Size exclusion chromatography: overview

Size exclusion chromatography (SEC), also known as gel filtration, is the simplest and mildest of all the chromatography techniques. SEC separates molecules by differences in size as they pass through a SEC resin packed in a column. Unlike techniques such as IEX or AC, molecules do not bind to the chromatography resin, which means that buffer composition does not directly affect resolution (the degree of separation between peaks). Consequently, a significant advantage of SEC is that conditions can be varied to suit the type of sample or the requirements for further purification, analysis, or storage without altering the separation. Download our SEC handbook now.

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. Watch this animation to visualize what happens inside a chromatography column during the SEC run.

When should I use size exclusion chromatography?

SEC can be categorized into three main application approaches:

  1. Preparative size exclusion chromatography: a high-resolution size-based separation of biomolecules with fractionation. Preparative SEC is performed to isolate one or more components of a sample. Separated components can be directly transferred to a suitable buffer for assay or storage. Small sample volumes of 0.5% to 4% of the total column volume are applied at low flow rates using long columns, often 60 cm or longer. As the separation takes place in only 1 CV, it is essential to have a well packed column for good results in SEC. For convenience and optimal performance, prepacked SEC columns are recommended.
     
  2. Analytical SEC: a high-resolution size-based separation without fractionation. Often, the analysis is connected to highly selective detectors such as mass spectrometers and photodiode arrays (DAD), as well as multiangle light scattering (MALS) or fluorescence detectors. Analytical SEC is performed to check the quality of the sample or to study the properties of a biomolecule. Small sample volumes, often 0.3% to 0.5% of the column volume are applied at low flow rates using long columns, typically 30 cm. Prepacked SEC columns are an excellent choice for ensuring reliable results and for convenience.
    Analytical SEC can also be used for rapid purity checks and screening. For this, shorter columns of typically 15 cm, which provide adequate resolution are used giving short cycle times together with small sample volume and low buffer consumption. Note that shorter columns typically used for purity check/screening give lower resolution than longer columns. Download our white paper to get started with analytical SEC.
     
  3. Desalting and buffer exchange: a group separation where small molecules such as salt or free labels are separated from a group of larger molecules such as proteins. Samples can be prepared for storage or for other chromatography techniques and assays. Large sample volumes—up to 30% of the total column volume—can be applied at high flow rates using broad, short columns. Check this presentation for an introduction to desalting.

Typical high-resolution SEC separation
Typical high-resolution SEC separation.
Typical group separation, desalting
Typical group separation, desalting.