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Characterisation of gel filtration media
The selectivity of a gel filtration medium depends solely on its pore size distribution and a selectivity curve is used to describe this.
Selectivity curves, independent on the conditions used are obtained by plotting Kd or Kav vs log Mr for series of macromolecules with different sizes (Fig 4.1).
Fig 4.1 Selectivity curve where Kav
is plotted against log Mr.
Kd is the distribution coefficient for the partitioning of a sample molecule between the mobile and stationary phases and is defined by the following expression:Kd is linearly related to Vr and represents the fraction of the pores available to a given sample molecule. It depends only on the gel filtration medium used and the size of the sample molecule.
Since Vi is difficult to measure it is usual to substitute the term (Vc-Vo) for Vi in the partition equation and call the result Kav instead of Kd.
Though not a true partition coefficient, Kav is also linearly related to Vr for a given GF medium.
Kav varies between 0 (Vr = V0) and slightly less than 1 (Vr approximately = Vc). Selectivity curves are usually fairly linear over the range Kav = 0.1 to Kav = 0.7. This part of the curve is used to define the fractionation range (Fig 4.2).
Fig 4.2. Definition of fractionation range and
exclusion limit for a gel filtration medium.
Another important characteristic of a GF medium is the Mr of the smallest molecule excluded from the pores called the exclusion limit. The exclusion limit is defined by a conventional as the Mr obtained from the intercept of the extrapolated linear part of the selectivity curve with the log Mr axis (Fig 4.2).
Figure 4.3 shows the linear parts of the selectivity curves for Superdex™ and Sephacryl™ media. Together they cover a Mr range from 100 to 8,000,000 for peptides and globular proteins.
As seen, Superdex has very steep selectivity curves and should be chosen for applications requiring the highest resolution.
Fig 4.3. Fractionation ranges for Superdex and Sephacryl gel filtration media.
The steeper the curve, the higher the resolution. |
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