Size exclusion chromatography is a type of liquid chromatography where the molecules in the sample are separated according to their size.

The stationary phase is normally a gel composed of spherical porous beads with a specific size distribution. Depending on the size of different molecules, they will be included or excluded from the pores within the beads. Those included in the pores will stay longer in the stationary phase before eluting, while those excluded will elute more rapidly as they will not be retained within the matrix. Therefore, smaller molecules will elute later than bigger ones (Figure 1).

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. Example of size exclusion chromatography.

The resolution of the chromatogram depends on a few factors.