exam
Gel Filtration
Gel filtration is a non-adsorptive chromatography technique that separates molecules on the basis of molecular size. Desalting and buffer exchange are two special examples of gel filtration that are widely used in many downstream bioprocesses. Desalting is used to completely remove or lower the concentration of salt or other low molecular weight components in the sample while buffer exchange replaces the sample buffer with a new buffer.
Gel filtration is one of the easiest chromatography methods to perform because samples are processed using an isocratic elution. In its analytical form, gel filtration (also known as size exclusion chromatography) can distinguish between molecules (e.g. proteins) with a molecular weight difference of less than a factor of 2 times. In this application, the porosity of the gel filtration media to be used is selected to provide high resolution in the molecular weight range of interest.
The more common applications for gel filtration are desalting and buffer exchange. In these applications, the size difference between the substances being separated is very large (i.e. proteins vs. salts). A gel filtration media is chosen that completely excludes the larger molecules while allowing the smaller molecules to freely diffuse into all of the pore spaces. The column is equilibrated with a buffer, which may be the same or different from that of the sample. Following application of the sample to the column, more of the column buffer (eluting buffer) is added to carry the sample molecules down the column. The larger molecules, which can’t enter the pores of the media, elute first from the column, followed by the smaller molecules that diffuse into the pores, slowing them down relative to the larger molecules. If the eluting buffer is different from the sample that was applied, the larger molecules will be displaced from the original salts and elute in this new buffer, completely separated from the original sample buffer.
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Gel filtration is a non-adsorptive chromatography technique that separates molecules on the basis of molecular size. Desalting and buffer exchange are two special examples of gel filtration that are widely used in many downstream bioprocesses. Desalting is used to completely remove or lower the concentration of salt or other low molecular weight components in the sample while buffer exchange replaces the sample buffer with a new buffer.
Gel filtration is one of the easiest chromatography methods to perform because samples are processed using an isocratic elution. In its analytical form, gel filtration (also known as size exclusion chromatography) can distinguish between molecules (e.g. proteins) with a molecular weight difference of less than a factor of 2 times. In this application, the porosity of the gel filtration media to be used is selected to provide high resolution in the molecular weight range of interest.
The more common applications for gel filtration are desalting and buffer exchange. In these applications, the size difference between the substances being separated is very large (i.e. proteins vs. salts). A gel filtration media is chosen that completely excludes the larger molecules while allowing the smaller molecules to freely diffuse into all of the pore spaces. The column is equilibrated with a buffer, which may be the same or different from that of the sample. Following application of the sample to the column, more of the column buffer (eluting buffer) is added to carry the sample molecules down the column. The larger molecules, which can’t enter the pores of the media, elute first from the column, followed by the smaller molecules that diffuse into the pores, slowing them down relative to the larger molecules. If the eluting buffer is different from the sample that was applied, the larger molecules will be displaced from the original salts and elute in this new buffer, completely separated from the original sample buffer.
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