Separation medium for chromatography of various biomolecules

Abstract

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 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. 
     
     
         2 . The separation medium of  claim 1 , wherein the porous material in the inner core has the same porosity as the porous material in the lid. 
     
     
         3 . The separation medium of  claim 2 , wherein the porosity of the inner core and lid allows penetration of a biomolecule with a molecular weight of less than 100 000 g/mol. 
     
     
         4 . The separation medium of  claim 3 , wherein the porosity of the inner core and lid allows penetration of a biomolecule with a molecular weight of less than 50 000 g/mol. 
     
     
         5 . The separation medium of  claim 2 , wherein the amount of charged ligands is higher in the lid than in the core. 
     
     
         6 . The separation medium of  claim 1 , wherein the porous material in the inner core has the larger porosity than the porous material in the lid. 
     
     
         7 . The separation medium of  claim 6 , wherein the porosity of the lid is such that biomolecules larger or equal to a molecular weight of 20 000, preferably 50 000 or 100 000 g/mol are excluded, and the porosity of the inner core is larger than the lid porosity. 
     
     
         8 . The separation medium of  claim 1 , wherein the porous material is derived from a synthetic polymer material, or from a natural polymer material. 
     
     
         9 . The separation medium of  claim 1 , wherein the charged ligands are —SO 3   − , —COO − , —N + (CH 3 ) 3  or —NH + (C 2 H 5 ) 2 . 
     
     
         10 . A method for biomolecule separation comprising applying a sample to the separation medium of  claim 1 , wherein large biomolecules are prevented from entering the medium by charge repulsion from the medium and small biomolecules are captured in the inner core. 
     
     
         11 . The method of  claim 10 , wherein the charge repulsion is caused by the charged ligands in the lid. 
     
     
         12 . The method of  claim 11 , wherein the charge repulsion, and thereby the prevention of entering of large biomolecules, is accompanied by a lower porosity (=steric hindrance) in the lid compared to the inner core. 
     
     
         13 . The method of  claim 10 , wherein large biomolecules are obtained in the flow through by charge repulsion and small biomolecules adsorbed in the inner core are eluted from the separation medium. 
     
     
         14 . The method of  claim 13 , wherein the small biomolecules are eluted with a salt gradient. 
     
     
         15 . The method of  claim 13 , wherein the small biomolecules are eluted with a salt gradient and/or a pH gradient, in such a way that a portion of the small biomolecules gradually adhere to the ligands in the lid on their way out from the separation medium. 
     
     
         16 . The method of  claim 10 , wherein the method is run in column format and the desired biomolecules are obtained in the flow-through. 
     
     
         17 . The method of  claim 10 , wherein the method is run in column format and the desired biomolecules are obtained by elution from the inner core of the separation medium.