Biological Analysis

Molecular Fluorescence Spectroscopy Analysis

Molecular fluorescence spectroscopic analysis is a method for qualitative or quantitative analysis of substances by utilizing the characteristics and intensity of fluorescence generated when molecules of certain substances are irradiated by light.

When a substance molecule absorbs photons of characteristic frequency, it transitions from the original ground state energy level to different vibrational energy levels of the electronic excited state. The excited state molecule consumes part of the energy by colliding with the surrounding molecules, rapidly drops to the lowest vibrational energy level of the first electronic excited state, and stays for about 10-9 seconds (10 minus 9 seconds), and then directly emits light with light. The form releases excess energy and drops to various vibrational energy levels of the electronic ground state, and the light emitted at this time is fluorescence.

The first necessary condition for generating fluorescence is that the molecule of the substance must have a structure that can absorb excitation light, usually a conjugated double bond structure; the second condition is that the molecule must have a certain degree of fluorescence efficiency, that is, after the fluorescent substance absorbs light. The ratio of the emitted fluorescence quantum number to the absorbed quantum number of excitation light. The wavelength and intensity of the excitation light are kept unchanged, and the fluorescence emitted by the fluorescent substance is irradiated on the detector through the emission monochromator, that is, scanning is performed, with the fluorescence wavelength as the abscissa and the fluorescence intensity as the ordinate. , which is the fluorescence spectrum, also known as the fluorescence emission spectrum.

Let the excitation light of different wavelengths excite the fluorescent substance to generate fluorescence, and let the fluorescence irradiate the detector with a fixed emission wavelength, and then take the excitation light wavelength as the abscissa and the fluorescence intensity as the ordinate. Fluorescence excitation spectroscopy. The shape of the fluorescence emission spectrum is independent of the wavelength of the excitation light.

For dilute solution (absorbance A=εcl≤0.05), its fluorescence intensity F=2.3jI0εcl. where j is the fluorescence efficiency of the fluorescent substance; I0 is the incident light intensity; ε is the molar absorption coefficient of the fluorescent substance, c is the concentration of the fluorescent substance, and l is the thickness of the sample cell. This formula shows that the fluorescence intensity is proportional to the concentration of the substance in a dilute solution (A≤0.05) and I0 and l remain unchanged. The fluorescence intensity of solution is also affected by solvent, temperature and pH value. It is also reduced due to the interaction of fluorescent substances and other solute molecules, a phenomenon known as fluorescence quenching.

Detection settings
An instrument for molecular fluorescence spectroscopic analysis is called a fluorescence spectrophotometer. It consists of 5 parts: light source; monochromator; sample cell; detector; display device.
Fluorescence excitation and emission spectra can be used to identify organic compounds. When cooled to 77K, a highly resolved low-temperature fluorescence spectrum can be obtained, which is helpful for identification. Synchronous scanning fluorescence method, 1-4 order derivative fluorescence spectrum and three-dimensional spectrum, etc. can also be used to identify multi-component fluorescent substances.