Biochemical Techniques and Instrumentation

2.2.1. Principle

Gel filtration is performed in a column having porous matrix support. The matrix support consists of porous gel beads or glass granules. The column is kept in equilibrium with the solvent mobile phase. The matrix support has two measurable liquid volumes. They are external (Vo) and internal (Vi) volumes. External or void volume means the volume of liquid occupying the spaces between the packing matrix beads. Internal volume means the volume occupied by the liquid within the pores of the matrix beads. The larger molecules do not enter the pores of the matrix beads and pass out quickly through the column.The smaller molecules enter the pores of the matrix beads and their flow is retarded as they equilibrate with the internal and external volumes.Their extent of availability in the pores depends on the porosity of the matrix beads and size of the molecules.

The partition behavior of a particular molecule is related to the total column volume (Vt) and the external or void volume (Vo).

K_av = (V_e-V_o) / (Vt – V_o)

where Vt - Total column volume, V_o - Void volume, V_e - Elution volume of the molecule, K_av - Partition coefficient

Total volume is the volume occupied by the matrix beads in the column and not the size of the beads. Partition coefficient defines the proportion of the bead pores that can be occupied by the molecule.

The molecular size of an unknown molecule can be determined by comparing the elution volume of the molecules with known molecular weights.