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Size exclusion chromatography. Gel Permeation Chromatography GPC Basic Systems




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1 INSTITUTION OF THE RUSSIAN ACADEMY OF SCIENCES INSTITUTE OF ELEMENTO-ORGANIC COMPOUNDS im. A.N. NESMEYANOV. SCIENTIFIC AND EDUCATIONAL CENTER FOR PHYSICS AND CHEMISTRY OF POLYMERS MOSCOW

2 Table of contents. BASES OF CHROMATOGRAPHY OF POLYMERS. Driving forces and modes of polymer chromatography. Chromatographic peak characteristics. The concept of theoretical plates..3 Fundamentals of the size-exclusion (gel-penetrating) chromatography method. CARRYING OUT PRACTICAL WORK ON THE ANALYSIS OF MWD OF THE POLYMER BY THE METHOD OF GEL PERMEATION CHROMATOGRAPHY 3. REFERENCES. BASICS OF POLYMER CHROMATOGRAPHY. Driving forces and modes of polymer chromatography. Chromatography is a method of separating substances by distributing between two phases, one of which is mobile and the other is immobile. The role of the mobile phase in liquid chromatography is played by a liquid (eluent) moving in channels between particles along a column filled with a porous material (see Fig.). Fig. Movement of a macromolecule in a chromatographic column: d k - the size of the channels between the particles of the stationary phase; dn - pore size; R is the size of the macromolecule; t s - time spent by the macromolecule in the pore, t m ​​- in the mobile phase. The stationary phase is the pores of the sorbent filled with liquid. The average velocity of movement of this phase along the axis of the column is equal to zero. The analyte moves along the axis of the column, moving along with the mobile phase and occasionally stopping when it enters the stationary phase. This process is illustrated in Fig., which schematically shows the jump-like motion of a macromolecule with size R through channels with size d corresponding to the particle size. Molecules stop in slit-like pores, the size of which corresponds in order of magnitude to the size of macromolecules. The time between successive stops can be written as:

3 t t s + t m + t k, () where t s is the residence time of the molecule in the stationary phase, t m ​​d is the time spent by the molecule in the mobile phase (D - D is the transverse diffusion coefficient, t k is the time of transition from the mobile phase to the stationary phase and vice versa). Usually in the processes of high performance liquid chromatography (Hgh Performance Lqud Chromatography in the English literature) in its analytical version, this time t k is much less than the first two and can be omitted in the formula (). If the number of stops during the movement along the column is sufficiently large, then the total time of the macromolecule movement along the column is sufficiently large as compared to the characteristic time of equilibrium establishment. In this case, to determine the probability of finding a macromolecule in a unit volume of the stationary phase with respect to the mobile phase (or the distribution coefficient K d equal to the ratio of concentrations in these phases), the methods of equilibrium thermodynamics can be used. Namely, the distribution coefficient will be determined by the free energy of the transition of the macromolecule from the mobile phase to the stationary phase: T S H G RT Kd exp exp () RT For a chain consisting of N segments, K exp(N µ), (3) d where µ is the change in the chemical potential segment. The distribution coefficient in chromatography is a fundamental concept and is defined as follows: VR V K d (4) Vt V t is the elution volume of the substances leaving together with the solvent front. From (3), one can immediately see that, depending on the sign of G, the macromolecules behave differently when they enter the pore (see figure): Fig.. if G>, then K d tends to with increasing length of the macromolecule the volume of elution also decreases). This corresponds to size exclusion chromatography. At G< K d экспоненциально растет с ростом ММ и это соответствует адсорбционному режиму хроматографии. Таким образом, оба режима хроматографии могут рассматриваться в рамках единого механизма и, более того, плавно меняя энергию взаимодействия сегмента с поверхностью сорбента за счет состава растворителя или температуры, можно обратимо переходить от одного режима к другому. Экспериментально это было впервые показано в работе Тенникова и др. . Точка (для данной пары полимер - сорбент - это состав растворителя и температура), соответствующая равенству G, при которой происходит компенсация энтропийных потерь и энергетического выигрыша при каждом соударении сегмента макромолекулы со стенкой поры называется критической точкой адсорбции или критическими условиями хроматографии. Как видим, в этих условиях не происходит деления по ММ и это обстоятельство является предпосылкой для использования режима критической хроматографии для исследования разных типов молекулярной неоднородности полимеров, таких как число функциональных групп на концах цепи, состав блоксополимеров, топология 3

4 (presence of branched or cyclic macromolecules). This chromatographic method is relatively new and some of the most interesting results of its application can be found, for example, in [,3,4]. Chromatography mode corresponding to condition G< широко применяется для разделения низкомолекулярных соединений и называется, в зависимости от химической природы функциональных групп на поверхности сорбента, адсорбционной, нормальнофазной, обращеннофазной, ионпарной и т.д. хроматографией. Для полимеров его применение ограничено областью слабых взаимодействий вблизи критических условий и областью олигомерных макромолекул, т.к. с ростом длины цепи мы переходим к практически необратимой адсорбции макромолекулы на колонке. Наиболее важным для полимеров является режим эсклюзионной хроматографии или, как его еще называют, гельпроникающей хроматографии. Этот режим более подробно будет рассмотрен в следующем разделе, а сейчас мы перейдем к описанию некоторых важнейших хроматографических характеристик... Характеристики хроматографического пика. Концепция теоретических тарелок. После прохождения через хроматографическую колонку узкой зоны какого-либо монодисперсного вещества, на выходе мы получаем расширенную зону в виде пика приблизительно гауссова по форме (в случае хорошо упакованной колонки и правильно выбранной скорости хроматографии). Причины расширения пика лежат в различных диффузионных процессах, сопровождающих движение молекул вдоль колонки (см. например, соотношение ()). Наиболее важные характеристики пика - объем элюирования или V R или объем удерживания (относится к центру пика) и дисперсия пика, т.е. второй центральный момент (см.рис.3): σ h V V dv R. (5) Справедливы следующие соотношения между величинами, показанными на рис.3: σ, 43W W b. (6) 4 Рис. 3. Модель гауссова пика. Параметры уширения пика. Часто все эти величины выражаются в единицах времени, тогда говорят о времени удерживания и т.д., однако, в этом случае скорость потока элюента должна быть строго фиксирована. Существует простая феноменологическая теория описания относительного вклада расширения зоны в хроматографическое разделение. Это - теория тарелок. Хроматографическая колонка мысленно делится на ряд последовательных зон, в каждой из которых достигается полное равновесие между растворенным веществом в подвижной и неподвижной фазе. Физическую основу этого подхода составляет скачкообразное движение, описанное в начале первого раздела, и число теоретических тарелок в колонке связано с числом остановок при попадании в неподвижную фазу за время движения данного вещества по колонке. Чем больше это число, тем больше число теоретических тарелок и тем выше эффективность колонки. Число теоретических тарелок определяется следующим образом: 4

5 VR N σ V 5.54 W R V 6 W R b. (7) Since this value changes with the elution volume, it is correct to use the unretained substance exiting at K d..3 to characterize the efficiency of the column. Fundamentals of the size-exclusion (gel-penetrating) chromatography method. Size exclusion chromatography (Sze Excluson Chromatography, SEC) or gel permeation chromatography (GPC, Gel Permeaton Chromatography, GPC) is implemented when the behavior of macromolecules in pores is determined by the entropy component of free energy, and the energy component is small compared to it. In this case, the distribution coefficient will depend exponentially on the ratio of macromolecule size and pore size. The scaling theory predicts the following regularities for the case of pores commensurate with the macromolecule size R K d Aexp D α, (8) 4/3 to depending on the adopted pore model (slit, capillary, strip) and the chain model (ideal or imperfect). Thus, the behavior of macromolecules under the conditions of size exclusion chromatography is determined by the chain size. The size of a macromolecule is determined by its chemical structure, the number of links in the chain (or molecular weight), topology (for example, the size of a branched macromolecule or macrocycle decreases compared to a linear macromolecule of the same chemical structure). In addition, the size of flexible macromolecules depends to a certain extent on the solvent used due to the excluded volume effect. However, the GPC method has become widely used in laboratory practice as a method of separation by molecular weights, determination of average molecular weights and molecular weight distributions (MWDs). The development of the method began in the mid-1950s, when the first wide-pore organic sorbents for high performance gel permeation chromatography were created. As can be seen from relations (8), the method is not absolute for determining molecular weights, but requires an appropriate calibration against standard (preferably narrowly dispersed) samples with known MW, relating the retention volume (or time) to MW. Figure 4 illustrates the calibration curves for polystyrene in terms of lg V R on Waters semi-rigid organic sorbents (crostyragel) with different pore sizes. For the analysis of any polymer by molecular weight, it is necessary to select a column with an appropriate pore size or a series of columns with different pores, or use a column with a mixture of sorbents with different pores (the Lnear column in the given example). Of course, in order to use the GPC method for the analysis of MWD, it is necessary to provide conditions for the implementation of the exclusion mechanism of separation, which is not complicated by the effects of the interaction of both middle and terminal links of the chain. We are talking about adsorption interaction from a nonpolar solvent or reversed-phase interaction of nonpolar chain fragments during chromatography of hydrophilic polymers in an aqueous medium. In addition, water-soluble polymers containing ionized groups are capable of strong electrostatic interactions and require especially careful selection of chromatography conditions. The selection of conditions includes the selection of a sorbent and solvent (eluent) suitable for a particular analysis in terms of chemical structure. 5

6 Recommendations can be found in the manuals of chromatographic equipment manufacturers, as well as in reference books and monographs (see, for example, ), 6 V R, ml Pic. 4. Calibration curves for µstyragel columns. The figure shows the corporate labeling of the columns with a value that characterizes the size of the sorbent pores, which is equal to the length of the extended polystyrene chain excluded from the pores for steric reasons. The chromatographic column is the heart of the liquid chromatograph. The chromatograph also includes a number of necessary additional devices:) an eluent supply system (pump) that provides a stable flow,) a sample injection system without stopping the flow (injector or autosampler), 3) a detector - a device that provides the formation of a signal proportional to the concentration of a substance at the column outlet (detectors are of various types, the most popular in gel permeation chromatography are refractometric and spectrophotometric detectors), and 4) data acquisition and processing systems based on a personal computer. In modern chromatographs, the operation of all parts of the chromatograph is often also controlled by means of a control program integrated with the data processing system. The polymer chromatogram obtained under size exclusion chromatography F(V) is a reflection of its molecular weight distribution function W(). By virtue of the law of conservation of matter: F V dv W d ). The real chromatogram is the result of the separation of the sample by MW when moving along the column and the simultaneous mixing of polymer homologues due to the blurring of the zones. Therefore, the function F(W) in relation (9) should be understood as a chromatogram corrected for PU. This function is a solution to the Fredholm integral equation of the first kind. There are quite a lot of ways to correct for PU. See, for example, . However, in modern high-performance chromatographic systems, in most cases, the contribution of PU to the chromatogram is small compared to MWD and can be neglected. The most important procedure is the calibration of the chromatograph according to the molecular weight of the polymer under study. If there are corresponding narrowly dispersed standards with different MM, the elution volumes (V R or Ve) are determined for them and a calibration dependence similar to that shown in Fig. 4 is built. Typically, the calibration relation is sought in the form (): n lg C V e () Polynomials of the first or third degree are most often used. Polynomials of odd degrees (3, 5, 7) most accurately describe the characteristic shape of the calibration curves with upper and lower MM limits. Sets of narrowly dispersed standards exist for such polymers as polystyrene, polyisoprene, polymethylmethacrylate,

7 polyethylene oxide, dextrans and some others. You can also use the method of universal calibration, first introduced into practice by Benoit and co-workers. The method is based on the fact that the hydrodynamic volume of macromolecules is proportional to the product of the intrinsic viscosity and the molecular weight of the polymer and can be used as a function of the elution volume as a universal parameter for different polymers. Then we build a universal gauge relation (), () lg η n BV e, () using a set of some standards and the well-known Mark-Kuhn-Houwink relation (3): η K a. (3) To pass from a relation of the form () to a calibration dependence () for the polymer under study, it is sufficient to use the corresponding Mark-Kuhn-Houwink relation, after which we obtain (4): lg n B V e + a lg K. (4) As a result, From the data of gel permeation chromatography, one can find the average molecular weights of various degrees of averaging, which, by definition, are the following values: () n - number average MM, W () d W d w z W d W d W d W d - weight average MM, - z-mean MM. The MM ratios of different degrees of averaging characterize the statistical width of the MMD. The most commonly used ratio is w / n, which is called the polydispersity index. 4. CARRYING OUT PRACTICAL WORK ON THE ANALYSIS OF MWD OF A POLYMER BY METHOD OF GEL PENETRATION CHROMATOGRAPHY Purpose of the work: To get acquainted with the operation of a liquid chromatograph, the method of conducting a chromatographic experiment, the method of calibrating a chromatograph according to narrowly dispersed polymer standards and calculating average molecular weights. Equipment:) Liquid chromatograph, consisting of a pump, an injector, a column thermostat, a column with a polymeric sorbent and a data processing system based on a personal computer.) A set of narrowly dispersed standards with different MM (polystyrene or polyethylene oxide). 3) Test sample with unknown molecular weights. Operation procedure:) Preparation of a solution of a mixture of standards. 7

8) Obtaining a chromatogram of the standards and determining their retention volumes (V e). 3) Construction of the calibration dependence in the form (). 4) Preparation of a solution of the investigated polymer. 5) Obtaining a chromatogram of the investigated polymer. 6) Calculation of the average MM of the sample. Figure 5 shows a typical example of a polymer sample chromatogram prepared for calculating the average MW, namely, a baseline is drawn that defines the beginning and end of the chromatogram, and then the chromatogram is divided into equal parts along the time axis, the so-called slices. n w z A, A A A, A A. 5. For each slice, its area A is determined and the molecular weight corresponding to its middle is calculated from the calibration dependence. The average molecular weights are then calculated: 8

9 3. LITERATURE. M. B. Tennikov, P. P. Nefedov, M. A. Lazareva, S. Ya. Comm., A, 977, v.9, N.3, with S.G.Entelis, V.V.Evreinov, A.I.Kuzaev, Reactive oligomers, M: Chemistry, T.M.Zimina, E.E. Kever, E.Yu. Melenevskaya, V.N. Zgonnik, B.G. Belenkiy, On the experimental verification of the concept of chromatographic "invisibility" in the critical chromatography of block copolymers, Vysokomolek. comm., A, 99, vol. 33, N6, with I.V. Comm., A, 997, v.39, N6, with A.M. Skvortsov, A.A. Gorbunov, Scaling theory of chromatography of linear and ring macromolecules, Vysokomolek. comm., A, vol. 8, N8, with B. G. Belenkiy, L. Z. Vilenchik, Chromatography of polymers, M: Chemistry, W. W. Yau, J. J. Krkland, D. D. Bly, orn Sze-Excluson Lqud Chromatography, New York: John Wley & Sons, E.L. Styskin, L.B. Itsikson, E.B. Braudo. Practical High Performance Liquid Chromatography. Moscow Ch Wu, Ed.Column Handbook for Sze Excluson Chromatography, N-Y: Academc Press..Z.Grubsc, R.Rempp, H.Benor, J. Polym. Sc., B, 967, v.5, p


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In this method, the analyzed solution is passed through a column filled with swollen granular gel (stationary phase). The gel particles consist of a macromolecular compound (HMC) having a network structure (flexible macromolecules are crosslinked by chemical bonds). For this reason, the swollen gel has a network structure, between the nodes of which there is a solvent.

The distribution of the interstitial space of the gel along the radii- the main characteristic of the gel used, it depends on the nature of the polymer and solvent, grid frequency and temperature.

The effect of separation of substances in the case of gel chromatography is due to the fact that molecules that differ in molar mass (length) are able to penetrate into the gel structure to different depths and stay in it for different times. Therefore, during elution, large molecules that are not able to penetrate deep into the gel granules come out of the column first, and the smallest molecules come out last. There is a kind of sieving of molecules through the interstitial space of the gel.

Chromatography is carried out as follows. The gel granules are placed in a glass column, allowed to swell in the solvent, and then the analyzed mixture of substances is fed into the column. Small molecules are evenly distributed over the entire volume of the granules, while larger molecules, being unable to penetrate inside, remain only in the solvent layer surrounding the granules (outer volume). Next, the column is washed with a solvent - eluent. As already noted, large molecules move through the column at a higher speed than small ones, the movement of which is constantly slowed down by diffusion deep into the granules of the stationary phase. As a result, the components of the mixture are eluted from the column in descending order of their molar mass. Samples (fractions) of the eluent leaving the column are taken for analysis. The experiment is greatly simplified if there is the possibility of continuous automatic analysis of the eluent.

For research, the gel should be chosen so that its affinity for the analyzed substances is minimal: in this case, the substances are able to freely mix along the column layer in accordance with the size of their molecules. The gel granules must have optimal dimensions: too small - contribute to the rapid establishment of diffusion equilibrium, but cause high hydraulic resistance of the column. The use of large granules gives low hydraulic resistance, but inhibits diffusion, increasing the time of release of the analyzed substances.

In addition, the granules must have a certain mechanical strength, otherwise their deformation in the column will lead to a drop in the elution rate.

The most widely used for gel chromatography is sephadex(dextran gel - a high molecular weight polysaccharide), formed by growing certain bacteria in a sucrose medium. Eight types of Sephadex are produced, differing in the degree of their swelling, it is resistant to alkalis and weak acids.

Consider a specific example of the separation of a mixture of starch and glucose on Sephadex G- 25. 2 cm 3 of an aqueous solution of starch and glucose were placed in a column with 87 g of gel and the mixture was eluted with a solution of common salt. The filtrate fractions were collected and their starch and glucose content was determined. Starch molecules practically did not penetrate into the gel granules, so starch was eluted first at an eluent flow rate of 32–44 ml, and glucose was eluted second at an eluent flow rate of 66–80 ml.

Based on the data obtained, a chromatogram was constructed. To do this, the concentration of substances in fractions was plotted along the ordinate axis, and the volume of the eluent (or fraction number) was plotted along the abscissa axis. Determined from the chromatogram retention volumes V/ is the total volume of the collected eluent until the fraction with the maximum concentration of the substance exits the column. From a particular column, a given substance is always eluted at the same V,. In the case under consideration, the retention volume for starch turned out to be 35 ml, and for glucose - 73 ml.

The retention volume of substances is reproduced quite accurately. Therefore, with the help of gel chromatography, it is also possible to solve the inverse problem - to determine the molar mass of unknown compounds by determining them V,. To do this, the column is first calibrated: the retention volumes of IUDs (standard polymers) with a known molar mass are determined. For this purpose, proteins with a known fixed molar mass are most often used to calibrate hydrophilic gels. In addition, for a number of globular proteins, in addition to the molar mass determined chemically, the size of their molecules is also known. Thus, using a column calibrated with known proteins, one can also get an idea of ​​the effective radius of the studied molecules.

5. Gel chromatography

Gel filtration (synonymous with gel chromatography) is a method for separating a mixture of substances with different molecular weights by filtering through various so-called cellular gels.

The stationary phase in gel chromatography is the solvent located in the pores of the gel, and the mobile phase is the solvent itself, i.e. both the mobile and stationary phases are the same substance or the same mixture of substances. The gel is prepared on the basis of, for example, dextran, polyacrylamide or other natural and synthetic compounds.

Unlike other chromatographic methods that use differences in the chemical properties of the substances to be separated, which manifest themselves during their distribution between the stationary and mobile phases, the separation is based on the sieve effect, which is characteristic of gels with a certain pore radius. The solvent (mobile phase) fills both the external volume between the gel grains and the internal pore volume. The volume of the solvent between the gel grains - V m is called the intermediate, transport or dead volume, and the internal pore volume - V p is considered as an object of the stationary phase. When a sample containing several types of ions or molecules with different sizes is introduced into the column, they tend to penetrate the pores from the mobile phase. Such penetration is due to the entropy distribution, since the concentration of molecules of the substances to be separated in the external solution is higher than in the pore space. But it becomes possible only if the size of the ions or molecules is less than the diameter of the pores.


Figure 5 General view of the calibration curve in gel chromatography:

1 - region of exclusion, where all molecules are larger than m 2 ;

2 - area of ​​penetration or separation, where the sizes of molecules lie in the range from m 1 and m 2;

3 - the area where there is a complete penetration of molecules with sizes less than m 1.

In the process of gel chromatography, large molecules can be separated, which are not adsorbed by the gel, since their sizes exceed the pore sizes, from small ones, which penetrate into the pores, and then can be eluted. Finer separations are also carried out, since the pore sizes can be controlled by changing, for example, the composition of the solvent and, as a consequence, the swelling of the gel. Gel chromatography can be performed in column and thin layer versions.

Used in practice, gels are usually divided into soft, semi-rigid and hard. Soft gels are high-molecular organic compounds with a small number of cross-links. The capacitance factor, equal to the ratio of the volume of the solvent inside the gel to its volume outside the gel, is 3 for them. When swelling, they significantly increase their own volume. These are sephadex or dextran gels, agar gels, starch, etc. They are used to separate mixtures of low molecular weight substances, often in a thin layer version. Chromatography on soft gels is called gel filtration.

Semi-rigid gels are obtained by polymerization. Styrogels, products of the copolymerization of styrene and divinylbenzene with a large number of cross-links, are widely used. The capacity factor of semi-rigid gels is in the range of 0.8 ... 1.2, their volume does not increase very significantly during swelling (1.2 ... 1.8 times). Chromatography on semi-rigid gels is called gel permeation chromatography.

Rigid gels include silica gels and often porous glasses, although they are not gels. Rigid gels have a small capacity factor (0.8...1.1) and a fixed pore size. These materials are used in high pressure gel chromatography.

Solvents for gel chromatography should dissolve all components of the mixture, wet the gel surface and not be adsorbed on it.

The practical application of gel chromatography is mainly associated with the separation of a mixture of high molecular weight compounds, although they are often used for the separation of low molecular weight compounds, since separation by this method is possible at room temperature.

6. High performance liquid chromatography (HPLC)

High performance liquid chromatography is the most efficient method for analyzing complex organic samples. The sample analysis process is divided into 2 stages:

separation of the sample into its constituent components;

· detection and measurement of the content of each component.


The problem of separation is solved using a chromatographic column, which is a tube filled with a sorbent. During the analysis, a liquid (eluent) of a certain composition is fed through a chromatographic column at a constant speed. An accurately measured sample dose is injected into this stream.

The components of the sample introduced into the chromatographic column, due to their different affinity to the sorbent of the column, move along it at different speeds and reach the detector sequentially at different times.

Thus, the chromatographic column is responsible for the selectivity and separation efficiency of the components. By selecting different types of columns, you can control the degree of separation of the analyzed substances. Compounds are identified by their retention time. The quantitative determination of each of the components is calculated based on the magnitude of the analytical signal measured using a detector connected to the output of the chromatographic column.

In the analysis of compounds with low MPCs (biogenic amines, polyaromatic hydrocarbons, hormones, toxins), the sensitivity and selectivity of the method becomes especially important due to the complexity of preparing real samples. The use of a fluorimetric detector makes it possible not only to reduce the limits of detection, but also to selectively isolate the analyzed substances against the background of the matrix and related components of the sample.

The HPLC method is used in sanitary and hygienic research, ecology, medicine, pharmaceuticals, petrochemistry, forensics, quality control and product certification.

The pump "Python" of the syringe type is used as the eluent supply unit, which has the following features:

· absence of pressure pulsations when supplying the solvent;

a wide range of volumetric flow rates;

large volume of the pump chamber;

Extensibility (the ability to combine several blocks to create a gradient system).

Different types of detectors can be used in the chromatographic system, for example, "Fluorat-02-2M" (spectral selection is carried out by filters) or "Fluorat-02 Panorama" (spectral selection is carried out by monochromators).

7. Application

Liquid chromatography is the most important physical and chemical research method in chemistry, biology, biochemistry, medicine, and biotechnology. It is used to analyze, separate, purify and isolate amino acids, peptides, proteins, enzymes, viruses, nucleotides, nucleic acids, carbohydrates, lipids, hormones, etc.; study of metabolic processes in living organisms of drugs; diagnostics in medicine; analysis of products of chemical and petrochemical synthesis, intermediates, dyes, fuels, lubricants, oils, wastewater; study of isotherms of sorption from solution, kinetics and selectivity of chemical. processes.

In the chemistry of macromolecular compounds and in the production of polymers, liquid chromatography is used to analyze the quality of monomers, study the molecular weight distribution and distribution by type of functionality of oligomers and polymers, which is necessary for product control. Liquid chromatography is also used in perfumery, the food industry, for the analysis of environmental pollution, and in forensics.


Conclusion

The beginning of the 20th century was marked by the discovery of the chromatographic method of analysis, which enriched and united various fields of science, without which the scientific progress of the 21st century is unthinkable. The introduction of chromatographic methods, and primarily liquid chromatography, into medicine has made it possible to solve many vital problems: the study of the degree of purity and stability of drugs, the preparative isolation of individual hormonal drugs (for example, insulin, interferon), the quantitative determination of neurotransmitters in biological objects: adrenaline, norepinephrine. The presence of these substances in a living organism is associated with the ability to memorize, learn, acquire any skills. HPLC identification of steroids, amino acids, amines, and other compounds proved to be extremely important in the diagnosis of certain hereditary diseases: myocardial infarction, diabetes, and various diseases of the nervous system. One of the urgent tasks of clinical medicine for express diagnostics is the so-called profile analysis of the components of a biological object, carried out by liquid chromatography, which makes it possible not to identify each peak, but to compare chromatogram profiles to conclude about the norm or pathology. The processing of a huge amount of information is carried out only with the use of a computer (the method is called the "pattern recognition method").


Bibliography

1. V. P. Vasiliev, Analytical Chemistry, in 2 books. Book. 2 Physical and chemical methods of analysis: Proc. for stud. universities studying chemical engineering. specialist. - 4th ed., stereotype. – M.: Bustard, 2004 – 384 p.

2. Moskvin L.N., Tsaritsyna L.G. Separation and concentration methods in analytical chemistry. - L.: Chemistry, 1991. - 256 p.

3. http://bibliofond.ru/view.aspx?id=43468

4. http://ru.wikipedia.org/wiki/Paper_chromatography

5. http://referats.qip.ru/referats/preview/93743/6

6. http://www.curemed.ru/medarticle/articles/12186.htm

7. http://www.lumex.ru/method.php?id=16

8. http://www.xumuk.ru/encyklopedia/1544.html

9. http://www.pereplet.ru/obrazovanie/stsoros/1110.html

size exclusion chromatography

Gel filtration or size exclusion chromatography(sieve, gel permeation, gel filtration chromatography) - a type of chromatography, during which the molecules of substances are separated in size due to their different ability to penetrate into the pores of the stationary phase. In this case, the largest molecules (higher molecular mass) capable of penetrating into the minimum number of pores of the stationary phase are the first to leave the column. Substances with small molecular sizes, which freely penetrate into the pores, come out last. Unlike adsorption chromatography, in gel filtration, the stationary phase remains chemically inert and does not interact with the substances to be separated.

Principle

A sample solution is introduced into the column, the volume of which is limiting for the quality of chromatography. For analytical separations, it should not exceed 0.1% of CV (total column volume), and for preparative purification, it should not exceed 8-10% of CV. The column is packed with powder, the particles or granules of which have pores of a certain diameter. Macromolecular substances that do not enter the pores pass between the granules, so their retention volume is equal to the volume of the column minus the volume of the stationary phase (the so-called free volume). They elute first. Molecules of medium size fit into the pores of the sorbent, but not completely. Therefore, their retention volume is slightly higher than the free volume. They elute second. The smallest molecules freely enter the pores together with solvent molecules. Therefore, their retention volume in the column is much higher than the free volume and approaches the total volume of the column (i.e. 100% CV). They elute last.

Sorbents

Gel - a heterogeneous system in which the mobile phase (usually water) is always inside the pores of a stationary or solid phase, called the gel matrix.

Low pressure

  • dextran,
  • sephadex,
  • sefakril,
  • sepharose,
  • superdex.

High pressure

  • polymethacrylate,

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See what "Size Exclusion Chromatography" is in other dictionaries:

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The physical basis of this method is very simple and clear. The investigated polymer solution flows through a column filled with a porous sorbent. The separation of component mixtures is based on the distribution of the substance between the mobile (flowing solvent) and stationary (solvent in the pores of the sorbent) phases, i.e., on the different ability of polymer macromolecules to penetrate into the pores of gel granules, hence the name of the method.

The surface of the sorbent granules is covered with many channels, depressions and other irregularities, conditionally called pores, the total volume of which is V„. The volume inaccessible to the solvent is called the dead volume. Let a solution flow past such a surface, the size of which is commensurate with the size of the pores or less than them. Some of these molecules penetrate into the pores if their concentration in the moving phase is greater than in the pores. When the solute zone leaves this area of ​​the sorbent, the concentration of molecules inside the gel pores becomes greater than outside, and the molecules again diffuse into the flow of the mobile phase. If the size of the molecules is larger than the size of the pores, then such a molecule passes by the gel granule without lingering, i.e., it is excluded (exclusion) from the pore space. Thus, larger macromolecules flow through the column faster. This means that different polydisperse sample molecules will exit the column at different times for different retention volumes. VR

VR= V0 +kvV„ >

Where Vo- volume of the mobile phase (current solvent); kv- pore volume distribution coefficient: for large macromolecules completely excluded from pores, kv = 0; for solvent molecules kv= 1),

Values VR depend mainly on temperature, the nature of the solvent and the concentration of the solution.

The behavior of a macromolecule in solution can be easily described in detail if its Gibbs energy is determined AG. If a macromolecule enters a pore, its entropy decreases. In the presence of interaction of segments of the macromolecule with the walls of the pore, the enthalpy changes: with attraction, the enthalpy decreases, and vice versa. Therefore, in the absence of adsorption AG > 0, with strong adsorption of macromolecules on the pore walls AG < 0. Accordingly, in the first case, size exclusion chromatography (size distribution) takes place, and in the second - adsorption; conditions at AG=0 called critical. Because in the region AG > 0, macromolecules are separated by size, analysis by molecular weights of linear polymers is possible. If the polymer is branched, the separation process becomes more complicated and depends on the type and number of branches, and in the case of copolymers, also on the composition and blockiness of the chain.

Gels of hydrophobic materials, such as polystyrene cross-linked with divinyl-benzene, have received the greatest use as sorbents: In such gels, the effects of adsorption of analyzed samples are almost completely absent. Recently, macroporous glasses have been widely used, which have a number of advantages compared to a polymeric sorbent (particle rigidity, pore size variation, chemical stability) and disadvantages (increased sorption of polymers on them).

The most commonly used solvents are tetra-hydrofuran (THF), chloroform, toluene, cyclohexane and mixtures thereof. Preference is given to THF, which, unlike toluene, does not form micelles or aggregates with polymer macromolecules and is transparent in the UV region of the spectrum. In addition, the efficiency of method 11IX using THF is maximum at rather low temperatures (35–45°C). However, during long-term storage, THF oxidizes with the formation of explosive peroxide compounds; therefore, it must be pre-purified. Using THF as a solvent, all rubber grades as well as thermoplastic elastomers can be analyzed. When carrying out the analysis of nitrile butadiene rubber, it is advisable to use a mixture of solvents, one of which has an affinity for the non-polar rubber unit, and the other for the polar one. If a refractive index detector is used, a necessary requirement for the solvent is the difference between the refractive indices of the solvent and the polymer.

For the first time, an instrument for gel chromatography poly analysis Merov was released by "Waters" in 1964, later five years after Method discoveries. Today, liquid chromatographs for analysis Molecular weight distribution (MWD) polymers are produced in all industrialized countries, in Russia chromatographs of the KhZh series are known. Among the latest modifications of foreign devices is a gel chromatograph manufactured by "Waters Chem. Div." with a viscometer to determine the molecular weight, MWD, as well as the degree of orientation of macromolecules. The carousel design of the device allows you to simultaneously test 16 samples.

The block diagram of the chromatograph includes: О Degasser block - serves to remove gases from the solvent and helps to maintain the same amount of solvent for a long time.

О Dispenser block - allows you to enter a sample of a given volume in time and work in automatic mode,

О In modern liquid chromatographs, the conversion of the chromatogram into the MWD of the polymer, including instrument calibration by molecular weight and correction for instrument broadening, is carried out using a computer. This allows us to calculate the differential and integral MWD and the average values ​​of the molecular weight using the accepted programs. Special microprocessors control the operation of the device blocks according to a given program.

An example of recording the conditions of an experiment conducted by the method of gel permeation chromatography. The installation consists of the following main elements; pump model 6000A, sampler U 6K and differential refractometer R 401. The unit also includes 3 separating columns ^ each 300 mm long and with an internal diameter of 8 mm. The columns are filled with SDV-Gel 5 which has a pore diameter of 103, 104 and 105 A (Polymer-Standard-Service, PSS, Mainz). The test temperature is 22° C. and the flow rate is 1.0 ml/min. Tetrahydrofuran is used as a solvent, the injection volume is 100 µl at a sample concentration of 6-10 g/l. Universal calibration is performed on polystyrene with a molecular weight of 104-106 g/mol.

GPC allows you to study subtle changes in the chemical structure of polymers and determine the total MWD, and therefore is widely used in polymer chemistry. In the industrial production of elastomers, the GPC method can be used for operational quality control of commercially available products and appropriate adjustment of the technological process, as well as in the development and improvement of obtaining elastomers with desired properties. Gel chromatographs can be included in automated process control systems with sampling for analysis directly from the reactor. The duration of the analysis, including sample preparation, is 20-30 minutes.