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Vezhkh systems for gel permeation chromatography. Size Exclusion Chromatography GPC Basic Systems




Gel chromatography as a method for determining molecular weight

Gel Permeation Chromatography is a type of column fractionation method in which the separation is carried out according to the molecular sieve principle. This principle was already known in the early 1950s, but it was only after Porat and Flodin rediscovered and widely used this method that it was recognized and widely used in scientific research. From that moment until 1964, more than 300 papers were published on this new fractionation method.

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 (of larger molecular mass) that are able to penetrate 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. In contrast to adsorption chromatography, in gel filtration, the stationary phase remains chemically inert and does not interact with the substances to be separated. 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. Molecules stop in slit-like pores, the size of which corresponds in order of magnitude to the size of macromolecules.

In size exclusion chromatography, molecules that are large in solution either do not penetrate at all, or penetrate only part of the pores of the sorbent (gel) and are washed out of the column earlier than small molecules. The ratio of the effective sizes of macromolecules and pores of the sorbent determines the distribution coefficient Kd, which determines the retention volume of the component VR in the column:

The effective size of a macromolecule in size exclusion chromatography is its hydrodynamic radius R, which together with the polymer molecular weight M determines the intrinsic viscosity of the polymer. The universal calibration dependence of V R on the product / equation (2) was first obtained experimentally by G. Benois, it has the form (Fig. 1):

where A and B are constants. Equation (2) is equally valid for linear and branched polymers, block and graft copolymers, and oligomers.

Rice. one.

molecular size exclusion chromatography

In the region from V 0 to V T (column volume available for the solvent and molecules below a certain size, corresponding to M min), the working dependence is linear (quasi-linear) in nature. Corresponding volumes V 0 and V T mol. masses represent the exclusion limits - M max (large molecules, do not penetrate into the pores of the sorbent) and M min, (molecules are small, completely penetrate into the pores of the sorbent). Sorbents with pores of the same size are theoretically capable of separating macromolecules within the limits commercial sorbents are characterized. To separate macromolecules in a large range of M, sorbents with a bimodal and trimodal pore size distribution are needed, providing a linear mol. mass calibration dependence in the range М = 10 2.5 - 10 6.5 . Maximum selectivity is achieved by increasing the volume of the sorbent's pore space for bimodal and trimodal sorbents, in addition, by optimal pore size distribution. It is important that when separating a mixture of macromolecules, their largest and smallest M should be within the limits of M MIN - M MAX characteristic of a given sorbent.

Mechanism of size exclusion chromatography. Size Exclusion Chromatography (SEC) or Gel Permeation Chromatography (GPC, Gel Permeation 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. Macromolecules in p-re are statistical. ensemble (statistical tangle). Their distribution between the porous sorbent and the solution is controlled by the change in the Gibbs energy during the transition of the macromolecule from the solution to the pores: where is the change in the enthalpy of the macromolecule due to the interaction. its segments with the surface of the sorbent (gel matrix); - decrease in entropy during the transition of the macromolecule from the solution to the pores; T - abs. t-ra. The separation of macromolecules occurs in the exclusion mode, when a K d , which depends on the ratio of the sizes of macromolecules and pores, is less than 1. To suppress the phenomena of ion exclusion and ion exchange sorption, which are undesirable for size exclusion chromatography, the surface of the sorbents is modified (to give it a neutral charge at pH > 4) , increase the ionic strength of the solvent, weakening the Coulomb interactions, add organic solvents, thereby shifting the pK of the polyelectrolyte or the isoelectric point of polyampholytes. On the other hand, ion exchange sorption and ion exclusion can be used to separate neutral macromolecules, polyanions, and polycations of the same size. Since the dissociation of polyelectrolytes increases with dilution of their solutions, during size exclusion chromatography, macromolecules at the edges of the chromatographic column, where their concentration is low, dissociate and move along the column not according to the laws of size exclusion chromatography, but according to the laws of ion exchange sorption and ion exclusion, depending on the charge of the sorbent surface and macromolecules, which leads to a distortion of the shape of the curve of the dependence of V and M (Fig. 2), and also makes it possible to diagnose the presence of one or another process.

Rice. 2. Size exclusion chromatography of neutral macromolecules (a) and polyelectrolytes: ion exclusion (b), ion exchange sorption (c)

Effects similar to ion-exchange sorption, but only to a lesser extent, can be observed during hydrophobic interactions of macromolecular segments with a sorbent surface modified by hydrophobic radicals or during electrostatic interactions of surface silanol hydroxy groups with functional groups of polar macromolecules. All these effects must be suppressed by size exclusion chromatography.

To analyze any polymer by molecular weight, it is necessary to select a column with a suitable pore size or a series of columns with different pores, or use a column with a mixture of sorbents with different pores (the Linear 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.

Size exclusion chromatography technique. To separate macromolecules in size exclusion chromatography, two types of columns are used: those operating in narrow = 10 2) and wide (= 10 4 - 10 5) ranges. Columns with a wide M range have a wide sorbent pore size distribution (bimodal, trimodal). This distribution is selected in such a way that, for a given degree of linearity of the calibration mol.-mass dependence and mass range, the greatest degree of selectivity is provided. Size exclusion chromatography is carried out using a chromatograph, the detector is a spectrophotometer or a flow refractometer with a sensitivity limit of 5 x 10 -8 units. refraction, which corresponds to a polymer concentration of 5-10 -5%. Typically, the instrument operates at room temperature, however, polyolefin size exclusion chromatography requires an elevated temperature, which increases separation selectivity, column efficiency, and analysis speed due to a decrease in the viscosity of the mobile phase. Modern chromatographs are equipped with an automatic device for preparation (polymer dissolution, solution filtration) and sample injection, a computer for interpreting the results of MMP analysis. The use of a combination of a refractive index detector and a photometer makes it possible to determine the MWD and branching indices without calibrating the chromatograph according to polymer standards. During gel filtration of proteins, it is necessary to take measures to prevent their adsorption on the sorbent and prevent their denaturation. Unlike size exclusion chromatography of synthetic polymers and oligomers, which is mainly used for analytical purposes, protein gel filtration is one of the most important methods for their isolation and purification.

Size exclusion chromatography uses macroporous inorganic or polymeric sorbents. For size exclusion chromatography of polar polymers, inorganic sorbents (silica gels and macroporous glasses) are modified with organosilicon radicals, and for size exclusion chromatography of hydrophilic polymers, with hydrophilic groups. Among the polymeric sorbents, styrene-divinyl-benzene sorbents are the most common (for size exclusion chromatography of high polymers and oligomers). For gel filtration of biopolymers, primarily proteins, hydrophilic polymer sorbents (sephadexes - dextrans with cross-links, as well as polyacrylamide gels) or macroporous silica gels modified with polysaccharides are used.

Size exclusion chromatography is effectively used in the development of new polymers, technological processes for their production, production control and standardization of polymers. Size exclusion chromatography is used to analyze the MWD of polymers, research, isolation and purification of polymers, including biopolymers.

Description

Together with the German company Polymer Standards Service (PSS) - one of the leading manufacturers of materials and equipment for gel permeation chromatography (GPC, GPC) or, in other words, size exclusion chromatography (SEC) - we offer complete solutions for the determination of average molecular weights polymers (natural, synthetic, biopolymers), molecular weight distribution and characteristics of polymeric macromolecules in solution. In this method, the separation of the analyte occurs not due to adsorption interactions with the stationary phase, but solely by the value of the hydrodynamic radius of macromolecules.

For the detection of components separated by molecular weight, at least one concentration detector (traditional HPLC refractive and spectrophotometric, evaporative light scattering detector), as well as special detectors for polymer analysis: viscometric, detector by laser light scattering. In combination with concentration detectors, these detectors make it possible to determine the absolute molecular weight, the conformation of macromolecules in solution, the radius of gyration, the hydrodynamic radius, the degree of branching, the constants of the Mark-Kuhn-Houwink equation, and virial coefficients. In the presence of calibration dependences, this system makes it possible to obtain comprehensive information about macromolecular objects and their behavior in solutions in just one analysis (~15 min), while the evaluation of these characteristics by traditional methods takes several days.

To process the measurement results, it is necessary to use special software. We offer flexible, modular HPLC systems for Gel Permeation Chromatography (GPC), including Prominence modules (pumps, column oven, autosamplers, refractive index detector) and specific modules from Polymer Standards Service (PSS), an authority on polymer HPLC analysis. To calculate the results of the analysis, it is possible to use both the Shimadzu GPC Option software integrated into the standard LabSolution LC program, and the use of PSS - WinGPC SW software products that support special detectors.

To work with mobile phases that are aggressive towards traditionally used capillaries and fittings (hexafluoroisopropanol, tetrahydrofuran), HPLC systems can be equipped with a special degasser, pumps and autosampler, the components of which are resistant to these solvents.

Basic systems for GPC

Basic HPLC system for GPC

A basic HPLC system for GPC can be configured with LC-20 Prominence units with one of the concentration detectors (spectrophotometric/diode array SPD-20A/SPD-M20A for UV-absorbing polymers, universal refractive index RID-20A and evaporative light scattering detector ELSD -LTII). This system, in the presence of suitable standards and calibration dependencies, makes it possible to determine the relative molecular weight of polymers, as well as to estimate the hydrodynamic sizes of macromolecules in solution.

Specifications of the main modules
Pump LC-20AD
Pump type Dual Parallel Micro Plunger Mechanism
Plunger chamber capacity 10 µl
Eluent flow rate range 0.0001-10 ml/min
Max pressure 40 MPa
Flow setting accuracy 1% or 0.5 µl (whichever is better)
Ripple 0.1 MPa (for water at 1.0 ml/min and 7 MPa)
Working mode constant flow, constant pressure
The pumps can be equipped with an additional device for automatic flushing of the plunger. The pumps are equipped with a leak sensor. The material of the pump plunger is resistant to aggressive media (sapphire).
Refractometric detector RID-20A
Radiation source Tungsten lamp, operating time 20000 hours
Refractive index range (RIU) 1,00 - 1,75
Temperature control of the optical unit 30 - 60С° with dual optical system temperature control
Operating range of flow rates Ability to work in a wide range of applications (from analytical mode to preparative chromatography) without changing the measuring cell: from 0.0001 to 20 ml/min in analytical mode; up to 150 ml/min in preparative mode
Noise 2.5×10 -9 RIU
Drifting 1×7 -7 RIU/hour
Linearity range 0.01-500×10 -6 in analytical mode
1.0-5000×10 -6 in preparative mode
Flow line switch solenoid valve
Max. operating pressure 2 MPa (20 kgf/cm²)
Cell volume 9 µl
Zero setting optical balance (optical zero);
auto-zero, zero fine-tuning by baseline shift
Column thermostat with forced air convection STO-20A
Controlled temperature range from 10C° above room temperature to 85C°
Temperature control accuracy 0.1C°
The internal volume of the thermostat 220×365×95mm (7.6L)
thermostat capacity 6 columns; in addition to the columns, 2 manual injectors, a gradient mixer, two high-pressure switching valves (6 or 7 ports), a conductometric cell can be installed
Capabilities linear temperature programming; tracking and saving to a file changes in column parameters, the number of analyzes, the amount of the past mobile phase (when installing the optional CMD device)
Performance monitoring solvent leakage sensor; overheat protection system

Light scattering detector

Multi-angle light scattering detector SLD7100 MALLS (PSS)

The SLD7100 MALLS (PSS) multi-angle light scatter detector allows you to measure static light scattering simultaneously at up to seven angles (35, 50, 75, 90, 105, 130, 145°) and determine the absolute values ​​of molecular weights, the true parameters of molecular weight distribution, estimate the size and conformation of macromolecules in solution. This detector eliminates the need for any standards and can also serve as a capacitance instrument (without an HPLC system) without any additional modifications.

Viscometric detector (PSS, Germany)

Viscometric detector DVD1260 (PSS)

The DVD1260 viscometric detector (PSS) when used as part of the LC-20 Prominence HPLC system, allows you to determine average molecular weights and molecular weight distribution parameters, using the universal calibration method, indispensable for macromolecules with complex and globular architecture, as well as the intrinsic viscosity, the constants of the Mark-Kuhn-Houwink equation, the degree of branching, virial coefficients and the conformation of macromolecules in solution, based on certain models already embedded in the software. The unique measuring cell of the detector is a four-arm asymmetric capillary bridge, which, unlike all analogues available on the market, does not contain delay cells (hold-up columns) - a special dilution tank is built into the comparative circuit, which makes it possible to reduce the analysis time by at least half and avoid the appearance of negative systemic peaks. The error of maintaining the temperature in the cell is less than 0.01 °C, which is the first critical factor in viscometric analysis.

Specifications:
Food 110 to 260 V; 50/60 Hz; 100 VA
Differential pressure range (DP) -0.6 kPa - 10.0 kPa
Inlet pressure range (IP) 0-150 kPa
Measuring cell volume 15 µl
Dilution compensation volume (reservoir) 70 ml
Shear rate (1.0 ml/min) < 2700 с -1
Noise level 0.2 Pa, differential pressure signal, 5 °C
analog output 1.0 V / 10 kPa FSD differential pressure
1.0 V / 200 kPa FSD inlet pressure
Total detector volume About 72ml (including tank)
Max. flow rate 1.5 ml/min
Temperature setting accuracy ±0.5 °C
temperature stability Not worse than 0.01 °C
Digital interface RS-232C, USB, Ethernet
Baud rate (baud) 1200 - 115200
Digital inputs Flushing, Zeroing, Injection, Error
Digital outputs Injection, Error
Weight About 4 kg
Dimensions (W, H, D) 160×175×640 mm

Accessories


For work in the GPC mode and construction of calibration dependences, we offer a wide range of speakers for GPC filled with gels (stationary phase) and eluents of a wide variety of chemical nature (polar and non-polar), intended for the analysis of both high molecular weight polymers and oligomers, as well as standard polymer objects.

Gel Permeation Chromatography (GPC, SEC) columns:

  • for any organic eluents: PSS SDV, GRAM, PFG, POLEFIN (up to 200 °C);
  • for aqueous eluents: PSS SUPREMA, NOVEMA, MCX PROTEEMA;
  • columns with monodisperse pore size distribution or mixed type for absolutely linear calibrations;
  • to determine low and high values ​​of MM;
  • ready-made sets of columns to expand the range of determined molecular weights;
  • for synthetic and biopolymers;
  • solutions from micro GPC to preparative systems;
  • columns for quick separations.

Columns can be supplied in any eluent of your choice.

Standards for gel permeation chromatography (GPC, SEC):

  • individual standard samples and ready-made sets of standards;
  • soluble in organic solvents:
    • polystyrene
    • poly(α-methylstyrene)
    • polymethyl methacrylate
    • poly(n-butyl methacrylate)
    • poly(tert-butyl methacrylate)
    • polybutadiene-1,4
    • polyisoprene-1,4
    • polyethylene
    • poly(2-vinylpyridine)
    • polydimethylsiloxane
    • polyethylene terephthalate
    • polyisobutylene
    • polylactide
  • soluble in aqueous systems:
    • dextran
    • pullulan
    • hydroxyethyl starch
    • polyethylene glycols and polyethylene oxides
    • Na-salt of polymethacrylic acid
    • Na-salt of polyacrylic acid
    • Na-salt of poly(p-styrenesulfonic acid)
    • polyvinyl alcohol
    • proteins
  • MALDI standards, validation kits for light scattering detectors (LSD) and viscometry;
  • deuterated polymers;
  • polymers and custom-made standards.

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

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.

Size exclusion chromatography is a variant of liquid chromatography in which separation occurs due to the distribution of molecules between the solvent inside the pores of the sorbent and the solvent flowing between its particles, i.e. the stationary phase is a porous body or gel, and the different retention of substances is due to differences in the size of the molecules of substances, their shape and ability to penetrate into the pores of the stationary phase. The name of the method reflects the mechanism of the process, from the English term "Size Exclusion", denoting a size exception. Gel Permeation Chromatography (GPC) is size exclusion chromatography in which a gel serves as the stationary phase.

Unlike other versions of HPLC, where separation occurs due to different interaction of components with the surface of the sorbent, the role of a solid filler in size exclusion chromatography is only to form pores of a certain size, and the stationary phase is a solvent that fills these pores.

The principal feature of the method is the possibility of separating molecules according to their size in solution in the range of almost any molecular weight - from 10 2 to 10 8 , which makes it indispensable for the study of synthetic macromolecular substances and biopolymers.

Consider the fundamental principles of the method. The volume of the exclusion column can be expressed as the sum of three terms:

V c \u003d V m+V i+ Vd,

where v m- dead volume (solvent volume between sorbent particles, in other words, the volume of the mobile phase); V i is the volume of pores occupied by the solvent (the volume of the stationary phase); V d is the volume of the sorbent matrix, excluding pores. The total volume of solvent in the column V t is the sum of the volumes of the mobile and stationary phases:

V t = V m+V i .

The retention of molecules in the size-exclusion column is determined by the probability of their diffusion into the pores and depends mainly on the ratio of the sizes of molecules and pores. The distribution coefficient K d, as in other versions of liquid chromatography, is the ratio of the concentrations of a substance in the stationary and mobile phases:

K d \u003d C 1 / C 0

Since the mobile and stationary phases have the same composition, then K d of a substance for which both phases are equally accessible is equal to one. This situation is realized for the smallest molecules (including solvent molecules), which penetrate into all pores and therefore move through the column most slowly. Their retained volume is equal to the total solvent volume V t . All molecules larger than the pore size of the sorbent cannot enter them (complete exclusion) and pass through the channels between the particles. They elute from the column with the same retention volume equal to the mobile phase volume V m. The partition coefficient for these molecules is zero.

The principle of sample separation and detection in size exclusion chromatography.
A - sample input; B - division by size; C - yield of large macromolecules;
D is the yield of small macromolecules.

The relationship between retention volume and molecular weight (or molecular size) of the sample is described by a partial calibration curve, i.e. each specific sorbent is characterized by its own calibration curve, according to which the area of ​​molecular weights separated on it is estimated. Point A corresponds to the exclusion limit, or dead volume of column V m. All molecules with a mass greater than at point A will elute with a single peak with a retention volume V m. Point B reflects the permeation limit, and all molecules whose mass is less than point B will also leave the column as a single peak with a retention volume V t . Between points A and B is the range of selective separation. The corresponding volume

V i= V t - V m

commonly referred to as the working volume of the column. Segment CD is a linear section of a partial calibration curve built in coordinates V R - lg M. This section is described by the equation

V R \u003d C 1 - C 2 lg M ,

where C 1 is the segment cut off on the y-axis by the continuation of the segment CD, C 2 is the tangent of the angle of inclination of this segment to the y-axis. The value of C 2 is called the separation capacity of the column, it is expressed as the number of milliliters of solvent per one order of magnitude of change in molecular weight. The larger the separating capacity, the more selective the separation in a given mass range. In the non-linear regions of the calibration curve (sections AC and BD), due to a decrease in C 2, the efficiency of fractionation decreases markedly. In addition, the non-linear relationship between lg M and V R significantly complicates data processing and reduces the accuracy of the results. Therefore, one tends to choose a column (or a set of columns) so that the separation of the analyzed polymer proceeds within the linear section of the calibration curve.

If any substance is eluted with a retained volume greater than V t , then this indicates the manifestation of other separation mechanisms (most often adsorption). Adsorption effects usually appear on rigid sorbents, but are sometimes also observed on semirigid gels, apparently due to an increased affinity for the gel matrix. An example is the adsorption of aromatic compounds on styrenedivinylbenzene gels.

Apparently, by changing the interaction parameters in the polymer-sorbent-solvent system, one can switch from the adsorption mechanism to the exclusion mechanism and vice versa. In general, size exclusion chromatography tends to completely suppress adsorption and other side effects, since they, especially when studying the molecular weight distribution (MWD) of polymers, can significantly distort the results of the analysis. One of the interfering factors is the hydrodynamic mode of chromatography, in which the role of the stationary phase is played by the walls of the column (channel) and the separation of a mixture of macromolecules or particles occurs due to the difference in the flow rates of the mobile phase along the axis of the capal and near its walls, as well as due to the distribution of particles to be separated over the cross section channels according to their size.

The fundamental differences of size exclusion chromatography from other options are the a priori known duration of the analysis in a particular system used, the possibility of predicting the order of elution of components by the size of their molecules, approximately the same peak width over the entire range of selective separation, and confidence in the yield of all sample components in a fairly short period of time corresponding to volume V t . This method is mainly used for the study of the MWD of polymers and the analysis of macromolecules of biological origin (proteins, nucleic acids, etc.), but these features make it extremely promising for the analysis of low molecular weight impurities in polymers and the preliminary separation of samples of unknown composition. This information greatly facilitates the choice of the best HPLC option for the analysis of a given sample. In addition, micropreparation size exclusion separation is often used as the first step in the separation of complex mixtures by a combination of different types of HPLC.

In polymer size exclusion chromatography, the most stringent requirements are imposed on the stability of the mobile phase flow. The accuracy of results in polymer size exclusion chromatography depends markedly on temperature. When it changes by 10°C, the error in determining the average molecular weights exceeds ±10%. Therefore, in this variant of HPLC, temperature control of the separation system is mandatory. As a rule, the accuracy of maintaining the temperature of ±1°C in the range up to 80-100°C is sufficient. In some cases, for example, in the analysis of polyethylene and polypropylene, the operating temperature is 135-150°C. The most common detector in polymer size exclusion chromatography is the differential refractometer.

The choice of sorbents that provide optimal conditions for solving a specific analytical problem is carried out in several stages. The gel matrix must be chemically inert, i.e. in the course of size exclusion chromatography, chemical binding of the separated macromolecules should not occur. When separating proteins, enzymes, nucleic acids in contact with the matrix, their denaturation should not occur. Initially, on the basis of data on the chemical composition or solubility of the analyzed substances, it is determined which version of the process should be used - chromatography in aqueous systems or in organic solvents, which largely determines the type of sorbent required. The separation of substances of low and medium polarity in organic solvents can be successfully carried out on both semirigid and rigid gels. The study of the MWD of hydrophobic polymers containing polar groups is more often carried out on columns with styrenedivinylbenzene gels, since in this case adsorption effects practically do not appear and the addition of modifiers to the mobile phase is not required, which greatly simplifies the preparation and regeneration of the solvent.

For work in aqueous systems, mainly rigid sorbents are used; sometimes very good results can be obtained with special types of semi-rigid gels. Then, according to the calibration curves or data on the fractionation range, a sorbent of the desired porosity is selected, taking into account the available information on the molecular weight of the sample. If the analyzed mixture contains substances that differ in molecular weight by no more than 2–2.5 orders of magnitude, then it is usually possible to separate them on columns with the same pore size. For a wider range of masses, sets of several columns with sorbents of different porosity should be used. An approximate calibration dependence in this case is obtained by adding the curves for individual sorbents.

Solvents used in size exclusion chromatography must meet the following basic requirements:

1) completely dissolve the sample at the separation temperature;

2) wet the surface of the sorbent and not impair the efficiency of the column;

3) prevent adsorption (and other interactions) of the substances to be separated with the surface of the sorbent;

4) provide the highest possible detection sensitivity;

5) have low viscosity and toxicity.

In addition, in the analysis of polymers, the thermodynamic quality of the solvent is essential: it is highly desirable that it be "good" with respect to the polymer to be separated and the gel matrix, i.e. concentration effects were most pronounced.


Chromatogram of polyethylene glycol oligomers obtained on a composite column 2(600x7.5) mm with TSK gel G2000PW, PF 0.05 M NaCl solution, flow rate 1 ml/min, pressure 2 MPa, temperature 40°C, refractometric detector.

The solubility of the sample is usually the main limiting factor limiting the range of suitable mobile phases. The best organic solvent for size exclusion chromatography of synthetic polymers in terms of a set of properties is THF. It has a unique dissolving power, low viscosity and toxicity, is more compatible with styrenedivinylbenzene gels than many other solvents, and, as a rule, provides high detection sensitivity when using a refractometer or UV detector in the region up to 220 nm. For the analysis of highly polar and insoluble in tetrahydrofuran polymers (polyamides, polyacrylonitrile, polyethylene terephthalate, polyurethanes, etc.), dimethylformamide or μ-cresol is usually used, and the separation of low polarity polymers, for example, various rubbers and polysiloxanes, is often carried out in toluene or chloroform. The latter is also one of the best solvents for working with an IR detector. about-Dichlorobenzene and 1,2,4-trichlorobenzene are used for high temperature chromatography of polyolefins (usually at 135°C), which are otherwise insoluble. These solvents have a very high refractive index, so it is sometimes useful to use them instead of tetrahydrofuran for the analysis of low refractive index polymers, which can improve the sensitivity of refractometer detection.

To prevent the oxidation of solvents and semi-rigid gels under high temperature size exclusion chromatography, about-dichlorobenzene and 1,2,4-trichlorobenzene add antioxidants (ionol, santonox R, etc.).

Rigid sorbents are compatible with any mobile phases with pH<8-8.5. При более высоких значениях рН силикагель начинает растворяться и колонка необратимо теряет эффективность. Стиролдивинилбензольные гели совместимы в основном с элюентами умеренной полярности. Для работы на колонках с μ-стирогелем (от 1000Å и выше) пригодны тетрагидрофуран, ароматические и хлорированные углеводороды, гексан, циклогексан, диоксан, трифторэтанол, гексафторпропанол и диметилформамид.

The degree of swelling of gel particles in different solvents is not the same, therefore, the replacement of the eluent in columns with these sorbents can lead to a decrease in efficiency due to a change in the volume of the gel and the formation of voids. When using unsuitable solvents (acetone, alcohols), the gel shrinks so strongly that the column is hopelessly damaged. For sorbents with a small pore size (such as μ-styrogel 100E and 500E), such shrinkage is observed both in polar and nonpolar solvents; therefore, they cannot also be used in saturated hydrocarbons, fluorinated alcohols, and dimethylformamide. A convenient, albeit very expensive, way out is to use separate sets of columns for each solvent used. For this purpose, some companies produce columns with the same pore size filled with different solvents - tetrahydrofuran, toluene, chloroform, and DMF.

During the separation of macromolecules, the main contribution to the smearing of the band is determined by hindered mass transfer. Unfortunately, many of the eluents used have a high viscosity. To reduce viscosity (as well as to improve solubility), size exclusion chromatography is often carried out at elevated temperatures, which greatly improves the efficiency of the chromatographic system.

The analysis of most polymers on rigid gels is often complicated by their adsorption. To suppress adsorption, solvents are usually used, which are adsorbed on the column packing more strongly than the analytes. If for some reason this is not possible, then the mobile phase is modified by adding 0.1-2% of a polar modifier, such as tetrahydrofuran. Much stronger modifiers are ethylene glycol and polyglycols with different molecular weights (PEG-200, PEG-400, carbovax 20 M). Sometimes, for example, in the analysis of polyacids in dimethylformamide, the addition of sufficiently strong acids is required. It should be noted that it is not always possible to completely eliminate adsorption by adding modifiers. In such cases, semi-rigid gels should be used. Some polymers dissolve well only in highly polar solvents (acetone, dimethyl sulfoxide, etc.) that are incompatible with styrene-divinylbenzene gels. When separating them on rigid sorbents, the choice of solvent is carried out in accordance with the general principles outlined above.

Size exclusion chromatography in aqueous media has its own characteristic features. Due to the specifics of many separable systems (proteins, enzymes, polysaccharides, polyelectrolytes, etc.) and the variety of sorbents used, there are many variations in the PF composition to suppress various undesirable effects. As sorbents, dextran gels (sephadexes), polyacrylamide, hydroxyacrylmethacrylate gels, agarose gels, etc. are used. ionic strength of the solution. The lower the pH value and the ionic strength of the solution, the more favorable the unfolded conformations of macromolecules become (the so-called polyelectrolyte swelling). In this case, the average sizes increase, which leads to a decrease in the retention volumes in the size exclusion chromatography mode. General methods of modification are the addition of various salts and the use of buffer solutions with a certain pH value. In particular, maintaining the pH<4 дает возможность подавить слабую ионообменную активность силикагелей, обусловленную присутствием на их поверхности кислых силанольных групп. Требуемая ионная сила подвижной фазы достигается при концентрации буферного раствора 0,05-0,6М; оптимальную концентрацию подбирают экспериментально. Для предотвращения ионообменной сорбции катионных соединений наиболее часто используют такой активный модификатор, как тетраметиламмонийфосфат при рН=3. Однако при разделении некоторых белков могут проявляться гидрофобные взаимодействия, в свою очередь осложняющие эксклюзионный механизм разделения. Те же эффекты иногда проявляются и при работе с дезактивированными гидрофильными сорбентами. Для их устранения к растворителю добавляют метанол. Иногда в водную подвижную фазу вводят полярные органические растворители, полигликоли, кислоты, основания и поверхностно-активные вещества.

The most important field of application of size exclusion chromatography is the study of macromolecular compounds. As applied to synthetic polymers, this method has taken a leading position in a short time for determining their molecular weight characteristics and is intensively used to study other types of heterogeneity. In the chemistry of biopolymers, size exclusion chromatography is widely used to fractionate macromolecules and determine their molecular weight.

A fundamental feature of size exclusion chromatography of high molecular weight synthetic polymers is the impossibility of separating a mixture into individual compounds. These substances are a mixture of polymer homologues with different degrees of polymerization and, accordingly, with different molecular weights M i. The molecular weight of such mixtures can be estimated by some average value, which depends on the method of averaging. Content of molecules of each molecular weight M i determined either by their numerical fraction in the total number of polymer molecules, or by mass fraction in their total mass. Typically, the polymer is characterized by the mean values ​​found by these methods, which are called, respectively, the number average M n and mass average M w molecular weight. M values n give, for example, cryoscopy, osmometry, ebullioscopy, and the values ​​of M w- light scattering and ultracentrifugation.

If we denote the number of molecules with molecular mass M i through N i, then the total mass of the polymer can be expressed in terms of Σ M i N i , the numerical fraction of molecules with mass M i through N i / Σ N i , and the mass fraction of molecules with mass M i- across f i=M i N i / Σ M i N i . To determine the part of the total polymer mass corresponding to these fractions, they are multiplied by M i .

By summing the obtained values ​​for all quantities, the average molecular weights are obtained:

M n = Σ 1 /( fi/M i ) = (Σ M i N i )/(Σ N i )

M w = Σ M i fi = (Σ M i 2 N i )/(Σ M i N i )

Ratio M w>/M n characterizes the polydispersity of the polymer.

In practice, the molecular weight of polymers is often determined by viscometry. The average viscosity molecular weight is found according to the Mark-Kuhn-Houwink equation:

[η ] = K η / M η a

where [η] - intrinsic viscosity; K η , and are constants for a given polymer-solvent system at a given temperature.

The value M η is described by the equation

M η = ( Σ M i a fi ) 1/a

As a rule, the average molecular weights satisfy the inequality

M w> M η > M n

Typically, a polymer sample is characterized by a set of values ​​M w, M η , M n and M w/M η , but this may not be enough. The MWD curves provide the most complete information on the molecular mass inhomogeneity of a sample. A typical chromatogram obtained during an exclusion separation is a fairly smooth curve with one or more maxima. From this curve, using the calibration dependence and the corresponding calculations, the values ​​of the average molecular characteristics and MWD of the polymer are determined in differential or integral form.