Size Exclusion Chromatography
Size Exclusion Chromatography Size Exclusion Chromatography (SEC) is the separation technique based on the molecular size of the components. Size exclusion chromatography is a kind of method to separate different size of molecules that put in solution. It was first discovered by two scientists who named Grant Henry Lathe and Colin R Ruthven.
Both of them received the John Scott Award for this fabulous invention. There are various applications for Size exclusion chromatography such as biochemical aspect and polymer synthesis.
For application in biochemical aspect, this technique can find out the quaternary structure of purified proteins which possess slow exchange times, since it can be carried out under native solution conditions and preserve macromolecular interactions. The reason why we use this technique for purification is Size exclusion chromatography is a low resolution chromatography method as it does not identify similar species very well. It can also test the tertiary structure of protein as it measures the hydrodynamic volume, allowing folded and unfolded versions of the same protein to be distinguished.
Besides using in biochemical research, it is able to find the distribution of the sizes of polymer molecules like if a solvent is chose and run, we can create a calibration curve to determine the sizes of polymer molecules in it. It is better to introduce the mobile phase and stationary phase first. Stationary phase is the solid absorbent or the pore(SEC) with solid support that allow sample across through it while the mobile phase is the sample percolate through or along to the stationary phase.
In SEC, separation is achieved by the differential exclusion from the pores of the packing material, of the sample molecules(mobile phase) as they pass through a bed of porous particles(stationary phase). For the principle of the SEC, molecules of different sizes can be separated by this technique because of differential time spent inside a solid phase particle which excludes entrance of relatively larger molecules, allows some entrance of medium-sized molecules, and allows free accessibility of the smallest molecules.
The particles contain pores with tunnels(stationary phase) in which the size can be controlled depending on the size of molecules(mobile phase) to be separated. Smaller molecules experience a more complex pathway to exit the particle than do larger molecules. Because molecules that have a large size compared to the pore size of the stationary phase have very little entrance into the pores, these larger sized molecules elute first from the column. Medium sized molecules are relatively large compared to the pore size of the solid phase and therefore may find some pores in which they enter and spend some time.
Smaller-sized molecules have more pores that are accessible to them and therefore spend more time inside the pores relative to larger-sized molecules. Therefore, smaller molecules elute last and larger molecules elute first in SEC. “Elute” is mean that the carrier of the mobile phase or the mobile phase from chromatographic bed emerge. For the pore size, which is the important part of stationary phase in SEC, solid phase materials used in SEC are usually classified based on their ability to separate different sizes of proteins.
Since size is a difficult item to accurately measure for a large molecule, the solid phase materials are identified with a molecular weight range instead and the weight is equated with size. All compounds with a molecular weight less than or equal to the lower number in the range will see the entire internal volume of the beads resulting in no selection and therefore no separation. All compounds with a molecular weight greater than or equal to the higher number in the range are completely excluded from the inside of a bead and therefore no separation is achieved.
Molecules with weights or sizes between these two extremes of the range can be separated. This is the numerical pore size range reported for each solid phase material used in SEC. The pore size used for a separation is dependent on the size range of the particular set of molecules to be separated. Smaller pore sizes are used for rapid desalting of proteins or for protein purification. Intermediate pore sizes are used to separate relatively small proteins. Very large pore sizes are used for purification of biological complexes.
For the factor that affect the SEC, first, the particles in solution do not have a fixed size, resulting in the probability that a particle that would otherwise be hampered by a pore passing right by it. Second, the stationary-phase particles are not ideally defined, both particles and pores may vary in size. . The stationary phase may also interact in undesirable ways with a particle and influence retention times, though great care is taken by column manufacturers to use stationary phases that are inert and minimize this issue.
Third, increasing the column length will enhance the resolution, and increasing the column diameter increases the capacity of the column. Proper column packing is important to maximize resolution: An over-packed column can collapse the pores in the beads, resulting in a loss of resolution. An under-packed column can reduce the relative surface area of the stationary phase accessible to smaller species, resulting in those species spending less time trapped in pores.
Unlike affinity chromatography techniques, a solvent head at the top of the column can drastically diminish resolution as the sample diffuses prior to loading, broadening the downstream elution. The advantages of this method include good separation of large molecules from the small molecules with a minimal volume of eluate, and that various solutions can be applied without interfering with the filtration process, all while preserving the biological activity of the particles to be separated.
Second, the technique is generally combined with others that further separate molecules by other characteristics, such as acidity, basicity, charge, and affinity for certain compounds. Third, with size exclusion chromatography, there are short and well-defined separation times and narrow bands, which lead to good sensitivity. The SEC is separated rapidly. Then, there is also no sample loss because solutes do not interact with the stationary phase. The stationary phase doesn’t have any absorbent that nteract with the sample and carry out the reaction with the sample. For the disadvantage of the this method , first is the molecular mass that we need to know. The SEC separation is base on the molecular size/ weight, like the gel electrophoresis. It is required to know that there are the range for different of the molecular size. If the difference of the molecular size in the mobile phase, it is not recommended to use this separation. So, before using the SEC, the molecular size of each sample in mobile phase are required to identify.
In addition, the accommodated of SEC is limited. The mobile phase can not be too big. The time scale of the chromatogram is short, and, in general, there has to be a 10% difference in molecular mass to have a good resolution Also, the pore size need to be determined, too small size or too big size will lead to the failure of the separation SEC. In the world, the chromatography is the separation of the sample base on the polar, size, acidity, basicity, charge, and affinity for certain compounds…
Size Exclusion Chromatography is the one of the chromatography that base on the size of the sample, which is similar to the principle of gel electrophoresis. One different point is the stationary phase, which is the column with the pores of the particles. Reference: (http://www. separations. us. tosohbioscience. com/ServiceSupport/TechSupport/ResourceCenter/PrinciplesofChromatography/SizeExclusion/) (http://www. asdlib. org/separations_pdfs/Size_Exclusion_Chromatography_Separations_Module-finalversion. pdf) (http://en. wikipedia. org/wiki/Size-exclusion_chromatography),goldbook