Gel permeation chromatography, also known as size exclusion chromatography, separates molecules by their size and shape. The stationary phase consists of porous beads. Small molecules enter the pores and are retained longer, eluting later, while large molecules are excluded from the pores and elute earlier. Common gels used include dextran, polyacrylamide, and agarose which have different pore sizes for separating biomolecules. Gel filtration chromatography is useful for purifying and determining the molecular weights of proteins.

1. Gel Permeation Chromatography
OR
Gel Filtration Chromatography
OR
Size/Gel/Molecule
Exclusion Chromatography
OR
Molecular Sieve Chromatography
Amandeep Singh
Assistant Professor
Department of Biotechnology
GSSDGS Khalsa College
Patiala

2. Principle
• It is a type of column chromatography.
• Gel filtration chromatography separates
molecules according to their size and shape.
• The stationary phase consists of beads containing
pores that span a relatively narrow size range.
• Smaller molecules spend more time inside the
beads than larger molecules and therefore elute
later (after a larger volume of mobile phase has
passed through the column).

3. Principle
Bead of
Stationary phase
Molecules smaller than the pore size
Molecules Larger than the pore size
Pore size
Do not enter pore
Elute out First
Enter inside pore
Elute out Last

4. Principle
• In more strict terms, principle of gel filtration
chromatography is based on distribution coefficient
(Kd), which is a function of molecular size.
• Kd=0, if molecule is larger than the pore size and
completely excluded from entering the bead.
• Kd=1, if molecule is smaller than the pore size and
penetrate in the pore of gel bead.
Kd=0 Kd=1
Molecule is larger Molecule is smaller
Can not penetrate the gel bead Can penetrate the gel bead
Completely excluded Completely retarded

5. Principle
Bead of
Stationary phase
Molecules smaller than the pore size
Molecules Larger than the pore size
Pore size
Do not enter pore
Elute out First
Enter inside pore
Elute out Last
Kd = 0 Kd = 1

6. Void Volume & Total Volume
• Void Volume = Volume of outer solvent i.e. the solvent
surrounding the gel bead. It is denoted by Vo
• The volume of solvent inside gel i.e. inner solvent is indicated
as Vi
• The effluent/elution volume (The volume of buffer required to
elute any given substance is known as the elution volume),
Ve is dependent upon these three variables. Thus
Ve=Vo+KdVi
Vo = void volume
Vi = solvent volume inside gel bead
Vt= total volume

7. Types of gels used
• The gels used as molecular sieves are cross linked
polymers.
• They are uncharged (or small number of ionic
groups) and inert i.e. don’t bind or react with the
materials being analyzed.
• Gel material should provide a wide choice of pore
and particle sizes.
• Should have uniform particle and pore size.
• Should have high mechanical rigidity.
• Three types of gels are used:

8. Types of gels cont…
1. Dextran: is a homopolysaccharide of glucose
residues.
• it’s prepared with various degrees of cross-linking
to control pore size.
• It’s bought as dry beads, the beads swell when water
is added.
• The trade name is sephadex.
• It’s mainly used for separation of small peptides and
globular proteins with small to average molecular
mass.

9. Types of gels cont…
2. Polyacrylamide:these gels are prepared by cross
linking acrylamide with N,N-methylene bis
acrylamide.
The pore size is determined by the degree of cross-
linking.
The separation properties of polyacrylamide gels are
mainly the same as those of dextrans.
They are sold as bio-gel P. They are available in wide
range of pore sizes.

10. Types of gels cont…
3. Agarose: linear polymers of D-galactose and 3,6
anhydro-1-galactose.
It forms a gel that’s held together with H bonds. It’s
dissolved in boiling water and forms a gel when it’s
cold.
The concentration of the material in the gel determines
the pore size.
The pores of agarose gel are much larger than those of
sephadex or bio-gel p.
It’s useful for analysis or separation of large globular
proteins or long linear molecules such as DNA

11. Column Preparation
• Dry gel first converted to swollen (hydrated) form in water
or weak salt solution.
• Higher the porosity of gel, higher the timing for
equilibration.
• Swelling time can be reduced by heating the gel slurry in
water bath for 1-5 hours.
• Slurry is poured in the column.
• Air bubbles are removed by connecting to vacuum pump.
• Sample application is done.
• Addition of Eluant to separate sample components.
• Blue dextran is added to calculate the void volume.

12. Advantages of Gel filtration
It’s the best method for separation of molecules
differing in molecular weight because:
1. It doesn’t depend on temperature, pH, ionic strength
and buffer composition. So separation can be
carried out under any conditions.
2. There is very little adsorption
3. There is less zonal spreading than in other
techniques.
4. The elution volume is related to the molecular
weight

13. Applications of gel filtration
• Purification of enzymes and other proteins.
• Estimation of molecular weight mainly for
globular proteins.