Definition
Quenching refers to any process that decreases the fluorescence intensity of a sample.
It can be due to various mechanisms, both dynamic and static.
Types of Quenching
Dynamic Quenching (Collisional Quenching)
Mechanism: Occurs when the excited fluorophore collides with a quencher molecule, returning to the ground state without emitting a photon.
Temperature Dependence: Increases with temperature due to higher collision frequencies.
Static Quenching
Mechanism: Formation of a non-fluorescent ground-state complex between the fluorophore and quencher before excitation.
Concentration Dependence: Increases with the concentration of quencher.
Self-Quenching and Self-Absorption
Self-Quenching: Fluorophore molecules quench each other's fluorescence at high concentrations.
Self-Absorption: Emitted photons are reabsorbed by other fluorophore molecules.
Energy Transfer
Förster Resonance Energy Transfer (FRET): Non-radiative transfer of excitation energy from a donor to an acceptor molecule.
Implications
Analysis: Quenching studies can provide information on molecular interactions and distances.
Applications: Used in biochemical assays to study protein folding, ligand binding, and conformational changes.
Factors Influencing Quenching:
Quencher Concentration: Higher concentrations enhance quenching efficiency.
Temperature: Increases dynamic quenching due to faster diffusion; reduces static quenching by lowering complex formation.
Solvent Viscosity: Higher viscosity slows mobility, reducing collisional quenching.
Spectral Overlap: Greater overlap improves FRET efficiency and energy transfer rates.