Lec 4 Size Exclusion Chromatography

8/3/2019 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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Lec 4 Size Exclusion Chromatography