exclusion-size chromatography ( SEC ), also known as molecular filter chromatography , is a chromatographic method in which the molecules in solution are separated by their size, and in some cases of molecular weight. These are usually applied to large molecules or complex macromolecules such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport samples through a column, this technique is known as gel filtration chromatography , versus name gel permeation chromatography, used when organic solvents are used as a mobile phase. Chromatographic columns are packaged with fine porous beads consisting of dextran polymers (Sephadex), agarose (Sepharose), or polyacrylamide (Sephacryl or BioGel P). The pore size of the beads is used to estimate the dimensions of macromolecules. SEC is a widely used method of polymer characterization because of its ability to provide a good molar mass distribution (Mw) for polymers.
Video Size-exclusion chromatography
Apps
The main application of gel-filtration chromatography is the fractionation of proteins and other water soluble polymers, whereas gel permeation chromatography is used to analyze the molecular weight distribution of organic soluble polymers. One technique should not be equated with gel electrophoresis, where an electric field is used to "pull" or "push" molecules through a gel depending on its electrical charge. The amount of solute time that remains in the pore depends on the pore size. Larger solutions will have access to smaller volumes and vice versa. Therefore, larger solutes will remain within the pores for longer periods of time compared to smaller solutes.
The use of other size exclusion chromatography is to check the stability and characteristics of natural organic matter in water. In this method, Margit B. Muller, Daniel Schmitt, and Fritz H. Frimmel tested water sources from different parts of the world to determine how stable natural organic materials are in a given period of time. Although many size exclusion chromatography is used to study natural organic matter, there are some limitations. One of these limitations includes that there is no standard molecular weight tag; thus, nothing can compare the result back. If proper molecular weight is required, other methods should be used.
Maps Size-exclusion chromatography
Benefits
The advantages of this method include the good separation of large molecules from small molecules with minimal volume, and that solutions can be applied without disturbing the filtration process, while maintaining the biological activity of the particles separately. This technique is generally combined with others that separate molecules with other characteristics, such as acidity, alkalinity, charge, and affinity for certain compounds. With size exclusion chromatography, there is a short and well-defined separation time and narrow band, leading to good sensitivity. Also no samples are lost because the solute does not interact with the stationary phase.
Another advantage to this experimental method is that in certain cases it is feasible to determine the approximate molecular weight of a compound. The shape and size of the compound (eluent) determines how the compound interacts with the gel (stationary phase). To determine the approximate molecular weight, the elution volume of the corresponding molecular weight compound is obtained and then plot "K av " vs "log (Mw)" is made, where K av = (V t -V o ) and Mw is the mass of the molecule. This plot acts as a calibration curve, which is used to estimate the molecular weight of the desired compound. The component V e represents the volume at which the intermediate molecule elutes like a molecule having partial access to the column beads. In addition, V t is the sum of the total volume between the beads and the volume within the beads. The component V o represents the volume at which the larger molecule elute, the elute at the beginning. The drawbacks are, for example, that only a limited number of bands can be accommodated because of the short chromatogram time scale, and, in general, there should be a 10% difference in molecular mass to have good resolution.
Discovery
This technique was invented by Grant Henry Lathe and Colin R Ruthven, who worked at Queen Charlotte Hospital, London. They then received the John Scott Award for this discovery. While Lathe and Ruthven use starch gel as a matrix, Jerker Porath and Per Flodin then introduce dextran gel; Other gels with fractionation properties of size include agarose and polyacrylamide. A brief review of this development has emerged.
There is also an attempt to fractionate high synthetic polymers; However, it was not until 1964, when JC Moore of Dow Chemical Company published his work on gel permeation chromatography (GPC) column preparation based on crosslinked polystyrene with controlled pore size, that a rapid increase of research activity in this regard. field starts. It was recognized immediately that with proper calibration, GPC was able to provide molar mass and molar mass distribution information for synthetic polymers. Since last information is difficult to obtain with other methods, GPC comes quickly into extensive use.
Theory and method
SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping smaller molecules in adsorption pores of adsorbent material ("stationary phase"). This process is usually done with columns, consisting of a vacuum tube packed tightly with very small porous polymer beads designed to have pores of different sizes. These pores can become depressed on the surface or channel through the beads. When the solution goes down the column, some particles enter into the pores. Larger particles can not enter many pores. The larger the particle, the faster the elution. The bigger molecules just pass through the pores because the molecules are too big to get into the pores. Therefore, larger molecules flow through columns faster than smaller molecules, that is, the smaller the molecule, the longer the retention time.
One requirement for the SEC is that the analyte does not interact with the stationary phase surface, with the difference in elution time between the analyte ideally based solely on the volume of solute the analytes can insert, rather than the chemical or electrostatic interaction with the stationary phase. Thus, small molecules that can penetrate every region of the stationary phase pore system can enter a total volume equal to the sum of all pore volume and interparticle volume. These small molecules elutes late (after the molecule has penetrated all the pore volume and interparticles - about 80% of the column volume). At the other extreme, very large molecules that can not penetrate smaller pores can enter only the interparticle volume (~ 35% of the column volume) and deceive beforehand when the volume of this phase of motion has passed through the column. The principle underlying the SEC is the particles of different sizes elute (filter) through the stationary phase at different rates. This results in separation of particle solutions by size. As long as all particles are loaded simultaneously or almost simultaneously, particles of the same size must elute together.
However, since there are various measures of macromolecular size (eg, radius of the circle and hydrodynamic radius), the fundamental problem in SEC theory has been the choice of precise molecular size parameters in which the molecules are different. separated types. Experimentalally, Benoit and colleagues found an excellent correlation between elution volume and dynamic-based molecular size, hydrodynamic volume, for several different chain architectures and chemical compositions. The observed correlation based on hydrodynamic volume becomes accepted as the basis of universal SEC calibration.
However, the use of hydrodynamic volume, size based on dynamic properties, in the interpretation of SEC data is not fully understood. This is because the SEC usually runs under low flow rate conditions where the hydrodynamic factor must have little effect on the separation. In fact, both computer theory and simulation assume the principle of thermodynamic separation: the separation process is determined by the equilibrium distribution (partition) of the solvated macromolecules between two phases --- the aqueous mass solution phase located in the interstitial space and the limited solution phase in the pores of the column packing material. Based on this theory, it has been shown that the size parameters relevant to the polymer partition in the pores are the average range dimensions (mean maximum projection to line). Although this problem is not yet fully resolved, it is likely that the mean range dimensions and hydrodynamic volume are strongly correlated.
Each size exclusion column has various separable molecular weights. The exception limit defines the molecular weight at the top end of the 'working' column range and where the molecule is too large to get stuck in the stationary phase. The lower end of the range is defined by the permeation boundary, which defines the molecular weight of a molecule small enough to penetrate all the pores of the stationary phase. All the molecules under the mass of these molecules are so small that they elute as single bands
The filtered solution collected at the end is known as eluat . The void volume includes particles that are too large to enter the medium, and the volume of the solvent is known as volume column .
Factors affecting filtering
In real life situations, the particles in the solution do not have a fixed size, resulting in the probability that a particle would otherwise be inhibited by the pores passing right by it. Also, stationary phase particles are not ideally defined; particles and pores can vary in size. The elution curve, therefore, resembles the Gaussian distribution. The stationary phase can also interact in unwanted ways with particles and affect retention times, although great attention is taken by the column manufacturers to use an inert phase of silence and minimize this problem.
Like other chromatographic forms, increasing the column length increases the resolution, and increasing the column diameter increases the column capacity. Proper column packing is important for maximum resolution: Excess columns can break down the pores within the beads, resulting in a loss of resolution. Unpacked columns can reduce the relative surface area of ​​the stationary area accessible by smaller species, so that the species spend less time trapped in the pores. Unlike affinity chromatography techniques, the solvent head at the top of the column can drastically reduce the resolution as the sample diffuses before loading, extending the downstream elution.
Analysis
In a simple manual column, eluents are collected in a constant volume, known as fractions. The more like particles in size the more likely they are in the same fraction and not detected separately. The more advanced columns solve this problem by continuously monitoring the eluent.
The collected fractions are often examined by spectroscopic techniques to determine the concentration of eluted particles. Common spectroscopic detection techniques are refractive index (RI) and ultraviolet (UV). When elaborating species similar to spectroscopy (such as during biological purification), other techniques may be needed to identify the contents of each fraction. It is also possible to analyze continuous flow of eluent with RI, LALL, Multi-Angle Laser Light Scattering MALS, UV, and/or viscosity measurements.
The elution volume (Ve) decreases roughly linearly with the molecular hydrodynamic volume logarithm. Columns are often calibrated using 4-5 standard samples (eg, proteins folded from known molecular weights), and samples containing very large molecules such as thyroglobulin to determine the volume of vacancies. (Blue dextran is not recommended for the determination of Vo as it is heterogeneous and can provide variable results) The standard elution volume is divided by the elution volume of thyroglobulin (Ve/Vo) and plotted against the log of the standard molecular weight '.
Applications
Biochemical Application
In general, the SEC is considered low-resolution chromatography because it does not distinguish very similar species very well, and is therefore often reserved for the final step of purification. This technique can determine the quaternary structure of a purified protein that has a slow exchange time, as it can be done under the conditions of the original solution, preserving macromolecular interactions. The SEC can also test the tertiary structure of proteins, as it measures the hydrodynamic volume (not molecular weight), allowing the same version of protein to be folded and folded to be distinguished. For example, the apparent hydrodynamic radius of a typical protein domain may be 14 ÃÆ'... and 36 ÃÆ'... for folded and unfolded shapes, respectively. The SEC allows the separation of these two forms, since the shape is folded some distance later because of its smaller size.
Polymer synthesis
SEC can be used as a measure of both the size and polydispersity of the synthesized polymer, ie the ability to find the size distribution of polymer molecules. If a standard of a known measure is run beforehand, then a calibration curve can be made to determine the size of an interesting polymer molecule in a solvent chosen for analysis (often THF). In alternative modes, techniques such as light scrolling and/or viscometry can be used online with the SEC to produce absolute molecular weights that do not rely on calibration with known molecular weight standards. Because of the difference in the size of two polymers with identical molecular weight, the method of determining absolute, in general, is more desirable. A typical SEC system can quickly (about half an hour) provide polymer chemical information on the size and sample polydispersity. SEC preparative can be used for polymer fractionation at analytic scale.
Weakness
In the SEC, the mass is not measured as the hydrodynamic volume of the polymer molecule, that is, how much space a particular polymer molecule takes when it is in solution. However, the approximate molecular weight can be calculated from the SEC data because the exact relationship between molecular weight and hydrodynamic volume for polystyrene can be found. For this, polystyrene is used as a standard. But the relationship between hydrodynamic volume and molecular weight is not the same for all polymers, so only estimation of the measurements can be obtained. Another disadvantage is the possibility of interaction between stationary and analytical phases. Each interaction leads to elution time later and thus mimics smaller analytical sizes.
When performing this method, the eluting molecular bands can be expanded. This can occur by turbulence caused by the flow of moving phase molecules passing through the stationary phase molecule. In addition, thermal diffusion of molecules and friction between glass wall molecules and eluent molecules contributes to the expansion of the tape. In addition to widespread, bands also overlap. As a result, eluen usually gets diluted. Some precautions can be taken to prevent the possibility of widespread bands. For example, one can apply samples in very narrow bands at the top of the column. The more concentrated the eluent, the more efficient the procedure. However, it is not always possible to concentrate eluent, which can be regarded as one more weakness.
Absolute size exclusion chromatography
Absolute size exclusion chromatography (ASEC) is a technique that combines dynamic light scattering instruments (DLS) with a size exclusion chromatography system for measuring the absolute size of proteins and macromolecules as they elute from the chromatographic system.
The absolute definition used here is that it does not require calibration to obtain hydrodynamic size, often referred to as the hydrodynamic diameter (D H in units nm). Macromolecular sizes are measured as they elute DLS instrument stream cells from a set of size exclusion columns. It should be noted that the hydrodynamic size of the molecule or particle is measured and not the molecular weight. For proteins, the Mark-Houwink type of calculation can be used to estimate the molecular weight of the hydrodynamic size.
The big advantage of DLS coupled with the SEC is the ability to get DLS resolution enhancements. The DLS batch is fast and simple and provides a direct measure of average size, but the DLS base resolution is 3 to 1 in diameter. Using SEC, protein and protein oligomers are separated, allowing oligomer resolution. Aggregation studies can also be done using ASEC. Although the aggregate concentration may not be calculated, the aggregate size can be measured, limited only by the maximum size eluted from the SEC column.
The limitations of ASEC include flow rate, concentration, and precision. Because the correlation function takes between 3-7 seconds to build properly, a limited number of data points can be collected across the peak.
See also
- Pegilasi
- gel permeation chromatography
References
External links
Source of the article : Wikipedia
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