PRINCIPLE AND APPLICATIONS OF GEL FITRATION

CHROMATOGRAPHY
 CHROMATOGRAPHY

• Chromatography literally means “colour writing”

• Chromatography was invented by Russian

botanist Mikhail Tsvet in 1900. He used it to
separate chlorophyll containing extracts of
plants.

• Chromatography is process in which separation

of mixture compounds using the stationary
phase and mobile phase.
 Techniques of chromatography

1.Plane chromatography.
2. Column chromatography.

Column chromatography are of different types:

 Affinity chromatography.
 Ion Exchange chromatography.
 Size Exclusive chromatography.
 Adsorption chromatography.
 Gel filtration chromatography

• Size-exclusion chromatography(SEC) also called Gel

filtration or Gel permeation chromatography is a
chromatographic method in which molecules in
solution are separated by their size, and in some cases
molecular weight.

• It is usually applied to larger molecules or

macromolecular complexes such as proteins and
industrial polymers
• Gel filtration medium is packed into a column to form
a packed bed.
Schematic of a size- exclusive chromatography
• The medium is a porous matrix in the form of
spherical partials that have been chosen for their
chemical and physical stability and inertness.
• A mixture of molecules dissolved in liquid (the
mobile phase) is applied to a chromatography
column which contains a solid support in the form of
microscopic spheres, or “beads” (the stationary
phase).
• The mass of beads within the column is often
referred to as the column bed.
• The beads act as “traps” or “sieves” and function to
filter small molecules which become temporarily
trapped within the pores.
 Column parameter

Vo = void Vt = total Vs= volume of

volume volume solvent held in
the pores. This is
normally
approximated to
Vt-Vo = volume
of beads
 Concept of Plates: Column Efficiency
• Column can be divided into a number of identical
segments such that one equilibrium distribution takes
place on each of these segments. Each of these
segments is called theoretical plate.
• Thus, the number of equilibrations taking place in a
column is equal to the number of plates that the column
possesses.
• The column which has a larger number of plates will be
better suited for resolving these compounds whereas a
column which has small number of plates will not be as
efficient.
• Therefore column efficiency is directly related to the
number of plates it possesses
 Types of gels used
• Sephadex Gel : It’s mainly used for separation of small
peptides and globular protein with small to average
molecular mass.its separate molecular weight limit up
to 5000-200000 Daltons.
• Polyacrylamide gel : This type of gel can be used to
separate molecules of up to 300000 Daltons,
• Agarose gel : Agarose gel produced from agar. It's useful
for analyses or separation of large globular proteins or
long linear molecules such as DNA.
• Agarose gel can be used to separate molecules and
particles up to a molecular weight more than 300000
Daltons.
 Separation procedures
1. Preparation of column for gel filtration
Swelling of the gel
Packing of the column
Washing with the buffer

2. Loading the sample onto the column

3. Eluting the sample and detecting of compounds

Theory
• If the mixture of molecules of different size is placed on the top
of such an equilibrated column.

• Molecules larger than the pore size can not enter the pores and
elute together as the first peak in the chromatogram.

• Different molecules therefore have different total transit times

through the column.

• Molecules that are smaller than the pore size can enter all
pores, and have the longest residence time on the column and
elute together as the last peak in the chromatogram.

• The collected fraction are often examined by spectroscopic

technique to determine the concentration of partial eluted.
 Aaa

Theoretical chromatogram of a high resolution

fractionation (UV absorbance)
Applications of Gel Filtration Chromatography

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 condition.

2. The elution volume is related to the molecular

weight.

3. Purification of biomolecules.

4. Estimation of molecular weight mainly for proteins.

 Molecular weight determination by gel filtration

• In gel filtration experiment if one determines the

effluent volume for a macromolecules, one can
calculate its distribution coefficient.

• If distribution coefficient of standard proteins of known

molecule weight are plotted against the log of their
molecular weights.

• The position of the distribution coefficient of the

unknown protein on the plot will lead to the
determination of its molecular weight.
Molecular weight and elution volume for proteins from a gel
permeation chromatography
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