Size Exclusion Chromatography

Lectures to B. Pharm III By Kefa Bosire.

Size Exclusion


Chromatographic separation based on their size, or ( hydrodynamic volume) Usually applied to large molecules or macromolecular complexes e.G proteins and industrial polymers

Size Exclusion Chromatography



Also known as gel filtration chromatography when an aqueous solution is used to transport the sample through the column used for fractionation of proteins and other water-soluble polymers Proteins, polysaccharides and nucleic acids
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Size Exclusion Chromatography



Biologists and biochemists typically use

1. 2. 3.

Polyacrylamide (bio-gel), Dextran (Sephadex), or Agarose (Sepharose) filtering under low pressure

Principle of SEC


Hollow tube tightly packed with very fine porous polymer beads with pores of different sizes. Depressions on the surface or channels through the bead. Larger particles cannot enter into as many pores hence less overall volume to 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 the volume outside of the beads using Blue Dextran. Inclusion (internal volume),Vi: space within the beads. amino acid linked to a fluorescent molecule e.g. dinotrophenyl or ascorbic acid. Elution volume, Ve volume required to "elute" a biomolecule from a SEC column, Ve should fall between the Vo and the Vi.

Size Exclusion
 

 

 

Particles are not ideally defined; Both particles and pores may vary in size hence elution resembles gaussian distributions Interaction with stationary phase Length will tighten the resolution, and increasing the column diameter increases the capacity 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 linearly with the logarithm of the molecular hydrodynamic volume (often assumed to be proportional to molecular weight).
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Size Exclusion


Columns often calibrated using 4-5 standard samples to get

1. 2.

Void volume Slope of the logarithmic dependence

Gel Permeation chromatography



Gel permeation chromatography used when an organic solvent is the mobile phase. Analyse the molecular weight distribution of organic-soluble polymers. Gel filtration chromatography generally refers to separation of proteins and other biological macromolecules on the basis of molecular size.
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Gel Permeation chromatography



Polymer chemists typically use

1. 2.

Silica or 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 the particles. Can be coupled with separation based on acidity, basicity, charge, and affinity. Can determine the quaternary structure of purified proteins. Can be carried out under native solution conditions.
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Size Exclusion


Can assay protein tertiary structure by the hydrodynamic volume distinguishing folded and unfolded versions of a protein. Measure of both the size and the polydispersity of a synthesised polymer (in about half an hour). Light scattering and/or viscometry can be used online with SEC to yield absolute molecular weights avoiding use of calibration with standards.
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Size Exclusion


Separation of small molecule contaminants from protein preps polishing steps during manufacture

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Shortcoming of SEC
 

 

Low resolution. Hydrophobic proteins may bind to the matrix and chose not to elute. May need ionic component buffer to shield active 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 a hypothetical, globular protein. Lower range: molecules see the entire internal volume of beads no selection below this size Upper range: molecules excluded from seeing inside 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 to prevent 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 and generation of fine particles during stirring. Rigid spherical particles. High flow throughput the gels at a low drop in pressure.
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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 used solvents and buffers. Applicability in wide range of ph and salt concentrations with minimal changes (swelling / shrinking) in the gel bed. High temperature stability allows sterilization 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 preswollen.
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Sephadex


Hydroxyalkyl ethers of hydroxyalkoxy polysaccharides prepared by reacting the corresponding polysaccharide first with an epoxide in an aqueous alkaline medium, then with an epoxide in the presence of a Lewis acid catalyst in a nonaqueous, nonalcoholic organic solvent. Resulting products are outstanding for liquid-gel chromatography
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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 mins without loss of properties, melting
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Sephacryl


Cross linking Dextran with N,Nmethylenebiscrylamide. (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 general purpose Insoluble unless degraded pH range 3-11 Hydrolyses <3 Autoclaved at 120C at neutral pH repeatedly Separates proteins and polysaccharides >dense matrix than sephadex

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Sephacryl


   

Excellent separations over a wide molecular weight range 5 kD to 1.5 million 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 Agarose which 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 of Sepharose


   

Stable H2O, salt solutions, enzymes 3,6anhydro-L-galactose bond. pH 4-9 Melts >40C Damaged when frozen Sterilised using diethylprocarbonate
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Properties of Sepharose
  

Chain held by H bonds Wet bead dia 45 200 microM Fractionation 10,000 40, 000,000

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Applications of SEC
   

Desalting Separation of biomolecules by size Phenols from Nucleic acids Removal of radio isotopes 135I

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Applications of SEC
Follow bioreactions separated in column Eg ezymes, cofactors, products  Mwt determinations  Separation of viruses


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End
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