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.

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.

 

Figure 1 – Simplified Perrin-Jablonski diagram describing the electronic levels of common organic molecules.

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 Flashlamp light source (LS-XeF) when excitation needs to be performed with power in deep-UV.

 

Fluorescence with Cuvettes

The Fluorescence Cuvette Pack is the ideal bundle for cuvette-based fluorescence measurements. This package combines a stable and high-power LED  FLEX PLUS spectrometer that is configured with 200-µm slit for achieving the best sensitivity and maximum detection efficiency at 500 nm. The cuvette holder has also an important role in the sensitivity and flexibility of this package. The multipurpose cuvette holder is set into a right-angle configuration and uses 12.7-mm diameter lenses to achieve a large sample area excitation and collection. These larger lenses are combined with enhancing mirrors to minimize the loss of excitation and emission energy. The multipurpose cuvette holder also supports a wide range of user-interchangeable filters. The package is completed with two 1000-µm core diameter optical fibers (each with 50-cm length) and the LightScan software.

 

Fluorescence with Flow Cells

The Fluorescence FLOW Pack is the ideal bundle to combine the flow injection analysis with fluorescence measurements. This package combines a stable and high-power LED with improved speed and performance FLEX PLUS spectrometer that is configured with 200-µm slit for achieving the best sensitivity and maximum detection efficiency at 500 nm. The package is completed with a Flow Cell FCFPE or FCFSS in a right angle configuration and with sapphire windows that are coupled through adjustable focus SMA 905 optical connectors to 1000-µm core diameter optical fibers (each with 50-cm length)