History of chromatography. History of the development of liquid chromatography

2. The emergence and development of chromatography

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

M.S. The color passed a solution of analytes and mobile phase through a column of adsorbent contained in a glass tube. In this regard, his method was called column chromatography. In 1938 N.A. Izmailov and M.S. Schreiber proposed modifying Tsvet's method and separating a mixture of substances on a plate coated with a thin layer of adsorbent. This is how thin-layer chromatography arose, allowing analysis with microquantities of a substance.

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

E.N. and G.B. Gapon in 1948 carried out what was expressed by M.S. Color the idea of ​​the possibility of chromatographic separation of a mixture of substances based on differences in solubility of sparingly soluble precipitates. Sediment chromatography appeared.

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

In the 60s, it became possible to synthesize both ionic and uncharged gels with strictly defined pore sizes. This made it possible to develop a version of chromatography, the essence of which is to separate a mixture of substances based on the difference in their ability to penetrate the gel - gel chromatography. This method allows you to separate mixtures of substances with different molecular weights.

Currently, chromatography has received significant development. Today, a variety of chromatography methods, especially in combination with other physical and physicochemical methods, help scientists and engineers solve a wide variety of, often very complex, problems in scientific research and technology.

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

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

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

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

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

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

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

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

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

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

At the end of the 60s. Interest in liquid chromatography has increased sharply. High performance liquid chromatography (HPLC) appeared. This was facilitated by the creation of highly sensitive detectors, new selective polymer sorbents, and new equipment that allows operation at high pressures. Currently, HPLC occupies a leading position among other chromatography methods and is implemented in various versions.

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

This allows rapid separation of complex mixtures of components.

Discovery history:

Birth of chromatography

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

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

The following advantages of chromatographic methods should be emphasized:

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

separation compared to static sorption methods and

extraction.

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

This makes it possible to selectively separate a wide range of

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

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

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

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

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

the process of separation into frontal, displacement and eluent.

Ion chromatography

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

low capacity. Widespread use of ion chromatography

due to a number of its advantages:

– the ability to determine a large number of inorganic and

organic ions, and also simultaneously determine cations and

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

pre-concentration;

– high selectivity and expressivity;

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

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

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

– possibility of complete automation of determination;

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

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

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

development of the method;

– lower separation efficiency compared to HPLC;

– the need for high corrosion resistance

chromatographic system, especially when determining

cations.

2.1 Development history:

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

2.3 Principles of ion separation in sorption processes

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

Ion chromatography is a variant of ion exchange column chromatography.

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

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

The following separation principles are used in ion chromatography:

Ion exchange.

Formation of ion pairs.

Ion exclusion.

Ion exchange

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

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

Formation of ion pairs

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

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

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

Ionic exclusion

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

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

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

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

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

Based on the fractionation principle:

Affinity chromatography

Gel filtration

Adsorption

Sedimentary

Adsorption-complexation

Distribution (normal phase, reverse phase).

According to the method of evolution:

Size exclusion chromatography

Chromatographic evolution

Frontal analysis

Ion exchange chromatography.

By location of the stationary phase:

Columnar chrome

Thick layer chromatography

Thin layer chromatography

Paper (film) chromatography.

According to the aggregate composition of the phases:

Supercritical fluid chromatography

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

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

By purpose of behavior:

Analytical

Preparative

Industrial.

By pressure in the chromatographic system:

High pressure

Low pressure.

2. History of the development of liquid chromatography.

Chromatography was discovered by M. S. Tsvet in 1903 in the form of a column liquid-adsorption method. This method used adsorbents with a grain size of more than 50-100 microns, the eluent passed through the column by gravity due to gravity, there were no flow detectors. Separation occurred slowly, within several hours, and in this mode liquid chromatography could not be used for analytical purposes. In 1965-1970, the efforts of specialists in various countries were aimed at creating rapid liquid chromatography. It was clear that to increase the rate of separation it was necessary to shorten the paths of external and internal diffusion. This could be achieved by reducing the diameter of the adsorbent grains. Filling the columns with small grains (5-10 μm) created a high inlet pressure, which required the use of high-pressure pumps. This is how high pressure liquid chromatography was born. With the transition to fine-fraction adsorbents, the efficiency of columns greatly increased (per unit length, hundreds of times higher than the efficiency of columns in gas chromatography), therefore modern rapid analytical liquid chromatography was called high-performance liquid chromatography (HPLC). Development of rigid fine-grained adsorbents (5 or 10 μm ), the creation of high-pressure pumps (over 200 atm) and flow-through detectors - all this ensured high performance of HPLC. In terms of separation times, it was not inferior to gas chromatography, and in areas of application it significantly surpassed it. This period of time began to be called the second birth of liquid chromatography, the revival, the period of its renaissance. One of the first commercial liquid chromatographs was the Du Pont model 820 (1968). This was preceded by the development of a series of detectors for liquid chromatography: a conductometric detector (1951), a heat of adsorption detector (1959), a refractometric detector (1962), and a UV detector (1966). ), liquid chromatograph/mass spectrometer system (1973), the first version of a diode array detector (1976) In 1969, I Halasz and I Sebastian proposed sorbents with chemically grafted alkyl chains (“brush sorbents”) with Si - O - C bonds. This connection turned out to be unstable. In 1970, J. Kirkland developed sorbents with more stable Si-O-Si bonds. For the sake of fairness, it should be noted that such a modification was proposed much earlier (1959) by K.D. Shcherbakova and A.V. Kiselev.

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

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

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

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

cottages of proteomics.

1. Introduction.

2. The emergence and development of chromatography.

3. Classification of chromatographic methods.

4. Chromatography on a solid stationary phase:

a) gas (gas adsorption) chromatography;

b) liquid (liquid adsorption) chromatography.

5. Chromatography on a liquid stationary phase:

a) gas-liquid chromatography;

b) gel chromatography.

6. Conclusion.

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

M. S. Tsvet, 1906

Introduction

Not only the chemist, but also many other specialists have to deal with the need to separate and analyze a mixture of substances.

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

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

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

2. The emergence and development of chromatography

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

M.S. The color passed a solution of analytes and mobile phase through a column of adsorbent contained in a glass tube. In this regard, his method was called column chromatography. In 1938 N.A. Izmailov and M.S. Schreiber proposed modifying Tsvet's method and separating a mixture of substances on a plate coated with a thin layer of adsorbent. This is how thin-layer chromatography arose, allowing analysis with microquantities of a substance.

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

E.N. and G.B. Gapon in 1948 carried out what was expressed by M.S. Color the idea of ​​the possibility of chromatographic separation of a mixture of substances based on differences in solubility of sparingly soluble precipitates. Sediment chromatography appeared.

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

In the 60s, it became possible to synthesize both ionic and uncharged gels with strictly defined pore sizes. This made it possible to develop a version of chromatography, the essence of which is to separate a mixture of substances based on the difference in their ability to penetrate the gel - gel chromatography. This method allows you to separate mixtures of substances with different molecular weights.

Currently, chromatography has received significant development. Today, a variety of chromatography methods, especially in combination with other physical and physicochemical methods, help scientists and engineers solve a wide variety of, often very complex, problems in scientific research and technology.

3. Classification of chromatographic methods

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

The classification can be based on various characteristics, namely:

1. state of aggregation of phases;

2. separation mechanism;

3. method of carrying out the process;

4. purpose of the process.

Classification according to the state of aggregation of phases:

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

Classification by separation mechanism.

Adsorption chromatography is based on the selective adsorption (absorption) of individual components of the analyzed mixture by 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 mobile ions of the adsorbent and electrolyte ions when passing a solution of the analyte through a column filled with an ion exchange substance (ion exchanger). Ion exchangers are insoluble inorganic and organic high-molecular compounds. Aluminum oxide, permutite, sulfonated coal and various synthetic organic ion exchange substances - ion exchange resins are used as ion exchangers.

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

Classification according to the method of carrying out the process.

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

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

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

Thin layer chromatography is a type of chromatography similar in its separation mechanism to paper chromatography. The difference between them is that instead of sheets of paper, the separation is carried out on plates coated with a thin layer of sorbent made from powdered aluminum oxide, cellulose, zeolites, silica gel, kieselguhr, etc. and holding a stationary solvent. The main advantage of thin-layer chromatography is the simplicity of the equipment, the simplicity and high speed of the experiment, the sufficient clarity of the separation of a mixture of substances, and the possibility of analyzing ultra-microquantities of a substance.

Classification according to the purpose of the chromatographic process.

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

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

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

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

4. Chromatography on a solid stationary phase

a) Gas (gas adsorption) chromatography

Gas chromatography is a chromatographic method in which the mobile phase is gas. Gas chromatography is most widely used for the separation, analysis and study of substances and their mixtures that pass into a vapor state without decomposition.

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

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

The gas chromatographic process is usually carried out in special instruments called gas chromatographs (Fig. 3). Each of them has a system for supplying a carrier gas flow, a system for preparing and introducing the mixture under study, a chromatographic column with a system for regulating its temperature, an analyzing system (detector) and a system for recording the results of separation and analysis (recorder).

Temperature is important in gas adsorption chromatography. Its role primarily lies in changing the sorption equilibrium in the gas-solid system. The degree of separation of the components of the mixture, the efficiency of the column, and the overall speed of analysis depend on the correct selection of the column temperature. There is a certain column temperature range within which chromatographic analysis is optimal. Typically, this temperature range is in the region close to the boiling point of the chemical compound being determined. When the boiling points of the mixture components differ greatly from each other, programming the column temperature is used.

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

Applications of gas adsorption chromatography

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

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

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

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

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

However, there are often cases when people simply neglect the information received. This primarily applies to smoking. Detailed gas chromatographic analysis has long established that the smoke of cigarettes and cigarettes contains up to 250 different hydrocarbons and their derivatives, of which about 50 are carcinogenic. This is why lung cancer is 10 times more common in smokers, but millions of people still continue to poison themselves, their colleagues and relatives.

Gas chromatography is widely used in medicine to determine the content of numerous drugs, determine the level of fatty acids, cholesterol, steroids, etc. in the patient's body. Such analyzes provide extremely important information about the state of a person’s health, the course of his illness, and the effectiveness of using certain medications.

Scientific research in metallurgy, microbiology, biochemistry, in the development of plant protection products and new drugs, in the creation of new polymers, building materials and in many other very diverse areas of practical human activity 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 hazardous to human health in water and air, the level of gasoline in the air at gas stations, the composition of vehicle exhaust gases in the air, etc.

This method is widely used as one of the main methods for monitoring environmental cleanliness.

Gas chromatography occupies an important place in our lives, providing us with a colossal amount of information. The national economy and research organizations use more than 20 thousand of a wide variety of gas chromatographs, which are indispensable assistants in solving many complex problems that researchers and engineers face every day.

b) Liquid (liquid adsorption) chromatography

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

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

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

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

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

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

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

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

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

Microadsorption detectors are universal, suitable for detecting both organic and inorganic substances. However, it is difficult to obtain sufficiently clear chromatograms from them, especially when the components of the mixture are incompletely separated.

5. Liquid stationary phase chromatography

a) Gas-liquid chromatography

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

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

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

The chromatography process can be schematically represented as follows. A mixture of gases or vapors of volatile liquids is introduced by a stream of carrier gas into a column filled with a stationary inert carrier on which a non-volatile liquid (stationary phase) is distributed. The gases and vapors being studied are absorbed by this liquid. Then the components of the mixture to be separated are selectively displaced in a certain order from the column.

Gas-liquid chromatography uses a number of detectors that specifically respond to any organic substance or to organic substances with a specific functional group. These include ionization detectors, electron capture detectors, thermionic, spectrophotometric and some other detectors.

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

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

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

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

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

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

b) Gel chromatography

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

Separation of a mixture of substances occurs if the sizes of the molecules of these substances are different, and the diameter of the pores of the gel grains is constant and can only pass through those molecules whose sizes are smaller than the diameter of the holes in the gel pores. When filtering a solution of the analyzed mixture, smaller molecules, penetrating into the pores of the gel, are retained in the solvent contained in these pores and move along the gel layer more slowly than large molecules that are not able to penetrate the pores. Thus, gel chromatography allows you to separate a mixture of substances depending on the size and molecular weight of the particles of these substances. This separation method is quite simple, fast and, most importantly, it allows you to separate mixtures of substances under milder conditions than other chromatographic methods.

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

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

Application of gel chromatography.

The main purpose of gel chromatography is to separate mixtures of high molecular weight compounds and determine the molecular weight distribution of polymers.

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

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

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

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

Conclusion

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

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

BIBLIOGRAPHY

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

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

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