Chromatography

Chromatography is a method of separating mixtures into its constituent substances, based on differences in the degree of absorptivity individual substances at their passing through the layer of the absorber.
Chromatography is used for qualitative and quantitative determination of substances in various mixtures, for diagnostic purposes (for example, changing the content of certain amino acids in the blood plasma, in the tissues of the liver and other organs, coming in a number of pathological conditions, and so on, and so on), as well as for preparative purposes when receiving many, including biologically active substances.
In clinical, sanitary-hygienic and pharmaceutical laboratories are widely used the following types of chromatography.

column chromatography
Fig. 1 Column chromatography: 1 - the solution being analyzed; 2 - layer sorbent; 3 - zone formed by substances that are part of the analyzed mixture.
Fig. 2. Chromatography on paper: 1 - a strip of paper; 2 - colored spots corresponding to the different substances, included in the analyzed solution; 3 - a drop of the analyzed solution; 4 - the starting line; 5 - solvent.

Column chromatography - is the transmission of the investigated solution containing several dissolved substances through a glass tube (column (Fig. 1), filled poroshkoobraznymi absorber (sorbent). Due to unequal absorptivity (sobiraemosti) of various substances is their separation. The better absorbed by a substance, the higher parts of the column it is delayed. Determination of the nature of the substances produced or on its own characteristic colouring substances, or passing through the column (after the split mix) solution of reagent - developer, forming with the analyzed substances specifically painted connection. The resulting layer of the sorbent with differently colored zones called the chromatogram. Substances adsorbed on a column that can be successively replaced (washed) and collected by the factions. This process is called elution.
Paper chromatography - is held on strips of special sorts of paper. Drop researched solution is applied at a certain distance from the edge of the paper strip (Fig. 2). Edge strips are placed in the appropriate solvent, which moves through the capillaries of paper along the strip. Thus there is a separation of substances: the worse is absorbed by the substance, the farther it will be from the start line. After the separation of a strip of dried and sprayed with chemical solutions, forming defined substances are characterized by colored compound.
Chromatography in a thin layer of the sorbent similar chromatography on paper. The difference is that in this case on the glass plate is applied in a thin layer of powdery sorbent (for example, aluminum oxide, silica, kaolin, ion-exchange resin). The technique of separation of the analyzed mixture on individual substances and qualitative methods of their definition is basically the same as when chromatography on paper.
In recent years in clinical and laboratory practice is increasingly began to penetrate the so-called automatic analyzers, devices, allowing to make chromatographic analysis with the help of automatic devices with simultaneous recording of the received results on a special tape.

Chromatography (from the Greek. chroma, chromatos - color, color and grapho - write; literally - svetopisi) - dynamic sorption method of separation of mixtures of substances. Proposed M. S. Color in 1903 for the analysis of plant pigments. Later became a universal method of separation of colored and colorless substance of any nature in the analytical and preparative purposes.
The method consists in the absorption original mix a small plot layer of granulated sorbent placed in the column, and subsequent spread of the zones components (elution) by passing through the layer of the sorbent additionally separated by a solvent or solution, not containing components of the analyzed mixture. When elution components of the mixture are moved by layer sorbent with a different speed and consequently dismantled on the sorbent in the form of isolated areas, and further elution sorbent consistently pass into the filtrate (Fig. 1). Chromatography allows to obtain pure substances, including biologically active (morphine, antibiotics, vitamins, hormones, enzymes and other). Instead of columns with the sorbent is often used deposited on a glass plate thin powder (silica, clay, Al2O3, MgO and other) is the thin layer chromatography (TLC) or a strip of filter paper (chromatography on paper). In this case, the components of the mixture form isolated spots; the ratio of the speed of their movement to the movement of the front of the solvent (value Rf) characterizes the nature of matter, space - its quantity. These methods allow to separate and analyze 10-8 - 10-6 g of substance. In the analytical aspect chromatography permits identification of the substance on the basis of comparison of speeds and his "witness" for layer sorbent and facilitates quantitative analysis of mixtures, with similar properties of the components.
There are adsorption chromatography, ion exchange, distribution and sedimentary; recently to chromatographic methods are identified as gel filtration method.
Adsorption chromatography (AH). In the basis of AH is the process of adsorption (see) of components of a mix sorbent. In AH using porous sorbents with a large specific surface (100-1000 m2/g). OH widely used for obtaining of medicinal substances, analysis of biological materials and other
Ion-exchange chromatography (ZIOC). Based on the absorption of ions sorbent in the heterogeneous chemical reaction ion exchange between the sorbent and analyzed solution. In ZIOC use natural and synthetic ion substances (see Ion), absorption which components of the mixture increases with increasing charge of the ion component, and ions equal amount of charge - with the increase of the radius of the ion. ZIOC is the most perfect method of qualitative and quantitative analysis of amino acids and peptides. Using an automatic analyser, the basis of which is the method of fractionation of amino acids on the sulfonic cation by Moore, Spackman, Stein, allows to analyze up to 60 nitrogen-containing compounds (Fig. 2); full analysis, for which enough 0,01-0,025 of umol each amino acid, is carried out within 3-5 hours.
For the separation of mixtures of high-molecular compounds (proteins, nucleic acids, acidic polysaccharides and other) using ion-exchange sefadex (C) or unstitched linear ion exchangers based on cellulose (QI). SI receive the introduction of the remnants of acids or bases in ready copolymer; received two cation carboxymethyl (KMC) and sulfoethylated (SES) and anion-dietilaminoetanola (DEAE-C). QI are the cellulose ethers with different ionogenic groups; widely used cation - carboxymethylcellulose, hospitaloleh, sulfoetoksilaty; anionites - dietilaminometiltselljuloza (DEAE-C, almost universal anion exchange resin), dietilaminometiltselljuloza (TIAA-C), weak-basic anion - Aktera-cellulose (Aktera-C), obtained by reaction of cellulose with epichlorohydrin and triethanolamine. Exchange capacity C more than QI, and is 3-4,5 mg-EQ/year
With the aid of QI, or C has been great progress in the field of extraction and purification of proteins, including enzymes, hormones, toxins and nucleic acids, major bacterial polysaccharides. DEAE-C or Aktera-C caused a thin layer on a plate - best sorbents for quick and very fine fractionation of nucleotides and nucleic acids.
Distribution chromatography (PX). The separation of components of mixtures when this chromatography arise from differences in the distribution coefficients between the stationary phase, held inert absorbent, and the mobile phase is a solvent or gas; in the latter case we speak about gas chromatography (GRH). PX - the most common analytical and preparative method of fractionation similar structure of substances. As an inert medium is used mainly polar substances - cellulose (in the form of sheets of filter paper), rarely - silica gel. Technology of chromatography paper is very simple; depending on the direction of the solvent can be one-dimensional, two-dimensional or circular chromatogram. The movement of a solvent on paper can be ascending, descending and horizontal ways. The analyzed mixture is applied on paper in the form of a drop volume 1-5 MKL containing 10-8 -10-6 g each analyte. Upward or downward flow of solvent moves the components of the mixture according to the paper, forming a chain (- dimensional chromatogram) or pattern (two-dimensional chromatogram) spots (Fig. 3). The content of each isolated component determined by conventional methods after extraction of substances from the "spot" or less directly on paper but the spot area, determined based on its color, radioactivity or substance absorption of ultraviolet rays.
HRH allows with utmost precision to analyze ultramicrotomist (10-11 - 10-9 g) substances which, in the context of the analysis can be converted to gas or vapor state. It is used for studying the processes of respiration and gas composition of blood, determination of sex hormones of adrenal cortex hormones, bile acids, fat-soluble vitamins, especially vitamins D (D2 - D3), lipids and some volatile components tissue - lower alcohols, thiols, ketones. In biochemistry power HRH used to identify substances that determine the smells of food products, such as coffee, cheese, fried meat, etc.


Sedimentary chromatography. When this chromatography is the education components of the mixture of insoluble deposits due to their chemical interaction with reagent, impregnating sorbent. In biochemical and clinical studies of sedimentary chromatography while use is not found.
Gel filtration (GF). The separation of substances by the method of gel filtration based on a mechanical phenomenon of molecular sieving; GF molecules smaller than the diameter of pores homogeneous porous swollen "sorbent", evenly distributed between "external" and "internal" solutions; large molecules do not get in the grain "sorbent" and, therefore, move around the column with the speed of the flow of solvent. In biochemical research, the most widespread porous copolymers partially depolimerizovannogo bacterial polysaccharide dextran with different number of hydrophobic bridges, educated remnants of glycerol (sefadex) or polyacrylamide (biogels). The increase in the content of "linking agent" reduces the pore size of the sieve and the swelling properties of copolymer, reducing the absorption of large molecules, usually substances with a large mole. weight. The use of sephadexes different brands allows to separate substances with mol. weighing from 200 to 200 000 and biogels - from 200 to 300 000. "Poorly sewn" gels are used for analysis, isolation and purification of proteins, polysaccharides, and nucleic acids. "Tightly stitched" gels allow to fractionate amino acids, peptides, sugars, nucleotides. Fine gels used to separate low-molecular substances from high-molecular and replace dialysis.
Considered are the types of chromatography widely implemented in version thin-layer chromatography (TLC), at which fine sorbent or media applied to a thickness of 0.1 - 0.3 mm) on the glass plate. Technology of obtaining of thin-layer chromatogram similar chromatography on paper; the advantage is speed of separation and high sensitivity. TLC) on silica gels, Al2O3, kieselguhr, normal and ion-exchange cellulose or cephalexan is the most perfect method microanalysis. TLC is used in biochemical and clinical studies for separation, identification and quantitative determination of amino acids, peptides, carbohydrates, nucleotides, hormones, fatty acids, proteins, and so on; for example, a mixture of ATP and ADP of 5·10-4 umol each can be divided in a thin layer Aktera-pulp for 4-5 minutes; the mix of the four nucleotides is divided in these conditions for 15 minutes In analytical chemistry used TLC, at which the most common sorbents are alumina and silica gel.

Fig. 1. Successive stages of the process chromatographic separation of a mixture of substances (a, b, C and d): I - primary chromatogram; II-III stages afterwards; at the bottom - the output curves substances (a, b and c).
Fig. 2. Chromatogram of free amino acids deproteinising blood plasma of healthy people (1.7 ml); column size H,9 cm; division neutral and acidic amino acids.
Fig. 3. Two-dimensional chromatogram of a mixture of essential amino acids included in the composition of proteins and biological liquids. The first solvent: butanol - acetic acid; second solvent: phenol - NH3. 1 - cysteine acid; 2 - aspartic acid; in - glutamic acid; 4 - serine; 5 - taurine; in - glycine; 7 - glutamine; 8 - threonine; 9 - alanine; 10 - beta-alanine; 11 - hydroxyproline; 12 - tyrosine; 13 - histidine; 14 - lysine; 15 - arginine; 16 - methionine-sulfoxide; 17 - V-aminobutyric acid; 18 b - aminoadamantane acid; 19 - valine; 20 - phenylalanine; 21 - isoleucine; 22 - leucine; 23 - Proline.