Size exclusion chromatography (SEC)

At SOLVE:

A typical use of SEC at SOLVE is as a high resolution quantitative method for monitoring degradation and oligomerization. When coupled to MALS it is possible to obtain molar mass distributions directly and thereby validate clients’ values obtain by SEC-methods that do not employ MALS. At SOLVE we have columns from different manufacturers, covering the size range from peptides to large polymers. The columns are connected to UV, RI, MALS, and FL detectors.

Principle:

Size exclusion chromatography (SEC), also referred to as gel permeation or gel filtration chromatography (GPC/GFC), is a separation methodology that separates macromolecules in solution according to differences in size. The separation is based on the ability of the macromolecule to access the pores of the stationary phase which are defined by the size of the molecule  that is made of. SEC is commonly used in connection with on-line detectors such as UV to monitor the amount of the size separated components as they are eluted from the column.

Applicability:

SEC is a commonly used method, applicable to a wide range of compounds, such as peptides, proteins, and polymers. SEC is applicable to compounds with molecular weight of ~ 1000 g/mol up to ~10·106 g/mol. Typical uses are for stability testing of proteins, aggregation detection, quality control, molar mass distribution determinations, etc.

Pros

  • High resolution
  • Quantitative (with UV or RI)
  • Especially well suited for <100 kDa compounds
  • Suited for standardization
  • Fractions can be collected

Cons

  • Limited size range (poor >1 – 10 MDa)
  • High surface area and shear rates
  • Molar mass and Mw determination through standards (unless coupled to MALS)
  • High salt buffer often required
  • Concentration changes during separation