Method and apparatus for separating polynucleotides using monolithic capillary columns
View Patent ↗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.
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:
(
N
/
L
)
=
(
5.54
/
L
)
(
t
R
w
0.5
)
2
wherein N is the number of theoretical plates, t R is the retention time of said standard determined during an isocratic elution, w 0.5 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.