Stairs. Entry group. Materials. Doors. Locks. Design

To separate the components of a mixture using paper chromatography, a drop of the sample being analyzed is placed on a strip of filter chromatography paper 2-4 cm from the end, and the end of the strip is immersed in a solvent, which begins to move along the paper under the action of capillary forces. To prevent dehydration of the paper, the mobile phase is usually saturated with water. When the mobile phase moves, the components of the test sample, applied to the paper near the start, are distributed between the moving solvent and the water film held by the cellulose. In this case, the components move at different speeds in the form of zones, the size of which is usually slightly larger than the size of the initial spot. Chromatography on paper is usually carried out in a closed vessel (Fig. 1) to avoid evaporation of the solvent during chromatography. In ascending chromatography, the upper end of a strip of paper is fixed in a holder, and the lower end is immersed in a solvent, which is poured into a low cuvette or Petri dish located at the bottom of the vessel in which chromatography is being carried out. For these purposes, you can also use a large measuring cylinder, onto the bottom of which the mobile phase is poured and covered with glass on top.

Rice. 1 - Chromatography on paper: A - ascending chromatogram; B - descending chromatogram; 1 - vessel for chromatography; 2 - reservoir with solvent; 3 - chromatographic paper; 4 - starting points; 5 - separated components; 6 - solvent front

In downward chromatography, the solvent moves down the paper from a solvent reservoir located at the top of the vessel. In this way, individual components can be eluted.

The manifestation of paper chromatograms is, in principle, no different from that described for thin-layer chromatograms.

The efficiency of paper chromatography depends on both the type of paper and the composition of the mobile phase. Paper grades differ in porosity, thickness, and degree of hydration. Based on the speed of movement of solvents, they distinguish between fast, medium and slow papers. The most common types of chromatographic papers are Leningrad paper, Whatman paper, etc.

The most common solvent systems: CH3COOH-H2O (15:85 volume), 1-butanol - CH3COOH-H20 (4:1:5), 2-propanol - NH3 (conc.) - H2O (9:1:2), 1 -butanol - 1.5 N. NH3 (1:1), phenol - water, etc. The composition of the mobile phase is usually selected experimentally or based on the data given in reference books or monographs on paper chromatography.

The use of ion exchange paper allows you to combine the advantages of paper chromatography and ion exchange. This paper is made by mixing ion exchange resin with the cellulose used to make paper.

Paper chromatography is of great importance for qualitative analysis. Its use in quantitative analysis is limited.

PAPER CHROMATOGRAPHY, a method of separation, identification and quantification of substances; one of the planar variants of liquid chromatography, in which special paper is used as a stationary phase or an inert carrier of the stationary phase. The paper chromatography method was proposed by A. Martin and R. Singh in 1944 for the analysis of mixtures of amino acids.

The separation of the components of the mixture occurs due to the distribution of substances between the stationary phase and the mobile phase (eluent); uniform movement of the eluent along the stationary phase layer is ensured by the capillary structure of the paper. The transfer of components by the eluent occurs at different rates in accordance with their distribution coefficient. As a result, on the chromatogram the substances form separate zones (spots), the position of which is characterized by the values ​​of R f - the relative speed of movement. Experimentally, the value of R f is determined as the ratio of the distance traveled by the substance to the distance traveled by the eluent during the same time; R f ≤1; the Rf value depends on the nature of the substance, the composition of the mobile phase, the type of paper, the experimental technique and should not depend on the concentration of the substance being determined and the presence of other substances. The colored zones on the chromatogram are observed visually, the uncolored zones are revealed with reagents that form colored or fluorescent compounds with the components of the mixture being separated. Identification of substances can be based on a comparison of the R f values ​​of the test and standard solutions. Quantitative analysis is carried out directly on the chromatogram (by spot size, absorption or reflection spectra, using densitometry, radiometry, etc.) or after separation (for example, by extraction) of the substance of the chromatographic zone from the cellulose base (spectrophotometric, fluorimetric, atomic absorption methods are used for determination and other methods).

Paper chromatograms can be obtained by ascending, descending or horizontal (radial) movement of the eluent; Using repeated separation, two-dimensional chromatograms are obtained. The separation is carried out in closed chambers (glasses, cylinders, etc.) saturated with vapors of the mobile phase. To separate hydrophilic compounds, special chromatographic paper (made of cellulose fibers) containing water or ion exchangers as a stationary phase is used. To separate water-insoluble compounds, paper is hydrophobized by acetylation or impregnation with hydrophobic substances (paraffin, rubber, organic reagents, etc.). Various solvents or their mixtures, aqueous solutions of organic and inorganic acids, alcohols, electrolytes, etc. are used as eluents.

The paper chromatography method can be used to analyze small quantities (10 -9 -10 -6 g) of chemical compounds of almost all classes. Due to its technical simplicity and accessibility, paper chromatography is used to detect easily separated substances, to check the identity of organic compounds, to determine trace elements in geochemical analysis, etc.

Lit.: Chromatography on paper. M., 1962; Chromatography. Practical application of the method. M., 1986. T. 1-2; Fundamentals of Analytical Chemistry / Edited by Yu. A. Zolotov. 2nd ed. M., 1999. Book. 1.

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 another 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, changes in the water content in the pores of the paper when storage conditions change, a very low chromatography speed (up to several days), and low reproducibility of results. These shortcomings seriously affect the spread of paper chromatography as a chromatographic method.

Thin layer chromatography

In this method, chromatography of substances occurs in a thin layer of sorbent deposited on a solid flat substrate. The separation in this method is mainly based on sorption-desorption. The use of various sorbents has made it possible to significantly expand and improve this method.

At the beginning of the method, the plates had to be made independently. But today, factory-made plates are mainly used, which have a fairly wide range of sizes, media, and substrates.

A modern chromatographic plate is made of glass, aluminum or 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, it is heavy in weight), the most widely used plates are those using aluminum foil or polymers as bases.

To fix the sorbent, gypsum, starch, silica sol, etc. are used, which hold the sorbent grains on the substrate. The thickness of the layer 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 hydrated silicic acid, formed by the action of mineral acids on sodium silicate and drying 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 adsorbs water on the surface and forms hydrogen bonds.

Alumina. Aluminum oxide is a weakly basic adsorbent and is used primarily for the separation of weakly basic and neutral compounds. The disadvantage of aluminum oxide plates is the mandatory activation of the surface before use in an oven at high temperatures (100-150 0 C) and the 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 in cases where more polar adsorbents do not provide effective separation.

Cellulose - Thin-layer plates coated with cellulose are very effective at separating complex organic molecules. The adsorbent consists mainly of cellulose beads with a diameter of up to 50 microns, fixed to a carrier with starch. But, as in paper chromatography, the rise of the solvent front occurs very slowly.

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 high molecular weight 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 separated components have low solubility (if the solubility of the substance is high, then the substances will move with the front, if solubility is low, they will remain at the start). When partition chromatography or when using reverse phases, the solubility of 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 toxic or deficient.

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

· The system should not cause chemical changes in the separated components.

· In the chosen system, the analytes must have different R f values ​​and be distributed along the entire length of the chromatogram. It is desirable that the R f values ​​lie in the range of 0.05-0.85.

· When choosing a system, it is also necessary to take into account the nature of the separated substances. Thus, when chromatography of substances with basic properties, the system should not have acidic properties and vice versa.

Preparation of plates

When using purchased plates, they must first be prepared for chromatography. This is due to the fact that plate adsorbents during storage absorb not only moisture, but also other substances contained in the air. When using unprepared plates during the chromatography process, a “dirt” front appears, which can interfere with the determination of substances with large R f values, and some substances, such as water, can change the composition of the mobile phase, thereby changing the obtained R f values.

Preliminary preparation of the plates consists of spreading 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, retain their properties for several months.

Paper chromatography. From the first word you understand that this is something related to paper; and the second word “chromatography” means “color” (chroma) and “write” (graphia). Add them up and you get "write with color on paper".

Paper chromatography is the most important test in science. By carefully analyzing the composition of a chemical by color, a scientist can easily identify the original substances. It's easy to see that the chromatography that's really worth studying is the one that works through capillary action, the way water spreads through paper.

Column - contains a chromatographic sorbent and performs the function of separating a mixture into individual components. Eluent - mobile phase: gas, liquid or (less commonly) supercritical fluid. The stationary phase is a solid phase or liquid bound on an inert carrier; in adsorption chromatography, it is a sorbent. A chromatogram is the result of recording the dependence of the concentration of components at the outlet of the column on time. Detector - a device for recording the concentration of mixture components at the outlet of the column. Chromatograph is a device for performing chromatography.

Descending chromatography A method in which the mobile phase moves downward Ascending chromatography A method in which the mobile phase moves upward Horizontal chromatography A method in which the mobile phase moves horizontally Circular chromatography A method in which the mobile phase moves from the middle of a circle to its circumference Flow chromatography A method in which in which the advancement of the mobile phase continues even after the front reaches the end of the paper Repeated chromatography A method in which, upon completion of the first advance of the mobile phase, the chromatogram is dried and the chromatography is repeated (sometimes several times) Manifestation Method of detecting substances in the chromatogram Carrier Chromatographic paper

Stationary (stationary) phase Phase fixed on a carrier Mobile (mobile) phase Phase that ensures the movement of separated substances along a carrier with a stationary phase Start Place on which the test sample is applied

In paper chromatography, special grades of paper are used, differing in numbers, and as they increase, the density of the paper increases. The paper retains water in its pores, which is the stationary liquid phase. The sample solution is applied in the form of drops onto a sheet of paper at some distance from the edge. After the solvent has evaporated, the edge of the sheet is placed in a sealed chamber containing a developer - a mobile liquid phase (for example, alcohols, ketones, phenols, carbon tetrachloride, chloroform and other mixtures thereof, as well as mixtures with inorganic solvents). In this case, the initial spot moves along the developer current and the mixture is separated into components. If the substances are not colored, then the chromatogram is developed, for example, by spraying with an indicator solution, examined in ultraviolet rays, etc. The ratio of the distance Rf traversed by spot I to the distance traversed by the front of the developer m, under the same experimental conditions, is a constant value; Rf values ​​vary for different substances and can be used to identify compounds.

Classification Paper chromatography, like chromatography in general, can be divided into distributional adsorption Normal (the method is used to separate lipophilic substances.) ion exchange reverse phase preparative analytical

Quantitative determinations of various substances in chromatogram spots are carried out using conventional analytical methods. There are: one-dimensional, two-dimensional, circular, column and electrophoretic chromatograms.

I. Adsorption chromatography is based on the selective adsorption of individual components of the analyzed mixture by appropriate adsorbents. When working with this method, the analyzed solution is passed through a column filled with small adsorbent grains. Adsorption chromatography is used to separate non-electrolytes, vapors and gases. II. Partition chromatography is based on the use of the difference in sorption coefficients of individual components of the analyzed mixture between two immiscible liquids. One of the liquids (immobile) is located in the pores of the porous substance (carrier), and the second (mobile) is another solvent that is immiscible with the first.

This solvent is passed through the column at low speed. Different values ​​of distribution coefficients provide different speeds of movement and separation of mixture components. The distribution coefficient of a substance between two immiscible solvents is the ratio of the concentration of a substance in a mobile solvent to the concentration of the same substance in a stationary solvent: (K = Spodv/Snepodv).

Sometimes, instead of a column, strips or sheets of filter paper that do not contain mineral impurities are used as a carrier for a stationary solvent. In this case, a drop of the test solution is applied to the edge of a strip of paper, which is suspended in a closed chamber, lowering its edge with a drop of the test solution applied to it into a vessel with a movable solvent (propellant), which, moving along the paper, wets it. In this case, each substance contained in the analyzed mixture moves at its inherent speed in the same direction as the mover.

A special type of partition chromatography is gas-liquid chromatography (GLC). Various non-volatile liquids deposited on an inert solid carrier are used as a stationary phase; gaseous nitrogen, hydrogen, helium, carbon dioxide, etc. are used as the mobile phase. The separation of mixtures by the GLC method is carried out in columns, which are tubes with an internal diameter of 1-6 mm and a length of 1-5 m, filled with an inert carrier, for example, diatomite impregnated with a non-volatile liquid, or steel and glass capillaries with a diameter of 0.2-0.3 mm and a length of 25-100 m with a liquid phase deposited on the walls of these capillaries (capillary gas-liquid chromatography).

. Ion exchange chromatography is based on the use of ion exchange processes occurring between mobile ions of the adsorbent and electrolyte ions when passing a solution of the analyzed substance through a column filled with an ion exchange substance (ion exchanger). Ion exchangers are insoluble inorganic and organic high-molecular compounds containing active (ionogenic) groups. Mobile ions of these groups are capable of exchanging cations or anions of the dissolved substance upon contact with electrolyte solutions. Aluminum oxide (for chromatography), permutin, sulfonated carbon and various ion exchange substances and ion exchange resins are used as ion exchangers. Ion exchangers are divided into cation exchangers capable of cation exchange (contain active groups: SO 3 H, COOH, OH); anion exchangers capable of anion exchange (active groups: NH 2, =NH); ampholytes are ion exchange substances with amphoteric properties.

IV. Sedimentary chromatography is based on the different solubility of precipitation formed by various components of the analyzed mixture with special reagents applied to a highly dispersed substance. The analyzed solutions are passed through a column filled with a porous substance (carrier). The carrier is impregnated with a precipitating reagent, which forms precipitates with different solubilities with the ions of the solution. The formed precipitates, depending on solubility, are located in a certain sequence along the height of the column.

V. Size exclusion (molecular sieve) chromatography is based on the different permeability of the component molecules into the stationary phase (highly porous nonionic gel). Size exclusion chromatography is divided into gel permeation chromatography (GPC), in which the eluent is a non-aqueous solvent, and gel filtration, in which the eluent is water.

By adding a drop of a mixture of red and blue ink to the center of a piece of moistened filter paper and carefully applying clean water drop by drop, you will soon get exactly the same picture. Below is a ring chromatogram on paper of a complex mixture of six different amino acids,

demonstrated by four different reagents. Above right is a two-dimensional chromatogram of an even more complex mixture of fourteen different amino acids. This chromatogram was obtained from one drop of a solution of a mixture of acids applied to the point indicated by a circle. Development was carried out alternately in two directions using different reagents. Each label spot belongs to one amino acid. By the color and position of the spot, one can accurately determine the nature of the substance. Top left is a chromatogram of an ordinary ink blot on blotting paper.

Development of chromatograms The development of components in a chromatogram is carried out using one of the methods given below. Physical methods (Visually, in daylight, mark the position of spots of colored substances on the chromatogram. In the presence of fluorescent substances, development is carried out in UV light.) Chemical methods (Chromatograms are developed with liquid and gaseous developers, using the reaction of compounds present on the chromatogram with a suitable developer reagent to form colored or fluorescent substance. Liquid developers are applied with a spray bottle or use reagents in aerosol packaging, gaseous ones are used by placing the chromatogram in the developer vapor.)

The chromatogram is placed horizontally on a sheet of filter paper or left suspended on a glass rod and the entire area of ​​the chromatogram is sprayed with as small drops (mist) of developer as possible, first on one side and then on the other. When developed with a gaseous developer, the chromatogram is suspended in a chamber in which a volatile reagent (for example, iodine crystals) is placed, or at the bottom of which the developer is produced chemically (for example, nitrogen oxides are obtained by adding solid sodium nitrite to a solution of hydrochloric acid).

Biological methods Chromatograms are developed using the biological activity of the substances being chromatographed. Qualitative assessment of the chromatogram consists in determining the position of the spot or band, which is characterized by the value R f = a/b, where a is the distance from the center of the sample spot to the starting line, mm; b distance from the solvent front to the starting line, mm, or the value of Rx: Rx= a/c, where c is the distance from the center of the spot of the reference substance to the starting line, mm.

Determination of the amount of the required component in the sample is carried out by comparing the size and color intensity of its spot with spots of a reference substance applied to paper in the concentration range specified in the regulatory technical documentation for the test reagent and processed under test conditions. The assessment is carried out visually or using equipment (for example, a densitometer, a device for scanning spots of components on paper), or by eluting the spots and subsequent photometric determination of the optical density of solutions. Chromatograms are stored under conditions that prevent the appearance of mutual impressions of chromatograms (for example, with filter paper pads). If the nature of the stains allows, then a layer of quick-drying varnish is applied to the chromatograms. If necessary, sketch the chromatogram outline or take photographs.

EXAMPLES OF EQUIPMENT FOR PAPER CHROMATOGRAPHY AND METHODS OF ITS USE Chamber for ascending and descending chromatography 1. Chamber for ascending and descending chromatography (Fig. 1) Draw. 2. Chamber for horizontal chromatography (Fig. 2) (Fig. 2) 1 chamber; 2 grid of glass rods; 3 glass rod for pressing the end of the chromatogram; 4 cover; 5 chromatogram; 6 solvent

Crap. 3. Chamber for circular chromatogram (two Petri dishes) (Fig. 3) 1 paper wick; 2.4 Petri dishes; 3rd circle chromatogram; 5 solvent Damn. 4. Methods for arranging a chromatogram for ascending chromatography (Fig. 4) 1 chromatogram; 2 chromatographic chamber; 3 solvent

Crap. 5. Method of inserting a paper chromatogram into the groove 1 chromatogram; 2 start; 3 glass rod; 4 bent stick for pressing the chromatogram in the groove; 5 groove

A two-dimensional chromatogram is obtained by separating spots of a one-dimensional chromatogram with another developer in a direction perpendicular to the first row of spots. On a circular chromatogram, a spot placed in the center of the sheet is blurred along concentric circles. In paper column chromatography, separation is carried out on paper disks tightly inserted into a cylindrical column. To obtain electrophoretic chromatograms, a paper sheet is impregnated with an electrolyte, fixed between the electrodes, the analyzed mixture is applied, the electrodes are connected to a direct current source, and at the same time a mobile solvent is applied to the paper in a direction perpendicular to the direction of the electric current lines.

In this method, the separation of components occurs due to their unequal distribution between the two liquid phases and different rates of movement of substances under the influence of an electric field. Paper chromatography is used to separate and analyze inorganic and organic substances in natural and industrial materials (for example, determine resins in petroleum products, rare earth elements in rocks and minerals).

Chromatographic methods are indispensable in food quality control. The nutritional value of products is determined by analyzing the amino acid composition of proteins, the isomeric composition of fatty acids and glycerides in fats, carbohydrates, organic acids and vitamins. In recent years, many of these analyzes have been performed using high-performance liquid chromatography. To assess the safety of products, food additives (preservatives, antioxidants, sweeteners, dyes, etc.) are identified, the freshness of the products is determined, the early stages of spoilage and acceptable shelf life are established.

In food products, chromatography methods can detect such contaminants as pesticides, nitrosamines, mycotoxins (aflatoxins, ochratoxin A, zearalenone, etc.), polynuclear aromatic compounds, biogenic amines, nitrates, etc. Contamination of food products is also possible due to the penetration of harmful substances from packaging materials, in particular vinyl chloride, benzene, plasticizers, etc. Anabolic steroids, hormones and other types of pharmaceuticals, the abuse of which is typical for intensive livestock farming, are determined in meat products. A separate area of ​​application of gas chromatography is analysis of the aroma composition of food products. Thousands of volatile components have been discovered, of which only a few dozen determine the nature of the smell, the rest give the smell and taste of the product its individuality.

In recent years, a new area of ​​enantioselective analysis of food components has emerged. By the ratio of optical isomers of amino acids, hydroxy acids and some other compounds, it is possible to unambiguously determine whether a given product is natural or contains synthetic imitators and additives. Enantiomeric analysis showed that microwave processing of food products, unlike severe thermal processing, does not lead to racemization of amino acids. However, all dairy products subjected to fermentation processes contain a lot of (non-toxic) D alanine and D aspartic acid, waste products of lactic acid bacteria.

Natural fats are dominated by cis isomers of fatty acids. Recently, it was discovered that trans isomers increase the content of low-density lipoproteins and reduce the concentration of high-density lipoproteins in the blood, which may contribute to the development of atherosclerosis. The development of a technique for gas chromatographic separation and analysis of all fatty acid isomers forced manufacturers to reduce the content of trans isomers of unsaturated acids in margarine several times.

Gas chromatography revealed many undesirable physiologically active biogenic amines in some cheeses, and these types of cheese were banned. In Japan, food products use L tryptophan obtained through genetic engineering and biotechnology. And when thousands of people were diagnosed with a previously unknown disease and dozens of patients died, chromatographic methods revealed that these tragic consequences were caused by the presence of toxic contaminants in tryptophan (60 impurities were identified). Wines, cognacs and other alcohol-containing products are subjected to gas chromatographic analysis.

Now let's go back to fake butter again. There is such an oil - “Peasant”. It looks like butter like butter. Smells like butter. Delicious. To begin with, it was decided to check how many triglycerides it contains. In real oil, triglycerides are the majority of all lipids. On average - 98%. To check, we use the thin layer chromatography method. We use Sorbfil plates. The simplest chromatographic system is benzene.

Paper chromatography method refers to plane chromatography, it is based on the distribution of analytes between two immiscible liquids.

In partition chromatography, the separation of substances occurs due to differences in the distribution coefficients of components between two immiscible liquids. The substance is present in both phases as a solution. The stationary phase is retained in the pores of the chromatographic paper without interacting with it; the paper acts as a carrier of the stationary phase.

Types of chromatography paper:

hydrophilic paper retains up to 22% water in its pores; stationary phase – water, mobile phase – organic solvent; Such paper is used to determine water-soluble substances.

hydrophobic paper repels water, so it is impregnated with a non-polar organic solvent (stationary phase); mobile phase – water; This paper is used to determine water-insoluble compounds (fat-soluble acids, vitamins).

The following requirements apply to chromatographic paper:

chemical purity;

chemical and adsorption neutrality with respect to the analyzed substances and mobile phase;

uniformity in density;

same direction of fibers.

To obtain a chromatogram, a drop of the mixture being analyzed is placed on paper. The paper is placed in the chromatographic chamber, its end is immersed in a vessel with the eluent. The solvent moves along the paper, the mixture of analytes is distributed between the mobile and stationary phases and is separated on the paper in the form of spots or stripes. The position of the component zones is determined by developing chromatographic paper with appropriate reagents, which form colored compounds with the components of the mixture being separated.

To quantify the ability to separate substances in a chromatographic system, the distribution coefficient K p is used - the ratio of the concentration of a substance in the stationary and mobile phases. Experimental determination of distribution coefficients in this method is impossible; to assess the ability to separate substances on paper, the displacement (mobility) coefficient R f is used. The displacement coefficient is equal to the ratio of the speed of movement of the substance () to the speed of movement of the mobile phase (
). Experimentally, the value of R f is found as the ratio of the distance X traveled by the substance to the distance X f traveled by the solvent from the start to the front line:

.

The coefficient Rf varies within the range 0 – 1.00. The value of Rf depends on the nature of the substance being determined, the type of chromatographic paper, the quality and nature of the solvent, the method of applying the sample, the experimental technique and temperature. The Rf coefficient does not depend on the concentration of the analyte and the presence of other components.

Identification The chromatogram is performed in the following ways:

visual comparison of the characteristic color of zones of substances in the studied and standard chromatograms;

by measuring the mobility coefficients R f for the standard and the analyte in a specific solvent. Chromatography and determination of R f for the test and standard mixtures are carried out on the same paper and in the same chamber under strictly identical conditions. By comparing the coefficients Rf, a conclusion is made about the presence of certain components in the analyzed mixture.

quantitation performed directly from the chromatogram or by washing (eluting) the analyte from the paper.

Methods of quantitative analysis:

visual comparison of the color intensity of spots on the test and standard chromatograms (semi-quantitative determination, accuracy 15–20%);

measuring the area of ​​the spot formed by a given component and finding the concentration of the substance using a calibration graph constructed for a series of standard solutions in the coordinates: spot area – concentration of the substance; determination accuracy 5 – 10%;

elution of the analyte from the surface of the chromatogram and spectrophotometric or fluorimetric measurement of the optical density of the eluate (A); the concentration of a substance in solution is calculated using the formula:

,

where K is the proportionality coefficient; S – spot area, measured previously, mm 2 ; determination accuracy 1%.

According to the chromatography method, there are ascending (Fig. 21), descending (Fig. 22), circular (Fig. 23), gradient and two-dimensional chromatography.

The paper chromatography method is widely used for the determination of inorganic compounds, amino acids, amines, proteins, carbohydrates, fatty acids, phenols, vitamins in the chemical, food, pharmaceutical industries, medicine, and biochemistry.

The method has found application in the analysis of almost all food products: in sugar production - for the determination of carbohydrates; in baking and confectionery - amino acids, organic acids, carbohydrates, polysaccharides and carbonyl compounds; in winemaking - organic acids and amino acids; in the production of milk and dairy products - amino acids; in the meat processing industry - phenols, fatty and volatile acids, amino acids and carbonyl compounds.