Non-polar media for polynucleotide separations

• US 20010023848A1
• Filed: 04/05/2001
• Published: 09/27/2001
• Est. Priority Date: 12/05/1997
• Status: Active Grant

• Current Assignee: ADS Biotec Inc.
• Original Assignee: Douglas T. Gjerde, Paul D. Taylor
• Inventors: Gjerde, Douglas T., Taylor, Paul D.
• Granted Patent: US 6,488,855 B2
• Time in Patent Office: Days
• US Class Current: 210/635
• CPC Class Codes: B01J 20/287 Non-polar phases; Reversed phases
• CPC Class Codes: B01J 20/3204 Inorganic carriers, supports or substrates
• CPC Class Codes: B01J 20/3225 involving a post-treatment of the coated or impregnated product
• CPC Class Codes: B01J 20/3227 by end-capping, i.e. with or after the introduction of functional or ligand groups
• CPC Class Codes: B01J 20/3268 Macromolecular compounds
• CPC Class Codes: B01J 20/328 Polymers on the carrier being further modified

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.

Claims

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, a 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.

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.

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.

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.