Separation mechanism in size-exclusion chromatography

Separation in size-exclusion chromatography requires careful matching of the pore size of the stationary phase material with the size of the molecules to be separated. Small molecules in a sample will be able to penetrate all the pores of the stationary phase and will elute with an elution volume, Fe, which is equal to the void volume of the column, Fm- Very large molecules will be excluded from all the pores of the stationary phase and will elute with an elution volume, which is equal to the interstitial volume of the Uquid between the particles, Fj. Molecules of intermediate size will be able to penetrate some but not all of the pores and will elute with an elution volume that is between the interstitial volume and the void volume of the column. The void volume is the total volume of the liquid in the column and is related to the interstitial volume and the pore volume, Fp by equation (3.38)  

The total volume of the column, Ftot, and its porosity, e, are given by equation (3.39) and (3.40), respectively  

It follows that the elution volume of a solute in size-exclusion chromatography is given equation (3.41)  

The main disadvantage of size-exclusion chromatography is its low peak capacity, which arises from the small elution volumes of the peaks. In addition because peak dispersion is small, extra-column contributions to band broadening in size-exclusion chromatography have to be kept to a minimum. The relative contribution of extra-column band broadening can be minimized by the use of small particles = 3-10 pm) and long (L = 50 100 cm), wide-bore (d = 8-10 mm) columns. 

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