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Chromatography

Chromatography is a method of separating mixtures into their constituent substances, based on differences in the degree of absorption of individual substances during their passage through the layer of the absorber.

Chromatography is used for the qualitative and quantitative determination of substances in various kinds of mixtures for diagnostic purposes (for example, changes in the content of individual amino acids in the blood plasma, in the tissues of the liver and other organs, occurring under a number of pathological conditions, etc., etc.). p.), as well as for preparative purposes in obtaining many, including biologically active substances.

The following types of chromatography are widely used in clinical, sanitary and pharmaceutical laboratories.

column chromatography
Fig. 1 Column chromatography: 1 — test solution; 2 - sorbent layer; 3 - zones formed by substances that are part of the analyzed mixture.
Fig. 2. Chromatography on paper: 1 - paper strip; 2 - colored spots corresponding to different substances that were part of the analyzed solution; 3 - a drop of the analyzed solution; 4 - start line; 5 - solvent .

Column chromatography is carried out by passing the test solution containing several solutes through a glass tube (column - fig. 1) filled with a powdered absorber (sorbent). Due to the unequal absorption (sorption) of various substances, they are separated. The better the substance is absorbed, the delayed it is in the higher parts of the column. The determination of the nature of a substance is made either by its own characteristic color of the substance, or by passing through a column (after separation of the mixture) a solution of a developer’s reagent, which forms specifically colored compounds with the analyzed substances. The sorbent layer thus obtained with differently colored zones is called a chromatogram. Substances sorbed on the column can be successively forced out (washed) and collected in fractions. This process is called elution.

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Paper chromatography is carried out on strips of special grades of paper. A drop of the test solution is applied at some distance from the edge of the paper strip (Fig. 2). The edge of the strip is placed in the appropriate solvent, which moves through the capillaries of the paper along the strip. When this occurs, the separation of substances: the worse the substance is absorbed, the further it will be from the start line. At the end of the separation, the strip is dried and sprayed with a solution of a reagent that forms characteristic colored compounds with the substances to be detected.

Chromatography in a thin layer of sorbent is similar to paper chromatography. The difference is that in this case a thin layer of powdered sorbent (for example, alumina, silica gel, kaolin, ion exchange resin) is applied on the glass plate. The technique of separating the analyzed mixture into individual substances and the methods for their qualitative determination are basically the same as for chromatography on paper.

In recent years, so-called automatic analyzers have become increasingly common in clinical and laboratory practice, devices that allow chromatographic analysis using automatic devices while simultaneously recording the results on a special tape.

Chromatography (from the Greek. Chroma, chromatos - color, color and grapho - write down; literally - color painting) - sorption dynamic method for separating mixtures of substances. Proposed by M. S. Tsvetom in 1903 for the analysis of plant pigments. Subsequently, it became a universal method for separating mixtures of colored and colorless substances of any nature for analytical and preparative purposes.

The method consists in absorbing the initial mixture by a small portion of a layer of granulated sorbent placed in a column, and subsequently expanding the zones of components (elution) by passing an eluent — solvent or solution not containing the components of the analyzed mixture — through a layer of sorbent. During elution, the components of the mixture move around the sorbent layer at different rates and, as a result, are separated on the sorbent in the form of isolated zones, and with further elution of the sorbent are successively transferred to the filtrate (Fig. 1). Chromatography allows to obtain pure substances, including biologically active (morphine, antibiotics, vitamins, hormones, enzymes, etc.). Instead of sorbent columns, a thin powder deposited on a glass plate (silica gel, clay, Al 2 O 3 , MgO, etc.) is often used. These are thin layer chromatography (TLC) or filter paper strips (paper chromatography). In this case, the components of the mixture form isolated spots; the ratio of the speeds of their movement to the speed of movement of the solvent front (the value of Rf) characterizes the nature of the substance, the area - its quantity. These methods allow you to separate and analyze 10 —8 - 10 —6 g of a substance. In the analytical aspect, chromatography identifies a substance based on a comparison of the speeds of its movement and the “witness” on the sorbent layer and facilitates the quantitative analysis of mixtures of similar components.


Fig. 1. The successive stages of the process of chromatographic separation of a mixture of substances (a, b, c and d): I - the primary chromatogram; II — III — successive stages of elution; below, the output curves of substances (a, b, and c).

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Distinguish chromatography adsorption, ion exchange, distribution and sedimentary; Recently, the method of gel filtration is referred to chromatographic methods.

Adsorption chromatography (AH). The basis of AH is the process of adsorption (see) of the mixture components by the sorbent. In AH, porous sorbents with a high specific surface area (100–1000 m 2 / g) are used. AH is widely used to obtain drugs, analysis of biological materials, etc.

Ion exchange chromatography (IOC). Based on the absorption of ions by the sorbent as a result of a heterogeneous chemical reaction of ion exchange between the sorbent and the analyzed solution. In IOC, natural and synthetic ion-exchanging substances are used (see Ionites), the absorption of which of the mixture components increases with an increase in the charge of the ion component, and for ions of equal magnitude the charge - with an increase in the ion radius. IOC is the most advanced method for the qualitative and quantitative analysis of amino acids and peptides. The use of an automatic analyzer based on the method of fractionation of amino acids on sulfonic cation exchangers according to Moore, Speckman, Stein allows you to analyze up to 60 nitrogen-containing compounds (Fig. 2); A complete analysis, for which 0.01–0.025 micromoles of each amino acid is sufficient, is carried out in 3-5 hours.


Fig. 2. Chromatogram of free amino acids of deproteinized blood plasma of a healthy person (1.7 ml); column size 69x0.9 cm; separation of neutral and acidic amino acids.

For the separation of mixtures of high-molecular compounds (proteins, nucleic acids, acidic polysaccharides, etc.) use ion-exchange Sephadex (SI) or unstitched linear ionites based on cellulose (CI). SI is obtained by introducing residues of an acid or base into the finished copolymer; Two cation exchangers — carboxymethyl (KMC) and sulfoethylsefadex (SES) and anion exchanger — diethylaminoethyl sethadex (DEAE-C) were obtained. CI are cellulose ethers with different ionic groups; cation exchangers are widely used - carboxymethylcellulose, phosphate cellulose, sulfoethyl cellulose; anion exchangers - diethylaminoethyl cellulose (DEAE-C, almost universal anion exchanger), triethylaminoethyl cellulose (TEAE-C), weakly basic anion exchanger - EKTEola-cellulose (EKTEola-C), obtained by the reaction of cellulose with epichlorohydrin and triethanolamine. The exchange capacity of SI is greater than QI, and is 3-4.5 mEq / g.

With the help of QI or SI, great success has been achieved in the field of protein isolation and purification, including enzymes, hormones, toxins, nucleic acids, and large bacterial polysaccharides. DEAE-C or EKTEOL-C, applied in a thin layer on plates, are the best sorbents for fast and very fine fractionation of nucleotides and nucleic acids.

Distribution chromatography (PX). The separation of the components of the mixtures in this chromatography is due to differences in their distribution coefficients between the stationary phase held by the inert sorbent and the mobile phase, either by solvent or gas; in the latter case, gas distribution chromatography (GRH) is referred to. PX is the most common analytical and preparative method of fractionation of substances of similar structure. As an inert carrier, predominantly polar substances are used - cellulose (in the form of sheets of filter paper), less often - silica gel. The paper chromatography technique is very simple; Depending on the direction of movement of the solvent, one-dimensional, two-dimensional, or circular chromatograms are distinguished. The movement of the solvent on the paper can be carried out in ascending, descending or horizontal ways. The analyzed mixture is applied to the paper in the form of a droplet with a volume of 1–5 μl containing 10–8 –10 –6 g of each analyte. The upward or downward flow of solvent moves the mixture components along the paper, forming a chain (one-dimensional chromatogram) or a pattern (two-dimensional chromatogram) of spots (Fig. 3). The content of each isolated component is determined by conventional methods after extraction of a substance from a “spot” or, less commonly, directly on paper but the spot area, which is established on the basis of its color, radioactivity, or absorption of ultraviolet rays by the substance.


Fig. 3. A two-dimensional chromatogram of a mixture of essential amino acids that make up proteins and biological fluids. The first solvent: butanol - acetic acid; second solvent: phenol - NH 3 . 1 - cysteic acid; 2 - aspartic acid; in - glutamic acid; 4 - serine; 5 - taurine; in - glycine; 7 - glutamine; 8 - threonine; 9 - alanine; 10 is β-alanine; 11 - hydroxyproline; 12 - tyrosine; 13 - histidine; 14 - lysine; 15 - arginine; 16 - methionine sulfoxide; 17 - ү-aminobutyric acid; 18 - β-aminoisobutyric acid; 19 - valine; 20 - phenylalanine; 21 - isoleucine; 22 - leucine; 23 - proline.

The GRH makes it possible with extreme accuracy to analyze ultramikrokolichestva (10 -11 - 10 -9 g) substances that under the conditions of the analysis can be converted to a gas or vapor state. It is used to study the processes of respiration and composition of blood gases, determination of sex hormones, adrenal cortex hormones, bile acids, fat-soluble vitamins, especially group D (D2 - D3) vitamins, lipids and some volatile components of tissues - lower alcohols, thiols, ketones. In the biochemistry of nutrition, GRH is used to identify substances that determine the smells of foods, such as coffee, cheese, roasted meat, etc.

Sediment chromatography . This chromatography results in the formation of insoluble precipitate mixture components as a result of their chemical interaction with the reagent impregnating the sorbent. In biochemical and clinical studies, sediment chromatography is not yet used.

Gel filtration (GF). The separation of substances by gel filtration is based on the mechanical phenomenon of molecular sieving; with HF, molecules with sizes smaller than the diameter of the pores of a homogeneous porous swollen “sorbent” are evenly distributed between the “external” and “internal” solutions; large molecules do not penetrate into the grain of the "sorbent" and, therefore, move along the column at the rate of flow of the solvent. In biochemical studies, porous copolymers of partially depolymerized bacterial polysaccharide dextran with various amounts of hydrophobic bridges formed by glycerol residues (sephadexes) or polyacrylamide (biogels) are the most common. Increasing the content of the "crosslinking agent" reduces the pore size of the sieve and the swelling capacity of the copolymer, reducing its absorption of large molecules, usually substances with a large mol. weight. The use of Sephadex different brands allows you to separate substances with mol. weighing from 200 to 200 000, and bio-gels - from 200 to 300 000. "Weakly cross-linked" gels are used for the analysis, isolation and purification of proteins, polysaccharides, nucleic acids. "Tightly stitched" gels allow the fractionation of amino acids, peptides, sugars, nucleotides. Small gels are used to separate low molecular weight from high molecular weight substances and replace dialysis.

The considered types of chromatography are widely implemented in the form of thin layer chromatography (TLC), in which the finely ground sorbent or carrier is applied with a layer (0.1 - 0.3 mm) on a glass plate. The technique for producing thin-layer chromatograms is similar to paper chromatography; the advantage is the speed of separation and high sensitivity. TLC on silica gel, Al 2 O 3 , diatomaceous earth, conventional and ion-exchange cellulose or Sephadex is the most advanced method of microanalysis. TLC is used in biochemical and clinical studies for the separation, identification and quantitative determination of amino acids, peptides, carbohydrates, nucleotides, hormones, fatty acids, proteins, etc .; for example, a mixture of ATP and ADP in the amount of 5 × 10–4 μmol each can be separated in a thin layer of ECTEA cellulose in 4–5 minutes; a mixture of four nucleotides is separated under these conditions in 15 minutes. In analytical chemistry used TLC, in which the most common sorbents are alumina and silica gel.