Gel Filtration

Chromatography
Presented by: Akshit Kapila
Adm. No. H-2019-05-010
  Refers to the chromatography method
which uses porous gel beads of
specific porosity to isolate components
Defination: depending upon their molecular sizes.
 Retains or excludes particles based on
the size differences, hydrophobicity and
molecular charges.
Principle:
• The principle of size
exclusion chromatography
depends on the isolation
of biomolecules relative to
their different molecular
weight or size
differences.
• The gel filtration
technique employs
spherical gel beads with
definite porosity as
the packing material in
the chromatography
column.
 Two Phases:
1. Mobile Phase.
2. Stationary Phase.
Phases: 1. Mobile phase: The solvent running through
the column is the mobile phase.
2. Stationary phase: The column packed
with microporous gel beads acts as the
stationary phase.
Procedure:
  Firstly, a column of spherical gel beads is prepared for the gel
filtration chromatography.
 The packed bed is equilibrated with a buffer.
 Then, the test solution (mobile phase) is eluted through the
column.
Procedure:  After that, the particles in the test sample will enter into or diffuse
out of the porous gel matrix (stationary phase).
(Continued)  The molecules with a small molecular size will enter the gel pores,
i.e. small molecules will cover a longer path or stay longer on the
column.
 Large molecules with large molecular sizes cannot get into the
pores of gel beads or easily pass through the column.
  Thus, the separation of the particles occurs at different intervals
by following isolation and identification of the components
separated.

Procedure:  Fractionation and desalting are the methods that facilitate the
separation of components in size exclusion chromatography.

(Continued) Desalting elutes the heavy molecules by keeping the

exclusion limit of the gel smaller. The smaller molecules enter
the gel pores while the larger particles leave the column.
Fractionation isolates the target molecules within the gel
matrix, whose molecular size must be within the gel’s
fractionation range.
 • For purifying biomolecules like enzymes,
polysaccharides, nucleic acids, proteins etc.
• Enables the renaturation of denatured
proteins.
Applications: • Aids in protein fractionation experiments.
• Examines the quaternary structure of purified
proteins.
 • Can separate biomolecules sensitive to varying
pH, temperature, the concentration of metal
ions etc.
• Takes minimal time to explicate the result.
• Gives a well-defined separation.
Advantages:  A small amount of test sample is enough to
conclude the results.
 The particles do not stick to the
chromatography medium
  To prevent particles from clogging within the
instrument, one needs to filter the test sample
before passing it through the gel filtration
column.
Limitations:  Any dust or particulate matter within the
instrument may interfere with the result
interpretation or give aberrant results.
 1. Gel Chromatography - Explanation, Principle, Applications and
FAQs (vedantu.com)
2. Wilson, K., Walker, J. (2018). Principles and Techniques of
Biochemistry and Molecular Biology (8 eds.). Cambridge
University Press: New York.
REFERENCES: 3. http://www.materials-talks.com/blog/2016/08/30/an-introducti
on-to-gel-permeation-chromatography-in-30-minutes/
4. https://chromatography.conferenceseries.com/events-list/appli
cations-of-chromatography
5. http://library.umac.mo/ebooks/b28050630.pdf
6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206469/
7. S.G. Prapulla, N.G. Karanth, in Encyclopedia of Food
Microbiology (Second Edition), 2014