When a fluorophore absorbs a photon of light, an energetically excited state is formed. The fate of this species is varied, depending upon the exact nature of the fluorophore and its surroundings, but the end result is deactivation (loss of energy) and return to the ground state. The main deactivation processes which occur are fluorescence (loss of energy by emission of a photon), internal conversion and vibrational relaxation (non-radiative loss of energy as heat to the surroundings), and intersystem crossing to the triplet manifold and subsequent non-radiative deactivation.
The fluorescence quantum yield (ΦF) is the ratio of photons absorbed to photons emitted through fluorescence. In other words the quantum yield gives the probability of the excited state being deactivated by fluorescence rather than by another, non-radiative mechanism.
The most reliable method for recording ΦF is the comparative method of Williams et al.,1 which involves the use of well characterised standard samples with known ΦF values. Essentially,
solutions of the standard and test samples with identical absorbance at the same excitation wavelength can be assumed to be absorbing the same number of photons. Hence, a simple ratio of the integrated fluorescence intensities of the two solutions (recorded under identical conditions) will yield the ratio of the quantum yield values. Since ΦF for the standard sample is known, it is trivial to calculate the ΦF for the test sample.
In practice, the measurement is slightly more complicated than this because it must take into account a number of considerations. For example:
• The presence of concentration effects, e.g. self-quenching;
• The use of different solvents for standard and test samples;
• The validity in using the standard sample and its ΦF value.
These considerations are answered by
• Working within a carefully chosen concentration range and acquiring data at a number of different absorbances (i.e. concentrations) and ensuring linearity across the concentration range;
• Including the solvent refractive indices within the ratio calculation;
• Cross-calibrating the standard sample with another, to ensure both are behaving as
expected and allowing their ΦF values to be used with confidence.
The measurement of ΦF values is challenging if the values are to be trusted. Incorrect quantum yields are all too easy to obtain!