Question

You are trying to separate proteins using gel filtration. You know that for gel filtration resin 1, the Kav values for your proteins are 0.38, 0.41 and 0.45 while for resin 2 the values of Kav of your proteins are 0.25, 0.45 and 0.6. Which column will give you better separation of the proteins, resin 1 or resin 2, and why?

Answer 1

Gel filtration is a seperating technique based on the size of the molecules. it is also called molecular exclusion or gel permeation chromatography. in this technique, the stationary phase consisits of porous beads with a well defined range of porous sizes. that is , the molecules within this molecular limit can be seperated. When an aqueous solution of molecules of various sizes is passed through a column containing these molecular sieves,small molecules can enter them but the large ones cannot. As a result, the big molecules are located in the solution around the beads while the smaller ones are able to penetrate the beads and the solution between them. The most important point is that large molecules flow more rapidly through this column. In this technique , there is a linear relationship between the relative elution volume of a substance ( the volume of the fractions in which the molecule is found) and the logarithm of its molecular mass.

Kav value represents the the ratio of elution volume of the solute - volume occupied by the gel phase. Particle size determines the void volume- Vo and the pore size determines the resolving range. Molecules larger than the largest pore are excluded, elute at Vo. Molecules that are smaller than the smallest pore are included, they sample both Vi and Vo elute at Vt=Vi + Vo. Molecules that can occupy some but not all of the pores elute at an intermediate volume Ve.

Partition coefficient Kav = (Ve-Vo)/(Vt-Vo).

Vt-total volume, Ve = elution volume of solute, Vo = void volume of column , Vs = volume of stationary phase (= Vi)
Vi = Vt-Vo-Vgel matrix.Vt-Vo- volume occupied by the gel space space includng gel matrix. For the resin 1, Kav values are 0.38, 0.41 and 0.45, where as, in resin 2,Kav values are 0.25, 0.45 and 0.6. Difference in Kav values for resin 1 is 0.03 where as for resin 2 it is 0.15, which is five times larger. There fore Resin 2 will have more seperation compared to resin 1.