Theory of Gel Chromatography

Mechanism of Separation

• The separation mechanism is based on the size (hydrodynamic volume) of molecules relative to the pore sizes of the gel beads in the stationary phase.

Exclusion:

• Molecules: Larger than the largest pores.

• Behavior: Excluded from entering the beads.

• Elution: Pass straight through, eluting first.

Partial Penetration:

• Molecules: Smaller than the largest pores but larger than the smallest.

• Behavior: Enter some pores partially.

• Elution: Delayed compared to excluded molecules.

Total Penetration:

• Molecules: Small enough to enter all pores.

• Behavior: Spend the most time within the column.

• Elution: Pass through the column last.

Elution Volume

• Definition: The volume at which a molecule elutes from the column.

• Relation to Size:

  • Larger Molecules: Smaller elution volume (elute first).

  • Smaller Molecules: Larger elution volume (elute last).

Partition Coefficient (Kav)

• Definition: Describes the behavior of a molecule in the column.

• Calculation:

• Ve: Elution volume of the molecule.

• V0: Column void volume (volume outside the pores).

• Vt: Total column volume.

Relation to Molecular Size:

• A calibration curve is created by plotting Kav​ values of standards with known molecular weights.

• The molecular weight of unknown molecules is determined using their elution volumes and corresponding Kav​ values.