Liquid Chromatography / HPLC Separation Modes: Steric Exclusion Chromatograply (SEC)

The main separation modes of liquid chromatography were described in previous posts entitled "Selection of the Liquid Chromatographic (LC) / HPLC Separation Mode" and "Which are the main liquid chromatography separation modes? / Liquid-Liquid Chromatography (LLC)".

Another separation mode in Liquid Chromatography is steric exclusion chromatography (SEC).
In steric exclusion chromatography(SEC) there is no interaction between the sample compounds and the column packing material. Instead, molecules selectively diffuse into and out of the pores of a porous medium.

The degree of retention depends on the size of the solute molecule relative to the size of the pore. Small molecules will pass through the smaller ports, intermediate size molecules will pass through some of the pores and be excluded from others while very large molecules may by completely excluded.

In a steric exclusion system, the larger molecules are retained less and therefore will elute from the column before the smaller molecules (Fig. 1a).
 

Fig.1a: Mechanism of the steric (size) exclusion process. Large polymer molecules (red dots) are not retained by the porous stationary phase and elute first. Small polymer molecules (green dots) are retained by the porous stationary phase (can enter into the porous stationary phase) and elute after the large molecules.

 The SEC technique is used by 15% of chromatographers, mainly for polymer characterization and for proteins (human serum proteins, DNA, crude oils). Steric exclusion chromatography has  found very wide application for separations in the life science and biochemical areas. Since compounds in most life systems are water-soluble and of high molecular weight, this form of chromatography is very applicable.  

In Fig. 1b a SEC analysis of a polymer and the obtained fractions are shown after separation.

 

Fig. 1b: A  SEC analysis of a polymer and the obtained fractions are shown after separation

There are two modes of  SEC:

Non-aqueous SEC [sometimes termed Gel Permeation Chromatography (GPC)] and  aqueous SEC [sometimes referred to an Gel Filtration Chromatography (GFC)].