Analysis & purification of proteins: SEC (Size Exclusion Chromatography), the separation of molecules according to their sizes

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.

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

	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
	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

Column size influence the separation

Increasing the length improves the resolution.

One column	Two columns

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