or that
show very weak fluorescence have Φ values that are essentially zero.

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Excitation and Emission Spectra

• Excitation spectrum: measuring fluorescence at a single emission wavelength
while the excitation wavelength is varied (scanned). Very similar in appearance
to the absorbance spectrum of the same molecule.

• Emission spectrum: measuring fluorescence at a single excitation wavelength
while the emission wavelength is varied (scanned). Excitation wavelength is
usually identical or close to the wavelength of maximum absorbance (λmax)
which is determined from the absorption spectrum of the compound.

• Excitation spectrum: variable λex and constant λem
Emission spectrum: constant λex and variable λem

• The wavelength of maximum emission (λem) is always longer (lower energy)
than the wavelength of maximum excitation (λex). The difference between the
two wavelengths is known as the Stokes Shift.

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