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Size exclusion chromatography

Size exclusion chromatography (SEC), sometimes called gel filtration chromatography, separates molecules based on their shape and particle size or molecular weight. The technique is used to purify many biological molecules and particles, including proteins, protein complexes, and viruses.

Size exclusion chromatography does not require a binding and elution protocol. It’s the mildest purification option and preserves the biological activity of target molecules. Since target molecules don’t bind to the chromatography resins, buffer composition has no direct impact on resolution. This allows researchers and biomanufacturers to vary conditions to match the type of sample.

How size exclusion chromatography works

Size exclusion chromatography resins comprise a porous matrix of spherical beads that lack reactivity and adsorption properties. Once a sample is loaded onto a size exclusion column, molecules that are larger than the pores are unable to diffuse into the beads. This causes these molecules to leave the column, or elute, first. Based on their size, biomolecules that are of average size penetrate the pores to different degrees.

If a molecule is smaller than the tiniest pore size in the resin, it enters the total pore volume and gets eluted last. SEC stationary phases have designed fractionation ranges that list the range of molecular weights that can pass through the pores. Lower fractionation ranges are well-suited for separation of small biomolecules like peptides, while higher ranges support separation of large proteins such as antibodies. Exclusion limits of a resin show the size of molecules that elute in the void volume.

For high-resolution protein purification, consider the following factors:

Resin selectivity is dependent on chromatography conditions, resin characteristics, and the interaction between sample and resin.

Size exclusion columns FAQs

Here are answers to some frequently asked questions about size exclusion columns.

How do you choose a column for size exclusion chromatography?

Prepacked size exclusion columns come with recommended running conditions for optimal results. Choose a column with a resin that has an appropriate fractionation range to achieve high-resolution separation. Your size exclusion columns should have a bed height of 30 to 100 cm for preparative separation. Also, choose column size based on the volume of sample that requires processing. You can use the Cytiva Purify app to help you select our chromatography columns and resins for SEC.

What is the resolution of size exclusion chromatography?

Resolution is the measure of the separation of two peaks of different retention times in a chromatographic separation.

Which molecules leave a size exclusion column first?

During gel filtration chromatography, larger biomolecules leave the chromatography column first. Smaller-sized molecules have more pores that are accessible to them, making them stay in the column longer.