Diapositive 1

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Size-exclusion chromatography (SEC) Gel
permeation chromatography (GPC) Gel Filtration
Chromatography (GFC)
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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
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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
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Stationary phase
Stationary phase is a material of controlled pore
size 10 lt pore diameter lt 500 nm NB silica
particles for adsorption and partition
chromatography lt 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
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Stationary phase
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)
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Mobile 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
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Mobile phase influences the retention mechanism
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
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Injection volume
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
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Detection
Refractive index detector (RID)
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 
Quantitative polymer analysis Number of monomer
units gt 10 RI is directly proportional to the
concentration of the polymer RI is practically
independent of the molecular weight
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Detection
UV-visible detection
When peptides or proteins are analysed, UV
absorption at 280 nm is more convenient
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Applications
Oligomers, polymers and macromolecules 500 lt
Molecular Weight lt 2.106 Da Synthetic and
Natural polymers Peptides, proteins Oligosaccharid
es, polysaccharides Nucleic acids Pre-fractionati
on
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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 proteins so SEC is
still an alternative
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Molecular weight distribution
Though they are subtle, differences such as those
could cause marked variations in the performance
of the polymer
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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)
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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
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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
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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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Efficiency of the analysis
Particle size is between 60 and 140 µm NB
particle size in adsorption and partition LC is
2-10 µm The smaller the particle size, the
larger the efficiency
High efficiency Thin peaks
Low efficiency Large peaks
No need for large separation for the peaks to be
resolved
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Peptide analysis
High resolution run of a peptide mix
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Application Archaeometry
Preservation of wood
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
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
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Application Archaeometry
Preservation of wood
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
Size exclusion chromatogram Refractive index
detection
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
Mortensen et al., J. Archaeological Sci., 34
(2007) 1211-1218
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