GEL-FILTRATION CHROMATOGRAPHY
INTRODUCTION
Gel filtration chromatography (some times referred to as molecular sieve chromatography) is a method that separates molecules according to their size and shape. The separation of the components in the sample mixture, with some exceptions, correlates with their molecular weights. In these cases, gel filtration can be used as an analytical method to determine the molecular weight of an uncharacterized molecule. Gel filtration is also an important preparative technique since it is often a chromatographic step in the purification of proteins, polysaccharides and nucleic acids.
Gel filtration chromatography is a separation based on size. It is also called molecular exclusion or gel permeation chromatography. In gel filtration chromatography, the stationary phase consists of porous beads with a well-defined range of pore sizes. The stationary phase for gel filtration is said to have a fractionation range, meaning that molecules within that molecular weight range can be separated.
Gel filtration chromatography (some times referred to as molecular sieve chromatography) is a method that separates molecules according to their size and shape. The separation of the components in the sample mixture, with some exceptions, correlates with their molecular weights. In these cases, gel filtration can be used as an analytical method to determine the molecular weight of an uncharacterized molecule. Gel filtration is also an important preparative technique since it is often a chromatographic step in the purification of proteins, polysaccharides and nucleic acids.
Gel filtration chromatography is a separation based on size. It is also called molecular exclusion or gel permeation chromatography. In gel filtration chromatography, the stationary phase consists of porous beads with a well-defined range of pore sizes. The stationary phase for gel filtration is said to have a fractionation range, meaning that molecules within that molecular weight range can be separated.
Background information
The basic components of the gel filtration experiment are the matrix, chromatography
column and the elution buffer. The matrix is the material in the column that is actually
the separation medium. It is the stationary phase of the chromatography. The column is
a tube with a frit and elution spout fitted at the bottom. The frit is a membrane or porous disk that supports and retains the matrix in the column but allows water and dissolved solutes to pass. The elution buffer is the mobile phase of the chromatography and flows through the matrix and out of the column. The column, with the matrix and applied sample, is “developed” by the elution buffer. This means that the molecules in the
sample are carried by the flow of buffer into the matrix where they are gradually
separated. The separated zones of molecules then flow out of the column where they
are collected for analysis. Filling the chromatography column with matrix is referred to as “packing”. The packed
matrix is called the “bed” and the volume it occupies is termed the “bed volume”. It is
very important not to allow the bed to run dry. Otherwise, cracks and fi ssures develop
and the matrix has to be removed and repacked. The gel filtration matrix consists ofmicroscopic beads that contain pores and internal channels. The larger the molecule, the more difficult it is for it to pass through the pores and penetrate the beads. Larger
molecules tend to fl ow around and in between the beads. The total volume of buffer between the beads is the “void volume”. Smaller molecules tend to spend more time in the maze of channels and pores in the bed. Consequently, the larger, higher molecular weight molecules are eluted from the column before smaller molecules. Larger
molecules take the faster, more direct path that involves less time in the beads. This is somewhat analogous to fi nding your way out of a complicated maze or simply walking around the outside of the maze and avoiding the whole situation entirely.
Molecules can have the same molecular weight but radically different shapes.
Molecules with a more compact shape, such as a sphere, will penetrate the beads more
easily than those having an elongated shape, like a rod. Therefore, a rod-like molecule
will elute before a spherical one of the same molecular weight.
column and the elution buffer. The matrix is the material in the column that is actually
the separation medium. It is the stationary phase of the chromatography. The column is
a tube with a frit and elution spout fitted at the bottom. The frit is a membrane or porous disk that supports and retains the matrix in the column but allows water and dissolved solutes to pass. The elution buffer is the mobile phase of the chromatography and flows through the matrix and out of the column. The column, with the matrix and applied sample, is “developed” by the elution buffer. This means that the molecules in the
sample are carried by the flow of buffer into the matrix where they are gradually
separated. The separated zones of molecules then flow out of the column where they
are collected for analysis. Filling the chromatography column with matrix is referred to as “packing”. The packed
matrix is called the “bed” and the volume it occupies is termed the “bed volume”. It is
very important not to allow the bed to run dry. Otherwise, cracks and fi ssures develop
and the matrix has to be removed and repacked. The gel filtration matrix consists ofmicroscopic beads that contain pores and internal channels. The larger the molecule, the more difficult it is for it to pass through the pores and penetrate the beads. Larger
molecules tend to fl ow around and in between the beads. The total volume of buffer between the beads is the “void volume”. Smaller molecules tend to spend more time in the maze of channels and pores in the bed. Consequently, the larger, higher molecular weight molecules are eluted from the column before smaller molecules. Larger
molecules take the faster, more direct path that involves less time in the beads. This is somewhat analogous to fi nding your way out of a complicated maze or simply walking around the outside of the maze and avoiding the whole situation entirely.
Molecules can have the same molecular weight but radically different shapes.
Molecules with a more compact shape, such as a sphere, will penetrate the beads more
easily than those having an elongated shape, like a rod. Therefore, a rod-like molecule
will elute before a spherical one of the same molecular weight.
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