Fluorescence quenching and Stern-Volmer equation

Abstract: in this article we describe some fluorescence quenching measures in some substances. The phenomenon of quenching occurs when some molecules absorb the energy of the fluorophores. This interesting phenomenon can be used in quantitative measurements for the determination of the quencher concentration and also, through the Stern-Volmer model, for the study of the temporal decay of the fluorescence.

Introduction

The quenching is the phenomenon through which the fluorescence is dampened; it is due to molecules that absorb energy (for example metal atoms or oxygen). If the quenching of the fluorescence is due to molecules adjacent to the emitter molecule, which are part of the same material, we speak of self-quenching.

Quenching can be static or collisional. Static quenching consists in the formation of a non-fluorescent complex between the fluorophore, i.e. the molecule that fluoresces, and the quencher, i.e. the molecule responsible for the phenomenon in question, which is located near the fluorophore. In collisional-type quenching, the quenchers, on the other hand, are scattered in solution, and when they collide with the fluorophore, they absorb its energy.

Quenchers are molecules with a high vibrational degree of freedom, such as water, which absorbs almost all the energy of the fluorophore, or paramagnetic ions or even ions of heavy atoms such as iodine, bromine, etc. Collision-type quenching is favored by the temperature, which evidently increases the kinetic energy. This reduces fluorescence and phosphorescence, this explain why fluorescence and phosphorescence occur mainly at low temperatures. The kinetics of the process is described by the Stern-Volmer equation.

From the study of fluorescence quenching, using the Stern-Volmer model, it is also possible to obtain quantitative indications on the decay time of the fluorescence itself.

Experimental Setup

The experimental setup consists of the system for fluorescence measurements, described in the previous post fluorescence spectroscopy. It consists of a cuvette holder, a led excitation source and a fiber optic spectrometer (we used a reconditioned B&W Tek). The apparatus is shown in figure 1.

Fig.1 – Setup for measuring the fluorescence spectrum

The excitation source of the fluorescence is a high quality LED driven by a driver which guarantees stability of the output current, in order to have a constant excitation light intensity during the measurements. We used two LEDs, one that emits in the near ultraviolet, for quinine measurements, and one that emits in the blue band, for fluorescein measurements. The UV LED is shown in figure 2, while in figures 3 and 4 the emission spectra of the two LEDs are reported.

Fig.2 – Excitation LED

Fig.3 – Emission spectrum of UV LED

Fig.4 – Emission spectrum of blue LED

Measurements on quinine quenched with NaCl

Fig.5 – Fluorescence spectra of quinine at various concentrations of the quencher NaCl

Fig.6 – Regression line of the Stern – Volmer equation for quinine fluorescence

Measurements on fluorescein quenched with KI

Fig.7 – Fluorescence spectra of fluorescein at various concentrations of the KI quencher

Fig.8 – Regression line of the Stern – Volmer equation for the fluorescence of fluorescein

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