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Methods and devices based on capillary monolithic columns, preferably consisting of an underivatized poly(styrene-divinylbenzene) monolith, for separating a mixture of polynucleotides by ion pair-reverse phase-high performance chromatography (IP-RP-HPLC). In various aspects of the method and device

Methods and devices based on capillary monolithic columns, preferably consisting of an underivatized poly(styrene-divinylbenzene) monolith, for separating a mixture of polynucleotides by ion pair-reverse phase-high performance chromatography (IP-RP-HPLC). In various aspects of the method and device the monolith is characterized by one or more of the following: the monolith is contained within a capillary tube; the monolith is immobilized by covalent attachment at the inner wall of the tube; the tube is devoid of retaining frits; the monolith is characterized by having above 10,000 theoretical plates per meter and preferably above 200,000 theoretical plates per meter; the method uses a mobile phase which is devoid of EDTA; the monolith has a surface morphology that is rugulose or brush-like; the chromatographic surfaces of the monolith are non-porous; the monolith has channels sufficiently large for convective flow of the mobile phase; the monolith is formed from a polymerization mixture including underivatized styrene, a crosslinking agent, and a porogen, wherein the porogen includes tetrahydrofuran. The monolith can be incorporated into a miniaturized chromatography system which can be coupled to a mass spectrometer for on-line separation and mass determination of single-or double-stranded polynucleotides.

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What is claimed is: 1. A device for separating a mixture of polynucleotides, said device comprising: a polymeric monolith having non-polar chromatographic surfaces devoid of micropores, wherein said monolith comprises an underivatized poly(styrene-divinylbenzene) matrix, wherein said monolith is co

What is claimed is: 1. A device for separating a mixture of polynucleotides, said device comprising: a polymeric monolith having non-polar chromatographic surfaces devoid of micropores, wherein said monolith comprises an underivatized poly(styrene-divinylbenzene) matrix, wherein said monolith is contained within a fused silica tube having an inner diameter in the range of 1 micrometer to 1000 micrometer, wherein said monolith is immobilized by covalent attachment at the inner wall of said tube. 2. A device of claim 1 wherein said tube is devoid of retaining frits. 3. A device of claim 1 wherein said monolith is characterized by having 100,000 to 200,000 theoretical plates per meter. 4. A device of claim 3 wherein said theoretical plates per meter is determined from the retention time of single stranded p(dT) 18 standard using the following equation: wherein N is the number of theoretical plates, tR is the retention time of said standard determined during an isocratic elution, w0.5 is the peak width at half height, and L is the length of the monolith in meters. 5. A device of claim 4 wherein said tube has an inner diameter of 200 micrometer and a length of 60 mm, wherein during said isocratic elution said monolith has a back pressure in the range of 180 to 200 bar, and a flow rate in the range of 2 to 3 μL/min at an elution temperature of 50° C. 6. A device of claim 1 wherein said monolith has a surface morphology, as determined by scanning electron microscopy, that resembles the surface morphology of octadecyl modified poly(styrene-divinylbenzene) particles, wherein said surface morphology of said monolith is rugulose. 7. A device of claim 1 wherein said monolith has channels sufficiently large for convective flow of said mobile phase. 8. A device for separating a mixture of polynucleotides, said device comprising: a polymeric monolith having nonpolar chromatographic surfaces, wherein said monolith comprises an underivatized poly(styrene-divinylbenzene) matrix and is devoid of micropores, wherein said monolith is contained within a fused silica tube, and wherein said monolith is immobilized by covalent attachment at the inner wall of said tube. 9. A device of claim 8 wherein said tube has an inner diameter in the range of 1 micrometer to 1000 micrometer. 10. A device of claim 8 wherein said tube is devoid of retaining frits. 11. A device of claim 8 wherein said monolith is characterized by having 10,000 to 200,000 theoretical plates per meter. 12. A device of claim 8 wherein said monolith has a surface morphology, as determined by scanning electron microscopy, that is brush-like. 13. A device of claim 8 wherein said monolith comprises an underivatized monolithic stationary phase. 14. A device of claim 8 wherein said monolith has a surface morphology, as determined by scanning electron microscopy, that resembles the surface morphology of octadecyl modified poly(styrene-divinylbenzene) particles, wherein said surface morphology of said monolith is rugulose. 15. A device of claim 8 wherein said monolith has channels sufficiently large for convective flow of said mobile phase. 16. A device for separating a mixture of polynucleotides, said device comprising: a polymeric monolith having non-polar chromatographic surfaces which are devoid of micropores, wherein said monolith comprises an underivatized poly(styrene-divinylbenzene) matrix, wherein said monolith is contained within a fused silica tube, wherein said tube has been silanized, and wherein said tube is devoid of retaining frits. 17. A device of claim 16 wherein said monolith is immobilized by covalent attachment at the inner wall of said tube. 18. A device of claim 16 wherein said monolith is characterized by having 100,000 to 200,000 theoretical plates per meter. 19. A device of claim 16 wherein said monolith has a surface morphology, as determined by scanning electron microscopy, that resembles the surface morphology of octadecyl modified poly (styrene-divinylbenzene) particles, wherein said surface morphology of said monolith is brush-like. 20. A device of claim 16 wherein said tube has an inner diameter in the range of 1 micrometer to 1000 micrometer. 21. A device of claim 16 wherein said monolith has a surface morphology, as determined by scanning electron microscopy, that resembles the surface morphology of octadecyl modified poly(styrene-divinylbenzene) particles, wherein said surface morphology of said monolith is rugulose. 22. A device for separating a mixture of polynucleotides, said device comprising: a polymeric monolith having non-polar chromatographic surfaces which are devoid of micropores, wherein said monolith comprises an underivatized polystyrene divinylbenzene matrix, wherein said monolith is contained within a tube having an inner diameter in the range of 1 micrometer to 1000 micrometer, wherein said monolith is characterized by having 10,000 to 200,000 theoretical plates per meter. 23. A device of claim 22 wherein said monolith is contained within a tube having an inner diameter in the range of 10 micrometer to 500 micrometer. 24. A device of claim 22 wherein said monolith is immobilized by covalent attachment at the inner wall of said tube. 25. A device of claim 24 wherein said tube is devoid of retaining frits. 26. A device for separating a mixture of polynucleotides, said device comprising: a polymeric monolith having non-polar chromatographic surfaces which are devoid of micropores, wherein said monolith comprises an underivatized poly(styrene-divinylbenzene) matrix, wherein said monolith is characterized by having at least 100, 000 theoretical plates per meter, wherein said monolith is contained within a silanized fused silica tube having an inner diameter in the range of 10 micrometer to 1000 micrometer, wherein said monolith is immobilized at the inner wall of said tube. 27. A device of claim 26 wherein said monolith is characterized by having 100,000 to 200,000 theoretical plates per meter. 28. A device of claim 26 wherein said monolith is contained within a tube having an inner diameter in the range of 1 micrometer to 1000 micrometer. 29. A device of claim 26 wherein said monolith has a surface morphology, as determined by scanning electron microscopy, that resembles the surface morphology of octadecyl modified poly(styrene-divinylbenzene) particles, wherein said surface morphology of said monolith is rugulose. 30. A miniaturized chromatographic system for separating a mixture of polynucleotides said system comprising the device of claim 26. 31. A device for separating a mixture of polynucleotides, said device comprising: a polymeric monolith having nonpolar chromatographic surfaces, wherein said monolith has a surface morphology, as determined by scanning electron microscopy, that resembles the surface morphology of octadecyl modified poly(styrene-divinylbenzene) particles, wherein said surface morphology of said monolith is rugulose and brush-like, wherein said monolith comprises an underivatized poly(styrene-divinylbenzene) matrix, wherein said monolith is contained within a fused silica tube having an inner diameter in the range of 1 micrometer to 1000 micrometer, wherein said monolith is immobilized at the inner wall of said tube, and wherein said surfaces of said monolith are non-porous. 32. A device of claim 31 wherein said tube is devoid of retaining frits. 33. A device of claim 31 wherein said monolith characterized by having 100,000 to 200,000 theoretical plates per meter. 34. A device of claim 31 wherein said tube has been silanized. 35. A device of claim 31 wherein said monolith is formed from a polymerization mixture including underivatized styrene, a crosslinking agent, and a porogen, wherein said porogen comprises tetrahydrofuran. 36. A device of claim 31 wherein said polynucleotides comprise double-stranded fragments having lengths in the range of 3 to 600 base pairs. 37. A system of claim 30 wherein said monolith is operatively coupled to a mass spectrometer. 38. A chromatographic device, said device comprising: a polymeric monolith having non-polar chromatographic surfaces wherein said surfaces are nonporous, wherein said monolith comprises an underivatized poly(styrene-divinylbenzene) matrix, wherein said monolith is contained within a silanized fused silica tube having an inner diameter in the range of 10 micrometer to 1000 micrometer, and wherein said monolith is immobilized at the inner wall of said tube.