Collisional quenching is a process that deactivates excited fluorophores through molecular collisions. This phenomenon impacts fluorescence by decreasing intensity and shortening lifetimes, with factors like temperature and viscosity playing crucial roles.

The Stern-Volmer relationship quantifies quenching effects, relating fluorescence intensities to quencher concentration. This powerful tool allows scientists to determine quenching efficiency, measure quencher accessibility, and study protein conformational changes in various applications.

Collisional Quenching

Process of collisional quenching

• Collisional quenching deactivates excited fluorophores through molecular collisions without chemical alteration (acetone quenching rhodamine B)
• Quenching mechanism transfers energy from excited fluorophore to quencher or involves electron transfer between them (oxygen quenching tryptophan fluorescence)
• Factors affecting collisional quenching include temperature, viscosity of medium, and diffusion rates of fluorophore and quencher (glycerol increases viscosity, slowing quenching)
• Impact on fluorescence decreases intensity and shortens lifetime (oxygen quenching reduces anthracene fluorescence intensity)
• Common quenchers encompass oxygen, halogens, and amines (iodide quenching of fluorescein)

Stern-Volmer relationship in quenching

• Stern-Volmer equation $F_0/F = 1 + K_{SV}[Q]$ relates fluorescence intensities with and without quencher
• Linear relationship between $F_0/F$ and $[Q]$ allows determination of Stern-Volmer constant $K_{SV}$
• Applications include:
  • Determining quenching efficiency (acrylamide quenching tryptophan in proteins)
  • Measuring quencher accessibility to fluorophore (iodide quenching surface-exposed tryptophans)
  • Studying protein conformational changes (tryptophan quenching in folded vs unfolded states)
  • Assessing membrane permeability (oxygen penetration in lipid bilayers)

Stern-Volmer Analysis

Analysis of Stern-Volmer plots

• Stern-Volmer plot displays $F_0/F$ ratio vs quencher concentration $[Q]$
• Slope of plot represents Stern-Volmer constant $K_{SV}$, higher slope indicates more efficient quenching
• Distinguishing quenching types:
  1. Dynamic quenching produces linear plot
  2. Static quenching shows upward curvature
• Temperature effects differ:
  • Dynamic quenching: $K_{SV}$ increases with temperature (faster diffusion)
  • Static quenching: $K_{SV}$ decreases with temperature (complex dissociation)
• Fluorescence lifetime measurements reveal:
  • Dynamic quenching: $\tau_0/\tau = F_0/F$ (lifetime changes)
  • Static quenching: $\tau_0/\tau = 1$ (lifetime remains constant)

Applications of Stern-Volmer equation

• $K_{SV}$ relates to quenching rate constant: $K_{SV} = k_q\tau_0$
• Calculate quenching rate constant: $k_q = K_{SV}/\tau_0$
• Determine quencher concentration: $[Q] = (F_0/F - 1)/K_{SV}$
• Fraction of accessible fluorophores found using modified equation: $F_0/(F_0 - F) = 1/(f_aK_{SV}[Q]) + 1/f_a$
• Quenching sphere of action modeled by: $F_0/F = (1 + K_{SV}[Q])exp(V[Q])$