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 hυ’ 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