GEL
CHROMATOGRAPHY
Submitted To- Dr. Pooja Chawla
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Presented By- Sharib Khan
Rajib Das
Sumit Pannu
Sitanshu Khilar
PRESENTATION OUTLINE
INTRODUCTION
PRINCIPLE OF SEPERATION
STATIONARY PHASE
MOBILE PHASE
SEPERATION PROCEDURE
DETECTORS
ADVANTAGE AND DISADVANTAGE
APPLICATION
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REFERENCE
GEL CHROMATOGRAPHY
Gel Chromatography (GC) is a chromatographic method
in which molecules are separated based on their size.
Gel Chromatography is also known as :-
Size Exclusion Chromatography
Gel Permeation Chromatography
Molecular Sieve Chromatography
Gel Filtration Chromatography
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BASIC DIFFERENCE BETWEEN GEL
PERMEATION & GEL FILTRATION
CHROMATOGRAPHY
When an organic solvent is used as a mobile phase, then it
is tend to called as gel permeation chromatography.
When an aqueous solution is used to transport the sample
through the column, the technique is known as gel filtration
chromatography.
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GEL
CHROMATOGRAP
HY COMPONENTS
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STATIONARY PHASE
Composed of semi-permeable, porous polymer gel beads with well defined
range of pore sizes Chemically inert.
Nature of the gel:-
Mechanically stable
Ideal porous structure
Wide pore size give low resolution
Uniform particle size
Types of gels used:-
The gels used as molecular sieves are cross linked polymers.
They are uncharged and inert i.e. don’t bind or react with the materials
being analyzed.
Three types of gels are used:-
Dextran (Sephadex)
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Agarose gel
Acrylamide gel (Synthetic gel)
MOBILE PHASE
•The mobile phase in Gel chromatography refers to the
solvent being continuously applied to the column or
stationary phase.
•The mobile phase acts as a carrier to the sample solution
Material Solvent
Synthetic elastomers Toluene
( polybutadiene , polyisoprene )
Styrene-Butadiene Rubber , Tetrahydrofuran (THF)
Epoxy resins
Polyolefins Tri- chloro -benzene
Polyurethane Di- methylformamide (DMF)
Proteins, polysaccharides Water / Buffers
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PRINCIPLE
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PRINCIPLE
It is a kind of chromatography technique based on the
difference of molecular weight and is one of the effective
and mild methods extensively used to isolate and analyze
the bio macromolecular substances.
The stationary phase consists of beads containing pores
that span a relatively narrow size range.
When the gel is packed into a column and percolated
with a solvent, it permits the large molecular weight
compounds to pass rapidly without penetration of the
pores.
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 9
column).
PRINCIPLE
Smaller molecules penetrate the particles to varying
extents depending upon their shape and size, there is thus
partition of the molecules between the liquid inside the gel
particles and that outside, the smaller the molecules, the
larger the percentage of liquid within the particles that is
available to them.
Molecules therefore leave the column in the order of
decreasing molecular size, the large size will leave the
column first followed by the smaller sizes depending on
their partition (shape and size) ranges.
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SEPARATION PROCEDURE
1- Preparation of column for gel filtration which involves
Swelling of the gel
Packing the column
Washing: After packing, several column volumes of
buffer solution is passed through the column to
remove any air bubbles and to test the column
homogeneity.
2- Loading the sample onto the column
3- Eluting the sample and detection of components
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DETECTORS
Concentration sensitive detectors
•Bulk Property Detectors- Refractive Index (RI) Detector
•Solute Property Detectors- Ultraviolet (UV)
Absorption Detector
•Evaporative Detectors- Evaporative Light Scattering
Detector (ELSD)
Light Scattering Detectors
•Low Angle Light Scattering (LALS) Detectors
•Multi Angle Light Scattering (MALS) detectors
• Viscosity Detectors- Differential Viscometers
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ADVANTAGES AND DISADVANTAGES
Advantages:
Short analysis time.
Well defined separation.
Narrow bands and good sensitivity.
There is no sample loss.
Small amount of mobile phase required.
The flow rate can be set.
Disadvantages:
Limited number of peaks that can be resolved within the short time scale of the GPC
run.
Filtrations must be performed before using the instrument to prevent dust and other
particulates from ruining the columns and interfering with the detectors.
The molecular masses of most of the chains will be too close for the GPC separation
to show anything more than broad peaks.
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APPLICATIONS OF GC
Proteins fractionation
Purification
Molecular weight determination.
Separation of sugar, proteins, peptides, rubbers and others
on the basis of their size.
This technique can be use to determine the quaternary
structure of purified proteins.
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REFERENCE
1. Hagel L. Gel‐filtration chromatography. Current
protocols in molecular biology. 1998. 44 :9-10.
2. Fagain C, Cummins PM, Connor BF. Gel-filtration
chromatography. In Protein Chromatography 2011. 25-
33.
3. Stanton P. Gel filtration chromatography. In Gel
Chromatography of Peptides and Proteins 2004. 55-73.
Springer, Totowa, NJ.
4. Yau W.W, Kirkland JJ, Bly DD. Modern size-exclusion
liquid chromatography. Wiley; 1979.
5. Cameronf BF. Gel filtration chromatography.
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Separation Science. 1971 6 :37-229.
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