Ultraviolet analyzer is the application of fluorescence technology. What is fluorescence technology? First, let's understand what fluorescence is.
When a substance is irradiated at room temperature with incident light of a certain wavelength (usually ultraviolet or X-rays), it goes into an excited state after absorbing light energy and is immediately de-excited and emitted again light The emitted light with a long wavelength (typically the wavelength is in the visible light band); and as soon as the incident light is stopped, the luminescence phenomenon disappears immediately.
The light emitted with this property is called fluorescence. As the name suggests, if you know what fluorescence is, you can think about what fluorescence technology is.
Fluorescence technology means that some substances, after being exposed to light of a certain wavelengthe were excited to emit light at a wavelength greater than the excitation wavelength in a very short time (10-8 seconds). for fluorescence. The application of this luminescence phenomenon in different aspects and related methods are called fluorescence techniques.
There are two types of phenomena in which substances emit fluorescence after irradiation with ultraviolet rays: The first is autofluorescence. It is autofluorescence; the second is induced fluorescence, that is, the object is stained with a fluorescent dye and then fluoresces by ultraviolet radiation, which is called induced fluorescence.
The application of fluorescence technology in biochemical and molecular biological research mainly includes the following aspects:
1. The quality of substances: different fluorescent substances have different excitation - and emission spectra, so that fluorescence can be used to identify substances. Fluorescence is more selective than absorption spectroscopy.
2. Quantitative Determination: Using the relationship that the fluorescence intensity is proportional to the sample concentration at a lower concentration, the content of fluorescent components in the sample can be analyzed quantitatively. This is often used to determine the content of amino acids, proteins and nucleic acids.
An advantage of quantitative fluorescence determination is its high sensitivity. For example, the limit of quantification of vitamin B2 can reach 1 ng/ml. This advantage significantly reduces the amount of sample required for the determination.
This quantitative determination method can also be applied to enzyme-catalyzed reactions. If the fluorescence intensity changes before and after the reaction, it can be used to determine the enzyme genehalts to be used and the rate of the enzyme reaction.
3. Study the physical and chemical properties of biological macromolecules, as well as their molecular structures and conformations: Parameters such as fluorescence excitation spectrum, emission spectrum, quantum yield and fluorescence lifetime are not only related to intramolecular fluorescence The structure of the chromophore itself is related and also highly dependent on the environment of the chromophore, that is, it is very sensitive to the environment.
This feature allows to study the properties and changes of the microenvironment where the fluorescent chromophore resides by measuring the changes of the above mentioned fluorescence parameters.
In this study, in addition to using the fluorescent chromophores (such as tryptophan, tyrosine, guanylic acid, etc., referred to as endogenous fluorescence), biological Macromolecules some specific fluorescent dye molecules covalently bound or adsorbed to a Certain parts of biological macromolecules and biological macromolecules can be studied by measuring the changes in the fluorescent properties of the dye molecules. Such dye molecules are called “fluorescent probes” and emit fluorescence, commonly referred to as extrinsic fluorescence.
The use of fluorescence probes has greatly expanded the scope of fluorescence technology in molecular biology.
4. The energy transfer phenomenon in biomacromolecules can be studied using parameters such as fluorescence lifetime and quantum yield: By studying this phenomenon, a lot of information can be obtained inside biomacromolecules.