Using SEC to elute proteins using a resin with a exclusion limit of 75 kDA and...

Using SEC to elute proteins using a resin with a exclusion limit of 75 kDA and 100kDa. Which would elute first and why? What is the order of elution

Protein 1 80kDa

Myoglobin 16.7

Cardiac Troponin 24

Cytochrome C 11.7

Expert Solution

Order of elution: Protein 1(80KDa)>Cardiac troponin(24KDa)>Myoglobin(16.7 KDa)> Cytochrome C(11.7 KDa)

Separation in size exclusion chromatography is based on stokes radius.

stokes radius is the radius of hypothetical sphere formed by trumbling of biomolecules in the solution.

stokes radius is directly proportional to molecular weight of the molecule. The molecule with the highest weight have highest stokes radius.

fraction of mobile phase which is present within the beads(stationary phase) is known as internal volume and volume which is present outside is known as void volume.

Kd value is the fraction of internal volume which is freely accessible for any separating biomolecule. Molecule with stokes radius larger than permissible limit of stationary phase has kd =0, and therefore have no interaction with the internal volume, and travel through the voids between the beads and eluted first whereas the smaller molecules have kd=1, which means they are accessible to the internal volume and travel from within the beads before elution.

gladiator answered 2 years ago

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