EP1027121A1 - Milieu chromatographique liquide ameliore utilise dans la separation de polynucleotides - Google Patents

Milieu chromatographique liquide ameliore utilise dans la separation de polynucleotides

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Publication number
EP1027121A1
EP1027121A1 EP98918701A EP98918701A EP1027121A1 EP 1027121 A1 EP1027121 A1 EP 1027121A1 EP 98918701 A EP98918701 A EP 98918701A EP 98918701 A EP98918701 A EP 98918701A EP 1027121 A1 EP1027121 A1 EP 1027121A1
Authority
EP
European Patent Office
Prior art keywords
bead
nonporous
beads
silica
polymer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98918701A
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German (de)
English (en)
Other versions
EP1027121A4 (fr
Inventor
Douglas T. Gjerde
Paul D. Taylor
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Precipio Inc
Original Assignee
Transgenomic Inc
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Filing date
Publication date
Application filed by Transgenomic Inc filed Critical Transgenomic Inc
Publication of EP1027121A1 publication Critical patent/EP1027121A1/fr
Publication of EP1027121A4 publication Critical patent/EP1027121A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/328—Polymers on the carrier being further modified
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/287—Non-polar phases; Reversed phases
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3225—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating involving a post-treatment of the coated or impregnated product
    • B01J20/3227—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating involving a post-treatment of the coated or impregnated product by end-capping, i.e. with or after the introduction of functional or ligand groups
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds

Definitions

  • the present invention is directed to the separation of polynucleotides using nonporous beads. More specifically, the invention is directed to the chromatographic separation of both single stranded and double stranded polynucleotides by chromatography using chromatographic columns containing nonporous beads, where the beads comprise either organic or inorganic particles which are coated with a polymer, or non-polar substituted polymer, and/or which have substantially all surface substrate groups substituted with a non-polar hydrocarbon or non-ionic substituted hydrocarbon.
  • Silica-based columns are by far the most common LC columns. Of these, reverse phase silica-based columns are preferred because they have high separation efficiencies, are mechanically stable, and a variety of functional groups can be easily attached for a variety of column selectivities. Although silica-based reverse phase column materials have performed adequately for separating single stranded DNA, these materials have not performed well for separating double stranded DNA. The peaks from double stranded DNA separations using silica-based materials are badly shaped or broad, or the double stranded DNA may not even elute. Separations can take up to several hours, or the resolution, peak symmetry, and sensitivity of the separation are poor.
  • one object of the present invention is to provide a chromatographic method for separating polynucleotides with improved separation and efficiency.
  • the method comprises flowing a mixture of polynucleotides having up to 1500 base pairs through a separation column containing beads having an average diameter of 0.5 to 100 microns, and separating the mixture of polynucleotides.
  • the beads comprise nonporous particles coated with a hydrocarbon or non-polar substituted polymer or having substantially all surface substrate groups reacted with a non-polar hydrocarbon or substituted hydrocarbon group. Precautions are taken during the production of the beads so that they are substantially free of multivalent cation contaminants and the beads are treated, for example by an acid wash treatment, to remove any residual surface metal contaminants.
  • the beads are characterized by having a DNA Separation Factor (as defined hereinbelow) of at least 0.05.
  • the beads are characterized by having a DNA Separation Factor of at least 0.5.
  • the separation is preferably by Matched Ion Polynucleotide Chromatography (MIPC) as defined hereinbelow.
  • MIPC Matched Ion Polynucleotide Chromatography
  • the beads preferably have an average diameter of about 1 - 5 microns.
  • the nonporous particle is preferably selected from silica, silica carbide, silica nitrite, titanium oxide,
  • the nonporous particle is most preferably silica, which preferably is substantially free from unreacted silanol groups.
  • the particles can be prepared by non-covalently bonded coatings, covalently bonded coatings, or reaction of the silanol groups with hydrocarbon groups.
  • the nonporous particle can be coated with a polymer.
  • the polymer is
  • polystyrenes preferably selected from polystyrenes, polymethacrylates, polyethylenes, polyurethanes, polypropylenes, polyamides, cellulose, polydimethyl siloxane, and polydialkyl siloxane.
  • the polymer is optionally unsubstituted or substituted with hydrocarbon groups or other groups having nonionic substituents.
  • the polymer can be optionally substituted with hydrocarbon groups having from 1 to 1 ,000,000 carbons, the hydrocarbon groups optionally being alkyl groups with from 1 to 100 carbons and preferably from 1 to 24 carbons. Hydrocarbon groups from 24 to 1 ,000,000 are described herein as hydrocarbon polymers and have the constituency of hydrocarbon groups as defined herein.
  • organosilanols e.g. HO-Si-R 3
  • alkoxy- e.g., RO-Si- R 3
  • the reaction of organosilanols (e.g. HO-Si-R 3 ) or alkoxy- (e.g., RO-Si- R 3 ) silanes with silica supports without polymerization can also produce good packings.
  • the method produces a dense monolayer of functional groups of alkyl or alkylsubstituted, ester, cyano, and other nonionic groups.
  • monofunctional dimethyl silanes X-Si(CH 3 ) 2 -R
  • Monochlorosilane reagents are preferred, if the required organic functionality can be prepared.
  • the nonporous particle is preferably endcapped with a tri(lower alkyl)chlorosilane (preferably a trimethylchlorosilane) to block residual reactive silanol sites following the coating or hydrocarbon substitution. Alternatively, all of the silanol sites can be reacted with an excess of the endcapping reagent to extinguish all reactive silanol groups. Endcapping of the nonporous particle can be effected by reaction of the nonporous particle with the corresponding hydrocarbon substituted silane, such as trialkyl chlorosilane (eg. trimethyl chlorosilane) or by reaction with the corresponding hydrocarbon substituted disilazane, such as dichloro-tetraalkyl-disilazane (eg. dichloro-tetramethyl- disilazane).
  • a hydrocarbon substituted silane such as trialkyl chlorosilane (eg. trimethyl chlorosilane)
  • disilazane such as dichloro
  • the method of the present invention can be used to separate double stranded polynucleotides having up to about 1500 to 2000 base pairs. In many cases, the method is used to separate polynucleotides having up to 600 bases or base pairs, or which have up to 5 to 80 bases or base pairs.
  • the method is performed at a temperature within the range of 20°C to 90°C to yield a back-pressure not greater than 10,000 psi.
  • the method also preferably employs an organic solvent that is water soluble.
  • the solvent is preferably selected from the group consisting of alcohols, nitriles, dimethylformamide, esters, and ethers.
  • the method also preferably employs a counter ion agent selected from trialkylamine acetate, trialkylamine carbonate, and trialkylamine phosphate.
  • the most preferred counter ion agent is triethylammonium acetate or triethylammonium hexafluoroisopropyl alcohol.
  • the method also preferably comprises supplying and feeding solutions entering the separation column with components having process solution- contacting surfaces which contact process solutions held therein or flowing therethrough.
  • the process solution-contacting surfaces are material which does not release multivalent cations into aqueous solutions held therein or flowing therethrough, so that the column and its contents are protected from multivalent cation contamination
  • the process solution-contacting surfaces are preferably material selected from the group consisting of titanium, coated stainless steel, passivated stainless steel, and organic polymer. Multivalent cations in eluent solutions and sample solutions entering the column are also preferably removed by contacting the solutions with multivalent cation capture resin before the solutions enter the column so as to protect the resin bed from multivalent cation contamination.
  • the multivalent capture resin is selected from cation exchange resin and chelating resin.
  • the column and process solutions held therein or flowing therethrough are preferably substantially free of multivalent cation contaminants.
  • the polynucleotides are separated by Matched Ion Polynucleotide Chromatography.
  • Also disclosed herein is a method for separating a mixture of polynucleotides, comprising flowing a mixture of polynucleotides having up to 1500 base pairs through a separation column containing beads having an average diameter of 0.5 to 100 microns, and separating the mixture of polynucleotides by Matched Ion Polynucleotide Chromatography.
  • the beads comprise nonporous particles coated with a polymer or having substantially all surface substrate groups reacted and/or endcapped with a non-polar hydrocarbon or substituted hydrocarbon group.
  • the beads are characterized by having a DNA Separation Factor of at least 0.05.
  • the column and process solutions held therein or flowing therethrough are substantially free of multivalent cation contaminants.
  • the method is performed at a temperature within the range of 20°C to 90°C to yield a back-pressure not greater than 10,000 psi.
  • An organic solvent that is water soluble is used in the performance of the method.
  • a bead comprising a nonporous particle coated with a polymer.
  • the bead has an average diameter of 0.5 to 100 microns and is characterized by having a DNA Separation Factor of at least 0.05. In a preferred embodiment, the bead is characterized by having a DNA Separation Factor of at least 0.5.
  • the bead preferably has a diameter of about 1 - 5 microns.
  • the nonporous particle is preferably selected from silica, silica carbide, silica nitrite, titanium oxide, aluminum oxide, zirconium oxide, carbon, insoluble polysaccharides such as cellulose, and diatomaceous earth, or any of these materials that have been modified to be nonporous.
  • the nonporous particle is most preferably silica, which preferably has minimum silanol groups.
  • the polymer is preferably selected from polystyrene, polymethacrylate, polyethylene, polyurethane, polypropylene, polyamide, cellulose, polydimethyl siloxane, and polydialkyl siloxane, and is preferably unsubstituted, alkylated, or alkyl or aryi substituted, or alkylated with a substituted alkyl group methyl-substituted, or ethyl-substituted.
  • the polymer can be alkylated with alkyl groups having 1 - 22 carbon atoms, preferably, 8 - 18 carbon atoms.
  • a bead comprising a nonporous particle having substantially all surface substrate groups reacted with a hydrocarbon group and then endcapped with a non-polar hydrocarbon or substituted hydrocarbon group, preferably a tri(lower alkyl)chlorosilane or tetra(lower alkyl)dichlorodisilazane.
  • the bead has an average diameter of 0.5 to 100 microns and is characterized by having a DNA Separation Factor of at least 0.05.
  • the bead preferably has a diameter of about 1 - 5 microns.
  • the nonporous particle is preferably selected from silica, silica carbide, silica nitrite, titanium oxide, aluminum oxide, zirconium oxide, carbon, insoluble polysaccharides such as cellulose, and diatomaceous earth, or any of these materials that have been modified to be nonporous.
  • the nonporous particle is most preferably silica, which preferably has minimum silanol groups. Endcapping of the nonporous particle can be effected by reaction of the nonporous particle with trimethyl chlorosilane or dichloro-tetraisopropyl- disilazane.
  • FIG. 1 is a schematic representation of how the DNA Separation
  • FIG. 2 is a schematic drawing of a cross-section of a representation of a reverse phase bead with a silica core and endcapping shielding.
  • FIG. 3 is a schematic drawing of a cross-section of a representation of a reverse phase bead with a silica core and polymer shielding.
  • FIG. 4 is a MIPC separation of pUC18 DNA-Haelll digestion fragments on a column containing alkylated poly(styrene-divinylbenzene) beads. Peaks are labeled with the number of base pairs of the eluted fragment.
  • FIG. 5 is a MIPC separation of pUC18 DNA-Haelll digestion fragments on a column containing nonporous 2.1 micron beads of underivatized poly(styrene-divinylbenzene).
  • FIG. 6 is a Van't Hoff plot of the retention factor 1/T /°K " '7with alkylated poly(styrene-divinylbenzene) beads showing positive enthalpy using acetonitrile as the solvent.
  • FIG. 7 is a Van't Hoff plot of the retention factor 1/T /°/ " '; with underivatized poly(styrene-divinylbenzene) beads showing positive enthalpy using acetonitrile as the solvent.
  • FIG. 8 is a Van't Hoff plot of the retention factor 1/Tj°K 1 ] with alkylated poly(styrene-divinylbenzene) beads showing negative enthalpy using methanol as the solvent.
  • FIG. 9 is a separation using alkylated beads and acetonitrile as solvent.
  • FIG. 10 is a separation using alkylated beads and 50.0% methanol as the solvent.
  • FIG. 11 is a separation using alkylated beads and 25.0% ethanol as the solvent.
  • FIG. 12 is a separation using alkylated beads and 25.0% vodka (Stolichnaya, 100 proof) as the solvent.
  • FIG. 13 is a separation using alkylated beads and 25.0% 1-propanol as the solvent.
  • FIG. 14 is a separation using alkylated beads and 25.0% 1-propanol as the solvent.
  • FIG. 15 is a separation using alkylated beads and 10.0% 2-propanol as the solvent.
  • FIG. 16 is a separation using alkylated beads and 10.0% 2-propanol as the solvent.
  • FIG. 17 is a separation using alkylated beads and 25.0% THF as the solvent.
  • FIG. 18 is an isocratic/gradient separation on non-alkylated poly(styrene-divinylbenzene) beads.
  • the subject matter of the present invention is the separation of polynucleotides by Matched Ion Polynucleotide Chromatography utilizing columns filled with nonporous beads having an average diameter of about 0.5 -100 microns; preferably, 1 - 10 microns; more preferably, 1 - 5 microns. Beads having an average diameter of 1.0 - 3.0 microns are most preferred.
  • MIPC Matched Ion Polynucleotide Chromatography
  • the beads are characterized as having a DNA Separation Factor of at least 0.5.
  • the performance of the beads of the present invention is demonstrated by high efficiency separation by MIPC of double stranded and single stranded DNA. We have found that the best criterion for measuring performance of the beads is a DNA Separation Factor. This is measured as the resolution of 257- and 267-base pair double stranded DNA fragments of a pUC18 DNA- Hae ⁇ restriction digest wherein the distance from the valley between the peaks to the top of one of the peaks, over the distance from the baseline to the valley. Referring to the schematic representation of FIG.
  • the DNA Separation Factor is determined by measuring the distance "a” from the baseline to the valley “e” between the peaks “b” and “c” and the distance “d” from the valley “e” to the top of one of the peaks “b” or “c”. If the peak heights are unequal, the highest peak is used to obtain “d.”
  • the DNA Separation Factor is the ratio of d/(a+d).
  • the peaks of 257- and 267-base pairs in this schematic representation are similar in height.
  • Operational beads of the present invention have a DNA Separation Factor of at least 0.05.
  • Preferred beads have a DNA Separation Factor of at least 0.5. In an optimal embodiment, the beads have a DNA Separation Factor of at least 0.95.
  • the beads which conform to the DNA Separation Factor as specified herein have a pore size which essentially excludes the polynucleotides being separated from entering the bead.
  • the term "nonporous" is defined to denote a bead which has surface pores having a diameter that is less than the size and shape of the smallest DNA fragment in the separation in the solvent medium used therein. Included in this definition are beads having these specified maximum size restrictions in their natural state or which have been treated to reduce their pore size to meet the maximum effective pore size required.
  • all beads which provide a DNA Separation Factor of at least 0.05 are intended to be included within the definition of "nonporous" beads.
  • the surface conformations of nonporous beads of the present invention can include depressions and shallow pit-like structures which do not interfere with the separation process.
  • a pretreatment of a porous bead to render it nonporous can be effected with any material which will fill the pores in the bead structure and which does not significantly interfere with the MIPC process.
  • Pores are open structures through which eluent and other materials can enter the bead structure. Pores are often interconnected so that fluid entering one pore can exit from another pore. We believe that pores having dimensions that allow movement of the polynucleotide into the interconnected pore structure and into the bead impair the resolution of separations or result separations that have very long retention times. In MIPC, however, the beads are "nonporous" and the polynucleotides do not enter the bead structure.
  • polynucleotide is defined as a linear polymer containing an indefinite number of nucleotides, linked from one ribose (or deoxyribose) to another via phosphoric residues.
  • the present invention can be used in the separation of RNA or of double- or single-stranded DNA. For purposes of simplifying the description of the invention, and not by way of limitation, the separation of double-stranded DNA will be described in the examples herein, it being understood that all polynucleotides are intended to be included within the scope of this invention.
  • Chromatographic efficiency of the column beads is predominantly influenced by the properties of surface and near-surface areas. For this reason, the following descriptions are related specifically to the close-to-the- surface region of the polymeric beads.
  • the main body and/or the center of such beads can exhibit entirely different chemistries and sets of physical properties from those observed at or near the surface of the polymeric beads of the present invention.
  • the beads of the invention comprise a nonporous particle which has non-polar molecules or a non-polar polymer attached to or coated on its surface.
  • the beads comprise nonporous particles which have been coated with a polymer or which have substantially all surface substrate groups reacted with a non-polar hydrocarbon or substituted hydrocarbon group, and any remaining surface substrate groups endcapped with a tri(lower alkyl)chlorosilane or tetra(lower alkyl)dichlorodisilazane as described above.
  • the nonporous particle is preferably an inorganic particle, but can be a nonporous organic particle.
  • the nonporous particle can be, for example, silica, silica carbide, silica nitrite, titanium oxide, aluminum oxide, zirconium oxide, carbon, insoluble polysaccharides such as cellulose, or diatomaceous earth, or any of these materials which have been modified to be nonporous.
  • Examples of carbon particles include diamond and graphite which have been treated to remove any interfering contaminants.
  • the preferred particles are essentially non-deformable and can withstand high pressures.
  • the nonporous particle is prepared by known procedures.
  • the preferred particle size is about 0.5 -100 microns; preferably, 1 - 10 microns; more preferably, 1 - 5 microns. Beads having an average diameter of 1.0 - 3.0 microns are most preferred.
  • the nonporous beads of the invention are characterized by having minimum exposed silanol groups after reaction with the coating or alkylation reagents.
  • Minimum silanol groups are needed to reduce the interaction of the DNA with the substrate and also to improve the stability of the material in a high pH and aqueous environment.
  • Silanol groups can be harmful because they can repel the negative charge of the DNA molecule, preventing or limiting the interaction of the DNA with the stationary phase of the column.
  • Another possible mechanism of interaction is that the silanol can act as ion exchange sites, taking up metals such as iron (III) or chromium (III). Iron (III) or other metals which are trapped on the column can distort the DNA peaks or even prevent DNA from being eluted from the column.
  • Silanol groups can be hydrolyzed by the aqueous-based eluent.
  • Hydrolysis will increase the polarity and reactivity of the stationary phase by exposing more silanol sites, or by exposing metals that can be present in the silica core. Hydrolysis will be more prevalent with increased silanol groups.
  • the effect of silanol groups on the DNA separation depends on which mechanism of interference is most prevalent. For example, iron (III) can become attached to the exposed silanol sites, depending on whether the iron (III) is present in the eluent, instrument or sample.
  • metals can only occur if metals are already present within the system or reagents. Metals present within the system or reagents can get trapped by ion exchange sites on the silica. However, if no metals are present within the system or reagents, then the silanol groups themselves can cause interference with DNA separations. Hydrolysis of the exposed silanol sites by the aqueous environment can expose metals that might be present in the silica core. Fully hydrolyzed silica contains a concentration of about 8 ⁇ moles of silanol groups per square meter of surface.
  • silanol groups At best, because of steric considerations, a maximum of about 4.5 ⁇ moles of silanol groups per square meter can be reacted, the remainder of the silanol being sterically shielded by the reacted groups. Minimum silanol groups is defined as reaching the theoretical limit of or having sufficient shield to prevent silanol groups from interfering with the separation.
  • Nonporous silica core particles Numerous methods exist for forming nonporous silica core particles. For example, sodium silicate solution poured into methanol will produce a suspension of finely divided spherical particles of sodium silicate. These particles are neutralized by reaction with acid. In this way, globular particles of silica gel are obtained having a diameter of about 1 - 2 microns.
  • Silica can be precipitated from organic liquids or from a vapor. At high temperature (about 2000°C), silica is vaporized, and the vapors can be condensed to form finely divided silica either by a reduction in temperature or by using an oxidizing gas. The synthesis and properties of silica are described by R. K. Her in The Chemistry of Silica, Solubility, Polymerization, Colloid and Surface Properties, and Biochemistry, John Wiley & Sons:New York (1979).
  • St ⁇ ber et al. described controlled growth of monodisperse silica spheres in the micron size range in J. Colloid and Interface Sci., 26:62-69 (1968).
  • St ⁇ ber et al. describe a system of chemical reactions which permit the controlled growth of spherical silica particles of uniform size by means of hydrolysis of alkyl silicates and subsequent condensation of silicic acid in alcoholic solutions.
  • Ammonia is used as a morphological catalyst. Particle sizes obtained in suspension range from less than 0.05 ⁇ m to 2 ⁇ m in diameter.
  • Nonporous silica core beads can be obtained from Micra Scientific (Northbrook, IL) and from Chemie Uetikkon (Lausanne, Switzerland).
  • the nonporous particle is coated with a polymer or reacted and endcapped so that substantially all surface substrate groups of the nonporous particle are blocked with a non-polar hydrocarbon or substituted hydrocarbon group. This can be accomplished by several methods.
  • the organic bonded-phase siloxane coating can be made as a monomolecular layer or as a polymerized multilayer coating.
  • Packings with so-called monomolecular organic layers are normally prepared by reacting the surface silanol groups of siliceous-base particles with mono-, di-, or trifunctional chloro-, dimethyl-, amino-, siloxy-, or alkoxy-silanes.
  • FIG. 2 is a schematic representation of a bead 20 having a silica core 22 and a monomolecular organic layer. (The figure does not necessarily reflect the morphology or pore structure of the beads of the invention and is meant for illustrative purposes only.)
  • organosilanols e.g., HO-Si-fi j
  • organoalkoxy- e.g.,
  • RO-S -R 3 silanes with silica supports without polymerization can also produce good packings. These reactions are relatively reproducible, provided that traces of water or other reactive species are absent. Unreacted, accessible silanols can be left after the initial reaction, but these can be removed by capping of the packing with chlorotrimethylsilane (providing the R groups do not react with the latter silane).
  • the nonporous particle is coated with a polymer coating.
  • Suitable polymers for use in coating the particle include chain reaction polymers and step reaction polymers, for example, polystyrene, polymethacrylate, polyethylene, polyurethane, polypropylene, polyamide, insoluble polysaccharides such as cellulose, polydimethyl siloxane, polydialkyi siloxane, and related materials.
  • the polymer coating can be attached to the nonporous particle by means of a multi-coating process so that complete shielding of the surface is achieved.
  • FIG. 3 is a schematic illustration of a coated bead 30 having a silica core 32 and polymer coating 34. (The figure does not necessarily reflect the morphology or pore structure of the beads of the invention and is meant for illustrative purposes only.)
  • An alternative method comprises a combination of covalent bonding with a vinyl-containing silane molecule and then polymerizing a coating on the surface of the particles.
  • a second coating can be applied if residual silanol groups or metal groups are present.
  • the silica surface is first modified by reaction with vinyltrichlorosilane, followed by polymerizing acrylic acid derivatives to and over the derivatized silica surface.
  • silica since the general approach does not depend on the chemistry of the underlying substrate, materials other than silica, for example, alumina and zirconia, can be modified and used under conditions for which silica is unsuitable, for example, with mobile phases outside the pH range 2 - 7.5.
  • presilanization decreases the number of active silanol groups, which are then further shielded by the polymeric film anchored over the surface.
  • reverse phase liquid chromatography these packings have shown improved chromatographic properties compared to monomeric, chemically bonded phases for the separation of basic solutes.
  • Polymer-encapsulated packings have a film thickness of about 1 nm to maintain reasonable mass transfer characteristics. A description of the this procedure has been published by H. Engelhart et al. (Chromatographia, 27:535 (1989)).
  • hydrocarbon as used herein is defined to include alkyl and alkyl substituted aryl groups, having from 1 to 1 ,000,000 carbons, the alkyl groups including straight chained, branch chained, cyclic, saturated, unsaturated nonionic functional groups of various types including, aldehyde, ketone, ester, ether, alkyl groups, and the like, and the aryl groups including as monocyclic, bicyclic, and tricyclic aromatic hydrocarbon groups including phenyl, naphthyl, and the like.
  • hydrocarbon substitution is conventional and well- known in the art and are not an aspect of this invention.
  • the hydrocarbon can also contain hydroxy, cyano, nitro groups, or the like which are considered to be non-polar, reverse phase functional groups.
  • the preferred hydrocarbon groups are alkyl groups, and the description of suitable substitution processes hereinbelow are presented as alkylation for purposes of simplification and not by way of limitation, it being understood that aryl substitution by conventional procedures are also intended to be included within the scope of this invention.
  • the polymer-coated beads can be alkylated by reaction with the corresponding alkyl halide such as the alkyl iodide.
  • Alkylation is achieved by mixing the polymer-coated beads with an alkyl halide in the presence of a Friedel-Crafts catalyst to effect electrophilic aromatic substitution on the aromatic rings at the surface of the polymer blend.
  • Suitable Friedel-Crafts catalysts are well-known in the art and include Lewis acids such as aluminum chloride, boron trifluoride, tin tetrachloride, etc.
  • Substitution with hydrocarbon groups having from 1 to 1 ,000,000 and preferably from 1 to 22 carbons can be effected by these processes. Hydrocarbon groups having from 23 to 1 ,000,000 carbons are referenced herein as hydrocarbon polymers.
  • Alkylation can be accomplished by a number of known synthesis procedures. These include Friedel-Crafts alkylation with an alkyl halide, attachment of an alkyl alcohol to a chloromethylated bead to form an ether, etc.
  • an alternative method of alkylation is to polymerize alkylated monomers to form an alkylated polymer coating on the nonporous particle.
  • the monomers will be substituted with alkyl groups having any number of carbon atoms, for example, from 1 to 100, 1 to 50 or 1 to 24, for example, depending upon the requirements of the separation variables.
  • the nonporous particle can be functionalized with an alkyl group or other non-polar functional group including cyano, ester, and other non-ionic groups, followed by a complete endcapping process to reduce silanol and metal interaction. Endcapping of the nonporous particle can be achieved by reacting the particle with trialkyl chlorosilane or tetraalkyl dichlorodisilazane, such as, for example, trimethyl chlorosilane or dichloro-tetraisopropyl-disilazane.
  • the rate and extent of the bonding reaction depends on the reactivity of the silane, choice of solvent and catalyst, time, temperature, and the ratio of reagents to substrate.
  • Reactive organosilanes with Cl, OH, OR, N(CH 3 ) 2 , OCOCF 3 , and enolates as leaving groups have been widely used.
  • the dimethylamine, trifluoroacetate, and enol ethers of pentane-2,4-dione are the most reactive leaving groups, although economy, availability, and familiarity result in the chlorosilanes and alkoxysilanes being the most widely used, particularly among commercial manufacturers.
  • reaction times tend to be long (12 - 72 hours), reaction temperatures moderately high (in most cases, around 100°C) and, in the case of chlorosilanes, an acid acceptor catalyst (e.g., pyridine) is used.
  • an acid acceptor catalyst e.g., pyridine
  • solvents employed are toluene and xylene, although other solvents, such as carbon tetrachloride, trichloroethane, and dimethylformamide (DMF), have been recommended as being superior. Since the bonding reactions are carried out by refluxing in an inert atmosphere, solvents are often selected based on their capacity to be a good solvent for the organosilanes and to attain the desired reaction temperature at reflux. Except for 3-cyanopropylsiloxane bonded phases, the high reactivity of chlorosilanes towards certain polar functional groups (e.g., OH, etc.) precludes the use of these groups for the preparation of polar, reverse phase bonded phases.
  • polar functional groups e.g., OH, etc.
  • Alkoxysilanes containing acidic or basic functional groups are autocatalytic and the bonded phases are usually prepared by refluxing the silane in an inert solvent at a temperature high enough to distill off the alcohol formed by the condensation reaction with the surface silanol groups.
  • Bonding of neutral, polar ligands generally requires the addition of a catalyst, such as toluene-4-sulfonic acid or triethylamine, in the presence of sufficient water to generate monolayer coverage of the silica.
  • a catalyst such as toluene-4-sulfonic acid or triethylamine
  • bonded phases are prepared from organosilanes containing a single functionalized ligand bonded to silicon, with the remaining groups being leaving groups and/or methyl groups, more highly substituted organosilanes can also be used.
  • Bifunctional organosilanes such as 1 ,3- dichlorotetraisopropyldisilazane, are able to react with surface silanol groups at both ends of the chain, forming a bonded phase that is more hydrolytically stable than bonded phases formed from conventional organosilanes.
  • the bidentate organosilanes have reactive sites that more closely match the spacing of the silanol groups on the silica surface and provide a higher bonded phase coverage than is achieved with dichlorosilanes with both leaving groups attached to the same silicon atom.
  • increasing the length of the alkyl group increases the hydrolytic stability of the bonded phase relative to that of the trimethylsilyl bonded ligands.
  • Increasing the chain length of the methyl groups increases the hydrolytic stability of the bonded phase, but reduces the phase coverage due to steric effects.
  • the use of monofunctional organosilanes containing one or two bulky groups, for example, isopropyl or t-butyl, on the silicon atom of the silane can become more important in the preparation of bonded phases for use at low pH.
  • the bulky alkyl groups provide better steric protection to the hydrolytically sensitive siloxane groups on the packing surface than does the methyl group.
  • the general process of coating and endcapping of a silica substrate is well-known technology. However, the general understanding of those who have used these materials is they are not suitable for high performance double stranded DNA separations.
  • the beads of this invention are formed by a more careful application of the coating and end-capping procedures to effect a thorough shielding of the silica core, the resulting beads having the ability to perform rapid separations of both single stranded and double stranded DNA which are equal to or better than those achieved using the alkylated nonporous polymer beads disclosed in U.S. Patent No. 5,585,236, for example.
  • the beads of the invention are also characterized by having low amounts of metal contaminants or other contaminants that can bind DNA.
  • the preferred beads of the present invention are characterized by having been subjected to precautions during production, including a decontamination treatment, such as an acid wash treatment, designed to substantially eliminate any multivalent cation contaminants (e.g. Fe(lll), Cr(lll), or colloidal metal contaminants). Only very pure, non-metal containing materials should be used in the production of the beads in order that the resulting beads will have minimum metal content.
  • chromatographic column To achieve high resolution chromatographic separations of polynucleotides, it is generally necessary to tightly pack the chromatographic column with the solid phase nonporous beads. Any known method of packing the column with a column packing material can be used in the present invention to obtain adequate high resolution separations.
  • a slurry of the beads is prepared using a solvent having a density equal to or less than the density of the beads.
  • the column is then filled with the bead slurry and vibrated or agitated to improve the packing density of the beads in the column. Mechanical vibration or sonification are typically used to improve packing density.
  • the separation method of the invention is generally applicable to the chromatographic separation of single stranded and double stranded polynucleotides of DNA and RNA.
  • Samples containing mixtures of polynucleotides can result from total synthesis of polynucleotides, cleavage of DNA or RNA with restriction endonucleases or with other enzymes or chemicals, as well as polynucleotide samples which have been multiplied and amplified using polymerase chain reaction techniques.
  • the method of the present invention can be used to separate double stranded polynucleotides having up to about 1500 to 2000 base pairs. In many cases, the method is used to separate polynucleotides having up to 600 bases or base pairs, or which have up to 5 to 80 bases or base pairs.
  • the separation is by MIPC.
  • the nonporous beads of the invention are used as a reverse phase material that will function with counter ion agents and a solvent gradient to produce the DNA separations.
  • the DNA fragments are matched with a counter ion agent and then subjected to reverse phase chromatography using the nonporous beads of the present invention.
  • Counter ion agents that are volatile such as trialkylamine acetate, trialkylamine carbonate, trialkylamine phosphate, etc., are preferred for use in the method of the invention, with triethylammonium acetate (TEAA) and triethylammonium hexafluoroisopropyl alcohol being most preferred.
  • TEAA triethylammonium acetate
  • TEAA triethylammonium hexafluoroisopropyl alcohol
  • the method is performed at a temperature within the range of 20°C to 90°C to yield a back-pressure not greater than 10,000 psi.
  • separation of single-stranded fragments should be performed at higher temperatures.
  • an organic solvent that is water soluble is preferably used, for example, alcohols, nitriles, dimethylformamide (DMF), esters, and ethers.
  • Water soluble solvents are defined as those which exist as a single phase with aqueous systems under all conditions of operation of the present invention.
  • Solvents which are particularly preferred for use in the method of this invention include methanol, ethanol, 1-propanol, 2-propanol, tetrahydrofuran (THF), and acetonitrile, with acetonitrile being most preferred.
  • the partition coefficient (K) and the retention factor (k) are related through the following equations:
  • phase volume ratio ( ⁇ ) is known.
  • silica beads coated with poly(styrene-divinylbenzene) also give a negative slope for a plot of In k versus 1/T, although the plot is slightly curved.
  • the retention factor k decreases with increasing temperature, indicating the retention mechanism is an exothermic process ( ⁇ H sorp ⁇ 0).
  • thermodynamic data reflect the relative affinity of the DNA-counter ion agent complex for the beads of the invention and the elution solvent.
  • An endothermic plot indicates a preference of the DNA complex for the bead.
  • An exothermic plot indicates a preference of the DNA complex for the solvent over the bead.
  • the plots shown herein are for alkylated and non-alkylated surfaces as described in the Examples. Most liquid chromatographic separations show exothermic plots.
  • the separation column and all process solutions held within the column or flowing through the column should be substantially free of multivalent cation contaminants (e.g. Fe(lll), Cr(lll), and colloidal metal contaminants).
  • the process solution-contacting surfaces of the system components are preferably material selected from the group consisting of titanium, coated stainless steel, passivated stainless steel, and organic polymer. Metals found in stainless steel, for example, do not harm the separation, unless they are in an oxidized or colloidal partially oxidized state. For example, 316 stainless steel frits are acceptable in column hardware, but surface oxidized stainless steel frits harm the DNA separation.
  • multivalent cations in eluent solutions and sample solutions entering the column can be removed by contacting these solutions with multivalent cation capture resin before the solutions enter the column to protect the resin bed from multivalent cation contamination.
  • the multivalent capture resin is preferably cation exchange resin and/or chelating resin.
  • EXAMPLE 1 C-18 Bonded Phase Standard Phase To a 1000-mL round bottomed flask, add 200 g of nonporous, 2 ⁇ m silica and one small stirring egg. Transfer flask with silica to an oven and heat at 125°C overnight (i.e., at least 8 hours). Have heating mantle and condenser set up.
  • the C-18 bonding reagent n-octadecyldimethylsilane
  • n-octadecyldimethylsilane is a waxy white solid to semi-solid at room temperature.
  • a heat gun note: pressure can build up in stored chlorosilane bottles, and they should be handled as if they were HCI, as upon contact with moisture, HCI is the side product).
  • EXAMPLE 2 CN Bonded Phase, Cyano Phase
  • a 1000-mL round-bottomed flask add 200 g of nonporous, 2 ⁇ m silica, one stirring egg, and place in an oven at 125°C overnight (i.e., at least 8 hours) to dry.
  • To the dried silica add 100 mL of the 3- cyanopropylmethyldichlorosilane, 10 mL of chloroform, 450 mL of toluene, and 50 mL of pyridine. Suspend the mixture and bring to reflux for 15 hours.
  • endcap by adding 20 mL of trimethylchlorosilane, 6 mL of hexamethyl-disilane, 350 mL of toluene, 10 mL of chloroform, and 25 mL of pyridine to the dried bonded phase, and bring to reflux for 6 hours. Cool the resulting mixture, transfer to a Buchner funnel, and wash with three 200-mL aliquots of methanol, followed by three 200-mL aliquots of acetone. Air dry for at least 0.5 hour, and then dry in the oven at 100°C overnight. Submit a sample for elemental analysis. The bonded phase is now ready for column packing.
  • Example 4 Acid Wash Treatment The procedures of Example 1 are repeated but the silica is washed with 500 mL of 100 mM HCI and then water prior to drying. The product is washed with 500 mL of 100 mM HCI after cooling and prior to the methanol wash.
  • EXAMPLE 5 The product of Example 1 is coated with 100 mL of dichloromethane containing 1 gram of divinylbenzene and 10 mg of benzoylperoxide. The dichloromethane is removed by rotary evaporation until the monomer is coated onto the beads. While rotating very slowly, the temperature is increased to 70°C for 8 hours. The product is washed with methanol. This procedure is repeated with the product of Example 4.
  • EXAMPLE 6 The procedure of Example 5 is repeated with stearyldivinyl benzene in place of divinylbenzene. This procedure is repeated with the product of Example 4.
  • EXAMPLE 7 The procedure of Example 5 is repeated with stearyldivinyl benzene in place of divinylbenzene. This procedure is repeated with the product of Example 4.
  • acetonitrile containing 2 g of a vinyl monomer (divinylbenzene, styrene, acrylonitrile, acrylic acid, butyl methacrylate, or 2-hydroxy methacrylate) are added and the mixture well dispersed.
  • a vinyl monomer divininylbenzene, styrene, acrylonitrile, acrylic acid, butyl methacrylate, or 2-hydroxy methacrylate
  • the products are extracted with acetonitrile and then acetone to remove unreacted monomers and oligomers from the particle.
  • the packing materials are dried at 80°C prior to packing.
  • EXAMPLE 8 Standard Procedure for Testing the Performance of Separation Media Separation particles are packed in an HPLC column and tested for their ability to separate a standard DNA mixture.
  • the standard mixture is a pUC18 DNA-Haelll digest (Sigma-Aldrich, D6293) which contains 11 fragments having 11 , 18, 80, 102, 174, 257, 267, 298, 434, 458, and 587 base pairs, respectively.
  • the standard is diluted with water and five ⁇ L,
  • a counter ion agent is selected from trialkylamine acetate, trialkylamine carbonate, trialkylamine phosphate, or any other type of cation that can form a matched ion with the polynucleotide anion.
  • FIG. 4 shows the high resolution of the standard DNA mixture using octadecyl modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads.
  • the separation was conducted under the following conditions: Eluent A: 0.1 M TEAA, pH 7.0; Eluent B: 0.1 M TEAA, 25% acetonitrile; Gradient:
  • the flow rate was 0.75 mlJmin, detection UV at 260 nm, column temp. 50°C.
  • the pH was 7.0.
  • FIG. 5 is a high resolution separation of the standard DNA mixture on a column containing nonporous 2.1 micron beads of underivatized poly(styrene-divinylbenzene).
  • EXAMPLE 9 This example demonstrates the high resolution separation of DNA restriction fragments using octadecyi modified, nonporous silica reverse phase material, as described in Example 1.
  • the experiment is conducted under the following conditions: Column: 50x4.6 mm i.d. Mobile phase: 0.1 M TEAA, pH 7.0. Gradient: 8.75 - 11.25% acetonitrile in 2 minutes, followed by 11.25 - 14.25% acetonitrile in 10 minutes, 14.5 - 15.25% acetonitrile in 4 minutes, and by 15.25 - 16.25% acetonitrile in 4 minutes. Flow rate 1 mlJmin. Column temperature: 50°C. Detection: UV at 254 nm.
  • Sample Mixture of 0.75 ⁇ g pBR322 DNA-tfaelll restriction digest and 0.65 ⁇ g ⁇ x174 DNA-H/nc ll restriction digest.
  • a high resolution separation is obtained by optimizing the concentration of triethylammonium acetate (TEAA), shape of the gradient curve, column temperature, and flow rate.
  • TEAA triethylammonium acetate
  • the gradient is optimized by decreasing the steepness of the gradient curve with increasing fragment lengths of DNA molecules.
  • the best separations of double-stranded DNA molecules are accomplished at about 30°C to 50°C. Denaturation of DNA at higher than about 50°C prevents utilization of higher column temperatures for double-stranded DNA fragments, although single- stranded DNA separations can be performed at temperatures up to 80°C and higher.
  • EXAMPLE 10 If the gradient delay volume is minimized, the separation of PCR products and hybrid DNA derived from various sources of DNA, including living and dead organisms (animal and plant), as well as parts of such organisms (e.g., blood cells, biopsies, sperm, etc.) on octadecyl modified, nonporous poly-(ethyivinylbenzene-divinylbenzene) coated beads can be achieved with run times under 2 minutes.
  • living and dead organisms animal and plant
  • parts of such organisms e.g., blood cells, biopsies, sperm, etc.
  • PCR products and hybrid DNA usually requires only separation and detection of one or two species of known length. Because of this, the resolution requirements are considerably less severe than for separations of DNA restriction fragments. Such less stringent resolution requirements allow the utilization of steep gradients and, consequently, lead to still shorter run times. The recovery rate for a DNA fragment containing 404 base pairs is about 97.5%.
  • PCR samples do not have to be desalted prior to analysis by MIPC. This represents a decisive advantage of MIPC over CE. With MIPC, it is thus possible to achieve a fully automated analysis of PCR samples if an automatic autosampler is utilized.
  • a fully automated version of the method of the invention can be used to discriminate (distinguish) normal from mutated genes, as well as to detect oncogenes, bacterial and viral genome polynucleotides (hepatitis C virus, HIV, tuberculosis) for diagnostic purposes.
  • adjustment of column temperature allows one to moderate the stringency of hybridization reactions or to separate heteroduplex from homoduplex DNA species.
  • Eluents are chosen to match the desorption ability of the elution solvent to the attraction properties of the bead to the DNA-counter ion complex. As the polarity of the bead decreases, a stronger (more organic) or higher concentration of solvent will be required. Weaker organic solvents such as methanol are generally required at higher concentrations than stronger organic solvents such as acetonitrile.
  • FIG. 9 shows the high resolution separation of DNA restriction fragments using octadecyl modified, nonporous poly(ethylvinylbenzene- divinylbenzene) beads.
  • the experiment was conducted under the following conditions. Column: 50 x 4.6 mm i.d.; mobile phase 0.1 M tetraethylacetic acid (TEAA), pH 7.2; gradient: 33-55% acetonitrile in 3 min, 55-66% acetonitrile in 7 min, 65% acetonitrile for 2.5 min; 65-100% acetonitrile in 1 min; and 100-35% acetonitrile in 1.5 min.
  • the flow rate was 0.75 mlJmin,
  • the separation shown in FIG. 13 was obtained using octadecyl modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows: Column: 50 x 4.6 mm i.d.; mobile phase 0.1 M TEAA, pH 7.3; gradient: 12-18% 0.1 M TEAA and 25.0% 1-propanol (Eluent B) in 3 min, 18- 22% B in 7 min, 22% B for 2.5 min; 22-100% B in 1 min; and 100-12% B in 1.5 min. The flow rate was 0.75 mlJmin, detection UV at 260 nm, and column
  • the separation shown in FIG. 14 was obtained using octadecyl modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows: Column: 50 x 4.6 mm i.d.; mobile phase 0.1 M TEAA, pH 7.3; gradient: 15-18% 0.1 M TEAA and 25.0% 1-propanol (Eluent B) in 2 min, 18- 21 % B in 8 min, 21 % B for 2.5 min; 21 -100% B in 1 min; and 100-15% B in 1.5 min.
  • the flow rate was 0.75 mlJmin, detection UV at 260 nm, and column
  • the separation shown in FIG. 15 was obtained using octadecyl modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows: Column: 50 x 4.6 mm i.d.; mobile phase 0.1 M TEAA, pH 7.3; gradient: 35-55% 0.1 M TEAA and 10.0% 2-propanol (Eluent B) in 3 min, 55- 65 % B in 10 min, 65% B for 2.5 min; 65-100% B in 1 min; and 100-35% B in 1.5 min.
  • the flow rate was 0.75 mlJmin, detection UV at 260 nm, and column temp. 51 °C.
  • the separation shown in FIG. 16 was obtained using octadecyl modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows: Column: 50 x 4.6 mm i.d.; mobile phase 0.05 M TEA 2 HP0 4 , pH 7.3; gradient: 35-55% 0.05 M TEA 2 HP0 4 and 10.0% 2-propanol (Eluent B) in 3 min, 55-65% B in 7 min, 65% B for 2.5 min; 65-100% B in 1 min; and 100- 65% B in 1.5 min.
  • the flow rate was 0.75 mlJmin, detection UV at 260 nm, and column temp. 51 °C.
  • the separation shown in FIG. 17 was obtained using octadecyl modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows: Column: 50 x 4.6 mm i.d.; mobile phase 0.1 M TEAA, pH 7.3; gradient: 6-9% 0.1 M TEAA and 25.0% THF (Eluent B) in 3 min, 9-11 % B in 7 min, 11 % B for 2.5 min; 11 -100% B in 1 min; and 100-6% B in 1.5 min. The flow rate was 0.75 mlJmin, detection UV at 260 nm, and column temp. 51 °C.
  • Isocratic/gradient separation of dsDNA The following is an isocratic/gradient separation of dsDNA on a polystyrene coated silica base material. Isocratic separations have not been performed in DNA separations because of the large differences in the selectivity of DNA/alkylammonium ion pair for beads. However, by using a combination of gradient and isocratic elution conditions, the resolving power of a system can be enhanced for a particular size range of DNA.
  • the range of 250-300 base pairs can be targeted by using an eluent of 0.1 M TEAA, and 14.25% acetonitrile at 0.75 mlJmin at 40°C on 50 x 4.6 mm crosslinked polystyrene coated silica reverse phase column, 2.0 micron.
  • the pUC18 DNA-Haelll digest was injected under isocratic conditions and 257, 267 and 298 base pairs DNA eluted completely resolved. Then the column was cleaned from larger fragments with 0.1 M TEAA/25% acetonitrile at 9 minutes.
  • FIG. 18 shows a separation using the same elution conditions but performed on a poly(styrene-divinylbenzene) polymer based column.
  • an initial isocratic step to condition the column
  • a gradient step to remove or target the first group of DNA at a particular size
  • an isocratic step to separate the target material of a different size range

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Abstract

L'invention porte sur des billes non poreuses dont le diamètre moyen est compris entre environ 0,5 et 100 microns et qui sont appropriées pour être utilisées dans la séparation chromatographique. Ces billes comprennent des particules non poreuses qui sont recouvertes d'un polymère dont presque tous les groupes de la surface du substrat sont coiffés aux extrémités par un groupe d'hydrocarbures non polaires ou substitués. Les billes assurent une séparation efficace des polynucléotides par chromatographie polynucléotidique à compatibilité ionique.
EP98918701A 1997-04-25 1998-04-24 Milieu chromatographique liquide ameliore utilise dans la separation de polynucleotides Withdrawn EP1027121A4 (fr)

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US6576133B2 (en) 1996-11-13 2003-06-10 Transgenomic, Inc Method and system for RNA analysis by matched ion polynucleotide chromatography
US6177559B1 (en) 1998-04-24 2001-01-23 Transgenomic, Inc. Process for separation of polynucleotide fragments
CA2308314A1 (fr) * 1997-10-30 1999-05-14 Transgenomic, Inc. Procede de separation des polynucleotides par chromatographie liquide a haute resolution
US6258264B1 (en) 1998-04-10 2001-07-10 Transgenomic, Inc. Non-polar media for polynucleotide separations
US6265168B1 (en) 1998-10-06 2001-07-24 Transgenomic, Inc. Apparatus and method for separating and purifying polynucleotides
AU777251B2 (en) * 2000-05-01 2004-10-07 Transgenomic, Inc. Polynucleotide separations on polymeric separation media
CA2396667A1 (fr) * 2000-05-26 2001-12-06 Benjamin L. Legendre, Jr. Appareil et procede permettant la separation et la purification de polynucleotides
US6521411B2 (en) 2000-09-28 2003-02-18 Transgenomic, Inc. Method and system for the preparation of cDNA
JP4069999B2 (ja) * 2001-03-13 2008-04-02 独立行政法人理化学研究所 ナノ被覆分子性材料
DE10120821A1 (de) * 2001-04-27 2002-10-31 Sachtleben Chemie Gmbh Mit Polyalken beschichtete, poröse Füllstoffe
WO2003031580A2 (fr) 2001-10-05 2003-04-17 Transgenomic, Inc. Procedes et compositions d'analyse de mutation par chromatographie en phase liquide
WO2012033194A1 (fr) * 2010-09-09 2012-03-15 ダイセル化学工業株式会社 Procédé servant à séparer un matériau biologique soluble dans l'eau
EP2570183A1 (fr) * 2011-09-15 2013-03-20 InstrAction GmbH Sorbant comportant à sa surface une unité aliphatique pour la purification des molécules organiques
TW201726917A (zh) * 2015-12-28 2017-08-01 昭和電工股份有限公司 分離分析方法
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