24.11.202227.05.2017

2. The emergence and development of chromatography

The emergence of chromatography as a scientific method is associated with the name of the outstanding Russian scientist Mikhail Semenovich Tsvet (1872 - 1919), who in 1903 discovered chromatography in the course of research into the mechanism of solar energy conversion in plant pigments. This year should be taken as the date of creation of the chromatographic method.

M.S. The color passed the solution of the analytes and the mobile phase through the adsorbent column in the glass tube. In this regard, his method was called column chromatography. In 1938 N.A. Izmailov and M.S. Schreiber suggested modifying the Color method and carrying out the separation of a mixture of substances on a plate covered with a thin layer of adsorbent. This is how thin-layer chromatography arose, which makes it possible to carry out analysis with a micro-amount of a substance.

In 1947 T.B. Gapon, E.N. Gapon and F.M. Shemyakin was the first to carry out the chromatographic separation of a mixture of ions in a solution, explaining it by the presence of an exchange reaction between the sorbent ions and the ions contained in the solution. Thus, another direction of chromatography was discovered - ion-exchange chromatography. Currently, ion-exchange chromatography is one of the most important areas of the chromatographic method.

E.N. and G.B. Gapon in 1948 implemented what M.S. Color the idea of the possibility of chromatographic separation of a mixture of substances based on differences in the solubility of sparingly soluble precipitates. Sedimentary chromatography appeared.

In 1957, M. Goley suggested applying a sorbent to the inner walls of a capillary tube - capillary chromatography. This option allows the analysis of microquantities of multicomponent mixtures.

In the 1960s, it became possible to synthesize both ionic and uncharged gels with strictly defined pore sizes. This made it possible to develop a version of chromatography, the essence of which is to separate a mixture of substances based on the difference in their ability to penetrate into the gel - gel chromatography. This method allows the separation of mixtures of substances with different molecular weights.

At present, chromatography has received significant development. Today, various methods of chromatography, especially in combination with other physical and physicochemical methods, help scientists and engineers to solve various, often very complex problems in scientific research and technology.

Dmitry Ivanovich Mendeleev: contribution to the development of chemistry

Dmitry Mendeleev was born on January 27 (February 8), 1834 in Tobolsk in the family of the director of the gymnasium and the trustee of public schools in the Tobolsk province, Ivan Pavlovich Mendeleev and Maria Dmitrievna Mendeleeva, nee Kornilieva ...

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1. History of the discovery and development of chromatography

2. Basic provisions

3. Classification of chromatographic methods of analysis

4. Adsorption chromatography. Thin layer chromatography

4.1 Experimental technique in thin layer chromatography

5. Gas chromatography

5.1 Gas adsorption chromatography

5.2 Gas-liquid chromatography

6. Partition chromatography. Paper chromatography

7. Sediment chromatography

7.1 Classification of sediment chromatography methods according to experimental technique

7.2 Sediment chromatography on paper

8. Ion exchange chromatography

Conclusion

Bibliography

1. HISTORYDISCOVERIES AND DEVELOPMENT OF CHROMATOGRAPHY

The discoverer of chromatography was a Russian scientist, botanist and physicochemist Mikhail Semyonovich Tsvet.

The discovery of chromatography dates back to the time Tsvet completed his master's thesis in St. Petersburg (1900 - 1902) and the first period of work in Warsaw (1902 - 1903). Investigating plant pigments, Tsvet passed a solution of a mixture of pigments that differed very little in color through a tube filled with an adsorbent - powdered calcium carbonate, and then washed the adsorbent with a pure solvent. The individual components of the mixture separated and formed colored bands. According to modern terminology, Tsvet discovered the developing variant of chromatography (developing liquid adsorption chromatography). Tsvet outlined the main results of research on the development of the variant of chromatography he created in the book Chromophylls in the Plant and Animal World (1910), which is his doctoral dissertation. chromatography gas sedimentation ion exchange

Tsvet widely used the chromatographic method not only for separating a mixture and establishing its multicomponent nature, but also for quantitative analysis, for this purpose he broke a glass column and cut an adsorbent column into layers. Tsvet developed apparatus for liquid chromatography, was the first to carry out chromatographic processes at reduced pressure (pumping out) and at some excess pressure, and developed recommendations for the preparation of efficient columns. In addition, he introduced many basic concepts and terms of the new method, such as "chromatography", "development", "displacement", "chromatogram", etc.

Chromatography was used very rarely at first, with a latent period of about 20 years during which only a very small number of reports of various applications of the method appeared. And only in 1931, R. Kuhn (Germany) A. Winterstein (Germany) and E. Lederer (France), who worked in the chemical laboratory (led by R. Kuhn) of the Emperor Wilhelm Institute for Medical Research in Heidelberg, managed to isolate a - and b-carotene from raw carotene and thereby demonstrate the value of discovering Color.

An important stage in the development of chromatography was the discovery by Soviet scientists N.A. Izmailov and M.S. Schreiber of the thin layer chromatography method (1938), which allows analysis with a trace amount of a substance.

The next important step was the discovery by A. Martin and R. Sing (England) of a variant of liquid partition chromatography using the example of the separation of acetyl derivatives of amino acids on a column filled with water-saturated silica gel using chloroform as a solvent (1940) . At the same time, it was noted that not only a liquid, but also a gas can be used as a mobile phase. A few years later, these scientists proposed to carry out the separation of amino acid derivatives on water-moistened paper with butanol as the mobile phase. They also implemented the first two-dimensional separation system. Martin and Sing received the Nobel Prize in Chemistry for their discovery of partition chromatography. (1952). Further, Martin and A. James carried out a variant of gas partition chromatography, separating mixtures on a mixed sorbent of silicone DS-550 and stearic acid (1952 - 1953). Since that time, the method of gas chromatography has received the most intensive development.

One of the variants of gas chromatography is chromatography, in which, in order to improve the separation of a mixture of gases, simultaneously with the movement of the mobile phase - gas, the sorbent and the mixture to be separated are affected by a moving temperature field having a certain gradient along the length (A.A. Zhukhovitsky et al., 1951) .

A significant contribution to the development of the chromatographic method was made by G. Schwab (Germany), who was the founder of ion-exchange chromatography (1937 - 1940). It was further developed in the works of Soviet scientists E.N. Gapon and T.B. Gapon, who carried out the chromatographic separation of a mixture of ions in solution (together with F.M. Shemyakin, 1947), and also implemented the idea expressed by Tsvet about the possibility of chromatographic separation of a mixture of substances based on the difference in the solubility of sparingly soluble precipitates (sedimentary chromatography, 1948).

The modern stage in the development of ion-exchange chromatography began in 1975 after the work of G. Small, T. Stevens and W. Bauman (USA), in which they proposed a new analytical method called ion chromatography (a variant of high-performance ion-exchange chromatography with conductometric detection).

Of exceptional importance was the creation by M. Golay (USA) of a capillary variant of chromatography (1956), in which a sorbent is applied to the inner walls of a capillary tube, which makes it possible to analyze microquantities of multicomponent mixtures.

At the end of the 60s. interest in liquid chromatography has risen sharply. High performance liquid chromatography (HPLC) was born. This was facilitated by the creation of highly sensitive detectors, new selective polymeric sorbents, and new equipment that makes it possible to operate at high pressures. Currently, HPLC occupies a leading position among other chromatography methods and is implemented in various versions.

2. MAIN PROVISIONS

Chromatography is a method of separation and determination of substances based on the distribution of components between two phases - mobile and stationary. The stationary (stationary) phase is a solid porous substance (often called a sorbent) or a liquid film deposited on a solid substance. The mobile phase is a liquid or gas flowing through a stationary phase, sometimes under pressure. The components of the analyzed mixture (sorbates) together with the mobile phase move along the stationary phase. It is usually placed in a glass or metal tube called a column. Depending on the strength of interaction with the sorbent surface (due to adsorption or some other mechanism), the components will move along the column at different speeds. Some components will remain in the upper layer of the sorbent, others, interacting with the sorbent to a lesser extent, will end up in the lower part of the column, and some will leave the column altogether with the mobile phase (such components are called unretained, and their retention time determines the “dead time” of the column) . In this way, complex mixtures of components are quickly separated. The following advantages of chromatographic methods should be emphasized:

1. The separation is dynamic in nature, and the acts of sorption-desorption of the separated components are repeated many times. This is the reason for the significantly higher efficiency of chromatographic separation compared to static methods of sorption and extraction.

2. When separating, various types of interaction between sorbates and the stationary phase are used: from purely physical to chemisorption. This makes it possible to selectively separate a wide range of substances.

3. Various additional fields (gravitational, electric, magnetic, etc.) can be imposed on the substances to be separated, which, by changing the separation conditions, expand the possibilities of chromatography.

4. Chromatography is a hybrid method that combines the simultaneous separation and determination of several components.

5. Chromatography allows solving both analytical problems (separation, identification, determination) and preparative ones (purification, isolation, concentration). The solution of these tasks can be combined by performing them in the “on line” mode.

6. Numerous methods are classified according to the state of aggregation of the phases, separation mechanism and separation technique. Chromatographic methods also differ in the way the separation process is carried out into frontal, displacement and eluent.

3. CLASSIFICATION OF CHROMATOGRAPHIC METHODS OF ANALYSIS

The classifications of chromatographic methods are based on principles that take into account the following various features of the separation process:

* differences in the state of aggregation of the phases of the chromatographic system used;

* differences in the nature of the interactions of the separated substances with the stationary phase;

* experimental differences in the way the chromatographic separation process is carried out.

Tables 1–3 show the main options for classifying known chromatographic methods.

Since the nature of the interactions of the compounds to be separated with the phases of various chromatographic systems can vary greatly, there are almost no objects for the separation of which it would not be possible to find a suitable stationary phase (solid or liquid) and systems of mobile solvents. The areas of application of the main variants of chromatography, depending on the molecular weight of the compounds under study, are given in Table. 4.

4. ADSORPTION CHROMATOGRAPHY. THIN LAYER CHROMATOGRAPHY

One of the most common methods of adsorption chromatography is thin layer chromatography (TLC) - a type of planar chromatography, in which the adsorbent is used in the form of a thin layer on a plate.

Principle and basic concepts of the TLC method. On a clean flat surface (a plate of glass, metal, plastic) in one way or another, a thin layer of sorbent is applied, which is most often fixed on the surface of the plate. The dimensions of the plate can be different (length and width - from 5 to 50 cm, although this is not necessary). On the surface of the plate, carefully so as not to damage the sorbent layer, mark (for example, with a pencil) the start line (at a distance of 2–3 cm from the bottom edge of the plate) and the finish line of the solvent.

Scheme for the separation of components A and B by TLC

A sample is applied to the start line of the plate (with a microsyringe, capillary) - a small amount of liquid containing a mixture of substances to be separated, for example, two substances A and B in a suitable solvent. The solvent is allowed to evaporate, after which the plate is immersed in the chromatographic chamber into the liquid phase of the PF, which is a solvent or mixture of solvents specially selected for this case. Under the action of capillary forces, the PF spontaneously moves along the NF from the starting line to the solvent front line, carrying with it components A and B of the sample, which move at different speeds. In the case under consideration, the affinity of component A for NP is less than the affinity for the same phase of component B, so component A moves faster than component B. After the mobile phase (solvent) reaches the solvent front line in time t, chromatography is interrupted, the plate is removed from the chromatographic chamber, and dried in air and determine the position of the spots of substances A and B on the surface of the plate. Spots (zones) usually have an oval or round shape. In the case under consideration, the spot of component A has moved from the start line to a distance l A , component B spot - at a distance l AT, and the solvent has traveled through a distance L.

Sometimes, simultaneously with the application of a sample of substances to be separated, small amounts of a standard substance, as well as witness substances (those that are presumably contained in the analyzed sample) are applied to the start line.

To characterize the components to be separated in the system, the mobility coefficient Rf (or Rf factor) is introduced:

R f=V 1 /V E= (l 1 /t)/ (L/t)=l 1 /L ,

where V 1 = l 1 / t and V E= L/ t - according to the speed of movement i- th component and solvent E; l 1 andL - path taken i- m component and solvent, respectively, t is the time required to move the solvent from the start line to the front line of the solvent. Distances l 1 count from the start line to the center of the spot of the corresponding component.

Usually the mobility coefficient is in the range R f =0 - 1. The optimal value is 0.3-0.7. Chromatography conditions are selected so that the value of R f differs from zero and one.

The mobility coefficient is an important characteristic of the sorbent-sorbate system. For reproducible and strictly constant chromatographic conditions R f = const.

The mobility coefficient Rf depends on a number of factors: the nature and quality of the solvent, its purity; the nature and quality of the sorbent (thin layer), uniformity of its granulation, layer thickness; sorbent activity (moisture content in it); experimental techniques (sample weights, lengths L of solvent run); the skill of the experimenter, etc. The constancy of reproduction of all these parameters in practice is sometimes difficult. To level the influence of the process conditions, the relative mobility coefficient is introduced Rs.

Rs=l/l st=R f/R f( st ) ,

where R f = l/ L; R f (st)= l st/ L; l cm - distance from the start line to the center of the standard spot.

The relative mobility coefficient Rs is a more objective characteristic of the mobility of a substance than the mobility coefficient R f .

As a standard, one often chooses a substance for which, under given conditions, R f ? 0.5. According to the chemical nature, the standard is chosen close to the substances to be separated. With the use of the standard, the value of Rs usually lies in the range Rs=0.1--10, the optimal limits are about 0.5--2.

For more reliable identification of the separated components, "witnesses" are used - reference substances, the presence of which is expected in the analyzed sample. If R f = R f (certificate), where R f and R f (certificate) are the mobility coefficients of this component and witness, respectively, then it can be more likely to assume that the witness substance is present in the mixture being chromatographed.

To characterize the separation of two components A and B under these conditions, the degree (criterion) of separation R (A / B) is introduced:

R (A / B) \u003d D l( =2D l ,

where D l- distance between the centers of the spots of components A and B; a(A) and a(B) are the diameters of spots A and B on the chromatogram, respectively.

The greater the value of R (A/B), the more clearly the spots of components A and B are separated on the chromatogram.

To evaluate the selectivity of the separation of two substances A and B, the separation factor is used a:

a=l B / l A.

If a=1, then components A and B are not separated.

To determine the degree of separation R (A/B) of components A and B.

4.1 Experimental technique in thin layer chromatography:

a) Sample application. The analyzed liquid sample is applied to the start line using a capillary, microsyringe, micropipette, carefully touching the sorbent layer (the spot diameter on the start line is usually from one to several millimeters). If several samples are applied to the start line, then the distance between the spots of the samples on the start line should not be less than 2 cm. If possible, use concentrated solutions. The spots are air dried and then chromatographed.

b) Chromatogram development (chromatography). The process is carried out in closed chromatographic chambers saturated with vapors of the solvent used as PF, for example, in a glass vessel covered with a lid on top.

Depending on the direction of movement of the PF, there are ascending, descending and horizontal chromatography.

In the variant of ascending chromatography, only plates with a fixed layer of sorbent are used. PF is poured onto the bottom of the chamber (a glass beaker of a suitable size with a glass lid can be used as the latter), the chromatographic plate is placed vertically or obliquely into the chamber so that the PF layer at the bottom of the chamber wets the bottom of the plate (below the start line by ~1.5 - 2 cm). The PF moves due to the action of capillary forces from the bottom up (against the force of gravity) relatively slowly.

Downward chromatography also uses only fixed bed plates. PF is fed from above and moves down along the plate sorbent layer. The force of gravity accelerates the motion of the PF. This option is implemented in the analysis of mixtures containing components that slowly move with the PF.

In a variant of horizontal chromatography, the plate is placed horizontally. Rectangular or round plates can be used. When using round plates (circular version of horizontal chromatography), the starting line is designated as a circle of suitable radius (~1.5-2 cm), on which samples are applied. A hole is cut in the center of the round plate, into which a wick is inserted to supply the PF. The latter moves along the sorbent layer from the center of the circle to its periphery. Chromatography is carried out in a closed chamber - a desiccator or in a Petri dish. With the circular version, up to several dozen samples can be analyzed simultaneously.

TLC methods use one-dimensional, two-dimensional, multiple (repeated), stepwise chromatography.

With a single chromatography, the analysis is carried out without changing the direction of the PF movement. This method is the most common.

Two-dimensional chromatography is usually used to analyze complex mixtures (proteins, amino acids, etc.). First, a preliminary separation of the mixture is carried out using the first PF 1 . On the chromatogram, spots are obtained not of individual substances, but of mixtures of several unseparated components. Then, a new start line is drawn through these spots, the plate is turned 90° and chromatographed again, but with the second PF 2, trying to finally separate the spots of mixtures into spots of individual components.

If the plate is square, then the sample is applied to the diagonal of this square near its lower corner. Sometimes two-dimensional chromatography is carried out with the same PF on a square plate.

Scheme illustrating the principle of two-dimensional chromatography:

a - chromatogram obtained with PF1;

b - chromatogram obtained with PF2

In multiple (repeated) chromatography, the process is carried out several times sequentially with the same PF (each time after the next drying) until the desired separation of the spots of the mixture components is obtained (usually no more than three times).

In the case of stepwise chromatography, the process is carried out with the same plate sequentially, using a new PF each time, until a distinct separation of the spots is achieved.

in) Chromatogram interpretation. If the spots on the chromatogram are colored, after drying the plates, the distance from the start line to the center of each spot is determined and the mobility coefficients are calculated. If the composition of the analyzed sample includes colorless substances that give uncolored, i.e. visually unidentifiable spots on the chromatogram, it is necessary to carry out detection these spots, for which chromatograms manifest.

The most common detection methods are described below.

Irradiation with ultraviolet light. It is used to detect fluorescent compounds (the spots glow when the plate is exposed to UV light) or non-fluorescent substances, but using a sorbent with a fluorescent indicator (the sorbent glows, the spots do not glow). In this way, for example, alkaloids, antibiotics, vitamins and other medicinal substances are detected.

Heat treatment. After chromatography, the plate dried after chromatography is carefully heated (up to ~200°C) to avoid darkening of the sorbent layer itself (for example, when a thin sorbent layer contains starch). In this case, spots usually appear in the form of brown zones (due to partial thermolysis of organic components).

Chemical processing. Chromatograms are often developed by treating them with reagents that form colored compounds with separable mixture components. For these purposes, various reagents are used: vapors of iodine, ammonia, bromine, sulfur dioxide, hydrogen sulfide, specially prepared solutions with which the plates are treated. Both universal and selective reagents are used (the concept of "universal" is rather arbitrary).

For example, concentrated sulfuric acid can serve as universal reagents (darkening of spots of organic compounds is observed when heated), an acidic aqueous solution of potassium permanganate (zones are observed in the form of brown spots on a purple background of the sorbent), a solution of phosphomolybdic acid when heated (blue spots appear on yellow background), etc.

As selective ones, for example, Dragendorf's reagent is used; Zimmermann's reagent; aqueous ammonia solution of copper sulfate (10% for CuSO 4 , 2% for ammonia); a mixture of ninhydrin C 9 H 4 O 3 H 2 O with ethanol and acetic acid.

The Dragendorff reagent is a solution of basic bismuth nitrate BiONO 3 , potassium iodide KJ and acetic acid in water. Used to determine amines, alkaloids, steroids.

The Zimmermann reagent is prepared by treating a 2% ethanol solution of dinitrobenzene with a KOH alkali solution, followed by heating the mixture at ~70–100°C. Used to detect steroids.

With the help of ninhydrin, spots of amines, amino acids, proteins and other compounds are detected.

Some other methods of detecting spots are also used. For example, their radioactivity is measured if some of the separated components are radioactive, or special additives of radioactive isotopes of elements that are part of the separated components of the mixture are introduced.

After detecting spots on the chromatogram, they are identified, i.e. determine which compound corresponds to a particular spot. For this, reference spots of "witnesses" are most often used. Sometimes the spots are identified by the value of the coefficients of mobility R f , comparing them with the values of R f known for the given conditions. However, such identification by the value of R f is often preliminary.

The color of fluorescent spots is also used for identification purposes, since different compounds fluoresce with different wavelengths (different colors).

In the chemical detection of spots, selective reagents give colored spots with compounds of a certain nature, which is also used for identification purposes.

Using the TLC method, one can not only discover, but also quantify the content of components in mixtures. To do this, either the spots themselves are analyzed on the chromatogram, or the separated components are extracted from the chromatogram in one way or another (extraction, elution with suitable solvents).

When analyzing spots, it is assumed that there is a certain relationship between the area of the spot and the content of a given substance (for example, the presence of a proportional or linear dependence), which is established by constructing a calibration graph by measuring the areas of spots of "witnesses" - standards with a known content of the analyzed component.

Sometimes the color intensity of spots is compared, assuming that the color intensity of a spot is proportional to the amount of a given colored component. Various methods are used to measure the color intensity.

When extracting the separated components from the chromatogram, a solution containing this component is obtained. The latter is then determined by one or another analytical method.

The relative error in the quantitative determination of the substance by TLC is 5-10%.

TLC is a pharmacopoeial method and is widely used for the analysis and quality control of various drugs.

5. GAS CHROMATOGRAPHY

Gas chromatography (GC) uses an inert gas (nitrogen, helium, hydrogen) as the mobile phase, called a carrier gas. The sample is fed in the form of vapors, the stationary phase is either a solid substance - a sorbent (gas-adsorption chromatography) or a high-boiling liquid deposited in a thin layer on a solid carrier (gas-liquid chromatography). Consider a variant of gas-liquid chromatography (GLC). Kieselguhr (diatomite) is used as a carrier - a kind of hydrated silica gel, it is often treated with reagents that convert Si-OH groups into Si-O-Si (CH 3) 3 groups, which increases the inertness of the carrier with respect to solvents. These are, for example, the carriers “Chromosorb W” and “Gazochrome Q”. In addition, glass microballoons, Teflon and other materials are used.

5.1 Gazo- adsorption chromatography

A feature of the gas adsorption chromatography (GAC) method is that adsorbents with a high specific surface area (10–1000 m 2 g -1) are used as the stationary phase, and the distribution of substances between the stationary and mobile phases is determined by the adsorption process. Adsorption of molecules from the gas phase, i.e. concentrated at the interface between the solid and gaseous phases, occurs due to intermolecular interactions (dispersion, orientation, induction), which are of an electrostatic nature. Perhaps, the formation of a hydrogen bond, and the contribution of this type of interaction to the retained volumes decreases significantly with increasing temperature.

For analytical practice, it is important that at a constant temperature the amount of adsorbed substance on the surface С s be proportional to the concentration of this substance in the gas phase С m:

C s = kc m (1)

those. so that the distribution occurs in accordance with the linear adsorption isotherm (to -- constant). In this case, each component moves along the column at a constant speed, independent of its concentration. The separation of substances is due to the different speed of their movement. Therefore, in GAC, the choice of an adsorbent is extremely important, the area and nature of the surface of which determine the selectivity (separation) at a given temperature.

As the temperature rises, the heat of adsorption decreases. DH/T, on which the retention depends, and, accordingly, t R . This is used in the practice of analysis. If compounds are separated that differ greatly in volatility at a constant temperature, then low-boiling substances elute quickly, high-boiling substances have a longer retention time, their peaks on the chromatogram will be lower and wider, and the analysis takes a long time. If, however, during chromatography, the column temperature is increased at a constant rate (temperature programming), then peaks close in width on the chromatogram will be evenly distributed.

Active carbons, silica gels, porous glass, and aluminum oxide are mainly used as adsorbents for HAC. The inhomogeneity of the surface of active adsorbents is responsible for the main disadvantages of the GAC method and the impossibility of determining strongly adsorbed polar molecules. However, it is possible to analyze mixtures of highly polar substances on geometrically and chemically homogeneous macroporous adsorbents. In recent years, adsorbents with a more or less uniform surface have been produced, such as porous polymers, macroporous silica gels (silochrome, porasil, spherosil), porous glasses, and zeolites.

The most widely used method of gas adsorption chromatography is to analyze mixtures of gases and low-boiling hydrocarbons that do not contain active functional groups. The adsorption isotherms of such molecules are close to linear. For example, for the separation of O 2 , N 2 , CO, CH 4 , CO 2 clay is successfully used. The column temperature is programmed to reduce analysis time by reducing the t R of high-boiling gases. On molecular sieves - highly porous natural or synthetic crystalline materials, all the pores of which are approximately the same size (0.4 - 1.5 nm), - hydrogen isotopes can be separated. Sorbents called porapaks are used to separate metal hydrides (Ge, As, Sn, Sb). The GAC method on columns with porous polymer sorbents or carbon molecular sieves is the fastest and most convenient way to determine water in inorganic and organic materials, such as solvents.

5.2 Gazo- liquid chromatography

In analytical practice, the method of gas-liquid chromatography (GLC) is more often used. This is due to the extreme diversity of liquid stationary phases, which facilitates the selection of a phase selective for a given analysis, with a linear distribution isotherm over a wider concentration range, which allows you to work with large samples, and easily obtain reproducible columns in terms of efficiency.

The mechanism of component distribution between the carrier and the stationary liquid phase is based on their dissolution in the liquid phase. Selectivity depends on two factors: the vapor pressure of the analyte and its activity coefficient in the liquid phase. According to Raoult's law, upon dissolution, the vapor pressure of a substance over a solution p i is directly proportional to its activity coefficient g mole fraction N i in solution and vapor pressure of a pure substance R° i at a given temperature:

p i = N i R ° I (2)

Since the concentration of the ith component in the equilibrium vapor phase is determined by its partial pressure, we can assume that,

P i ~ c m , and N i ~ c s then

and the selectivity coefficient:

Thus, the lower the boiling point of a substance (the greater P 0 i), the weaker it is retained in the chromatographic column.

If the boiling points of substances are the same, then differences in interaction with the stationary liquid phase are used to separate them: the stronger the interaction, the lower the activity coefficient and the greater the retention.

Stationary liquid phases . To ensure the selectivity of the column, it is important to choose the correct stationary liquid phase. This phase should be a good solvent for the components of the mixture (if the solubility is low, the components leave the column very quickly), non-volatile (so that it does not evaporate at the operating temperature of the column), chemically inert, should have a low viscosity (otherwise the diffusion process slows down) and when applied to the carrier to form a uniform film, firmly bound to it. The separating power of the stationary phase for the components of this sample should be maximum.

There are three types of liquid phases: non-polar (saturated hydrocarbons, etc.), moderately polar (esters, nitriles, etc.) and polar (polyglycols, hydroxylamines, etc.).

Knowing the properties of the stationary liquid phase and the nature of the substances to be separated, for example, class, structure, it is possible to quickly select a selective liquid phase suitable for separating a given mixture. In this case, it should be taken into account that the retention time of the components will be acceptable for analysis if the polarities of the stationary phase and the substance of the analyzed sample are close. For solutes of close polarity, the order of elution usually correlates with boiling points, and if the temperature difference is large enough, complete separation is possible. To separate near-boiling substances of different polarity, a stationary phase is used, which selectively retains one or more components due to dipole-dipole interaction. As the polarity of the liquid phase increases, the retention time of polar compounds increases.

For uniform application of the liquid phase on a solid carrier, it is mixed with a highly volatile solvent, such as ether. A solid carrier is added to this solution. The mixture is heated, the solvent evaporates, the liquid phase remains on the carrier. The dry carrier thus coated with the stationary liquid phase is filled into the column, taking care to avoid the formation of voids. For uniform packing, a gas jet is passed through the column and at the same time the column is tapped to seal the packing. Then, before attaching to the detector, the column is heated to a temperature of 50 ° C above that at which it is supposed to be used. In this case, there may be losses of the liquid phase, but the column enters a stable operating mode.

Carriers of stationary liquid phases. Solid carriers for dispersing the stationary liquid phase in the form of a homogeneous thin film must be mechanically strong with a moderate specific surface area (20 m 2 /g), small and uniform particle size, and also be inert enough to allow adsorption at the solid-gaseous interface. phases was minimal. The lowest adsorption is observed on carriers of silanized chromosorb, glass beads and fluoropaque (fluorocarbon polymer). In addition, solid carriers should not react to temperature rise and should be easily wetted by the liquid phase. In gas chromatography of chelates, silanized white diatomite carriers, diatomite silica, or kieselguhr, are most often used as a solid carrier. Diatomaceous earth is a micro-amorphous, water-containing silica. Such carriers include chromosorb W, gas chrome Q, chromaton N, etc. In addition, glass beads and teflon are used.

Chemically bonded phases. Often, modified carriers are used, covalently bonded to the liquid phase. In this case, the stationary liquid phase is more firmly held on the surface even at the highest column temperatures. For example, a diatomaceous earth carrier is treated with chlorosilane with a long chain substituent having a certain polarity. The chemically bonded stationary phase is more efficient.

6. DISTRIBUTION CHROMATOGRAPHY. PAPER CHROMATOGRAPHY (PAPER CHROMATOGRAPHY)

Partition chromatography is based on the use of differences in the solubility of a partitioned substance in two contacting immiscible liquid phases. Both phases - PF and NF - are liquid phases. When the liquid PF moves along the liquid NF, the chromatographed substances are continuously redistributed between both liquid phases.

Partition chromatography is paper chromatography (or chromatography on paper) in its normal form. In this method, instead of plates with a thin layer of sorbent used in TLC, special chromatographic paper is used, along which, impregnating it, liquid PF moves during chromatography from the start line to the finish line of the solvent.

Distinguish normal phase and reversed phase paper chromatography.

In the variant normal-phase paper chromatography liquid NF is water adsorbed in the form of a thin layer on the fibers and located in the pores hydrophilic paper (up to 25% by weight). This bound water in its structure and physical state is very different from ordinary liquid water. The components of the separated mixtures dissolve in it.

The role of the PF moving over the paper is played by another liquid phase, for example, an organic liquid with the addition of acids and water. Before chromatography, liquid organic PF is saturated with water so that PF does not dissolve the water adsorbed on the fibers of the hydrophilic chromatographic paper.

Chromatographic paper is produced by the industry. It must meet a number of requirements: it must be prepared from high-quality fibrous cotton varieties, be uniform in density and thickness, in the direction of fiber orientation, chemically clean and inert with respect to NF and separable components.

In the normal-phase variant, liquid mixtures composed of various solvents are most often used as PF. A classic example of such a PF is a mixture of acetic acid, n-butanol and water in a 1:4:5 volume ratio. Solvents such as ethyl acetate, chloroform, benzene, etc. are also used.

In the variant reverse phase In paper chromatography, liquid NF is an organic solvent, while liquid PF is water, aqueous or alcoholic solutions, and mixtures of acids with alcohols. The process is carried out using hydrophobic chromatographic paper. It is obtained by treating (impregnating) paper with naphthalene, silicone oils, paraffin, etc. Non-polar and low-polar organic solvents are sorbed on the fibers of hydrophobic paper and penetrate into its pores, forming a thin layer of liquid NF. Water is not retained on such paper and does not wet it.

The paper chromatography technique is in general the same as in the TLC method. Usually, a pot of the analyzed solution containing a mixture of substances to be separated is applied to a strip of chromatographic paper at the start line. After the solvent has evaporated, the paper below the start line is immersed in the PF, placing the paper vertically (hanging it). Close the chamber with a lid and carry out chromatography until the PF reaches the solvent front line indicated on the paper. After that, the process is interrupted, the paper is dried in air, and stains are detected and the components of the mixture are identified.

Paper chromatography, like the TLC method, is used in both qualitative and quantitative analysis.

Various methods are used to quantify the content of a particular component of a mixture:

1) they proceed from the presence of a certain relationship (proportional, linear) between the amount of substance in the spot and the area of the spot (often, a calibration graph is preliminarily built);

2) weigh the cut out spot with the substance and clean paper of the same area, and then find the mass of the substance to be determined by the difference;

3) take into account the relationship between the intensity of the color of the spot and the content in it of the determined component that gives the color to the spot.

In some cases, the substances contained in the spots are extracted with some solvent and then the extract is analyzed.

Paper chromatography is a pharmacopoeial method used to separate mixtures containing both inorganic and organic substances. The method is accessible, easy to perform, but in general it is inferior to the more modern TLC method, which uses a thin layer of sorbent.

7. SEDIMENT CHROMATOGRAPHY

Sedimentary chromatography is mainly used for the separation and identification of inorganic ions in mixtures.

The essence of the method. Sedimentary chromatography is based on the use of chemical reactions of precipitation of the separated components of a mixture with a precipitant, which is part of the NF. The separation is carried out due to the unequal solubility of the resulting compounds, which are transferred by the mobile phase at different rates: less soluble substances are transferred from the PF more slowly than more soluble ones.

The application of the method can be illustrated by the example of the separation of halide ions: chloride ions Cl - , bromide ions Br - and iodide ions I - simultaneously contained in the analyzed aqueous solution. To do this, use a chromatographic column (which is a glass tube with a tap at the bottom) filled with a sorbent. The latter consists of their media - aluminum oxide Al 2 O 3 or silicon SiO 2 impregnated with a solution of silver nitrate AgNO 3 (the content of silver nitrate is about 10% by weight of the mass of the sorbent carrier).

An aqueous solution containing a mixture of anions to be separated is passed through a chromatographic column. These anions interact with silver cations Ag + , forming sparingly soluble precipitates of silver halides:

Ag + + I - > AgIv (yellow)

Ag + + Br - > AgBrv (cream)

Ag + + Cl - > AgClv (white)

The solubility of silver halides in water increases in the sequence:

Agl (K ° \u003d 8.3 * 10 -17)< АgВг (К° = 5,3*10 -13) < AgCl (K°= 1,78*10 -10),

where values of solubility products at room temperature are given in parentheses. Therefore, at first a yellow precipitate of silver iodide will form, as the least soluble on the chromatogram, a yellow (upper) zone will be observed. A cream-coloured silver bromide precipitate zone (intermediate zone) then forms. Lastly, a white precipitate of silver chloride is formed - the lower white zone, which darkens in the light due to the photochemical decomposition of silver chloride with the release of finely dispersed metallic silver.

The result is a primary sedimentary chromatogram.

For a clearer separation of the zones, after obtaining the primary chromatogram, a pure solvent is passed through the column until a secondary sedimentary chromatogram is obtained with a clear separation of the precipitation zones.

In the example described, the precipitant was a part of the NF, and a solution containing a mixture of ions to be separated was passed through the column. On the contrary, it is possible to pass the solution of the precipitant through the column, in the NF of which the ions to be chromatographed are located. In this case, however, mixed zones are formed.

Scheme for the separation of Cl-, Br- and I- ions in a chromatographic column by sedimentary chromatography.

7.1 Classification of sediment chromatography methods according to experimental technique

I usually distinguish columnar sedimentary chromatography carried out in chromatographic columns, and planar sedimentary chromatography, implemented on paper or in a thin layer of sorbent.

As sorbents in sedimentary chromatography, mixtures of inert carriers with a precipitant are used; sorbents that retain precipitants in the form of ions (ion-exchange resins) or in the form of molecules (activated carbon); paper impregnated with a precipitant solution.

The most commonly chosen carriers are silica gel, starch, oxides of aluminium, calcium, barium sulfate, ion exchange resins, etc. The carrier is used in a finely dispersed state with a particle size of about 0.02-0.10 mm.

As precipitants, such reagents are used that form sparingly soluble precipitates with chromatographic ions, for example, sodium iodide NaI, sodium sulfide Na 2 S, silver sulfate Ag 2 SO 4, potassium ferrocyanide K 4, oxyquinoline, pyridine, etc.

Usually, when using the method of sedimentary column chromatography, after passing a pure solvent through a column, clearly separated zones are obtained, each of which contains only one component (in the case when the solubilities of the precipitates differ by at least three times). The method has good reproducibility of results.

In the case of the formation of colorless precipitates, the chromatogram is developed either by passing a developer solution through the column, which gives colored reaction products with precipitates, or by immediately introducing the developer into PF or NF.

7.2 Sediment chromatography on paper

Let us consider the essence of this method on the example of the analysis of an aqueous solution containing a mixture of copper cations Cu 2+ ? iron Fe 3+ and aluminum Al 3+.

In the center of a sheet of paper impregnated with a solution of a precipitant - potassium ferrocyanide K 4 , the analyzed aqueous solution is applied by a capillary. Copper ions Cu 2+ and iron Fe 2+ interact with ferrocyanide ions to form sparingly soluble precipitates:

2Cu 2+ + 4- > Cu 2 (brown)

4Fe 3+ + 3 4->Fe4 (blue)

Since copper (II) ferrocyanide is less soluble than iron (III) ferrocyanide, a precipitate of copper (II) ferrocyanide is first precipitated, forming a central brown zone. A blue precipitate of iron(III) ferrocyanide then forms, giving a blue zone. The aluminum ions migrate to the periphery, giving a colorless zone because they do not form colored aluminum ferrocyanide.

Scheme of separation of Cu2+, Fe3+ and Al3+ by sediment chromatography.

In this way, a primary chromatogram is obtained in which the precipitation zones partially overlap.

Then a secondary chromatogram is obtained. To do this, a suitable solvent (in this case, an aqueous solution of ammonia) is applied with a capillary to the center of the primary chromatogram. The solvent spontaneously moves from the center of the paper to the periphery, carrying with it the precipitates, which move at different speeds: the zone of more soluble iron ferrocyanide precipitate moves faster than the zone of less soluble copper ferrocyanide precipitate. At this stage, due to the difference in the speeds of movement of the zones, they are more clearly separated.

To open the aluminum ions that form a colorless peripheral zone, the secondary chromatogram is shown - sprayed (from a spray bottle) with a solution of alizarin, an organic reagent that forms pink reaction products with aluminum ions. Get the outer pink ring.

8. ION EXCHANGE CHROMATOGRAPHY

In ion-exchange chromatography, the separation of mixture components is achieved due to the reversible interaction of ionizable substances with the ionic groups of the sorbent. Preservation of the electrical neutrality of the sorbent is ensured by the presence of counterions capable of ion exchange located in close proximity to the surface. The ion of the introduced sample, interacting with the fixed charge of the sorbent, is exchanged with the counterion. Substances with different affinities for a fixed charge are separated on anion exchangers or on cation exchangers. Anion exchangers have positively charged groups on the surface and sorb anions from the mobile phase. Cation exchangers respectively contain groups with a negative charge interacting with cations.

As a mobile phase, aqueous solutions of salts of acids, bases and solvents such as liquid ammonia are used, i.e. solvent systems having a high dielectric constant and a strong tendency to ionize the compounds. Usually they work with buffer solutions that allow you to adjust the pH value.

During chromatographic separation, the ions of the analyte compete with the ions contained in the eluent, seeking to interact with oppositely charged groups of the sorbent. It follows that ion exchange chromatography can be used to separate any compounds that can be ionized in any way. It is possible to analyze even neutral sugar molecules in the form of their complexes with the borate ion.

Ion-exchange chromatography is indispensable for the separation of highly polar substances, which cannot be analyzed by GLC without conversion into derivatives. These compounds include amino acids, peptides, sugars.

Ion-exchange chromatography is widely used in medicine, biology, biochemistry, for environmental control, in the analysis of the content of drugs and their metabolites in blood and urine, pesticides in food raw materials, as well as for the separation of inorganic compounds, including radioisotopes, lanthanides, actinides, etc. The analysis of biopolymers (proteins, nucleic acids, etc.), which usually took hours or days, using ion exchange chromatography is carried out in 20-40 minutes with better separation. The use of ion exchange chromatography in biology has made it possible to observe samples directly in biological media, reducing the possibility of rearrangement or isomerization, which can lead to misinterpretation of the final result. It is interesting to use this method to control changes in biological fluids. The use of porous weak anion exchangers based on silica gel made it possible to separate the peptides. The ion exchange mechanism can be represented as the following equations:

for anion exchange X - + R + Y - - Y - + R + X -

for cation exchange X + + R - Y + - Y + + R - X +

In the first case, the sample ion X - competes with the mobile phase ion Y - for the ionic centers R + of the ion exchanger, and in the second case, the cations of the sample X + enter into competition with the mobile phase ions Y + for the ionic centers R - .

Naturally, sample ions that weakly interact with the ion exchanger will be weakly retained on the column during this competition and are the first to be washed out from it, and, conversely, more strongly retained ions will be the last to elute from the column. Usually, secondary interactions of a nonionic nature occur due to adsorption or hydrogen bonding of the sample with the nonionic part of the matrix or due to the limited solubility of the sample in the mobile phase.

Separation of specific substances depends primarily on the choice of the most suitable sorbent and mobile phase. As stationary phases in ion-exchange chromatography, ion-exchange resins and silica gels with grafted ionogenic groups are used.

Polystyrene ion-exchange resins for HPLC with a grain size of 10 μm or less have selectivity and stability, but their network structure, characterized by a distance between grid nodes of 1.5 nm, which is much smaller than the pore size of silica gel used for adsorption chromatography (10 nm), slows down mass transfer and, therefore, significantly reduces efficiency. The ion exchange resins used in HPLC are mainly copolymers of styrene and divinylbenzene. Usually add 8-12% of the latter. The greater the content of divinylbenzene, the greater the rigidity and strength of the polymer, the higher the capacity and, as a rule, the selectivity, and the lower the swelling.

2.1 Development history:

The study of ion-exchange processes began already at the beginning of the 19th century. from observations on the influence of soils on the chemical composition of salt solutions in contact with it. At the end of the 1940s, G. Thompson noted that the soil absorbs ammonia from the applied organic fertilizers; the corresponding experiments were carried out by their York specialist D. Spence. The first results of D. Spence's experiments were published by G. Thompson in 1850. The article notes that "the first discovery of highly important properties of the soil can almost fail as useful for agriculture" and his last works were published in 1852 and 1855.

2.3 Principles of ion separation in sorption processes

Ion-exchange chromatography refers to liquid-solid-phase chromatography in which the mobile phase is a liquid (eluent) and the stationary phase is a solid (ion exchanger). The method of ion-exchange chromatography is based on the dynamic process of replacing ions associated with the stationary phase with eluent ions entering the column. The separation occurs due to the different affinity of the ions in the mixture to the ion exchanger, which leads to different rates of their movement through the column.

Ion chromatography is a variant of ion exchange column chromatography.

According to the recommendations of IUPAC (1993), the terms ion exchange (IOC) and ion (IC) chromatography are defined as follows. "Ion exchange chromatography is based on the difference in ion exchange interactions for individual analytes. If ions are separated and can be detected using a conductometric detector or indirect UV detection, then it is called ion chromatography."

Modern (2005) formulation: "Ion chromatography includes all high-performance liquid chromatographic (HPLC) column separations of ions, combined with direct detection in a flow detector and quantitative processing of the resulting analytical signals." This definition characterizes ion chromatography regardless of the separation mechanism and detection method, and thus separates it from classical ion exchange.

In ion chromatography, the following separation principles apply:

ion exchange.

Formation of ion pairs.

Exclusion of ions.

Ion exchange

Ion exchange is a reversible heterogeneous reaction of equivalent exchange of ions in the ion exchanger phase (counterions) with eluent ions. The counterions are held by the functional groups of the ion exchanger due to electrostatic forces. Typically, in cationic chromatography, these groups are sulfonic acid groups; in the case of anion chromatography, quaternary ammonium bases. On fig. 1 shows a diagram of the process of exchange of cations and anions. The ions of the analyte are designated as A, the ions of the eluent competing with them for exchange centers are designated as E.

Rice. 1. Ion exchange of cations (A+) and anions (A-) for eluent ions (E+ or E-) with the participation of a cation exchanger containing functional sulfo groups - SO3-, and an anion exchanger (groups of a quaternary ammonium base -N + R3).

Ion pair formation

To implement this separation mechanism, ion-pair reagents are used, which are added to the eluent solution. Such reagents are anionic or cationic surfactants, for example alkylsulfonic acids or tetraalkylammonium salts.

Together with oppositely charged analyte ions, the ions of this ion-pair reagent form an uncharged ion pair, which can be retained on the stationary phase due to intermolecular interactions. The separation is carried out due to the difference in the constants of formation of ion pairs and the degree of their adsorption on the sorbent matrix. On fig. 2 shows a static ion-exchange model in ion-pair chromatography after adsorption of the reagent on the stationary phase. This separation principle applies to both anions and cations.

Rice. 2. Ion-exchange model in ion-pair chromatography.

Ionic exclusion

Ion exclusion chromatography (IEC). mainly used to separate weak acids or bases. IEC is most important for the determination of carboxylic and amino acids, phenols, and carbohydrates.

On fig. Figure 3 shows the principle of IEC separation using R–COOH acids as an example.

Rice. Fig. 3. Scheme for the separation of carboxylic acids R–COOH using ion exclusion chromatography.

In ion exclusion chromatography, a fully sulfonated cation exchanger containing hydrogen ions (counterions) is often used as the stationary phase. In an aqueous solution of the eluent, the sulfonic acid groups of the ion exchanger are hydrated. The hydration shell is limited by an imaginary negatively charged membrane (Donnan's membrane). The membrane is only permeable to non-dissociated molecules (eg water).

Organic carboxylic acids can be separated if strong mineral acids are used as eluent. Due to the low values of the acidity constants, carboxylic acids are present in such solutions in an undissociated form. These forms can pass through the Donnan membrane and be adsorbed onto the stationary phase.

History of chromatography. History of the discovery of chromatography

2. The emergence and development of chromatography

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