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Size exclusion chromatography

Size exclusion chromatography (SEC) or gel filtration chromatography is a separation technique that isolates biomolecules based on their size, shape, or molecular weight (Mr). You can use SEC to isolate macromolecular complexes like proteins and polysaccharides. Buffer composition has no direct effect on the resolution, allowing researchers and biomanufacturers to vary conditions to match the sample type. The two common uses of SEC are desalting and fractionation. Used to purify protein or nucleic acid samples, desalting or buffer exchange requires a gel with a smaller exclusion limit than the molecule of interest. Fractionation separates molecules based on molecular weight in a gel matrix.

Principle of size exclusion chromatography

Size exclusion chromatography uses resins composed of agarose, polyacrylamide, or dextran polymer beads. Unlike resins that use other chromatographic separation methods, size exclusion resins do not directly bind target molecules. Separation of biomolecules occurs when particles of varying sizes are included or excluded from the pores in the matrix. After introducing a sample to the size exclusion columns, molecules that are larger than the pores cannot diffuse through the resin. Molecules smaller than the pore size pass through the column.

SEC stationary phases have specified fractionation ranges that outline the molecular weight ranges that can be separated by a given matrix. For small biomolecules such as peptides, consider using size exclusion resins with low fractionation ranges. Larger particles, like antibodies, require stationary phases with higher fractionation ranges. To ensure high resolution in your purification processes, consider:

  • Particle size
  • Resin bead pore size
  • Column dimensions
  • Column packing
  • System volumes
  • Flow rates

Size exclusion resin FAQs

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

What does size exclusion chromatography do?

Size exclusion chromatography separates biomolecules according to their molecular size using filtration through a resin that contains spherical beads.

What is a size exclusion resin?

Size exclusion resins are stationary phases that help isolate biomolecules based on their shape or size and molecular weight. We offer size exclusion resins in bulk and in prepacked columns.

What is the exclusion limit of an SEC resin?

Exclusion limit and fractionation range are important parameters for size exclusion resins. The exclusion limit of the resin tells you the largest molecule size that can pass through the matrix pores and elute in the void volume. The fractionation range defines the range of molecular weights that have partial access to the pores of the particle and can be separated.

How do you select a resin for size exclusion chromatography?

Select resins based on the resolution and yield you need. We offer resins for preparative and analytical SEC. Check out our Purify app for help selecting a size exclusion resin or other chromatography products.