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Size Exclusion Chromatography

Size exclusion chromatography - SEC - is a type of liquid chromatography where the molecules in the sample are separated according to their molecular size. All types of liquid chromatography involve a mobile or stationary phase. The mobile phase is the mixture's solvent and propels the compounds in the mixture through the stationary phase. While the stationary phase is immobile and has different properties to separate different compounds in the mixture.

With SEC, the stationary phase is a gel or resin composed of spherical beads with a range of pore sizes. Smaller molecules will enter the smaller pores and thus have a larger volume of the stationary phase to travel through. As a result, smaller molecules will elute or exit the column last.

Principle of separation molecules in size exclusion chromatography. First, a sample mixture consisting of molecules of different sizes - big red, medium green, and small blue spheres - is injected into the column filled with big grey spheres - porous packing. As time passes, molecules travel through the packed column from the top to the bottom, big red spheres coming down faster than the smaller ones. First large solutes, red spheres, are eluted. Then, medium solutes, green spheres, are eluted. Finally, small solutes, blue spheres, are eluted. Those elutions are depicted on the elution curve below the diagram. Three peaks are visible, the first peak, with smallest retention time, for big spheres, second, with bigger retention time, for medium spheres, and last with biggest retention time, for smallest molecules.

Figure 1. Diagram explaining the premise behind size exclusion chromatography and an example dataset.

The resolution of the chromatogram depends on a few factors.

Referred from:

Article
Liquid-liquid purification
Article
Chromatography resolution