Gel Filtration Chromatography
Applications
Gel filtration chromatography, a
type of size exclusion chromatography, can be used to either fractionate
molecules and complexes in a sample into fractions with a particular size range,
to remove all molecules larger than a particular size from the sample, or a
combination of both operations. Gel filtration chromatography can be used to
separate compounds such as small molecules, proteins, protein complexes,
polysaccharides, and nucleic acids when in aqueous solution. When an organic
solvent is used as the mobile phase, the process is instead referred to as gel
permeation chromatography.
Gel filtration chromatography can
also be used for:
1. Fractionation
of molecules and complexes within a predetermined size range
2. Size
analysis and determination
3. Removal
of large proteins and complexes
4. Buffer
exchange
5. Desalting
6. Removal
of small molecules such as nucleotides, primers, dyes, and contaminants
7. Assessment
of sample purity
8. Separation
of bound from unbound radioisotopes
Gel filtration chromatography media
for all of the above uses are available in prepacked gravity flow columns, spin
columns, low-pressure and medium-pressure chromatography columns, and bottled
resins.
Gel Filtration Chromatography
Mechanism
In a gel filtration chromatography
column, the stationary phase is composed of a porous matrix, and the mobile
phase is the buffer that flows in between the matrix beads. The beads have a
defined pore size range, known as the fractionation range. Molecules and
complexes that are too large to enter the pores stay in the mobile phase and
move through the column with the flow of the buffer. Smaller molecules and
complexes that are able to move into the pores enter the stationary phase and
move through the gel filtration column by a longer path through pores of the
beads.
Any molecule or complex that is
above the fractionation range for a particular gel filtration chromatography
column will move through the column faster than any molecule that can enter the
stationary phase. Therefore, any constituent in the sample that is above the
fractionation range will elute first (in the void volume) before anything that
is in the fractionation range. The minimum size that will remain in the mobile
phase and not enter the stationary phase is known as the exclusion limit.
Bio-Rad offers gel filtration chromatography media and columns with exclusion
limits ranging over three orders of magnitude, from 100 daltons to 100,000
daltons (100 kDa).
Molecules and complexes that can
enter the stationary phase will be fractionated according to their sizes.
Smaller molecules will migrate deep into the pores and will be retarded more
than larger molecules that do not so easily enter the pores, and are thus
eluted from the column more quickly. This difference in pore migration leads to
fractionation of components by size with the largest eluting first.
In gel filtration chromatography
columns designed for desalting, buffer exchange, and the removal of small
molecules such as nucleotides, the salts and small compounds readily enter the
pores, are retarded, and migrate more slowly through the column than the larger
proteins or nucleic acids. Therefore, the components of interest in the sample
are eluted in advance of salts, nucleotides, etc. DNA cleanup kits using this
mechanism often contain gel filtration spin columns.
Resolution, here defined as the
sharpness of the boundaries between size fractions, is determined by bead size
and a number of other factors. Smaller bead size generally yields higher
resolution in a gel filtration chromatography column. Compact molecules diffuse
through the stationary phase faster than linear molecules. Size exclusion,
fractionation range, and elution rate are affected by buffer composition, ionic
strength, and pH. For the fractionation of complex mixtures of proteins,
elution times and size exclusion limits may need to be determined empirically.
Gel Filtration Chromatography Media
An important criterion for gel
filtration chromatography media is that media is inert and that nothing in the
sample or any buffer binds to the media. Another consideration is the type of
gel filtration column being used and whether it is used in a pressurized
chromatography system or gravity flow or spin columns. If a pressurized
chromatography system is being used, both the column and the media must be able
to tolerate the pressure and flow rates used.
Commonly used media for gel
filtration chromatography are based on agarose or polyacrylamide beads,
dextrose for gravity or low-pressure systems, and polymeric resins for
medium-pressure systems. The choice of media depends on the properties of the
components to be separated and other experimental factors. The following are
general considerations when determining the choice of gel filtration
chromatography media:
l
Fractionation range
l
Size exclusion limit
l
Operating pressure
l
Flow rate
l
Sample viscosity
l
pH range
l
Autoclavability
Tolerance for water-miscible organic
solvents; some samples may be more soluble in a water-organic mix
Tolerance for detergents, chaotropic
agents, formamide, etc.
Operating temperature
The types of samples, choice of
media, and the chromatography system setup will determine which parameters are
the most important for a given purification application.
this symbol highlights
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