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Basics of Size Exclusion Chromatography (SEC)

Synge and Tiselius were the first to report the concept of chromatographic size-based separations; they noted that small molecules could be excluded from the small pores of zeolites as a function of their molecular size. 1,2  Wheaton and Bauman reported the first examples of size-based separations by liquid chromatography during their work on ion exclusion chromatography. 3

In size exclusion chromatography (SEC) separation is not based on interactions between the analyte and the column surface; separation relies on the degree of inclusion or exclusion of an analyte from the pores within the stationary phase.  The stationary phase pores have different accessibility for molecules depending on their size (hydrodynamic volume), i.e. larger molecules are generally poorly retained and elute first as they cannot move into the pores within the stationary phase, whereas smaller molecules can diffuse into the pores to a greater extent, depending on their size, and will therefore be eluted later, with the smallest molecules eluting last.

The ‘size’ of a biomolecule can depend upon the molecular weight, degree of folding, and degree of hydration, so it should be noted that although elution order typically follows molecular weight, the true mechanism of SEC is based on size in solution.  Proteins can come in several shapes, most are compact, however, some are cylindrical.  Cylindrical proteins have a larger hydrodynamic radius in solution and will elute earlier (even if they do not necessarily have the highest MW).  Different mobile phases can also affect elution order due to hydration of proteins in solution, resulting in changes in hydrodynamic radius or radius of gyration.

Size exclusion chromatography is also referred to as:

  • Gel filtration chromatography - low pressure aqueous applications
  • Gel permeation chromatography - analysis of synthetic polymers in organic eluents

Additional names used for SEC are; exclusion chromatography, steric-exclusion chromatography, restricted-diffusion chromatography, and liquid-exclusion chromatography.

 

Figure 1: Typical elution and chromatogram seen in size exclusion chromatography (SEC).

 
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