Theory of Gel Chromatography

Theory of Gel Chromatography describes separation based on molecular size as smaller molecules enter gel pores.

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.

1. Exclusion:

   • Molecules: Larger than the largest pores.
   • Behavior: Excluded from entering the beads.
   • Elution: Pass straight through, eluting first.

2. Partial Penetration:

   • Molecules: Smaller than the largest pores but larger than the smallest.
   • Behavior: Enter some pores partially.
   • Elution: Delayed compared to excluded molecules.

3. 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:

$K_{av} = \frac{V_e – V_0}{V_t – V_0}$

• • V_e: Elution volume of the molecule.
  • V₀: Column void volume (volume outside the pores).
  • V_t: 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.

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