Gel Filtration Chromatography Assignment - Desklib
Added on 2020-05-16
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Running head: GEL FILTRATION CHROMATOGRAPHYGel Filtration ChromatographyName of the StudentName of the UniversityAuthor Note
1GEL FILTRATION CHROMATOGRAPHYIntroduction and BackgroundThe principal method that is used for the separation and purification of thebiomolecules in biochemical laboratories is chromatography. In chromatographic separation,partition occurs in between two phases one is mobile phase and another one is stationaryphase. Gel filtration chromatography or size exclusion chromatography is a type ofchromatography (Murray, Granner, Mayes and Rodwell 2009). It is used to separate proteinmolecules based on stokes radius. Stoke radius is the measured diameter of the sphereoccupied by the protein when they tumble in the solution. Stoke radius can also be defined asthe function of molecular mass and shape (Murray, Granner, Mayes and Rodwell 2009). Asprotein with large stroke radius are too large to enter inside the porous bed (stationary phase)and are thus eroded away (mobile phase) where as protein with smaller stoke radius enter theindentation of the porous bed and remain there until they are drifted back by the mobilephase. The protein thus separated emerge from the gel filtration column in the descendingorder of their measured stroke radii (Murray, Granner, Mayes and Rodwell 2009; Nelson,Lehninger and Cox 2008).The following report gives a detailed analysis of concept behind the gel filtrationchromatography along with the potential advantages and disadvantages associated with it.The report further illustrates the mechanism of gel filtration chromatography viadiagrammatic representations and two video links. After illustrating the concept, the reporttends to shed light on the practical application of this biochemical procedure in researchalong with different porous beds with their size limits, which are commonly employed asstationary phase. At the end, the report will provide a detailed breakup of the formula used tocalculate the protein size in gel filtration chromatography.
2GEL FILTRATION CHROMATOGRAPHYWhat's it used forGel filtration chromatography is the simplest technique used for the separation of thecomplex component by according to their molecule size only, measuring by how dynamicthey penetrate the pore of the stationary phase, this chromatography is also know a sizeexclusion chromatography or molecular exclusion chromatography. This type of methodpermits to purified polypeptide from other polypeptides based to their size by movingthrough the gel porous into the column. Gel filtration is application use to analyze theseparating of molecules size such as protein, polysaccharides -and nucleic acids by separatingand removing large proteins and complexes.Separation and removing molecular weight proteins and complexesThis is used for the separation of the molecules according to their molecular weight,the proteins are aqueous in solution (mobile phase), the protein solution will be transfer to thetop of chromatography column, these columns composed of a gel bed of porous beads, this isknown as (stationary phase).During the analyzing gel filtration the smaller molecules able to penetrate the bed ofporous of beads, and will take time and elute last, while the larger molecules will not be ableto enter bed of the porous of beads and pass through the sides of the gap between the porousand elute first.The substance has been chosen for the fraction and the recognition of the molecularsize depending on the size rage of the molecules within the sample. The molecules passthrough the column in the pores including their aqueous solution to complete the elution.Subsequently, the molecule is measured. The volume is known as the total value TV.During the separation process of the protein large molecules will elute first by passingthrough the sides of the gel of the stationary phase. However, the small molecules will take
3GEL FILTRATION CHROMATOGRAPHYsome time to elute as they enter the pore of the beads. There are many other technique orways that can be purified the protein. Nevertheless, this process is of the ways to carry out. Size analyzing and determiningGel chromatography is one of the methods that is used to analyze in the attempt todetermine the size of the protein.This process separates the protein based on their size,solution and their shape as they pass through the stationary phase or gel pores. Usuallyprotein has three-dimensional polymers of dextran, agarose or silica which is composed poresof controlled size ranges to give free movement to the liquid phase and limited diffusion ofthe protein to be separated.Desalting and Buffer exchangeDesalt and buffer exchange of gel filtration is used for the separation of themacromolecules from the smaller size molecules. Desalting and the buffer exchange are oneof those applications used gel filtration chromatography. Both desalt and buffer exchangeuses the same substance. The process to extract salt from the sample is known as “desalt” Thebuffer exchange occurs when extracting the salt from the test. There are different types of saltexchanges. In chromatography desalt is balanced with water while the buffer exchange isbalanced with salt. Buffer exchange is a method used to exchange a set of buffer salts insubstance to another. The applications used for desalting is the salt extraction from proteinsolution extracting phenol or unincorporated nucleotides from nucleic acid preparation.Buffer exchange is generally used to place a protein solution into a more appropriatebuffer before the subsequent applications such as:1.Electrophoresis2.Ion exchange
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