Separation medium for chromatography of various biomolecules
View Patent ↗The present invention relates to a separation medium, comprising an inner core of a porous material provided with charged ligands, and an outer lid comprising a porous material provided with charged ligands, wherein the charge of the ligands in the inner core is opposite that of the charge of the ligands in the lid. The present invention also relates to a method for biomolecule separation comprising applying a sample to the above separation medium, wherein large molecules are prevented from entering the medium by charge repulsion from the medium and small molecules are captured in the inner core.
1. A method for separation of first and second biomolecules comprising:
charging a sample comprising the first and second biomolecules, where the second biomolecules are larger than the first biomolecules, so that the first and second biomolecules have a charge; and
applying the sample to a separation medium comprising an outer lid and an inner core positioned within the outer lid,
wherein the outer lid comprises ligands charged the same as the first and the second biomolecules and has a first porosity smaller than the second biomolecules,
wherein the inner core comprises ligands charged opposite the first and the second biomolecules and has a second porosity smaller than the second biomolecules,
wherein the second biomolecules are charge repulsed from the separation medium, and
wherein the first porosity and the second porosity allow penetration of the first biomolecules but not the second biomolecules into the inner core.
2. The method of claim 1 , wherein the first porosity is smaller than the second porosity preventing entry of the second biomolecules through the first porosity.
3. The method of claim 1 , wherein the second biomolecules are obtained in column flow-through by charge repulsion and the first biomolecules are charge attracted into the inner core and then eluted from the separation medium.
4. The method of claim 3 , wherein the first biomolecules are eluted with a salt gradient.
5. The method of claim 3 , wherein the first biomolecules are eluted with a salt gradient or a pH gradient, such that a portion of the first biomolecules gradually adhere to the ligands in the outer lid as they are eluted from the inner core.
6. The method of claim 3 , wherein the second biomolecules are obtained by elution from the inner core of the separation medium.
7. The method of claim 1 , wherein the first and second porosities allow penetration of first biomolecules having a molecular weight of less than 100 000 g/mol.
8. The method of claim 7 , wherein the first and second porosities allow penetration of the first biomolecules having a molecular weight of less than 50 000 g/mol.
9. The method of claim 1 , wherein the outer lid has a higher ligand density than the inner core.
10. The method of claim 1 , wherein the first porosity is larger than the second porosity preventing the second biomolecules entry through the first porosity.
11. The method of claim 1 , wherein the second porosity is such that first biomolecules having a molecular weight approximately equal to or greater than 20 000 g/mol, preferably 50 000 or 100 000 g/mol, are excluded.