Fluorescence
Fluorescence spectroscopy is an extremely useful and ultrasensitive analytical technique to analyze the fluorescence emission of certain molecules that arises from the excitation with ultraviolet or visible light.
The fluorescent properties of these molecules can be detected using spectrofluorometers, a spectroscopic instrument designed with a right-angle configuration to measure the various parameters of fluorescence, including its intensity and wavelength distribution over the optical range.
What is Fluorescence?
Fluorescence is a form of luminescence (emission of light that is not associated with a rise in temperature) that results from the transition between the two lowest electronic states (the first excited state and the ground state), of the same nature.
When a molecule is irradiated with ultraviolet and visible light, it might be temporarily raised from low energy and equilibrium state (ground state) to high energy and non-equilibrium state (excited state), where it has an excess of energy. The probability of finding this molecule in one possible excited state (almost all molecules encountered in our life exist in a singlet state, where all electrons in the molecule are spin paired) depends on the transition probabilities and the excitation wavelength. The Perrin-Jablonski diagram is a convenient way for resuming the physical processes occurring since the molecule absorbs light until it returns once again to the ground state, see Figure 1.
Figure 1 – Simplified Perrin-Jablonski diagram describing the electronic levels of common organic molecules.
Being a non-equilibrium state, the amount of time that the molecule stays in S1 is usually too short (around 10-9 seconds). Depending on the structure of the excited molecule, a radiative transition from S1 might occur by the spontaneous emission of a photon. This process is called fluorescence emission. The fluorescence emission spectrum is always found at higher wavelengths than the absorption spectrum because of the energy loss in the excited state due to a number of energy transfer processes.
Typical setups for Fluorescence Measurements
Sensitivity is a key factor when performing fluorescence measurements.
To achieve high sensitivity in our compact and modular spectrometers, it is important to combine, on the emission detection side, the high sensitivity of a back-thinned CCD detector (choose the thermoelectrically cooled version for getting the ultimate sensitivity), which has a maximum quantum efficiency of around 80% (at the peak), with collecting lens and large entrance slits (200 µm).
On the excitation side, a high-power light source is crucial. Sarspec has two great light sources capable of delivering a stable and high-power excitation: LEDs light source (LS-LED) for narrow bandpass excitation over the UV-Vis-NIR wavelength range and Xenon Flash lamp light source (LS-XeF) when excitation needs to be performed with power in deep-UV.