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