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 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 dependencies, makes it possible to determine the relative molecular weight of polymers, as well as to estimate the hydrodynamic sizes of macromolecules in solution.
| 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 |
| Possibilities | 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.
| 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 reservoir) |
| 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.
size exclusion chromatography
Gel filtration or size exclusion chromatography(sieve, gel permeation, gel filtration chromatography) - a type of chromatography, during which the molecules of substances are separated in size due to their different ability to penetrate into the pores of the stationary phase. In this case, the largest molecules (higher molecular mass) capable of penetrating into the minimum number of pores of the stationary phase are the first to leave the column. Substances with small molecular sizes, which freely penetrate into the pores, come out last. Unlike adsorption chromatography, in gel filtration, the stationary phase remains chemically inert and does not interact with the substances to be separated.
Principle
A sample solution is introduced into the column, the volume of which is limiting for the quality of chromatography. For analytical separations, it should not exceed 0.1% of CV (total column volume), and for preparative purification, it should not exceed 8-10% of CV. The column is packed with powder, the particles or granules of which have pores of a certain diameter. Macromolecular substances that do not enter the pores pass between the granules, so their retention volume is equal to the volume of the column minus the volume of the stationary phase (the so-called free volume). They elute first. Molecules of medium size fit into the pores of the sorbent, but not completely. Therefore, their retention volume is slightly higher than the free volume. They elute second. The smallest molecules freely enter the pores together with solvent molecules. Therefore, their retention volume in the column is much higher than the free volume and approaches the total volume of the column (i.e. 100% CV). They elute last.
Sorbents
Gel - a heterogeneous system in which the mobile phase (usually water) is always inside the pores of a stationary or solid phase, called the gel matrix.
Low pressure
- dextran,
- sephadex,
- sefakril,
- sepharose,
- superdex.
High pressure
- polymethacrylate,
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See what "Size Exclusion Chromatography" is in other dictionaries:
- (sieve chromatography), liquid chromatography based on acc. the ability of molecules of different sizes to penetrate into the pores of a nonionic gel, which serves as a stationary phase. There are gel permeation chromatography (eluent org. solvent) and gel ... Chemical Encyclopedia
size exclusion chromatography- ekskliuzinė chromatografija statusas T sritis chemija apibrėžtis Skysčių chromatografija, pagrįsta medžiagos molekulių pasiskirstymu tarp porose esančio ir judančio tirpiklio. atitikmenys: engl. exclusion chromatography. exclusive ... ... Chemijos terminų aiskinamasis žodynas
- (from other Greek ... Wikipedia
- (from the Greek chroma, genitive case chromatos color, paint and ... graphics is a physicochemical method for separating and analyzing mixtures based on the distribution of their components between two phases, stationary and mobile (eluent), flowing through ... ... Great Soviet Encyclopedia
A type of chromatography in which the liquid (eluent) serves as the mobile phase, and it is the stationary phase. sorbent, tv. a carrier with a liquid or gel applied to its surface. Carried out in a column filled with a sorbent (column chromatography), on a flat ... ... Natural science. encyclopedic Dictionary
This is chromatography in which the mobile phase is a liquid. Liquid chromatography is divided into liquid adsorption (separation of compounds occurs due to their different ability to adsorb and desorb from the surface ... ... Wikipedia
gel permeation chromatography- Gel Permeation Chromatography Explanatory English-Russian Dictionary of Nanotechnology. - M.
Typical setup for manual column chromatography. A glass column, equipped with a tap at the bottom to control the speed of the process, is packed with a solid phase (white), the tank is filled with liquid eluent at the top, in the upper part of the solid phase ... ... Wikipedia
Devices that measure the content (concentration) of one or more. components in liquid media; J. a. often also referred to as devices for determining St in liquids (viscometers, density meters, etc.). Distinguish Zh. and. laboratory and industrial (for ... ... Chemical Encyclopedia
See Size Exclusion Chromatography... Chemical Encyclopedia
In this method, the analyzed solution is passed through a column filled with swollen granular gel (stationary phase). The gel particles consist of a macromolecular compound (HMC) having a network structure (flexible macromolecules are crosslinked by chemical bonds). For this reason, the swollen gel has a network structure, between the nodes of which there is a solvent.
The distribution of the interstitial space of the gel along the radii- the main characteristic of the gel used, it depends on the nature of the polymer and solvent, grid frequency and temperature.
The effect of separation of substances in the case of gel chromatography is due to the fact that molecules that differ in molar mass (length) are able to penetrate into the gel structure to different depths and stay in it for different times. Therefore, during elution, large molecules that are not able to penetrate deep into the gel granules come out of the column first, and the smallest molecules come out last. There is a kind of sieving of molecules through the interstitial space of the gel.
Chromatography is carried out as follows. The gel granules are placed in a glass column, allowed to swell in the solvent, and then the analyzed mixture of substances is fed into the column. Small molecules are evenly distributed throughout the entire volume of the granules, while larger molecules, being unable to penetrate inside, remain only in the solvent layer (external volume) surrounding the granules. Next, the column is washed with a solvent - eluent. As already noted, large molecules move through the column at a higher speed than small ones, the movement of which is constantly slowed down by diffusion deep into the granules of the stationary phase. As a result, the components of the mixture are eluted from the column in descending order of their molar mass. Samples (fractions) of the eluent leaving the column are taken for analysis. The experiment is greatly simplified if there is the possibility of continuous automatic analysis of the eluent.
For research, the gel should be chosen so that its affinity for the analyzed substances is minimal: in this case, the substances are able to freely mix along the column layer in accordance with the size of their molecules. The gel granules must have optimal dimensions: too small - contribute to the rapid establishment of diffusion equilibrium, but cause high hydraulic resistance of the column. The use of large granules gives low hydraulic resistance, but inhibits diffusion, increasing the time of release of the analyzed substances.
In addition, the granules must have a certain mechanical strength, otherwise their deformation in the column will lead to a drop in the elution rate.
The most widely used for gel chromatography is sephadex(dextran gel - a high molecular weight polysaccharide), formed by growing certain bacteria in a sucrose medium. Eight types of Sephadex are produced, differing in the degree of their swelling, it is resistant to alkalis and weak acids.
Consider a specific example of the separation of a mixture of starch and glucose on Sephadex G- 25. 2 cm 3 of an aqueous solution of starch and glucose were placed in a column with 87 g of gel and the mixture was eluted with a solution of common salt. The filtrate fractions were collected and their starch and glucose content was determined. Starch molecules practically did not penetrate into the gel granules; therefore, starch was eluted first at an eluent flow rate of 32–44 ml, and glucose was eluted second at an eluent flow rate of 66–80 ml.
Based on the data obtained, a chromatogram was constructed. To do this, the concentration of substances in the fractions was plotted along the ordinate axis, and the volume of the eluent (or fraction number) was plotted along the abscissa axis. Determined from the chromatogram retention volumes V/ is the total volume of the collected eluent until the fraction with the maximum concentration of the substance exits the column. From a particular column, a given substance is always eluted at the same V,. In the case under consideration, the retention volume for starch turned out to be 35 ml, and for glucose - 73 ml.
The retention volume of substances is reproduced quite accurately. Therefore, with the help of gel chromatography, it is also possible to solve the inverse problem - to determine the molar mass of unknown compounds by determining them V,. To do this, the column is first calibrated: the retention volumes of IUDs (standard polymers) with a known molar mass are determined. For this purpose, proteins with a known fixed molar mass are most often used to calibrate hydrophilic gels. In addition, for a number of globular proteins, in addition to the molar mass determined chemically, the size of their molecules is also known. Thus, using a column calibrated with known proteins, one can also get an idea of the effective radius of the studied molecules.
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1 INSTITUTION OF THE RUSSIAN ACADEMY OF SCIENCES INSTITUTE OF ELEMENTO-ORGANIC COMPOUNDS im. A.N. NESMEYANOV. SCIENTIFIC AND EDUCATIONAL CENTER FOR PHYSICS AND CHEMISTRY OF POLYMERS MOSCOW
2 Table of contents. BASES OF CHROMATOGRAPHY OF POLYMERS. Driving forces and modes of polymer chromatography. Chromatographic peak characteristics. The concept of theoretical plates..3 Fundamentals of size exclusion (gel permeation) chromatography. CARRYING OUT PRACTICAL WORK ON THE ANALYSIS OF MWD OF THE POLYMER BY THE METHOD OF GEL PERMEATION CHROMATOGRAPHY 3. REFERENCES. BASICS OF POLYMER CHROMATOGRAPHY. Driving forces and modes of polymer chromatography. Chromatography is a method of separating substances by distributing between two phases, one of which is mobile and the other is immobile. The role of the mobile phase in liquid chromatography is played by a liquid (eluent) moving in channels between particles along a column filled with a porous material (see Fig.). Fig. Movement of a macromolecule in a chromatographic column: d k - the size of the channels between the particles of the stationary phase; dn - pore size; R is the size of the macromolecule; t s - time spent by the macromolecule in the pore, t m - in the mobile phase. The stationary phase is the pores of the sorbent filled with liquid. The average velocity of movement of this phase along the axis of the column is equal to zero. The analyte moves along the axis of the column, moving along with the mobile phase and occasionally stopping when it enters the stationary phase. This process is illustrated in Fig., which schematically shows the jump-like motion of a macromolecule with size R through channels with size d corresponding to the particle size. Molecules stop in slit-like pores, the size of which corresponds in order of magnitude to the size of macromolecules. The time between successive stops can be written as:
3 t t s + t m + t k, () where t s is the residence time of the molecule in the stationary phase, t m d is the time spent by the molecule in the mobile phase (D - D is the transverse diffusion coefficient, t k is the time of transition from the mobile phase to the stationary phase and vice versa). Usually in the processes of high performance liquid chromatography (Hgh Performance Lqud Chromatography in the English literature) in its analytical version, this time t k is much less than the first two and can be omitted in the formula (). If the number of stops during the movement along the column is sufficiently large, then the total time of the macromolecule movement along the column is sufficiently large as compared to the characteristic time of equilibrium establishment. In this case, to determine the probability of finding a macromolecule in a unit volume of the stationary phase with respect to the mobile phase (or the distribution coefficient K d equal to the ratio of concentrations in these phases), the methods of equilibrium thermodynamics can be used. Namely, the distribution coefficient will be determined by the free energy of the transition of the macromolecule from the mobile phase to the stationary phase: T S H G RT Kd exp exp () RT For a chain consisting of N segments, K exp(N µ), (3) d where µ is the change in the chemical potential segment. The distribution coefficient in chromatography is a fundamental concept and is defined as follows: VR V K d (4) Vt V t is the elution volume of the substances leaving together with the solvent front. From (3), one can immediately see that, depending on the sign of G, the macromolecules behave differently when they enter the pore (see figure): Fig.. if G>, then K d tends to with increasing macromolecule length (in this case the volume of elution also decreases). This corresponds to size exclusion chromatography. At G< K d экспоненциально растет с ростом ММ и это соответствует адсорбционному режиму хроматографии. Таким образом, оба режима хроматографии могут рассматриваться в рамках единого механизма и, более того, плавно меняя энергию взаимодействия сегмента с поверхностью сорбента за счет состава растворителя или температуры, можно обратимо переходить от одного режима к другому. Экспериментально это было впервые показано в работе Тенникова и др. . Точка (для данной пары полимер - сорбент - это состав растворителя и температура), соответствующая равенству G, при которой происходит компенсация энтропийных потерь и энергетического выигрыша при каждом соударении сегмента макромолекулы со стенкой поры называется критической точкой адсорбции или критическими условиями хроматографии. Как видим, в этих условиях не происходит деления по ММ и это обстоятельство является предпосылкой для использования режима критической хроматографии для исследования разных типов молекулярной неоднородности полимеров, таких как число функциональных групп на концах цепи, состав блоксополимеров, топология 3
4 (presence of branched or cyclic macromolecules). This chromatographic method is relatively new and some of the most interesting results of its application can be found, for example, in [,3,4]. Chromatography mode corresponding to condition G< широко применяется для разделения низкомолекулярных соединений и называется, в зависимости от химической природы функциональных групп на поверхности сорбента, адсорбционной, нормальнофазной, обращеннофазной, ионпарной и т.д. хроматографией. Для полимеров его применение ограничено областью слабых взаимодействий вблизи критических условий и областью олигомерных макромолекул, т.к. с ростом длины цепи мы переходим к практически необратимой адсорбции макромолекулы на колонке. Наиболее важным для полимеров является режим эсклюзионной хроматографии или, как его еще называют, гельпроникающей хроматографии. Этот режим более подробно будет рассмотрен в следующем разделе, а сейчас мы перейдем к описанию некоторых важнейших хроматографических характеристик... Характеристики хроматографического пика. Концепция теоретических тарелок. После прохождения через хроматографическую колонку узкой зоны какого-либо монодисперсного вещества, на выходе мы получаем расширенную зону в виде пика приблизительно гауссова по форме (в случае хорошо упакованной колонки и правильно выбранной скорости хроматографии). Причины расширения пика лежат в различных диффузионных процессах, сопровождающих движение молекул вдоль колонки (см. например, соотношение ()). Наиболее важные характеристики пика - объем элюирования или V R или объем удерживания (относится к центру пика) и дисперсия пика, т.е. второй центральный момент (см.рис.3): σ h V V dv R. (5) Справедливы следующие соотношения между величинами, показанными на рис.3: σ, 43W W b. (6) 4 Рис. 3. Модель гауссова пика. Параметры уширения пика. Часто все эти величины выражаются в единицах времени, тогда говорят о времени удерживания и т.д., однако, в этом случае скорость потока элюента должна быть строго фиксирована. Существует простая феноменологическая теория описания относительного вклада расширения зоны в хроматографическое разделение. Это - теория тарелок. Хроматографическая колонка мысленно делится на ряд последовательных зон, в каждой из которых достигается полное равновесие между растворенным веществом в подвижной и неподвижной фазе. Физическую основу этого подхода составляет скачкообразное движение, описанное в начале первого раздела, и число теоретических тарелок в колонке связано с числом остановок при попадании в неподвижную фазу за время движения данного вещества по колонке. Чем больше это число, тем больше число теоретических тарелок и тем выше эффективность колонки. Число теоретических тарелок определяется следующим образом: 4
5 VR N σ V 5.54 W R V 6 W R b. (7) Since this value changes with the elution volume, it is correct to use the unretained substance exiting at K d..3 to characterize the efficiency of the column. Fundamentals of the size-exclusion (gel-penetrating) chromatography method. Size exclusion chromatography (Sze Excluson Chromatography, SEC) or gel permeation chromatography (GPC, Gel Permeaton Chromatography, GPC) is implemented when the behavior of macromolecules in pores is determined by the entropy component of free energy, and the energy component is small compared to it. In this case, the distribution coefficient will depend exponentially on the ratio of macromolecule size and pore size. The scaling theory predicts the following regularities for the case of pores commensurate with the macromolecule size R K d Aexp D α, (8) 4/3 to depending on the adopted pore model (slit, capillary, strip) and the chain model (ideal or imperfect). Thus, the behavior of macromolecules under the conditions of size exclusion chromatography is determined by the chain size. The size of a macromolecule is determined by its chemical structure, the number of links in the chain (or molecular weight), topology (for example, the size of a branched macromolecule or macrocycle decreases compared to a linear macromolecule of the same chemical structure). In addition, the size of flexible macromolecules depends to a certain extent on the solvent used due to the excluded volume effect. However, the GPC method has become widely used in laboratory practice as a method of separation by molecular weights, determination of average molecular weights and molecular weight distributions (MWDs). The development of the method began in the mid-1950s, when the first wide-pore organic sorbents for high performance gel permeation chromatography were created. As can be seen from relations (8), the method is not absolute for determining molecular weights, but requires an appropriate calibration against standard (preferably narrowly dispersed) samples with known MW, relating the retention volume (or time) to MW. Figure 4 illustrates the calibration curves for polystyrene in terms of lg V R on Waters semi-rigid organic sorbents (crostyragel) with different pore sizes. For the analysis of any polymer by molecular weight, it is necessary to select a column with an appropriate pore size or a series of columns with different pores, or use a column with a mixture of sorbents with different pores (the Lnear column in the given example). Of course, in order to use the GPC method for the analysis of MWD, it is necessary to provide conditions for the implementation of the exclusion mechanism of separation, which is not complicated by the effects of the interaction of both middle and terminal links of the chain. We are talking about adsorption interaction from a nonpolar solvent or reversed-phase interaction of nonpolar chain fragments during chromatography of hydrophilic polymers in an aqueous medium. In addition, water-soluble polymers containing ionized groups are capable of strong electrostatic interactions and require particularly careful selection of chromatography conditions. The selection of conditions includes the selection of a sorbent and solvent (eluent) suitable for a particular analysis in terms of chemical structure. 5
6 Recommendations can be found in the manuals of chromatographic equipment manufacturers, as well as in reference books and monographs (see, for example, ), 6 V R, ml Pic. 4. Calibration curves for µstyragel columns. The figure shows the corporate labeling of the columns with a value that characterizes the size of the sorbent pores, which is equal to the length of the extended polystyrene chain excluded from the pores for steric reasons. The chromatographic column is the heart of the liquid chromatograph. The chromatograph also includes a number of necessary additional devices:) an eluent supply system (pump) that provides a stable flow,) a sample injection system without stopping the flow (injector or autosampler), 3) a detector - a device that provides the formation of a signal proportional to the concentration of a substance at the 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 the data processing system. The polymer chromatogram obtained under size exclusion chromatography F(V) is a reflection of its molecular weight distribution function W(). By virtue of the law of conservation of matter: F V dv W d ). The real chromatogram is the result of the separation of the sample by MW when moving along the column and the simultaneous mixing of polymer homologues due to the blurring of the zones. Therefore, the function F(W) in relation (9) should be understood as a chromatogram corrected for PU. This function is a solution to the Fredholm integral equation of the first kind. There are quite a lot of ways to correct for PU. See, for example, . However, in modern high-performance chromatographic systems, in most cases, the contribution of PU to the chromatogram is small compared to MWD and can be neglected. The most important procedure is the calibration of the chromatograph according to the molecular weight of the polymer under study. If there are corresponding narrowly dispersed standards with different MM, the elution volumes (V R or Ve) are determined for them and a calibration dependence similar to that shown in Fig. 4 is built. Typically, the calibration relation is sought in the form (): n lg C V e () Polynomials of the first or third degree are most often used. Polynomials of odd degrees (3, 5, 7) most accurately describe the characteristic shape of the calibration curves with upper and lower MM limits. Sets of narrowly dispersed standards exist for such polymers as polystyrene, polyisoprene, polymethylmethacrylate,
7 polyethylene oxide, dextrans and some others. You can also use the method of universal calibration, first introduced into practice by Benoit and co-workers. The method is based on the fact that the hydrodynamic volume of macromolecules is proportional to the product of the intrinsic viscosity and the molecular weight of the polymer and can be used as a function of the elution volume as a universal parameter for different polymers. Then we 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 B V e + a lg K. (4) As a result, From the data of gel permeation chromatography, one can find the average molecular weights of various degrees of averaging, which, by definition, are the following values: () n - number average MM, W () d W d w z W d W d W d W d - weight average MM, - z-mean MM. The MM ratios of different degrees of averaging characterize the statistical width of the MMD. The most commonly used ratio is w / n, which is called the polydispersity index. 4. CARRYING OUT PRACTICAL WORK ON THE ANALYSIS OF MWD OF A POLYMER BY METHOD OF GEL PENETRATION CHROMATOGRAPHY Purpose of the work: To get acquainted with the operation of a liquid chromatograph, the method of conducting a chromatographic experiment, the method of calibrating a chromatograph according to narrowly dispersed polymer standards and calculating average molecular weights. Equipment:) Liquid chromatograph, consisting of a pump, an injector, a column thermostat, a column with a polymeric sorbent and a data processing system based on a personal computer.) A set of narrowly dispersed standards with different MM (polystyrene or polyethylene oxide). 3) Test sample with unknown molecular weights. Operation procedure:) Preparation of a solution of a mixture of standards. 7
8) Obtaining a chromatogram of the standards and determining their retention volumes (V e). 3) Construction of the calibration dependence in the form (). 4) Preparation of a solution of the investigated polymer. 5) Obtaining a chromatogram of the investigated polymer. 6) Calculation of the average MM of the sample. Figure 5 shows a typical example of a polymer sample chromatogram prepared for calculating the average MW, namely, a baseline is drawn that defines the beginning and end of the chromatogram, and then the chromatogram is divided into equal parts along the time axis, the so-called slices. n w z A, A A A, A A. 5. For each slice, its area A is determined and the molecular weight corresponding to its middle is calculated from the calibration dependence. The average molecular weights are then calculated: 8
9 3. LITERATURE. M. B. Tennikov, P. P. Nefedov, M. A. Lazareva, S. Ya. Comm., A, 977, v.9, N.3, with S.G.Entelis, V.V.Evreinov, A.I.Kuzaev, Reactive oligomers, M: Chemistry, T.M.Zimina, E.E. Kever, E.Yu. Melenevskaya, V.N. comm., A, 99, vol. 33, N6, with I.V. Comm., A, 997, v.39, N6, with A.M. Skvortsov, A.A. Gorbunov, Scaling theory of chromatography of linear and ring macromolecules, Vysokomolek. comm., A, vol. 8, N8, with B. G. Belenkiy, L. Z. Vilenchik, Chromatography of polymers, M: Chemistry, W. W. Yau, J. J. Krkland, D. D. Bly, orn Sze-Excluson Lqud Chromatography, New York: John Wley & Sons, E.L. Styskin, L.B. Itsikson, E.B. Braudo. Practical High Performance Liquid Chromatography. Moscow Ch Wu, Ed.Column Handbook for Sze Excluson Chromatography, N-Y: Academc Press..Z.Grubsc, R.Rempp, H.Benor, J. Polym. Sc., B, 967, v.5, p
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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.
Chromatographic instrument. 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 similar 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 k n is small, F of this chromatographic method is less than in adsorption chromatography.
Gel chromatography (or gel permeation chromatography) is a type 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 during diffusion through membranes, on both sides of which the concentrations of these molecules are different, do not differ too 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 (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 different 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.
