GEL CHROMATOGRAPHY
By
Sumaiyya Naz
[Link] Final Year
Sai Meer College of Pharmacy
Presentation Date:-
CONTENTS-
• Introduction of Gel Chromatography
• Principle of separation
• Theory
• Instrumentation
• Applications
INTRODUCTION-
It is also called as Gel permeation, Exclusion,
Molecular sieve chromatography.
It is a simple and reliable method for
separating molecules according to their size.
It uses porous material as stationary phase
and liquid as a mobile phase.
The smaller molecules travel faster than larger
molecules and get separated.
PRINCIPLE-
• A mixture of molecules dissolved in liquid is applied to a
chromatography column which contains a solid support in the
form of microscopic spheres or “beads”.
• The mass of beads within the column is often referred to as the
column bed.
• The beads acts as “traps” or “sieves” and function to filter small
molecules which become temporarily trapped within the pores.
• Larger molecules pass around or are excluded from the beads.
• Large sample molecules cannot or can only partially penetrate the
pores, whereas smaller molecules can access most or all pores.
• Thus, larger molecules elute first, smaller molecules elute later,
while molecules that can access all the pores elute last from the
column.
• Particles of different sizes will elute through a stationary phase at
different rates.
THEORY-
• Total volume of column packed with gel
that has been swelled by water or other
solvent is given by-
Vt = Vg + V1 + Vo
Where, Vt = total bed volume
Vg = volume occupied by solid matrix of gel
V1 = volume of solvent held in pores
Vo = free volume outside the gel particles
INSTRUMENTATION-
1. Stationary phase-
• It is semi-permeable, porous beads with well-
defined range of pore sizes.
• Beads are cross-linked polymers.
• Smaller pore sizes are used for rapid desalting of
protein or for protein purifications, while
intermediate pore size are used to separate
repeatedly small size proteins.
• Very large pore size used for purifications of
biological complexes.
• Ex- Dextran, polyacrylamides and dextran
polyacrylamides.
2. Mobile phase-
• The choice of mobile phase to be used in any separation will
depend on the type of separation to be achieved and
component to be separated.
• It composed of a liquid used to dissolve the biomolecules to
make the mobile phase permitting high detection response
and wet the packing surface.
• The most common eluents for polymers that dissolve at
room temp. are tetrahydrofuran, chloroform, dimethyl
formamide, etc.
3. Column-
• It consists of a straight tube with a bed support (glass wool)
at bottom.
• The bed support allows only the liquid to pass thrugh
without disturbing the bed material.
4. Pumps-
• A highly constant flow rate has to be maintained
during the entire chromatogram.
• A change in flow rate of only 0.1% can cause an error
in molar mass of upto 10%.
• Most pumps can only reproduce the flow rate to 0.2-
0.3%.
• In-line filters in the solvent reservoir may prevent
particles from coming into pump heads, which might
damage the valves or the pump seals.
• Ex- Syringe pumps, reciprocating pumps.
5. Detectors-
i. Conc. Sensitive detector-
Bulk property detector- Refractive Index detector.
Solute property detector- UV absorption detector.
Evaporative detector- Evaporative Light Scattering Detector.
ii. Molar mass sensitive detector-
Light scattering detector- Low angle scattering detector.
Viscosity detector- Differential viscometers.
iii. Other-
Flame ionization detector.
APPLICATIONS-
1. Purification- of biological macromolecule like protein,
polysaccharide, nucleic acid, etc.
2. Mol. Wt. determination- The chromatogram results from a
separation based on the size and mol. Wt. of sample
molecule.
3. Protein binding studies- It is used to study the reversible
binding of a ligand to a macromolecule such as protein
including receptor protein.
4. Solution conc.- Solution of high mol. Wt. substances can be
concentrated by addition of dry sephadex G-25.
5. Desalting- By use of a column of Sephadex G-25, solutions of
high mol. Wt. compound may be desalted. [The separation of
large molecule of biological origin from inorganic & ionisable
species is termed as desalting.]
