Transcript GEL FILTRATION CHROMATOGRAPHY Size Exclusion

GEL FILTRATION CHROMATOGRAPHY
Size Exclusion Chromatography
What is gel filtration chromatography?
• Separation of molecules on the basis of molecular
size and shape
• Separation of proteins and nucleic acids
• Molecular sieve chromatography - Natural or
synthetic zeolites of metallic alumina-silicates
• Controlled pore glass chromatography - Porous
glass granules
Principle
• Separation based on size, shape and molecular weight
• Mechanism - Molecular sieve properties of porous
materials
• Porous materials – Gel beads or Porous glass granules
packed into a glass column and act as molecular sieve
• Ability of molecules to enter the pores within the beads or
granules
• Beads are made of different materials with different porosity
and form 3D network of cross-linked chains
GEL FILTRATION ASSEMBLY
Materials
• Gels
• Majority available in two particle size grades
– viz. coarse (300-100 m) and fine (20-80 m).
• Available in bead form than powder form.
– Cross-linked dextrans (Sephadex)
– Agarose (Sepharose, Bio Gel A)
– Polyacrylamide (Bio Gel P)
– Polyacryloyl morphine (Enzocryl gel)
– Polystyrene (Bio Beads S)
• Porous glass granules
– Bio Glass
– Porous silica as Porasil.
Dextran gels
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Cross-linked polysaccharide, epichlorohydrin
Hydrophilic character
Swells in aqueous media.
Stable in water, salt solution, organic solvents, alkali and
weak acidic solution
• Molecular size exclusion limit is 200 – 800 Kda
Agarose gel
• Agar (polysaccharide with D-galactose and 3,6
anhydro L-galactose units)
• Hydrophilic and absence of charged groups.
• Molecular size exclusion limit several million daltons.
• Compatible with cell aqueous buffers
• Stable at pH 4-10
• Mostly incorporated with bacteriostatic agent (0.2%
sodium azide)
• To study viruses, nucleic acids and polysaccharides
Polyacrylamide gels
• Polymerization of acrylamide and methylene bis
acrylamide form this gel
• Stable in aqueous buffers at pH 1-10
• Molecular size exclusion limit is 1.8 to 400 Kda
Porous glass gravels
• Manufactured from borosilicate glass.
• Molecular size exclusion limit is 3000 to 9 million daltons
Procedure
Column Packing
• First gel is converted to swollen
form i.e. gel in weak salt solution.
Swelling time
• Sephadex G-25 and G-50 –
Overnight
• Sephadex G-75 – 1 day
• Sephadex G-100 – 2 days
• Sephadex G-200 – 3 days
• Swelling time reduced by heating
gel in boiling water bath for 1-5 h.
• Space between the polymer chains
is increased due to swelling.
• Gel is then packed into column
Procedure…..
Sample application
• Sample (mixture of larger and smaller molecules)
• Application depends on the column size
• Smaller molecules enter the gel material and their flow is
retarded
• Larger molecules pass down rapidly because they are
unable to penetrate the gel molecules
• Smaller molecules later fall down slowly.
Procedure….
• Retardation depends upon
– Size of the particles
– Adsorption property of the gel
• Void volume is measured by use of completely
excluded compound
(Blue dextran – high MW polysaccharide)
• Effluent volume of particular compound enables to
calculate its distribution coefficient.
Void volume
Measured by use of
completely excluded
compound
(Blue dextran – high
MW polysaccharide)
Effluent volume of
particular compound
enables to calculate its
distribution coefficient
Thin layer gel filtration
• Separation of hydrophilic substances such as proteins,
peptides and nucleic acids
• Swollen gel is spread on a thin plate and placed in air
tight container
• Then connected to reservoir at either end by filter paper
bridges
• Plates are inclined at 20o and equilibrated for minimum
12 h
• Sample is applied as spot or band and the plate is
developed
• Spots are then detected.
Applications
• Purification of biological macromolecules
– Proteins, enzymes, antibodies, hormones and
nucleic acids, etc.
• Determination of molecular weight
– Proteins and enzymes.
• Desalting
– Amino acids from proteins
– Phenol from nucleic acids
– Ammonium sulphate from proteins
– Monosaccharides from polysaccharides
• Study the protein binding structures
• Solution concentrations