To follow our set of articles speaking about proteins purification technics, today we are focusing on the SEC (Size Exclusion Chromatography), separation of molecules according to their size.

In the CEP strategy (Capture, Enhance, Polishing) among all the technics available, SEC is the most often used.

Chromatographic technics

 

Protein Analysis & Purification

SEC – Size Exclusion Chromatography

With this technic molecules are discriminated according to their size. The elution order is inversely proportional to the proteins hydrodynamic radius (the biggest molecules are excluded first).
The chromatographic resins (polymeric, silica or agarose) is modified in order to avoid any interactions with proteins. Thus elution is strictly isocratic.
To get the highest efficiency the stationary phase porosity must be strictly defined.

Numerous mobile phase are possible but all of them must:

  • Solubilize the sample
  • Gel swelling
  • Detector compatibility
  • Isocratic

Buffers ex: TRIS, phosphate, NaCl, & neutral detergent

 

Analytical Purification Technics : SEC

Advantages

  • Speed & efficiency for desalting
  • Mild conditions & high yield
  • Numerous buffers can be used
  • Aggregates elimination
  • Several columns can be used in series to increase the resolution

Disadvantages

  • Limited sample Volume
  • Sample diluted
  • Long technic
SEC

Used for:

  • Study of the aggregation & folding of proteins
  • Protein desalting (ex. after digestion or chemical modification)

Other names:

  • Gel filtration (GFC) (in buffer)
  • Gel permeation chromatography (GPC) (in solvent)

 

SEC pathway

1 - Spherical particles packed into a column 2 - Sample applied 3 - Buffer mobile phase & sample move through column, molecules diffuse in & out of matrix 4 - Large molecules leave the column first followed by smaller molecules in order of size, molecules larger than the matrix pores straight through
1 – Spherical particles packed into a column 2 – Sample applied 3 – Buffer mobile phase & sample move through column, molecules diffuse in & out of matrix 4 – Large molecules leave the column first followed by smaller molecules in order of size, molecules larger than the matrix pores straight through

SEC pathway

SEC pathway 2

 

Column size influence the separation

Increasing the length improves the resolution.

One column

Two columns

Influence de la taille des colonnes sur la séparation des protéines - 1 colonne Influence de la taille des colonnes sur la séparation des protéines - 2 colonnes

Typical bed length
Analytical/Micropurification column: 300mm
Preparative column: 600 – 1000mm

 

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