lec 4 size exclusion chromatography
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Size Exclusion
Chromatography
Lectures to B. Pharm III
By
Kefa Bosire.
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Size Exclusion
Chromatographic separation based on
their size, or ( hydrodynamic volume)
Usually applied to large molecules ormacromolecular complexes e.G
proteins and industrial polymers
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Size Exclusion
Chromatography
Also known as gel filtration
chromatography when an aqueoussolution is used to transport the samplethrough the column used for fractionation
of proteins and other water-solublepolymers
Proteins, polysaccharides and nucleic acids
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Size Exclusion
Chromatography
Biologists and biochemists typicallyuse
1. Polyacrylamide (bio-gel),
2. Dextran (Sephadex), or
3. Agarose (Sepharose) filtering under lowpressure
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Principle ofSEC
Hollow tube tightly packed with very
fine porous polymer beads with pores ofdifferent sizes.
Depressions on the surface or channels
through the bead. Larger particles cannot enter into as
many pores hence less overall volumeto traverse and the faster the elution.
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Size Exclusion Collected at the end is known as the eluent.
Void volume, Vo consists of any particles
too large to enter the medium, represents thevolume outside of the beads using BlueDextran.
Inclusion (internal volume),Vi: space within
the beads. amino acid linked to a fluorescentmolecule e.g. dinotrophenyl or ascorbic acid.
Elution volume,Ve volume required to "elute"a biomolecule from a SEC column,Ve should
fall between theVo and theVi.
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Size Exclusion
Particles are not ideally defined;
Both particles and pores may vary in sizehence elution resembles gaussian distributions
Interaction with stationary phase
Length will tighten the resolution, and
increasing the column diameter increases thecapacity of the column
Over packed column can collapse the pores
Under packed column can reduce the relative
surface area to trap small particles
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Size Exclusion
Collected fractions often examined by
spectroscopic techniques Refractive index (RI), evaporative light
scattering (ELS), and ultraviolet (UV)
Elution volume decreases roughly linearlywith the logarithm of the molecularhydrodynamic volume (often assumed to beproportional to molecular weight).
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Size Exclusion
Columns often calibrated using 4-5standard samples to get
1. Void volume
2.
Slope of the logarithmic dependence
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Gel Permeation
chromatography
Gel permeation chromatography used
when an organic solvent is the mobile phase.
Analyse the molecular weight distribution oforganic-soluble polymers.
Gel filtration chromatography generallyrefers to separation of proteins and otherbiological macromolecules on the basis ofmolecular size.
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Gel Permeation
chromatography
Polymer chemists typically use1. Silica or
2. Cross-linked polystyrene medium
Under a higher pressure
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Size Exclusion Various solutions can be applied without
interfering with the filtration process.
Preserving the biological activity of theparticles.
Can be coupled with separation based onacidity, basicity, charge, and affinity.
Can determine the quaternary structure ofpurified proteins.
Can be carried out under native solutionconditions.
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Size Exclusion
Can assay protein tertiary structure by thehydrodynamic volume distinguishing foldedand unfolded versions of a protein.
Measure of both the size and thepolydispersity of a synthesised polymer (inabout half an hour).
Light scattering and/or viscometry can beused online with SEC to yield absolutemolecular weights avoiding use of calibrationwith standards.
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Size Exclusion
Separation of small moleculecontaminants from protein preps -polishing steps during manufacture
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Shortcoming ofSEC
Low resolution.
Hydrophobic proteins may bind to the matrixand chose not to elute.
May need ionic component buffer to shieldactive charges on gel particles.
Need to know recovery ration before loading. Needs good pouring technique and
maintenance.
May need overnight for swelling of matrix.
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General Gel Classification
Resins are usually classified based on their
capacity to separate different sizes of ahypothetical, globular protein.
Lower range: molecules see the entireinternal volume of beads no selection belowthis size
Upper range: molecules excluded from seeinginside of a beads allowing no separation
Linear range: between the two extremes
allowing decent separation of molecules
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General Gel Classification
Soft gels
Semi rigid gels
Rigid gels
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Soft Gels
Cross linked
Imbibe large solvent volumes (MP)usually aqueous
Deform easily forming voids, bending
Need capping at top of column toprevent out swelling from column
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Semi Rigid Gels
About double volume Resilient to Pressure under wide range
Applicable in HPLC
Usable with organic solvents
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Rigid Gels
Usually glass.
Fixed pore size.
Exhibit adsorptive properties.
Less efficient. Increased peak broadening compared
to semi rigid gels.
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Bead Cellulose
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Bead Cellulose Excellent mechanical stability
even at gel with large pores. Easy to
handle. Resistant to destruction andgeneration of fine particles duringstirring.
Rigid spherical particles. High flowthroughput the gels at a low drop inpressure.
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Bead Cellulose Narrow particle size distribution in several
degrees converting all ranges common for this
sort of gel. High chemical resistance and
compatibility with most commonly usedsolvents and buffers. Applicability in widerange of ph and salt concentrations withminimal changes (swelling / shrinking) in thegel bed.
High temperature stability allowssterilization by autoclaving.
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Bead Cellulose High hydrophility. A number of free
hydroxy groups in matrix allows preparation
of derivatives or enzyme immobilization. High porosity of matrix allows good
capacity and recovery.
High selectivity of separation.
Easy-to-ready. Gels are supplied pre-swollen.
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Sephadex Hydroxyalkyl ethers of hydroxyalkoxy
polysaccharides prepared by reacting the
corresponding polysaccharide first with anepoxide in an aqueous alkaline medium,then with an epoxide in the presence of a
Lewis acid catalyst in a nonaqueous,nonalcoholic organic solvent. Resultingproducts are outstanding for liquid-gelchromatography
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Sephadex
C3H5ClO Epichlorohydrin
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Sephadex
Very polar (several OH grps)
Swells in H2O,electrolytes, DMSO,formamide
Large variety by linkage types
Generally insoluble unless degraded Stable
Autoclavable at 120C for upto 30 minswithout loss of properties, melting
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Sephacryl
Cross linking Dextran with N,N-
methylenebiscrylamide.
(See hand notes).
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The polymerization of acrylamide with the
crosslinking agent bisacrylamide.
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Sephacryl
Used mainly in biologicals generalpurpose
Insoluble unless degraded
pH range 3-11 Hydrolyses dense matrix than sephadex
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Sephacryl
Excellent separations over a widemolecular weight range 5kD to 1.5million D
High reproducibility due to high stability
Easy to pack, run, and maintain High flow rates and recoveries
Well-suited to industrial- scale use
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Sepharose
Bead formed from heated Agarosewhich forms sepharose on cooling
Sepharose blue Cross linked form
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Sepharose
D-Galactose 3,6-Anhydro-L-Galactose
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Properties ofSepharose
Stable H2O, salt solutions, enzymes
3,6-anhydro-L-galactose bond.
pH 4-9
Melts >40C
Damaged when frozen
Sterilised using diethylprocarbonate
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Properties ofSepharose Chain held by H bonds
Wet bead dia 45 200 microM
Fractionation 10,000 40, 000,000
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Applications ofSEC Desalting
Separation of biomolecules by size
Phenols from Nucleic acids
Removal of radio isotopes 135I
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Applications ofSEC Follow bioreactions separated in column
Eg ezymes, cofactors, products
Mwt determinations
Separation of viruses
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End
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