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CHROMATOGRAPHY
INTRODUCTION
PRINCIPLE OF SEPERATION
STATIONARY PHASE
MOBILE PHASE
SEPERATION PROCEDURE
DETECTORS
APPLICATION
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REFERENCE
GEL CHROMATOGRAPHY
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BASIC DIFFERENCE BETWEEN GEL
PERMEATION & 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
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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.
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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
Proteins fractionation
Purification
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REFERENCE