Mechanism of Separation:
The fundamental mechanism of separation in gel chromatography is based on the size (or hydrodynamic volume) of the molecules in relation to the pore sizes of the gel beads in the stationary phase.
Molecules are separated based on their ability to penetrate these pores:
Exclusion:
Molecules larger than the largest pores in the gel beads are excluded from entering the beads and therefore pass straight through the column. They elute first.
Partial Penetration:
Molecules smaller than the largest pores but larger than the smallest ones can penetrate some of the pores, delaying their elution.
Total Penetration
The smallest molecules can enter all the pores, leading them to spend the most time within the column, and they elute last.
Elution Volume:
The volume at which a molecule is eluted from the column (elution volume) is related to its size.
Larger molecules, which are excluded from the pores, have a smaller elution volume, while smaller molecules that penetrate more pores have a larger elution volume.
Partition Coefficient:
The partition coefficient Kav is often used to describe the behavior of a molecule in the column.
It's defined as:
Relation to Molecular Size:
A calibration curve can be prepared using standard molecules with known molecular weights.
This curve, which plots the logarithm of the molecular weight against Kav, can then be used to determine the molecular weight of unknown molecules based on their elution volumes.