Post on 19-Dec-2015
Size-exclusion chromatography (SEC)
Gel permeation chromatography (GPC)
Gel Filtration Chromatography (GFC)
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 samplesNo specific skills required, no expensive material
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
Stationary phase
Stationary phase is a material of controlled pore size10 < pore diameter < 500 nm
NB: silica particles for adsorption and partition chromatography < 500 Å
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
Choice is based on:
Pore diameter and distribution, according to size of solutesExclusion 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
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
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 Stokes’radiusGiven the molecules are the same MW, the molecules with the largest
Stokes’radius will elute first
Steric exclusion of species depends on their hydrodynamic dimensions
Mobile phase influences the retention mechanism
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
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 detectorBased 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 0.0001°C for optimum performance
One of the least sensitive detectors« Choice of last resort »
Refractive index detector (RID)
Detection
When peptides or proteins are analysed, UV absorption at 280 nm
is more convenient
UV-visible detection
Oligomers, polymers and macromolecules
500 < Molecular Weight < 2.106 Da
Synthetic and Natural polymersPeptides, proteinsOligosaccharides, polysaccharidesNucleic acids
Pre-fractionation
Applications
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 proteinsso SEC is still an alternative
Applications
Though they are subtle, differences such as those could cause marked variations
in the performance of the polymer
Molecular weight distribution
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)
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
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
Pre-fractionation
Pre-fractionation of the components of a chewing gum formulation
Pre-fractionation
Pre-fractionation of a reaction mixture
No need for large separation for the peaks to be resolved
Efficiency of the analysis
Low efficiencyLarge peaks
High efficiencyThin peaks
Particle size is between 60 and 140 μmNB: particle size in adsorption and partition LC is 2-10 μm
The smaller the particle size, the larger the efficiency
High resolution run of a peptide mix
Peptide analysis
Application: Archaeometry
Viking ships from the 11th 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
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