A brief history of the development of liquid chromatography. History of the discovery of chromatography Solid stationary phase chromatography

The discoverer of chromatography was the Russian scientist, botanist and physical chemist 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). While studying plant pigments, Tsvet passed a solution of a mixture of very slightly different pigments 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 stripes. According to modern terminology, Tsvet discovered a developing version of chromatography (developing liquid adsorption chromatography). Tsvet outlined the main results of research on the development of the version of chromatography he created in the book “Chromophylls in the Plant and Animal World” (1910), which is his doctoral dissertation. chromatography gas sediment ion exchange

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

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

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 microquantities of a substance.

The next important step was the discovery by A. Martin and R. Synge (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 silica gel saturated with water, using chloroform as a solvent (1940). At the same time, it was noted that not only liquid, but also 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 Singh received the Nobel Prize in Chemistry for their discovery of partition chromatography. (1952). Next, Martin and A. James carried out a version of gas distribution chromatography, separating mixtures on a mixed sorbent of silicone DS-550 and stearic acid (1952 - 1953). Since that time, the gas chromatography method has received the most intensive development.

One of the variants of gas chromatography is chrothermography, in which, to improve the separation of a mixture of gases, simultaneously with the movement of the mobile phase - gas, the sorbent and the mixture being 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 solubility of sparingly soluble sediments (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 an employee of the Perkin-Elmer company, M. Golay (USA), of a capillary version 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 increased sharply. High performance liquid chromatography (HPLC) appeared. This was facilitated by the creation of highly sensitive detectors, new selective polymer sorbents, and new equipment that allows operation at high pressures. Currently, HPLC occupies a leading position among other chromatography methods and is implemented in various versions.

Chromatographic techniques prevail among others when monitoring the air quality of work areas in industry and industrial hygiene; they form the basis of the vast majority of toxicological studies; Using gas chromatography, doctors were able to study the “sick building syndrome” - poor health and some diseases caused by the presence in the air of residential premises and office buildings of a large number of harmful chemicals released from synthetic materials (carpets, paths, panels, linoleum, upholstery and etc.), mastics, varnishes, dressings and other household chemical products, as well as gas emissions during the operation of laser printers and gas heaters.[...]

The process of chromatographic separation is based on sorption, which we encounter in everyday life - the absorption of substances by a solid surface (adsorption) or the dissolution of gases and liquids in liquid solvents (absorption). The most well-known application of adsorption is air purification in gas masks: the adsorbent (active carbon) filling the gas mask box retains harmful impurities or chemical agents contained in the air. Absorption is characteristic of many biological processes, in particular the respiration process. The absorption of oxygen by hemoglobin in the blood in the lungs is also, to a certain extent, a chromatographic process, since this involves the sorption separation of oxygen from other gases present in the inhaled air. Unfortunately, harmful impurities in the air are also absorbed by the blood and sometimes irreversibly.[...]

The person who was the first to correctly explain the process of sorption (phenomena that occur when a substance moves along a sorbent layer) was the Russian scientist Mikhail Semenovich Tsvet. Using these phenomena, he created a remarkable analytical method, showed its wide possibilities and gave a name that to this day we use to designate not only the method, but also the process itself and the scientific discipline that studies it.[...]

But since different substances were extracted differently by benzene from the adsorbent (chalk), they descended through the tube at different speeds. Therefore, the original green ring, descending, gradually expanded and was divided into several colored rings. In the end there were six of these rings: the top one was yellow, then olive green, then dark green and three yellow.[...]

Tsvet removed a layer of chalk from the tube, cut it into cylinders, each of which contained its own colored ring. Now it was possible to extract substances from the adsorbent with alcohol and examine them. As a result, the scientist showed that chlorophyll is not an individual compound, but a mixture of two substances that separated on a chalk column and gave olive green and dark green rings. The remaining substances were xanthophylls.[...]

Color called the multi-colored picture obtained when separating substances a chromatogram, and the method itself (based on the separation of substances according to their tendency to adsorption) chromatographic adsorption analysis, or chromatography.[...]

Before 1914, Tsvet published several articles on chromatography, but after his work the method was not widely developed. Only in 1931 did Kuhn, Winterstein and Lederer reproduce Tsvet’s initial experiments (using examples of the separation of carotene from carrots, dandelion petals and chicken egg yolk). Such a long oblivion of the now classic research was largely due to the negative reviews of the authorities of that time, who could not understand the full depth of the young scientist’s discovery.[...]

For the development of the method of partition chromatography and its various variants, Martin and Singh were awarded the Nobel Prize in 1952. It was from this moment that the modern stage of development of gas chromatography began (1951-1952), when A. A. Zhukhovitsky and his colleagues (Russia) proposed chrothermography, and A. Martin and A. James - gas-liquid chromatography, with the help of which they succeeded in separating a mixture of fatty acids on a column with a diatomite carrier (Celite-545) impregnated with paraffin oil with the addition of stearic acid. Such a sorbent absorbs analyzed substances much weaker than, for example, active carbon or aluminum oxide, so James and Martin were able to separate volatile organic acids in a gas flow - nitrogen.[...]

Since then, gas chromatography has become one of the most common methods of analysis, with which you can study an extremely wide range of substances - from gases to high molecular weight liquids and metals.[...]

Some terminology issues regarding the classification of chromatographic methods should be clarified. In its simplest case, the term “gas chromatography” refers to an analysis method in which the separation of a mixture of substances in a chromatographic column is carried out in a gas stream (carrier gas) continuously passed through the column. Gas adsorption (separation on an adsorbent - carbon, silica gel or aluminum oxide) and gas-liquid (separation on a sorbent - a solid carrier coated with a liquid - a stationary liquid phase) - these are all variants of gas chromatography.

Based on the fractionation principle:

Affinity chromatography

Gel filtration

Adsorption

Sedimentary

Adsorption-complexation

Distribution (normal phase, reverse phase).

According to the method of evolution:

Size exclusion chromatography

Chromatographic evolution

Frontal analysis

Ion exchange chromatography.

By location of the stationary phase:

Columnar chrome

Thick layer chromatography

Thin layer chromatography

Paper (film) chromatography.

According to the aggregate composition of the phases:

Supercritical fluid chromatography

Liquid chromatography (liquid-gel, liquid-liquid, liquid-solid phase)

Gas chromatography (gas-solid-phase, gas-liquid).

By purpose of behavior:

Analytical

Preparative

Industrial.

By pressure in the chromatographic system:

High pressure

Low pressure.

2. History of the development of liquid chromatography.

Chromatography was discovered by M. S. Tsvet in 1903 in the form of a column liquid-adsorption method. This method used adsorbents with a grain size of more than 50-100 microns, the eluent passed through the column by gravity due to gravity, there were no flow detectors. Separation occurred slowly, within 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 aimed at creating rapid liquid chromatography. It was clear that to increase the rate of separation it was necessary to shorten the paths of external and internal diffusion. This could be achieved by reducing the diameter of the adsorbent grains. Filling the columns with small grains (5-10 μm) created a high inlet pressure, which required the use of high-pressure pumps.

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

The modern stage of HPLC: Currently, HPLC is the leading

positions among other chromatography methods both in terms of the volume of equipment produced (more than 40,000 chromatographs per year worth more than 2 billion dollars) and in the number of publications (5-6 thousand publications per year).

The role of HPLC is also great in such vital areas of science and production as biology, biotechnology, food industry, medicine, pharmaceuticals, forensic examination, environmental pollution control, etc. HPLC has played a major role in deciphering the human genome, in recent years years successfully solves problems

cottages of proteomics.

Chromatography is a method of separation and determination of substances based on the distribution of components between two phases - mobile and stationary. The 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 mixture being analyzed (sorbates), together with the mobile phase, move along the stationary phase. It is usually placed in a glass or metal tube called a column. Depending on the strength of interaction with the sorbent surface (due to adsorption or some other mechanism), the components will move along the column at different speeds. Some components will remain in the upper layer of the sorbent, others, interacting with the sorbent to a lesser extent, will end up in the lower part of the column, and some will completely leave the column along with the mobile phase (such components are called unretained, and their retention time determines the “dead time” of the column) .

This allows rapid separation of complex mixtures of components.

Discovery history:

Birth of chromatography

In the evening of this day, at a meeting of the biological department of the Warsaw Society of Naturalists, assistant of the department of anatomy and physiology of plants Mikhail Semenovich Tsvet made a report “On a new category of adsorption phenomena and their application to biochemical analysis.”

Unfortunately, M.S. Tsvet, being a botanist by training, did not adequately appreciate the chemical analytical aspect of his discovery and published little of his work in chemical journals. Subsequently, it was the chemists who appreciated the real scale of the proposed M.S. The color chromatographic method has become the most common method of analytical chemistry.

The following advantages of chromatographic methods should be emphasized:

1. Separation is dynamic in nature, and the acts of sorption-desorption of the separated components are repeated many times. This is due to the significantly greater efficiency of chromatographic

separation compared to static sorption methods and

extraction.

2. During separation, 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

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

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

5. Chromatography allows you to solve both analytical problems (separation, identification, determination) and preparative ones (purification, isolation, concentration). The solution to these problems can be combined by performing them online.

Numerous methods are classified according to the state of aggregation of the phases, the separation mechanism and the separation technique.

Chromatographic methods also differ in the way they are carried out.

the process of separation into frontal, displacement and eluent.

Ion chromatography

Ion chromatography is a high-performance liquid chromatography for the separation of cations and anions on ion exchangers

low capacity. Widespread use of ion chromatography

due to a number of its advantages:

– the ability to determine a large number of inorganic and

organic ions, and also simultaneously determine cations and

– high detection sensitivity (up to 1 ng/ml without

pre-concentration;

– high selectivity and expressivity;

– small volume of the analyzed sample (no more than 2 ml of sample);

– wide range of detectable concentrations (from 1 ng/ml to

– the possibility of using various detectors and their combinations, which allows for selectivity and short determination time;

– possibility of complete automation of determination;

– in many cases, a complete lack of preliminary sample preparation.

However, like any analytical method, ion chromatography is not without its disadvantages, which include:

– the complexity of the synthesis of ion exchangers, which greatly complicates

development of the method;

– lower separation efficiency compared to HPLC;

– the need for high corrosion resistance

chromatographic system, especially when determining

cations.

2.1 Development history:

The study of ion exchange processes began already at the beginning of the 19th century. from observations of the influence of soils on the chemical composition of salt solutions in contact with it. At the end of the 40s, G. Thompson noted that the soil absorbs ammonia from applied organic fertilizers; corresponding experiments were carried out by their York specialist D. Spence. The first results of D. Spence's experiments were published by G. Thompson in 1850. The article notes that “the first discovery of highly important soil properties may almost fail as useful for agriculture” and his last works were published in 1852 and 1855.

2.3 Principles of ion separation in sorption processes

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

Ion chromatography is a variant of ion exchange column chromatography.

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

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

The following separation principles are used in ion chromatography:

Ion exchange.

Formation of ion pairs.

Ion exclusion.

Ion exchange

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

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

Formation of ion pairs

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

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

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

Ionic exclusion

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

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

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

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

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

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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. Sedimentary chromatography

7.1 Classification of sediment chromatography methods according to experimental technique

7.2 Sedimentary paper chromatography

8. Ion exchange chromatography

Conclusion

Bibliography

1. STORYDISCOVERY AND DEVELOPMENT OF CHROMATOGRAPHY

The discoverer of chromatography was the Russian scientist, botanist and physical chemist Mikhail Semyonovich Tsvet.

2. BASIC POINTS

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

2. During separation, 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.

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

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

3. CLASSIFICATION OF CHROMATOGRAPHIC ANALYSIS METHODS

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 interactions of the separated substances with the stationary phase;

* experimental differences in the methods of carrying out the chromatographic separation process.

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

Since the nature of the interactions of the compounds being separated with the phases of different 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 mobile solvent systems. 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 adsorption chromatography methods is thin layer chromatography (TLC), a type of plane chromatography in which the adsorbent is used as a thin layer on a plate.

Principle and basic concepts of the TLC method. A thin layer of sorbent is applied in one way or another to a clean flat surface (a plate made of glass, metal, plastic), which is most often fixed to 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 separating 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 the 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 in 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 NP 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, therefore 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. The spots (zones) usually have an oval or round shape. In the case under consideration, the spot of component A moved from the start line to a distance l A , component B spot - at a distance l IN, and the solvent passed through the distance L.

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

To characterize the separated components 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 movement speed i- th component and solvent E; l 1 AndL - the path traveled i- m component and solvent, respectively, t is the time required to move the solvent from the start line to the solvent front line. Distances l 1 count from the start line to the center of the spot of the corresponding component.

Typically, the mobility coefficient lies within the range R f =0 - 1. The optimal value is 0.3-0.7. Chromatographic conditions are selected so that the R f value 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), the uniformity of its graining, the thickness of the layer; sorbent activity (moisture content); experimental techniques (sample masses, solvent travel length L); experimenter's skill, etc. Consistently reproducing all these parameters in practice is sometimes difficult. To level out the influence of process conditions, a 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 Rf.

A substance is often chosen as a standard for which, under given conditions, Rf? 0.5. Based on its chemical nature, the standard is chosen to be close to the substances being separated. Using 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 separated components, “witnesses” are used - reference substances, the presence of which is assumed in the analyzed sample. If R f = R f (width), where R f and R f (width) are the mobility coefficients of a given component and witness, respectively, then it can be more likely to be assumed that the witness substance is present in the chromatographed mixture.

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) = D l( =2D l ,

where D l- the 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 in the chromatogram.

To assess the selectivity of separation of two substances A and B, the separation coefficient 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 starting line using a capillary, microsyringe, micropipette, carefully touching the sorbent layer (the diameter of the spot on the starting line is usually from one to several millimeters). If several samples are applied to the starting line, then the distance between the sample spots on the starting line should not be less than 2 cm. If possible, use concentrated solutions. The spots are dried in air, after which chromatography is carried out.

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

Depending on the direction of movement of the PF, they are distinguished ascending, descending And horizontal chromatography.

In the ascending chromatography version, only plates with an attached sorbent layer 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 lower part of the plate (below the starting line by ~1.5 - 2 cm). The PF moves due to the action of capillary forces from bottom to top (against gravity) relatively slowly.

In the variant of descending chromatography, only plates with a fixed layer are also used. The PF is fed from above and moves down along the sorbent layer of the plate. Gravity accelerates the movement of the PF. This option is implemented when analyzing mixtures containing components that move slowly with the PF.

In a version of horizontal chromatography, the plate is placed horizontally. You can use rectangular or round plates. When using round plates (circular version of horizontal chromatography), the starting line is designated as a circle of suitable radius (~1.5-2 cm), onto which samples are applied. A hole is cut out in the center of the round plate into which a wick is inserted to supply 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 option, up to several dozen samples can be analyzed simultaneously.

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

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

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

Diagram 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 clear separation of the spots is achieved.

V) Interpretation of chromatograms. If the spots on the chromatogram are colored, after drying the plates, determine the distance from the starting line to the center of each spot and calculate the mobility coefficients. If the analyzed sample contains colorless substances that give uncolored substances, i.e. spots that are not visually identifiable on the chromatogram, it is necessary to detection these spots, why are chromatograms manifest.

The most common detection methods are described below.

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

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

Chemical treatment. Often, chromatograms are developed by treating them with reagents that form colored compounds with the separated components of mixtures. 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 quite arbitrary).

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

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

Dragendorff's reagent is a solution of basic bismuth nitrate BiONO 3, potassium iodide KJ and acetic acid in water. Used for the determination of amines, alkaloids, steroids.

Zimmermann's 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.

Ninhydrin is used to detect stains of amines, amino acids, proteins and other compounds.

Some other methods of spot detection are also used. For example, their radioactivity is measured if some of the separated components are radioactive, or special additions of radioactive isotopes of the elements included in 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 purpose, reference spots of “witnesses” are most often used. Sometimes spots are identified by the magnitude of the mobility coefficients Rf, comparing them with the values of Rf known for given conditions. However, such identification based on the R f value is often preliminary.

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

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

Using the TLC method, it is possible not only to discover, but also to quantify the content of components in mixtures. To do this, either analyze the spots themselves on the chromatogram, or extract the separated components 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 spot area and the content of a given substance (for example, the presence of a proportional or linear relationship), which is established by constructing a calibration graph by measuring the areas of “witness” spots - 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 techniques are used to measure color intensity.

When the separated components are extracted 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 a substance by TLC is 5-10%.

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

5. GAS CHROMATOGRAPHY

In gas chromatography (GC), an inert gas (nitrogen, helium, hydrogen), called a carrier gas, is used as a mobile phase. The sample is supplied in the form of vapor; the stationary phase is either a solid substance - a sorbent (gas adsorption chromatography) or a high-boiling liquid applied in a thin layer to a solid carrier (gas-liquid chromatography). Let's consider the option of gas-liquid chromatography (GLC). Kieselguhr (diatomite), a type of hydrated silica gel, is used as a carrier; 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 microbeads, Teflon and other materials are used.

5.1 Gaso- adsorption chromatography

The peculiarity 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) of an electrostatic nature. The formation of a hydrogen bond is possible, 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 C s is proportional to the concentration of this substance in the gas phase C m:

C s = ks 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 different speeds of their movement. Therefore, in GAS, the choice of an adsorbent is extremely important, the area and nature of the surface of which determine selectivity (separation) at a given temperature.

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

Active carbons, silica gels, porous glass, and aluminum oxide are mainly used as adsorbents for GAS. The heterogeneity 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, mixtures of highly polar substances can be analyzed 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 gas adsorption chromatography method is most widely used for the analysis of 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, clayey ones are successfully used to separate O 2, N 2, CO, CH 4, CO 2. 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 pores of which have approximately the same size (0.4-1.5 nm) - hydrogen isotopes can be separated. Sorbents called porapaks are used for the separation of metal hydrides (Ge, As, Sn, Sb). The GAS 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, for example, in solvents.

5.2 Gaso- liquid chromatography

In analytical practice, the gas-liquid chromatography (GLC) method is more often used. This is due to the extreme diversity of liquid stationary phases, which facilitates the selection of a selective phase for a given analysis, the linearity of the distribution isotherm over a wider range of concentrations, which allows working with large samples, and the ease of obtaining reproducible column performance.

The mechanism of distribution of components 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, when dissolving, the vapor pressure of a substance above the solution is 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 Р° I (2)

Since the concentration of the i-th 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 higher P 0 i), the weaker it is retained in the chromatographic column.

If the boiling points of the 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 column selectivity, it is important to select the correct stationary liquid phase. This phase must be a good solvent for the components of the mixture (if the solubility is low, the components leave the column very quickly), non-volatile (so that it does not evaporate at the operating temperature of the column), chemically inert, must have a low viscosity (otherwise the diffusion process slows down) and when applied to the carrier forms a uniform film firmly bonded to it. The separating ability of the stationary phase for the components of a given 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 being separated, for example, class, structure, it is possible to quickly select a selective liquid phase suitable for separating a given mixture. 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 similar polarity, the order of elution usually correlates with boiling points, and if the temperature difference is large enough, complete separation is possible. To separate close-boiling substances of different polarity, a stationary phase is used, which selectively retains one or more components due to dipole-dipole interaction. With increasing polarity of the liquid phase, the retention time of polar compounds increases.

To uniformly apply the liquid phase to 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, and the liquid phase remains on the carrier. The dry carrier with the stationary liquid phase deposited in this way is filled into the column, trying to avoid the formation of voids. To ensure uniform packing, a gas stream is passed through the column and at the same time the column is tapped to compact the packing. The column is then heated to a temperature 50°C above that at which it is intended to be used before being connected to the detector. 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-gas interface phases was minimal. The lowest adsorption is observed on carriers made of silanized chromosorb, glass granules and fluoropak (fluorocarbon polymer). In addition, solid carriers should not react to increased temperature and should be easily wetted by the liquid phase. In gas chromatography of chelates, silanized white diatomite carriers - diatomite silica, or kieselguhr - are most often used as a solid carrier. Diatomite is microamorphous silicon dioxide containing water. Such carriers include Chromosorb W, Gasochrome Q, Chroton N, etc. In addition, glass beads and Teflon are used.

Chemically bound phases. Often modified carriers are used, covalently bound to the liquid phase. In this case, the stationary liquid phase is more firmly retained on the surface even at the highest temperatures of the column. For example, a diatomite support is treated with chlorosilane with a long-chain substituent having a certain polarity. A chemically bonded stationary phase is more effective.

6. DISTRIBUTION CHROMATOGRAPHY. PAPER CHROMATOGRAPHY (CHROMATOGRAPHY ON PAPER)

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

Partition chromatography includes paper chromatography (or paper chromatography) in its usual variants. In this method, instead of plates with a thin layer of sorbent used for 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 reverse-phase paper chromatography.

In option normal phase paper chromatography liquid NF is water sorbed 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 is very different in structure and physical state from ordinary liquid water. The components of the separated mixtures dissolve in it.

The role of the PF moving along 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 the PF does not dissolve the water sorbed on the fibers of hydrophilic chromatography paper.

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

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

In option reverse phase In paper chromatography, liquid NP is an organic solvent, while the role of liquid PF is water, aqueous or alcohol solutions, or mixtures of acids and alcohols. The process is carried out using hydrophobic chromatography 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 does not hold onto such paper and does not wet it.

The paper chromatography technique is generally the same as the TLC method. Typically, a pot of the analyzed solution containing a mixture of the substances to be separated is placed on a strip of chromatographic paper on the starting line. After the solvent has evaporated, the paper below the starting 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 marked on the paper. After this, 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.

To quantitatively determine the content of one or another component of a mixture, various methods are used:

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 first constructed);

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

3) take into account the relationship between the intensity of the stain color and the content of the determined component in it, which imparts color to the stain.

In some cases, the substances contained in the stains 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 and simple to perform, but in general it is inferior to the more modern TLC method, which uses a thin layer of sorbent.

7. SEDIMENTARY CHROMATOGRAPHY

The method of sedimentary chromatography is used primarily for the separation and identification of inorganic ions included 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 precipitating reagent included in the NF. Separation is carried out due to the unequal solubility of the resulting compounds, which are transferred by the mobile phase at different speeds: 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 stopcock at the bottom) filled with a sorbent. The latter consists of a carrier - 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 separated anions is passed through a chromatographic column. These anions interact with silver cations Ag +, forming poorly 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 following order:

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

where the values of the solubility products at room temperature are given in parentheses. Therefore, first a yellow precipitate of silver iodide will form, and as the least soluble, a yellow (upper) zone will be observed on the chromatogram. A zone of cream-colored silver bromide precipitate (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 fine metallic silver.

The result is a primary sediment chromatogram.

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

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

Scheme of separation of Cl-, Br- and I- ions in a chromatographic column using sediment chromatography.

7.1 Classification of sediment chromatography methods according to experimental technique

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

Mixtures of inert carriers with a precipitant are used as sorbents in sedimentary chromatography; sorbents that retain precipitants in the form of ions (ion exchange resins) or in the form of molecules (activated carbon); paper soaked in a precipitant solution.

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

Reagents used as precipitants are those that form poorly soluble precipitates with chromatographed ions, for example, sodium iodide NaI, sodium sulfide Na 2 S, silver sulfate Ag 2 SO 4, potassium ferrocyanide K 4, oxyquinoline, pyridine, etc.

Typically, when using the method of column sediment 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 is characterized by good reproducibility of results.

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

7.2 Sedimentary paper chromatography

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

The analyzed aqueous solution is applied to the center of a sheet of paper impregnated with a solution of a precipitant - potassium ferrocyanide K4. Copper ions Cu 2+ and iron Fe 2+ interact with ferrocyanide ions to form poorly 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 forms first, forming a central brown zone. A blue precipitate of iron(III) ferrocyanide then forms, giving the blue zone. The aluminum ions move to the periphery, giving a colorless zone as they do not form colored aluminum ferrocyanide.

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

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

A secondary chromatogram is then 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 the more soluble precipitate of iron ferrocyanide moves faster than the zone of the less soluble precipitate of copper ferrocyanide. At this stage, due to the difference in the speeds of movement of the zones, they are more clearly separated.

To discover aluminum ions forming a colorless peripheral zone, a secondary chromatogram is developed - 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 through the reversible interaction of ionizing 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, exchanges with the counterion. Substances with different affinities for fixed charges are separated into anion exchangers or cation exchangers. Anion exchangers have positively charged groups on the surface and absorb anions from the mobile phase. Cation exchangers accordingly contain groups with a negative charge that interact with cations.

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

During chromatographic separation, analyte ions compete with ions contained in the eluent, tending 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 some way. It is possible to analyze even neutral sugar molecules in the form of their complexes with borate ions.

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

Ion exchange chromatography is widely used in medicine, biology, biochemistry, for environmental monitoring, in the analysis of the content of drugs and their metabolites in the blood and urine, pesticides in food raw materials, as well as for the separation of inorganic compounds, including radioisotopes, lanthanides, actinides, etc. The analysis of biopolymers (proteins, nucleic acids, etc.), which usually took hours or days, is carried out using ion exchange chromatography 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 incorrect interpretation of the final result. It is interesting to use this method to monitor changes occurring in biological fluids. The use of porous weak anion exchangers based on silica gel allowed the separation of peptides. The ion exchange mechanism can be represented in the form of 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 R + ion centers of the ion exchanger, and in the second case, sample X + cations compete with the mobile phase Y + ions for the R - ionic centers.

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

The separation of specific substances depends primarily on the choice of the most suitable sorbent and mobile phase. Ion exchange resins and silica gels with grafted ionogenic groups are used as stationary phases in ion exchange chromatography.

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 significantly 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 8-12% of the latter is added. The higher the divinylbenzene content, the greater the rigidity and strength of the polymer, the higher the capacity and, as a rule, selectivity, and the less swelling.

A brief history of the development of liquid chromatography. History of the discovery of chromatography Solid stationary phase chromatography

2. History of the development of liquid chromatography.

2.1 Development history:

2.3 Principles of ion separation in sorption processes

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