Dear Mohamed,

the bimolecular quenching is typically quantified in terms of:

k(obs) = k(0) + k(q) x [Q]

k(0) denotes the intrinsic excited-state decay rate constant in ABSENCE of a quencher, that is, in your case k(0) = 5 x 10(6) / s

k(obs) denotes the  actually measured decay rate constant, that is, in your case k(obs) = 10 x 10(6) / s

k(q), which is the desired bimolecular quenching constant, then derives as k(q) = (k(obs) - k(0))/[Q], that is, in your case

k(q) = (5 x 10(6))/0.01 = 5 x 10(8) / M / s

One note of caution: As your quenching data relies on only ONE single quencher concentration, the result will contain a huge intrinsic uncertainty. One should rather add a more complete concentration dependence.

Best regards

Gerald

Hi Mohamed,

If you are using steady-state fluorescence to measure fluorescence quenching, you can determine the bimolecular quenching rate by constructing a Stern-Volmer plot.

Plotting F0/F against quencher concentration, ideally (ignoring static quenching) you will get a straight line.  The line will take the linear form:

F0/F  =  1 +  kqt0 [Q]

F0 is the fluorescence intensity in absence of quencher, F is the fluorescence intensity at the quencher concentration [Q], the y-intercept will be 1, the slope of the line will be kqt0, where kq is the bimolecular quencning constant and t0 is the fluorescence lifetime of the fluorophore in absence of quencher.

If you have significant curvature in the plotted data, then you have a problem with static quenching resulting from the formation of ground state complexes.  One way to avoid issues with static quenching is to measure fluorescence lifetimes and plot t0/t against quencher concentration.  The line generated will take the same linear form.

t0/t  =  1 + kqt0 [Q]

I hope this helps.

Best

John

John E. Hansen helpful answer. Thanks!

Is it possible to calculate the quenching rate constant in the absence of a lifetime measurement value?