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The separation mechanism
The ability of gel filtration to separate molecules according to size resides with the highly porous structure of gel filtration media and is basically a question of accessible volumes.
In a column all sample molecules have access to the liquid between the beads . This volume is called the void volume in gel filtration and equals ~30% of the column volume.
Gel filtration media contain pores allowing the sample molecules to penetrate into the gel filtration beads to different degrees depending on size. Together the volume of these pores form the pore volume.
The non-porous part of the beads is called the backbone and is inaccessible for the sample molecules. For a good GF matrix the volume of the backbone is around 3-5% of the column volume of a well- packed column.
Fig 1.1. Scanning electron micrograph
of an agarose gel. Magnification x 50,000.
Ref. Anders S. Medin,PhD Thesis,
Uppsala University 1995.
Fig 1.2. The different volumes accessible to
sample molecules. |
Partitioning between the mobile and the stationary phase
In the gel filtration column, sample molecules are partitioned between the eluent (the mobile phase) and the accessible pores of the gel filtration beads (the stationary phase). This partitioning acts to establish a dynamic equilibrium of sample molecules between the mobile and the stationary phases and is driven exclusively by diffusion (Fig 1.3).
Fig 1.3
Transport along the column
The mobile phase transports the sample molecules down the column, but acts only on the sample molecules present in the mobile phase. The molecules present in the pores are "stationary" and escape this type of transportation.
However, this transport creates an uneven distribution of “stationary” and “mobile” sample molecules, which the partitioning mechanism tries to correct. The effect of this is a mass transfer of sample molecules from the mobile phase to the pores at the front of the sample zone (Fig 1.4), a mass transfer in the opposite direction at the rear end of the zone (Fig 1.5) and an apparent retardation of the sample zone.
The larger the part of sample molecules in the pores, the larger the retardation.
Fig 1.4
Fig 1.5
A certain deviation from equilibrium is needed in order to make the sample zone move down the column.
This, however, is unavoidably associated with broadening of the sample zone. |
Elution volumes
The migration rate of a sample zone in any chromatographic situation depends on the fraction of the sample molecules present in the mobile phase.
Molecules excluded from the pores of the stationary phase (100% in the mobile phase) move down the column at the same speed as the mobile phase. They will consequently leave the column after one void volume ( V0 ) of mobile phase has passed through the column.
Molecules with partial access to the pores will be retarded in relation to their respective degree of access to the pores: in other words they will elute from the column in order of decreasing sizes.
Molecules with full access to the pores will all move down the column at the same speed and remain unseparated from each other. Using a GF medium with a backbone volume of ~5% they leave the column after slightly less than one column volume of mobile phase has passed through the column.
Fig 1.6. The three categories of accessible
volumes are used for different purposes. |
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