MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION
GENERAL PHARMACOPEIAN AUTHORIZATION
ChromatographyOFS.1.2.1.2.0002.15
on paper Instead of Art. GFXI, issue 1
The chromatographic process that occurs on a sheet of filter paper while moving through its capillaries and the surface of the mobile phase is called paper chromatography.
The stationary phase is either the paper itself or substances previously deposited on its fibers. The mechanism of paper chromatography can be distributive or adsorption. The movement of the mobile phase occurs either only under the action of capillary forces (ascending chromatography), or under the action of capillary forces and gravity (descending chromatography).
During chromatography, the analytes form adsorption zones on paper in the form of round or oval spots or stripes, depending on the method of application (point or stripe). The set of adsorption zones obtained by chromatography of a given analyzed sample is called a chromatogram.
The mobility of a substance during chromatography is characterized by the retention factor R f , (see ).
Application area
Paper chromatography can be used to establish the identity, purity, and quantification of an analyte.
Authenticity is confirmed by simultaneous chromatography of the analyte and reference substance on the same sheet of paper. If the samples are identical, then the adsorption zones corresponding to them on the chromatograms have the same shape and equal values R f. For identification, it is sometimes advisable to chromatograph a mixture of equal amounts of the analyte and standard substance. One spot should be observed on the chromatogram, which characterizes the adsorption zone. Chromatography conditions should be chosen so that the values R f were different from 0 and did not exceed 1. In addition, to confirm the authenticity of the analyte, specific conditions for its detection specified in the monograph can be used.
When testing for purity, impurities and the main substance must have different values. R f. The degree of purity of the analyte can be judged by the magnitude and intensity of the color (or absorption) of the adsorption zones of impurities found on the chromatogram. The content of impurities can be determined semi-quantitatively. To do this, a certain amount of the analyte and several samples of the determined impurity (witness) with different, precisely known concentrations are simultaneously chromatographed on one sheet of paper. The impurity content in the analyzed sample is estimated by comparing its adsorption zone on the chromatogram by the combination of the area of the adsorption zone and the intensity of color (or absorption) with the adsorption zones of the witness.
For quantitative analysis, special instruments are used that allow measurements in the ultraviolet and visible regions of the spectrum. To process chromatograms in the visible region of the spectrum, it is advisable to use flatbed scanners and appropriate software.
it is also possible to carry out the quantitative determination of substances after their extraction from the chromatogram. To do this, the adsorption zones are cut out and the substance to be determined is extracted with a suitable solvent. The content of the analyte in the extract or dry residue after distillation of the solvent is found by any method suitable for determining low concentrations and taking into account the structure of the analyte.
A sample of the test mixture can be applied either pointwise to the start line, or in the form of a uniform strip along the entire start line, while the primary adsorption zone will have the form of a spot, and in the second case, a strip parallel to the start line.
Equipment
To carry out chromatography on paper, sealed chambers are used, made of an inert material and allowing one to observe the progress of the separation process with the chamber lid closed. Often, glass jars or cylinders with a polished lid and additionally sealed are used as chambers. The lid may have inlets (gates) for adding solvent or relieving excess pressure in the chamber.
When carrying out downward chromatography, a vessel for the mobile phase (boat) is placed in the upper part of the chamber. The boat must contain a volume of mobile phase sufficient to carry out at least one chromatography. The length of the boat should exceed the width of the sheet of chromatographic paper. The chamber must be equipped with devices for fixing a sheet of chromatographic paper in the working position and for introducing the mobile phase into the boat.
In ascending chromatography, the mobile phase is either placed in a boat placed at the bottom of the chamber or poured onto the bottom of the chamber.
The inner walls of the chamber are covered with filter paper, which contributes to its faster and more complete saturation with solvent vapors that are part of the mobile phase.
The preparation of equipment, chromatographic paper and mobile phase is given in pharmacopoeial monographs.
Chromatographic Separation Techniques
Paper chromatography can be one-dimensional or two-dimensional.
One-dimensional paper chromatography involves point application or application in the form of a strip of a sample of the mixture under study to the start line and subsequent chromatography in one direction (Fig. 1).
Figure 1 - Exemplary scheme of one-dimensional paper chromatography
Two-dimensional chromatography involves the sequential passage of the mobile phase in two perpendicular directions, which allows for a clearer separation of the mixture of analyzed substances (Fig. 2).
Figure 2 - An exemplary scheme of two-dimensional paper chromatography
Downward chromatography
At the bottom of the chromatographic chamber, place the stationary or mobile phase in an amount sufficient to form a layer 2.5 cm deep. The chamber is closed and left to saturate for 24 hours at a constant temperature. In some cases, when using sufficiently volatile solvents, this time can be reduced.
Solutions of substances are applied to the start line with a micropipette or microsyringe so that the distance between the points of application of individual samples is at least 3 cm. If the minimum amount of the analyzed solution required for chromatography can form a primary adsorption zone on paper in the form of a spot exceeding 10 mm in diameter , the application is carried out in several stages, preventing excessive diffusion at the start line by drying. After applying the solutions of the analytes and drying the resulting primary chromatogram, the paper strip is fixed in the working position in the chamber and left for 1.5 hours. the starting line was only one, as smooth as possible, inflection. At the end of the exposure, chromatography begins, for which the mobile phase is poured into the boat. Chromatography is usually terminated when the mobile phase front approaches the bottom end of the paper strip. If there are no special instructions in the monograph, the strip is removed from the chamber and dried in air, marking the final position of the mobile phase front with a graphite pencil. The detection of adsorption zones is carried out according to the instructions of the pharmacopoeial article.
Rising chromatography
For ascending chromatography on paper, chambers are used in which the vessel (boat) with the mobile phase is located at the bottom or at the bottom. A strip of chromatographic paper is fixed at the top of the chamber so as to allow the lower end of the strip to be immersed in the boat with the mobile phase. In the working position of the strip, the start line should be 2–3 cm from the surface of the mobile phase. Otherwise, the working methods for ascending chromatography on paper do not differ from those described above.
Preparation of phases and paper
In the preparation of immiscible solvent systems that are jointly used in chromatography as a mobile and stationary phase, it is necessary to ensure their mutual saturation, for example, by shaking in a separating funnel. At the same time, the monograph must indicate which of the phases is used for chromatography.
Filter paper of the desired density of the "chromatography" qualification is cut in a direction perpendicular or parallel to the fibers into sheets (strips) whose length is approximately equal to the height of the chamber. The width of these bands can be approximately determined by the formula: A = 3(TO+ 1), where A– strip width (cm), TO is the number of chromatograms per strip.
On each strip of paper, a straight line, called the start line, is drawn with a graphite pencil to indicate the places where the chromatographed substances are applied. The distance from the end of the paper strip to the start line is chosen so that when the paper is immersed in the boat, direct contact of the substances applied to the start line with the liquid in the boat is excluded.
On the sheets of filter paper prepared in this way, if indicated in the monograph, the stationary phase is applied. To do this, the corresponding non-volatile solvents (formamide, propylene glycol, etc.) are mixed with highly volatile solvents, and sheets of paper to be processed are immersed in the resulting mixture for 1–2 s. The excess of the mixture is removed from the surface of the sheets by compressing them between two layers of filter paper, after which the volatile component of the mixture is removed by drying in air for 15–20 minutes.
If an aqueous solution of non-volatile substances is recommended as the stationary phase, then the paper is treated with this solution, dried as described above, and kept in a chamber containing water vapor before chromatography. The volatile components of the stationary phases are applied to the paper by keeping it in phase vapor in the chamber immediately before chromatography.
Detection of adsorption zones
At the end of chromatography, the chromatograms are dried to remove traces of solvents and viewed in visible or ultraviolet light (at a certain detector wavelength specified in the Pharmacopoeia Monograph), while marking adsorption zones. In some cases, to detect adsorption zones, the chromatogram is subjected to treatment with special reagents that form colored reaction products with the analyzed substances by spraying or immersing in a reagent solution.
The method for detecting adsorption zones must be indicated in the relevant pharmacopoeial monograph for the medicinal product.
In paper chromatography, the stationary liquid phase is water adsorbed by paper fibers in an amount of up to 20%, or another polar solvent; butyl alcohol, collidine, phenol, cresols are most often used as the mobile phase. The carrier is good filtered paper, sufficiently uniform in thickness and density.
To separate the mixture by paper chromatography, a drop of the test solution is applied to a strip of filtered paper 15-20 mm wide and 300-500 mm long at a distance of 20-30 mm from the end. The end of the strip is immersed in an appropriate organic solvent, previously saturated with water, and the entire apparatus is placed in a sealed chamber, the atmosphere in which is saturated with vapors of the organic solvent and water. The movement of the solvent along the strip of paper, due to capillary forces, provides the development of the chromatogram, with individual zones moving at different speeds.
2D paper chromatography
Even more precise results are obtained using the so-called two-dimensional paper chromatography. For this option, not strips of filtered paper are used, but rectangles approximately 400X500 mm in size. A drop of the test solution is applied near one of the vertices of the rectangle, and the chromatogram is developed twice with different solvents, for example, phenol and collidine, first with one solvent, and then, after turning through 90°, with another.
Chromatogram development methods
Rising chromatography. The paper is immersed at the bottom end into the mobile phase. The rise of the liquid occurs under the action of capillary forces.
"+" The device is simple, it is possible to quantify the results;
"-" Gravity and capillary forces act in opposite directions; the suction rate after rising to 20 cm drops sharply. Applicable for substances with sufficiently large differences in Rf values
Downward chromatography. The paper is immersed in the mobile phase with its upper end. The flow of liquid occurs under the action of gravity.
"+" - rapid passage of the mobile phase; no restriction on the length of the run of spots (flow chromatogram); it is possible to separate substances with slightly different Rf values and quantify the results.
"-" - The device is more complicated than for ascending chromatography.
Rice. 5.
Radial-horizontal chromatography. The mobile phase is continuously applied to the center of a round sheet of paper.
"+" - Fast execution, the zones are narrow and sharply defined; greater completeness of separation than for the first methods.
«-» - Only a qualitative assessment of the results is possible; the use of "witnesses" is possible only with the so-called "secret method" (ie, when dividing paper into sectors).
Mobile phase preparation
The simplest case of paper chromatography, ascending chromatography with water as the ascending phase, is described below.
The components of the selected solvent system are mixed in the indicated ratio in a separating funnel. The two immiscible phases are brought to mutual saturation by shaking; the organic phase acts as the mobile phase.
Substance application
From paper of a certain grade, a strip is cut, the size of which corresponds to the size of the cylinder used for chromatography. At a distance of 3 cm from the bottom edge, a marking line is applied with a pencil. On this line, 2 - 2.5 cm apart and from the edges, the strips mark the starting points. Each starting point is applied with a special pipette; in this case, spots about 1 cm in diameter are formed. The solvent is then allowed to evaporate.
thin layer chromatography solvent paper
Manifestation
The mobile phase is poured into the bottom of the cylinder and a strip of paper is suspended. Leave it hanging overnight, and then the lower edge of the strip is immersed 0.5 cm into the mobile phase. After the solvent rises by 20–25 cm, the strip is removed, the position of the solvent front is marked with a pencil, and the chromatogram is dried.
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 the stationary phase or inert carrier of the stationary phase. The paper chromatography method was proposed by A. Martin and R. Sing 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 (eluent); uniform advance of the eluent along the stationary phase layer is ensured by the capillary structure of the paper. The components are transported by the eluent at different rates according to their partition 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 during the same time by the eluent; R f ≤1; the value of Rf depends on the nature of the substance, the composition of the mobile phase, the type of paper, and the technique of the experiment and should not depend on the concentration of the analyte and the presence of other substances. The colored zones on the chromatogram are observed visually, the uncolored zones are shown by 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 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. 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.
Paper chromatography can be used to analyze small amounts (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 separable 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.
Partition chromatography. Paper chromatography. Sedimentary chromatography. The concept of osit (exclusion) chromatography. Gel chromatography.
Partition chromatography.
a chromatographic method in which the stationary (stationary) phase is chemically bonded to the surface of a stationary carrier. The mobile phase is a liquid that serves as a solvent, or a gas (gas chromatography). Separation occurs due to the difference in polarity of the substances being separated. In partition chromatography, a carrier is impregnated with one of the solvents (“stationary solvent”) and the other solvent (“mobile”) is passed through the carrier column. Most often, water or other polar liquids (sulfuric acid, methyl alcohol, etc.) are taken as an immobile solvent; as a mobile solvent - less polar liquids that do not mix with the first in all proportions. A portion of the investigated mixture of substances, dissolved in a mobile solvent, is injected into the column, and after the solution is absorbed by the upper part of the column, the column is washed with a pure mobile solvent. During the washing process, the substances of the mixture are continuously redistributed between two immiscible liquid phases. Since different components of the mixture have different distribution coefficients, the speed of movement of the individual components is different. The component of the mixture that has the highest distribution coefficient will have the highest movement speed: C
C = fixed
those. the ratio of the concentration of a solute in the mobile phase to its concentration in the stationary phase.
One of the main conditions for obtaining a clear separation of the mixture by partition chromatography is the practical absence of any interaction between the components of the mixture and the carrier. If this condition is met, then the complete separation of the mixture occurs when the column is washed. The number of carriers suitable for partition chromatography is extremely limited. More or less satisfactory qualities are possessed by such carriers as specially prepared silica gel, purified starch, cellulose.
Paper chromatography.
a strip of filter paper 30-50 cm long and 1.5 cm wide is taken. A drop of a mixture of analyzed substances is applied to one of the ends of this strip at a certain distance from the edge. This end of the paper is then dipped into a bath containing an organic solvent saturated with water. With the slow advance of the solvent through the pores of the paper, a continuous redistribution of the substances of the mixture between the two liquid phases occurs. If different components of the mixture have different distribution coefficients, then the speed of advancement of the individual components of the mixture will be different. The movement of the mobile solvent on the paper can be either downward or upward. After chromatography is completed, the strip of paper is dried and then developed with a reagent giving a color reaction with the compounds to be analyzed. The resulting chromatogram is a collection of colored spots arranged in a certain order along a strip of paper.
Rice. 22. 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 down chromatography, the solvent moves down the paper from the solvent reservoir at the top of the vessel. In this way, the individual components can be eluted. The most common solvent systems are: CH3COOH-H2O (15:85 vol), 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 manuals or monographs on paper chromatography.
Sedimentary chromatography- a chromatography method based on the ability of the substances to be separated to form poorly soluble compounds with different solubility products. The stationary phase is an inert carrier coated with a layer of precipitant; the separated substances that are in the mobile phase interact with the precipitant and form poorly soluble substances - precipitation. With further passage of the solvent, they occur in turn: the dissolution of these precipitates, the transfer of the substance along the layer of the stationary phase, precipitation again, etc. In this case, the rate of movement of the precipitate along the stationary phase is proportional to its solubility product (PR). The chromatogram in this case will be the distribution of precipitation over the carrier layer. An example is the separation of halide ions on a support (silica gel, cellulose, etc.) impregnated with a silver salt. It is possible to use their unequal solubility in different solvents or in solutions with different ionic strength to separate precipitates. It is implemented both in the column and in the planar version.
Gel filtration or size exclusion chromatography(sieve, gel permeation, gel filtration chromatography) - a type of chromatography, during which the molecules of substances are separated in size due to their different ability to penetrate into the pores of the stationary phase. In this case, the largest molecules (higher molecular mass) capable of penetrating into the minimum number of pores of the stationary phase are the first to leave the column. Substances with small molecular sizes, which freely penetrate into the pores, come out last. In contrast to adsorption chromatography, in gel filtration, the stationary phase remains chemically inert and does not interact with the substances to be separated. A sample solution is introduced into the column, the volume of which is limiting for the quality of chromatography. For analytical separations, it should not exceed 0.1% of CV (total column volume), and for preparative purification, it should not exceed 8-10% of CV. The column is packed with powder, the particles or granules of which have pores of a certain diameter. Macromolecular substances that do not enter the pores pass between the granules, so their retention volume is equal to the volume of the column minus the volume of the stationary phase (the so-called free volume). They elute first. Molecules of medium size fit into the pores of the sorbent, but not completely. Therefore, their retention volume is slightly higher than the free volume. They elute second. The smallest molecules freely enter the pores together with solvent molecules. Therefore, their retention volume in the column is much higher than the free volume and approaches the total volume of the column (i.e. 100% CV). They elute last.
Qualitative chemical analysis. Classification of qualitative analysis methods (fractional and systematic, macro-, semi-micro-, micro-, ultra-microanalysis). Analytical reactions and reagents used in qualitative analysis (specific, selective, group). The use of qualitative analysis in pharmacy.
Qualitative Analysis- identification (detection) of the components of the analyzed substances and an approximate estimate of the amount of their content in substances and materials. The components can be atoms and ions, isotopes of elements and individual nuclides, molecules, functional groups and radicals, phases, etc.
Classification of methods The method of analysis is chosen depending on the expected content of the substance and on the limit of detection of the applied reaction. Currently, when studying qualitative chemical analysis in educational laboratories, semi-microanalysis is used.