Electrostatic Forces in Dynamic Fluorescence Quenching

The quenching constant for the iodide quenching of the fluorescence of riboflavin phosphate was measured at pH 5 and at pH 8. The value of the constant at the lower pH (where the phosphate group is unionized), k‘= 23 M‐1 was independent of the ionic strength, μ. The ratio k“/k’= f(k” the quenching constant at pH 8, where the phosphate group carries a single negative charge) was found to be 0.70 at μ=0.025 and 0.79 at μ= 0.045. The fact that f < 1 is interpreted as due to electrostatic repulsion between the dye molecule, when ionized, and the iodide ion. Due to counter ion screening the effect is less accentuated at higher μ. The quenching constant for riboflavin (which does not contain the ionizable phosphate group) was found to have the same value, 30 M‐1 both at pH 5 and at pH 8, and with no dependence on μ.