Principles of Biochemistry

Gel filtration chromatography

Principle

Gel filtration chromatography, also called size-exclusion or gel permeation chromatography, separates molecules based on the differences in their size. The sample is applied on top of the column containing porous beads. As the molecules pass through the column of porous beads of cross linked agarose, they get separated as follows

• Large molecules cannot enter the pores and elute as the first peak in the chromatogram. They elute fast and this is called total exclusion.
• Intermediate molecules may enter the pores and may have an average residence time in the particles depending on their size and shape and different total transit times through the column. This portion of a chromatogram is called the selective permeation region.
• Small molecules enter the pores and have the longest residence time on the column and elute together as the last peak in the chromatogram.
• This last peak in the chromatogram is the total permeation limit.