Development of chromatography. Chromatography as a research method

The chromatography is a Russian scientist, botany and physicochemist Mikhail Semenovich Color.

The discovery of chromatography refers to the time of completion of the work on the master's thesis in St. Petersburg (1900 - 1902) and the first period of work in Warsaw (1902 - 1903). Exploring the pigments of plants, the color passed the solution of the mixture very little differing in color of pigments through the tube filled with an adsorbent - powdered calcium carbonate, and was then washed then the adsorbent with a clean solvent. Separate components of the mixture were divided and formed colored stripes. According to modern terminology, the color discovered the developing variant of chromatography (developing liquid adsorption chromatography). The main results of research on the development of the chromatography created by him. Color outlined in the book "Chromophylls in the plant and animal world" (1910), which is his doctoral dissertation. Chromatography Gas sedimentary ion exchange

The color has widely used the chromatographic method not only for the separation of the mixture and establishing it multicomponency, but also for quantitative analysis, for this purpose it broke the glass column and cut the adsorbent column to the layers. The color has developed equipment for liquid chromatography, for the first time carried out chromatographic processes under reduced pressure (pumping) and at some overpressure, developed recommendations for the preparation of efficient columns. In addition, he introduced many basic concepts and terms of a new method, such as "chromatography", "manifestation", "displacement", "chromatogram", etc.

Chromatography was first used very rarely, its hidden period lasted about 20 years, during which only a very small number of messages about various applications of the method appeared. And only in 1931, R. Kunu (Germany) A. Winterstein (Germany) and E. Ledterre (France), who worked in a chemical laboratory (led by R. Kun), the Institute of Emperor Wilhelm on Medical Research in Heidelberg, managed to allocate this method a - and B-carotene from raw carotene and thereby demonstrate the value of the color opening.

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

The next important step was the discovery of A. Martin and R. Sing (England) of the variant of liquid distribution chromatography on the example of 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 can be used as a mobile phase, but also gas. A few years later, these scientists were proposed to carry out the separation of derivatives of amino acids on moistened with water with butanol as the mobile phase. They also implemented the first two-dimensional separation system. For the opening of the distribution version of chromatography, Martin and Sing received the Nobel Prize in Chemistry. (1952). Further, Martin and A. James carried out a variant of gas distribution chromatography, separating the mixture on a mixed sorbent from DS-550 silicone and stearic acid (1952 - 1953). From this time, the most intensive development was obtained by gas chromatography method.

One of the variants of gas chromatography is chromantography, in which to improve the separation of the gas mixture simultaneously with the movement of the moving phase - gas, affect the sorbent and the separated mixture with a moving temperature field having a certain gradient in length (A.A. Zhukhovitsky and Sotr., 1951) .

A noticeable contribution to the development of the chromatographic method was introduced by the city of Schwab (Germany), which was the founder of ion exchange chromatography (1937 - 1940). She received further development in the works of Soviet scientists E.N. Gapona and TB Gapona, which conducted a chromatographic separation of a mixture of ions in the solution (together with F.M. Shemyakin, 1947), and also carried out the idea of \u200b\u200bthe possibility of chromatographic separation of a mixture of substances based on the difference in solubility solubility (sedimentary chromatography, 1948).

The modern stage in the development of ion exchange chromatography began in 1975 after the work of the city of Smallla, T. Stevens and W. Bauman (USA), in which they proposed a new analytical method called ion chromatography (an option for highly efficient ion exchange chromatography with condacometric detection).

The creation of the company "Perkin-Elmer" M. Golley (USA) of the capillary variant of chromatography (1956), in which the sorbent is applied to the inner walls of the capillary tube, which allows to analyze microcolism of multicomponent mixtures.

In the late 60s. Increased interest in liquid chromatography. There was highly efficient liquid chromatography (HPLC). This was facilitated by the creation of highly sensitive detectors, new selective polymer sorbents, new equipment, allowing to operate at high pressures. Currently, HPLC occupies a leading position among other chromatography methods and implemented in various versions.

Chromatography is a method of separation and determination of substances based on the distribution of components between the two phases-lining and fixed. The fixed (stationary) phase serves a solid porous substance (often called a sorbent) or a liquid film applied to a solid. The mobile phase is a liquid or gas flowing through a fixed phase, sometimes under pressure. The components of the mixture of the mixture (core) together with the movable phase move along the stationary phase. It is usually placed in a glass or metal tube, called column. Depending on the interaction force with the sorbent surface (due to adsorption or by any other mechanism), the components will move along the column at different speeds. Some components will remain in the upper layer of the sorbent, others, to a lesser extent interacting with the sorbent, will be at the bottom of the column, and some will leave the column together with the movable phase (such components are unpaid, and their holding time determines the "dead time" speakers) .

Thus, the rapid separation of complex mixtures of components occurs.

History Opening:

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 Plant Mikhail Semenovich was made by the report "On a new category of adsorption phenomena and on the use of them to biochemical analysis."

Unfortunately, M.S. Tsvet, being on the formation of Botany, did not appreciate the chemical analytical aspect of his opening as a properly, and little published his work in chemical magazines. Subsequently, it was chemists that appreciated the real scale of the proposed M.S. The color of the chromatographic method, which has become the most common method of analytical chemistry.

The following details of chromatographic methods should be emphasized:

1. The separation is dynamic, and the acts of sorption-desorption of shared components are repeated repeated. This causes significantly greater efficiency of chromatographic

separation compared to static sorption methods and

extraction.

2. During separation, various types of interaction of the sorbates and stationary phases are used: from purely physical to hemosorption.

This causes the possibility of selective separation of a wide range

3. On the separated substances you can apply various additional fields (gravitational, electric, magnetic, etc.), which, changing the conditions of separation, expand the possibilities of chromatography.

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

5. Chromatography allows you to solve both analytical tasks (separation, identification, definition) and preparative (cleaning, selection, concentration). Solving these tasks can be combined by performing them in "Online" mode.

Numerous methods are classified by the aggregate state of the phases, the separation mechanism and the technique of separation.

Chromatographic methods differ in the method of carrying out

the separation process on the front, crucial and eluent.

Ionic chromatography

Ion chromatography is highly efficient liquid chromatography for separating cations and anions on ion exchangers

low capacity. Widespread ion chromatography

due to the number of her advantages:

- the ability to determine a large number of inorganic and

organic ions, as well as simultaneously define cations and

- High sensitivity of determination (up to 1 ng / ml without

preliminary concentration;

- high selectivity and expressivity;

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

- a wide range of defined concentrations (from1 ng / ml to

- the possibility of using various detectors and their combinations, which makes it possible to provide selectivity and small time definition;

- the ability to fully automate the definition;

- In many cases, the complete absence of preliminary sample preparation.

At the same time, as any analytical method, ion chromatography is not devoid of deficiencies, which can be attributed to:

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

development of the method;

- lower efficiency of separation compared to HPLC 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 at the beginning of the XIX century. With observations about the effect of soils on the chemical composition of salt solutions in contact with it. In the late 1940s, Tompson noted that the soil absorbs ammonia from the entered organic fertilizers, the relevant experiments were carried out by the specialist of their York D. Spence. The first results of the experiments D. Spence were published by G. Thompson in 1850. The article notes that "the first discovery of the high-quality properties of the soil can hardly fail as useful for agriculture" and its last work was published e in 1852 and 1855.

2.3 Principles of separation of ions in sorption processes

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

Ion chromatography is a variant of column ion exchange chromatography.

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

Modern (2005) wording: "ion chromatography includes all high-performance liquid chromatographic (HPLC) separation of ions in the columns, combined with direct detection in the flow detector and the quantitative processing of the analytical signals obtained." This definition characterizes ion chromatography without reference to the separation mechanism and the detection method and thereby separates it from the classical ion exchange.

The following principles of separation apply in ion chromatography:

Ion exchange.

Education of ion pairs.

Exclusion ions.

Ion exchange

Ion exchange is a reversible heterogeneous response of equivalent ionization ions in the ionate phase (counterions), eluent's unions. Anti -iaons are held by the functional groups of ionets at the expense of electrostatic forces. As a rule, in cationic chromatography these groups are groups of sulfonic acids; In the case of anionic chromatography - quaternary ammonium grounds. In fig. 1 shows the scheme of the process of exchange of cations and anions. The ions of the substance determined are marked as a, eluent ions competing with them for exchange centers - E.

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

Education of ion par

To implement this separation mechanism, ion-paired reagents are used, which are added to the eluent solution. Such reagents are anionic or cationic surfactants, for example, alkyl sulphonic acids or tetraalklammonium salts.

Together with the oppositely charged ion-defined ions, the ions of this ion-pair reagent form an unchargeable ionic pair, which can be held on the fixed phase due to intermolecular interactions. The separation is carried out due to the difference in the constants of the formation of ionic pairs and the degrees of their adsorption on the sorbent matrix. In fig. 2 shows a static ion exchange model in ion-pair chromatography after adsorption of the reagent on the fixed phase. This separation principle is used both for anions and cations.

Fig. 2.. Ion exchange model in ion-steam chromatography.

Ionic exclusion

IonEecClusion chromatography (IEX). Basically, it is used to separate weak acids or bases. The greatest value of IEX has to determine carboxylic and amino acids, phenols, carbohydrates.

In fig. 3 shows the principle of separation using IEX on the example of R-COOH acids.

Fig. 3. R-COOH carboxylic acid separation scheme using ionoecklusion chromatography.

In ionoeckskoluzion chromatography as a fixed phase, a fully sulfted cation-exchanger, containing ion-water (counterions), is often used. In an aqueous solution of eluent, sulfonic acid groups of ionis are hydrated. The hydrate shell is limited to an imaginary negatively charged membrane (Donnan membrane). The membrane is permeable only for non-expocted molecules (for example, water).

Organic carboxylic acids can be separated if strong mineral acids are used as eluent. Due to the low values \u200b\u200bof the acidity constants, carboxylic acids are present in such solutions in the unfair form. These forms can pass through the DonNan membrane and adsorbed on the fixed phase.

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1. History of the opening and development of chromatography

2. Basic provisions

3. Classification of chromatographic analysis methods

4. Adsorption chromatography. Thin layer chromatography

4.1 Technique of experiment in thin-layer chromatography

5. Gas chromatography

5.1 Gas-adsorption chromatography

5.2 Gas-liquid chromatography

6. Distribution chromatography. Paper chromatography

7. Sediment chromatography

7.1 Classification of methods of sedimentary chromatography on the technique of experiment

7.2 Sediment chromatography on paper

8. ion exchange chromatography

Conclusion

Bibliography

1. HISTORYChromatography discoveries and development

The chromatography is a Russian scientist, botany and physicochemist Mikhail Semenovich Color.

2. Basic provisions

Chromatography is a method of separation and determination of substances based on the distribution of components between the two phases - movable and fixed. The fixed (stationary) phase serves a solid porous substance (often called a sorbent) or a liquid film applied to a solid. The mobile phase is a liquid or gas flowing through a fixed phase, sometimes under pressure. The components of the mixture of the mixture (core) together with the movable phase move along the stationary phase. It is usually placed in a glass or metal tube, called column. Depending on the interaction force with the sorbent surface (due to adsorption or by any other mechanism), the components will move along the column at different speeds. Some components will remain in the upper layer of the sorbent, others, to a lesser extent interacting with the sorbent, will be at the bottom of the column, and some will leave the column together with the movable phase (such components are unpaid, and their holding time determines the "dead time" speakers) . Thus, the rapid separation of complex mixtures of components occurs. The following details of chromatographic methods should be emphasized:

1. The separation is dynamic, and the acts of sorption-desorption of shared components are repeated repeated. This causes significantly greater efficiency of chromatographic separation compared to static methods of sorption and extraction.

2. During separation, various types of interaction of the sorbates and stationary phases are used: from purely physical to hemosorption. This causes the possibility of selective separation of a wide circle of substances.

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

5. Chromatography allows you to solve both analytical tasks (separation, identification, definition) and preparative (cleaning, selection, concentration). Solution of these tasks can be combined by performing them in "On Line" mode.

6. Numerous methods are classified by the aggregate state of the phases, the separation mechanism and the separation technique. Chromatographic methods differ in the method of carrying out the separation process to the front, crucible and eluent.

3. Classification of chromatographic analysis methods

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

* Differences in the aggregate state of the phases of the chromatographic system used;

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

* Experimental differences in the methods for the process of chromatographic separation.

In Tables 1? 3 shows the basic variants of the classification of known chromatographic methods.

Since the nature of the interactions of the separated compounds with 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 fixed phase (solid or liquid) and rolling solvent systems. The scope of the basic variants of chromatography depending on the molecular weight of the compounds under study is given in Table. four.

4. Adsorption chromatography. Thin layer chromatography

One of the most common adsorption chromatography techniques is thin-layer chromatography (TLC) - a type of plane chromatography, at which the adsorbent is used as a thin layer on the plate.

The principle and the basic concepts of the TLC method. On a clean flat surface (plate of glass, metal, plastics) in one way or another, a thin layer of the sorbent is applied, which is most often fixed on the surface of the plate. The dimensions of the plate may be different (length and width - from 5 to 50 cm, although it is not necessary). On the surface of the plate, carefully, not to damage the sorbent layer, outlined (for example, a pencil) of the start line (at a distance of 2-3 cm from the lower edge of the plate) and the line of the solvent finish.

Scheme of separation of components A and in the TLC method

On the start line, the plate is applied (by a micro-shirt, capillary) sample - a small amount of fluid containing a mixture of separable substances, for example, two substances A and B in a suitable solvent. It is possible to evaporate the solvent, after which the plate is immersed in the chromatographic chamber in the liquid phase of the PF, which is a specially selected solvent or a solvent mixture for a given case. Under the action of the capillary forces of PF spontaneously moves along the NF from the starting line to the line of the solvent front, caring with them components A and in samples that move at different speeds. In this case, the affinity of the component A to the NF is less affinity for the same phase of the component B, so the component A is moving faster than the component B. After reaching the moving phase (solvent) of the solvent line, chromatography is interrupted, the plate is removed from the chromatographic chamber, dried in air and determine the position of stains of substances A and in on the surface of the plate. Spots (zones) usually have an oval or round shape. In the case under consideration, the stain component A moved from the start line to the distance l. A. , spot component in - for distance l. IN, and the solvent passed through the distance L..

Sometimes simultaneously with the application of samples of separated substances on the start line, small amounts of the standard of standard are applied, as well as witness substances (those that are allegedly contained in the analyzed sample).

To characterize the shared components, the RF mobility (or RF factor) is introduced in the system:

R. f.\u003d V. 1 / V. E.\u003d (L. 1 / T) / (L / T) \u003d L 1 / L. ,

where V. 1 = l. 1 / t. and V. E.= L./ t. - accordingly the speed of movement i.- th component and solvent E; l. 1 andL. - path passed i.- the 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 squeeze from the start line to the center of the spot of the corresponding component.

Usually, the coefficient of mobility lies in the redistribution R. f. =0 - 1. The optimal value is 0.3-0.7, chromatography conditions are selected so that the value R f differs from zero and units.

The coefficient of mobility is an important characteristic of the Sorbent Sorbate system. For reproducible and strictly constant chromatography conditions R. f. = const.

RF mobility coefficient depends on a number of factors: the nature and quality of the solvent, its purity; nature and quality of the sorbent (thin layer), uniformity of its grains, layer thickness; the activity of the sorbent (content in it moisture); techniques of the experiment (mass samples, the length of the moon of the solvent); Experimentator skill, etc. The constancy of reproducing all these parameters in practice is sometimes difficult. To level the impact of the conditions of the process, the relative mobility factor is introduced RS..

RS \u003d L / L art\u003d R. f./ R. f ( art ) ,

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

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

As a standard, such a substance is often chosen for which in these conditions R f? 0.5. In the chemical nature, the standard is selected close to the separated substances. Using the standard, the value of RS usually lies within RS \u003d 0.1--10, the optimal limits are about 0.5--2.

For more reliable identification of shared components use "Witnesses" - reference substances, the presence of which is assumed in the analyzed sample. If R f \u003d r f (svid), wherein r f and r f (svid) - respectively, the coefficients of the mobility of this component and a witness, then can most likely assume that the test substance is present in the chromatographic mixture.

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

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

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

The greater the value R (A / B), the clearer the stains of the components A and B are separated on the chromatogram.

To estimate the selectivity of separation of two substances A and B use the separation coefficient but:

a \u003d.l. B. / l. A.

If a a \u003d 1,that components A and B are not divided.

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

4.1 Experimental technique in thin layer chromatography:

but) Sample applying. The analyzed liquid sample is applied to the line of the start using the capillary, micro-shit, micropipettes, carefully touching the sorbent layer (the diameter of the stain on the start line is usually from one to a few millimeters). If a few samples are applied to the start line, then the distance between the stains of the samples on the start line should not be less than 2 cm. If possible, concentrated solutions are used. Spots are dried in air, after which chromatography is carried out.

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

Depending on the direction of movement, the PF is distinguished ascending, descending and horizontal chromatography.

In the form of ascending chromatography, only plates with a fixed sorbent layer are used. PF is poured onto the bottom of the chamber (as the latter, a glass chemical glass of suitable size can be used with a glass lid), the chromatographic plate is placed vertically or obliquely into the chamber so that the PF layer on the bottom of the camera wet 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 gravity) relatively slowly.

In the descending chromatography variant, only plates with a fixed layer also apply. PF is fed from above and moves down along the plate sorbent layer. The power of gravity accelerates the movement of the PF. This embodiment is implemented when analyzing mixtures containing components, slowly moving with PF.

In the embodiment of horizontal chromatography, the plate is placed horizontally. You can use rectangular or round plates. When applying round plates (circular variant of horizontal chromatography), the starting line is denoted as a circle of a suitable radius (~ 1.5-2 cm), which samples are applied. In the center of the round plate, the hole is cut into which the 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 - the desiccator or in Petri Cooker. With a circular version, you can simultaneously analyze up to several dozen samples.

In TLC methods, one-dimensional, two-dimensional, multiple (re-), step chromatography is used.

In one-time chromatography, the analysis is carried out without changing the direction of movement of the PF. This method is most common.

Two-dimensional chromatography is usually used to analyze complex mixtures (proteins, amino acids, etc.), the mixture is first carried out using the first PF 1. The chromatogram is obtained spots not individual substances, and mixtures of several inseparable components. Then, through these stains, a new start line is carried out, the plate is turned on 90 ° and is chromatographed again, but already with the second PF 2, striving to finally split the stains with mixtures on the stains 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.

The scheme illustrating the principle of two-dimensional chromatography:

a - chromatogram obtained from PF1;

b - chromatogram obtained from PF2

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

In the case of step chromatography, the process is carried out with the same plate sequentially using each time a new PF, until a clear separation of stains is reached.

in) Decoding chromatograms. If the stains on the chromatogram are painted, after drying the plates, determine the distance from the start line to the center of each spot and calculate the coefficients of mobility. If the composition of the analyzed sample consists of colorless substances, giving unpainted, i.e. Visually not identified spots on chromatogram, you need to spend detection these spots for which chromatogram show.

The most common detection methods are described below.

Irradiation with ultraviolet light.It is used to detect fluorescent compounds (spots are glowing during the irradiation of the UV light plate) or petrolevascular substances, but using a sorbent with a fluorescent indicator (the sorbent is lit, the spots will not be lit). Thus detected, for example, alkaloids, antibiotics, vitamins and other medicinal substances.

Heat treatment.The plate dried after chromatography is gently heated (up to ~ 200 ° C), avoiding the darkening of the sorbent itself (for example, when the thin layer of the sorbent contains starch). At the same time, the stains are usually manifested in the form of brown zones (due to partial thermolysis of organic components).

Chemical processing.Often chromatograms exhibit, processing them with reagents that form colored compounds with the shared components of the mixtures. For these purposes, various reagents are used: a pair of iodine, ammonia, bromine, sulfur dioxide, hydrogen sulfide, specially prepared solutions that are treated with plates. Apply both universal and selective reagents (the concept of "universal" is sufficiently conditionally).

Universal reagentamimogoot serve, for example, concentrated sulfuric acid (when heated, there is a darkening of organic compounds), a sour aqueous solution of potassium permanganate (zones are observed in the form of brown spots on a purple sorbent background), a solution of phosphorous-molybdenum acid during heating (blue stains appear on yellow background), etc.

As selective use, for example, the reagent of Dragendorf; Zimmerman reagent; aqueous ammonium solution of copper sulfate (10% of Cuso 4, 2% in ammonia); A mixture of ninhydrin C 9 H 4 O 3 H 2 O with ethanol and acetic acid.

The dragandorf reagent is a solution of the main bondo 3 bonding nitrate, KJ potassium iodide and acetic acid in water. Used to determine the amines, alkaloids, steroids.

The zimmerman reagent is prepared by treating the alkali solution KOH 2% ethanol solution of dinitrobenzene, followed by the heating of the mixture at ~ 70-100 ° C. Apply to detect steroids.

With the help of ningidrin, the stains of amines, amino acids, proteins and other connections are detected.

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

After detecting spots on the chromatogram, they are identified, i.e. It is determined by which the compound corresponds to this or that spot. For this purpose, the reference spots of "Witnesses" are most often used. Sometimes stains are identified by the magnitude of the mobility coefficients R F, comparing them with the values \u200b\u200bof R f known for these conditions. However, such identification of R F is often preliminary.

The color of fluorescent spots is also used for identification purposes, since various compounds are fluorescent by radiation of various wavelengths (different colors).

With chemical detection of spots, selective reagents give painted stains with compounds of certain nature, which is also used for identification purposes.

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

When analyzing, the stains imply the existence of a certain connection between the spots area and the content of this substance (for example, the presence of a proportional or linear dependence), which is established by the method of constructing a graduation graph, measuring the spots of the "Witnesses" spots with a known content of the component being analyzed.

Sometimes they compare the intensity of the color of the stains, believing that the intensity of the color stains is proportional to the number of this painted component. For measuring intensity, different techniques are used.

When removing 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 of the quantitative determination of the substance by the TLC is 5-10%.

TLC is a pharmacopoeial method and is widely used for analyzing and controlling the quality of various drugs.

5. Gas chromatography

In gas chromatography (GC), inert gas (nitrogen, helium, hydrogen), called gas carrier, is used as the mobile phase. The sample is fed in the form of a vapor, a fixed phase serves or a solid - a sorbent (gas-adsorption chromatography) or a high-boiling liquid applied by a thin layer on a solid carrier (gas-liquid chromatography). Consider the option of gas-liquid chromatography (GLC). Kizelgur (diatomitis) is used as a carrier - a type of hydrated silica gel is used, often treated with reagents that translate the Si-OH groups in the Si-O-Si group (CH 3) 3, which increases the inertness of the carrier with respect to solvents. These are, for example, the carriers of the "chromosorb W" and "gasohromq". In addition, glass microsists, teflon and other materials are used.

5.1 Gaza- adsorption chromatography

The peculiarity of the method of gas pump chromatography (GA) is that as a fixed phase, adsorbents with a high specific surface (10--1000 m 2 g -1) are used, and the distribution of substances between fixed and moving phases is determined by the adsorption process. Adsorption of molecules from the gas phase, i.e. Concentrated on the surface of the separation of solid and gaseous phases, occurs due to intermolecular interactions (dispersion, orientational, induction) having electrostatic nature. It is possible, the formation of hydrogen bonds, and the contribution of this type of interaction into the retained volumes is significantly reduced with increasing temperature.

For analytical practice, it is important that at a constant temperature, the amount of adsorbed substance on the surface with S was proportional to the concentration of this substance in the gas phase with M:

C. s. = kC. m. (1)

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

The heat of adsorption decreases with increasing temperature DH / T.from which detection depends and, accordingly t. R. . This is used in the practice of analysis. If compounds are separated highly differing in volatility at a constant temperature, then low-boiling substances are eluted quickly, high-boiling has a greater time, their peaks on the chromatogram will be lower and wider, the analysis takes a lot of time. If, in the process of chromatography, increase the temperature of the column with a constant speed (temperature programming), then peaks close by the width on the chromatogram will be placed evenly.

As adsorbents for the gi, active coals, silica gels, porous glass, aluminum oxide are used mainly. The inhomogeneity of the surface of active adsorbents is due to the main disadvantages of the methods of the GAH and the impossibility of determining highly adsorbed polar molecules. However, on geometrically and chemically homogeneous macroporous adsorbents, it is possible to analyze mixtures of strong-polar substances. In recent years, adsorbents in recently produce adsorbents with a more or less homogeneous surface, such as porous polymers, macroporous silica gels (satlohrom, struck, spray), porous glasses, zeolites.

The most widely large method of gas pump chromatography is used to analyze mixtures of gases and low-boiling hydrocarbons that do not contain active functional groups. Adsorption isotherms of such molecules are close to linear. For example, for separation of 2, N 2, CO, CH 4, CO 2 is successfully used by clay. The column temperature is programmed to reduce the analysis time by reducing T R high-boiling gases. On molecular sints - highly pharmaceutical natural or synthetic crystalline materials, all the pores of which have about the same dimensions (0.4--1.5 nm), - can be divided by hydrogen isotopes. Sorbents, called hunters, are used to separate metal hydrides (GE, AS, SN, SB). The GA method on columns with porous polymeric sorbents or carbon molecular sizes is the fastest and most convenient way to determine water in inorganic and organic materials, for example in solvents.

5.2 Gaza- 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 fixed phases, which makes it easier to choose a selective phase for this analysis, with a linearity of distribution isotherms in a wider concentration area, which allows you to work with large samples, and easily obtaining reproducible columns.

The mechanism of distribution of components between the carrier and a fixed liquid phase is based on dissolving them in the liquid phase. Selectivity depends on two factors: steam elasticity of the determined substance and its activity coefficient in the liquid phase. According to the law of Raoul, when dissolving the elasticity of a steam of a substance over a solution p. i. directly proportional to its coefficient of activity G molar fraction N. i. in solution and pressure of the vapor of pure substance R ° i. At this temperature:

p i \u003d n i p ° I (2)

Since the concentration of the i-th component in the equilibrium steam phase is determined by its partial pressure, you can accept that

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

and selectivity coefficient:

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

If the boiling point of substances are the same, then the differences in interaction with a fixed liquid phase are used to separate them: the stronger the interaction, the less the activity coefficient and more retention.

Fixed liquid phases . To ensure the selectivity of the column, it is important to correctly select the fixed liquid phase. This phase should be a good solvent for the mixture components (if the solubility is small, the components extend from the column very quickly), non-volatile (so as not to evaporate at the operating temperature of the column), chemically inert, must have a slight viscosity (otherwise the diffusion process slows down) and when applied to The carrier to form a uniform film is firmly connected with it. The separation capacity of the fixed phase for the components of this sample must be maximum.

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

Knowing the properties of a fixed liquid phase and the nature of separable substances, such as a class, structure, it is possible to quickly choose a selective liquid phase to separate this mixture. It should be borne in mind that the holding time of the components will be acceptable for analysis, if the polarity of the stationary phase and the substance of the analyzed sample is close. For dissolved substances with a close polarity, the elution order usually correlates with boiling temperatures, and if the temperature difference is large enough, it is possible to complete separation. For the separation of close-boiling substances of different polarity, the stationary phase is used, selectively holding one or more components due to the dipole - dipole interaction. With an increase in the polarity of the liquid phase, the retention time of polar compounds increases.

To uniformly applying the liquid phase to the solid carrier, it is mixed with a 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. A dry carrier with a fixed liquid phase applied in this way is filled with a column, trying to avoid the formation of voids. For uniform packaging through the column, the gas stream is passed and simultaneously tapping on the column for packing seals. Then, before attaching to the detector, the column is heated to a temperature of 50 ° C above the one at which it is supposed to be used. In this case, there may be losses of the liquid phase, but the column is included in the stable operating mode.

Media of fixed liquid phases. Solid media for dispersion of a fixed liquid phase in the form of a homogeneous thin film must be mechanically durable with a moderate specific surface area (20m 2 / g), small and equal particle size, and also be enough inert to adsorption on the surface of the solid and gaseous phases It was minimal. The lowest adsorption is observed on carriers of silaneized chromosorba, glass granules and fluoropac (fluorocarbon polymer). In addition, solid carriers should not respond to an increase in temperature and should be easily made with a liquid phase. In the gas chromatography of chelates as a solid carrier, the silaneered white diatomitis carriers are most often used - diatomitic silica, or Kizelgour. Ditomitis is a micronomorphic, containing water, silicon dioxide. Such carriers include chromosorb W, gasohrom Q, chromaton N, et al. In addition, glass balls and teflon use.

Chemically related phases. Often use modified carriers, covalently associated with a liquid phase. In this case, the stationary liquid phase is more firmly held on the surface even at the highest column temperatures. For example, the diatomitic carrier is treated with a chlorosilane with a long chain substituent, which has a certain polarity. Chemically coupled fixed phase is more efficient.

6. Distribution chromatography. Paper chromatography (chromatography on paper)

Distribution chromatography is based on the use of differences in solubility of the distributed substance in two contacting unintegrating liquid phases. Both phases - PF and NF are liquid phases. When moving liquid PF along the liquid NF, chromatographic substances are continuously redistributed between both liquid phases.

Distribution chromatography refers paper chromatugraphic (or chromatography on paper) in its usual options. In this method, instead of plates with a thin layer of sorbent used at TLC, a special chromatographic paper is used, according to which, impregnating it, liquid PF is moved during chromatography from the start line to the solvent finish line.

Distinguish normal phase and faithful paper chromatography.

In the embodiment normal phase paper chromatography of liquid NF is water, sorbed in the form of a thin layer on the fibers and in the pores hydrophilic paper (up to 25% by weight). This bound water in its structure and physical state is very different from conventional liquid water. In it, the components of the shared mixtures are dissolved.

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

Chromatographic paper is manufactured by industry. It must meet a number of requirements: prepare from high-quality fibrous cotton varieties, to be homogeneous in density and thickness, in the direction of orientation of fibers, chemically clean and inert relative to the NF and the shared components.

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

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

Technique of paper chromatography in general terms is the same as in the TLC method. Usually, a kashpo of an analyzed solution containing a mixture of shared substances is applied to the lane of chromatographic paper on the start line of the start. After evaporation of the solvent, the paper below the start line is immersed in PF, placing paper vertically (hanging it). Close the camera with a lid and chromatography until the PF does not reach the line of the solvent front designated on paper. After that, the process is interrupted, the paper is dried in air and detecting spots and identifying the components of the mixture.

Paper chromatography is similar to the TLC method used both in qualitative and quantitative analysis.

To quantify the content of one or another mixture component, various methods are used:

1) proceed from the presence of a certain dependence (proportional, linear) between the amount of substance in the stain and spots area (often the calibration schedule is pre-built);

2) weigh the cut stain with the substance and the same pure paper on the area, and then in terms of the difference they find a mass of the substance determined;

3) take into account the relationship between the intensity of the stain color and the content in it in it, which gives the stain color.

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

Paper chromatography is a pharmacopoeia method, used to separate mixtures containing both inorganic and organic substances. The method is available, simple to fulfill, but in general it is inferior to a more modern TLC method, in which a thin layer of the sorbent is applied.

7. Sedimentary chromatography

The method of sediment chromatography is used mainly for the separation and identification of inorganic ions that make up the mixtures.

The essence of the method. Sediment chromatography is based on the use of chemical reactions of precipitation of shared components of a mixture with a reagent-precipient, which is part of the NF. The separation is carried out due to the unequal solubility of the formed compounds that are transferred to the movable phase at different speeds: less soluble substances are transferred to the PF slower than soluble.

You can illustrate the use of the method on the example of the separation of halide ions: CL-ion chloride, bromide-ion-ion-ion and iodide-ion-ion-ion-ion, simultaneously contained in the analyzed aqueous solution. To do this, use a chromatographic column (representing a glass tube with a crane at the bottom) filled with a sorbent. The latter consists of their carrier - aluminum oxide Al 2 O 3 or SiO 2 silicon, impregnated with a silver nitrate solution of AGNO 3 (the content of silver nitrate is about 10% by weight from the mass of the carrier sorbent).

An aqueous solution containing a mixture of shared anions is passed through the chromatographic column. These anions interact with AG + silver cations, forming so-soluble silver halide preciputes:

AG + + I -\u003e Agiv (Yellow)

AG + + BR -\u003e AgBRV (cream)

AG + + CL -\u003e 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 in brackets are the values \u200b\u200bof solubility products at room temperature. Therefore, at first, the yellow precipitate of silver iodide will be formed, as the least soluble on the chromatogram will be observed a yellow (top) zone. The axis of silver silver bromide (intermediate zone) is then formed. Lastly, a white precipitate of silver chloride is formed - the lower white zone is dark due to the photochemical decomposition of silver chloride with the release of fine metallic silver.

As a result, a primary sedimentary chromatogram is obtained.

For a more clear separation of zones, after obtaining the primary chromatogram, a clean solvent is passed through the column to obtain a secondary sedimentary chromatogram with a clear separation of precipitation zones.

In the example described, the precipitator was part of the NF, and a solution containing a mixture of shared ions was passed through the column. It is possible, on the contrary, to pass the solution of the precipitator through the column in the NF which are chromatographic ions. At the same time, however, mixed zones are formed.

The scheme of separation of CL-, BR- and I- ions in the chromatographic column by the method of sedimentary chromatography.

7.1 Classification of methods of sedimentary chromatography on the technique of experiment

Usually distinguished column sedimentary chromatography conducted in chromatographic columns and plane Sediment chromatography implemented on paper or in a thin layer of sorbent.

As sorbents in sedimentary chromatography, mixtures of inert media with an inspirator are used; sorbents holding precipitators in the form of ions (ion exchange resins) or in the form of molecules (activated carbon); Paper impregnated with a precipitant solution.

The carriers most often choose silica gel, starch, aluminum oxides, calcium, barium sulfate, ion exchange resins, etc. The carrier is used in fine-dispersed state with dimensions of about 0.02-0.10 mm.

As precipitants, such reagents that form low-soluble precipitates with chromatographic ions are used, for example, sodium iodide NAI, sulfide sodium Na 2 S, silver sulfate AG 2 SO 4, potassium ferrocyanide K 4, oxychinoline, pyridine, etc.

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

In the case of the formation of colorless areas of precipitation, the chromatogram is shown or passing through a column-developer-developer, which gives with precipitates painted reaction products, or immediately introducing a developer in the PF or in the NF.

7.2 Sediment chromatography on paper

Consider the essence of this method on the example of the analysis of aqueous solution containing a mixture of copper cations Cu 2+? Iron FE 3+ and aluminum Al 3+.

In the center of the paper sheet impregnated with a precipitant solution - potassium ferrocyanide K 4, the capillary is applied with an analyzed aqueous solution. CU 2+ and iron ions Fe 2+ interact with ferrocyanide ions with the formation of poorly soluble precipitation:

2CU 2+ + 4-\u003e Cu 2 (brown)

4Fe 3+ + 3 4-\u003e Fe4 (Blue)

Since the copper (II) ferrocyanide is less soluble than ferrocyanide of iron (III), then the precipitate of the copper (II) ferrocyanide is distinguished, forming a central brown zone. Then the blue precipitate of ferrocyanide iron (III), which gives the blue zone is formed. Aluminum ions are moved to the periphery, giving a colorless zone, as they do not form a painted aluminum ferrocyanide.

The division scheme Cu2 +, Fe3 + and Al3 + by the method of sedimentary chromatography.

In this way, the primary chromatogram is obtained, where the precipitation zones are partially overlapped.

Then the secondary chromatogram is obtained. For this, a suitable solvent (in the case under consideration is an ammonia aqueous solution) is applied by the capillary to the center of the primary chromatogram. The solvent spontaneously moves from the center of the paper to the periphery, carried away with itself and precipitates, which move at different speeds: the zone of more soluble sediment of ferrocyanide iron moves faster zone of a less soluble sediment of the copper ferrocyanide. At this stage, due to the difference in the speeds of displacement zones, their more clear separation occurs.

To open aluminum ions, forming a colorless peripheral zone, a secondary chromatogram exhibit - spray (from a spray) solution of alizhar - an organic reagent forming with aluminum ions Pink reaction products. Get an external rose ring.

8. Ion exchange chromatography

In ion exchange chromatography, the separation of the mixture components is achieved by reversible interaction of ionizing substances with ion sorbent groups. The preservation of the e-referral 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 having different affinity for a fixed charge are divided into anionics or on cation. Anionates have positively charged groups on the surface and sorbite from the mobile phase of anions. Cationias respectively contain groups with a negative charge, interacting with cations.

As a mobile phase, aqueous solutions of acid salts, bases and solvents of the type of liquid ammonia are used, i.e. Systems of solvents having a high meaning of dielectric constant and a large tendency to ionize compounds. Typically operate with buffer solutions, allowing adjusting the pH value.

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

Ion exchange chromatography is indispensable in the separation of you-juice substances that cannot be analyzed by the GLC without translation to derivatives. Such compounds include amino acids, peptides, sugar.

Ion-exchange chromatography is widely used in medicine, biology, biochemistry, for environmental control, when analyzing the content of drugs and their metabolites in the blood and urine, eradicates in food raw materials, as well as for the separation of inorganic compounds, including radioisotopes, lantanoids, actinoids, etc. . Analysis of biopolymers (proteins, nucleic acids, etc.), which usually spent hours or days, with the help of ion exchange chromatography are carried out in 20-40 minutes with a better separation. The use of ion exchange chromatography in biology made it possible to observe the samples directly in biosructures, reducing the possibility of regrouping or isomerization, which can lead to improper interpretation of the final result. It is interesting to use this method to control changes occurring with biological fluids. The use of porous weak anionic exchanges on a silica gel basis made it possible to divide peptides. The mechanism of ion exchange can be represented as the following equations:

for anion exchange x - + r + y - - y - + r + x -

for cationic exchange X + + R - Y + - Y + + R - X +

In the first case, the X-sample ion is competing with the ion of the movable phase Y - for the ion centers R + ion exchanger, and in the second to competition with the ions of the mobile phase y + for ion centers R are the cations of the sample X +.

Naturally, the sample ions that are weakly interacting with the ion exchanger, with this competition will be weakly kept on the column and are first washed away from it and, on the contrary, more strongly retained ions will elute from the column last. Usually, secondary interactions of non-ionic nature are arising due to adsorption or hydrogen bonds of the sample with the non-ionic part of the matrix or due to the limited solubility of the sample in the movable phase.

The separation of specific substances depends primarily on the choice of the most suitable sorbent and the mobile phase. As a fixed phase in ion exchange chromatography, ion exchange resins and silica gels with grafted ionic groups are used.

Polystyrene ion exchange resins for HPLC grain of 10 μm and less possess selectivity and stability, but the mesh structure of them, characterized by the distance between the mesh nodes of 1.5 nm, which is significantly less than the size of the pores used for silica gel adsorption chromatography (10 nm), slows the mass transfer and, therefore, , significantly reduces efficiency. Ion-exchange resins used in HPLC are mainly copolymers of styrene and divinyl benzene. Typically add 8-12% of the latter. The greater the di-vinylbenzene content, the greater the rigidity and strength of the polymer, above the capacity and, as a rule, selectivity and the smaller swelling.

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Many discoveries of the past century are obliged to Russian scientist Mikhail Color and its method of chromatographic analysis. A large number of outstanding researchers must him with their successes, and many and Nobel Prize!

"... Without Michael's work, we will have nothing to do with all the" pigments ", there would be nothing to do ..." - here is the opinion of one famous English scientist.

Mikhail Semenovich Color (1872-1919) - the son of Italian and the 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 Physics and Mathematics Faculty of Geneva University. Representing the dissertation "Study of the physiology of the cell. Materials for the knowledge of the movement of protoplasm, plasma membranes and chloroplasts" Color in October 1896 received a doctor of the doctor of natural sciences. In December of the same year, he comes to St. Petersburg.

Mikhail did not know that the scholarship of the University of Geneva is not recognized in Russia. Therefore, he had to work at the famous botany Andrei Sergeyevich Famincin, who also studied chlorophyll, can be said on bird rights. In St. Petersburg, the color got acquainted with other outstanding botany and plant physiologists: I.P. Borodin, M.S. Voronin, A.N. Beketovo. It was a brilliant society of original rich in ideas of thinkers and skillful experimenters. Color continued its research of chloroplasts, preparing at the same time to new master's exams and to defending the thesis. He passed the exam in 1899, and he defended his master's thesis at the University of Kazan on September 23, 1901.

Since November 1901, the color has been working as an assistant office of the Anatomy Department and Plant Physiology in Warsaw University. At the XI Congress of Naturalists and Doctors, Mikhail Semenovich made the report "Methods and objectives of physiological research Chlorophyll", in which for the first time reported on the method of adsorption chromatography.

Mikhail Semenovich has solved the problem of separating green leaf pigments, and they are very close by properties. In addition, there are other, very bright, pigments - carotenoids in the leaves. It is thanks to carotenoids and on the autumn yellow, orange, crimson leaves appear. However, while chlorophylls are not destroyed, it was almost impossible to separate them from carotenoids.

How notes Yu.G. Chirkov, "Apparently, the opening of the color was a reaction to the existing methods of rude and murderous methods of their separation. Here is one of the techniques.

First, chlorophyll alcohol extract was mined, then her three hours were boiling guil with the addition of strong alkali (caustic potassium). As a result, chlorophyll decomposes into composite parts - green and yellow pigments.

But in the process of manufacturing this potion (almost alchemical manipulations), natural chlorophyll could collapse. And then the researcher would have dealt with pieces of pigments, and even with the products of their chemical transformation. "

About how the great discovery happened, writes S.E. Shnol: "He took the glass tube, filled it with a powder of chalk and poured a little alcohol extract of the leaf extract on the top layer, and the top layer of the chalk column was the same color. And then MS began to pour on top in Tube with chalk pure alcohol. Drop per drop. Another portion of the solvent was eluted with pigments from the grain grains, which moved down the tube. There, the fresh gram graves adsorbed pigments and in turn gave them to new portions of the solvent. By virtue of several different adsorption strength (ease of elution) Missed with a movable solvent, different pigments moved along the chalk column at different speeds and formed a homogeneous painted stripes of pure substances in the chalk column. It was beautiful. The bright green strip, the strip of yellow yellow - these are two types of chlorophylls - and bright yellow-orange barotinoids. M.S. called this picture chromatogram. "

"The color showed, - writes chirkov - that when sheep-dissolved vegetable pigments through a layer of colorless porous sorbent, individual pigments are located in the form of painted zones - each pigment has its own color or at least a shade. Sorbent powder (it can be chalk, sugar powder ...) adsorb (superficially absorbs: Latin adSorbere means "swallow") different pigments with unequal power: some can "slip" with a solution current further, others will be detained closer. Thus, the layer-in-law painted color sorbent color called chromatogram, and method - chromatography. "

Thus, the seemingly intractable task was solved. The method was ingenious simple. It is not at all similar to bulky, demanding a large number of reagents complex procedures applied before.

Maybe this simplicity caused the fact that most of the contemporaries or did not perceive this amazing discovery, or, which is still sad, rebelled against his author.

But time all put into place. Color invented chromatography for chlorophyll studies. He first allocated a substance called Chlorophyll Alpha and Chlorophyll Beta. It turned out to be suitable for research not only pigments, but also of colorless, unpainted mixes - proteins, carbohydrates. For the sixties of the twentieth century chromatography, several thousand studies were devoted. Chromatography has become a universal method.

"... The principle of chromatographic separation of substances, open by M. Color, underlies the set of various methods of chromatographic analysis. Without its use, most of the achievements in the science and technique of the 20th century would be impossible ...

The basis of all this is one common idea. She is simple. This is essentially the idea of \u200b\u200bgeometric progression. Let there be two substances very close in all its properties. Neither deposition nor extraction nor adsorption is divided into a noticeable extent. Let one substance 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 from the content of the other. We treat the adsorbent by any solvent so that desorption (disconnection) and elution (flushing) of both substances and both of them would move from the adsorbent to the solvent, and we move 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 0.99 from the content of the second, that is, the part is adsorbed, equal to 0.99 x 0.99 \u003d 0.98 from the initial amount. Once again, we will carry out elution and again adsorption - now the share of the first substance will be 0.98 x 0.99 \u003d 0.97 from the content of the second. In order for the content of the first substance to the next portion of the adsorbent, only 1 percent of the content of the second, it will be necessary to repeat the absorption-elution cycle of about 200 times ...

The idea of \u200b\u200bmultiple conversation for the separation of substances can be modified into a multiple redistribution of the mixture of substances in the system of non-dry solvents. This is the basis of distribution chromatography. The same idea underlies the current methods of electrophoresis when the mixture of substances moves at different speeds of various adsorbents in the electric field.

1. INTRODUCTION.

2. The emergence and development of chromatography.

3. Classification of chromatographic methods.

4. Chromatography on a solid fixed phase:

a) gas (gas-adsorption) chromatography;

b) Liquid (liquid adsorption) chromatography.

5. Chromatography on the liquid fixed phase:

a) gas-liquid chromatography;

b) gel chromatography.

6. Conclusion.

As the rays of the spectrum, the various components of the mixture of pigments are naturally distributed in the carbon dioxide column, allowing its qualitative and quantitative determination. The drug obtained in this way I call the chromatogram, and the proposed method - chromatographic.

M. S. Color, 1906

Introduction

With the need to divide and analyze the mixture of substances, it is necessary to face not only the chemist, but also to many other specialists.

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

Chromatography is a physico-chemical method of separation and analysis of mixtures of gases, vapors, liquids or solutes and determining the physicochemical properties of individual substances based on the distribution of the shared components of the mixtures between the two phases: movable and fixed. Substances that make up the fixed phase are called sorbents. The fixed phase can be solid and liquid. The mobile phase is a flow of liquid or gas, filtered through the sorbent layer. The mobile phase performs the functions of the solvent and the carrier of the analyzed mixture of substances translated into the gaseous or liquid state.

There are two types of sorption: adsorption - absorption of solids and absorption - dissolution of gases and liquids in liquid solvents.

2. It has arisenchromatography and Development and Development

The occurrence of chromatography as a scientific method is associated with the name of the outstanding Russian scientist Mikhail Semenovich colors (1872 - 1919), which in 1903 opened chromatography during the study of the mechanism for transformation of solar energy in plant pigments. This year should be considered the date of creation of the chromatographic method.

M.S. The color passed the solution of the analyzed substances and the movable phase through the adsorbent pillar located in a glass tube. In this regard, its method received the name of column chromatography. In 1938 N.A. Izmailov and M.S. Schreiber offered to modify the color method and carry out the separation of the mixture of substances on the plate covered with a thin layer of the adsorbent. Thus, thin-layer chromatography appeared, which makes it possible to analyze the microcolism of the substance.

In 1947. TB Gapon, E.N. Gapon and F.M. Shemyakin for the first time carried out a chromatographic separation of a mixture of ions in solution, explaining its presence of an exchange reaction between sorbent ions and ions contained in solution. Thus, another direction of chromatography was opened - ion exchange chromatography. Currently, ion exchange chromatography is one of the most important directions of chromatographic method.

E.N. and G. B. Gapon in 1948 implemented expressed by M.S. The idea of \u200b\u200bthe possibility of chromatographic separation of a mixture of substances based on the difference in solubility of hard-soluble precipitation. There was a sediment chromatography.

In 1957, M. Golay proposed to apply a sorbent on the inner walls of the capillary tube - capillary chromatography. This variant allows you to analyze microcolism of multicomponent mixtures.

In the 60s, there was an opportunity to synthesize both ionic and uncharged gels, which have strictly defined pore sizes. This made it possible to develop a chromatography option, the essence of which consists in separating the mixture of substances on the basis of the difference in their ability to penetrate gel - gel chromatography. This method allows you to separate the mixtures of substances with different molecular weight.

Currently, chromatography has gained substantial development. Today, various methods of chromatography, especially in combination with other physical and physicochemical methods, help researchers and engineers solve the most different, often very complex tasks in scientific research and technique.

3. Classifikchromatographic methods

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

The basis of the classification can be put on various signs, namely:

1. The aggregate state of the phases;

2. The separation mechanism;

3. The method of conducting the process;

4. The purpose of the process.

Classification by the aggregate state of the phases:

gas (movable phase - gas), gas-liquid (mobile phase - gas, fixed phase - liquid), liquid (movable phase - liquid) chromatography.

Classification by separation mechanism.

Adsorption chromatography is based on selective adsorption (absorption) of individual components of the mixed mixture with appropriate 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 the movable ions of the adsorbent and the electrolyte ions when the solution of the analyte was analyzed through the column filled with ion exchange substance (ionite). Ionites are insoluble inorganic and organic high molecular weight compounds. Aluminum oxide is used as ionites, permutitis, sulfouggol and a variety of synthetic organic ion exchange substances - ion-exchange resins.

Sedimentary chromatography is based on different solubility of precipitation formed by the components of the analyzed mixture with special reagents. For example, when the solution of the mixture of NG (II) and PB salts is passed through a column with a carrier pre-impregnated with Ki solution, 2 colored layers are formed: upper, painted in orange-red (HGI 2), and bottom, painted in yellow (PBI 2).

Classification according to the process of conducting the process.

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

Paper chromatography is a type of chromatography, in which strips or sheets of filter paper not containing mineral impurities are used as a carrier for a fixed solvent instead of a speaker. In this case, a drop of a test solution, for example, a mixture of solutions of FE salts (III) and CO (II), is applied to the edge of the paper strips. The paper is suspended in a closed chamber (Fig. 1), lowering its edge with a drop of the test solution to it into a vessel with a movable solvent, for example with n-butyl alcohol. Movable solvent, moving through paper, wets it. In this case, each substance contained in the analyzed mixture with the rate inherent in it moves in the same direction as the solvent. Upon completion of the separation of ions, the paper is dried and then sprayed with a reagent, in this case with a solution K 4, which forms painted compounds with separable substances (blue - with iron ions, green - with cobalt ions). The zones formed with all this in the form of painted stains allow you to establish the presence of individual components.

Paper chromatography in combination with the use of organic reagents makes it possible to carry out a qualitative analysis of complex mixtures of cations and anions. On one chromatogram, with the help of one reagent, a number of substances can be detected, since it is characterized by not only the corresponding staining, but also a certain location location on the chromatogram.

Thin-layer chromatography is a type of chromatography in its separation mechanism similar to paper chromatography. The difference between them lies in the fact that instead of sheets of paper, the separation is carried out on plates coated with a thin layer of sorbent made of powdered aluminum oxide, cellulose, zeolites, silica gel, kizelur, etc. and holding a fixed solvent. The main advantage of thin-layer chromatography is the easyness of the equipment, simplicity and high velocity of the experiment, sufficient clarity of the separation of the mixture of substances and in the ability to analyze ultramicrocolism of the substance.

Classification for the purpose of the chromatographic process.

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

Preparative chromatography - type of chromatography, in which the separation of the mixture of substances is produced in preparative purposes, i.e. For more or less significant amounts of substances in pure, free from impurities. The task of preparative chromatography may also be concentrating and subsequent release from the mixture of substances contained in the form of microstrums to the main substance.

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

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

4. ChromatographerfIX on a solid fixed phase

but)Gas (G.azo adsorbovative) Chromatography

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

One of the variants of gas chromatography is gas-adsorption chromatography - this is a method in which a fixed phase is a solid adsorbent.

In gas chromatography as the mobile phase (gas carrier) uses inert gas: helium, nitrogen, argon, significantly less than hydrogen and carbon dioxide. Sometimes the carrier gas serves a pair of volatile fluids.

The gas chromatographic process is usually carried out in special devices called gas chromatographs (Fig. 3). In each of them, there is a gas carrier stream system, a system of preparation and insertion of the mixture under study, a chromatographic column with a temperature control system, analyzing the system (detector) and a system for registering the results of separation and analysis (recorder).

The temperature in gas-adsorption chromatography has a temperature. Its role primarily consists in changing the sorption equilibrium in the gas system - a solid. From the correct selection of the temperature of the column depends, the degree of separation of the components of the mixture, and the efficiency of the column, and the total rate of analysis. There is some temperature range of the column in which chromatographic analysis is optimal. Typically, this temperature interval is located in a region close to the boiling point of the chemical compound. When the boiling points of the mixture components differ greatly between themselves, apply the temperature of the column temperature.

The chromatographic column separation is the most important, but preliminary operation of the entire process of gas chromatographic analysis. Above the binary mixtures (gas-carrier gas - component) fall into the detecting device. Here there is a conversion of changes in the concentrations of components over time into an electrical signal, recorded using a special system in the form of a curve, called chromatogram. The results of the whole experience are largely dependent on the right choice of the type of detector, its design. There are several classifications of detectors. Different differential and integral detectors. Differential detectors register the instantaneous value of one of the characteristics (concentration or stream) in time. Integral detectors summarize the amount of substance for a certain period of time. The detectors are also used in the principle of action, sensitivity and purpose: thermoconductometric, ionization, spectroscopic, mass spectrometric, capulometric, and many others.

Application of gas-adsorption chromatography

Gas-adsorption chromatography is used in the chemical and petrochemical industry for analyzing the products of chemical and petrochemical synthesis, the composition of oil fractions, determining the purity of the reagents and the content of key products at different stages of technological processes, etc.

Analysis of permanent gases and light hydrocarbons, including isomers, gas chromatography occupies 5 to 6 minutes. Previously, on traditional gas analyzers, this analysis lasted 5 to 6 hours. All this led to the fact that gas chromatography began to be widely used not only in research institutes and control and measuring laboratories, but also included in the system of comprehensive automation of industrial enterprises.

Today, gas chromatography is used in the search for oil and gas fields, allowing to determine the content of organic substances that indicate the proximity of oil and gas fields selected from soil samples.

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

Gas chromatography allows you to get valuable and unique information about the composition of food smells, such as cheese, coffee, caviar, brandy, etc. Sometimes the information received by gas chromatographic analysis is not happy. For example, often in food products, it is unnecessary than pesticides or fruit juice contains trichloroethylene, which, contrary to prohibitions, was used to increase the degree of carotene from fruits, etc. But it is this information that protects human health.

However, there are often cases when people just neglect the information received. First of all it refers to smoking. A detailed gas chromatographic analysis has long established that smoke cigarettes and cigarette contains up to 250 different hydrocarbons and their derivatives, of which about 50 have a carcinogenic effect. That is why smokers have lung cancer in 10 times more often, but still millions of people continue to poison themselves, their colleagues and relatives.

Gas chromatography is widely used in medicine to determine the content of numerous drugs, determining the level of fatty acids, cholesterol, steroids, etc. In the body of the patient. Such analyzes provide extremely important information on the state of human health, the course of its illness, the effectiveness of the use of certain drugs.

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

Gas chromatography is successfully used to determine the content of polycyclic aromatic compounds, dangerous to human health, in water and in the air, gasoline level in the air of planes of gas 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 occupies an important place in our lives, providing us with a colossal amount of information. In the national economy and in research organizations, more than 20 thousand of various gas chromatographs are used, which are indispensable assistants in solving many complex tasks, daily arising from researchers and engineers.

b)Liquid (liquid adsorption)chromatography

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

One embodiment of liquid chromatography is liquid-adsorption chromatography - this is a method in which a fixed phase is a solid adsorbent.

Although liquid chromatography was opened earlier than gas, it only in the second half of the twentieth century entered the period exclusively intensive development. Currently, according to the degree of development of the theory of chromatographic process and techniques of instrumental design, according to efficiency and the separation rate, it is unlikely to give way to the gas chromatographic separation method. At the same time, each of these two main types of chromatography has its own primary scope. If gas chromatography is mainly suitable for analysis, separation and research of chemicals with a molecular weight of 500 - 600, then liquid chromatography can be used for substances with a molecular weight of several hundred and several million, including extremely complex macromolecules of polymers, proteins and nucleic acids. At the same time, the opposition of various chromatographic methods is inherently devoid of common sense, since chromatographic methods are successfully complemented by each other, and to the very task of a specific study, it is necessary to approach differently, namely, what chromatographic method allows you to solve it with more speed, informativeness and with less cost.

As in gas chromatography, detectors are used in modern liquid chromatography, which can continuously fix the concentration of the determined substance in the flow of fluid flowing out of the column.

Universal detector for liquid chromatography does not exist. Therefore, in each particular case, it should be selected in the greatest union detector. Ultraviolet, refractometric, micro-drug detectors, refractive-ionization detectors were greatest.

Spectrometric detectors. Detectors of this type are highly sensitive selective devices, allowing very small substances concentrations in the stream of the liquid phase. Their testimony depends little on temperature fluctuations and other random changes of the medium. One of the important features of spectrometric detectors is the transparency of the most solvents used in the liquid-adsorption chromatography in the working area of \u200b\u200bwavelengths.

Most often used absorption in UV, less often in the IR region. In the UV region, appliances operating in a wide range are used - from 200 nm to the visible part of the spectrum, or on certain wavelengths, most often at 280 and 254 nm. Mercury low pressure bulbs (254 nm), medium pressure (280 nm) and corresponding filters are used as sources of radiation.

Micro Persorption detectors. The basis of micro-purpose detectors is based on the release of heat during adsorption of the substance on the adsorbent, which is filled with the detector cell. It is measured, however, not heat, but the temperature of the adsorbent, to which it is heated as a result of adsorption.

A micro-purpose detector is a fairly highly sensitive tool. Its sensitivity depends primarily from the heat of adsorption.

Micro Perseter detectors are universal, suitable for detecting both organic and inorganic substances. At the same time, they are difficult to obtain quite clear chromatograms, especially with the incomplete separation of the mixture components.

5. Chromatographeratia on liquid fixed phase

a) gas-liquid chromatography

Gas-liquid chromatography - a gas chromatographic method in which a fixed phase is a young liquid applied to a solid carrier.

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

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

The chromatographic 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 is distributed (fixed phase). The studied gases and pairs are absorbed by this liquid. Then the components of the shared mixture are selectively displaced in a specific order from the column.

In gas-liquid chromatography, a number of detectors are used specifically reacting to any organic substances or on organic substances with a specific functional group. These include ionization detectors, electronic capture detectors, thermal, spectrophotometric and some other detectors.

Flame-ionization detector (PID). The work of the PID is based on the fact that the organic substances falling into the flame of the hydrogen burner are subject to ionization, as a result of which the ionization is simultaneously an ionization, which is simultaneously an ionization chamber, which is proportional to the number of charged particles.

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

When working with the PID, 3 gas is used: gas carrier (helium or nitrogen), hydrogen and air. All 3 gas must have a high degree of purity.

Argon detector. In the argon detector, ionization is caused by the collision of the molecules of the determined substance with metastable argon atoms resulting from the effects of radioactive in-radiation.

Thermal detector. The principle of the thermo-ion detector is that alkali metal salts, evaporating in the flame burner, are selectively reacting with compounds containing halogens or phosphorus. In the absence of such compounds in the ionization chamber of the detector, an equilibrium of alkali metal atoms is established. The presence of phosphorne atoms due to their reaction with alkali metal atoms violates this balance and causes the appearance in the ionic current chamber.

Since the thermal detector has the highest sensitivity to phosphorus-containing connections, it received the name of phosphate. This detector is mainly used to analyze phosphorodinorganic pesticides, insecticides and a number of biologically active compounds.

b)Gel chromatografIA

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

The separation of the mixture of substances occurs in the event that the dimensions of the molecules of these substances are different, and the diameter of the gel grains is constant and only those molecules can pass, the dimensions of which are smaller than the diameter of the gel pores. When filtering the solution of a mixture of the mixture, smaller molecules penetrated into the pores of the gel, are delayed in the solvent contained in these pores, and move along the gel layer slower than large molecules that are not capable of penetrating the pores. Thus, gel chromatography allows you to separate the 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 you to separate mixtures of substances under silt conditions than other chromatographic methods.

If the gel granules fill the column and then pour a solution of various substances with different molecular weight into it, then when the solution is moved along the gel layer in the column, this mixture will take place.

The initial period of experience: applying the solution of the analyzed mixture on the gel layer in the column. The second stage - gel does not interfere with the diffusion of small molecules in the pores, the largest molecules remain in solution surrounding the gel granules. When washed with a layer of gel with a pure solvent, large molecules start moving at a speed close to the speed of the solvent movement, while small molecules must first predefund from the inner age of the gel into the volume between the grains and as a result of this are delayed and washed away with the solvent later. A mixture of substances according to their molecular weight occurs. Washing substances from the column occurs in order to reduce their molecular weight.

The use of gel chromatography.

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

At the same time, an equally gel chromatography is used to separate the mixture of substances of medium molecular weight and even low molecular weight connections. In this case, it is important that gel chromatography makes it possible to separate at room temperatures, which favorably distinguishes it from gas-liquid chromatography requiring heating to transfer the analyzed substances into the steam phase.

The separation of the mixture of substances by gel chromatography is possible and when the molecular weights of the analyzed substances are very close or even equal. In this case, the interaction of solutes with gel is used. This interaction may be so significant, which reduces the differences in the sizes of molecules. If the nature of interaction with the gel for different substances is non-IDAs, 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 set by the number of iodine defined during the analysis.

The above examples of the use of gel chromatography show its ample opportunities for solving a wide variety of analytical tasks.

Conclusion

As a scientific method of learning around us, chromatography is constantly developing and improving. Today it is applied so often and so widely in scientific research, medicine, molecular biology, biochemistry, technology and the national economy, which is very difficult to find the area of \u200b\u200bknowledge in which chromatography would not be used.

Chromatography as a study method with its exceptional capabilities is a powerful factor in the knowledge and transformation of a complicating world in the interests of creating acceptable human habitat conditions on our planet.

With p and with about toL and t e r and t u r s

1. Ivazov B.V. Introduction to chromatography. - M.: Higher. Sk., 1983 - s. 8-18, 48-68, 88-233.

2. Kreshkov A.P. Basics of analytical chemistry. Theoretical basis. Qualitative analysis, the first book, ed.4-e, recreation. M., "Chemistry", 1976 - p. 119-125.

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