HROMATOGRAFYYa, stratographic analysis — the physical and chemical method of division of liquid or gas mixtures based on distribution of their components between two immiscible phases one of to-rykh is not mobile (permanently), another — is mobile and continuously proceeds through a motionless phase. The chromatography is one of the most widely applied analytical methods in biochemistry and medical chemistry, toxicology, pharmacy. The method of chromatographic fractionation is irreplaceable in the analysis of pharmaceuticals, definition of resistance of pharmaceuticals to various influences, the analysis of mixes and substances of a natural origin, is suitable for division of polymers on a pier. to the weight (weight), structure and a microstructure (including on stereostructure). The chromatography is widely used in clinical diagnostic laboratories.
Development of bioorganic chemistry, physical chemistry of polymers and molecular biology gave to a chromatography a new object for a research — high-molecular compounds (see). There was a need for division of biopolymers, nucleic acids (see), proteins (see), and also viruses (see), phages (see the Bacteriophage), ribosomes (see) etc. The large role in it is played by methods of fractionation of biopolymers on ion-exchange cellulose and the affine chromatography based on biospecific sorption. Essential development is gained by methods of the analysis of substances and especially polymers, in to-rykh various hromatografichesky methods are combined with each other, and also with vi-skozimetrichesky (see Viscosity), sedimentation (see Sedimentation) and spectroscopic (see Spectroscopy) methods of the analysis.
Unlike other methods of division which are also based on distribution of substances between phases (e.g., extractions), the chromatography is a dynamic method since division happens in a flow of a mobile phase. Distinctiveness of hromatografichesky methods is their universality, i.e. a possibility of their use for division of liquid and gaseous organic and inorganic compounds of various structure in a wide interval of their concentration. As a mobile phase gas or liquid, as stationary (motionless) — liquid or solid matter acts most often. Division of components happens owing to distinction in speeds of their adsorption (see), dissolution or reaction to mobile and motionless phases.
The term «chromatography» was entered by M. S. Tsvet into 1901 — 1903 g for designation of separation process of vegetable pigments (see) during a transmission of the extracts supporting them through a glass column with calcium carbonate, and distribution in a column of differently painted components was called by it hromatogrammy. Offered M. S. Tsvet the main options of a method and developed the corresponding equipment. M. S experiences. Colors were reproduced only in 1941. After that the chromatography began to be improved in high gear against the background of development of the theory of adsorption and ion exchange (see. Ion exchange reactions) and progress in synthesis and use of new effective inorganic and organic sorbents, including ion-exchange resins (see Ionites). Technology of elution analysis was at the same time improved and the new principles of sorption division of substances were developed.
As it was told above, the hromatografichesky system consists of mobile and motionless phases. Components of the divided mix move through a porous stationary phase under the influence of the moving liquid or gas making a mobile phase. In nek-ry cases as a stationary phase serves tone-koizmelchennoye or the granulated solid matter placed in a narrow glass or metal tube (column), the mobile phase is passed sometimes through a column under pressure. In other methods the crushed occluding solid matter applied with a thin coat on a glass plate or special hromatografichesky paper can be a stationary phase. Movement of a mobile phase through solid matter is caused by capillary or gravitational forces. As a rule, solid matter does not take part in division, and only holds a stationary liquid phase. For chromatographic fractionation of mixes use also films from porous materials and special grades of paper. As a stationary phase motionless liquid, not mixing up with a mobile phase can serve.
Thanks to a tendency to achievement of a thermodynamic equilibrium (see Thermodynamics) between phases there is an exchange of molecules of the divided substances which is carried out by the directed diffusion flows (see Diffusion) from one phase in another and proceeding against the background of accidental movements of molecules in each of phases. In a motionless phase this movement has exclusively thermal character (see the Molecule), and in mobile — is connected also with a hydra (aero) dynamic conditions (see the Hydrodynamics engineer) currents of solution (see Solutions) or gas mixture (see Gaza).
At the movement along hromatografichesky system of a molecule of the divided substances a part of time are in a motionless phase (their average speed in the direction of the movement is equal to zero), and a part of time — in mobile where they move with a speed equal to the speed of the movement of a phase. Transition of molecules from a mobile phase in motionless is called sorption, and the return — a desorption. In the presence of the adsorptive interaction of a molecule are adsorbed on a surface of a sorbent (in the adsorption chromatography); for lack of the adsorptive interaction sorption comes down to stay of molecules in a motionless phase. If chromatographic fractionation is conducted with use of a porous sorbent, then molecules are late in its pore space as, e.g., in gel-fil.tratsionnoy of a chromatography (see a fir-tree filtering).
All types of chromatographic fractionation are based on different extent of distribution of solutes between mobile and stationary phases. The established balance quantitatively is described by a constant, temperature-dependent and called by a distribution coefficient. The distribution coefficient is equal to the relation of concentration of the analyzed substance in a motionless phase to its concentration in a mobile phase. Depending on a type of a chromatography it is called a distribution coefficient (in gas-liquid, or distribution, and ion-exchange chromatography), coefficient of adsorption (in the adsorption chromatography) and a permeability coefficient (in a molecular sitovoy of a chromatography). This parameter determines the speed of movement of substances by a layer of a sorbent. Speed of movement of substances at chromatographic fractionation is in these conditions size constant and characteristic for each substance. It is estimated in size RF, by regions. represents the relation of distance from the starting line of a hromatogramma to the center of the zone occupied by this substance at any moment to the distance passed by the front of solvent.
Depending on the chemical nature of molecules defining interaction with a sorbent, or from a ratio of their sizes spend different time in phases of hromatografichesky system with pore sizes of a sorbent of a molecule of the chromatographed substances. At the same time more occluded molecules spend in a motionless phase more time, than less occluded. As a result they move along hromatografichesky system more slowly, than and unmixing of substances on the components making them in the course of a hromatografirovaniye speaks. Along with it there is a washing out of a zone of each formulation component. It is a consequence of thermal agitation of molecules and the accidental nature (stochasticity) of hromatografichesky process. If the speed of washing out of zones of formulation components is less than difference of speeds of their movement along hromatografichesky system, then these components can be allocated from mix hromatografichesky pu? I eat.
Depending on the purposes of division distinguish the analytical chromatography used for the analysis of composition of complex mixtures, preparative chromatography — for receiving the substances which are in mix or in pure form and the chromatography applied for the purpose of physical. - chemical researches.
The most important indicators reflecting physical. - chemical essence and features of a stratographic analysis, the following is: 1) aggregate state of the divided substances — gas (steam) or liquid; 2) the nature of a sorbent — solid matter or liquid; 3) the nature of interaction between a sorbent and the divided substances — distribution of molecules or ions between two phases, formation of coordination connections in a phase or on surfaces of a sorbent, course of redoxreactions at contact of the divided substances with a sorbent; 4) technology of implementation of the analysis — in a column, a capillary, on paper, in a thin coat of a sorbent.
A variety of hromatografichesky methods does not allow to classify them by any one criterion. The same method of a stratographic analysis can be applied in various options. So, the sedimentary hromatogramma can be received in a column with a sorbent, on paper or in gels. A certain principle of division (e.g., distribution of molecules between phases) often is the cornerstone of various methods of a stratographic analysis. At the same time, napr, in a thin-layer chromatography, practically any principle of division — sorption, distribution, ion-exchange, etc. is possible. Put different methods and options of a chromatography into practice. It can differ in a form of a sorption layer, a way of movement of the analyzed mix, aggregate state of mobile and motionless phases, etc. E.g., depending on a form of a layer of a sorbent distinguish plane and column chromatography.
In a plane chromatography the sorbent in the form of a thin homogeneous coat is distributed on a plate (a chromatography in a thin coat, a thin-layer chromatography, TGX) or as a sorption layer serves special paper (paper chromatography). In comparison with paper chromatography of TSH was widely adopted more thanks to the simplicity and a possibility of division of small amounts of multi-component mixtures. As a sorbent in most cases apply silica gel to TSH, as a motionless phase usually serves water, and mobile — various solvents. TSH differs in big speed of division of substances, resistance to heating and a nek-eye to the reagents destroying paper and also high sensitivity. Thanks to these qualities the chromatography in a thin coat became one of the most widespread analytical methods applied in pharmacy, toxicology and biochemistry. The classical technique of a plane chromatography consists of the following operations: 1)
Nan sengiye of the analyzed test on a layer of a sorbent; 2) division of formulation components into separate zones in a flow of a mobile phase; 3) detection of zones on a layer of a sorbent (it is frequent by means of the reagent forming colored compounds with the divided substances); 4) quantitative assessment of the received division, including determination of content of substance in zones on a hromatogramma.
In column chromatography the sorbent is placed in special tubes — columns. Distinguish stuffed (nozzle) and capillary columns. In the first all volume of a tube is filled with a sorbent. Secondly — the sorbent is applied only on a surface of internal walls of a column. On a way of movement of the divided mix along a layer of a sorbent distinguish the following methods of column chromatography: frontal,
displacement, show l ny (elyuentny). If test serves as a mobile phase, then division call frontal. The displacement column chromatography is based on use of the elements having bigger affinity to a motionless phase than to solute. In elution column chromatography the mobile phase performs only transport function in relation to solute.
In the simplest option of column chromatography — the frontal analysis test is continuously given to a column. At the same time on a sorbent zones with consistently increasing number of components are formed. The least occluded substance moves ahead with greater speed and forms the feed zone or the front. The second zone contains mix of the second substance with the first, and the third zone represents mix already of three substances: the third, second and first etc. In the frontal analysis only one slabosorbi-ruyushchiysya a component is received in pure form. This method for analytical division is used a little. Much more often it is applied to concoction of light or heavy formulation components.
In displacement column chromatography test is brought in a column once and then formulation components move on a layer of a sorbent due to their replacement from a motionless phase by eluent, to-ry is occluded more actively than any of formulation components. At the same time the mobile phase forces out the most strongly occluded component, to-ry, in turn, forces out the following, less occluded component, etc. The zones corresponding to formulation components are as a result formed. Each of components can be allocated in pure form. However this allocation cannot be quantitative because of an overshoot and imposing of zones, to-rye are only ideally divided completely. This method as well as the method of a frontal chromatography, is a little used in analytical practice, sometimes it is applied to the preparative purposes.
At elution (elyuentny) column chromatography the mobile phase is occluded much less, than any of components of the divided mix therefore it serves only for movement of solute along hromatografichesky system. The analyzed mix is entered periodically. Division of components of the defined mix in this case happens thanks to their different affinity to a motionless phase and, therefore, thanks to different speeds of their movement along system. At the choice of optimal conditions of division it is possible to divide completely all components even of the most complex mixtures. Thanks to high performance of division this method was widely adopted and almost completely forced out other options of a chromatography.
If to register change of concentration of solute on escaping of hromatografichesky system and to represent this change graphically as function of time, on graphics a series of symmetric peaks will turn out. Such schedules which are also called by hromatogramma use both in qualitative, and in quantification: position of peak can
be used for identification of a component of test, and the space on a curve occupied by this peak characterizes concentration of substance.
As a mobile phase the chromatography shares on gas and liquid. The gas chromatography (gas adsorption, gas-liquid) is applied to division of volatile substances, solution chromatography — to the analysis and fractionation of thermolabile and non-volatile substances.
In gas chromatography carrier gas (usually hydrogen, helium or carbon dioxide) comes to a column of the gas-chromatograph. On the way of gas by means of the syringe pipette or a glass ampoule enter a certain amount of the studied substance (mix). Components of the studied mix are absorbed by adsorbent according to their sorption properties. At a further transmission of hectare for - the carrier the absorbed substances begin to be desorbed and to leave consistently a column. Depending on aggregate state of a motionless phase distinguish two types of gas chromatography: gas adsorption, in this case the column is filled with solid matter — adsorbent, and gas-liquid, at a cut the column is filled with grains of solid substance carrier with a liquid film.
In solution chromatography as a mobile phase use liquid. Depending on aggregate state of a motionless phase in the liquid and adsorptive and liquid liquid chromatography there can also be options.
As the sorbents used as a motionless phase and on the nature of the interactions causing distribution of components between mobile and motionless phases it is possible to allocate the following main types of a chromatography: the adsorptive; distribution; sedimentary; ion-exchange; affine; molecular sitovaya, or gel - filtrational; oxidation-reduction.
The adsorption chromatography is based on various adsorbability of components of mixes connected with features of their structure and sostAva. The section -
ny arises owing to an insignificant difference in adsorbability or in kinetics of sorption and a desorption of the analyzed divided substances. At the movement of the divided mix in a liquid or gaseous phase through the fixed bed of the sorbent consisting of discrete elements (the granules or fibers possessing a big total surface), elementary acts of sorption and a desorption repeatedly repeat. Repeated repetition of these processes is idiosyncrasy of a hromatografichesky method, it creates necessary conditions for division of components of complex mixtures with very close properties. Sorption ability depends both on the chemical nature of this or that formulation component, and on chemical and physical composition of the adsorbent. As adsorbents apply alumina, active coal, silica gel, au oxyquinoline and other substances. Efficiency of a hromatografichesky method is defined by various sorption ability of substances and speed of advance of zones during the washing of a column solvent. In practice often use consecutive washing away of substance solvents with gradually increasing stripping ability. In this case formulation components are consistently desorbed and washed away from system.
The partition chromatography is based on various solubility of the divided components in two immiscible solvents, one of to-rykh is applied on the surface of the macroporous firm carrier, indifferent to the chromatographed substances, and forms a stationary phase. After saturation of the carrier motionless solvent through system pass the second solvent with the analyzed mix dissolved in it and then the same solvent wash out a column. At the same time formulation components move with various speeds, inversely proportional to their distribution coefficients. The technique of columnar distribution chromatographic fractionation almost does not differ from the technique of the adsorptive division described above: washing out (eluating) a column solvent, collect separate portions of eluate, to-ry then it is analyzed by various methods (see E Lucia). The partition chromatography along with the adsorption gas chromatography is used also in the analysis of gases. In this case as a motionless phase apply the high-boiling liquid on adsorbent; division of gases results from their various solubility in this liquid film.
The sedimentary chromatography is based on process of formation of slightly soluble rainfall. In a heating-up period of a sedimentary hromatogramma there is a repeated repetition of process of education and dissolution of rainfall. At the same time distinctions in solubility of the formed rainfall cause division of the chromatographed substances. Sedimentary hromatogramma can be received on a column, hromatografichesky paper and in a thin coat. The columns applied at a sedimentary chromatography contain the carrier and a precipitator. For preparation of columns small used up substance carrier is impregnated with solution of substance precipitator and dried up (dry columns) or impregnate the carrier with solution of a precipitator and not dried up (wet columns). As the carrier use indifferent in relation to a wasp of dibodies yu and to the divided mixes the substance having the good filtering ability. Substances carriers can be as high-disperse (silica gel, aluminum oxide), and coarse-dispersion (fixed white). In sedimentary paper chromatography a role of the carrier is played by hromatografichesky paper. In vertical sedimentary hromatogramma of draft are evenly distributed on height of a zone, forming clear equal boundary. It gives the chance to use the size of zones as criterion at quantitative definition of substance. The sedimentary chromatography is applied in qualitative and quantification, to division and release of substances, concoction of substances, etc.
The ion-exchange chromatography is based on the phenomenon of exchange of the ions which are in solution and the ions adsorbed by solid adsorbent i.e. on distinction of constants of ion-exchange balance between two phases. The Hromatografichesky column is filled anion or cation exchangers — the solid matters having ion-exchange groups. As such adsorbents apply ion-exchange resins, or ionites (see) — the high-molecular compounds bearing the dissociating groups attached to a structural skeleton of pitch and giving it properties of acid (cation exchangers) or the bases (anion exchangers). Also amphoteric ionites, or ampholytes (see), capable to absorb both cations, and anions are applied. The method of the selection eluating is widely applied to division of ions. Selecting the corresponding structure and acidity of the eluating solution, it is possible to delete one ions from a column, leaving others in the adsorbed state. Ion-exchange columns with success are used for division of organic matters. Ions of streptomycin, e.g., are capable to replace ions of sodium in cation exchanger. Amino acids are occluded by anion exchangers and elyuirovana can be solution of ammonia. At the same time different amino acids are found in different portions of eluate. E.g., for anion exchanger of a vofatit an order of replacement of amino acids during the eluating following: at first it is eluated asparaginic to - that, then serine, glutaminic to - that, alanine, valine, a leucine.
The affine chromatography which is applied to the selection release and purification of biologically active agents, in particular enzymes is based on the specific interactions connected with biol. function of the emitted substance, including enzyme. As a sorbent use the ligands (the molecules and ions connected with a certain ion of substance carrier) immobilized on the carrier, to-rye selectively connect only certain enzymes. Communication between a molecule of enzyme and a ligand is carried out due to not covalent interactions. The multilateral contact between molecules of enzyme and a ligand resulting in sharp selectivity of sorption is provided with the certain spatial relationship of functional groups of enzyme connected with its unique spatial structure (see Conformation).
Molekulyarno - sitovy, or gel - filtratsion-naya a chromatography is used for division of substances on a pier. to scales. Development of this method is closely connected with creation of gels (see), during the use to-rykh sorption processes are almost excluded. Division at gel filter-tsionnoy of a chromatography is caused molecular sitovym by effect of gel thanks to various particle sizes of the divided substances and a time of the gel. At a transmission of substances with various a pier. scales (i.e. with various volumes of molecules) through gel with a time of a certain molecular size with large volume are late only in a time of the big size. Therefore they are washed away from a column before molecules of the small size, for to-rykh well bigger number and, therefore, bigger bulk volume of a time. Thus, substances quit the hromatografichesky system as reduction a pier. scales. Gel-filtrational of chrome togas a raffia is applied to division of substances with big a pier. scales. In biochemistry and polymer chemistry by means of this method desalinate and concentrate solutions, study kinetics of chemical changes, define chain-length distributions of polymers in their mix.
The Oxidation-reduction chromatography (oxychromatography) is based on education and distribution of zones of column chromatograms, containing reagent — an oxidizer or a reducer according to various ability of the analyzed substances to oxidation or recovery. Along with distribution of molecules or ions between two phases in an oxidation-reduction chromatography use oxidation reactions — recovery, in to-rykh ions and molecules of the substances capable to be oxidized or recovered participate. Oxidation or recovery of substances in a column happens in the sequence corresponding to their redox potential (see). On an oxidizing column first of all substance with the lowest value of redox potential reacts; on a recovery column — substance with the highest value of this potential. The method of an oxidation-reduction chromatography is used for high-quality detection of the substances forming the products painted oxidized or recovered.
Chromatographs. Special devices are used to a stratographic analysis — chromatographs, circuit and constructive decisions to-rykh are caused by specifics of this or that method of a stratographic analysis.
Chromatographs as the used mobile phase divide into two main classes: liquid and gas. Liquid chromatographs can work under the pressure up to 30,6 mm w.g. (300 Pas), gas — to 0,2 mm w.g. (2 Pas). Depending on features of a motionless phase chromatographs happen columnar and thin layer. The intermediate place is taken by capillary chromatographs, in to-rykh a thin coat of a sorbent apply on an internal wall of a capillary. To destination chromatographs divide on analytical and preparative. The first of them are intended for qualitative and quantification of complex mixtures, the second — for receiving a hromatograficheska of pure components of such mixes.
Chromatographs, is a floor z at yushchy
as a mobile phase gas, call gas. Specific elements of gas chromatographs are dividing columns, system of detecting, the device for input of tests and thermostats.
The components of gas test divided in a column shall be registered by the system of detecting consisting of the detector of this or that type, the measuring scheme and the self-recording device. The main requirements, to-rye show to modern detectors, a small inertance, high sensitivity, big dynamic range of the measured concentration are. In gas chromatographs several detectors usually are established and works.
There are detectors on heat conductivity, flame and ionization, konduktometrichesky, temperature and flame, etc. The greatest distribution was gained by flame ionization detectors and detectors on heat conductivity, the last apply in that case when heat conductivity of the analyzed substances and carrier gas significantly differ from each other. Ionization of molecules in the flame ionization detector happens in a zone of a hydrogen flame. Threshold sensitivity of flame ionization detectors for organic matters reaches 10 "4 mol.
In radio ionization detectors ionization of the defined molecules is carried out by means of radioisotopes (e.g., 3H or 1:47). The detector on cross-section of ionization is based on distinction of effective section of ionization of molecules of carrier gas and the analyzed components, and the detector on electron capture that the nek-ry substances having big affinity to electrons (e.g., haloid derivatives), are capable to form the negative ions causing sharp reduction of ionization current at ionization. The most sensitive and universal is the helium detector, ionization of components analiziruyemokh! tests in Krom occurs due to energy of metastable atoms of helium sufficient for ionization practically of all organic and inorganic compounds and stable gases.
In fotoioiizatsionny detectors ionization is carried out at the expense of the Uv-radiation received from special sources.
The most perfect chromatographs have systems of identification of components in size of the volumes withheld by a sorbent. On condition of constancy of gas flow rate retention volume is defined as the work of rate of volume flow of the gas given to reference conditions on retention time of a component.
Dosers of various types are intended for introduction to a dividing column of the analyzed tests of the set volumes with a necessary accuracy. In a lab. to practice in the analysis of gases and vapors apply obvshny microsyringes, to-rye are easy to use, allow to change easily the volume of test and to quickly enter it into a column. As automatic dosers rotary cranes with the channels or the calibrated tanks calibrated on volume are most often used. The calibrated ekhmkost under a certain pressure is filled with test from a flow of the analyzed gas or steam-gas mix, and then at turn of the crane is blown by carrier gas and by that it is entered into a dividing column.
Dosing of tests of a liquefied gas or steam can be carried out also by means of glass ampoules which are entered into special tight devices and collapse where their contents evaporate.
For providing optimal heating environments of division chromatographs are supplied with systems of thermostating of dividing columns with programmable change of temperature.
In the USSR various models of gas chromatographs are produced (JIXM-80; 3700, «Colour»). For the research purposes with success the laboratory universal JIXM-80 chromatograph can be used. The chromatograph is equipped a set of detectors, has nozzle and capillary columns. Division of the analyzed mixes can be made both in isothermal conditions, and at programmable rise in temperature to 75 — 300 °.
The chromatographs using various liquids as a mobile phase call liquid. For ensuring high performance of division in liquid chromatographs the calibrated columns from stainless steel are used.
Feature of a liquid chromatograph is ensuring continuous feeding of the solvent liquid playing a role of a mobile phase. Use the special pumps which are continuously giving solvent to a hromatografichesky column without fluctuations of pressure to this purpose. Usually it is piston pumps with a uniform cycle of giving, pumps syringes with the volumes sufficient for work during several running cycles, or besporshnevy giving of a liquid phase. In case of need pumps can perform functions of the device for gradient eluating. The most typical device for gradient giving of a mobile phase consists of two programmnoupravlyaemy pumps and the mixing chamber. The camera shall have rather small volume that from the beginning of mixing before emergence of a gradient of concentration of the eluating structure in an entrance to a column there passed the small time term.
The detector installed at the exit of a column provides quantitative definition of concentration of the divided formulation components in test, and the recorder registers qualitative and quantitative structure of test in the form of a hromatogramma. Detectors for liquid chromatographs shall have small volume (apprx. 5 mkl), to provide a possibility of measurements in the big dynamic range, to have good linearity, small dependence on the speed of current of a mobile phase and temperature. In liquid chromatographs the greatest distribution was gained by UF-spektrofoto-metricheskiye, flyuorimetrichesky, refraktometrichesky, polarimetric detectors, detectors on warmth of adsorption, etc. UF-spektrofotometrichesky and flyuorimetrichesky detectors differ in the highest sensitivity. With their help it is possible to determine the content of substance in test with concentration to 5 * 10-10 g/ml.
The most widespread devices for measurement of a flow rate of a mobile phase are rotameters and bubble otmetchik of time. The last provide the accuracy of definition reaching about 1% that it is enough for the majority of the definitions which are carried out by methods of solution chromatography (including and for definition of keeping of volumes of various samples).
In the USSR the release of several models of liquid chromatographs is mastered. The simplest and acceptable for medical researches is the chromatograph Millikhr. The device has the spektrofotometrichesky detector and microcolumns providing a possibility of the analysis of tests with capacities from 2 mkl. The Swedish firms «LKB» and «Pharmacia» release chromatographs and special columns for solution chromatography under big pressure [5,1 mm w.g. (50 Pas) — 30,6 mm w.g. (300 Pas)] high-molecular polymers, in particular proteins.
For carrying out division in a thin coat of a sorbent, and also on paper the set of the special equipment for a thin-layer chromatography of KTH-01 is developed. The equipment for preparation is included in this package hromatografichesky plates, drawing tests, manifestation hromatogramm in the chemical way, viewing hromatogramm (see Densitometryya), including in UF-light, and their photoregistration.
For carrying out qualitative and quantification of substances, marked isotopes, in addition provided the scintillation hromatografichesky USH-1 installation (see Scintillation).
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Nabivanetsb. And. and Mazurenko E. A. Stratographic analysis, Kiev, 1979; Roberts T. A radio chromatography, the lane with English, M., 1981; R at d e N to about B. A. Capillary chromatography, M., 1978;
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