(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`11111111111111111111111111111111111111111111111111111111111111111111111111111111
`
`(43) International Publication Date
`2 August 2001 (02.08.2001)
`
`PCT
`
`(10) International Publication Number
`WO 01/55713 A2
`
`(51) International Patent Classification7:
`
`G01N 30/96
`
`(21) International Application Number: PCT/US01/02539
`
`(22) International Filing Date: 25 January 2001 (25.01.2001)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/178,553
`
`26 January 2000 (26.01.2000) US
`
`(71) Applicant: TRANSGENOMIC, INC. [US/US]; 2032
`Concourse Drive, San Jose, CA 95131 (US).
`
`(72) Inventors: HUBER, Christian; -- (**). OBERAR(cid:173)
`CHER, Herbert; -- (**). PREMSTALLER, Andreas; -(cid:173)
`(**).
`
`(74) Agents: WALKER, William, B. et al.; Transgenomic,
`Inc., 2032 Concourse Drive, San Jose, CA 95131 (US).
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ,
`DE, DK, DM, DZ, EE, ES, Fl, GB, GD, GE, GH, GM, HR,
`HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
`LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ,
`NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM,
`TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`patent (AT, BE, CH, CY, DE, DK, ES, Fl, FR, GB, GR, IE,
`IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
`CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
`
`(54) Title: METHOD AND APPARATUS FOR SEPARATING POLYNUCLEOTIDES USING MONOLITHIC CAPILLARY
`COLUMNS
`
`iiiiiiii
`
`[Continued on next page]
`
`0.7 -
`
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`..... E
`"' c
`c
`Cl.l~
`i:lO
`,_N
`
`.. .... -;::;
`
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`....
`Q
`en
`ao
`0
`ao
`
`.... ..,
`....
`..., -...,
`
`-
`- ------------------------------------------------------------------------------------------
`-iiiiiiii
`== ---!!!!!!!!
`iiiiiiii -iiiiiiii
`--iiiiiiii
`iiiiiiii ----
`
`(57) Abstract: Methods and devices
`based
`on
`capillary monolithic
`columns, preferably consisting of an
`underivatized
`poly(styrene-divinyl(cid:173)
`benzene) 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
`the
`at the inner wall of the tube;
`tube is devoid of retaining frits; the
`v~ 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;
`
`I
`15
`
`~
`,..-.!
`
`0 • 0
`
`V
`
`1\.u
`'""''
`
`0
`
`J :.., ~ W '.- ~ ~.,
`
`11
`
`1
`5
`
`1
`1 0
`time [min]
`
`&;
`lf)
`..........
`S
`0 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
`
`MTX1013
`
`

`

`WO 01/55713 A2
`
`11111111111111111111111111111111111111111111111111111111111111111111111111111111
`
`Published:
`For two-letter codes and other abbreviations, refer to the "Guid-
`without international search report and to be republished ance Notes on Codes and Abbreviations" appearing at the begin-
`upon receipt of that report
`ning of each regular issue of the PCT Gazette.
`
`2
`
`

`

`wo 01/55713
`
`PCT/USOl/02539
`
`METHOD AND APPARATUS FOR SEPARATING POL YNUCLEOTIDES USING
`
`TITLE OF THE INVENTION
`
`MONOLITHIC CAPILLARY COLUMNS
`FIELD OF THE INVENTION
`
`The present invention relates to methods and devices for analyzing
`
`polynucleotides. In particular, the invention relates to the use of monolithic capillary
`
`columns for use in high-performance liquid chromatography of single and double-
`
`stranded polynucleotides.
`BACKGROUND OF THE INVENTION
`
`Genetics and proteomics depend on the ability to analyze complex mixtures of
`
`biological origin with high sensitivity and maximum selectivity. Especially the rapid
`
`development of miniaturized techniques in analytical chemistry (He et al. Anal. Chern.
`
`70:3790-3797 (1998)) has had a profound impact on the modern practice of analyzing
`
`1
`2
`3
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`4
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`5
`6
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`7
`
`8
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`9
`10
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`11
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`12
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`13
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`14
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`biological samples of high complexity (Novotny J. Chromatogr. B 689: 55-70 (1997)).
`15
`16 Several techniques based on the principle of differential migration (Rathore et al. J.
`
`17 Chromatogr. A 743: 231-246 (1996)) were developed after the introduction of fused
`silica capillaries to analytical chemistry (Dandeneau et al. HRC & CC: 2:351 (1979)), in
`18
`
`19
`
`20
`
`particular capillary liquid chromatography (CLC) (Hirata et al. J. Chromatogr. 186:521-
`
`528 (1979)), capillary electrophoresis (CE) (Jorgenson et al. J. Chromatogr. 218:209-
`
`216 (1981 )), and capillary electrochromatography (CEC) (Jorgenson et al. J.
`21
`22 Chromatogr. 218:209-216 (1981)).
`
`23
`
`24
`
`25
`26
`
`Columns packed with microparticulate sorbents have been successfully applied as
`
`separation media in high-performance liquid chromatography (HPLC). Despite many
`
`advantages, HPLC columns packed with microparticulate, porous stationary phases
`have some limitations, such as the relatively large void volume between the packed
`
`particles and the slow diffusional mass transfer of solutes into and out of the stagnant
`27
`28 mobile phase present in the pores of the separation medium (Martin eta. Biochem J.
`
`29
`
`30
`
`31
`
`32
`
`35:1358 ( 1941 ); Unger et al in Packings and Staionary Phases in Chromatographic
`
`Techniques, Unger Ed: Marcel Dekker: New York, p. 75 (1990)).
`
`One approach to alleviate the problem of restricted mass transfer and intraparticular
`
`void volume is the concept of monolithic chromatographic beds, where the separation
`
`33 medium consists of a continuous rod of a rigid, polymer which has no interstitial volume
`34
`but only internal porosity consisting of micropores and macropores. Monolithic
`
`35
`
`separation columns are becoming more widely used in HPLC of biomolecules.
`1
`
`3
`
`

`

`1
`2
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`3
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`6
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`7
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`8
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`1 0
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`11
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`12
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`13
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`14
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`15
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`16
`17
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`18
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`19
`20
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`21
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`23
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`24
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`25
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`26
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`27
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`28
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`29
`
`wo 01/55713
`WO 97/19347 relates to a method and device for separating one or several organic
`substances in a sample. The chromatographic device comprises a monolith prepared in
`
`PCT/USOl/02539
`
`an emulsion system containing at least 75% by weight of water phase. Separations of
`
`polynucleotides were not disclosed.
`
`U.S. 5,334,310 relates to a monolith containing small pores having diameters less
`
`than about 200 nm and large pores with diameters greater that about 600 nm. The
`
`columns were equipped with end fittings. No separations of polynucleotides were
`
`demonstrated.
`
`WO 00/15778 relates to polymeric monolithic beds for resolving mixtures containing
`
`polynucleotides. However, single-stranded molecules were poorly resolved using the
`
`column. The columns had inner diameters (I D) of greater than 4mm and were equipped
`
`retaining frits. The mobile phase buffers included EDTA. Useful separations of DNA
`
`fragments by IP-RP-HPLC using underivatized polystyrene/divinylbenzene monolithic
`
`columns could not be achieved and such columns were not recommended.
`
`There is a need for improved monolithic columns and methods for the separation
`
`of polynucleotides.
`
`SUMMARY OF THE INVENTION
`
`In one aspect, the present invention provides a method for separating a mixture
`
`of polynucleotides. The method includes applying the mixture of polynucleotides to a
`polymeric monolith having non-polar chromatographic surfaces and eluting the mixture
`
`of polynucleotides with a mobile phase including a counterion agent and an organic
`solvent, wherein the monolith is an underivatized poly(styrene-divinylbenzene) matrix.
`
`In the method, the monolith preferably is contained within a fused silica tube having an
`
`inner diameter in the range of 1 to 1000 micrometer and the monolith is immobilized by
`
`covalent attachment at the inner wall of the tube. The tube is preferably devoid of
`
`retaining frits. In preferred embodiments of this aspect of the invention, the monolith is
`
`characterized by having 100,000 to 200,000 theoretical plates per meter. The
`
`theoretical plates per meter can determined from the retention time of single stranded
`p(dT)1 8 standard using the following equation:
`2
`(NIL)= (5.54/ L)(!..B._J
`
`Wo.s
`
`30
`
`31
`
`32
`
`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. In one embodiment, during the isocratic
`
`2
`
`4
`
`

`

`WO 01/55713
`
`PCT/USOl/02539
`
`elution, the back pressure was about 180 to 200 bar, at a flow rate in the range of 2 to 3
`
`IJL/ min and at an elution temperature of 50°C for a monolith having an ID of 200
`
`1
`
`2
`
`3 micrometer and a length of 60 mm. The method can be performed using a mobile
`
`4
`
`5
`
`6
`
`phase which is devoid of EDT A. The preferred monolith has a surface morphology, as
`
`determined by scanning electron microscopy, that resembles the surface morphology of
`
`octadecyl modified poly(styrene-divinylbenzene) particles, wherein the surface
`
`7 morphology of the monolith is rugulose. Additionally, the preferred monolith has a
`
`8
`
`9
`
`surface morphology, as determined by scanning electron microscopy, that resembles
`
`the surface morphology of octadecyl modified poly(styrene-divinylbenzene) particles,
`
`10 wherein the surface morphology of the monolith is brush-like. The monolith can be
`
`11
`
`12
`
`13
`
`14
`
`15
`
`formed from a polymerization mixture including underivatized styrene, a crosslinking
`
`agent, and a porogen, wherein the porogen includes tetrahydrofuran. A preferred
`
`porogen includes a mixture of tetrahydrofuran and decanol. In the method, the
`
`polynucleotides can include double-stranded fragments having lengths in the range of 3
`
`to 600 base pairs. The method can further include analyzing eluted polynucleotides by
`
`16 mass spectral analysis. In the method, the monolith preferably has a back pressure in
`
`17
`
`the range of about 20 to about 300 bar, and typically in the range of about 70 to about
`
`18. 200 bar. The method can be performed at a monolith temperature in the range of about
`
`19
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`20°C to about 90°C.
`
`In another aspect, the invention concerns a method for separating a mixture of
`
`polynucleotides. The method includes applying the mixture of polynucleotides to a
`
`polymeric monolith having non-polar chromatographic surfaces and eluting the mixture
`
`of polynucleotides with a mobile phase comprising a counterion agent and an organic
`
`solvent. In a preferred embodiment, the monolith comprises an underivatized
`
`poly(styrene-divinylbenzene) matrix. In this aspect of the invention, the monolith is
`
`preferably contained within a fused silica tube, and the monolith is immobilized by
`
`covalent attachment at the inner wall of the tube. The tube can have an inner diameter
`
`in the range of 10 micrometer to 1 000 micrometer, and preferably in the range of 1
`
`29 micrometer to 1000 micrometer. The tube is preferably devoid of retaining frits. In
`
`30
`
`31
`
`32
`
`33
`
`34
`
`35
`
`certain embodiments, the monolith is characterized by having 10,000 to 200,000
`
`theoretical plates per meter and preferably characterized by having 100,000 to 200,000
`
`theoretical plates per meter. During the elution, the mobile phase preferably is devoid of
`
`EDTA. The preferred monolith has a surface morphology, as determined by scanning
`
`electron microscopy, that resembles the surface morphology of octadecyl modified
`
`poly(styrene-divinylbenzene) particles, wherein the surface morphology of the monolith
`3
`
`5
`
`

`

`WO 01/55713
`PCT/USOl/02539
`is rugulose. The monolith can be formed from a polymerization mixture including
`underivatized styrene, a crosslinking agent, and a porogen, wherein the porogen
`
`comprises tetrahydrofuran.
`
`In another aspect, the invention provides a method for separating a mixture of
`
`polynucleotides. The method includes applying the mixture of polynucleotides to a
`
`polymeric monolith having non-polar chromatographic surfaces and eluting the mixture
`
`of polynucleotides with a mobile phase comprising a counterion agent and an organic
`
`solvent, wherein the monolith comprises an underivatized poly(styrene-divinylbenzene)
`
`1
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9 matrix, wherein the monolith is contained within a fused silica tube, and wherein the
`
`10
`
`tube is devoid of retaining frits, wherein the tube has an inner diameter in the range of 1
`
`11 micrometer to 1000 micrometer, and wherein the polynucleotides are double-stranded
`
`12
`
`fragments having lengths in the range of 3 to 600 base pairs. During the elution, the
`
`13 mobile phase preferably is devoid of EDTA. The monolith preferably is immobilized by
`covalent attachment at the inner wall of the tube. In certain embodiments, the monolith
`14
`
`15
`
`16
`17
`
`18
`
`is characterized by having 50,000 to 200,000 theoretical plates per meter. In preferred
`
`embodiments, the monolith is characterized by having greater than about 190,000
`theoretical plates per meter. The preferred monolith has a surface morphology, as
`
`determined by scanning electron microscopy, that resembles the surface morphology of
`
`octadecyl modified poly(styrene-divinylbenzene) particles, wherein the surface
`19
`20 morphology of the monolith is rugulose.
`
`21
`
`22
`
`23
`24
`
`25
`
`26
`
`27
`
`28
`
`29
`
`30
`
`31
`
`In a further aspect, the invention provides a method for separating a mixture of
`
`polynucleotides. The method includes applying the mixture of polynucleotides to a
`
`polymeric monolith having non-polar chromatographic surfaces and eluting said mixture
`of polynucleotides with a mobile phase comprising a counterion agent and an organic
`
`solvent, wherein the monolith is characterized by having 10,000 to 200,000 theoretical
`
`plates per meter, wherein the monolith includes an underivatized poly(styrene-
`
`divinylbenzene) matrix, wherein the monolith is contained within a fused silica tube
`
`having an inner diameter in the range of 1 micrometer to 1000 micrometer, and wherein
`
`the monolith is immobilized by covalent attachment at the inner wall of the tube. In a
`
`preferred embodiment, the theoretical plates per meter is determined from the retention
`time of single stranded p(dT)18 standard using the following equation:
`(NIL)= (5.54/ L)(~J 2
`
`Wo.s
`
`4
`
`6
`
`

`

`WO 01/55713
`PCT/USOl/02539
`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. The tube preferably is devoid of retaining frits.
`
`In the method, the mobile phase preferably is devoid of EDT A. In a preferred
`
`embodiment, the monolith has a surface morphology, as determined by scanning
`
`electron microscopy, that resembles the surface morphology of octadecyl modified
`
`poly(styrene-divinylbenzene) particles, wherein the surface morphology of said monolith
`
`is rugulose. Also in a preferred embodiment, the monolith has a surface morphology, as
`
`determined by scanning electron microscopy, that resembles the surface morphology of
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`octadecyl modified poly(styrene-divinylbenzene) particles, wherein the surface
`
`11 morphology of said monolith is brush-like.
`
`12
`
`13
`
`14
`
`15
`
`16
`17
`
`18
`19
`
`In a yet further aspect, the invention concerns a method for separating a mixture
`
`of polynucleotides. The method includes applying the mixture of polynucleotides to a
`
`polymeric monolith having non-polar chromatographic surfaces and eluting the mixture
`
`of polynucleotides with a mobile phase comprising a counterion agent and an organic
`
`solvent, and wherein the mobile phase is devoid of EDT A. In this aspect, the monolith
`preferably is contained within a fused silica tube having an inner diameter in the range
`
`of 10 micrometer to 1 000 micrometer. The monolith preferably is immobilized by
`covalent attachment at the inner wall of the tube. The tube preferably is devoid of
`
`20
`
`retaining frits. In certain embodiments of this aspect of the invention, the monolith is
`
`characterized by having 10,000 to 200,000 theoretical plates per meter. The preferred
`21
`22 monolith has a surface morphology, as determined by scanning electron microscopy,
`
`23
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`24
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`25
`
`26
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`27
`
`28
`
`29
`
`30
`
`31
`
`32
`
`33
`
`34
`
`35
`
`that resembles the surface morphology of octadecyl modified poly(styrene-
`
`divinylbenzene) particles, wherein the surface morphology of the monolith is rugulose.
`
`The preferred monolith comprises an underivatized poly(styrene-divinylbenzene) matrix.
`In a still further aspect, the invention provides a method for separating a mixture
`of polynucleotides. The method includes applying the mixture of polynucleotides to a
`
`polymeric monolith having non-polar chromatographic surfaces and eluting the mixture
`
`of polynucleotides with a mobile phase comprising a counterion agent and an organic
`
`solvent, wherein the monolith has a surface morphology, as determined by scanning
`
`electron microscopy, that resembles the surface morphology of octadecyl modified
`
`poly(styrene-divinylbenzene) particles, wherein the surface morphology of the monolith
`
`is rugulose, and wherein the monolith comprises an underivatized poly(styrene-
`
`divinylbenzene) matrix. The mobile phase preferably is devoid of EDT A. In preferred
`
`embodiments, the monolith can be characterized by one or more of the following: the
`5
`
`7
`
`

`

`WO 01/55713
`
`PCT/USOl/02539
`
`1 monolith is contained within a fused silica tube having an inner diameter in the range of
`2
`1 micrometer to 1 000 micrometer; the monolith is immobilized by covalent attachment at
`
`3
`
`4
`
`5
`
`6
`
`7
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`8
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`9
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`1 0
`
`11
`
`12
`
`the inner wall of the tube; and, the tube is devoid of retaining frits. In preferred
`
`embodiments, the monolith is characterized by having 100,000 to 200,000 theoretical
`
`plates per meter.
`
`In a related aspect, the invention provides a method for separating a mixture of
`
`polynucleotides. In this aspect, the method includes applying the mixture of
`
`polynucleotides to a polymeric monolith having non-polar chromatographic surfaces and
`
`eluting the mixture of polynucleotides with a mobile phase comprising a counterion
`
`agent and an organic solvent, wherein the monolith comprises an underivatized
`
`poly(styrene-divinylbenzene) matrix, wherein the monolith is contained within a fused
`
`silica tube having an inner diameter in the range of 1 micrometer to 1000 micrometer,
`
`13 wherein the monolith is immobilized at the inner wall of the tube, and wherein the tube is
`
`14
`
`15
`
`devoid of retaining frits. Preferred embodiments of this aspect of the invention can
`
`include one or more of the following: the mobile phase is devoid of EDTA; the monolith
`
`is characterized by having 100,000 to 200,000 theoretical plates per meter; and, the
`16
`17 monolith has a surface morphology, as determined by scanning electron microscopy,
`
`18
`
`19
`20
`
`21
`
`that resembles the surface morphology of octadecyl modified poly(styrene-
`
`divinylbenzene) particles, wherein the surface morphology of the monolith is rugulose.
`The monolith can be formed from a polymerization mixture including underivatized
`
`styrene, a crosslinking agent, and a porogen, wherein the porogen comprises
`
`tetrahydrofuran. The method can further include analyzing eluted polynucleotides by
`22
`23 mass spectral analysis.
`
`24
`
`25
`26
`
`27
`
`In an additional aspect, the invention provides a device for separating a mixture
`
`of polynucleotides. The device includes a polymeric monolith having non-polar
`chromatographic surfaces, wherein the monolith is contained within a fused silica tube
`
`having an inner diameter in the range of 1 micrometer to 1000 micrometer, wherein the
`
`28 monolith is immobilized by covalent attachment at the inner wall of the tube, and
`
`29 wherein the monolith comprises an underivatized poly(styrene-divinylbenzene) matrix.
`
`30
`
`31
`
`32
`
`33
`34
`
`Preferred embodiments of this aspect of the invention can be further characterized by
`
`the following: the tube is devoid of retaining frits; the monolith is characterized by having
`
`100,000 to 200,000 theoretical plates per meter. The theoretical plates per meter
`preferably is determined from the retention time of single stranded p(dT)18 standard
`using the following equation:
`
`6
`
`8
`
`

`

`wo 01/55713
`(NIL)= (5.54/ L)(!.J!__J 2
`
`Wo.s
`
`PCT/USOl/02539
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`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. During the isocratic elution the monolith
`
`preferably has a back pressure of 180 to 200 bar, and a flow rate in the range of 2 to 3
`
`IJLI min at an elution temperature of 50°C. Preferred embodiments of the device can be
`
`characterized by one or more of the following: the chromatographic surfaces of the
`
`7 monolith are non-porous; the monolith has channels sufficiently large for convective flow
`
`8
`
`9
`
`10
`
`11
`
`12
`
`of said mobile phase; and, the monolith can be formed from a polymerization mixture
`
`including underivatized styrene, a crosslinking agent, and a porogen, wherein the
`
`porogen comprises tetrahydrofuran.
`
`In a further yet aspect, the invention concerns a device for separating a mixture
`
`of polynucleotides. The device includes a polymeric monolith having non-polar
`
`chromatographic surfaces, wherein the monolith is contained within a fused silica tube,
`13
`14 wherein the monolith is immobilized by covalent attachment at the inner wall of the tube,
`
`and wherein the monolith comprises an underivatized poly(styrene-divinylbenzene)
`15
`16 matrix. Preferred embodiments can include one or more of the following features: the
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`17
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`tube has an inner diameter in the range of 1 micrometer to 1000 micrometer; the tube is
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`devoid of retaining frits; the monolith is characterized by having 10,000 to 200,000
`theoretical plates per meter; the monolith compri'ses an underivatized monolithic
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`stationary phase; the monolith has a surface morph<;>logy, as determined by scanning
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`electron microscopy, that resembles the surface morphology of octadecyl modified
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`poly(styrene-divinylbenzene) particles, wherein the surface morphology of the monolith
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`is rugulose; the chromatographic surfaces of the monolith are non-porous; and the
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`particles have channels sufficiently large for convective flow of the mobile phase. The
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`25 monolith can be formed from a polymerization mixture including underivatized styrene, a
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`26
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`27
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`28
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`29
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`crosslinking agent, and a porogen, wherein the porogen comprises tetrahydrofuran.
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`In another aspect, the invention concerns a device for separating a mixture of
`
`polynucleotides. The device includes a polymeric monolith having non-polar
`
`chromatographic surfaces, wherein the monolith is contained within a fused silica tube,
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`30 wherein the tube is devoid of retaining frits, and wherein the monolith comprises an
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`31
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`32
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`underivatized poly(styrene-divinylbenzene) matrix. Preferred embodiments of this
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`aspect of the invention can further include one or more of the following: the monolith is
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`WO 01/55713
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`immobilized by covalent attachment at the inner wall of said tube; the monolith is
`
`characterized by having 100,000 to 200,000 theoretical plates per meter; the tube has
`
`an inner diameter in the range of 1 micrometer to 1000 micrometer; and, the monolith
`
`has a surface morphology, as determined by scanning electron microscopy, that
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`resembles the surface morphology of octadecyl modified poly(styrene-divinylbenzene)
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`particles, wherein the surface morphology of the monolith is rugulose. The monolith can
`
`be formed from a polymerization mixture including underivatized styrene, a crosslinking
`
`agent, and a porogen, wherein the porogen comprises tetrahydrofuran.
`
`In a related aspect, the invention provides a device for separating a mixture of
`polynucleotides. The device includes a polymeric monolith having non-polar
`
`chromatographic surfaces, wherein the monolith is characterized by having 100,000 to
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`200,000 theoretical plates per meter, wherein the monolith is contained within a fused
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`silica tube having an inner diameter in the range of 1 micrometer to 1000 micrometer,
`and wherein the tube has been silianized. Preferred embodiments of this aspect of the
`
`invention can further include one or more of the following: the monolith is immobilized
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`by covalent attachment at the inner wall of the tube; the tube is devoid of retaining frits;
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`1
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`2
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`3
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`5
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`6
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`9
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`11
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`14
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`15
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`16
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`the monolith comprises an underivatized poly(styrene-divinylbenzene) matrix; and, the
`17
`18 monolith has a surface morphology, as determined by scanning electron microscopy,
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`19
`20
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`21
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`22
`23
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`24
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`25
`26
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`27
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`28
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`29
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`30
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`that resembles the surface morphology of octadecyl modified poly(styrene-
`divinylbenzene) particles, wherein the surface morphology of the monolith is rugulose.
`
`The monolith can be formed from a polymerization mixture including underivatized
`
`styrene, a crosslinking agent, and a porogen, wherein the porogen comprises
`tetrahydrofuran.
`
`In an important aspect, the invention provides a device for separating a mixture
`
`of polynucleotides. The device includes a polymeric monolith having non-polar
`chromatographic surfaces, wherein the monolith comprises an underivatized
`
`poly(styrene-divinylbenzene) matrix, and wherein the monolith is characterized by
`
`having 10,000 to 200,000 theoretical plates per meter. Preferred embodiments of this
`
`aspect of the invention can further include one or more of the following: the monolith is
`
`contained within a tube having an inner diameter in the range of 1 micrometer to 1000
`
`31 micrometer; the monolith is immobilized at the inner wall of the tube; the tube is devoid
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`32
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`33
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`of retaining frits; and, the monolith has a surface morphology, as determined by
`
`scanning electron microscopy, that resembles the surface morphology of octadecyl
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`34 modified poly(styrene-divinylbenzene) particles, wherein the surface morphology of the
`35 monolith is rugulose.
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`WO 01/55713
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`In another aspect, the invention provides a miniaturized chromatographic system
`for separating a mixture of polynucleotides. The device includes a polymeric monolith
`
`having non-polar chromatographic surfaces, wherein the monolith comprises an
`
`underivatized poly(styrene-divinylbenzene) matrix, wherein the monolith is
`
`characterized by having at least 100,000 theoretical plates per meter, wherein the
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`1
`2
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`3
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`4
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`5
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`6 monolith is contained within a tube having an inner diameter in the range of 10
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`7 micrometer to 1000 micrometer, and wherein the monolith is immobilized at the inner
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`8 wall of the tube. Preferred embodiments of this aspect of the invention can further
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`9
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`10
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`include one or more of the following: the tube is devoid of retaining frits; the monolith is
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`contained within a tube having an inner diameter in the range of 1 micrometer to 1000
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`11 micrometer; the monolith has a surface morphology, as determined by scanning
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`12
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`13
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`14
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`electron microscopy, that resembles the surface morphology of octadecyl modified
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`poly(styrene-divinylbenzene) particles, wherein the surface morphology of the monolith
`
`is rugulose; and wherein the monolith has a surface morphology, as determined by
`
`scanning electron microscopy, that resembles the surface morphology of octadecyl
`15
`16 modified poly(styrene-divinylbenzene) particles, wherein the surface morphology of the
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`17 monolith is brush-like. The monolith can be formed from a polymerization mixture
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`18
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`19
`20
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`21
`22
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`including underivatized styrene, a crosslinking agent, and a porogen, wherein the
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`porogen comprises tetrahydrofuran.
`In an additional aspect, the invention concerns a miniaturized chromatographic
`
`system for separating a mixture of polynucleotides. The system preferably includes a
`device which includes a polymeric monolith having non-polar chromatographic surfaces,
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`23 wherein the monolith comprises an underivatized poly(styrene-divinylbenzene) matrix,
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`24 wherein the monolith is characterized by having at least 100,000 theoretical plates per
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`25 meter, wherein the monolith is contained within a tube having an inner diameter in the
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`26
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`27
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`range of 10 micrometer to 1000 micrometer, and wherein the monolith is immobilized at
`
`the inner wall of the tube. In the system, the monolith can be operatively coupled to a
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`28 mass spectrometer.
`29
`30
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`In a further aspect, the invention concerns a device for separating a mixture of
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`31
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`32
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`33
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`polynucleotides. The device includes a polymeric monolith having non-polar
`
`chromatographic surfaces, wherein the monolith has a surface morphology, as
`
`determined by scanning electron microscopy, that resembles the surface morphology of
`
`octadecyl modified poly(styrene-divinylbenzene) particles, wherein the surface
`34
`35 morphology of the monolith is rugulose and brush-like, wherein the monolith is
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`WO 01/55713
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`contained within a fused silica tube having an inner diameter in the range of 1
`1
`2 micrometer to 1000 micrometer, and wherein the monolith is immobilized at the inner
`
`3 wall of said tube. Preferred embodiments of this aspect of the invention can further
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`4
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`5
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`6
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`7
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`8
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`9
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`1 0
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`11
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`12
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`include one or more of the following: the tube is devoid of retaining frits; the monolith is
`
`characterized by having 100,000 to 200,000 theoretical plates per meter; the monolith
`
`comprises an underivatized poly(styrene-divinylbenzene) matrix; and, the surface of
`
`said monolith is non-porous. The monolith can be formed from a polymerization mixture
`
`including underivatized styrene, a crosslinking agent, and a porogen, wherein the
`
`porogen comprises tetrahydrofuran. The polynucleotides can include double-stranded
`
`fragments having lengths in the range of 3 to 2000 base pairs, and preferably 3 to 600
`
`base pairs.
`
`In a final aspect, the invention concerns a chromatographic device: The device
`
`13
`includes a polymeric monolith having non-polar chromatographic surfaces, wherein the
`14 monolith comprises an underivatized poly(styrene-divinylbenzene) matrix, wherein the
`
`15 monolith is characterized by having at least 10,000 theoretical plates per meter, wherein
`
`16
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`17
`18
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`19
`20
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`the monolith is contained within a silanized fused silica tube having an inner diameter in
`
`the range of 10 micrometer to 1000 micrometer, and wherein the monolith is
`immobilized at the inner wall of the tube. Preferred embodiments of this aspect of the
`
`invention can be further characterized by the following: the tube is devoid of retaining
`frits; the monolith is characterized by having 100,000 to 200,000 theoretical plates per
`
`21 meter; the monolith has a surface morphology, as determined by scanning electron
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`22 microscopy, that resembles the surface morphology of octadecyl modified poly(styrene-
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`23
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`24
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`25
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`26
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`27
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`28
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`29
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`30
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`31
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`32
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`divinylbenzene) particles, wherein the surface morphology of the monolith is rugulose;
`
`and wherein the monolith has a surface morphology, as determined by scanning
`
`electron microscopy, that resembles the surface morphology of octadecyl modified
`
`poly(styrene-divinylbenzene) particles, wherein the surface morphology of the monolith
`
`is brush-like. The monolith can be formed from a polymerization mixture including
`
`underivatized styrene, a crosslinking