Difference between adsorption and gel permeation chromatography. Gel chromatography. Basic HPLC System for GPC

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) 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 interaction 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%. Normally, the instrument operates at room temperature, however, polyolefin size exclusion chromatography requires elevated temperatures, 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 polymer 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 for 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 a concentration detector, 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 thermostat, 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 with respect to 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 dependences, 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
Opportunities	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, 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 method of universal calibration, 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 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:

Nutrition	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.

Gel Permeation Chromatography is probably the most commonly used method, as it is the simplest method for separating polysaccharides having a wide range of molecular weights. At the same time, it makes it possible to determine the molecular weights of polysaccharides. When mild detection conditions are applicable, this method is especially useful for unstable biological materials.
Device for chromatographic. Gel Permeation Chromatography (GPC) is a technique in which the separation of polymer molecules is based on the different volumes within porous gel particles that are available to different sized solute molecules.
Gel permeation chromatography is a type of column fractionation method in which fractionation is carried out by the molecular sieve method, based on the ability of molecules to penetrate into the pores of the adsorbent of a certain size. As adsorbents in this method, materials are used that do not have charges and ionogenic groups, which have a precisely specified pore size (see Chap. These requirements are best met by specially prepared copolymers of styrene with divinylbenzene, which form gels when swollen.
Scheme of work in the recycle mode. Gel permeation chromatography is used primarily as a method for determining the molecular weight distribution of polymeric substances, while gel filtration chromatography is mainly a preparative separation method, but both methods are suitable in both cases. When determining the molecular weight distribution, it is necessary to establish a relationship between the chromatogram and the molecular size, or more correctly, the molecular weight.
Gel permeation chromatography, with size exclusion chromatograph.
Gel Permeation Chromatography is a size exclusion raffia chromatography in which the stationary phase is a gel.
Gel permeation chromatography is a type of column fractionation method in which separation is carried out according to the molecular sieve principle. This principle was already known in the early 50s, but only after Porat and Flodin rediscovered and widely used this method, 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.
Separation of amino acids by ion-exchange chromatography. Gel permeation chromatography also allows the characterization of phenol-formaldehyde resins.
Scheme of operation in recycle mode (10]. Gel permeation chromatography is used mainly as a method for determining the molecular weight distribution of polymeric substances, while gel filtration chromatography is mainly a preparative separation method, but both methods are suitable in both cases. When determining the molecular weight distribution, it is necessary to establish a relationship between the chromatogram and the molecular size, or more correctly, the molecular weight.
Gel Permeation Chromatography (GPC) is a method for separating molecules based on their size differences. This method is known as gel chromatography, size exclusion and molecular sieve chromatography. The latter name most fully reflects the essence of the method, however, the term gel permeation chromatography is more widely used in the literature.

Gel Permeation Chromatography (GPC) is a technique in which the separation of polymer molecules is based on the different volumes within porous gel particles that are available to varying sizes of solute molecules.
Gel Permeation Chromatography (GPC) is a technique that uses highly porous, non-ionic gel beads to separate polydisperse polymers in solution. According to the developed theories and models of GPC fractionation, the determining factor of separation is not the molecular weight, but the hydrodynamic volume of the molecule.
Gel permeation chromatography is based on the ability of macromolecules of different lengths, and hence different molecular weights, to penetrate into a porous component to different depths. The column is packed with porous glass or a highly cross-linked swollen polymer gel, the polymer is added to the top of the column, and then the column is washed with a solvent. Smaller molecules penetrate much deeper into the pores and are retained in the column during the elution process much longer than larger macromolecules.
Gel permeation chromatography makes it possible not only to fractionate mixtures of oligomers, but also to determine their average molecular weights and molecular weight distributions. In this case, the numerical values ​​of the constants of the Mark-Kuhn equation differ little from the coefficients for a Gaussian coil in a theta solvent.
Gel permeation chromatography of nucleic acid components was performed on cross-linked dextran gels (sephadex) (Sephadex, Pharmacia, Uppsala, Sweden) and polyacrylamide gels (biogels) (Bio-Gel, Bio-Rad Labs Richmond, Calif. In addition, gels have ion-exchange and adsorption properties, showing an increased affinity for aromatic and heterocyclic compounds.
Gel permeation chromatography also shows adsorption of purine bases on the gel matrix.
RTF of oligobutadienes and copolymers of butadiene with acrylic acid and acrylonitrile according to data 3. The use of gel permeation chromatography (GPC) in the classical version for evaluating the RTF of oligomers is still limited. The separation of molecules of close molecular weights but different functionality by GPC is based on the change in the root-mean-square distance between the ends of macromolecules g/2 in solution, depending on the nature and molecular weight of the end groups. The cyclization and branching of molecules, which lead to its decrease by a factor of 15 - 2, in comparison with linear molecules of the same molecular weight, is especially strongly affected by the value of r g) /.
The mechanism of gel permeation chromatography is essentially the same for high and low crosslink density, although significant differences can be observed in practice. The gel particles in the column are suspended in a solvent. The channels between the gel particles are much larger than the pore sizes inside the gel granules, so the solvent only flows in the space between the gel granules. Molecules of the dissolved substance, depending on their size, penetrate into the pores of the gel to different depths and move almost without restrictions in the solvent contained in the gel granules.
The mechanism of gel permeation chromatography as presented here is based on the assumption of diffusion equilibrium. In other words, it is assumed that the time of distribution of the solute molecules between the space external to the gel particles and the pore volume accessible to these molecules is rather small. The time interval during which the zone containing the solute molecules passes through the gel particles is usually much longer than the half-period of reaching equilibrium by diffusion of the solute molecules into the gel granules.
In gel permeation chromatography, the substance is characterized by the value of K and, as in conventional chromatography. The K value is independent of column size and can therefore be used to compare GPC data obtained on different columns.
In gel permeation chromatography, a polymer solution is introduced into a liquid (eluent) that moves through a column filled with a sorbent. At the outlet of the column, the solution is divided into fractions (zones) in accordance with the size of the macromolecules. The time elapsed from the moment the solution was introduced into the eluent to the moment the given zone left the column is called the retention time, and the volume of eluent that passed through the column during this time is called the retention volume.
Displacement chromatography of polyurethane. Determination of molecular weight. The method of gel permeation chromatography was used to determine the molecular weight distribution in polyurethane samples dissolved in tetrahydrofuran.

The principle of gel permeation chromatography can be used to separate substances that differ significantly in the size of their molecules. The pore size of the sorbent used should be commensurate with the size of the molecules of the substances to be separated. The separating power of the material depends on the distribution of pores. Substances whose molecules are so large that they cannot penetrate the pores pass through the column at the same rate as the mobile phase. The smaller the molecules of the substances to be separated, the larger the volume of pores they can penetrate and the more they will lag behind the front of the mobile phase. Gel permeation chromatography is used mainly for the analysis of substances of a macromolecular nature.
In gel permeation chromatography, 0 characterizes molecules and substances that cannot penetrate the gel pores in the column; in adsorption chromatography, substances that, although they penetrate almost the entire volume of pores, are not retained due to interaction with the surface of the sorbent. The capacitance coefficient characterizes the processes of interaction of the substance being separated with the mobile and stationary phases and, therefore, is a thermodynamic quantity.
In gel permeation chromatography, macroporous silica gels, porous glasses, and organic polymer gels are used as column fillers. Materials of the same type, differing in their porosity, are designed to separate substances with molecules of different sizes.
In gel permeation chromatography, the mobile phase is in most cases the only solvent. The choice of solvent must be carried out taking into account the solubility of the polymer in it and, at the same time, so that in the mobile phase used, the interactions of the substances to be separated with the stationary phase are minimal. Tetrahydrofuran is most often used to separate hydrophilic water-soluble polymers.
Schematic representation of a swollen gel. In gel permeation chromatography, the sorption activity of the components and the interfacial mass transfer associated with it are determined only by the diffusion mobility of macromolecules and the ratio of their sizes to the pore sizes.
For gel permeation chromatography, gel chromatographs are used, consisting of a set of chromatographic columns filled with an appropriate sorbent (macroporous glasses, styrogels, etc.).
In gel permeation chromatography, in addition to regularities of a general chromatographic nature, there are specific features associated primarily with the properties of polymer solutions that are the object of study, with a variety of these objects, sorbents, and analysis conditions. All this naturally complicates the construction of a general theoretical scheme. Therefore, researchers working in the field of GPC were forced at the first stages of the development of the method to develop particular theoretical concepts, within which they found an explanation for individual patterns observed in the experiment. This made it possible to set up the experiment more competently, optimize its mode, and interpret the results.
Gel permeation chromatography of these polymers was carried out and calibration curves were obtained to determine their molecular weight.
The processing of gel permeation chromatography data requires the determination of three characteristics of the system: the reliability of the data obtained, the calibration of the system, and its resolution. These three characteristics are interrelated and must ultimately be established by direct measurements. After this is done, one can further use indirect data on the invariance of the specified characteristics of the system.
In the gel permeation chromatography method, a polymer sample is separated according to the size of its macromolecules. As long as we are talking about molecules that differ only in molecular weights, separation efficiency is determined solely by molecular weight. But even such a simple situation can become more complicated if the molecules of a chemically inhomogeneous polymer sample contain groups that are solvated to varying degrees. Then, despite the same molecular weights, some chains may have large molar volumes.
Gel Permeation Chromatography analyzes a wide range of materials, and its advantages such as simplicity and high efficiency contribute to the rapid spread of the method. The effectiveness of the method is most clearly manifested in the analysis of natural substances, the molecular weight of which varies over a wide range.
Dependence of the height equivalent to a theoretical plate on the diameter of sorbent grains for different types of sorbents with different packing methods. O - surface-porous sorbent. dK - 2 1 mm, manual packaging.. - surface-porous sorbent, dK 7 9 mm, machine packaging. f-surface-porous sorbent, dK 7 9 mm, manual packing. c - silica gel, balanced suspension. f - microspherical silica gel. stabilized suspension. P - diatomaceous earth, tampon packaging. A - microspherical silica gel, stabilized suspension.| GPC of narrowly dispersed polystyrene standards on a column (250 X 0 20 mm with silica gel (Fp 0 20 mm, dp 5 - 6 μm. 1 - Mw 2 - 10. 2 - Mw 5 MO4. 3 - D w 4. Since the gel -penetrating chromatography kn is small, F of this chromatographic method is less than in adsorption chromatography.
Gel chromatography (or gel permeation chromatography) is a variant of liquid chromatography in which the solute is partitioned between the free solvent surrounding the gel beads and the solvent inside the gel beads. Since the gel is a swollen structured system with pores of different sizes, the separation in this type of chromatography depends on the ratio of the sizes of the molecules of the substances being separated and the sizes of the gel pores. In addition to the size of the molecules, which can be assumed to be proportional to the molecular weights, the shape of the molecules plays an important role in gel chromatography. This factor is especially important for polymer solutions, in which, with the same molecular weight, molecules can take a different shape (spherical or other arbitrary) in accordance with their conformation and, as a result, behave differently in the column. Further reasoning is valid for molecules having a spherical shape.

GPC (for gel permeation chromatography) , which serve exclusively for analytical purposes and have a total length of 370 cm. (The principle of operation of this chromatograph, in which the molecular weight distribution of synthetic polymers is determined almost completely automatically, is described on p. can also be created to work with water-soluble polymers, which will greatly facilitate the task of determining the molecular weight.
However, the wide use of gel permeation chromatography is hindered by a small range of porous gels and the impossibility of separating asphaltenes taking into account their chemical nature. According to this method, on ion-exchange resins (amberlite-27 and amberlite-15), asphaltenes were separated into four acidic (386% of the original), four basic (166%) and neutral (413%) fractions. Then, by gel permeation chromatography, they are separated into fractions having the same molecular size. This method revealed a significant polarity of asphaltenes isolated from Romashkino oil.
Three-point interaction model proposed by Dalglish. In principle, in gel permeation chromatography (also called size exclusion or sieve), which is especially important in protein chemistry, separation is carried out mainly due to the difference in the steric sizes of the molecules: large molecules, since they are not able to diffuse into the small pores of the matrix, elute faster, than small molecules.
The mechanism of gel permeation chromatography discussed above seems to be fully confirmed by experiment. In most cases, a change in the flow rate does not affect the eluting volume, which indicates a very close approach of the system to equilibrium conditions. It should also be noted that the above picture is a very rough approximation to reality. On fig. 5 - 1 indicate the molecules of the solute, which, having a very small size, can diffuse through all the pores of the matrix and even in places where the pores are narrowed. At the same time, among the molecules of the dissolved substance, there are molecules whose large dimensions allow them to penetrate only into pores of certain sizes located only on the outer shell of the gel granules. However, there must be molecules with intermediate sizes that can pass through the bottlenecks in the pores, although at a much slower speed due to interaction with the channel walls. Craig convincingly showed that the rates of passage of solute molecules in the process of diffusion through membranes, on both sides of which the concentrations of these molecules are different, do not differ much if the pores of the membranes are significantly larger than the sizes of the diffusing molecules. However, diffusion rates turn out to be a sensitive measure of molecular dimensions for those molecules whose dimensions are only slightly smaller than the pore diameter. Obviously, by their nature, the processes of differential diffusion and gel permeation chromatography are close to each other.
In gel permeation chromatography fractionation, a wide variety of gels are used or attempted to be used. As a rule, these gels are polymers with varying degrees of crosslinking and usually swell in the solvents in which they are prepared. Examples include dextrans used in aqueous solutions and polystyrenes used when working in organic solvents. Contrary to conventional wisdom, swelling has not been shown to play a significant role, but permeability or degree of porosity is a very important indicator of gel quality. Vaughan made extensive studies of various gels and other porous materials and showed that swollen silica gel (Monsanto's Santocel A) allows very efficient fractionation of polystyrene in benzene. Silica gel is a hydrophilic substance and therefore, of course, does not swell in benzene.
Without dwelling on the theory of gel permeation chromatography, we note that the permeability of particles depends on the porosity and on the method of obtaining the jelly. The most widely used jellies at present include: for aqueous solutions, epichlorohydrin-crosslinked dextran (a biologically synthesized carbohydrate) and cross-linked polyacrylamide, and for non-aqueous solutions, polystyrene cross-linked with divinylbenzene.
Acrylonitrile and ABS copolymers were studied by gel permeation chromatography and calibration curves were obtained for various solvents. The methods used in this work for the analysis of ABS copolymers will be described below. In this work, methods were developed for determining the insoluble polymer (gel), soluble polymer and the total amount of non-polymer additives, as well as methods for determining the bound acrylonitrile, butadiene and styrene both in the initial polymer and in the isolated insoluble polymer (gel) and in the soluble polymer fraction. . All these techniques are also applicable to the analysis of intermediate samples of the grafted ABS copolymer, as well as mixtures of this copolymer with a low molecular weight styrene-acrylonitrile polymer, which are used in the production of ABS.
In this work, polycarbonates synthesized by various methods were studied by gel permeation chromatography. The authors of the work came to the conclusion that this method is the best for the analysis of end groups. Polycarbonate was also fractionated by gel permeation chromatography. Polycarbonates were fractionated from methylene chloride by sequential precipitation. This calibration was further confirmed by membrane osmometry and light scattering measurements. The experimental viscosity values ​​have shown that the Kurata-Stockmeyer-Roy ratio is suitable for interpreting the molecular stretching of polycarbonate in methylene chloride.
In a general description of the process of gel permeation chromatography, one should proceed from the theoretical concepts of chromatography and sorption dynamics modified in an appropriate way, taking into account the specifics of polymer solutions. It is convenient to consider a chromatographic system as a two-phase system, meaning that the mobile phase is a set of channels formed by voids between sorbent particles, and the immobile phase is the pore space of the sorbent.
When determining the MMP by gel-penetrating chromatography, the solution of the polymer is passed through a column with a packing in the form of a cross-linked polymer swollen in the solution. The speed of movement of macromolecules in the column depends on their mol.
Size exclusion chromatography is subdivided into gel permeation chromatography (GPC) and gel filtration chromatography.
Fractionation of an alkaline extract from spruce holocellulose by ion-exchange chromatography. For fractionation, gel permeation chromatography is often used.

transcript

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: TSHG 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 VK 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 size of the macromolecule RK 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 is reduced 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. Nevertheless, 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 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. 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 Lnear column in the 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. five

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 outlet of the column (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 a 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: FV dv W d (9) To pass from the chromatogram to the MWD function, it is necessary to have a calibration function V f (), then the desired function will be WF (f) df () d These relationships are written without taking into account instrumental broadening (PU ). 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 appropriate 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 construct 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 BV 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 dwz 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 POLYMER BY METHOD OF GEL PERMEABILITY CHROMATOGRAPHY Purpose of 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. com., A, v.8, N8, with B.G. Belenky, L.Z. Vilenchik, Chromatography of polymers, M: Chemistry, WWYau, JJKrkland, DDBly, 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

ESTABLISHMENT OF THE RUSSIAN ACADEMY OF SCIENCES INSTITUTE OF ELEMENTORGANIC COMPOUNDS im. A.N. NESMEYANOV. RESEARCH AND EDUCATIONAL CENTER FOR PHYSICS AND CHEMISTRY OF POLYMERS Blagodatskikh I.V. LIQUID CHROMATOGRAPHY OF POLYMERS

1 Macromolecular compounds (Lysenko EA) Lecture 7. Fractionation of macromolecules 2 1. The concept of fractionation. 2. Preparative fractionation. 3. Method of turbidimetric titration. 4. Gel-penetrating

Laboratory work 7b Chromatographic determination of the composition of the gas phase of soils. Chromatography (from the Greek chroma, genitive chromatos color, paint) is a physicochemical method of separation and analysis

8. Questions 1. Define chromatography. 2. What features of chromatography make it possible to achieve a better separation of substances with similar properties compared to other separation methods. 3. List

Moscow Institute of Physics and Technology (State University) Department of Molecular Physics Physical research methods Lecture Gas chromatography Theory and principles Dolgoprudny, November

04.07 Moscow Institute of Physics and Technology Department of Molecular and Biological Physics Physical research methods Lecture 8 Chromatography Dolgoprudny, April 6, 07 Plan. History of occurrence

Moscow Institute of Physics and Technology (State University)) Department of Molecular Physics Physical Methods of Research Lecture 0 Gas Chromatography Dolgoprudny, November 5, 0g. Plan. History

Analytical chemistry 4th semester, Lecture 17. Module 3. Chromatography and other methods of analysis. Chromatography. Principle and classification of methods. 1. The principle of chromatographic separation. Stationary and mobile

Discovery of chromatography (1903) MIKHAIL SEMENOVICH TsVET (1872-1919) Main stages in the development of chromatography 1903 Discovery of chromatography (Tsvet M.S.) 1938 Thin-layer or planar chromatography (Izmailov

Moscow Institute of Physics and Technology (State University) Department of Molecular and Biological Physics Physical research methods Lecture 7 Gas and liquid chromatography. Practical

CHAPTER 7 GAS-LIQUID CHROMATOGRAPHY As a method of analysis, chromatography was proposed by the Russian botanist MS Tsvet to solve the particular problem of determining the components of chlorophyll. The method turned out to be universal.

Moscow Institute of Physics and Technology Department of Molecular and Biological Physics Physical Research Methods Lecture 9 Gas Chromatography Experimental Technique and Methods Dolgoprudny, April 3

Topic 5. Fundamentals of rheology. Viscosity of polymer solutions. Theoretical part. Viscous liquids and solutions of macromolecular substances (NMWs) are divided into Newtonian and non-Newtonian according to the nature of the flow. Newtonian

Benefits of Agilent AdvanceBio SEC SEC columns for biopharmaceutical analysis Comparing columns from different vendors to improve data quality Technical Overview

Moscow Institute of Physics and Technology (State University) Department of Molecular and Biological Physics Physical research methods Lecture 9 Liquid chromatography Methods and technology

Journal of Analytical Chemistry, 5, volume 6, 7, p. 73-78 UDC 543.544 Simulation of gas chromatography for a given dependence of Henry's constant on temperature. 5g Prudkovsky A.G. Institute of Geochemistry and Analytical

Agilent AdvanceBio SEC Size Exclusion Chromatography Columns for Aggregation Analysis: Instrument Compatibility Technical Overview Introduction Agilent AdvanceBio SEC columns are a new family

MULTI DETECTOR GEL PERMEATION CHROMATOGRAPHY for POLYMER ANALYSIS K. Svirsky, Agilent Technologies, [email protected] Gel Permeation Chromatography is the only chromatographic technique that

ANNOTATION of the work program of the discipline "Introduction to chromatographic methods of analysis" in the direction of preparation 04.03.01 Chemistry on the profile of training "Analytical chemistry" 1. The objectives of mastering the discipline

46. ​​CHROMATOGRAPHIC SEPARATION METHODS Chromatographic separation methods are multi-stage separation methods in which the sample components are distributed between two phases, stationary and mobile. motionless

MINISTRY OF EDUCATION AND SCIENCE OF RUSSIA NATIONAL RESEARCH TOMSK STATE UNIVERSITY FACULTY OF CHEMISTRY Annotated work program of the discipline Chromatographic methods of analysis Area of ​​study

Scientific and technological company SINTEKO METHOD OF QUANTITATIVE CHEMICAL ANALYSIS OF COFFEE AND TEA FOR CAFFEINE CONTENT BY METHOD OF LIQUID CHROMATOGRAPHY. DZERZHINSK 1997 1 This document is distributed

Lecture 7 (9.05.05) TRANSFER PROCESSES IN GAS

FEDERAL AGENCY FOR EDUCATION State Educational Institution of Higher Professional Education “Ural State University. A.M. Gorky" IONTS "Ecology and nature management"

Macromolecular compounds (Lysenko E.A.) Lecture 5 (-Temperature). - temperature and ideality of the solution. - temperature and phase equilibria. 3. - temperature and sizes of macromolecular coils. .. Influence

Lecture 6 Chromatographic methods of analysis Lecture plan 1. Concepts and terms of chromatography. 2. Classification of chromatographic methods of analysis. Chromatographic equipment. 3. Types of chromatography: gas,

Theory of real matter. Science presents a large number of theories or laws of a real gas. The most famous van der Waals real gas law, which increases the accuracy of the behavior description

BELARUSIAN STATE UNIVERSITY FACULTY OF CHEMISTRY DEPARTMENT OF ANALYTICAL CHEMISTRY

Lecture 7. SURFACE PHENOMENA 1. Surface tension 1.1. surface energy. Until now, we have not taken into account the existence of an interface between different media*. However, its presence can be very

Viscoelasticity of polymeric liquids. Basic properties of polymeric liquids. Highly entangled polymer fluids include polymer melts, concentrated solutions, and semi-diluted

Moscow Institute of Physics and Technology (State University)) Department of Molecular Physics Physical research methods Lecture 9 Chromatography. Introduction Dolgoprudny, 9 October 0g. Plan.

ANALYTICAL CHEMISTRY UDC 543.544 ADSORPTION CHROMATOGRAPHY IN BIOGAS ANALYSIS 1999 M.V. Nikolaev Research Institute of Chemistry, UNN N.I. Lobachevsky L.P. Prokhorova Nizhny Novgorod aeration station A technique has been developed

MODERN PREPARATIVE FLASH CHROMATOGRAPHY Part 2* A.Abolin, Ph.D., "GalaChem" [email protected] P.-F. Ikar, Interchim (France) We continue to publish materials on modern methods of preparative

Quick Guide to Selecting Columns and Standards for Gel Permeation Chromatography SELECTION GUIDE Introduction Gel Permeation Chromatography (GPC) is a technique for evaluating molecular weight distribution

MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION GENERAL PHARMACOPEIAL AUTHORIZATION Chromatography OFS. SP XI, issue 1 Chromatography is a method for separating mixtures of substances, based

Agilent Software for Gel Permeation Chromatography One-Stop Solution for Fast and Easy Polymer Analysis Key Features Introduction Agilent Technologies

2.2.29. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY High performance liquid chromatography (HPLC) is a separation technique based on the differential distribution of substances between two immiscible

Yaroslavl State Pedagogical University. K. D. Ushinsky Department of General Physics Laboratory of Molecular Physics Laboratory work 5 Study of statistical regularities on the Galton board

Lecture 3. FREE SURFACE ENERGY OF THE PHASE DIVISION Surface forces. Surface Tension Consider a system containing liquid and vapor in equilibrium with it. Density distribution in the system

2 Methods of analysis: 1. Chemical methods. Chemical equilibrium and its use in analysis. Acid-base balance. The strength of acids and bases, the patterns of their change. Hammet function. calculation

Lecture 7 Branched chain reactions. Critical phenomena in branched chain reactions. E.-K. pp. 38-383, 389-39. A simple expression for the rate of formation of radicals: d r f(p) g(p) (1)

Lecture 6 Lukyanov I.V. Transport phenomena in gases. Contents: 1. The length of the free path of molecules. 2. Distribution of molecules over their mean free paths. 3. Diffusion. 4. Viscosity of the gas (internal friction).

Federal State Budgetary Institution of Science "Kirov Research Institute of Hematology and Blood Transfusion of the Federal Medical and Biological Agency" 3.3.2. Medical immunobiological

1. Explanatory note 1.1. Requirements for students The student must have the following initial competencies: basic provisions of mathematical and natural sciences; master the skills of independent

1 LECTURE 10 Two systems in diffusion contact. chemical potential. Phase equilibrium condition. Heat of transition. Clausius-Clapeyron formula. Two systems in diffusion contact Equilibrium state

1. A list of competencies indicating the stages (levels) of their formation. PC-1: the ability to use knowledge of the theoretical, methodological, procedural and organizational foundations of forensic science, forensics

Topic. Physico-chemistry of surface phenomena. Adsorption. Surface phenomena manifest themselves in heterogeneous systems, i.e. systems where there is an interface between components. Surface phenomena

TOMSK STATE UNIVERSITY Faculty of Physics STUDYING THE VISCOSITY COEFFICIENTS OF A LIQUID BY THE STOKES METHOD Guidelines for performing laboratory work Tomsk 2014 Reviewed and approved

Highly elastic polymer meshes. polymer meshes. Polymer networks consist of long polymer chains linked together and thus forming a giant three-dimensional macromolecule. All polymer

Gas chromatography 1 Substance requirements 1. Volatility 2. Thermal stability (substance must evaporate without decomposition) 3. Inertness Gas chromatograph scheme 1 2 3 4 5 1. Carrier gas cylinder

The curriculum is based on the OSVO 1-31 05 01 2013 educational standard and the HEI curriculum G 31 153/ac. 2013 COMPILER: V.A.Vinarsky, Associate Professor, Candidate of Chemical Sciences, Associate Professor RECOMMENDED

MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION GENERAL PHARMACOPIES AUTHORIZATION Chromatography on paper OFS.1.2.1.2.0002.15 Instead of Art. SP XI, issue 1 The chromatographic process occurring on the filter sheet

Ministry of Education and Science of the Russian Federation Federal Agency for Education State Educational Institution of Higher Professional Education "UFA STATE OIL

AGILENT POLYMER STANDARDS FOR GPC/SEC Contents POLYMER STANDARDS FOR GPC...3 InfinityLab EasiVial...5 InfinityLab EasiCal...8 Polystyrene Standards...9 Standards

GROUP P A C O M P A N I B I O C H I M M A K Z A C R B I O 1 1 9 8 9 9, Russia, Moscow, Lenin Tel./Fax (0 9 5 ) 939-59-67, tel. 939- I N S T R U C T I A ​​on the use of the Analytical Kit MOSCOW

Theory of Ion Chromatography: A Universal Approach to Describing Peak Parameters 1998 A.G. Prudkovskii, A.M. Dolgonosov V. I. Vernadsky Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences, 117975

Moscow Institute of Physics and Technology (State University) Department of Molecular and Biological Physics Physical research methods Lecture 8 Detectors in chromatography Liquid chromatography

MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION GENERAL PHARMACOPEIAL AUTHORIZATION Electrophoresis OFS.1.2.1.0021.15 Instead of Art. SP XI, issue 1 Electrophoresis method of analysis based on the ability of charged particles,

1 Macromolecular compounds (Lysenko E.A.) Lecture 10. Thermomechanical analysis of amorphous polymers. 2 1. Basic concepts of mechanical analysis of physical bodies. 2. Thermomechanical curves of amorphous polymers

5 PHYSICAL EQUILIBRIUM IN SOLUTIONS 5 Partial molar values ​​of mixture components Consideration of the thermodynamic properties of a mixture of ideal gases leads to the relationship Ф = Σ Ф, (5) n where Ф is any extensive

6. Moscow Institute of Physics and Technology (State University) Department of Molecular Physics Physical research methods Lecture Gas chromatography. Technical implementation Liquid chromatography

Macromolecular compounds (Lysenko EA) Lecture 4. Phase equilibria in polymer solutions. Dissolution kinetics. Concentration modes. Equation of state of polymer solution. . Phase equilibria

Laboratory work. Determination of the content of C 8 arenes in the gasoline fraction Knowledge of the hydrocarbon (HC) composition of oils and condensates at the molecular level is of great importance both for petrochemistry

Ideal polymer chain. Ideal polymer chain. An ideal chain is a model chain in which the so-called bulk interactions are neglected, i.e. interactions of links removed along the chain.

Laboratory work 1.17 STUDYING THE LAW OF NORMAL DISTRIBUTION OF RANDOM VARIABLES M.V. Kozintseva Purpose of the work: to study the distribution of random variables on a mechanical model (Galton board). The task:

BELARUSIAN STATE UNIVERSITY APPROVED Dean of the Faculty of Chemistry D.V. Sviridov 2011 Registration UD- /r SOLUTIONS OF POLYMERS Curriculum in the specialty 1-31 05 01 Chemistry (by directions)

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 sorbent surface, 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 the separated particles 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 to study 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 styrene-divinylbenzene 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 and are sometimes useful in place of tetrahydrofuran for the analysis of low refractive 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, so 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.