Gel filtration

Size exclusion
or
Gel permeation
or
Molecular exclusion
 PRINCIPLE
 Molecules in the solution

 Are separated depending on their size

Because, molecules are separated based on
the differences in their size.
GEL FILTRATION - MECHANISM

separated
depending
on their size
Gel matrix used:

• should be chemically and physically stable

• selected on the basis of:

 bead size
 size of the molecules to be separated

Beads are uncharged and inert

i.e. don’t react with the materials being fractionated

GEL FILTRATION – RESIN – Type 1:
Sephadex

GEL FILTRATION – RESIN – Type 2:

Polyacrylamide

GEL FILTRATION – RESIN – Type 3:

Agarose
 GEL FILTRATION – RESIN – Type 1
• Sephadex: it is a cross-linked dextran used as a resin.

• Dextran (Glucose units of the polysaccharide chains).

• Macroscopic beads.

• Size of the bead ranges between 20 and 300 µm.

• Beads have varying degree of porosity.

• Sephadex beads have Well defined range of pore sizes Molecules of that
molecular weight range can be separated. G-25 1000-5,000
G-50 1500–30,000
• For Ex: Sephadex G 75
• (Has a Fraction Range of 3000–80,000 Da) G-75 3000–80,000
G-100 4000–150,000
• Sephadex G-25, is a very common gel filtration material.

• It has the capacity to separate molecules with molecular weights from 1000 to 5000 Da.

• Molecules with molecular weights more than 5000 Da will be excluded from the beads
and
• Molecules with molecular weight less than 1000 Da will be completely included.

• Therefore, a mixture containing:

 molecules of varying size
 can be fractionated
 on a Sephadex G-25 column
 by gel filtration chromatography
 GEL FILTRATION – RESIN – Type 2

Polyacrylamide:
• This comes under the trade name of bio-gel P.

Prepared by:
• Cross linking acrylamide
and
• N, N’-methylene-bisacrylamide.

Its pore size is determined by:

• the degree of cross linking
and
• this is available in varying range of pore sizes.
 GEL FILTRATION – RESIN – Type 3

• Agarose is a linear polymer of

D-galactose and 3, 6 anhydro-1-galactose.

• Pore size is determined by its concentration in the gel.

• The pore size of agarose gels is much larger than sephadex or bio-gel P.

• This is useful for the separation of large globular proteins and DNA.
• Column holds the stationary phase and
the mobile phase carries the sample.

• The sample is applied on the top of the

column containing porous beads.

• As the molecules pass through the

column of porous beads of sephadex,
they get separated.
•Large molecules cannot
enter the pores and elute as
the first peak in the
chromatogram.

•They elute fast, and this is

called total exclusion.
•Intermediate molecule may enter the pores
and may have an average residence time in
the particular depending on their size and
shape.

•Different molecules therefore have different

total transit time through the column.

•This portion of chromatogram is called the

selective permeation region.
•Small molecules enter the pores
and have the longest residence time
in the column, and elute out together
as the last peak in the
chromatogram.

•This last peak in the chromatogram

is total permeation limit.
 PROCEDURE:
•Fix the column vertically to a stand.

•Equilibrate the column with 4ml of gel filtration buffer.

•Drain out the buffer completely / Allow the buffer to flow out completely.

•Load the sample on to the column, along the sides of the column.

•Keep filling the column with the buffer, till all the coloured
biomolecules have eluted out.

•Collect the coloured fractions in different tubes.

•Fix the bottom cap, and then the top cap to stop the flow of the buffer
store at 40oC for the next use.
 COLLECTION OF SAMPLES:
•The blue coloured component is Dextran having a
molecular weight of 2000000 daltons.

•These are very large molecules that exit fast from the column,
•and are collected as the first fraction.

•The brownish-red component collected as the second fraction

is haemoglobin having a molecular weight of 64500 daltons.

•These molecules are of intermediate size and they enter the pores,
but their retention time is slow.

• These molecules take an average time to exit the column.

•The pink coloured component is vitamin B12,

•having a molecular weight of 376 daltons.

•They are very small molecules that enter or penetrate the pores of the beads,
and are retained in the beads for a longer time.

•They take a long time to exit the column, and are collected as the third fraction.
SCHEMATIC DIAGRAM OF GEL FILTRATION CHROMATOGRAPHY -
1