Size-exclusion chromatography (SEC) Gel permeation chromatography (GPC) Gel Filtration...

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Transcript of Size-exclusion chromatography (SEC) Gel permeation chromatography (GPC) Gel Filtration...

  • Slide 1
  • Size-exclusion chromatography (SEC) Gel permeation chromatography (GPC) Gel Filtration Chromatography (GFC)
  • Slide 2
  • Size-exclusion chromatography Retention is only determined by size Interactions with SP and MP are identical for all solutes Larger species will elute first they can not pass through as many pores so their path is shorter Employed for over 40 years Simple, economical, rapid, highly reproducible, gentle on the samples No specific skills required, no expensive material
  • Slide 3
  • Retention mechansim Large molecules cannot enter gel and are excluded. They have less volume to traverse and elute sooner Smaller molecules can enter the pores, they are not excluded. They have more volume to traverse and they elute later
  • Slide 4
  • Stationary phase Stationary phase is a material of controlled pore size 10 < pore diameter < 500 nm NB: silica particles for adsorption and partition chromatography < 500 Scanning electron micrograph of an agarose gel Deactivated silica, bonded or not, dextran, agarose or polyacrylamide Columns can be obtained that will separate specific size ranges
  • Slide 5
  • Choice is based on: Pore diameter and distribution, according to size of solutes Exclusion limit: defines MW of the smallest molecule that cannot penetrate the pores (between 20 and 3000 kDa) Total pore volume (defines volume of solvent required for most retained solutes) Rigidity (polymers or bonded silica) Solvent compatibility (silica gel for synthetic polymers, polymers for bio-macromolecules) Particle diameter (depends on required resolution) Stationary phase
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  • Organic solvent (Gel permeation) Buffered aqueous (Gel Filtration) = Compatible with physiological conditions = OK for biopolymers Choice is based on: Solubilising power Viscosity Compatibility with detection method Mobile phase
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  • Solvation phenomena Configuration of the species depends on the mobile phase Vary the binding conditions such as pH, temperature and salt concentration to influence the size and shape of the proteins Elution depends on Stokesradius Given the molecules are the same MW, the molecules with the largest Stokesradius will elute first Steric exclusion of species depends on their hydrodynamic dimensions Mobile phase influences the retention mechanism
  • Slide 8
  • Care for volume of sample injected: too high a volume sample leads to reduced resolution too small a volume leads to high sample dilution and poor recovery Selecting an appropriate column size Generally, the column would be 4-20 times the sample volume Injection volume
  • Slide 9
  • Detection Quantitative polymer analysis Number of monomer units > 10 RI is directly proportional to the concentration of the polymer RI is practically independent of the molecular weight Bulk property detector Based on refraction of light as it passes from one media to another Presence of a solute changes the refraction index of the solvent T must be maintained to 0.0001C for optimum performance One of the least sensitive detectors Choice of last resort Refractive index detector (RID)
  • Slide 10
  • Detection When peptides or proteins are analysed, UV absorption at 280 nm is more convenient UV-visible detection
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  • Oligomers, polymers and macromolecules 500 < Molecular Weight < 2.10 6 Da Synthetic and Natural polymers Peptides, proteins Oligosaccharides, polysaccharides Nucleic acids Pre-fractionation Applications
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  • Determination of physico-chemical parameters (average molecular mass, branching index, intrinsic viscosity) Diversity of the molecular weights of proteins in biological tissues and extracts One of the first methods that appeared to measure MW of proteins until 1969 when SDS-PAGE appeared! Polyacrylamide gel can be denaturating to certain proteins so SEC is still an alternative Applications
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  • Though they are subtle, differences such as those could cause marked variations in the performance of the polymer Molecular weight distribution
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  • Group-separation mode Buffer exchange of a protein sample is frequently necessary not only between purification steps, but also prior to further analysis The presence of salts mostly disturbs the MALDI-TOF MS signal Proper purification or desalting procedures must be employed Compared to dialysis, SEC is more rapid (a few minutes vs. several hours)
  • Slide 15
  • Protein desalting (Buffer exchange) Salts are small, enter the pores completely and are thus slowed down They are last to elute or displaced by the water molecules The column is pre-equilibrated with several column volumes of the preferred buffer, i.e. the buffer into which one wishes to transfer the protein The sample is then added to the column and allowed to enter the resin bed Additional preferred buffer is applied to the column and the emering fractions are collected
  • Slide 16
  • Protein desalting (Buffer exchange) Desalting can be extended to Removal of low molecular weight sugars, such as lactose from whey Removal of certain agents used for solubilizing proteins, such as urea and guanidine salts The contaminant can be left on the separating device, an important feature when working with toxic or radioactive substances
  • Slide 17
  • Pre-fractionation Pre-fractionation of the components of a chewing gum formulation
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  • Pre-fractionation Pre-fractionation of a reaction mixture
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  • No need for large separation for the peaks to be resolved Efficiency of the analysis Low efficiency Large peaks High efficiency Thin peaks Particle size is between 60 and 140 m NB: particle size in adsorption and partition LC is 2-10 m The smaller the particle size, the larger the efficiency
  • Slide 20
  • High resolution run of a peptide mix Peptide analysis
  • Slide 21
  • Application: Archaeometry Viking ships from the 11 th century were impregnated with PEG in the 1960s Study of the MW, amount and integrity of the polymeric layer were needed Mortensen et al., J. Archaeological Sci., 34 (2007) 1211-1218 Wood will bend and crack if it is dried without preservation Water in the wood must be replaced by something hat does not evaporate PEG has proven useful for this purpose Preservation of wood
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  • Application: Archaeometry Preservation of wood PEG 600 is the major PEG component in the ship which makes this object sensitive to changes in air humidity since PEG 600 is hydroscopic Sample extracted from the ship: Cylinder, 5 cm long, 2.5 cm diameter, sliced into 5 mm thick disks Soxhlet extraction with chloroform during 3 h Mortensen et al., J. Archaeological Sci., 34 (2007) 1211-1218 Size exclusion chromatogram Refractive index detection
  • Slide 23