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Luminescent analysis

Luminescent analysis is a combination of analysis methods based on the observation of luminescence. To excite luminescence (see), the object under study is exposed to ultraviolet light (a quartz mercury lamp ). Observation of the glow is made in a dark room visually or with the help of special devices (fluorometers).

Qualitative luminescent analysis is based on the difference in the color of luminescence produced by substances of different chemical nature; quantitative luminescence analysis — on measuring the luminescence intensity using fluorometers or by recording the luminescence spectra with special spectrographs. Luminescent analysis is widely used to determine vitamins, hormones, antibiotics, carcinogenic substances, medicinal substances, etc. in various materials, including biological objects (blood, urine, tissues, etc.).

Fluorescent analysis in hygiene. In the sanitary-hygienic practice, fluorescent analysis has become widespread in assessing the quality of food products (the presence of impurities, food spoilage). Depending on the freshness of meat, fish and other food products, different shades and intensities of the luminescent glow appear. For analysis, you can use the aqueous extract of the products (the crushed sample is mixed with a diet, water in the ratio of 1:10, and shaken). The hoods are placed in a fluorometer, which allows you to measure the intensity of luminescence. For example, the luminescence intensity of an extract from fresh meat (fresh or chilled) is so small that it cannot be measured in the device. In extracts of meat, conditionally suitable, the luminescence intensity is significantly higher and ranges from 18 to 30 conventional units. Instrument readings in excess of 30 characterize meat as stale. When determining the freshness of fish and fish products, quantitative methods of luminescence analysis allow to fix the initial stage of damage to the product.

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A luminescent method for the determination of proteins in milk is proposed, with the help of which, without the use of reagents, an analysis can be performed in 3-5 minutes. (instead of 8-12 hours according to the Kjeldahl method). The method is based on measuring the integral intensity of the tryptophan luminescence of milk proteins, diluted ten times with water, when excited by ultraviolet light (wavelength 250-290 microns). Fluorescent analysis can also be used to determine water quality. The luminescent luminescence of waters is caused by the organic substances contained in them, as well as organisms living in water, algae cells and the remains of aquatic plants. For clear waters of water pipes and artesian wells, weak-violet luminescence is characteristic. The water of the Moscow and Leningrad aqueducts luminesces very weakly; distilled water practically does not glow. Samples of waters with a bluish and bluish tint of light are characteristic of waters with varying degrees of pollution. Waters with high chromaticity glow in yellow-green. Food industry waste waters are distinguished by a bright (blue, purple, blue, reddish) glow. The luminescence of humic compounds masks the luminescence of organic compounds trapped in water along with pollution.

Quantitative luminescence analysis is based on the relationship that exists between the luminescence intensity and the concentration of the luminescent substance. At low concentrations of a substance in a solution, the luminescence intensity is proportional to its content. At high concentrations of the luminescent substance, this proportionality is disturbed. The technique of quantitative luminescent analysis consists in the empirical determination of the relationship between the concentration of the analyte and the intensity of the luminescent glow. Pre-establish the same dependence for a series of standard solutions with a previously known amount of the substance to be determined. According to the data obtained during the measurement of a series of standard solutions, a calibration graph is constructed, according to which the concentration of a substance is determined by the intensity of the luminescent radiation of the analyzed solution. In a number of methods for quantitative luminescence analysis, not the previously prepared calibration schedule is used, but the intensity of the luminescence of a standard solution determined during the analysis.

Luminescent analysis can be used to determine carcinogenic substances, uranium in various objects of the external environment, insecticides and petroleum products in the water of open water bodies, etc. See also Fluorescence microscopy.

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Luminescent analysis - a set of analysis methods based on the phenomenon of luminescence (see).

The advantage of the methods of luminescence analysis is a very high sensitivity. Thus, the fluorescein dye can be detected by its luminescence at a concentration in the solution of about 10 -8 mol / l, and by the absorption of light on a spectrophotometer - only in concentrations of 10 -5 —10 -6 mol / l. In a wide range of concentrations that do not exceed the maximum, at which the absorption (absorption) of the exciting light and the reabsorption of the luminescent glow become significant, the luminescence intensity of a substance is proportional to its concentration. Therefore, by measuring the intensity of the luminescence of a solution with a known concentration of a substance, one can then easily determine the concentrations of this substance in the studied solutions. The advantages of luminescent analysis methods are even more pronounced when working with multicomponent media, if at the same time only the substance to be detected fluoresces or at least there is a spectral region (it can be distinguished using optical filters) in which it mostly fluoresces. For this purpose photoelectric fluorimeters are used.

To excite the luminescence of substances absorbing in the ultraviolet part of the spectrum, mercury-quartz, hydrogen and deuterium lamps are used, emitting a significant part of the light power in this part of the spectrum. For the excitation of substances that absorb visible waves, use optical incandescent lamps with small, "point" spirals. Isolation of a given spectral region, which is well absorbed by the test compound (and little absorbed by other luminescent substances, if any) from the total light flux, is achieved by using various optical filters. Interference filters are the most selective, which can pass a band just 10 mmk wide. Unknown compounds are determined by removing the luminescence spectra using spectrofluorimeters.

A significant number of unstained organic compounds characterized by absorption in the ultraviolet part of the spectrum (for example, proteins, amino acids, nucleic acids, carbohydrates and their metabolites) give a broad, less specific luminescence band in the range of close (ultraviolet, violet and blue) wavelengths; their identification by luminescence spectra is often difficult. In these cases, the studied material is frozen to —100a (sometimes freezing can be replaced by suspending in oil); at the same time, the luminescence spectra are narrowed and reveal a specific fine structure.

Sometimes the luminescence spectra react sensitively to minor changes in the state of a substance (aggregation, changes in the redox potential, changes in molecules associated with changes in pH, changes in the secondary structure, etc.). This also changes other characteristics of the compounds. So, during chlorophyll aggregation, its luminescent luminescence shifts towards the long waves, and the luminescence yield and its duration fall several times. Pyridine nucleotide ceases to luminesce when it is transferred from the reduced to the oxidized state. The fluorescence of flavins strongly depends on the pH of the medium, etc.

In addition to direct methods of luminescent analysis, there are a number of indirect. For example, normally non-fluorescent blood acquires this property when sulfuric acid is added to it. Oxygen is known as an effective luminescence quencher for many compounds; This phenomenon is used as an indicator for oxygen content.

Special methods of luminescent analysis are also being developed. For example, the insect fireflies emit the enzyme luciferase, which can oxidize luciferin in the presence of adenosine triphosphate with the emission of a chemiluminescent glow. The intensity of the latter when taken in excess of the luciferin-luciferase system is proportional to the concentration of adenosine triphosphate in the suspension under study. When nucleic acids are stained with proflavine dyes, the fluorescent characteristics of the latter change greatly with changes in the structure of the biopolymer. Luminescent analysis methods have recently been used in the study of compounds belonging to the classes of purines, porphyrins (especially chlorophylls), vitamins, steroid hormones, amino acids, proteins, as well as various drugs. In addition, fluorescent analysis is used in fluorescent paper chromatography, with fluorochromes, titrations and fluorescence microscopy (see).