Fluorescence Spectroscopy - Analysis of Data

Analysis of Data

At low concentrations the fluorescence intensity will generally be proportional to the concentration of the fluorophore.

Unlike in UV/visible spectroscopy, ‘standard’, device independent spectra are not easily attained. Several factors influence and distort the spectra, and corrections are necessary to attain ‘true’, i.e. machine-independent, spectra. The different types of distortions will here be classified as being either instrument- or sample-related. Firstly, the distortion arising from the instrument is discussed. As a start, the light source intensity and wavelength characteristics varies over time during each experiment and between each experiment. Furthermore, no lamp has a constant intensity at all wavelengths. To correct this, a beam splitter can be applied after the excitation monochromator or filter to direct a portion of the light to a reference detector.

Additionally, the transmission efficiency of monochromators and filters must be taken into account. These may also change over time. The transmission efficiency of the monochromator also varies depending on wavelength. This is the reason that an optional reference detector should be placed after the excitation monochromator or filter. The percentage of the fluorescence picked up by the detector is also dependent upon the system. Furthermore, the detector quantum efficiency, that is, the percentage of photons detected, varies between different detectors, with wavelength and with time, as the detector inevitably deteriorates.

Two other topics that must be considered include the optics used to direct the radiation and the means of holding or containing the sample material (called a cuvette or cell). For most UV, visible, and NIR measurements the use of precision quartz cuvettes is necessary. In both cases, it is important to select materials that have relatively little absorption in the wavelength range of interest. Quartz is ideal because it transmits from 200 nm-2500 nm; higher grade quartz can even transmit up to 3500 nm, whereas the absorption properties of other materials can mask the fluorescence from the sample.

Correction of all these instrumental factors for getting a ‘standard’ spectrum is a tedious process, which is only applied in practice when it is strictly necessary. This is the case when measuring the quantum yield or when finding the wavelength with the highest emission intensity for instance.

As mentioned earlier, distortions arise from the sample as well. Therefore some aspects of the sample must be taken into account too. Firstly, photodecomposition may decrease the intensity of fluorescence over time. Scattering of light must also be taken into account. The most significant types of scattering in this context are Rayleigh and Raman scattering. Light scattered by Rayleigh scattering has the same wavelength as the incident light, whereas in Raman scattering the scattered light changes wavelength usually to longer wavelengths. Raman scattering is the result of a virtual electronic state induced by the excitation light. From this virtual state, the molecules may relax back to a vibrational level other than the vibrational ground state. In fluorescence spectra, it is always seen at a constant wavenumber difference relative to the excitation wavenumber e.g. the peak appears at a wavenumber 3600 cm−1 lower than the excitation light in water.

Other aspects to consider are the inner filter effects. These include reabsorption. Reabsorption happens because another molecule or part of a macromolecule absorbs at the wavelengths at which the fluorophore emits radiation. If this is the case, some or all of the photons emitted by the fluorophore may be absorbed again. Another inner filter effect occurs because of high concentrations of absorbing molecules, including the fluorophore. The result is that the intensity of the excitation light is not constant throughout the solution. Resultingly, only a small percentage of the excitation light reaches the fluorophores that are visible for the detection system. The inner filter effects change the spectrum and intensity of the emitted light and they must therefore be considered when analysing the emission spectrum of fluorescent light.