Gel Filtration (Size-Exclusion Chromatography)

Gel filtration of protein is the separation of protein based on size and shape of the protein. The protein sample will pass through a column packed with gel beads. Each bead contains pores that have size that is comparable to the size of the protein molecules that we want to separate. Each bead has its own working range, 10kD to 600 kD. If the protein molecule us too large, it will get eluted from the column as a void volume. Only the smaller protein can e fractionated.

In gel filtration, the gel matrix should be physically and chemically stable. It is also paramount that the gel matrix should not have adsorptive properties, which means inert. In order to prevent the adsorptive property of the gel matrix, eluent of certain ionic strength should be used. However, the pH and composition of the eluent is not important as the separation technique is based on size and shape. In short, upon application of the eluent, the structure and activity of the protein should be preserved. In the separation of folded protein, chaotropic agent such as urea can be used for fractionation of the proteins.

Factor affecting the outcome of gel filtration:

1. Sample volume
   
2. Column length
   

It should be noted that the ration of sample volume to column length would affect the resolution of the gel filtration. Larger ratio would result in decreased resolution. Hence, in order to obtain high resolution, it is wise to either apply smaller sample volume or increase the column length. Sample concentration would not affect the resolution of the chromatography. However, it is inevitable that gel filtration process would result in dilution of the sample. Hence, the sample should be as concentrated as possible. However, it should be noted that the concentration of the sample should be within the limit of the viscosity, as well as solubility of the column.