Size exclusion chromatography

What is size exclusion chromatography?

Size exclusion chromatography (SEC), also known as gel filtration, is the simplest and mildest of all the chromatography techniques. SEC separates molecules on the basis of differences in size.

Size exclusion chromatography can be applied in two distinct ways: group separations and high-resolution fractionation of biomolecules.

How does size exclusion chromatography work?

SEC resins consist of a porous matrix of spherical particles that lack reactivity and adsorptive properties. After sample has been applied, molecules larger than the pores are unable to diffuse into the beads, so they elute first. Molecules that range in size between the very big and very small can penetrate the pores to varying degrees based on their size. If a molecule is smaller than the smallest of the pores in the resin, it will be able to enter the total pore volume. Molecules that enter the total pore volume are eluted last. Samples are eluted isocratically so there is no need to use different buffers during the separation.

When should I use size exclusion chromatography?

In a group separation, the sample components are separated into two major groups according to size range. Group separation can be used to remove high-or low-molecular weight impurities (such as for example phenol red from culture fluids) or for desalting and buffer exchange.

Group separation

In a high-resolution fractionation, the components of a sample are separated according to differences in their molecular size. High-resolution fractionation can be used to isolate one or more components, to separate monomers from aggregates, or to perform a molecular weight distribution analysis.

Hi-res fractionation


Size Exclusion Chromatography (pdf)
Selection guide: Size exclusion chromatography columns and media (pdf)
Application note: Size exclusion chromatography analysis of papain-cleaved monoclonal antibody using Superdex 200 Increase columns (pdf)

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