However, a gas version of TLC was also carried out. Fine-grained, Al 2 O 3, and others are used as fine-grained ones. These are covered with glass, foil or plates; to fix the layer is used, or others. Prom-Stu produces ready-made plates with an already fixed layer. The eluents are usually mixtures of org. solutions, water solutions, to-t, complex-forming, etc. in-in. Depending on the choice of chromatographic systems (composition of mobile and stationary phases) in separation in-in main processes can play a role. In practice, several are often implemented simultaneously. separation mechanisms.

Depending on the position of the plate and the direction of the eluent flow, ascending, descending and horizontal TLC are distinguished. According to the technique of work, frontal analysis is distinguished (when the analyzed mixture serves as the mobile phase) and the commonly used elution variant. Also used are "circular" (when the analyzed solution and solvent are sequentially fed into the center of the plate) and "anti-circular" TLC (when the analyzed solution is applied around the circumference and the eluent moves from the periphery to the center of the plate), TLC under (when p -solvent under is passed through a layer covered with tightly pressed), as well as TLC under the conditions of a gradient of t-ry, composition, etc. In the so-called. two-dimensional TLC chromatographic. the process is carried out sequentially in two mutually perpendicular directions with decomp. eluents, which increases the separation efficiency. For the same purpose, multiple elution in one direction is used.

In the elution variant, drops (1-5 μl) of the analyzed solution are applied to the layer and the edge of the plate is immersed in the eluent, which is located at the bottom of a hermetically sealed glass chamber. The eluent moves along the layer under the action of capillary and gravitational forces; the analyzed mixture moves in the same direction. As a result of repeated repetition and in accordance with the coefficient. distributions in the selected one are separated and arranged on the plate in separate zones.

After the process is completed, the plate is removed from the chamber, dried and separated zones are found in their own. coloring or after spraying them with solutions that form colored or fluorescent spotswith the components of the mixture to be separated. Radioactive substances are detected autoradiographically (by exposure to a plate superimposed on a plate). Also applied. and active methods of detection. The resulting picture of the distribution of chromatographic. zones called chromatogram (see Fig.).

Chromatogram obtained by separating a mixture of three components using the thin layer method.

The position of the chromatographic The zones on the chromatogram are characterized by the value of R f - the ratio of the path l i traversed by the center of the zone of the i-th component from the start line to the path l traversed by the eluent: R f = l i /l; R f 1. The value of R f depends on the coefficient. distribution () and on the ratio of the volumes of the mobile and stationary phases.

The separation in TLC is influenced by a number of factors - the composition and properties of the eluent, the nature, and, t-ra, the dimensions and thickness of the layer, the dimensions of the chamber. Therefore, to obtain reproducible results, it is necessary to carefully standardize the experimental conditions. Compliance with this requirement allows you to set R f with relative. standard deviation 0.03. Under standard conditions, R f is constant for a given in-va and is used for the latter.

The number of component in the chromatographic. the zone is determined directly on the layer by the area of ​​\u200b\u200bthe zone (usually its diameter varies from 3 to 10 mm) or the intensity of its color (). Also use automatic scanning instruments that measure the absorption, transmission or reflection of light, or chromatographic. zones. The separated zones can be scraped off the plate along with the layer, the component can be extracted into a solvent and analyzed by a suitable method (fluorescence, atomic absorption, atomic fluorescence, radiometric analysis, etc.). The error of quantitative determination is usually 5-10%; limits of detection in-in in the zones -10 -3 -10 -2 μg (for colored derivatives) and 10 -10 -10 -9 μg (using).

Advantages of TLC: simplicity, cost-effectiveness, availability of equipment, rapidity (separation time 10-100 min), high performance and separation efficiency, clarity of separation results, ease of detection of chromatographic. zones.

TLC is used for separation and analysis of both org. and inorg. in-in: almost all inorg.

Chromatography in modern chemistry

One of the important tasks of modern chemistry is reliable and accurate analysis organic matter, often similar in structure and properties. Without this, it is impossible to conduct chemical, biochemical and medical research, this is largely based ecological methods analysis environment, forensic examination, as well as chemical, oil, gas, food, medical industries and many other sectors of the national economy.

One of the most sensitive methods is chromatographic analysis, first proposed by the Russian scientist M.S. Tsvet at the beginning of the 20th century. and by the end of the century turned into a powerful tool, without which both synthetics and chemists working in other areas can no longer do.

Color separation was carried out in the column shown in fig. 1. A mixture of substances A, B and C - natural pigments, originally located in the zone e,- is separated by adding the appropriate solvent D (eluent) into separate zones.

As always, it all started, it would seem, from the simplest thing that any schoolboy could do. In previous years, schoolchildren, including the author of this article, wrote in ink. And if the blotter fell on the ink stain, then one could notice that the ink solution was divided into several “fronts” on it. Chromatography is based on the distribution of one of several substances between two, as they say, phases (for example, between solid and gas, between two liquids, etc.), and one of the phases is constantly moving, that is, it is mobile.

This means that such a phase, for example, a gas or a liquid, is constantly advancing, breaking the equilibrium. Moreover, the better this or that substance is sorbed (absorbed) or dissolved in the stationary phase, the slower its movement rate, and, conversely, the less the compound is sorbed, i.e., has a lower affinity for the stationary phase, the greater the movement speed. As a result, as shown in Fig. 2, if at first we have a mixture of compounds, then gradually all of them, pushed by the mobile phase, move to the “finish” with different speeds and eventually separate.

Rice. 2. The basic principle of chromatographic separation: NF is a layer of stationary phase covering the inner surface of the capillary tube T, through which the mobile phase (MP) flows. Component A 1 of the separated mixture has a high affinity for the mobile phase, and component A 2 - for the stationary phase. A "1 and A" 2 are the positions of the zones of the same components after a period of time during which the chromatographic separation occurred in the direction indicated by the arrow

In practice, a sample of a mixture of substances is injected, for example, with a syringe, into the layer of the stationary phase, and then the various compounds that make up the mixture, together with the mobile phase (eluent), move along the layer, driven by this phase. The speed of movement depends on the amount of interaction (affinity) of the components in the stationary and mobile phases, and as a result, separation of the components is achieved.

After separation, all components must be identified and quantified. This is the general scheme of chromatography.

It should be noted that this modern method makes it possible to determine the content of tens and hundreds of different compounds in a mixture within a few minutes, even in negligible, “trace” amounts of ~10–8%.

Let us consider in more detail the chromatographic method of analysis. Chromatographic systems can be divided according to the following principles:

– state of aggregation of the mobile and stationary phases;
– geometric characteristics of the system;
– the mechanism of interaction between the separated substance and phases.

A gas or liquid is used as the mobile phase. Solids or liquids are used as stationary, or stationary, phases.

According to the arrangement of phases, chromatographic systems are divided into two groups: planar and column.

The latter, in turn, are divided into:

- packed, filled with granular solid material (small balls), either being a separating medium or serving as a carrier of a stationary liquid phase;
- capillary, the inner walls of which are covered with a film of an immobile liquid or a layer of a solid adsorbent (absorber).

The interaction between the substance to be separated and the phases of the chromatographic system can be carried out either on the surface of the phase or in the bulk. In the first case, chromatography is called adsorption, in the second distribution.

The mechanisms of separation of molecules in chromatographic systems are most often reduced to the following:

– the stationary phase physically absorbs (sorbs) the substances to be separated;
- the stationary phase chemically interacts with the separated substances;
- the stationary phase dissolves the substances to be separated from the solution in an immiscible solvent;
- the stationary phase has a porous structure, which hinders the diffusion of molecules of the substances to be separated in this phase.

Chromatography, which began with self-made devices such as a strip of paper dipped in a solvent, is now represented by the most complex instrumental systems based on modern precise or precision principles and equipped with computer software. Suffice it to say that one of the best computer firms, Hewlett-Packard, simultaneously produces modern chromatographs.

The scheme of the chromatography process is, in essence, very simple and is shown in Fig. 3. Further, approximately in this sequence, the principle of operation of the chromatograph will be considered.

Main types of chromatography

The main types of chromatography include adsorption, ion exchange, liquid, paper, thin layer, gel filtration and affinity chromatography.

Adsorption chromatography. In this case, the separation of substances is carried out due to the selective (selective) adsorption of substances on the stationary phase. Such selective adsorption is due to the affinity of one or another compound for the solid adsorbent (stationary phase), and this, in turn, is determined by the polar interactions of their molecules. Therefore, chromatography of this type is often used in the analysis of compounds whose properties are determined by the number and type of polar groups. Adsorption chromatography includes ion-exchange, liquid, paper, thin-layer and gas-adsorption chromatography. Gas adsorption chromatography is described in more detail in the Eluent Analysis section.

Ion exchange chromatography. used as the stationary phase. ion exchange resins(Fig. 4) both in columns and as a thin layer on a plate or paper. Separation is usually carried out in aqueous media, so this method is used mainly in inorganic chemistry, although mixed solvents are also used. The driving force of separation in this case is the different affinity of the separated ions of the solution to ion-exchange centers of opposite polarity in the stationary phase.

liquid chromatography. In this case, the stationary phase is a liquid. The most common case is the adsorption version of liquid column chromatography. An example of the separation of natural pigments is shown in fig. 5.

Rice. 5. Chromatographic separation of natural pigments (flavones and isoflavones)

Paper chromatography. Strips or sheets of paper are used as the stationary phase (Fig. 6). Separation occurs according to the adsorption mechanism, and sometimes it is carried out in two perpendicular directions.

Thin layer chromatography is any system in which the stationary phase is a thin layer, in particular a layer of alumina (2 mm thick) in the form of a paste deposited on a glass plate. An example of such a system and the separation results are shown in Fig. 7.

Gel filtration, or molecular sieve, chromatography. The principle of separation in such systems is somewhat different than in previous cases. The stationary phase are materials, usually gels, with strictly controlled porosity, as a result of which some components of the mixture, in accordance with the size and shape of the molecules, can penetrate between the gel particles, while others cannot. Most often, this type of chromatography is used to separate macromolecular compounds. One of the applications of this method is the determination of the molecular masses of the substances to be separated, which are often necessary for chemical studies (Fig. 8).

affinity chromatography. This type of chromatography is based on the interaction between a substance, on the one hand, capable of reacting with the isolated compound, and on the other hand, associated with a solid carrier of the stationary phase. Such a substance has an affinity for the isolated compound and is called an affinity ligand.

Most often, this method is used in biochemical analysis. For example, when biological antigen objects containing proteins are passed through cellulose activated with cyanogen bromide, they are specifically retained, as shown in Scheme 1.

According to another method, to attach proteins to the hydroxyl group of cellulose, the latter is first treated with 2-amino-4,6-dichloro- Sim-triazine, and then the product of their interaction reacts with the amino group of the protein according to scheme 2:

Of course, the number of chromatography methods is not limited to those listed above. Chromatography is often combined with other physicochemical methods, such as mass spectrometry, but this article aims to acquaint the reader only with the general principles of chromatography. Therefore, we will further consider the processing of chromatography results.

Chromatogram development methods

Development is the process of transfer of separated substances by the mobile phase. Development can be done in three main ways: frontal analysis, displacement and elution. Elution is the most widely used.

Frontal analysis. This is the simplest case, since here the sample serves as the mobile phase. It is continuously added to the system, so large sample volumes are needed. The results are shown in fig. nine.

The formation of several zones is due to the different affinities of different components for the stationary phase. The cutting edge is called the front, hence the name. The first zone contains only the least retained substance A, which moves the fastest. The second zone contains substance A and B. The third zone is a mixture of substances A, B and C. In the frontal analysis, only component A is obtained in liquid form.

displacement analysis. In this case, the mobile phase has a greater affinity for the stationary phase than the substance to be separated. A small sample is introduced into the stationary phase. But due to the high affinity, the mobile phase displaces and pushes all the components. It displaces the most strongly sorbed component C, which, in turn, displaces substance B, which displaces the least sorbed component A. In contrast to frontal analysis, this method can be used to obtain all the main components in an individual (liquid) form.

eluent analysis. The mobile phase to move the solute is passed through the chromatographic system. The separation occurs due to the different affinity of the mixture components to the stationary phase and, consequently, due to different speeds of their movement. A small volume sample is introduced into the chromatographic system. As a result, zones with components will gradually form separate sections separated by pure eluent. Due to the high separation efficiency, the method has become the most widely used and has largely replaced other separation options. Therefore, we will further consider the theory and hardware design of this method.

A bit of theory. It is often convenient to consider chromatographic processes as a series of extraction processes; in this case, substances with very similar properties can be separated, since hundreds and even thousands of extraction cycles occur quickly and simultaneously during chromatographic processes.

To evaluate the efficiency of chromatographic processes, based on the theoretical concept of distillation (by analogy with the separation of oil in distillation columns, where the theoretical plate corresponds to the part of the distillation column in which vapor and liquid are in equilibrium), the concept is introduced "height equivalent to theoretical plate"(VETT). The chromatographic column is thus considered as a set of hypothetical layers (plates). HETP is usually understood as such a layer thickness that is necessary for the mixture coming from the previous layer to come into equilibrium with the average concentration of the substance in the mobile phase of this layer. It can be described by the following formula:

HETT = L/N,

where L- column length, N is the number of theoretical plates.

HETP is a summary characteristic of the separation of substances. However, separating the components of the mixture is important, but not sufficient. It is necessary to identify each component and determine its amount in the sample. This is usually done by processing chromatograms - the dependence of the signal intensity, proportional to the concentration of the substance, on the separation time. Some examples of chromatograms are shown in fig. 10, 11.

The time from the moment the sample is injected into the column until the moment the maximum peak is registered is called retention time (tR). Under optimal conditions, it does not depend on the amount of injected sample and, taking into account the geometrical parameters of the column, is determined by the structure of a particular compound, i.e., it is a qualitative characteristic of the components. The quantitative content of the component is characterized by the magnitude of the peak, more precisely by its area. Count peak area usually done automatically with an integrator instrument that records both retention time and peak area. Modern equipment allows you to immediately obtain a computer printout indicating the content of all components of the mixture to be separated.

The work of the chromatograph. The installation diagram of the simplest gas chromatograph is shown in fig. 12. It consists of a gas cylinder containing a mobile inert phase (carrier gas), most often helium, nitrogen, argon, etc. Using a reducer that reduces the gas pressure to the required level, the carrier gas enters the column, which is a tube filled with sorbent or other chromatographic material that plays the role of a stationary phase.

Rice. 12. Scheme of operation of a gas chromatograph:
1 - balloon high pressure with carrier gas; 2 – flow stabilizer; 3 and 3" - manometers; 4 - chromatographic column; 5 - sample injection device; 6 - thermostat; 7 - detector; 8 - recorder; 9 - flow meter

The chromatographic column is the "heart" of the chromatograph, since it is in it that the mixtures are separated. Columns are most often made of glass; there are steel, teflon, and also capillary columns. A sample injection device is installed near the gas inlet to the column. Most often, a sample is injected with a syringe, piercing the rubber membrane. The analyzed mixture is separated in the column and enters the detector - a device that converts the separation results into a form convenient for registration.

One of the most used detectors is a katharometer, the principle of which is based on measuring the heat capacity of different bodies.

On fig. 13 shows a diagram of a katharometer. A metal spiral (resistance thread) is placed in a cylindrical cavity, which heats up as a result of the passage of a direct electric current through it. When a carrier gas flows through it at a constant speed, the temperature of the coil remains constant. However, if the composition of the gas changes with the appearance of the eluted substance, then the temperature of the coil changes, which is recorded by the device.

Another common detector is the flame ionization detector, the scheme of which is shown in Fig. 14. It is much more sensitive than a katharometer, but requires the supply of not only carrier gas, but also hydrogen. The carrier gas leaving the column, containing the eluent, mixes with hydrogen and passes into the nozzle of the detector burner. The flame ionizes the eluent molecules, as a result of which the electrical resistance between the electrodes decreases and the current increases.

In liquid chromatography, spectrophotometric detectors are used (in the visible, UV and IR regions), as well as refractometric detectors based on measuring the refractive indices of solutions.

Those are in in general terms basics of chromatographic analysis. Of course, the article contains only general principles chromatography, and often they are simply labeled. In fact, the "kitchen" of this method is quite large and complex. the main objective this article, according to the author, to draw the attention of young readers to this powerful method.

Those wishing to learn more about this area can use the literature below.

Literature

Zhukhovitsky A.A., Turkeltaub N.M. Gas chromatography. M.: Gostoptekhizdat, 1962, 240 p.;
Sakodynsky K.I., Kiselev A.V., Iogansen A.V. and etc. Physico-chemical application gas chromatography. M.: Chemistry, 1973,
254 p.;
Liquid column chromatography. In 3 volumes. Ed. Z. Deyla, K. Maceka, J. Janaka. Moscow: Mir, 1972;
Berezkin V.G., Alishoev V.R., Nemirovskaya I.B.. Gas chromatography in polymer chemistry. M.: Nauka, 1972, 287 p.;
Morozov A.A. Chromatography in inorganic analysis. M.: Higher. school, 1972, 233 pp.;
Berezkin V.G., Bochkov A.S.. Quantitative thin layer chromatography. M.: Nauka, 1980, 183 p.;
Laboratory guide to chromatographic and related methods. In 2 volumes. Ed. O. Mikesh. Moscow: Mir, 1982, vol. 1–2, 783 pp.;
Chromatographic analysis of the environment. Ed. R. Coffin. M.: Mir, 1979, 606 p.;
Kirchner Yu. Thin layer chromatography. In 2 vol. M.: Mir, 1981, vol. 1, 615 pp., vol. 2, 523 pp.;
Extraction chromatography. Ed. T.Brown, G.Gersini. M.: Mir, 1978, 627 p.

V.V. Safonov,
professor of the Moscow
state textile
academy. A.N. Kosygina

MZRF

FESMU

Department of General, Physical and Colloid Chemistry

abstract

Thin layer chromatography. Application in pharmacy

Completed by: student of group 201-F

Danilov D.I.

Checked by: Nemov V.A.

Khabarovsk, 2005

PLAN:

Introduction

Physical and chemical bases of TLC

Partition chromatography on paper

Fundamentals of thin layer chromatography

• sorbents
• solvents
• plate preparation
• test solution application technique

Chromatography

Drying plates.

Identification of separated substances

Application of the TLC method in pharmacy

• Quantitative determination of triterpene saponins by HPTLC using scanning densitometry
• The study of the lipid and flavonoid composition of samples of some species of the genus Chin (Lathyrus.)

Conclusion

Literature

Introduction

Thin layer chromatography (TLC, TLC) is one of the most used methods of chromatographic analysis, but the least popularized.
Despite the significant shortcomings that existed until recently, it is widely used for qualitative analysis mixtures, mainly due to the cheapness and speed of obtaining results. Thin layer chromatography (TLC) was originally developed for the separation of lipids. Although paper chromatography is faster than column chromatography, it has the disadvantage that paper can only be made from cellulose-based materials, making it unsuitable for separating non-polar substances. Thin layer chromatography retains all the advantages of paper chromatography, but allows the use of any material that can be finely ground and then a homogeneous layer is obtained. It can be inorganic substances, such as silica gel, alumina, diatomaceous earth and magnesium silicate, as well as organic substances, in particular cellulose, polyamides and polyethylene powder.

Physical and chemical bases of thin-layer chromatography.

The basis of thin layer chromatography is the adsorption method, although partition chromatography is also found.
The adsorption method is based on the difference in the degree of sorption-desorption of the separated components on the stationary phase. Adsorption is carried out due to van der Waals forces, which is the basis of physical adsorption, polymolecular (formation of several layers of the adsorbate on the surface of the adsorbent) and chemisorption (chemical interaction of the adsorbent and adsorbate).
For efficient sorption-desorption processes, it is necessary big square, which imposes certain requirements on the adsorbent. At large surface phase separation is a rapid establishment of equilibrium between the phases of the components of the mixture and effective separation.
Another type of thin layer chromatography used in thin layer chromatography is partition liquid chromatography.
In partition chromatography, both phases - mobile and stationary - are liquids that do not mix with each other. The separation of substances is based on the difference in their distribution coefficients between these phases.
For the first time the method of thin layer chromatography declared itself as "Paper thin layer chromatography", which was based on the partition method of separation of components.

Partition chromatography on paper.

Due to the fact that the chromatographic paper used in this method (special grades of filter paper) contains water (20-22%) in the pores, organic solvents are used as the other phase.
The use of chromatography on paper has a number of significant disadvantages: the dependence of the separation process on the composition and properties of the paper, the change in the water content in the pores of the paper with changes in storage conditions, a very low chromatography speed (up to several days), and low reproducibility of the results. These shortcomings seriously affect the spread of paper chromatography as a chromatographic method.
Therefore, the appearance of chromatography in a thin layer of a sorbent, i.e., thin layer chromatography, can be considered natural.

Fundamentals of thin layer chromatography.

In the TLC method, the chromatography of substances occurs in a thin layer of a sorbent deposited on a solid flat substrate. Separation in this method mainly occurs on the basis of sorption-desorption.
The use of various sorbents made it possible to significantly expand and improve this method.
At the beginning of the appearance of the method, the plates had to be made independently. But today, prefabricated plates are mainly used, which have a fairly wide range both in size and carriers, and in substrates.
A modern chromatographic plate is a base made of glass, aluminum or a polymer (for example, polyterephthalate). Due to the fact that the glass base is becoming less popular (it often breaks, it is impossible to divide the plate into several parts without damaging the sorbent layer, heavy in weight), plates based on aluminum foil or polymers are most widely used.
To fix the sorbent, gypsum, starch, silicasol, etc. are used, which hold the sorbent grains on the substrate. The layer thickness can be different (100 or more microns), but the most important criterion is that the layer must be uniform in thickness anywhere on the chromatographic plate.

Sorbents

The most common sorbent is silica gel.
Silica gel is a hydrated silicic acid formed by the action of mineral acids on sodium silicate and drying of the resulting sol. After grinding the sol, a fraction of a certain grain size is used (indicated on the plate, usually 5-20 microns).
Silica gel is a polar sorbent, in which -OH groups serve as active centers. It easily sorbs water on the surface and forms hydrogen bonds.
Alumina. Alumina is a weakly basic adsorbent and is mainly used to separate weakly basic and neutral compounds. The disadvantage of plates on aluminum oxide is the mandatory activation of the surface before use in a drying cabinet when high temperature(100-150 0 C) and low adsorption capacity of the layer compared to silica gel.
Diatomaceous earth is an adsorbent obtained from natural minerals: diatomaceous earths. The sorbent has hydrophilic properties, but a lower adsorption capacity of the layer compared to silica gel.
Magnesium silicate is less polar than silica gel and is usually used when more polar adsorbents do not give effective separation.
Cellulose - thin-layer cellulose coated plates are very effective in separating complex organic molecules. The adsorbent is mainly cellulose balls with a diameter of up to 50 microns, fixed on the carrier with starch. But as in paper chromatography, the rise of the solvent front is very slow.
In ion-exchange chromatographic plates, ion-exchange resins containing quaternary ammonium or active sulfo groups involved in ion exchange are used as an adsorbent. Thin layer chromatography with this type of plates is carried out with mobile phases containing strong acids or alkalis. These plates are effective for separating macromolecular and amphoteric compounds.

The above sorbents are the most common, but in addition to these, there are many substances used as sorbents. These are talc, calcium sulfate, starch, etc.
At the same time, even the already mentioned sorbents can be modified to give them new sorption properties (impregnation of sorbents with reagents, for example, AgNO 3 , creation of plates with reversed phase). It is this variety of possible phases at minimal cost that makes it possible to use TLC for chromatography of a huge number of substances.

Solvents

In thin layer chromatography, either pure substances (ethyl acetate, benzene, etc.) or mixtures of substances (systems) in a certain ratio are used as the mobile phase.
The selection of the mobile phase (system) is carried out according to the following rules:

· Choose a system in which the components to be separated have low solubility (if the solubility of the substance is high, then the substances will move with the front, if the solubility is low, they will remain at the start). With partition chromatography or when using reversed phases, the solubility of the substances must be higher in the mobile phase than in the stationary phase.

· The composition of the system must be constant and easily reproducible.

· The solvent or system components must not be poisonous or deficient.

· The system must completely separate substances of similar structure, and the differences in Rf must be at least 0.05.

· The system must not cause chemical changes in the components to be separated.

In the selected system, the analytes must have various meanings Rf and distributed over the entire length of the chromatogram. It is desirable that the Rf values ​​lie in the range of 0.05-0.85.

· When choosing a system, the nature of the substances to be separated must also be taken into account. So, when chromatographing substances with basic properties, the system should not have acidic properties and vice versa.

Plate preparation

When using purchased plates, they must first be prepared for chromatography. This is due to the fact that during storage, plate adsorbents absorb not only moisture, but also other substances contained in the air. When using unprepared plates during chromatography, a “dirt” front appears, which can interfere with the determination of substances with large Rf values, and some substances, such as water, can change the composition of the mobile phase, thereby changing the resulting Rf values.
Preliminary preparation of the plates consists in dispersing the plates with a pure solvent to the entire height of the plate (methanol, benzene, diethyl ether), followed by drying the plate in an oven at a temperature of 110-120 0C for 0.5-1 hour. In this way, several plates can be prepared at once, and when stored in a dry, sealed place, they retain their properties for several months.

Technique for application of test solutions.

As it turns out, the application of the test substance is not such a complicated operation, but at the same time, it greatly affects the results of chromatography.
Often, either liquid analytes or solutions of solids are tested, without any prior sample preparation.
Therefore, it is always necessary to remember a number of points that seriously affect the results of the separation.
The most important is the concentration of applied substances. In TLC, it is customary to apply concentrations of solutions of about 1%. But on the other hand, the sensitivity of the method allows you to determine substances with much lower concentrations.
If the total concentration of the components in the test substance is unknown, or the concentration is known but this type of substance has not yet been chromatographed, it is necessary to determine what amount of the test solution is sufficient for high-quality chromatography. There are several methods to determine this.
First, you need to apply several spots of chromatographic solutions, equal in size, but with different amounts (for example, 1, 2, 5 µl) and after chromatography, study the shape and size of the separated spots.
So, with a properly selected concentration, the shape of the separated substances is the same as the shape applied to the start line. If the separated spots are larger than the spot at the start, then the applied concentration is too high. The appearance of "tails", the irregular shape of separated spots on the plate, can also indicate a high concentration, but can be caused by an incorrectly selected chromatographic system, or by chemical interaction of the separated components.
By selecting the amount of the deposited substance and the system of solvents, it is possible to achieve complete separation on one plate of up to ten components in the substances under study. It is convenient to apply samples on a special table with stencils and heating. Spotting is carried out on the "start line" 1-2 cm from the bottom edge of the plate. This is necessary so that when the plate is lowered into the system, the samples do not dissolve in it, and all the deposited substance is subjected to chromatography.
Application of solutions is carried out either with a microsyringe or graduated capillaries. The size of the applied spot should not exceed 4 mm. This is due to the fact that with a larger spot size, a change in shape occurs under the action of physical forces, and the boundaries of the separated components may overlap.
The application of the test substances to the plates should not be accompanied by the destruction of the sorbent (which has a rather strong effect on the quality of separation), so the drop should be applied by touching the needle or capillary against the sorbent layer, and not by pressing. The size of the resulting spot is affected not only by the amount of solution applied, but also by the polarity of the solvent and its boiling point. So, when applying the same substance in different solvents, the resulting spot in which methanol was used as a solvent will be larger than the spot from the chloroform solution. on the other hand, when the substrate is heated, the evaporation of solvents will be more intense and the spot size will also decrease.
Of course, it is easier to use a hair dryer when applying to dry stains, but only if there is complete confidence that the applied substances will not oxidize under the action of hot air.
The distance between the applied spots should be about 2 cm.
Sometimes, during chromatography on plates, an edge effect is observed, as a result of which the spots are not located on the same line, but look like a horseshoe, or diagonally. To eliminate this effect, each spot can be "provided" with its own track, separating the applied sample from the others by removing the sorbent line. This is best done under the ruler with a sharp object (such as a scalpel) but be careful not to remove too much sorbent.
After applying the test substances to the plate, it is necessary to achieve complete removal of solvents, since even a small amount of solvent in the test substance can affect the separation and even change the composition of the chromatographic system.
Removal of solvents is usually carried out by natural drying of the plates for 5-10 minutes, either by heating with a hair dryer or in an oven.

Chromatography

Thin layer chromatography has several methods, mainly related to the type of movement of solvents.

Upward thin layer chromatography

Downward thin layer chromatography

Horizontal thin layer chromatography

· Radial thin layer chromatography.

Upstream thin layer chromatography

This type of chromatography is the most common and is based on the fact that the front of the chromatographic system rises along the plate under the action of capillary forces, i.e. the front of the chromatographic system moves from bottom to top. For this method, the simplest equipment is used, since any container with a flat bottom and a tight-fitting lid, into which a chromatographic plate can be freely placed, can be used as a chromatographic chamber.
The method of ascending thin layer chromatography has a number of disadvantages. For example, the speed of the rise of the front along the plate occurs unevenly, i.e. in the lower part it is the highest, and as the front rises it decreases. This is due to the fact that in the upper part of the chamber the saturation with solvent vapors is less, therefore the solvent from the chromatographic plate evaporates more intensively, therefore its concentration decreases and the speed of movement slows down. To eliminate this shortcoming, strips of filter paper are attached along the walls of the chromatographic chamber, along which the ascending chromatographic system saturates the chamber with vapor throughout the entire volume.
Some chromatographic chambers have a division into two trays at the bottom. This improvement allows not only to reduce the consumption of the chromatographic system (less volume is required to obtain the required height of the chromatographic system), but also to use an additional cuvette for the solvent, which increases the saturation vapor pressure in the chamber.
The need to monitor the solvent front can also be considered a disadvantage, since the solvent front line can “run away” to the upper edge. In this case, it is no longer possible to determine the actual value of Rf.

Downward thin layer chromatography

This chromatography method is based on the fact that the front of the chromatographic system descends over the plate mainly under the action of gravity, i.e. the mobile phase front moves from top to bottom.
For this method, a cuvette with a chromatographic system is attached to the upper part of the chromatographic chamber, from which a solvent enters the chromatographic plate using a wick, which flows down and the sample is chromatographed.
The disadvantages of this method include the complexity of the equipment. This method is mainly used in paper chromatography.

Horizontal thin layer chromatography

This method is the most complex in hardware design but the most convenient. So, in the chromatographic chamber, the plate is placed horizontally and the system is fed to one edge of the plate using a wick. The solvent front moves in the opposite direction.
There is another trick to simplify the camera as much as possible. To do this, an aluminum-based chromatographic plate is slightly bent and placed in the chamber. In this case, the system will act from two sides at the same time. Only aluminum-backed plates are suitable for this purpose, since the plastic and glass bases are "inflexible", i.e. does not retain its shape.
The advantages of this method include the fact that in a horizontal cell the vapor saturation of the system occurs much faster, the front velocity is constant. And when chromatographing from both sides, the front does not "run away"

Radial thin layer chromatography.

Radial thin-layer chromatography consists in the fact that the test substance is applied to the center of the plate and the system is fed there, which moves from the center to the edge of the plate.

Drying plates.

After the process of separating the test substances, the plates are dried. This is also an important process, since if there are even traces of solvent on the plate, it is possible to obtain incorrect chromatographic results.
If the chromatographic system included only low-boiling components, then natural drying for 3-5 minutes is sufficient. If the system includes high-boiling liquids (alcohols, water, organic acids, etc.), the plates should be dried for at least 10 minutes or the plate should be placed in an oven.

Identification of separated substances.

The dried plate is a chromatogram of the studied substances. If the substances are colored, then identification begins with determining the color of the separated substances.
But in most cases, the separated substances are colorless and a simple visual comparison is not possible.
For thin layer chromatography, there are several types of qualitative analysis (identification) of separated substances:

· Visual methods and determination of Rf of separated substances.

Color reactions.

· Comparison with witnesses.

· Physical and chemical methods of identification.

Let us consider in more detail each type of qualitative analysis in thin layer chromatography.

Physical Methods

Visual methods are mainly used to locate spots of separated substances on a chromatographic plate. To do this, the plate is viewed both in visible light and using ultraviolet light (mainly light with a wavelength of 366 and 254 nm)
This is the first stage of identification, which determines the quality of the selected conditions and the results of chromatography.
Thus, having determined the quality of chromatography (the absence of "tails" of the substances to be separated or the overlapping of their spots, the correct shape and size, the absence of merging of chromatographic tracks, etc.) and deeming the separation suitable for further research, determine the Rf of the identified spots.

Rf value.

One of the main indicators in TLC is Rf. This parameter is analogous to the retention time and depends both on the properties of the substances to be separated, the composition of the mobile phase and sorbent, and on the physical parameters.
The determination of the Rf value is carried out as the ratio of the distance passed by the substance to the distance passed by the solvent front

The Rf value is a dimensionless value and has a value from 0 to 1. However, in the literature, such indicators as hRf, Rf × 100 are often found, which are the same Rf, but multiplied by 100, in order not to operate with decimal values.
The value of Rf is not affected by the distance traveled by the solvent front, however, many methods describe the passage of the front at a distance of 10 cm. This is used only to facilitate the calculation of Rf.
In practice, at the beginning, the distance passed by the solvent front is determined: from the start line (and not from the edge of the plate) to the place where the front was at the end of chromatography. Then the distance from the start line to the spot of the separated substance is determined. This is where the size of the spot affects! After all, if the stain has round shape and small size, then the resulting Rf has a clear value. And if the resulting spot has a large size or irregular shape, then when determining the Rf of such a spot, the error can reach 0.1!
In the case of partition chromatography, the distribution coefficient of a substance and its Rf is related by the relationship:

where Sp and Sn- cross-sectional areas of the mobile and stationary phases.
As we can see, the distribution coefficient, at a constant ratio Sp/Sn is a quantity proportionally dependent on Rf , and can be determined through it.

color reactions.

Color reactions in thin layer chromatography are used extremely widely. They serve not only to determine the location of the separated components (treatment with sulfuric acid, iodine vapor), but also to determine both the class of substances and identification (in the presence of individual reactions).
We will not consider here this huge variety of colored qualitative reactions, we will only say that if all qualitative reactions coincide and the obtained Rf values ​​of the substance in three various systems with literature data, the substance is identified. Although, in my opinion, additional confirmation is needed by research by another physicochemical method.

Witness comparison.

When conducting studies of substances with the expected composition, the chromatography method is used with witness- known substance. This method is used when chromatographic conditions are difficult to withstand, there is no literature data on Rf for a given system or adsorbent, the use of a gradient method, etc. And when carrying out color reactions, you can compare not only colors, but also shades of the substances under study and witnesses, which is also important.
On the other hand, this method requires additional costs for witnesses.

Methods of quantitative analysis

Quantitative analysis in thin layer chromatography has several types, characterizing each stage of the development of the method. And although some methods can only be applied as semi-quantitative, they are still used in practice.

visual comparison method. As mentioned above, the color intensity of the spot and its size depend on the amount of the chromatographed substance. Therefore, visual quantification is based on several techniques.
Dilution method. This method consists in the fact that for each substance the limiting concentration is determined at which the substance cannot be determined by the chromatographic method. When chromatographing the test substance, the dilution is carried out until it ceases to appear on the plate.
The content of substance C, determined by this method, is found by the formula:

where n-dilution, a- the concentration of a substance at which it does not appear during chromatography.
Method for determining the spot area. If the same volumes of test substances and witnesses are applied, then the areas of spots obtained after chromatography are proportional to the logarithm of the concentration of the substance. S=a ln c+b

where a and b are empirical coefficients determined experimentally.
If the spot of the separated substance has sharp boundaries, then the area of ​​the spot can be determined by the gravimetric method (cut out the spot and weighed), measured with a planimeter. This method gives an error of up to 10-15%.
However, it has a number of significant drawbacks. The first and most significant is that in this way it is possible to determine the concentration of colored substances or those having fluorescence in the UV region (254, 366 nm). This disadvantage can be eliminated by adding various phosphors to the sorbent, which increases the error of determination.
Treatment of plates with developing substances (reagents) can also be used (for example, using filter paper impregnated with a developing reagent, followed by contact with a chromatographic plate and further determination of the area of ​​the developed substance on it), but the determination error is also high.
The need for a more reliable quantification result led to the use of instrumental methods.
Elution method. This method consists in the fact that the separated substance is washed off the sorbent with a solvent and its concentration is determined by other methods - photometric, polarographic, etc. This is a fairly accurate method, but only under the condition of quantitative isolation of the separated substance. Due to the high labor intensity, the method is used quite rarely and is unacceptable for a large number of samples under study.
Photographic method The definition consists in photographing the plates with the separated substance and further determining the degree of blackening, using disintometers.
radiographic method similar to photometric, only with the difference that the blackening of the plate is determined, caused by the radiation of the separated substance. This method is used only in the determination of substances with labeled atoms.
Photodesynthometric method can be used without isolating the substance from the plate and is based on determining not only the area of ​​the spot, but also its intensity.
This is the most accurate method for determining the concentration of substances, as it allows, when using calibration graphs, to carry out fairly accurate quantitative determinations of all separated substances (up to 2-10%) directly on the plate in a short period of time.
It is not surprising that with the development of thin-layer chromatography, the use of disintometers increases, the sensitivity and, consequently, the accuracy of determining the concentration of separated substances increases and approaches the accuracy of high-performance liquid chromatography.

Rice. 1. Typical chamber for developing a thin layer chromatographic plate

1. lid
2. glass chamber
3. TLC plate
4. sorbent
5. sampling site
6. solvent

Typical TLC chromatogram of fatty acid methyl esters.

On the chromatogram FAME= fatty acids methyl esters = methyl esters of fatty acids. Start= point of application of mixtures to be separated.

The chromatogram was made on a Sorbfil plate. The system is benzene. Manifestation - charring after spraying with sulfuric acid.

Dot 1 - methyl esters of fatty acids. Dot 2 - methylation products of total lipids.

Arrow the direction of motion of the solvent front (system) is shown. The solvent front during chromatography moved up to the upper edge of the plate.

Application of the TLC method in pharmacy

The great importance of chromatographic methods for pharmacy is due to the fact that in the production of drugs, in many cases, the preliminary isolation of natural or synthetic products in pure form is required. Analysis is also often based on the separation of mixtures into components. Let us consider two examples of the application of the TLC method, proving its importance in the analysis and production of medicinal substances.

Quantitative Determination of Triterpene Saponins by HPTLC Using Scanning Densitometry

Triterpene saponins (glycosides) are the active ingredients of many drugs.

Most of the currently used methods for the quantitative determination of triterpene saponins are based on acid hydrolysis of the latter with further determination of aglycone, most often by titrimetric, less often by spectral methods of analysis.

Similar methods based on the destruction of saponin molecules have a number of disadvantages. They are lengthy and do not allow a quantitative assessment of the ratio of individual saponins in total saponin-containing preparations.

In most cases, the authors limit themselves, as a rule, to a qualitative assessment using the TLC method, the most accessible and simple chromatographic method of analysis. The use of TLC to determine the quantitative content of components is constrained by the lack of scanning densitometers.

This paper presents the results of quantitative TLC determination of some triterpene saponins, derivatives of oleanolic acid, in pharmaceutical preparations and extracts from plant materials.

Triterpene saponins of Manchurian aralia (aralosides) were chosen as objects of study. the qualitative determination of which in various objects is covered in the works, as well as triterpene saponins of sugar beet - substances with pre-established pharmacological activity. Both are derivatives of oleanolic acid with a small (no more than four) amount of sugar residues, which suggests their similar behavior in a thin layer of sorbent.

As standards, we used the sum of aralosides isolated from Saparal tablets and the sum of sugar beet saponins isolated from freshly harvested rhizomes. For application to the plate, aqueous-alcoholic (80% ethanol) solutions of saponins were prepared with the content of the latter 0.4–2.0 mg/ml. Chromatography was carried out using plates for TLC "Silufol" (Czech Republic) 15 x 15 cm, "Armsorb" for HPTLC (Armenia) 6 x 10 cm and "Sorbfil" (Russia) 10 x 10 cm. The height of the front rise, sufficient for complete separation, was 10.6 and 6 cm, respectively. The samples were applied using a MSH-10 microsyringe (Russia) onto a plate heated to 40°C. The optimal volume of the applied sample was 3–5 μl. The application was carried out in several stages so that the diameter of the starting spot did not exceed 2 mm.

Chromatography was carried out at a temperature of 20 - 25 °C. At the end of elution, the plates were dried in air and treated with a detecting reagent using a laboratory glass sprayer. The zones were scanned using a Shimadzu CS-9000" scanning densitometer (Japan). The quality of the zones obtained by chromatography in the three most commonly recommended elution systems was compared: I. chloroform - methanol - water (30:17:3): II. n-butanol - ethanol - ammonia (7:2:5) III. n-butanol - water - acetic acid (4:5:1), upper layer IV. benzene - ethyl acetate (1:1) (for sugar saponins beets).

A 25% alcohol solution of phosphotungstic acid was used as detecting reagents (crimson stains of saponins on a white background). most commonly used for quantitative TLC determinations of such compounds and a 10% alcoholic solution of phosphomolybdic acid, recommended for the development of triterpenoid zones (saponin zones are dark blue on a yellow background). Treatment of plates with ammonia vapor in the latter case makes it possible to discolor the background and increase the contrast of spots.

As a result of the studies carried out, the optimal conditions for the chromatographic process for densitometric determination were chosen. The best of the three types of plates were recognized by "Armsorb" for HPTLC. high efficiency The process is facilitated by a thin (II0 μm) and homogeneous in fractional composition (5-10 μm) layer of silica gel, which provides good separation and minimal blurring of the zones even when the solvent front rises by 6 cm. Sorbfil plates are almost as good as them in separation ability, but the elution time on them is almost twice as long. Plates "Silufol" at a sufficiently high elution rate allow you to separate the components with a longer chromatographic path, which leads to some blurring of the zones, but their use is also possible.

The elution can be carried out with sufficient quality in any of the first three elution systems. I gives a gain in time, IV allows you to get a better separation of sugar beet saponins than the first three.

Both detection reagents give a fairly stable staining of the zones when scanning is carried out within 1 - 2 hours from the moment of development. After this period, the saponin zones on the plates treated with phosphotungstic acid change color from crimson to violet, which can lead to a distortion of the quantitative analysis results during scanning. Treatment with phosphomolybdic acid is more preferable in this case. When stored in a place protected from light, the plates with zones developed by this reagent gave completely reproducible results after several months from the moment of development.

The limit of detection of saponins was 5 μg in the sample when developing with phosphotungstic acid and 0.5 μg in the sample when developing with phosphomolybdic acid. Treatment with ammonia vapor made it possible to reduce the detection limit of saponins in the latter case to 0.2 μg per sample.

Quantitative determination of saponins by TLC using scanning densitometry was carried out on Armsorb plates (the chromatography speed in this case was 2 times higher) or "Sorbfil", in the II and III elution system (as containing less toxic components). Zones were detected with a 10% solution of phosphomolybdic acid. The volume of the applied sample is not more than 5 μl, the height of the eluent front is 6 cm, the elution time is 30 - 60 minutes. Scanning wavelength λ = 675 nm. After processing the plates, aralia and sugar beet saponins appear in the form of three zones of different intensity.

The general view of the densitograms obtained during scanning is shown in fig. one.

Comparison of chromatograms of saponins isolated from tablets "Saparal" (Fig. 1, a) and "Aralia tincture" (Fig. 1,6) allows us to note the different ratio 44

aralosides A, B and C, which are part of these dosage forms. A similar variability in the ratio of individual saponins in raw materials, depending on the growing conditions of the plant, was noted earlier. The ratio of saponins in finished dosage forms easily assessed using the obtained densitograms. Since there are 3 zones corresponding to saponins on the chromatogram, and three peaks on the densitogram, respectively, the areas of all peaks were summarized when constructing the calibration curve. The error in this case was lower than in calculations based on the parameters of the peak of one of the components, taking into account the variability of their ratio (Fig. 2).

Rice. I. Densitograms obtained by scanning TLC plates: a- Aralia saponins isolated from Saparal tablets; 6 - aralia tincture saponins; in- sugar beet saponins. The total content of saponins in the sample is 5 μg. A, B, C- peaks of saponins; 0 - start line; f- Front line.

Rice. Fig. 2. Calibration dependence of the sum of areas of peakon saponins on the chromatogram on their content in the sample. / - Aralia saponins; 2 - sugar beet saponins. On the abscissa axis - the content of saponins in the sample (mcg), on the ordinate axis - the sum of peak areas (cm 2).

The calibration dependence of the sum of the areas of the peaks in the densitogram on the content of the substance in the sample was obtained by chromatography of a series of standard solutions with a known content of saponins. The initial solution was prepared by dissolving in 80% ethanol an exact weight of saponins, dried to a constant weight. A series of working solutions were prepared by sequential dilution of the initial 80% ethanol. The content of saponins in them was 0.04 - 2.0 mg/ml (0.2 - 10 µg in a sample with a sample volume of 5 µl). This concentration range can be considered optimal for scanning. The minimum content of saponins determined by this method is 0.2 µg/sample. The relative standard deviation in this case does not exceed 0.03.

The resulting calibration dependencies are non-linear, which is fully consistent with the Kubelka-Munk theory, which takes into account the absorption and scattering of light by the sorbent. In a narrow range of low concentrations, the dependences can be considered as linear (0.2 - 2.0 µg/sample). The non-linear part of the curves can be linearized by converting the amount of substance in the sample and the peak area into reciprocals and takes the form shown in Fig. 3.

Rice. 3. Dependence of the reciprocal of the sum of the areas of the peaks of saponins on the chromatogram (1/S 10 -1) on their reciprocal value of the content in the sample (1/m - 10 -1). 1 - Aralia saponins; 2 - sugar beet saponins.

Using the obtained calibration dependence, the content of saponins in the aralia tincture was determined. 5 ml of the tincture was diluted with 70% ethanol in a 25 ml volumetric flask. 5 μl of the resulting solution was applied to the starting line of the "Armsorb" plates. 5 μl of a standard solution of aralosides with a concentration of 1 mg/ml (5 μg in the sample) was applied to the adjacent point. The plates were processed as described above.

The difference between the obtained results and the results of the determination according to FS 42-1647-93 (5.3 mg/ml) was 6–7% towards overestimation, which can be explained by the loss of saponins during the multi-stage preparation of the sample according to FS (table).

The method described above was used to determine saponins in tablets "Saparal" and in plant materials (roots of Manchurian aralia and rhizomes of sugar beet) with preliminary exhaustive extraction of saponins from tablets from raw materials - 80% hot ethanol. Deviations from the results of the determination according to FS 42-1755 - 81 for tablets (0.040 g) are within the same limits as for tincture (table).

Thus, the possibility of express quantitative determination of some triterpene saponins, derivatives of oleanolic acid in pharmaceuticals and plant materials by HPTLC with subsequent quantitative evaluation of the obtained zones using scanning densitometry has been shown.

The results of the determination correlate quite well with the results of the determination by PS. The technique makes it possible to combine the determination of the authenticity of preparations by TLC (by FS) with the subsequent scanning of the zones of the components on the plates and their quantitative assessment. Chromatograms obtained during scanning also make it possible to determine the ratio of individual saponins in the analyzed objects.

The results of the quantitative determination of araloses inCTofik-from aralia (I) and tablets "Saparal" (2) by the TGC method using scanning dsnsptoietrnn /" = 0.95, and - 5,/=2,78,/=4

STUDY OF THE LIPID AND FLAVONOID COMPOSITION OF SAMPLES OF SOME SPECIES OF THE GENUS CHINA ( Lathyrus .)

From many representatives of the legume family, such as alfalfa, lupine, clover, vetch, flavonoids have been isolated that have a wide spectrum of action: anti-inflammatory, wound healing, vascular strengthening, etc. Isoflavones were isolated from red clover: biochanin A - 0.8% and formononetin - 0.78%, which have estrogenic activity.

We have studied the flavonoid composition in certain species of the genus rank: ch. sowing (I), schlugovaya (II), ch.

With a view to the possible use of the lipid complex in food additives and drugs, we also studied the complete fractional composition of lipids in the grass and seeds of the chin.

Materials and methods

The isolation of the lipid fraction from dry raw materials was carried out according to the method of Bligh and Dyer.

To a sample (0.2 g) of dry plant material was added 1.6 ml of distilled water and kept for a day in the cold. Then 6 ml of a mixture of chloroform - methanol (1:2) was added and left for 3 days, after which it was centrifuged at 8 thousand rpm - 15 minutes. 2 ml of water and 2 ml of chloroform were added to the clear supernatant. The resulting 2-phase system was separated in a separating funnel. The chloroform layer was washed twice with methanol and evaporated to dryness on a rotary evaporator. The residue was dried in a vacuum desiccator, then diluted with chloroform to a concentration of 10 mg/ml, and the solution was stored in stabilized chloroform at + 4°C.

Qualitative analysis of the lipid fraction was carried out using TLC on Kizelgel 60 (254) plates in the following solvent systems:

A. For neutral lipids: 1) Hexane - benzene (9:1); 2) Hexane - ether - acetic acid (90:10:1).

B. For polar lipids (phospholipids): 1) chloroform - acetone - methanol - acetic acid - water (6:8:2:2:1), acidic; 2) chloroform - meta-

nol - 26% ammonia (65:25:5), basic; 3) chloroform - methanol - water (65:25:4), neutral.

When determining the qualitative composition of phospholipids, their identification was carried out using various developers (iodine vapor, ninhydrin, Dragendorff's reagent, sulfuric acid) and using Rf standards.

The above set of systems and reagents made it possible to carry out an exhaustive analysis of lipid fractions isolated from chin samples.

To study the flavonoid composition, taking into account the phases of vegetation, the following samples were selected: I (flowering phase); IV (flowering phase); III (sochevnik) phase of budding; II (flowering phase); II (fruiting phase); II (vegetation phase before flowering).

The isolation of flavonoids from dry raw materials was carried out according to a method that ensures their exhaustive extraction.

For this purpose, 1 g of the crushed herb was placed in a reflux flask, filled with 20 ml of 70% ethanol, and boiled for 20 min in a water bath. The extract was cooled, filtered through a glass Schott filter, and evaporated to dryness on a rotary evaporator. The residue was dissolved in ethyl alcohol to a final concentration of 10 mg/ml. Determination of the total content of flavonoids was carried out using rutin as a standard. The results of this study are shown in table. 3.

The qualitative composition of flavonoids was determined by TLC on Kieselgel 60(254) plates from Merck, in the solvent system n-butanol - acetic acid - water (6:1:2). Detector - sulfuric acid at UV (254 nm) - flavonoid spots lilac color on a green background.

Table 1

The total content of flavonoids in raw materials (grass) of various types of chines (in%, in terms of absolutely dry weight)

Rice. Fig. 1. TLC of flavonoid composition of selected species of the genus Chin. C - the amount of witnesses of flavonoids: ononin - Rf 0.28; routine - R.0.48; luteolin-glucoside - Rf 0.58; formononetin - Rf 0.64; quercetin - Rf0,79: luteolin - Rf 0.82; biochanin A - Rf 0.85; apigenin - Rf 0,92.

Rice. Fig. 2. TLC of flavonoids of meadow grass in different phases of vegetation. IIa - flowering phase; IIb - fruiting phase: IIc - vegetation phase before flowering. C - the amount of witnesses of flavonoids: ononin - Rf f 0.28; routine - Rf 0.48; luteolin-glucoside - Rf 0.58; formononetin - Rf 0.64; quercetin - Rf 0.79; luteolin - R ( 0.82; biochanin A - Rf 0.85; apigenin - Rf 0,92.

When studying the flavonoid composition of II in different phases of vegetation, it was noted that in addition to rutin and quercetin, ononin and formononetin are present in a noticeable amount in the extract. Other flavonoids were found in trace amounts.

Thus, it is shown that in different types of chin, in different phases of vegetation, both glycosides and aglycones are contained - ononin, rutin, luteolin-glucoside and their aglycones: formononetin, quercetin and luteolin, and their composition varies depending on the type of plant, and in one plant (meadow rank) depending on the vegetation phase.

table 2

The quantitative content of the main flavonoids in individual representatives of the genus chiny (in %, in terms of absolutely dry weight)

In II, both ononin and formononetin were found in the vegetative period. During the period of flowering and fruiting, the amount of ononin noticeably decreases, and the amount of formononetin increases.

The quantitative content of the main flavonoids in the studied samples was determined by quantitative TLC under the same conditions. The results of this study are shown in table. 2.

As follows from the data in Table. 2, different kinds Chins are a rich source of bloflavonoids, due to which these plants exhibit one of the types of biological activity.

Conclusion:

One of the important tasks of modern chemistry is the reliable and accurate analysis of organic substances, often similar in structure and properties. Without this, it is impossible to carry out chemical, biochemical and medical research, ecological methods of environmental analysis, forensic examination, as well as chemical, oil, gas, food, medical industries and many other sectors of the national economy are largely based on this. Only a small part of the methods and techniques of thin layer chromatography is given here. But as you can see from this little, thin layer chromatography has significant and serious possibilities, combined with convenience and simplicity.