Quenching of Fluorescence

Quenching of Fluorescence:

Use of the Stern-Volmer Equation

Introduction

Excitation of some chemical compounds when radiated with visible or UV light results in electronic transitions to higher energy levels. The extent to which light of various wavelengths absorbed constitutes the absorption spectrum of the compound.

The spacing between levels in the two electronic states can be measured by either absorption or emission spectroscopy. Emission occurs following an absorption event if the upper state is not relaxed by non-radiative collisional process (called quenching). Dynamic quenching, also called collisional quenching, requires contact between the excited species and the quenching agent (Q). Dynamic quenching occurs as rapidly as the collision partners can diffuse together. The rate is temperature and viscosity dependent. The quencher concentration must be high enough that there is a high probability of collision between the excited species and the quencher during the lifetime of the exited state.

When fluorescein is illuminated with light that has a wavelength of 490 nm an electronic transition occurs. At room temperature most molecules in their lowest vibrational level of the ground electronic state and on absorption of light reside in the lowest vibrational state of the lowest excited state (level 0 of state S1). The molecule relaxes releasing a quantum of light as it returns to any one of vibrational-rotational levels of the ground state.

The process of excitation of molecule X by a photon of light is represented by:

where hП... is a photon of light and X* is the excited molecule. When the molecule undergoes the process of emission by dropping back to ground state, it is represented by:

This is the emission of fluorescence, where hП...’ is the emitted photon, with first order rate constant kf. Some of the X* is deactivated before it can emit a photon.

This is internal quenching. If the quenching substance, Q, is present it will result in further deactivation by interaction with X* and the fluorescence intensity will be further reduced.

(kQ is the rate constant).

In the absence of Q and under conditions of steady illumination and no irreversible photochemical reactions, a steady-state is reached, where by:

where Ia is the rate of light absorption. The X* molecules that fluoresce from a fraction of of the total number produced

This is called the fluorescence of quantum efficiency or quantum yield of fluorescence.

In the presence of the quenching agent Q, the process will compete with processes from (2) and (3) and a new steady state is reached:

Therefore, the quantum yield becomes

The ratio of equations (6) and (8) gives the Stern-Volmer equation

The intensity (I) of the fluorescent light is proportional to the quantum yield. As a result, the ratio of the intensity in the absence of quencher (I0) to that in the presence of a given concentration of quencher (I) is

In terms of П„, the equation becomes

Graphing I0/I with respect to the concentration of quencher will yield a line with slope kQП„i. The rate constant can then be determined if П„i is found by other means. The rate of the reaction is dependant on the rate at which the two substances diffuse together. This rate constant, the diffusion controlled bimolecular rate constant, k2, depend on the viscosity and temperature of the solvent. K2 is given by

where rA is equal to 6.0 Ð"... and is the fluorescent molecule radius and rB is equal to 2.2 Ð"... and is the quencher molecule radius. In this experiment rA is for fluorescein and rB is for the iodine. Equation (12) is for neutral species. The correction for a charged species is given by

where

For (14), T is the absolute temperature; ZA is the charge on the fluorescein (which is 2 for disodium salt); AB is the charge on iodine (which is -1); e is the electron charge in esu; Оµ is the dielectric constant of water at 25ЛљC (which is 78.54); k is the boltzman constant; and a is the distance from the carbon atom of fluorescein to an iodine molecule perpendicular to the plane of fluorescein (which is 4 Ð"...).

The Bronstead equation (14) has been used with frequent success to correct the rate constants for ion-ion

Procedure

A 2.5M stock solution of KI and must be prepared as well as a 5 ОјM fluorescein stock solution from solid fluorescein and 0.001M KOH. The fluorescein solution will be diluted to 1 ОјM.

The absorption and emission maxima for fluorescein for 490 nm and 515 nm, respectively, and the excitation and emission monochromators of spectrophotofluorimeter should be set to these values.

2.5 ml of the 1 ОјM fluorescein solution are pipeted into the cuvette to be used and the cuvette is placed in the sample chamber. After a 15-20 minute warm-up time for the instrument, the dark current and zero are adjusted. The excitation and emission shutters are opened and the instrument is adjusted to give 100% fluorescence intensity (I0) with the sample. The shutters are closed and subsequent measurements are made after the addition of various amounts of quencher. After the addition, the solution must be carefully mixed without loss of the liquid from the cuvette. The fluorescence intensity, I, is read following the addition. Readings should be made for the following total volumes added 2.5 M KI: 5, 10, 15, 20, 30, 40, 50, 60, 85, and 110 Ојl, corresponding to a range of iodide concentrations ranging from 0.005 M to 0.10 M.

The quenching constant should be measured as a function of viscosity. For this purpose, sufficient sucrose is dissolved in 3 aliquots of the 1 ОјM