Fluorescence: The physical background

Fluorescence is the spontaneous emission of light following excitation of a material.

When a chemical compound is exposed to light, the compound can absorb the energy from the light, which stimulates the electrons and raises an electron to an energetically higher state. Various basic transition types, referred to as photophysical primary processes, can take place between the energy states. These processes, which lead from the absorption of stimulation light to emission, are often represented with the aid of Jablonski diagrams:

FluoreszenzRgb

Both the absolute position of the individual energy states and the probability of transitions between the individual energy levels are specific to a given substance. The excited state at the S1 level is relatively long lasting, with the result that it may engender a number of competing processes, as listed below:

  • Further internal conversions with subsequent radiationless deactivation,
  • Emission of radiation from the S1 state into the S0 base state (this radiation is called fluorescence.),
  • Photoreactions, in which the “electronically” activated molecule forms other, non-fluorescing reaction products, and
  • Conversion to the triplet state with subsequent deactivation. Emission from T1 to S0 (phosphorescence) only occurs if a further intercombination and subsequent radiationless deactivation do not occur.

The emitted fluorescence spectrum is usually shifted toward longer wavelengths due to radiationless energy losses in the electronically excited state (Stokes shift).
Fluorescence quanta may not be emitted if the excitation energy:

  • is lost due to collisions with other molecules (radiationless deactivation, concentration quenching),
  • is transferred to other molecules in the form of electronic energy (sensitised, also delayed fluorescence; these molecules can function as fluorescence quenchers, but also as fluorescence amplifiers), or
  • following prior conversion into the triplet state, passes to a long-lived level and is emitted as a phosphorescence light quantum.

Thus, the property of fluorescence is heavily influenced by its chemical environment, such as a solvent, for example. This influence affects the fluorescence yield and / or the duration of the excited state. This change is caused by the radiationless deactivation of the excited state of the fluorophor (quenching).

The most important types of quenching processes are dynamic and static fluorescence quenching. In dynamic (collisional) fluorescence quenching, the excited molecule is reduced from the S1 state to the S0 base state by energy exchange with the quencher. This exchange of energy must take place during the time in which the fluorescing molecule is in the excited state. In static quenching, the dye molecule and the quencher form a complex in the base state, which does not fluoresce even in the excited state, but instead returns to the S0 base state without radiating. A feature shared by both quenching processes is that their effectiveness depends on the concentration of quencher molecules, and that direct molecular contact between the fluorophor and the quencher is essential for quenching to take place.

One possible way to improve the fluorescence yield and at the same time reduce quenching processes is to reduce the mobility of the fluorophors and the quenchers in the solution by lowering the temperature. Such an increase in fluorescence caused by lowering the temperature is called the Arrhenius behaviour of the fluorophor. Different Arrhenius parameters are often used to localise the individual excited states. (Bowen and Cook, 1953; Bowen and West, 1955; Lampert and Phillips, 1985).

Bowen, E.J., Cook, R.J. (1953) Temperature Coefficients of Fluorescence. J. Chem. Soc. 3059-3061
Bowen, E.J., West, K. (1955) Solvent Quenching of the Fluorescence of Anthracene. Chem. Soc. 4394-4395
Lampert, R.A., Phillips, D. (1985) Photophysics of meso-Substituted Anthracenes. J. Chem. Soc., Faraday Trans. 81, 383-393