Size Exclusion Chromatography (SEC)

At SOLVE:

Typical uses of SEC at SOLVE is as a high resolution quantitative method for monitoring degradation and oligomerization. Together with MALS it may also give molar mass distributions directly and validate clients SEC-methods that do not employ MALS. At SOLVE we have columns from a wide range of 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 which separate macromolecules in solution according to differences in size. The separation is based on the ability of macromolecules to have different abilities to access the pores of the stationary phase, which is based on the size of the molecules. 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 of 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
  • 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 through the use of standards (unless MALS is used)
  • High salt buffer often required
  • Concentration changes during separation