Development of chromatography. History of the development of liquid chromatography. Chromatography on a solid stationary phase

Many discoveries of the past century are due to the Russian scientist Mikhail Tsvet and his method of chromatographic analysis. A large number of outstanding researchers owe him their successes, and many Nobel Prizes!

"...Without the work of Michael Tsveta, we, all the "pigmenters", would have nothing to do ..." - this is the opinion of one famous English scientist.

Mikhail Semenovich Tsvet (1872–1919) - the son of an Italian woman and a Russian intellectual. He was born in Italy in the city of Asti, not far from Turin. In 1891, Mikhail graduated from the Geneva Gymnasium and entered the Faculty of Physics and Mathematics at the University of Geneva. In October 1896, after presenting his dissertation "Investigation of cell physiology. Materials for the knowledge of the movement of protoplasm, plasma membranes and chloroplasts", Tsvet received a diploma of a doctor of natural sciences. In December of the same year, he arrives in St. Petersburg.

Mikhail did not know that a degree from the University of Geneva is not recognized in Russia. Therefore, he had to work for the famous botanist Andrey Sergeevich Famintsin, who also studied chlorophyll, one might say, on a bird's right. In St. Petersburg, Tsvet met other outstanding botanists and plant physiologists: I.P. Borodin, M.S. Voronin, A.N. Beketov. It was a brilliant society of original, thoughtful thinkers and skilled experimenters. Tsvet continued his research on chloroplasts, while at the same time preparing for new master's examinations and for the defense of his dissertation. He passed the exams in 1899, and he defended his master's thesis at Kazan University on September 23, 1901.

Since November 1901, Tsvet has been working as an assistant at the Department of Plant Anatomy and Physiology at the University of Warsaw. At the XI Congress of Naturalists and Physicians, Mikhail Semenovich made a report "Methods and tasks of the physiological study of chlorophyll", in which he first reported on the method of adsorption chromatography.

Mikhail Semenovich solved the problem of separating green leaf pigments for a long time, and they are very similar in properties. In addition, the leaves contain other, very bright, pigments - carotenoids. It is thanks to carotenoids that yellow, orange, purple leaves appear in autumn. However, until the chlorophylls were destroyed, it was almost impossible to separate them from the carotenoids.

As Yu.G. Chirkov, “apparently, the discovery of Color was a reaction to the methods of their separation that were then crude and deadly for pigments. Here is one of the methods.

First, an alcoholic extract of chlorophyll was extracted, then it was boiled for three hours with the addition of strong alkali (caustic potassium) to the solution. As a result, chlorophyll decomposes into its constituent parts - green and yellow pigments.

But after all, in the process of making this potion (almost alchemical manipulations), natural chlorophyll could be destroyed. And then the researcher would have to deal with pieces of pigments, and even with the products of their chemical transformation.

S.E. writes about how the great discovery happened. Shnol: "He took a glass tube, filled it with chalk powder and poured a little alcoholic extract of the leaves on the top layer. The extract was brown-green in color, and the top layer of the chalk column became the same color. And then M.S. began to pour drops from above into drop by drop another portion of the solvent eluted the pigments from the grains of chalk, which moved down the tube, where the fresh grains of chalk adsorbed the pigments and, in turn, gave them to new portions of the solvent. entrained by the mobile solvent, different pigments moved along the chalk column at different speeds and formed uniform colored bands of pure substances in the chalk column.It was beautiful.A bright green band, a band slightly yellower than green - these are two types of chlorophylls - and a bright yellow-orange band of carotenoids. MS called this picture a chromatogram."

“The color showed,” writes Chirkov, “that when plant pigments dissolved in a liquid are passed through a layer of a colorless porous sorbent, individual pigments are arranged in the form of colored zones - each pigment has its own color or at least a shade. Sorbent powder (it can be chalk, powdered sugar ...) adsorbs (superficially absorbs: Latin adsorbere means "swallow") different pigments with unequal strength: some can "slip" further with the current of the solution, others will be delayed closer. Color called the chromatogram, and the method - chromatography.

Thus, a seemingly insurmountable problem was solved. The method turned out to be ingeniously simple. It is nothing like the cumbersome, reagent-intensive complex procedures used before.

Perhaps this simplicity was the reason that most of his contemporaries either did not accept this amazing discovery, or, even sadder, sharply rebelled against its author.

But time put everything in its place. Color invented chromatography for chlorophyll research. He first isolated a substance that he called chlorophyll alpha and chlorophyll beta. It turned out to be suitable for studying not only pigments, but also colorless, uncolored mixtures - proteins, carbohydrates. By the sixties of the twentieth century, several thousand studies had already been devoted to chromatography. Chromatography has become a universal method.

"... The principle of chromatographic separation of substances, discovered by M. Tsvet, underlies many different methods of chromatographic analysis. Without its use, most of the achievements in science and technology of the 20th century would not have been possible ...

At the heart of all this is one general idea. She is simple. This is essentially the idea of a geometric progression. Let there be two substances very similar in all their properties. Neither precipitation, nor extraction, nor adsorption can separate them to a noticeable degree. Let one substance be adsorbed on the surface, for example, calcium carbonate (i.e., less than 1 percent).

In other words, its content on the adsorbent will be 0.99 of the content of another. Let us treat the adsorbent with some solvent so that desorption (detachment) and elution (washing out) of both substances occur and both of them pass from the adsorbent to the solvent, and transfer this resulting solution to a fresh portion of the adsorbent. Then the proportion of the first substance on the surface of the adsorbent will again be equal to 0.99 of the content of the second, i.e., a part equal to 0.99 x 0.99 = 0.98 of the initial amount is adsorbed. Once again, we will carry out the elution and adsorption again - now the proportion of the first substance will be 0.98 x 0.99 \u003d 0.97 of the content of the second. In order for the content of the first substance on the next portion of the adsorbent to be only 1 percent of the content of the second, it will be necessary to repeat the adsorption-elution cycle about 200 times...

The idea of multiple re-adsorption to separate substances can be modified into multiple redistribution of a mixture of substances in a system of immiscible solvents. This is the basis of partition chromatography. The same idea underlies modern electrophoresis methods, when a mixture of substances moves at different speeds over various adsorbents in an electric field.

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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. STORYDISCOVERIES 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 options.

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 top layer sorbent, others interacting with the sorbent to a lesser extent, will end up in the lower part of the column, and some will even leave the column together 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 IN, 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.

V) 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).

Universal reagents can be, for example, concentrated sulfuric acid(darkening of spots of organic compounds is observed upon heating), an acidic aqueous solution of potassium permanganate (zones are observed as brown spots on a purple background of the sorbent), a solution of phosphorus-molybdic acid upon heating (blue spots appear on a 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 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, must have a low viscosity (otherwise the diffusion process slows down) and, when applied to the carrier, form a uniform film, firmly associated with him. 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 support. 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, to control environment, 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. Analysis of biopolymers (proteins, nucleic acids, etc.), for which usually spent 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.

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1 Short story development of liquid chromatography Chromatography was discovered by M.S. Tsvet in 1903 in the form of a liquid-adsorption column method. In this method, adsorbents with a grain size of more than µm were used, the eluent (solvent) passed through the column by gravity due to gravity, there were no flow detectors. Separation proceeded slowly, over several hours, and in this mode, liquid chromatography could not be used for analytical purposes. In the years the efforts of specialists in various countries were directed to the creation of express liquid chromatography. It was clear that in order to increase the separation rate, it was necessary to shorten the paths of external and internal diffusion. This could be achieved by reducing the diameter of adsorbent grains. Filling the columns with fine grains (5-10 μm) created a high inlet pressure, which required the use of high-pressure pumps. This is how high-pressure liquid chromatography was born. With the transition to adsorbents of a fine fraction, the efficiency of the columns greatly increased, therefore, modern express analytical liquid chromatography was called high-performance liquid chromatography (HPLC). The development of rigid fine-grained adsorbents (5 or 10 microns), the creation of high-pressure pumps (over 200 atm.) and flow detectors all ensured high performance HPLC. In terms of separation times, it was not inferior to gas chromatography, and in terms of areas of application it significantly surpassed it. Currently, HPLC occupies a leading position among other chromatography methods both in terms of the volume of equipment produced (more than chromatographs per year worth more than 2 billion dollars) and in terms of the number of publications (5-6 thousand publications per year). Modern HPLC is implemented in various versions. These options allow the separation of various mixtures of molecules (including mixtures of all types of isomers); macromolecules of synthetic and biopolymers (including viruses and molecules with masses up to several million); ions and stable radicals. The role of HPLC is also great in such vital areas of science and production as biology, biotechnology, food industry, medicine, pharmaceuticals, forensic medical examination, environmental pollution control, etc. HPLC has played one of the main roles in deciphering the human genome, in recent has been successfully solving problems of proteomics for years.

2 HPLC variants used in the last decade Variants Reverse-phase Normal-phase Ionic Ion-pair Ion-exchange Exclusionary Gel filtration Ligand-exchange Chiral Affinity Immune Micellar Hydrophobic Silver Reverse-phase Liquid-liquid Extraction Donor-acceptor Variants Complexing Inversion exclusion Nonlinear Capil lar micropipette multidimensional perfusion displacement ultrafast Turbulent Continuous Countercurrent Centrifuge Moving Bed High Temperature Membrane HPLC Theory Chromatographic Process: Retention, Elution, Separation A chromatographic column is a tube filled with a simple adsorbent through which a solvent flows continuously. The adsorbent (sorbent, column filler) is held in the column by filters, it is immobile and therefore is called the stationary phase. The solvent moving relative to the sorbent is called the mobile phase (in some cases, the eluent). When moving along the column, the molecules of the substance (sorbates) diffuse inside the pores of the sorbent and, as a result of intermolecular interactions of one type or another, are adsorbed on the surface of the stationary phase. The time during which the molecules are in the adsorbed state is determined by the strength of the intermolecular interaction of sorbates with the sorbent. With very weak sorption, the molecules spend almost all the time in

3 solution of the mobile phase and therefore move down the column at a speed only slightly inferior to the speed of the mobile phase. On the contrary, with very strong sorption, the molecules hardly leave the surface and the rate of their movement along the column is negligible. From the point of view of chromatography, of greater interest are such conditions under which the adsorption force is intermediate and the rate of movement of sorbates through the column is 2–10 times lower than the rate of movement of the mobile phase. The phenomenon of slow movement of molecules relative to the movement of the mobile phase in chromatography is called retention. If the sorption constants of substances are different, then their average velocity along the column will also be different. Thus, the main goal of separation chromatography is achieved. Naturally, in practice, single molecules are not introduced into the column. If at least several molecules of different types are introduced into the column, then the average rates of movement of sorbate molecules are still different. In addition, the speeds of movement of individual molecules of each type deviate in one direction or another from the average value for this type. Sorbate molecules, initially introduced into the column in the form of an instantaneous pulse, leave it in a wider zone. Such non-identity of the speeds of movement of identical molecules in chromatography is called smearing. This undesirable phenomenon leads to the fact that among the molecules of one substance there may also be molecules of another, the speed of which is close to the speed of the fastest molecules of the first. As a result, the zones of substances may partially overlap one another and the separation will be incomplete. The processes of retention and blurring are the subject of the theory of chromatography. Some Basic Terms and Definitions A chromatogram is a curve showing the concentration of compounds exiting a column with a mobile phase flow as a function of time from the start of separation.

4 A chromatogram usually consists of a baseline and peaks. In chromatographic instruments, as a rule, there is no direct measurement of the concentration of a substance in the mobile phase, but with the help of a special detector unit, any physical quantity that is functionally related to the concentration (electrical conductivity, optical density, etc.) is measured. The baseline corresponds to the period of time during which the detector registers only the signal from the mobile phase. The peak curve, ideally approaching a Gaussian distribution curve, describes a gradual increase in concentration at the outlet of the column and its subsequent decrease. The time when the maximum peak appears on the chromatogram is called the retention time (t R). Under constant operating conditions and the composition of the phases of the chromatographic system, the retention time is a constant value for a given substance. Sometimes a peak is recorded in the initial part of the chromatogram, the nature of which is associated with a short-term imbalance in the column during sample injection. This peak corresponds to the retention time of the non-sorbable substance (t 0). Comparative thermodynamic characterization of two separable peaks of substances gives the relative retention or selectivity. This value indicates the ability of a given chromatographic system to separate a given pair of substances. The retention times and all quantities derived from them are essentially thermodynamic characteristics of the process. However, the result is determined by the combined influence of thermodynamic and kinetic factors. If in a chromatographic system of a given composition at

At a given temperature, the values of t R for two substances are the same (or =1.0), then no change in the geometry of the column will lead to the separation of this pair. But, on the other hand, the difference in the values of t R does not automatically mean that the separation, and even more so the good one, will be achieved. To do this, the column used must have sufficiently high kinetic characteristics. The acts of sorption-desorption must be performed at high speed in order to realize the potential for separation, which is indicated by the difference in t R. The main kinetic characteristic of the process is the height h, equivalent to a theoretical plate (HETP). This value corresponds to the height of the sorbent layer, during the passage of which the act of sorption-desorption takes place on average once. It essentially reflects the quality of the sorbent used, the quality of column filling, and the correct choice of the chromatography mode. The reciprocal of the number of theoretical plates N is used to evaluate the quality of the column. The number of theoretical plates is a measure of column efficiency. Blurring of Chromatographic Zones The mixture to be separated is introduced into the column in the form of a narrow pulse, and its volume compared to the volume of the column can be neglected. As the molecules of the substances to be separated move with the flow of the mobile phase, the pulse gradually expands, while the concentration of the substances to be separated in it decreases. main reason of this process is that the speed of movement along the column of individual molecules differs from the average speed characteristic of this compound. From the point of view of the final useful result of the chromatographic process of achieving separation of molecules of various types, the spreading of zones is highly undesirable, at least for the following reasons. First, intense erosion leads to partial overlapping of the zones of various compounds and, therefore, it is necessary to impose more stringent requirements on the selectivity of the system. Moreover, even if in one case or another it is possible to provide increased selectivity, the total separating power is low. Another negative consequence of smearing is a decrease in the concentration of sorbate in the center of the zone, leading to a decrease in the sensitivity of the analysis. A measure of the intensity of erosion processes is the height equivalent to a theoretical plate. The value of h is determined by a number of particular processes. 1) Inhomogeneity of the flow of the mobile phase. The sorbent in the column forms a system of channels through which the mobile phase flows. The finer the sorbent particles, the closer to each other the path length of the mobile phase molecules, the smaller the difference in the time of molecules of one zone passing through the column, and the less the zone blurring.

6 2) Molecular diffusion in mobile and stationary phases. The greater the flow rate, the less blur due to this reason. 3) The rate of mass transfer is the time of sorption or ion exchange. The greater the flow rate, the greater the blur due to this reason. Clearly, to reduce h, it is necessary to use sorbent particles of smaller diameter. Unfortunately, this path can only be used up to a certain limit, which is dictated by technical considerations. The pressure drop in the column is related to other process parameters by the following relationship: where r is the flow resistance parameter, p is the pressure drop, U is the flow rate, L is the column length, and d is the size of the sorbent particles. With increased pressure, the cost and complexity of the equipment increases dramatically. Therefore, HPLC d p =3-10 μm. To increase efficiency, less viscous solvents are preferred, as they have a higher diffusion coefficient and lower column resistance. In HPLC, the theory of smearing of chromatographic zones has been more or less completed by now. The development of this theory made it possible to realize in practice the efficiency of the columns close to the theoretical one. Thus, when using sorbents with a grain diameter of less than 3 μm, efficiency up to theoretical plates per meter of column length was obtained. Much attention of chromatographers is paid to studies of separation selectivity. In HPLC, in contrast to gas chromatography, selectivity is determined by both the nature of the sorbent and the nature of the eluent. Work continues to study the interaction of the substance-solvent, which correlates with free energy sorption. Hot topics in HPLC theory are computer optimization of the separation process. As noted above, the main efforts of chromatographers are currently focused on the theoretical study of separation selectivity issues. There are dozens of publications on the study of the relationship between the structure of molecules and their retention on sorbents of various chemical nature and in multidimensional chromatography. To improve separation selectivity, both in gas chromatography and HPLC, the steric factor is widely used when cyclodextrins, crown ethers, and liquid crystals are used for the selective separation of isomers.

7 The achievements in the theory of separation of optical isomers, both in gas and liquid chromatography, are impressive. Results are obtained at the level of discovery, showing the possibility of separation of optical isomers upon contacts at two points (the surface of an achiral adsorbent can serve as the third point of contact). The development of the theory of polymer chromatography under critical conditions continues successfully. Progress has been made in establishing the relationship between chromatographic retention parameters and the biological and chemical activity of molecules. This is especially promising for the pharmaceutical industry when looking for new types of drugs. In recent years, there has been a growing interest in the problem of the influence of temperature on the entire separation process in HPLC. A high-temperature HPLC has been proposed and equipment for temperature programming in this method is being developed. The work on optimizing the separation while simultaneously varying the temperature and strength of the eluent looks promising. The influence of an electric field applied along the column on the retention and erosion of corticosteroids on columns with a porous carbon adsorbent, as well as the effect of a magnetic field on retention on columns filled with magnetic particles by steel balls coated with polytetrafluoroethylene, were studied. Sorbents for HPLC A wide range of sorbents has been developed and produced for HPLC. About 100 firms all over the world produce more than 300 types of sorbents. However, the real assortment is much narrower, since the sorbents of many companies are identical in the chemical nature of the surface and differ only in names. Relative share of application of different HPLC methods on different sorbents Chromatography method/type Percentage of sorbent users Reverse-phase 50.4 Silica gel with grafted groups С С 8 15.9 Phenyl 7.1 С 4 2.3 С 1 -С 2 1.1

8 Normal-phase 24.1 Silica gel with grafted groups CN- 8.9 Silica gel 8.5 MN 2-4.7 Diol 2 Ion-exchange and ionic 14 Anions 7.4 Cations 6.6 Exclusive 6.7 Aqueous 3.5 Non-aqueous 3 .2 Chiral 2.8 Hydrophobic 1.1 Other 1.1 The most commonly used pure silica gels and silica gels with grafted non-polar and polar groups. Sorbents based on oxides of aluminium, zirconium, titanium, etc. have been developed and continue to be developed. The share of application of various sorbents in HPLC is as follows: silica gels 70%, porous polymers (copolymer of styrene and divinylbenzene, polymethacrylates, cellulose, etc.) 20%, porous carbon sorbents, titanium oxide, zirconium oxide 4%, aluminum oxide 1%. In analytical practice, reverse phase chromatography (more than 70%) using silica gel with grafted С18 and С8 alkyl groups finds the greatest use. Despite their widespread use, these sorbents have a number of disadvantages, the main of which is insufficient chemical stability. At pH< 3 происходит гидролиз связи =Si О Si=, а при рн >10 dissolves the silica gel base, especially at elevated temperatures. These sorbents are nonselective in the separation of polar compounds and isomers. Substances of a basic nature elute, as a rule, in the form of unsymmetrical peaks due to interaction with residual hydroxyl groups. The properties of silica gel materials strongly depend on the purity, geometric and chemical nature of silica gel, the method of grafting alkyl groups, etc. In recent years, research has been actively carried out to eliminate these shortcomings. First of all, the production of initial silica gels has been significantly improved, which made it possible to reproducibly obtain spherical particles with an insignificant content of

9 heavy metals. Complete binding of the hydroxyl groups on the silica gel surface is never achieved. Residual hydroxyl groups lead to undesirable interactions and unsymmetrical peaks in compounds consisting of small polar molecules. To eliminate the influence of residual silanols, it was proposed to close (block) them with bulkier isopropyl or isobutyl groups. An example of such a sorbent is Zorbax Stable Bond. Bidentate substituents are also used when two adjacent alkyl chains are linked to silicon atoms through 3-4 methylene groups. This "bridge" closes the residual hydroxyl groups and such phases are stable even at elevated pH.< 12 (Zorbax Extend-С18). Силикагели со средними размерами пор, 80, 100, 120 Å, применяют для разделения низкомолекулярных соединений, силикагели с порами 300 Å и более для разделения макробиомолекул. Как известно, поверхность белковой глобулы богата гидрофильными аминокислотами, но в то же время содержит немало (до половины от их общего содержания) гидрофобных остатков, нередко образующих скопления (" гроздья"). Такие гидрофобные зоны, развитые в большей или меньшей степени, представляют характерную особенность структуры каждого белка, на чем и основан метод гидрофобной хроматографии. Соответствующие сорбенты синтезируют, включая гидрофобные группировки в гидрофильную матрицу, например в поперечносшитую агарозу сефарозу. По такому принципу построены, в частности, октил- и фенилсефароза: При пропускании белкового раствора через фенилсефарозу гидрофобные участки поверхности белков образуют контакты с фенильнымн группами, вытесняя прилегающие к этим структурам молекулы воды. Число и прочность таких контактов весьма различны у разных белков. Повышению их прочности способствует сорбция белков из концентрированных растворов солей, например

10 ammonium sulfate. A smooth decrease in the salt concentration in the solution flowing through the column with phenylsepharose leads to successive desorption of proteins. Sorbents obtained by adding hydrophobic alkyl radicals of various lengths to macroporous silica act in a similar way. They, being rigid, are particularly suitable for operation at elevated pressure under high performance liquid chromatography (HPLC, English HPLC). Those that contain long C18 hydrocarbon chains are of little use for protein separation due to too strong, often irreversible binding, but can be used for peptide chromatography. The best results are obtained by chromatography of proteins on sorbents containing shorter C 4 -C 8 hydrocarbon chains. Often hydrophobic chromatography is combined with other effects. For example, the addition of diamines of various lengths to cyanogen bromide-activated Sepharose gives sorbents that contain hydrobic hydrocarbon chains along with two cationic groups. Combining the features of a hydrophobic sorbent and anion exchanger in one chromatographic material enriches its possibilities. The method described above for carrying out chromatography on a hydrophobic sorbent is by no means the only one possible. For the sorption of proteins, it is not necessary to introduce elevated salt concentrations into the solution, and for elution, the addition of organic solvents, a pH shift, can be used. In some cases, when protein binding is based on a combination of hydrophobic and ionic interactions, elution with salt solutions gives good results. We also note that signs of hydrophobic chromatography are also found in other methods of protein separation, especially in affinity chromatography. Every year at the Pittsburgh Conference and Exhibition in the USA, dozens of new HPLC sorbents are presented and new directions and trends can be judged from them. Companies offer a wide range of columns: column lengths vary from 10 to 250 mm, and internal diameters from 1 to 50 mm. Equipment for HPLC Modern liquid chromatographs are available in three versions: block-modular, monoblock and intermediate (modular design in a single block). The choice of configuration of a modular device is determined by the analytical task. The modular system allows you to quickly and easily assemble a specific system at minimal cost. On the basis of a flexible block-modular system, it is possible to create both simple devices and complex ones, with building blocks, suitable for solving routine technological problems and performing complex research measurements.

11 The monobloc system is advantageous in some cases in the case of specialized specific tasks. The integrated system with replaceable blocks has similar advantages. Currently, the following types of liquid chromatographs are commercially produced: high pressure (closed systems), gradient, isocratic, preparative, ion, size exclusion, low pressure(open systems), multidimensional, on-line analyzers, high temperature continuous, counterflow, moving bed, amino acid analyzers. These liquid chromatographs may include the following detection systems: Variable Wavelength, Fixed Wavelength (with Filters), Scanning, Photodiode Array, Refractometric, Fluorescence, Electrochemical, Conductometric, Amperometric, Light Scattering, Chemiluminescent , mass spectrometric, chiral, microcolumn, radioactive, IR spectroscopic, flame ionization, etc. HPLC with micro- and nanocolumns is being developed. Chromatograph layout: 1-pump 2-sample injection unit 3-chromatographic column 4-detector 5-registrar 6-column thermostat 7-elluent preparation unit 8-elluate drain or fraction collector

12 HPLC applications HPLC methods have become official methods in the pharmacopoeias of various countries, in the EPA (US Agency for Environmental Pollution Analysis), in GOSTs and recommendations for the analysis of many harmful compounds. When monitoring environmental pollution by HPLC methods, oil products are determined in surface and drinking waters; pesticides in water, soil; phthalates in water; aromatic amines and polynuclear aromatic compounds in food and water; phenol, chlorophenol and nitrophenols in drinking water; nitrosamines in food; heavy metals in water, soil and food; mycotoxins (aflatoxins, zearalenone, etc.) in food and feed and many other pollutants. Early diagnosis of diseases by analysis of biochemical markers HPLC is increasingly used to determine biochemical markers and metabolites in the mass medical examination of the population and the detection of dangerous diseases. Usually, for the diagnosis of diseases, it is sufficient to determine only markers, however, in some cases, it is required to determine the metabolic profile of the level of many components. Biological markers are relatively small molecules: catecholamines, amino acids (homocysteine), indoles, nucleosides, porphyrins, sugars, steroids, hormones, vitamins, pterins and lipids. In some cases, large molecules are also used: enzymes, proteins, nucleic acids. The profile of physiological fluid concentrations in patients with various diseases can differ significantly from the profile healthy people. This indicator must also be determined in patients with hereditary metabolic disorders. In addition, profile analyzes are carried out in the case of oncological, cardiovascular, mental and neurological diseases, as well as diabetes and porphyriasis. The profile of body fluid concentrations is determined in patients with certain symptoms, but it does not give an accurate diagnosis of the disease. It has recently been shown that altered nucleosides appear in the profile of AIDS patients. For the analysis of objects such as complex and multicomponent biological fluids, it is high performance liquid chromatography that is suitable, which has clear advantages over gas chromatography due to the instability of many biologically active compounds at elevated temperatures. The content of many markers in biological fluids is at the level of g, therefore, their determination requires highly sensitive and selective detectors, in particular, amperometric and fluorescent ones. It is desirable that the analyzes

13 completed quickly, within 5-20 minutes. Currently, with the analysis of biochemical markers in medical centers around the world, more than 200 metabolic diseases are already being detected. Studies and analyzes in biochemistry HPLC is most widely used for the separation of biological compounds: proteins, enzymes, sugars, lipids, amino acids, peptides, vitamins, etc. In connection with the development of proteomics, interest in the separation and analysis of proteins, peptides and amino acids has sharply increased. The HPLC method is used to study drug-membrane and drug-protein interactions, and to assess the degree of protein oxidation. The established relationship between chromatographic parameters and biological properties allows a more conscious search for new drugs and other biologically active compounds in pharmaceuticals. HPLC is one of the most important methods for studying drug metabolites, separating and isolating allergens, and studying pharmacokinetic processes. The separation of enantiomeric medicinal substances has been mastered on an industrial scale.

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1. Introduction.

2. The emergence and development of chromatography.

3. Classification of chromatographic methods.

4. Chromatography on a solid stationary phase:

a) gas (gas-adsorption) chromatography;

b) liquid (liquid adsorption) chromatography.

5. Chromatography on liquid stationary phase:

a) gas-liquid chromatography;

b) gel chromatography.

6. Conclusion.

Like the rays of the spectrum, various components of the mixture of pigments are regularly distributed in a column of calcium carbonate, allowing their qualitative and quantitative determination. I call the preparation obtained in this way a chromatogram, and the proposed method - chromatographic.

M. S. Tsvet, 1906

Introduction

The need to separate and analyze a mixture of substances is faced not only by a chemist, but also by many other specialists.

In a powerful arsenal of chemical and physicochemical methods of separation, analysis, study of the structure and properties of individual chemical compounds and their complex mixtures, one of the leading places is occupied by chromatography.

Chromatography is a physicochemical method for separating and analyzing mixtures of gases, vapors, liquids or solutes and determining the physicochemical properties of individual substances, based on the distribution of the separated components of mixtures between two phases: mobile and stationary. The substances that make up the stationary phase are called sorbents. The stationary phase can be solid or liquid. The mobile phase is a liquid or gas flow filtered through a sorbent bed. The mobile phase performs the functions of a solvent and a carrier of the analyzed mixture of substances, transferred to a gaseous or liquid state.

There are two types of sorption: adsorption - the absorption of substances by a solid surface and absorption - the dissolution of gases and liquids in liquid solvents.

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 opened - 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 variant of chromatography, the essence of which is the separation of 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.

3. Classification of chromatographic methods

The variety of modifications and variants of the chromatography method requires their systematization or classification.

The classification can be based on various criteria, namely:

1. state of aggregation of phases;

2. separation mechanism;

3. method of carrying out the process;

4. the purpose of the process.

Classification according to the state of aggregation of phases:

gas (mobile phase - gas), gas-liquid (mobile phase - gas, stationary phase - liquid), liquid (mobile phase - liquid) chromatography.

Classification according to the separation mechanism.

Adsorption chromatography is based on the selective adsorption (absorption) of individual components of the analyzed mixture by the corresponding adsorbents. Adsorption chromatography is divided into liquid (liquid adsorption chromatography) and gas (gas adsorption chromatography).

Ion-exchange chromatography is based on the use of ion-exchange processes occurring between mobile adsorbent ions and electrolyte ions when a solution of the analyte is passed through a column filled with an ion-exchange substance (ion exchanger). Ion exchangers are insoluble inorganic and organic macromolecular compounds. As ion exchangers, aluminum oxide, permutite, sulfonated coal and a variety of synthetic organic ion-exchange substances - ion-exchange resins are used.

Sedimentary chromatography is based on the different solubility of precipitates formed by the components of the analyzed mixture with special reagents. For example, when a solution of a mixture of salts of Hg (II) and Pb is passed through a column with a carrier pre-impregnated with a solution of KI, 2 colored layers are formed: the upper, colored orange-red (HgI 2), and the lower, colored yellow (PbI 2).

Classification according to the method of carrying out the process.

Column chromatography is a type of chromatography in which a column is used as a carrier for an immobile solvent.

Paper chromatography is a type of chromatography in which, instead of a column, strips or sheets of filter paper that do not contain mineral impurities are used as a carrier for an immobile solvent. In this case, a drop of the test solution, for example, a mixture of solutions of Fe (III) and Co (II) salts, is applied to the edge of the paper strip. The paper is suspended in a closed chamber (Fig. 1), lowering its edge with a drop of the test solution applied to it into a vessel with a mobile solvent, for example, n-butyl alcohol. The mobile solvent, moving through the paper, wets it. In this case, each substance contained in the analyzed mixture moves with its inherent speed in the same direction as the solvent. At the end of the separation of ions, the paper is dried and then sprayed with a reagent, in this case a K 4 solution, which forms colored compounds with the substances to be separated (blue - with iron ions, green - with cobalt ions). The resulting zones in the form of colored spots make it possible to establish the presence of individual components.

Paper chromatography combined with the use of organic reagents allows qualitative analysis of complex mixtures of cations and anions. A number of substances can be detected on one chromatogram using one reagent, since each substance is characterized not only by the corresponding coloration, but also by a certain location on the chromatogram.

Thin layer chromatography is a type of chromatography similar to paper chromatography in its separation mechanism. The difference between them is that instead of sheets of paper, separation is carried out on plates coated with a thin layer of sorbent made from powdered alumina, cellulose, zeolites, silica gel, diatomaceous earth, etc. and retaining the immobile solvent. The main advantage of thin layer chromatography is the simplicity of the apparatus, the simplicity and high speed of the experiment, the sufficient clarity of the separation of a mixture of substances, and the possibility of analyzing ultramicroquantities of a substance.

Classification according to the purpose of the chromatographic process.

Chromatography is of the greatest importance as a method of qualitative and quantitative analysis of mixtures of substances (analytical chromatography).

Preparative chromatography is a type of chromatography in which the separation of a mixture of substances is carried out for preparative purposes, i.e. to obtain more or less significant quantities of substances in a pure form, free from impurities. The task of preparative chromatography can also be the concentration and subsequent isolation from a mixture of substances contained in the form of microimpurities to the main substance.

Non-analytical chromatography is a type of chromatography that is used as a scientific research method. It is used to study the properties of systems, such as solutions, the kinetics of chemical processes, the properties of catalysts and adsorbents.

So, chromatography is a universal method for analyzing mixtures of substances, obtaining substances in pure form, as well as a method for studying the properties of systems.

4. Chromatography on a solid stationary phase

a) Gas (gas-adsorption) chromatography

Gas chromatography is a chromatographic method in which the mobile phase is a gas. Gas chromatography has received the greatest application for the separation, analysis and study of substances and their mixtures, passing without decomposition into a vapor state.

One of the options for gas chromatography is gas adsorption chromatography - a method in which the stationary phase is a solid adsorbent.

In gas chromatography, an inert gas is used as a mobile phase (carrier gas): helium, nitrogen, argon, much less often hydrogen and carbon dioxide. Sometimes the carrier gas is a vapor of volatile liquids.

The gas chromatographic process is usually carried out in special instruments called gas chromatographs (Fig. 3). Each of them has a carrier gas flow supply system, a test mixture preparation and injection system, a chromatographic column with a temperature control system, an analyzing system (detector), and a system for recording separation and analysis results (registrar).

Temperature is of great importance in gas adsorption chromatography. Its role, first of all, is to change the sorption equilibrium in the gas-solid system. The degree of separation of the mixture components, the efficiency of the column, and the overall speed of analysis depend on the correct selection of the column temperature. There is a certain temperature range of the column in which the chromatographic analysis is optimal. Typically, this temperature range is in the region close to the boiling point of the determined chemical compound. When the boiling points of the mixture components are very different from each other, column temperature programming is used.

Separation in a chromatographic column is the most important, but preliminary operation of the entire process of gas chromatographic analysis. As a rule, binary mixtures (carrier gas - component) leaving the column enter the detecting device. Here, changes in the concentrations of components over time are converted into an electrical signal, which is recorded using a special system in the form of a curve called a chromatogram. The results of the entire experiment largely depend on the correct choice of the type of detector and its design. There are several classifications of detectors. There are differential and integral detectors. Differential detectors record the instantaneous value of one of the characteristics (concentration or flow) over time. Integral detectors summarize the amount of a substance over a certain period of time. Also used are detectors of various principles of operation, sensitivity and purpose: thermal conductometric, ionization, spectroscopic, mass spectrometric, coulometric and many others.

Application of gas adsorption chromatography

Gas adsorption chromatography is used in the chemical and petrochemical industries to analyze the products of chemical and petrochemical synthesis, the composition of oil fractions, determine the purity of reagents and the content of key products at different stages of technological processes, etc.

Analysis of permanent gases and light hydrocarbons, including isomers, by gas chromatography takes 5 - 6 minutes. Previously, on traditional gas analyzers, this analysis lasted 5-6 hours. All this led to the fact that gas chromatography began to be widely used not only in research institutes and control and measurement laboratories, but also became part of the integrated automation systems of industrial enterprises.

Today, gas chromatography is also used in the search for oil and gas fields, making it possible to determine the content of organic substances taken from soils, indicating the proximity of oil and gas fields.

Gas chromatography is successfully used in forensics, where it is used to establish the identity of samples of blood stains, gasoline, oils, counterfeiting of expensive food products, etc. Very often, gas chromatography is used to determine the alcohol content in the blood of car drivers. A few drops of blood from a finger is enough to find out how much, when and what kind of alcoholic drink he drank.

Gas chromatography allows us to obtain valuable and unique information about the composition of odors in food products such as cheese, coffee, caviar, cognac, etc. Sometimes the information obtained by gas chromatographic analysis does not please us. For example, it is not uncommon for food products to contain excessive amounts of pesticides or fruit juice contains trichlorethylene, which, contrary to prohibitions, was used to increase the degree of extraction of carotene from fruits, etc. But it is this information that protects human health.

However, it is not uncommon for people to simply neglect the information received. First of all, this applies to smoking. A detailed gas chromatographic analysis has long established that the smoke of cigarettes and cigarettes contains up to 250 different hydrocarbons and their derivatives, of which about 50 have a carcinogenic effect. That is why lung cancer is 10 times more common in smokers, but still millions of people continue to poison themselves, their colleagues and relatives.

Gas chromatography is widely used in medicine for the determination of the content of numerous drugs, the determination of the level of fatty acids, cholesterol, steroids, etc. in the patient's body. Such analyzes provide extremely important information about the state of human health, the course of his illness, the effectiveness of the use of certain drugs.

Scientific research in metallurgy, microbiology, biochemistry, in the development of plant protection products and new drugs, in the creation of new polymers, building materials and in many other very diverse areas of practical human activity it is impossible to imagine without such a powerful analytical method as gas chromatography.

Gas chromatography has been successfully used to determine the content of polycyclic aromatic compounds hazardous to human health in water and air, the level of gasoline in the air of filling stations, the composition of car exhaust gases in the air, etc.

This method is widely used as one of the main methods for monitoring the purity of the environment.

Gas chromatography takes important place in our lives, providing us with a colossal amount of information. More than 20 thousand of various gas chromatographs are used in the national economy and in research organizations, which are indispensable assistants in solving many challenging tasks that confront researchers and engineers every day.

b) Liquid (liquid adsorption) chromatography

Liquid chromatography is a group of chromatography variants in which the mobile phase is a liquid.

One of the variants of liquid chromatography is liquid adsorption chromatography - a method in which the stationary phase is a solid adsorbent.

Although liquid chromatography was discovered before gas chromatography, it only entered a period of exceptionally intensive development only in the second half of the 20th century. At present, in terms of the degree of development of the theory of the chromatographic process and the technique of instrumentation, in terms of efficiency and speed of separation, it is hardly inferior to the gas chromatographic separation method. However, each of these two main types of chromatography has its own preferred area of application. If gas chromatography is suitable mainly for the analysis, separation and study of chemicals with a molecular weight of 500 - 600, then liquid chromatography can be used for substances with a molecular weight from several hundreds to several millions, including extremely complex macromolecules of polymers, proteins and nucleic acids. However, the opposition of different chromatographic methods is inherently devoid of common sense, since chromatographic methods successfully complement each other, and the very task of a particular study must be approached differently, namely, which chromatographic method allows solving it with greater speed, information content and at lower costs.

As in gas chromatography, modern liquid chromatography uses detectors that allow you to continuously record the concentration of the analyte in the liquid stream flowing from the column.

There is no single universal detector for liquid chromatography. Therefore, in each specific case, the most suitable detector should be selected. The most widespread are ultraviolet, refractometric, microadsorption and transport flame ionization detectors.

Spectrometric detectors. Detectors of this type are highly sensitive selective devices, which make it possible to determine very small concentrations of substances in a liquid phase flow. Their readings depend little on temperature fluctuations and other random changes in the environment. One of the important features of spectrometric detectors is the transparency of most solvents used in liquid adsorption chromatography in the working wavelength range.

Most often, absorption is used in the UV region, less often in the IR region. In the UV region, devices are used that operate in a wide range - from 200 nm to the visible part of the spectrum, or at certain wavelengths, most often at 280 and 254 nm. As sources of radiation, mercury lamps of low pressure (254 nm), medium pressure (280 nm) and appropriate filters are used.

Microadsorption detectors. The action of microadsorption detectors is based on the release of heat during the adsorption of a substance on an adsorbent that fills the detector cell. However, it is not the heat that is measured, but the temperature of the adsorbent, to which it is heated as a result of adsorption.

The microadsorption detector is a rather highly sensitive instrument. Its sensitivity depends primarily on the heat of adsorption.

Microadsorption detectors are universal, suitable for detecting both organic and inorganic substances. However, it is difficult to obtain sufficiently clear chromatograms on them, especially in the case of incomplete separation of the mixture components.

5. Chromatography on liquid stationary phase

a) Gas-liquid chromatography

Gas-liquid chromatography is a gas chromatographic method in which the stationary phase is a low-volatility liquid deposited on a solid carrier.

This type of chromatography is used to separate gases and vapors of liquids.

The main difference between gas-liquid and gas-adsorption chromatography is that in the first case, the method is based on the use of the process of dissolution and subsequent evaporation of gas or vapor from a liquid film held by a solid inert carrier; in the second case, the separation process is based on the adsorption and subsequent desorption of a gas or vapor on the surface of a solid substance - an adsorbent.

The chromatography process can be schematically represented as follows. A mixture of gases or vapors of volatile liquids is injected with a carrier gas flow into a column filled with a fixed inert carrier, on which a non-volatile liquid (stationary phase) is distributed. The investigated gases and vapors are absorbed by this liquid. The components of the mixture to be separated are then selectively expelled in a certain order from the column.

In gas-liquid chromatography, a number of detectors are used that react specifically to any organic substances or to organic substances with a certain functional group. These include ionization detectors, electron capture detectors, thermionic, spectrophotometric and some other detectors.

Flame ionization detector (FID). The operation of the FID is based on the fact that organic substances entering the flame of a hydrogen burner undergo ionization, as a result of which an ionization current arises in the detector chamber, which is also an ionization chamber, the strength of which is proportional to the number of charged particles.

The FID is sensitive only to organic compounds and is not sensitive or very weakly sensitive to gases such as air, sulfur and carbon oxides, hydrogen sulfide, ammonia, carbon disulfide, water vapor and a number of other inorganic compounds. The insensitivity of the FID to air allows it to be used to determine air pollution by various organic substances.

There are 3 gases used in FID operation: carrier gas (helium or nitrogen), hydrogen and air. All 3 gases must be of high purity.

Argon detector. In an argon detector, ionization is caused by the collision of molecules of the analyte with metastable argon atoms formed as a result of exposure to radioactive B radiation.

Thermionic detector. The principle of operation of a thermionic detector is that alkali metal salts, evaporating in a burner flame, selectively react with compounds containing halogens or phosphorus. In the absence of such compounds, an equilibrium of alkali metal atoms is established in the ionization chamber of the detector. The presence of phosphorus atoms, due to their reaction with alkali metal atoms, disturbs this equilibrium and causes an ion current to appear in the chamber.

Since the thermionic detector has the highest sensitivity to phosphorus-containing compounds, it is called phosphorus. This detector is mainly used for the analysis of organophosphate pesticides, insecticides and a number of biologically active compounds.

b) Gel chromatography

Gel chromatography (gel filtration) is a method for separating mixtures of substances with different molecular weights by filtering the analyzed solution through cross-linked cellular gels.

The separation of a mixture of substances occurs if the sizes of the molecules of these substances are different, and the diameter of the pores of the gel grains is constant and can pass only those molecules whose dimensions are smaller than the diameter of the holes in the gel pores. When filtering a solution of the analyzed mixture, smaller molecules, penetrating into the pores of the gel, are retained in the solvent contained in these pores, and move along the gel layer more slowly than large molecules that are unable to penetrate into the pores. Thus, gel chromatography makes it possible to separate a mixture of substances depending on the size and molecular weight of the particles of these substances. This separation method is quite simple, fast, and, most importantly, it allows the separation of mixtures of substances under milder conditions than other chromatographic methods.

If you fill a column with gel granules and then pour a solution into it various substances with different molecular weights, then when the solution moves along the gel layer in the column, this mixture will separate.

The initial period of the experiment: applying a solution of the analyzed mixture to the gel layer in the column. The second stage - the gel does not prevent the diffusion of small molecules into the pores, while large molecules remain in the solution surrounding the gel granules. When the gel layer is washed with a pure solvent, large molecules begin to move at a speed close to the speed of the solvent, while small molecules must first diffuse from the internal pores of the gel into the volume between the grains and, as a result, are retained and washed out by the solvent later. A mixture of substances is separated according to their molecular weight. Substances are washed out of the column in order of decreasing molecular weight.

Application of gel chromatography.

The main purpose of gel chromatography is the separation of mixtures of macromolecular compounds and the determination of the molecular weight distribution of polymers.

However, gel chromatography is equally used to separate mixtures of substances of medium molecular weight and even low molecular weight compounds. In this case, it is of great importance that gel chromatography allows separation at room temperature, which compares favorably with gas-liquid chromatography, which requires heating to transfer the analytes to the vapor phase.

Separation of a mixture of substances by gel chromatography is also possible when the molecular weights of the analyzed substances are very close or even equal. In this case, the interaction of solutes with the gel is used. This interaction can be so significant that it cancels out the differences in the sizes of the molecules. If the nature of the interaction with the gel is different for different substances, this difference can be used to separate the mixture of interest.

An example is the use of gel chromatography for the diagnosis of thyroid diseases. The diagnosis is established by the amount of iodine determined during the analysis.

The given examples of the application of gel chromatography show its wide possibilities for solving a wide variety of analytical problems.

Conclusion

As a scientific method of understanding the world around us, chromatography is constantly evolving and improving. Today it is used so often and so widely in scientific research, medicine, molecular biology, biochemistry, technology and the national economy that it is very difficult to find a field of knowledge in which chromatography would not be used.

Chromatography as a research method with its exceptional capabilities is a powerful factor in understanding and transforming the increasingly complex world in the interests of creating acceptable living conditions for humans on our planet.

BIBLIOGRAPHY

1. Aivazov B.V. Introduction to chromatography. - M.: Vyssh.shk., 1983 - p. 8-18, 48-68, 88-233.

2. Kreshkov A.P. Fundamentals of analytical chemistry. Theoretical basis. Qualitative analysis, book one, 4th ed., revised. M., "Chemistry", 1976 - p. 119-125.

3. Sakodynsky K.I., Orekhov B.I. Chromatography in science and technology. - M.: Knowledge, 1982 - p. 3-20, 28-38, 58-59.

Introduction

High performance liquid chromatography (HPLC) is one of the effective methods for separating complex mixtures of substances, which is widely used in both analytical chemistry and chemical technology. The basis of chromatographic separation is the participation of the components of the mixture being separated in a complex system of van der Waals interactions (mainly intermolecular) at the phase boundary. As a method of analysis, HPLC is part of a group of methods, which, due to the complexity of the objects under study, includes the preliminary separation of the initial complex mixture into relatively simple ones. The resulting simple mixtures are then analyzed by conventional physicochemical methods or by special methods developed for chromatography.

History of the development of liquid chromatography

Chromatography was discovered by M.S. Tsvet in 1903 in the form of a liquid-adsorption column method. In this method, adsorbents with a grain size of more than 50–100 μm were used, the eluent passed through the column by gravity due to gravity, there were no flow detectors. Separation proceeded slowly, over several hours, and in this mode, liquid chromatography could not be used for analytical purposes. In 1965-1970. the efforts of specialists in various countries were directed to the creation of express liquid chromatography. It was clear that in order to increase the separation rate, it was necessary to shorten the paths of external and internal diffusion. This could be achieved by reducing the diameter of adsorbent grains. Filling the columns with fine grains (5-10 μm) created a high inlet pressure, which required the use of high-pressure pumps. This is how high-pressure liquid chromatography was born. With the transition to adsorbents of a fine fraction, the efficiency of columns greatly increased (per unit length, hundreds of times higher than the efficiency of columns in gas chromatography), so modern express analytical liquid chromatography was called high-performance liquid chromatography (HPLC). The development of rigid fine-grained adsorbents (5 or 10 µm), the creation of high-pressure pumps (over 200 atm.) and flow detectors - all this ensured high HPLC performance. In terms of separation times, it was not inferior to gas chromatography, and in terms of areas of application it significantly surpassed it. This period of time began to be called the second birth of liquid chromatography, the revival, the period of its renaissance.

One of the first commercial liquid chromatographs was the DuPont Model 820 (1968). This was preceded by the development of a series of detectors for liquid chromatography: a conductometric detector (1951), a heat of adsorption detector (1959), a refractive index detector (1962), a UV detector (1966), a liquid chromatograph / mass spectrometer system (1973), the first version of the detector on diode array (1976).

In 1969, I. Halash and I. Sebastian proposed sorbents with chemically grafted alkyl chains ("brush sorbents") with Si--O--C bonds. This relationship turned out to be unstable. In 1970, J. Kirkland developed sorbents with more stable Si--O-Si bonds. For the sake of justice, it should be noted that such a modification was proposed much earlier (1959) by K.D. Shcherbakova and A.V. Kiselev.

In our country, liquid chromatographs were developed in 1969--1972, these are the Tsvet-1-69, Tsvet-304 and KhG-1301 models.

Development of chromatography. History of the development of liquid chromatography. Chromatography on a solid stationary phase

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History of the development of liquid chromatography