Fluorescence Quenching Any process which decreases the fluorescence intensity of a sample

Collision/ Dynamic

Quenching

Static Quenching

Apparent Quenching

Collision returns fluorophore to G.S. without photon emission, Quencher must diffuse to fluorophore during lifetime of excited state.

Binding, Complex formed is non-fluorescent

Optical density, turbidity, etc not useful

Molecular oxygen best Paramagnetic, spin-orbit coupling, intersystem crossing to long-lived, easily quenched triplet state. Iodine, Bromine (heavy atoms)

Amines, chlorinated hydrocarbons excited-state charge-transfer complex. Fluorescence from complex is quenched in polar solvents.

F0/F = 1 + KDτ0[Q]

F0/F = 1 + KS[Q] Stern-Volmer Eqn.

intercept slope

1 -- KS or KD

KD = Kqτ0 Fluorescence lifetime in the absence of quencher

Bimolecular quenching constant

F0/F

[Q]

Deviation from Linearity

Linearity all fluorophores are equally accessible to quenchers

Bend towards x-axis Quenching starts to saturate because few of the fluorophore molecules are inaccessible (How many Trp residues are on the surface of protein?)

Bend towards y-axis Combination of Static quenching and Dynamic quenching (second order in [Q])

F0/F = (1 + KS[Q])(1 + KD[Q])

F0/F = 1 + Kapp[Q]

Kapp = ((F0/F) – 1) / [Q] = KS + KD + KSKD [Q]

KS + KD -- KSKD

Kapp

[Q]

In this case, quenching is either Static or Dynamic but not both. How do we decide which mechanism is at play? Static – Dynamic τ0/τ = 1 -- τ0/τ = F0/F Slope falls wit T – rises with T Absorption spectra changes – no change

The Bimolecular Quenching constant reflects: 1. Efficiency of Quenching 2. Diffusion Coefficient of Quencher 3. Accessibility of Fluorophore to Quencher

localization, membrane permeability

Here, we can see a change in protein conformation due to substrate binding because the extent of quenching changes.

Application of Quenching