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 SEC is coupled to MALS we can obtain  the molar mass of components directly, which allows our customers to validate molar mass  values obtain by SEC-methods that do not employ MALS. We have columns from different manufacturers, covering the size range from peptides to large polymers. We use UV, RI, MALS, and FL detection.

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. On-line detectors such as UV 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 detector
  • High salt buffer often required
  • Concentration changes during separation