Non-polar media for polynucloetide separations

Abstract

Nonporous beads having an average diameter of about 0.5-100 microns are suitable for chromatographic separation of mixtures of polynucleotides when the beads comprise a nonporous particle which are coated with a polymer or which have substantially all surface substrate groups endcapped with a non-polar hydrocarbon or substituted hydrocarbon group. The beads provide efficient separation of polynucleotides using Matched Ion Polynucleotide Chromatography.

The invention claimed is:  
     
         1 . A method for separating a mixture of polynucleotides, comprising applying a mixture of polynucleotides having up to 1500 base pairs to a separation medium, the separation surfaces of said medium coated with a hydrocarbon or non-polar hydrocarbon substituted polymer, or having substantially all polar groups reacted with a non-polar hydrocarbon or substituted hydrocarbon group, wherein said surfaces are non-polar; and eluting said mixture of polynucleotides.  
     
     
         2 . A method of  claim 1  wherein said medium is characterized by having a DNA Separation Factor of at least 0.05.  
     
     
         3 . A method of  claim 1  wherein said medium is characterized by having a Mutation Separation Factor of at least 0.1.  
     
     
         4 . A method of  claim 1  including eluting said mixture with a mobile phase comprising a counterion agent and an organic solvent, wherein said organic solvent is water soluble.  
     
     
         5 . A method of  claim 4 , wherein said solvent is selected from the group consisting of alcohol, nitrile, dimethylformamide, tetrahydrofuran, ester, ether, and mixtures of one or more thereof.  
     
     
         6 . A method of  claim 4  wherein said counterion agent is selected from the group consisting of lower alkyl primary amine, lower alkyl secondary amine, lower alkyl tertiary amine, lower alkyl trialkyammonium salt, quaternary ammonium salt, and mixtures of one or more thereof.  
     
     
         7 . A method of  claim 4  wherein said counterion agent is selected from the group consisting of octylammonium acetate, octadimethylammonium acetate, decylammonium acetate, octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, propyldiethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, triethylammonium hexafluoroisopropyl alcohol, and mixtures of one or more thereof.  
     
     
         8 . A method of  claim 4  wherein said counterion agent includes an anion, said anion is selected from the group comprising acetate, carbonate, phosphate, sulfate, nitrate, propionate, formate, chloride, and bromide.  
     
     
         9 . A method of  claim 1  wherein said separation is by Matched Ion Polynucleotide Chromatography.  
     
     
         10 . The medium of  claim 1  wherein said medium is subjected to an acid wash treatment in order to substantially remove multivalent cation contaminants from said surface.  
     
     
         11 . A method of  claim 1  wherein said medium comprises beads having an average diameter of 0.5 to 100 microns, said beads comprising nonporous particles.  
     
     
         12 . A method of  claim 4  wherein said beads are characterized by having a Mutation Separation Factor of at least 0.1.  
     
     
         13 . A method of  claim 11  wherein said beads are characterized by having a DNA Separation Factor of at least 0.05.  
     
     
         14 . A method of  claim 11  wherein said nonporous particles are a member selected from the group consisting of silica, silica carbide, silica nitrite, titanium oxide, aluminum oxide, zirconium oxide, carbon, insoluble polysaccharide, and diatomaceous earth.  
     
     
         15 . A method of  claim 11  wherein said nonporous particles are silica.  
     
     
         16 . A method of  claim 15  wherein said nonporous beads are substantially free from unreacted silanol groups.  
     
     
         17 . A method of  claim 11  wherein said separation is made by capillary electrochromatography.  
     
     
         18 . A method of  claim 11  wherein said separation is made by thin layer chromatography or high-speed thin layer chromatography.  
     
     
         19 . A method of  claim 1  wherein the non-polar surfaces are the surfaces of interstitial spaces of a silica gel monolith.  
     
     
         20 . A method of  claim 19  wherein said monolith has been subjected to an acid wash treatment in order to substantially remove multivalent cation contaminants.  
     
     
         21 . A method of  claim 19  wherein said monolith is characterized by having a DNA Separation Factor of at least 0.05.  
     
     
         22 . A method of  claim 19  wherein said monolith is characterized by having a Mutation Separation Factor of at least 0.1.  
     
     
         23 . A method of  claim 19  wherein said surfaces are substantially free form unreacted silanol groups.  
     
     
         24 . A method of  claim 19  wherein said monolith has substantially all separation surface substrate groups endcapped with a non-polar hydrocarbon or substituted hydrocarbon group.  
     
     
         25 . A method of  claim 19  including eluting said mixture with a mobile phase comprising a counterion agent and an organic solvent, wherein said organic solvent is water soluble.  
     
     
         26 . A method of  claim 19  wherein said separation is by Matched Ion Polynucleotide Chromatography.  
     
     
         27 . A bead comprising a nonporous particle coated with a polymer, wherein said bead has an average diameter of 0.5 to 100 microns and wherein said bead is characterized by having a Mutation Separation Factor of at least 0.1.  
     
     
         28 . A bead of  claim 27  wherein said bead is characterized by having a DNA Separation Factor of 0.05.  
     
     
         29 . A bead of  claim 27  wherein said bead is subjected to an acid wash treatment in order to substantially remove multivalent cation contaminants.  
     
     
         30 . A bead of  claim 27  wherein said nonporous particle is selected from the group consisting of silica, silica carbide, silica nitrite, titanium oxide, aluminum oxide, zirconium oxide, carbon, insoluble polysaccharides, and diatomaceous earth.  
     
     
         31 . A bead of  claim 27  wherein said nonporous particle is nonporous silica reacted to make a reverse phase material.  
     
     
         32 . A bead of  claim 31  wherein said nonporous bead is substantially free from unreacted silanol groups.  
     
     
         33 . A bead comprising a nonporous particle having substantially all surface substrate groups endcapped with a non-polar hydrocarbon or substituted hydrocarbon group, wherein said bead has an average diameter of 0.5 to 100 microns and wherein said bead is characterized by having a Mutation Separation Factor of at least 0.1 using Matched Ion Polynucleotide Chromatography.  
     
     
         34 . A method for treating the bead of  claim 27  in order to improve the resolution of polynucleotides separated using said bead, comprising contacting a solution containing a multivalent cation binding agent with said bead, wherein said solution has a temperature of about 50° C. to 90° C.  
     
     
         35 . A method for storing the bead of  claim 27  in order to improve the resolution of polynucleotides separated using said bead, comprising contacting a solution containing a multivalent cation binding agent with said beads prior to storing said bead.  
     
     
         36 . A silica gel monolith having non-polar interstitial separation surfaces wherein said monolith has been subjected to an acid wash treatment in order to substantially remove multivalent cation contaminants from said surfaces.  
     
     
         37 . A silica gel monolith of  claim 36  having substantially all separation surface substrate groups endcapped with a non-polar hydrocarbon or substituted hydrocarbon group.  
     
     
         38 . A monolith of  claim 36  characterized by having a DNA Separation Factor of at least 0.05.  
     
     
         39 . A monolith of  claim 36  characterized by having a Mutation Separation Factor of at least 0.1.  
     
     
         40 . A method for treating the monolith of  claim 36  in order to improve the resolution of polynucleotides separated using said monolith, comprising contacting a solution containing a multivalent cation binding agent with said surfaces, wherein said solution has a temperature of about 50° C. to 90° C.  
     
     
         41 . A method for storing the monolith of  claim 36  in order to improve the resolution of polynucleotides separated using said monolith, comprising contacting a solution containing a multivalent cation binding agent with the separation surfaces of said monolith prior to storing said monolith.