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.