Polymeric ion exchange-reversed phase-mixed-mode chromatography stationary phase for analysis of proteins and nucleic acids

Abstract

A chromatographic media for separating biopolymers, the chromatographic media having hydrophobic and ionic retention modes, the chromatographic media comprising porous substrate particles including a hydrophobic polymer resin and ion exchange functional groups copolymerized with the hydrophobic polymer resin or grafted to the surface of the porous substrate, wherein the ion exchange functional groups are not greater than about 5 mol% of the porous substrate particles.

Description

POLYMERIC ION EXCHANGE-REVERSED PHASE-MIXED-MODE CHROMATOGRAPHY STATIONARY PHASE FOR ANALYSIS OF PROTEINS AND NUCLEIC ACIDSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to US Provisional Application S / N 63 / 614,752, filed December 26, 2023, the entire contents of which is incorporated herein by reference.FIELD

[0002] The present disclosure generally relates to the field of chromatography including polymeric ion exchange-reverse phase-mixed-mode chromatography stationary phase for analysis of proteins and nucleic acids.INTRODUCTION

[0003] Ion-pair reversed phase chromatography is widely used for the characterization of proteins and nucleic acids, such as for use as pharmaceuticals. Ionpair reversed phase chromatography can be coupled with UV as well as mass spectrometry for characterization of the main product as well as impurities in the pharmaceutical product. However, due to the complex nature of protein and nucleic acid therapy, biologies (both mAb and mRNA) are biopolymers which are composed of many structurally and chemically related variants. These variants are often not well resolved. To better separate these variants, multiple modes of chromatography can be used including reversed phase chromatography, ion-exchange chromatography, size exclusion chromatography and hydrophobic interaction chromatography. These modes are used in separate experiments or are coupled inline to obtain separation in one run (multidimension-LC). From the foregoing it will be appreciated that a need exists for improved separation columns and methods.SUMMARY

[0004] In a first aspect, a chromatographic media for separating biopolymers has hydrophobic and ionic retention modes. The chromatographic media includes poroussubstrate particles including a hydrophobic polymer resin and ion exchange functional groups copolymcrizcd with the hydrophobic polymer resin, wherein the ion exchange functional groups are not greater than about 5 mol% of the porous substrate particles.

[0005] In various embodiments of the first aspect, the ion exchange functional groups are not greater than about 4 mol% of the porous substrate particles, such as not greater than about 3 mol% of the porous substrate particles, such as not greater than about 2 mol% of the porous substrate particles.

[0006] In various embodiments of the first aspect, the ion exchange functional groups can be not less than about 0.01 mol% of the porous substrate particles.

[0007] In various embodiments of the first aspect, the hydrophobic polymer resin includes vinylbenzene or derivatives thereof, such as ethylvinylbenzene and crosslinker di vinyl benzene.

[0008] In various embodiments of the first aspect, the ion exchange functional groups include functional groups with positive charge. In particular’ embodiments, the ion exchange functional groups originate from H2C=C(CH3)CO2RINR2R3 where Ri is an alkyl group and R2 and R3 are H or alkyl groups, such as 2-(N,N-Diethylamino)ethyl methacrylate. In particular embodiments, the porous substrate particles include between about 0.001 and about 0.5 wt% nitrogen, such as between about 0.003 and about 0.4 wt% nitrogen, even between about 0.001 and about 0.3 wt% nitrogen.

[0009] In various embodiments of the first aspect, the ion exchange functional groups include functional groups with negative charge, such as methacrylic acid or acrylic acid.

[0010] In various embodiments of the first aspect, porous substrate particles are monodisperse having a particle diameter coefficient of variation (CV) % of less than 15% when measured by Coulter Counter analysis, such as less than 10%, such as less than 5%, even less than 2.5%.

[0011] In various embodiments of the first aspect, the porous substrate particles have a specific surface area, as determined using Brunaucr-Emmctt-Tcllcr (BET) surface area analysis, of at least about 10 m2 / g, such as at least about 50 m2 / g. In particular embodiments, the specific surface area is not greater than about 250 m2 / g.

[0012] In various embodiments of the first aspect, the biopolymers include oligonucleotides, polynucleotides, peptides, proteins, or any combination thereof.

[0013] In a second aspect, a method of preparing a chromatographic media having hydrophobic and ionic retention modes includes copolymerizing a hydrophobic polymer resin monomer and ion exchange functional groups monomer to form monodisperse porous particles, wherein the ion exchange functional groups monomer is in an amount of less than 5 mol%.

[0014] In various embodiments of the second aspect, the ion exchange functional groups are not greater than about 4 mol% of the porous substrate particles, such as not greater than about 3 mol% of the porous substrate particles, such as not greater than about 2 mol% of the porous substrate particles, even not less than about 0.01 mol% of the porous substrate particles.

[0015] In various embodiments of the second aspect, the porous substrate particles include between about 0.001 and about 0.5 wt% nitrogen, such as between about 0.003 and about 0.4 wt% nitrogen, even between about 0.001 and about 0.3 wt% nitrogen.

[0016] In various embodiments of the second aspect, the hydrophobic polymer resin includes vinylbenzene or derivatives thereof, such as the hydrophobic polymer resin includes ethylvinylbenzene.

[0017] In various embodiments of the second aspect, the ion exchange functional groups include functional groups with positive charge. In particular embodiments, the ion exchange functional groups originate from H2C=C(CH3)CO2RINR2R3 where Ri is an alkyl group and R2 and R3 are H or alkyl groups, such as 2-(N,N-Diethylamino)ethyl methacrylate.

[0018] In various embodiments of the second aspect, the ion exchange functional groups include functional groups with negative charge. In particular embodiments, the ion exchange functional groups include methacrylic acid or acrylic acid.

[0019] In various embodiments of the second aspect, porous substrate particles are monodisperse having a particle diameter coefficient of variation (CV) % of less than 15% when measured by Coulter Counter analysis, such as less than 10%, such as less than 5%, even less than 2.5%.

[0020] In various embodiments of the second aspect, the porous substrate particles have a specific surface area, as determined using Brunauer-Emmett-Teller (BET) surface area analysis, of at least about 10 m2 / g, such as at least about 50 m2 / g. In particular embodiments, the specific surface area is not greater than about 250 m2 / g.

[0021] In various embodiments of the second aspect, the biopolymers include oligonucleotides, polynucleotides, peptides, proteins, or any combination thereof.

[0022] In a third aspect, a chromatographic media for separating biopolymers has hydrophobic and ionic retention modes. The chromatographic media includes porous substrate particles including a hydrophobic polymer resin; and ion exchange functional groups grafted to the surface of the porous substrate, wherein the ion exchange functional groups are up to 5 mol% of the chromatographic media.

[0023] In various embodiments of the third aspect, the ion exchange functional groups are not greater than about 4 mol% of the porous substrate particles, such as not greater than about 3 mol% of the porous substrate particles, such as not greater than about 2 mol% of the porous substrate particles, even not less than about 0.01 mol% of the porous substrate particles.

[0024] In various embodiments of the third aspect, the porous substrate particles include between about 0.001 and about 0.5 wt% nitrogen, such as between about 0.003 and about 0.4 wt% nitrogen.

[0025] The chromatographic media for separating biopolymers of claim 63 wherein the porous substrate particles include between about 0.001 and about 0.3 wt% nitrogen.

[0026] In various embodiments of the third aspect, the hydrophobic polymer resin includes vinylbenzene or derivatives thereof, such as ethylvinylbenzene.

[0027] In various embodiments of the third aspect, the ion exchange functional groups include functional groups with positive charge. In particular embodiments, the ion exchange functional groups originate from H2C=C(CH3)CO2RINR2R3 where Ri is an alkyl group and R2 and R3 are H or alkyl groups, such as 2-(N,N-Diethylamino)ethyl methacrylate.

[0028] In various embodiments of the third aspect, the ion exchange functional groups include functional groups with negative charge. In particular embodiments, the ion exchange functional groups include methacrylic acid or acrylic acid.

[0029] In various embodiments of the third aspect, porous substrate particles are monodisperse having a particle diameter coefficient of variation (CV) % of less than 15% when measured by Coulter Counter analysis, such as less than 10%, such as less than 5%, such as less than 2.5%.

[0030] In various embodiments of the third aspect, the porous substrate particles have a specific surface area, as determined using Brunauer-Emmett-Teller (BET) surface area analysis, of at least about 10 m2 / g, such as at least about 50 m2 / g. In particular embodiments, the specific surface area is not greater than about 250 m2 / g.

[0031] In various embodiments of the third aspect, the biopolymers include oligonucleotides, polynucleotides, peptides, proteins, or any combination thereof.

[0032] In a fourth aspect, a method of preparing a chromatographic media having hydrophobic and ionic retention modes includes polymerizing a hydrophobic polymer resin monomer to form porous particles; and grafting an ion exchange functional group monomer to the surface of the monodisperse, porous particles to form weakly chargedporous substrate particles wherein the ion exchange functional groups monomer is in an amount of less than 5 mol%.

[0033] In various embodiments of the fourth aspect, the ion exchange functional groups are not greater than about 4 mol% of the porous substrate particles, such as about 3 mol% of the porous substrate particles, such as about 2 mol% of the porous substrate particles, even not less than about 0.01 mol% of the porous substrate particles.

[0034] In various embodiments of the fourth aspect, the porous substrate particles include between about 0.001 and about 0.5 wt% nitrogen, such as between about 0.003 and about 0.4 wt% nitrogen, even between about 0.001 and about 0.3 wt% nitrogen.

[0035] In various embodiments of the fourth aspect, the hydrophobic polymer resin monomer includes vinylbenzene or derivatives thereof, such as ethylvinylbenzene.

[0036] In various embodiments of the fourth aspect, the ion exchange functional group monomer include functional groups with positive charge. In particular embodiments, the ion exchange functional groups originate from H2C=C(CH3)CO2RINR2R3 where Ri is an alkyl group and R2 and R3 are H or alkyl groups, such as 2-(N,N-Diethylamino)ethyl methacrylate.

[0037] In various embodiments of the fourth aspect, the ion exchange functional group monomer includes functional groups with negative charge. In particular embodiments, the ion exchange functional group monomer include methacrylic acid or acrylic acid.

[0038] In various embodiments of the fourth aspect, porous substrate particles are monodisperse having a particle diameter coefficient of variation (CV) of less than 15% when measured by Coulter Counter analysis, such as less than 10%, such as less than 5%, such as less than 2.5%.

[0039] In various embodiments of the fourth aspect, the porous substrate particles have a specific surface area, as determined using Brunauer-Emmett-Teller (BET) surfacearea analysis, of at least about 10 m2 / g, such as at least about 50 m2 / g. In particular embodiments, the specific surface area is not greater than about 250 m2 / g.

[0040] In various embodiments of the fourth aspect, the biopolymers include oligonucleotides, polynucleotides, peptides, proteins, or any combination thereof.

[0041] In a fifth aspect, a method includes loading a biopolymer sample onto a chromatographic media having hydrophobic and ionic retention modes, the chromatographic media including weakly charged porous particles, the weakly charged porous particles including hydrophobic polymer resin subunits and ion exchange functional group subunits, the ion exchange functional group subunits being less than 5 mol% of the weakly charged porous particles, the biopolymer sample containing a plurality of biopolymer species; and eluting the biopolymer sample from the chromatographic media by applying gradient of solvent conditions to the chromatographic media to cause the separation of the plurality of biopolymers species.

[0042] In various embodiments of the fifth aspect, the ion exchange functional groups are not greater than about 4 mol% of the porous substrate particles, such as not greater than about 3 mol% of the porous substrate particles, such as not greater than about 2 mol% of the porous substrate particles, even not less than about 0.01 mol% of the porous substrate particles.

[0043] In various embodiments of the fifth aspect, the porous substrate particles include between about 0.001 and about 0.5 wt% nitrogen, such as between about 0.003 and about 0.4 wt% nitrogen, even between about 0.001 and about 0.3 wt% nitrogen.

[0044] In various embodiments of the fifth aspect, the solvent does not include an ion pairing reagent.

[0045] In various embodiments of the fifth aspect, the gradient of solvent conditions includes varying the solvent, varying the ionic strength, varying the pH, or any combination thereof. In particular embodiments, varying the solvent includes increasing or decreasing the amount of an organic solvent.

[0046] In various embodiments of the fifth aspect, the biopolymers are amino acid biopolymers.

[0047] In various embodiments of the fifth aspect, the ionic retention modes are negatively charged.

[0048] In various embodiments of the fifth aspect, the mobile phase includes an acid, such as formic acid, acetic acid, citric acid, other mass spec compatible acids, or any combination thereof.

[0049] In various embodiments of the fifth aspect, the ion exchange functional group subunits include methacrylic acid or acrylic acid.

[0050] In various embodiments of the fifth aspect, the biopolymers are nucleic acid biopolymers.

[0051] In various embodiments of the fifth aspect, the ionic retention modes are positively charged.

[0052] In various embodiments of the fifth aspect, the mobile phase includes ammonium acetate, ammonium bicarbonate, ammonium citrate, ammonium, other mass spectrometry compatible buffers, or any combination thereof.

[0053] In various embodiments of the fifth aspect, the ion exchange functional groups originate from H2C=C(CH3)CO2RINR2R3 where Ri is an alkyl group and R2 and R3 are H or alkyl groups, such as 2-(N,N-Diethylamino)ethyl methacrylate .

[0054] In various embodiments of the fifth aspect, the method further includes detecting one or more of the biopolymer species by mass spectroscopy.

[0055] In various embodiments of the fifth aspect, the hydrophobic polymer resin includes vinylbenzene or derivatives thereof, such as ethylvinylbenzene.

[0056] In various embodiments of the fifth aspect, porous substrate particles are monodisperse having a particle diameter coefficient of variation (CV) % of less than 15%when measured by Coulter Counter analysis, such as less than 10%, such as less than 5%, such as less than 2.5%.

[0057] In various embodiments of the fifth aspect, the porous substrate particles have a specific surface area, as determined using Brunauer-Emmett-Teller (BET) surface area analysis, of at least about 10 m2 / g, such as at least about 50 m2 / g. In particular embodiments, the surface area is not greater than about 250 m2 / g.

[0058] In various embodiments of the fifth aspect, the biopolymers include oligonucleotides, polynucleotides, peptides, proteins, or any combination thereof.DRAWINGS

[0059] For a more complete understanding of the principles disclosed herein, and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings and exhibits, in which:

[0060] Figure 1A is a block diagram of an exemplary chromatography system, in accordance with various embodiments.

[0061] Figure IB is a block diagram of an exemplary mass spectrometry system, in accordance with various embodiments.

[0062] Figures 2A and 2B are diagrams illustrating the use of ion pairing agents with a reverse phase chromatography stationary phase to separate biopolymers, in accordance with various embodiments.

[0063] Figures 3A and 3B are diagrams illustrating the use of an ion exchange reverse phase mixed mode chromatography stationary phase to separate biopolymers, in accordance with various embodiments.

[0064] Figure 4 is a flow diagram illustrating a method of making an ion exchange reverse phase mixed mode chromatography stationary phase by copolymerization, in accordance with various embodiments.

[0065] Figure 5 is a flow diagram illustrating a method of making an ion exchange reverse phase mixed mode chromatography stationary phase by grafting, in accordance with various embodiments.

[0066] Figure 6 is a flow diagram illustrating a method of separating nucleic acids, in accordance with various embodiments.

[0067] Figure 7 is a flow diagram illustrating a method of separating proteins, in accordance with various embodiments.

[0068] It is to be understood that the figures are not necessarily drawn to scale, nor are the objects in the figures necessarily drawn to scale in relationship to one another. The figures are depictions that are intended to bring clarity and understanding to various embodiments of apparatuses, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Moreover, it should be appreciated that the drawings are not intended to limit the scope of the present teachings in any way.DESCRIPTION OF VARIOUS EMBODIMENTS

[0069] Embodiments of systems and methods for ion isolation are described herein and in the accompanying exhibits.

[0070] The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way.

[0071] In this detailed description of the various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the embodiments disclosed. One skilled in the art will appreciate, however, that these various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Furthermore, one skilled in the art can readily appreciate that the specific sequences in which methods are presented and performed are illustrative and it is contemplated that the sequences can be varied and still remain within the spirit and scope of the various embodiments disclosed herein.

[0072] All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. Unless described otherwise, all technical and scientific terms used herein have a meaning as is commonly understood by one of ordinary skill in the ail to which the various embodiments described herein belongs.

[0073] It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, pressures, flow rates, cross-sectional areas, etc. discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present teachings.

[0074] As used herein, "a" or "an" also may refer to "at least one" or "one or more." Also, the use of “or” is inclusive, such that the phrase “A or B” is true when “A” is true, “B” is true, or both “A” and “B” are true. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0075] A “system” sets forth a set of components, real or abstract, comprising a whole where each component interacts with or is related to at least one other component within the whole.

[0076] As used herein, biopolymers are a class of biological molecules that consist of monomeric units that are covalently bonded, generally in chains, to form larger molecules. Biopolymers can be classified by the type of subunit and length.Biopolymers can include nucleic acids, such as oligonucleotides and polynucleotides. Oligonucleotides, used herein to refer to chains of 3 to 12 nucleotides, and polynucleotides, used herein to refer to chains of 13 or more nucleotides, are biopolymers with nucleotides, such as deoxyribonucleotides and ribonucleotides, for the subunit.Bioplymers can also include peptides or proteins. Peptides, used herein to refer to chains of 3 to 50 amino acids, and polypeptides, used herein to refer to chains of 51 or more amino acids, are biopolymers with amino acid subunits. Polysaccharides, used herein to refer to chains of 5 or more monosaccharides, are biopolymers with monosaccharide subunits, such as glucose, fructose, and glyceraldehyde. Lipid polymers, such as triglycerides, phospholipids, and waxes, are biopolymers with lipid subunits.

[0077] Figure 1A depicts a liquid chromatography system 100 according to one aspect of the invention. The liquid chromatography system 100 comprises an analytical pump 102 to pump a solvent through the system 100. The system 100 comprises a sample reservoir 104 comprising a sample to be analyzed. The system 100 further comprises a separation column 106 and a detector 108. System 100 also comprises a controller 610.

[0078] The liquid chromatography system 100 is adapted to retrieve a sample from the sample reservoir 104. The sample can then be introduced into the system

[0079] The liquid chromatography system 100 is further adapted to introduce the sample into the separation column 106.

[0080] The system 100 is also adapted to inject the sample into the separation column 106 by means of the analytical flow. This can be done by guiding the sample by means of the analytical pump 102. The separation column 106 can separate the sample into component species based on retention time within the separation column 106. After separation of the sample by the separation column 106, the separated components can be detected by a detector 108. In some embodiments, the detector 108 can be an optical detector, such as an absorption detector, refractive index detector, a fluorescence detector, or the like. In other embodiments, the detector 108 can be a conductivity detector or an electrochemical detector. In yet other embodiments, the detector 108 can be a mass spectrometer.

[0081] In various embodiments, the separation column 106 generally consists of a tube packed with a stationary phase medium. The stationary phase medium can affect the time it takes for a compound to travel through the column (retention time). The effectcan be different for different compounds, such that individual components of a sample can be separated based on their respective retention times. There arc a variety of stationary phase mediums, including porous materials, ionic materials, polar materials, non-polar materials, and the like. Porous materials can affect retention time based on the size of a molecule and the ability of the molecule to enter the porous material. Ionic materials can affect retention time based on charge attraction or repulsion between the ionic material and the compounds. Polar and non-polar materials can affect retention time based on the hydrophobicity or hydrophilicity of the compounds.

[0082] Various embodiments of mass spectrometry platform 150 can include components as displayed in the block diagram of Figure IB. In various embodiments, mass spectrometry platform 150 can operate as a detector 108 of system 100. In various embodiments, elements of Figure IB can be incorporated into mass spectrometry platform 150. According to various embodiments, mass spectrometer 150 can include an ion source 152, a mass analyzer 154, an ion detector 156, and a controller 158.

[0083] In various embodiments, the ion source 152 generates a plurality of ions from a sample. The ion source can include, but is not limited to, a matrix assisted laser desorption / ionization (MALDI) source, electrospray ionization (ESI) source, atmospheric pressure chemical ionization (APCI) source, atmospheric pressure photoionization source (APPI), inductively coupled plasma (ICP) source, electron ionization source, chemical ionization source, photoionization source, glow discharge ionization source, thermospray ionization source, and the like.

[0084] In various embodiments, the mass analyzer 154 can separate ions based on a mass-to-charge ratio of the ions. For example, the mass analyzer 154 can include a quadrupole mass filter analyzer, a quadrupole ion trap analyzer, a time-of-flight (TOF) analyzer, an electrostatic trap (e.g., Orbitrap) mass analyzer, Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer, and the like. In various embodiments, the mass analyzer 154 can also be configured to fragment the ions using collision induced dissociation (CID) electron transfer dissociation (ETD), electron capture dissociation(ECD), photo induced dissociation (PTD), surface induced dissociation (SID), and the like, and further separate the fragmented ions based on the mass-to-chargc ratio.

[0085] In various embodiments, the ion detector 156 can detect ions. For example, the ion detector 156 can include an electron multiplier, a Faraday cup, and the like. Ions leaving the mass analyzer can be detected by the ion detector. In various embodiments, the ion detector can be quantitative, such that an accurate count of the ions can be determined. In various embodiments, such as for an electrostatic trap (e.g. ORBITRAP) mass analyzer, the mass analyzer 154 and the ion detector 156 can be combined into a single device.

[0086] In various embodiments, the controller 158 can communicate with the ion source 152, the mass analyzer 154, and the ion detector 156. For example, the controller 158 can configure the ion source or enable / disable the ion source. Additionally, the controller 158 can configure the mass analyzer 154 to select a particular mass range to detect. Further, the controller 158 can adjust the sensitivity of the ion detector 156, such as by adjusting the gain. Additionally, the controller 158 can adjust the polarity of the ion detector 156 based on the polarity of the ions being detected. For example, the ion detector 156 can be configured to detect positive ions or be configured to detected negative ions.

[0087] Reversed phase chromatography, in particular ion pair reversed phase chromatography, is widely used for the analysis of proteins and nucleic acids. In an ion pair reversed phase chromatography, small amounts of ion-pairing reagents are added to the mobile phase resulting in increasing retention of strongly polar compounds. FIGs. 2A and 2B illustrate the use of exemplary ion pairing reagents trifluoroacetate (TFA) and triethylamine (TEA), respectively. While ion-pairing reagents in the mobile phase sharpens the protein and nucleic acid analyte peaks, ion-pairing reagents are not compatible with mass spectrometry detection due their ion-suppression property. It is desirable to incorporate the ion-pair property into the stationary phase and mitigate the undesirable side effect of free ion-pairing reagents.

[0088] An alternative way to enhance the resolving power in a single separation run is to create a mixed-mode stationary phase by incorporating more than one mode of functional groups, resulting in mixed-mode separation. Mixed-mode separation has been used for small molecule separations as well as protein and nucleic acid separations. Most of mixed-mode stationary phases including both reversed phase and ion exchange mechanisms utilize silica substrate. However, silica substrate is less stable in high pH condition which is often applied to the nucleic acid separation. Furthermore, the commonly used silica substrate does not contain large enough pores suitable for large proteins and long nucleic acids separation. FIGs. 3A and 3B illustrate the use of mixedmode stationary phase which can be used without the need for an ion-pairing agent.

[0089] Disclosed herein are novel mixed-mode chromatography stationary phases for reversed phase separation of proteins and nucleic acids. These novel mixed-mode chromatography stationary phases consist of hydrophobic polymer resins with large pores and a small amount of ion exchange functional groups. The ion exchange sites can be incorporated into the resin by either co-polymerizing the charged monomers during the resin synthesis or by grafting onto the surface of the resin post-resin synthesis.

[0090] In various embodiments, the mixed-mode chromatography stationary phases consist of particles that are monodisperse. “Monodisperse” refers herein to particles having a low coefficient of variation (CV) of a specific parameter (for example, particle diameter), for example a CV of less than 20%, for example less than 15%, preferably less 10% and sometimes less than 5%, even less than about 2.5%. CV, when referred to in the claims of this specification is defined as 100 time (standard deviation) divided by average where “average”" is mean particle diameter and standard deviation is standard deviation in particle size. The disclosure also includes embodiments where the “average1is either the Z-average or mode particle diameter. In accordance with usual practice, CV is calculated on the main mode, that is the main peak, thereby excluding minor peaks relating to aggregates. Thus, some particles below or above mode size may be discounted in the calculation which may for example be based on about 90% of total particle number (of detectable particles that is). Such a determination of CV is performable on a Coulter Counter (electrical sensing zone method).

[0091] In various embodiments, the mixed-mode chromatography stationary phases can consist of polymer particles comprising subunits of neutral hydrophobic monomers, such as vinylbenzene (styrene) or derivatives thereof, such as ethylvinylbenzene, and subunits of ion exchange functional groups that can be either an anion exchangers or cation exchangers. Caition exchanger functional groups can include ion exchange functional groups with a negative charge, such as acrylic acid or methacrylic acid, such that the cation exchanger functional groups can interact with cationic compounds having a positive charge.. Anion exchanger functional groups can include ion exchange functional groups with a positive charge, such as ion exchange functional groups derived from monomers with a formula H2C=C(CH3)CO2RJNR2R3 where Ri is an alkyl group and R2 and R3 are H or alkyl groups, such as 2-(N,N-Diethylamino)ethyl methacrylate such that the cation exchanger functional groups can interact with cationic compounds having a negative charge.

[0092] In various embodiments, the polymer particles can contain a relatively small amount of subunits of ion exchange functional groups, such as not greater than about 5 mol% of the ionic functional monomers relative to the total monomer that are incorporated into the porous substrate particles, such as not greater than about 4 mol%, such as not greater than about 3 mol%, even not greater than about 2 mol%. In various embodiments, the ion exchange functional monomers can be not less than about 0.01 mol%, such as not less than about 0.02 mol%, even not less than about 0.03 mol%.

[0093] In various embodiments, the mixed-mode chromatography stationary phases consist of particles that are weakly charged. “Weakly charged” refers herein to particles with a low percentage of ion exchange functional groups. In various embodiments, the nitrogen content of the particles can be used to determine the amount of ion exchange functional groups for nitrogen containing functional groups. In various embodiment, the porous substrate particles can include between about 0.001 wt% N and about 0.5 wt% N, such as between about 0.003 wt% N and about 0.4 wt% N, even 0.001 wt% N and about 0.3 wt% N.

[0094] In various embodiments, the mixed-mode chromatography stationary phases can consist of polymer particles that arc stable over a pH range from about 0 to 14, such as from about 1 to 12. Additionally, the polymer particles can be stable up to a temperature of at least about 100°C, such as at least about 120°C.

[0095] In various embodiments, the mixed-mode chromatography stationary phases can consist of polymer particles having a mean particle diameter of between about 2 pm to about 50 pm. The polymer particles can have pores having an average pore size diameter of at least about 100 A, such as at least about 125 A. Generally, the average pore size diameter is not greater than about 500 A. The polymer particles can have a specific surface area, as determined using Brunauer-Emmett-Teller (BET) surface area analysis, of at least about 10 m2 / g, such as at least about 50 m2 / g. Generally, the specific surface area is not greater than about 250 m2 / g.

[0096] Fig. 4 illustrates a method of making weakly charged, monodisperse polymer particles by copolymerization. At 402, hydrophobic polymer resin monomers are provided, and, at 404, ion exchange functional group monomers are provided. The hydrophobic polymer resin monomers can be neutral hydrophobic monomers, such as vinylbenzene (styrene) or derivatives thereof, such as ethylvinylbenzene.

[0097] The ion exchange functional group monomers can be cither an anion exchangers or cation exchangers. Cation exchanger functional group monomers can include ion exchange functional groups with a negative charge, such as acrylic acid or methacrylic acid, such that the cation exchanger functional groups can interact with cationic compounds having a positive charge. Anion exchanger functional group monomers can include ion exchange functional groups with a positive charge, such as ion exchange functional groups derived from monomers with a formula H2C=C(CH3)CO2RINR2R3 where Ri is an alkyl group and R2 and R3 are H or alkyl groups, such as 2-(N,N-Diethylamino)ethyl methacrylate, such that the anion exchanger functional groups can interact with anionic compounds having a negative charge.

[0098] In various embodiments, the mixture of hydrophobic polymer resin monomers and ion exchange functional group monomers can contain a relatively smallamount of ion exchange functional group monomers, such as not greater than about 5 mol% of the ion exchange functional group monomers, such as not greater than about 4 mol%, such as not greater than about 3 mol%, even not greater than about 2 mol%. In various embodiments, the ion exchange functional group monomers can be not less than about 0.01 mol%.

[0099] At 406, the hydrophobic polymer resin monomers and the ion exchange functional group monomers can be copolymerized to form particles. The particles can be monodisperse, hydrophobic, and contain large pores. The particles can have a particle diameter CV of less than 20%, for example less than 15%, preferably less 10% and sometimes less than 5%, even less than about 2.5%. The particles can have a mean particle diameter of between about 2 pm to about 50 pm. The polymer particles can have pores having an average pore size diameter of at least about 100 A, such as at least about125 A. Generally, the average pore size diameter is not greater than about 500 A. The particles can have a specific surface area, as determined using Brunauer-Emmett-Teller (BET) surface area analysis, of at least about 10 m2 / g, such as at least about 50 m2 / g. Generally, the specific surface area is not greater than about 250 m2 / g.

[0100] In various embodiments, inert solvents (proogens) can be used to increase the porosity and pore size of the particles. Porogens can include hexanol, heptanol, octanol, heptane and mixtures thereof. The total amount of porogens can be from about 40 v% to about 60v% of the polymerization mixture.

[0101] Due to the relatively low amount of ion exchange functional group monomers relative to the hydrophobic polymer resin monomers, the particles can be weakly charged.

[0102] At 408, the weakly charged monodisperse polymer particles can be packed into a chromatography column.

[0103] Fig. 5 illustrates an alternate method of making weakly charged, monodisperse polymer particles by grafting. At 502, hydrophobic polymer resin monomers can be polymerized to form monodisperse polymer particles. Thehydrophobic polymer resin monomers can be neutral hydrophobic monomers, such as vinylbcnzcnc (styrene) or derivatives thereof, such as cthylvinylbcnzcnc and crosslinkcr such as divinyl benzene. The polymer particles can be monodisperse, hydrophobic, and contain large pores. The particles can have a particle diameter CV of less than 20%, for example less than 15%, preferably less 10% and sometimes less than 5%, even less than about 2.5%. The particles can have a mean particle diameter of between about 2 pm to about 50 pm. The polymer particles can have pores having an average pore size diameter of at least about 100 A, such as at least about 125 A. Generally, the average pore size diameter is not greater than about 500 A. The particles can have a specific surface area, as determined using Brunauer-Emmett-Teller (BET) surface area analysis, of at least about 10 m2 / g, such as at least about 50 m2 / g. Generally, the specific surface area is not greater than about 250 m2 / g.100104] In various embodiments, inert solvents (proogens) can be used to increase the porosity and pore size of the particles. Porogens can include hexanol, heptanol, octanol, heptane and mixtures thereof. The total amount of porogens can be from about 40v% to about 60v% of the polymerization mixture.

[0105] At 504, ion exchange functional group monomers can be grafted onto the surface of the monodisperse polymer particles to form weakly charged monodisperse polymer particles. The ion exchange functional group monomers can be either an anion exchangers or cation exchangers. Cation exchanger functional group monomers can include ion exchange functional groups with a negative charge, such as acrylic acid or methacrylic acid, such that the cation exchanger functional groups can interact with cationic compounds having a positive charge. Anion exchanger functional group monomers can include ion exchange functional groups with a positive charge, such as ion exchange functional groups derived from monomers with a formula H2C=C(CH3)CO2RINR.2R3 where Ri is an alkyl group and R2 and R3 are H or alkyl groups, such as 2-(N,N-Diethylamino)ethyl methacrylate, such that the anion exchanger functional groups can interact with anionic compounds having a negative charge.

[0106] The amount of ion exchange functional group monomers can be not greater than about 5 mol% of the ion exchange functional group monomers, such as not greater than about 4 mol%, such as not greater than about 3 mol%, even not greater than about 2 mol%. In various embodiments, the amount of ion exchange functional group monomers can be not greater than about 1 mol%, such as not greater than about 0.5 mol%, even not greater than about 0.25 mol%. In various embodiments, the ion exchange functional group monomers can be not less than about 0.001 mol%, such as not less than about 0.01 mol%.

[0107] At 506, the weakly charged monodisperse polymer particles can be packed into a chromatography column.

[0108] Figure 6 is a flow diagram illustrating a method 600 of analyzing a sample containing a plurality of nucleic acid species, particularly large nucleic acids. Generally, the nucleic acid species includes a number of negatively charged phosphate groups connecting the nucleotides.

[0109] At 602, the sample is injected into the chromatography column. Depending on the sample, various pre-injection sample preparation steps can be taken. In various embodiments, a solid or semi-solid sample may be ground up and suspended in a solution to extract the nucleic acid species. The liquid solution can be separated from the solid material and injected into the column. In other embodiments, the sample can be a liquid sample and can be injected directly onto the column without significant pre-injection sample preparation.

[0110] The column can include a mixed mode stationary phase. Mixed mode stationary phases include functional groups of with two or more different properties, such as cation exchange functional groups, anion exchange functional groups, hydrophobic groups, and the like. In particular embodiments, the mixed mode stationary phase includes hydrophobic groups and ion exchange functional groups, such as cationic functional groups. The cationic functional groups can interact with the negatively charged phosphate groups of the nucleic acid species. The mixed mode stationary phase can have a low percentage of ion exchange functional groups such thatmixed mode stationary phase is weakly charged. The nucleic acid species can be retained on the column by interaction with the functional groups.

[0111] At 604, the nucleic acid species can be eluted from the column with an eluent. The eluent can include water and an organic solvent, such as acetonitrile. In particular embodiments, the protein species can be eluted by applying a gradient, such as varying the solvent composition, varying the ionic strength, varying the pH, or any combination thereof.

[0112] At 606, the output of a detector can be recorded over time, and at 608, the detector output can be used to identify or quantify the nucleic acid species present in the sample. In particular embodiments, the detector can be a mass spectrometer.

[0113] Figure 7 is a flow diagram illustrating a method 700 of analyzing a sample containing a plurality of protein species, particularly large proteins. Generally, proteins can have both positively charged groups and negatively charged groups. Further, the groups may behave in a pH dependent manner, shifting between a charged state and a neutral state as the pH is changed. That is, the relative amount of positively charged groups to negatively charged groups can be altered by charging the pH of the solution.

[0114] At 702, the sample is injected into the chromatography column. Depending on the sample, various pre-injection sample preparation steps can be taken. In various embodiments, a solid or semi-solid sample may be ground up and suspended in a solution to extract the protein species. The liquid solution can be separated from the solid material and injected into the column. In other embodiments, the sample can be a liquid sample and can be injected directly onto the column without significant pre-injection sample preparation.

[0115] The column can include a mixed mode stationary phase. Mixed mode stationary phases include functional groups with two or more different properties, such as cation exchange functional groups, anion exchange functional groups, hydrophobic groups, and the like. In particular- embodiments, the mixed mode stationary phase includes hydrophobic groups and ion exchange functional groups, such as either cationicfunctional groups or anionic functional groups to interact with the negatively charged groups of the protein or the positively charged groups of the protein, respectively. The mixed mode stationary phase can have a low percentage of ion exchange functional groups such that mixed mode stationary phase is weakly charged. The protein species can be retained on the column by interaction with the functional groups.

[0116] At 704, the protein species can be eluted from the column with an eluent. The eluent can include water and an organic solvent, such as acetonitrile. In particular embodiments, the protein species can be eluted by applying a gradient, such as varying solvent composition, varying the ionic strength, varying the pH, or any combination thereof. In various embodiments, the eluent can include an acid, such as formic acid, acetic acid, citric acid, other mass spec compatible acids, or any combination thereof.

[0117] At 706, the output of a detector can be recorded over time, and at 708, the detector output can be used to identify or quantify the protein species present in the sample. In particular embodiments, the detector can be a mass spectrometer.

[0118] While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the ail.

[0119] Further, in describing various embodiments, the specification may have presented a method and / or process as a particular’ sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed to the method and / or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the various embodiments.

[0120] Any of the operations that form part of the embodiments described herein arc useful machine operations. The embodiments, described herein, also relate to a device or an apparatus for performing these operations. The systems and methods described herein can be specially constructed for the required purposes or it may be a general purpose computer selectively activated or configured by a computer program stored in the computer. In particular, various general purpose machines may be used with computer programs written in accordance with the teachings herein, or it may be more convenient to construct a more specialized apparatus to perform the required operations.EXAMPLES

[0121] Example 1

[0122] 1695.7 g of water, 10.1 g of sodium dodecyl sulphate (SDS), 169.7 g of acetone and 169.1 g of dioctanoyl peroxide was emulsified using an Ultra Turrax, for 5 minutes followed by homogenization using a high-pressure homogenizer APV GAULIN LAB 60 with a pressure of 30 MPa in the first step and 10 MPa in the second step for 15 minutes.

[0123] After homogenization 531.6 g emulsion was charged with 166.3 g of a monodisperse polystyrene seed suspension (0.63 pm, 8.97 weight % dry content in water).

[0124] After heating to 26°C and stirring for 21 hours 162.7 g of activated seed suspension were charged to 1097.3 g of an emulsion containing 873.3 g water, 4.4 g SDS, 14.5 g styrene, 109.9 g 63% divinylbenzene (DVB) (i.e., 63% DVB and 47% ethyl vinyl benzene isomeric blend), 66.7g 1-octanol and 28.5 g heptane. The mixture was homogenized using an Ultra Turrax for 5 minutes followed by homogenization using a high-pressure homogenizer APV GAULIN LAB 60 with a pressure of 30 MPa in the first step and 10 MPa in the second step for 5 minutes.

[0125] After swelling for 23 hours at 25 °C, 66.7 g 1-octanol was added. After 1 hour 673.9 g water was charged to the reactor and the dispersion was heated to 70°C and polymerized at 70°C for 10 hours, yielding a suspension of particles having a diameter of 2.5pm (SEM)

[0126] 330 g of particle suspension in 1 -liter centrifugal flasks was added methanol and the particles were separated from the liquid phase by centrifugation and the supernatant was discharged. The particles were added methanol and left on a shaker for 15 minutes before separating particles by centrifugation, the washing procedure was repeated once with methanol, five times with BuAc (butyl acetate) and five times with methanol before drying at 50°C in a vacuum oven over night.

[0127] The resulting particles (Phase 1 ) had a specific surface area of 20 m2 / g (BET) and a mean diameter of 2.5pm (SEM) and a particle diameter CV of 2.5% (Coulter Counter).

[0128] Example 2

[0129] 1420.4 g of water, 8.54 g of sodium dodecyl sulphate (SDS), 142.2g of acetone and 142.1 g of dioctanoyl peroxide was emulsified using an Ultra Turrax, for 5 minutes followed by homogenization using a high-pressure homogenizer APV GAULIN LAB 60 with a pressure of 30 MPa in the first step and 10 MPa in the second step for 14 minutes.

[0130] After homogenization 439.8 g emulsion was charged with 58.5 g of a monodisperse polystyrene seed suspension (0.50 pm, 15.7 weight % dry content in water).

[0131] After heating to 26°C and stirring for 21 hours 113.2 g of activated seed suspension were charged to 1154.1 g of an emulsion containing 917.6 g water, 4.5 g SDS, 17.3 g styrene, 132.5 g 63% divinylbenzene (DVB) (i.e., 63% DVB and 47% ethyl vinyl benzene isomeric blend), 57.6g 1 -hexanol and 24.6 g heptane. The mixture was homogenized using an Ultra Turrax for 5 minutes followed by homogenization using a high-pressure homogenizer APV GAULIN LAB 60 with a pressure of 30 MPa in the first step and 10 MPa in the second step for a total of four run-through of the dispersion volume and 245pl 2- (N, N-diethylamino) ethyl methacrylate was added to the mixture.

[0132] After swelling for 22 hours at 25 °C, 57.6 g 1-hexanol was added. After 1 hour 673.9 g water was charged to the reactor and the dispersion was heated to 70°C and polymerized at 70°C for 10 hours, yielding a suspension of particles having a diameter of 2.4pm (CC).

[0133] 450 g of particle suspension in 1 -liter centrifugal flasks was added methanol and the particles were separated from the liquid phase by centrifugation and the supernatant was discharged. The particles were added methanol and left on a shaker for 15 minutes before separating particles by centrifugation, the washing procedure wasrepeated twice with methanol, five times with BuAc four times with methanol and twice with acetone before drying at 50°C in a vacuum oven over night.

[0134] The resulting particles (Phase 2) had a specific surface area of 136 m2 / g (BET), a mean particle diameter of 2.4pm (Coulter Counter), a particle diameter CV of 1.7%, and nitrogen content 0.13 wt% (combustion analysis).

[0135] Example 3

[0136] Porous particles were prepared as in Example 2 using 73 pL of 2-(N,N- Diethylamino)ethyl methacrylate.

[0137] The resulting particles (Phase 3) had a specific surface area of 135 m2 / g (BET), a mean particle diameter of 2.4pm (Coulter Counter), a particle diameter CV of 1.4%, and nitrogen content 0.08 wt% (combustion analysis).

[0138] Example 4

[0139] Porous particles were prepared as in Example 2 using 817 pL of 2-(N,N- Diethylamino)ethyl methacrylate.

[0140] The resulting particles (Phase 4) had a specific surface area of 202 m2 / g (BET), a mean particle diameter of 2.4pm (Coulter Counter), a particle diameter CV of 1.8%, and nitrogen content 0.18 wt% (combustion analysis).

[0141] Example 5

[0142] Porous particles were prepared as in Example 2 using 2.45 mL of 2-(N,N- Diethylamino)ethyl methacrylate.

[0143] The resulting particles (Phase 5) had a specific surface area of 181 m2 / g (BET), a mean particle diameter of 2.4pm (Coulter Counter), a particle diameter CV of 2.1%, and nitrogen content 0.23 wt% (combustion analysis).

[0144] Example 6

[0145] Porous particles were prepared as in Example 2 using 7.35 mL of 2-(N,N- Dicthylamino)cthyl methacrylate.

[0146] The resulting particles (Phase 6) had a specific surface area of 266 m2 / g (BET), a mean particle diameter of 2.4pm (Coulter Counter), a particle diameter CV of 1.8%, and nitrogen content 0.36 wt% (combustion analysis).

[0147] Example 7

[0148] 1419.7 g of water, 8.5 g of sodium dodecyl sulphate (SDS), 142.3g of acetone and 142.1 g of dioctanoyl peroxide was emulsified using an Ultra Turrax, for 5 minutes followed by homogenization using a high-pressure homogenizer APV GAULIN LAB 60 with a pressure of 30 MPa in the first step and 10 MPa in the second step for 10 minutes.

[0149] After homogenization 459.5g emulsion was charged with 61.1 g of a monodisperse polystyrene seed suspension (0.50 pm, 15.7 weight % dry content in water).

[0150] After heating to 26°C and stirring for 21 hours 113.2 g of activated seed suspension were charged to 1153.6 g of an emulsion containing 917.0 g water, 4.5 g SDS, 17.3 g styrene, 132.5 g 63% divinylbenzene (DVB) (i.e., 63% DVB and 47% ethyl vinyl benzene isomeric blend), 57.6g 1-hexanol and 24.6 g heptane. The mixture was homogenized using an Ultra Turrax for 5 minutes followed by homogenization using a high-pressure homogenizer APV GAULIN LAB 60 with a pressure of 30 MPa in the first step and 10 MPa in the second step for 7 minutes.

[0151] After swelling for 23 hours at 25 °C, 735pl methacrylic acid and 57.6 g 1- hexanol was added. After 1 hour 674.1 g water was charged to the reactor and the dispersion was heated to 70°C and polymerized at 70°C for 10 hours, yielding a suspension of particles having a diameter of 2.4pm (Coulter Counter).

[0152] 395 g of particle suspension in 1 -liter centrifugal flasks was added methanol and the particles were separated from the liquid phase by centrifugation and thesupernatant was discharged. The particles were added methanol and left on a shaker for 15 minutes before separating particles by centrifugation, the washing procedure was repeated twice with methanol, five times with BuAc four times with methanol and twice with acetone before drying at 50°C in a vacuum oven over night.

[0153] The resulting particles (Phase 7) had a specific surface area of 188 m2 / g, a mean particle diameter of 2.4pm (Coulter Counter) and a particle diameter CV of 1.8%.

[0154] Example 8

[0155] Porous particles were prepared as in Example 7 using 147 mL of methacrylic acid.

[0156] The resulting particles (Phase 8) had a specific surface area of 151 m2 / g (BET), a mean particle diameter of 2.4pm (Coulter Counter), and a particle diameter CV of 1.5%.

[0157] Example 9

[0158] Porous particles were prepared as in Example 7 using 294 mL of methacrylic acid.

[0159] The resulting particles (Phase 9) had a specific surface area of 164 m2 / g (BET), a mean particle diameter of 2.4pm (Coulter Counter), and a particle diameter CV of 1.7%,

[0160] Example 10

[0161] Porous particles were prepared as in Example 7 using 2.94 mL of methacrylic acid.

[0162] The resulting particles (Phase 10) had a specific surface area of 270 m2 / g (BET), a mean particle diameter of 2.4pm (Coulter Counter), and a particle diameter CV of 1.7%.

[0163] Example 11

[0164] 1432.7 g of water, 8.5 g of sodium dodecyl sulphate (SDS), 1 2 g of acetone and 142 g of dioctanoyl peroxide was emulsified using an Ultra Turrax. for 5 minutes followed by homogenization using a high-pressure homogenizer APV GAULIN LAB 60 with a pressure of 30 MPa in the first step and 10 MPa in the second step for 20 minutes.

[0165] After homogenization 309.5 g emulsion was charged with 41.2 g of a monodisperse polystyrene seed suspension (0.50 pm, 15.7 weight % dry content in water).

[0166] After heating to 26°C and stirring for 20 hours, 113.3 g of activated seed suspension were charged to 1156.4 g of an emulsion containing 919.6 g water, 4.5 g SDS, 17.3 g styrene, 132.7 g 63% divinylbenzene (DVB) (i.e., 63% DVB and 47% ethyl vinyl benzene isomeric blend), 57.6g 1-hexanol and 24.6 g heptane. The mixture was homogenized using an Ultra Turrax for 5 minutes followed by homogenization using a high-pressure homogenizer APV GAULIN LAB 60 with a pressure of 30 MPa in the first step and 10 MPa for a total of four run-through of the dispersion volume.

[0167] After swelling for 20 hours at 25 °C, 57.6 g 1-hexanol was added. After 1 hour 677 g water was charged to the reactor and the dispersion was heated to 70°C and polymerized at 70°C for 10 hours, yielding a suspension of particles having a diameter of 2.38pm (Coulter Counter).

[0168] 350 g of particle suspension in 1 -liter centrifugal flasks was added methanol and the particles were separated from the liquid phase by centrifugation and the supernatant was discharged. The particles were added methanol and left on a shaker for 15 minutes before separating particles by centrifugation, the washing procedure was repeated twice with methanol, five times with BuAc, four times with methanol and twice with acetone before drying at 50°C in a vacuum oven over night.

[0169] The resulting particles (Phase 11) had a specific surface area of 5 m2 / g (BET), a particle mean diameter of 2.38 pm (Coulter Counter) and a particle diameter CV of 1.4%.

[0170] Example 12

[0171] 1432.7 g of water, 8.5 g of sodium dodecyl sulphate (SDS), 1 2 g of acetone and 142 g of dioctanoyl peroxide was emulsified using an Ultra Turrax. for 5 minutes followed by homogenization using a high-pressure homogenizer APV GAULIN LAB 60 with a pressure of 30 MPa in the first step and 10 MPa in the second step for 20 minutes.

[0172] After homogenization 226.5 g emulsion was charged with 54.2 g of a monodisperse polystyrene seed suspension (0.63 pm, 8.80 weight % dry content in water).

[0173] After heating to 26°C and stirring for 20 hours 255.5 g of activated seed suspension were charged to 1015.3 g of an emulsion containing 791.4 g water, 3.9 g SDS, 15.8 g styrene, 120.6 g 63% divinylbenzene (DVB) (i.e., 63% DVB and 47% ethyl vinyl benzene isomeric blend), 58.5 1-hexanol and 25.1 g heptane. The mixture was homogenized using an Ultra Turrax for 5 minutes followed by homogenization using a high-pressure homogenizer APV GAULIN LAB 60 with a pressure of 30 MPa in the first step and 10 MPa for a total of three run-through of the dispersion volume.

[0174] After swelling for 18 hours at 25 °C, 59.1 g 1-hexanol was added. After 1 hour 671 g water was charged to the reactor and the dispersion was heated to 70°C and polymerized at 70°C for 10 hours, yielding a suspension of particles having a diameter of 2.23pm (Coulter Counter).

[0175] 350 g of particle suspension in 1 -liter centrifugal flasks was added methanol and the particles were separated from the liquid phase by centrifugation and the supernatant was discharged. The particles were added methanol and left on a shaker for 15 minutes before separating particles by centrifugation, the washing procedure was repeated twice with methanol, five times with BuAc, four times with methanol and twice with acetone before drying at 50°C in a vacuum oven over night.

[0176] The resulting particles (Phase 12) had a specific surface area of 106 m2 / g (BET), a particle mean diameter of 2.23 pm (Coulter Counter) and a particle diameter CV of 1.8%.

[0177] Example 13

[0178] Dried porous particles from Example 1 (0.5 g) were suspended in 10 mL of a 4:1 mixture of methanol: water. VA-044 azo initiator (0.19 g, Wako Chemicals USA) was added. The mixture was sparged with nitrogen, sealed, and heated at 65 °C for 26 h. The resulting grafted particles were collected on filter paper, washed successively with water, 0.1 M HC1, and methanol, and then dried at 50 °C. The nitrogen content of the product was 0.06 wt%.

[0179] Example 14

[0180] Dried porous particles from Example 1 (0.5 g) were suspended in 10 mL of a 4:1 mixture of methanol: water. VA-044 azo initiator (0.19 g, Wako Chemicals USA) and N-[3-(dimethylamino)propyl]methacrylamide (120 pL, Sigma-Aldrich) were added. The mixture was sparged with nitrogen, sealed, and heated at 65 °C for 26 h. The resulting grafted particles were collected on filter paper, washed successively with water, 0.1 M HC1, and methanol, and then dried at 50 °C. The nitrogen content of the product was 0.10 wt%.

[0181] Example 15

[0182] Dried porous particles from Example 1 (0.5 g) were suspended in 10 mL of a 4:1 mixture of methanol: water. V-501 azo initiator (0.04 g, Wako Chemicals USA) was added. The mixture was sparged with nitrogen, sealed, and heated at 65 °C for 22 h. The resulting grafted particles were collected on filter paper, washed successively with water, 0.1 M HC1, and methanol, and then dried at 50 °C. The nitrogen content of the product was 0.05 wt%.

[0183] Example 16

[0184] Dried porous particles from Example 1 (0.5 g) were suspended in 10 mL of a 1:1 mixture of methanol: water. Ammonium persulfate (0.05 g, Sigma- Aldrich), TMEDA (30 pL, Sigma- Aldrich), and N-[3-(dimethylamino)propyl]methacrylamide (5 pL, Sigma-Aldrich) were added. The mixture was sparged with nitrogen, sealed, and heated at 50 °C for 22 h. The resulting grafted particles were collected on filter paper,washed successively with water, 0.1 M HC1, and methanol, and then dried at 50 °C. The nitrogen content of the product was 0.03 wt%.

[0185] Example 17

[0186] Dried porous particles from Example 1 (0.5 g) were suspended in 10 mL of a 1:1 mixture of acetonitrile: water. Potassium permanganate (0.1 g, JT Baker) was added. The mixture was stirred at ambient temperature for 20 h and then heated at 50 °C for 3 h. The resulting particles were collected on filter paper, washed successively with 1:1 acetonitrile: water, 5% hydrogen peroxide in 0.1 M HC1, and methanol, and then dried at 50 °C.

[0187] Example 18

[0188] Dried porous particles from Example 17 (0.2 g) were suspended in 10 mL of 40% dimethylamine in water (Sigma- Aldrich) and stirred at ambient temperature for 20 h. The resulting particles were collected on filter paper, washed successively with 1:1 acetonitrile: water, 1:1 acetonitrile :2M HC1, and methanol, and then dried at 50 °C. The nitrogen content of the product was 0.30 wt%.

[0189] Example 19

[0190] Dried porous particles from Example 17 (0.2 g) were suspended in a 10 mL mixture of 1:1 acetonitile: water containing 0.05 g disodium phosphate and 0.15g sodium sulfite, and stirred at ambient temperature for 20 h. The resulting particles were collected on filter paper, washed successively with 1:1 acetonitrile: water, 1:1 acetonitrile :2M HC1, and methanol, and then dried at 50 °C. The sulfur content of the product was 0.03 wt%.

[0191] Example 20

[0192] Dried porous particles from Example 1 (1 g) were suspended in 10 mL acetonitrile. V-65 azo initiator (0.02 g, Wako Chemicals USA), 55% divinylbenzene (250 pL, Sigma- Aldrich), and glycidyl methacrylate (5 pL, Sigma- Aldrich) were added. The mixture was sparged with nitrogen, sealed, and heated at 55 °C for 24 h. The resultinggrafted particles were collected on filter paper, washed successively with acetonitrile and methanol, and then dried at 50 °C.

[0193] Example 21

[0194] Dried porous particles from Example 20 (0.5 g) were suspended in 10 mL of 50% trimethylamine in water (Sigma- Aldrich) and stirred at ambient temperature for 24 h. The resulting particles were collected on filter paper, washed successively with 1:1 acetonitrile: water, 1:1 acetonitrile :2M HC1, and methanol, and then dried at 50 °C. The nitrogen content of the product was 0.24 wt%.

[0195] Example 22

[0196] Dried porous particles from Example 20 (0.5 g) were suspended in a 10 mL mixture of 1:1 2-propanol:0.1M NaOH. Sodium 3-mercapto-l -propanesulfonate (2.4 g, Sigma-Aldrich) was added. The mixture was stirred at 75°C for 24 h. The resulting particles were collected on filter paper, washed successively with 1:1 acetonitrile: water, 1:1 acetonitrile :2M HC1, and methanol, and then dried at 50 °C. The sulfur content of the product was 0.08 wt%.

[0197] Example 23

[0198] A 2.0 pL sample of oligonucleotides consisting of (GATC)3-IO is separated on an LC system using a 2.1 x 50 mm column at 60°C and a flow rate of 0.4 mL / min and a UV (260 nm) detector. Eluent conditions are 100% Mobile Phase A at 0 minutes to 91% Mobile Phase A with 9% Mobile Phase B at 9 minutes. Mobile Phase A is 0.1 M triethylammonium acetate (TEAA) at pH 7 and Mobile Phase B is acetonitrile. FIG 8A shows the chromatogram using Phase 12 as a stationary phase. FIG 8B shows the chromatogram using Phase 2 as a stationary phase. FIG 8C shows the chromatogram using Phase 4 as a stationary phase. FIG 8D shows the chromatogram using Phase 5 as a stationary phase.

[0199] FIG 12 shows a selectivity vs capacity analysis comparing Phase 2 (0.15% amine), Phase 4 (0.5% amine), Phase 5 (1.5% amine), and Phase 6 (4.5% amine)compared to Phase 12. As the % amine increases, the separation between compounds (selectivity) increases.

[0200] Example 24

[0201] A 2.0 pL sample of oligonucleotides consisting of (GATC)3-io is separated on an LC system using a 2.1 x 50 mm column at 60°C and a flow rate of 0.4 mL / min and a UV (260 nm) detector. Eluent conditions are 100% Mobile Phase A at 0 minutes to 91% Mobile Phase A with 9% Mobile Phase B at 9 minutes. Mobile Phase A is 0.1 M ammonium acetate at pH 7 and Mobile Phase B is acetonitrile. FIG 9A shows the chromatogram using Phase 12 as a stationary phase. FIG 9B shows the chromatogram using Phase 4 as a stationary phase.

[0202] Example 25

[0203] A 0.5 pL sample of containing Ribonuclease A, Cytochrome C, Lysozyme, and mAb is separated on an LC system using a 2.1 x 50 mm column at 80°C and a flow rate of 0.5 mL / min and a UV (280 nm) detector. Eluent conditions are 90% Mobile Phase A and 10% Mobile Phase B at 0 minutes to 60% Mobile Phase A with 40% Mobile Phase B at 2.5 minutes and maintaining 60% Mobile Phase A with 40% Mobile Phase B until 4.0 minutes. Mobile Phase A is 0.1% formic and Mobile Phase B is 0.1% formic acid, 90% acetonitrile, and 9.9% H2O. FIG 10A shows the chromatogram using Phase 12 as a stationary phase. FIG 10B shows the chromatogram using Phase 8 as a stationary phase. FIG 10C shows the chromatogram using Phase 9 as a stationary phase. FIG 10D shows the chromatogram using Phase 7 as a stationary phase.

[0204] Example 26

[0205] A 0.5 pL sample of containing Ribonuclease A, Cytochrome C, Lysozyme, and mAb is separated on an LC system using a 2.1 x 50 mm column at 80°C and a flow rate of 0.5 mL / min and a UV (280 nm) detector. Eluent conditions are 90% Mobile Phase A and 10% Mobile Phase B at 0 minutes to 60% Mobile Phase A with 40% Mobile Phase B at 2.5 minutes and maintaining 60% Mobile Phase A with 40% Mobile Phase B until 4.0 minutes. Mobile Phase A is 0.1% trifluoroacetic acid (TFA) and Mobile PhaseB is 0.1 % trifluoroacetic acid, 90% acetonitrile, and 9.9% H2O. FIG 11 A shows the chromatogram using Phase 12 as a stationary phase. FIG 1 IB shows the chromatogram using Phase 8 as a stationary phase. FIG 11C shows the chromatogram using Phase 9 as a stationary phase. FIG 1 ID shows the chromatogram using Phase 7 as a stationary phase.

Claims

WHAT IS CLAIMED IS:

1. A chromatographic media for separating biopolymers, the chromatographic media having hydrophobic and ionic retention modes, the chromatographic media comprising: porous substrate particles including a hydrophobic polymer resin and ion exchange functional groups copolymerized with the hydrophobic polymer resin, wherein the ion exchange functional groups are not greater than about 5 mol% of the porous substrate particles.

2. The chromatographic media for separating biopolymers of claim 1 wherein the ion exchange functional groups are not greater than about 4 mol% of the porous substrate particles.

3. The chromatographic media for separating biopolymers of claim 2 wherein the ion exchange functional groups are not greater than about 3 mol% of the porous substrate particles.

4. The chromatographic media for separating biopolymers of claim 3 wherein the ion exchange functional groups are not greater than about 2 mol% of the porous substrate particles.

5. The chromatographic media for separating biopolymers of claim 1 wherein the ion exchange functional groups are not less than about 0.01 mol% of the porous substrate particles.

6. The chromatographic media for separating biopolymers of claim 1 wherein the hydrophobic polymer resin includes vinylbenzene or derivatives thereof.

7. The chromatographic media for separating biopolymers of claim 6 wherein the hydrophobic polymer resin includes ethylvinylbenzene.

8. The chromatographic media for separating biopolymers of claim 1 wherein the ion exchange functional groups include functional groups with positive charge.

9. The chromatographic media for separating biopolymers of claim 8 wherein the ion exchange functional groups originate from H2C=C(CH3)CO2RINR2R3 where Ri is an alkyl group and R2 and R3 are H or alkyl groups.

10. The chromatographic media for separating biopolymers of claim 9 wherein the ion exchange functional groups include 2-(N,N-Diethylamino)ethyl methacrylate.

11. The chromatographic media for separating biopolymers of claim 8 wherein the porous substrate particles include between about 0.001 and about 0.5 wt% nitrogen.

12. The chromatographic media for separating biopolymers of claim 11 wherein the porous substrate particles include between about 0.003 and about 0.4 wt% nitrogen.

13. The chromatographic media for separating biopolymers of claim 12 wherein the porous substrate particles include between about 0.001 and about 0.3 wt% nitrogen.

14. The chromatographic media for separating biopolymers of claim 1 wherein the ion exchange functional groups include functional groups with negative charge.

15. The chromatographic media for separating biopolymers of claim 11 wherein the ion exchange functional groups include methacrylic acid or acrylic acid.

16. The chromatographic media for separating biopolymers of claim 1 wherein porous substrate particles are monodisperse having a particle diameter coefficient of variation (CV) % of less than 15% when measured by Coulter Counter analysis.

17. The chromatographic media for separating biopolymers of claim 16 wherein the particle diameter coefficient of variation (CV) % is less than 10% when measured by Coulter Counter analysis.

18. The chromatographic media for separating biopolymers of claim 17 wherein the particle diameter coefficient of variation (CV) % is less than 5% when measured by Coulter Counter analysis.

19. The chromatographic media for separating biopolymers of claim 18 wherein the particle diameter coefficient of variation (CV) % is less than 2.5% when measured by Coulter Counter analysis.

20. The chromatographic media for separating biopolymers of claim 1 wherein the porous substrate particles have a specific surface area of at least about 10 m2 / g.

21. The chromatographic media for separating biopolymers of claim 20 wherein the specific surface area is at least about 50 m2 / g.

22. The chromatographic media for separating biopolymers of claim 20 wherein the specific surface area is not greater than about 250 m2 / g.

23. The chromatographic media for separating biopolymers of claim 1, wherein the biopolymers include oligonucleotides, polynucleotides, peptides, proteins, or any combination thereof.

24. A method of preparing a chromatographic media having hydrophobic and ionic retention modes comprising: copolymerizing a hydrophobic polymer resin monomer and ion exchange functional groups monomer to form monodisperse porous particles, wherein the ion exchange functional groups monomer is in an amount of less than 5 mol%.

25. The method of claim 24 wherein the ion exchange functional groups are not greater than about 4 mol% of the porous substrate particles.

26. The method of claim 25 wherein the ion exchange functional groups are not greater than about 3 mol% of the porous substrate particles.

27. The method of claim 26 wherein the ion exchange functional groups are not greater than about 2 mol% of the porous substrate particles.

28. The method of claim 24 wherein the ion exchange functional groups are not less than about 0.01 mol% of the porous substrate particles.

29. The method of claim 24 wherein the porous substrate particles include between about 0.001 and about 0.5 wt% nitrogen.

30. The method of claim 29 wherein the porous substrate particles include between about 0.003 and about 0.4 wt% nitrogen.

31. The method of claim 30 wherein the porous substrate particles include between about 0.001 and about 0.3 wt% nitrogen.

32. The method of claim 24 wherein the hydrophobic polymer resin includes vinylbenzene or derivatives thereof.

33. The method of claim 25 wherein the hydrophobic polymer resin includes ethylvinylbenzene.

34. The method of claim 24 wherein the ion exchange functional groups include functional groups with positive charge.

35. The method of claim 34 wherein the ion exchange functional groups originate from H2C=C(CH3)CO2RINR2R3 where Ri is an alkyl group and R and R3 are H or alkyl groups.

36. The method of claim 35 wherein the ion exchange functional groups include 2-(N,N-Diethylamino)ethyl methacrylate.

37. The method of claim 24 wherein the ion exchange functional groups include functional groups with negative charge.

38. The method of claim 37 wherein the ion exchange functional groups include methacrylic acid or acrylic acid.

39. The method of claim 24 wherein porous substrate particles are monodisperse having a particle diameter coefficient of variation (CV) % of less than 15% when measured by Coulter Counter analysis.

40. The method of claim 39 wherein the particle diameter coefficient of variation (CV) % is less than 10% when measured by Coulter Counter analysis.

41. The method of claim 40 wherein the particle diameter coefficient of variation (CV) % is less than 5% when measured by Coulter Counter analysis.

42. The method of claim 41 wherein the particle diameter coefficient of variation (CV) is less than 2.5% when measured by Coulter Counter analysis.

43. The method of claim 24 wherein the porous substrate particles have a specific surface area of at least about 10 m2 / g.

44. The method of claim 43 wherein the specific surface area is at least about 50 m2 / g.

45. The method of claim 43 wherein the specific surface area is not greater than about 250 m2 / g.

46. The method of claim 24 wherein the biopolymers include oligonucleotides, polynucleotides, peptides, proteins, or any combination thereof.

47. A chromatographic media for separating biopolymers, the chromatographic media having hydrophobic and ionic retention modes, the chromatographic media comprising: porous substrate particles including a hydrophobic polymer resin; and ion exchange functional groups grafted to the surface of the porous substrate, wherein the ion exchange functional groups are up to 5 mol% of the chromatographic media.

48. The chromatographic media for separating biopolymers of claim 47 wherein the ion exchange functional groups arc not greater than about 4 mol% of the porous substrate particles.

49. The chromatographic media for separating biopolymers of claim 48 wherein the ion exchange functional groups are not greater than about 3 mol% of the porous substrate particles.

50. The chromatographic media for separating biopolymers of claim 49 wherein the ion exchange functional groups are not greater than about 2 mol% of the porous substrate particles.

51. The chromatographic media for separating biopolymers of claim 47 wherein the ion exchange functional groups are not less than about 0.01 mol% of the porous substrate particles.

52. The chromatographic media for separating biopolymers of claim 47 wherein the porous substrate particles include between about 0.001 and about 0.5 wt% nitrogen.

53. The chromatographic media for separating biopolymers of claim 52 wherein the porous substrate particles include between about 0.003 and about 0.4 wt% nitrogen.

54. The chromatographic media for separating biopolymers of claim 53 wherein the porous substrate particles include between about 0.001 and about 0.3 wt% nitrogen.

55. The chromatographic media for separating biopolymers of claim 47 wherein the hydrophobic polymer resin includes vinylbenzene or derivatives thereof.

56. The chromatographic media for separating biopolymers of claim 48 wherein the hydrophobic polymer resin includes ethylvinylbenzene.

57. The chromatographic media for separating biopolymers of claim 47 wherein the ion exchange functional groups include functional groups with positive charge.

58. The chromatographic media for separating biopolymers of claim 57 wherein the ion exchange functional groups originate fromH2C=C(CH3)CO2R i NR2R3 where Ri is an alkyl group and R2 and R3 are H or alkyl groups.

59. The chromatographic media for separating biopolymers of claim 58wherein the ion exchange functional groups include 2-(N,N-Diethylamino)ethyl methacrylate.

60. The chromatographic media for separating biopolymers of claim 47 wherein the ion exchange functional groups include functional groups with negative charge.

61. The chromatographic media for separating biopolymers of claim 60 wherein the ion exchange functional groups include methacrylic acid or acrylic acid.

62. The chromatographic media for separating biopolymers of claim 47 wherein porous substrate particles are monodisperse having a particle diameter coefficient of variation (CV) % of less than 15% when measured by Coulter Counter analysis.

63. The chromatographic media for separating biopolymers of claim 62 wherein the particle diameter coefficient of variation (CV) % is less than 10% when measured by Coulter Counter analysis.

64. The chromatographic media for separating biopolymers of claim 63 wherein the particle diameter coefficient of variation (CV) % is less than 5% when measured by Coulter Counter analysis.

65. The chromatographic media for separating biopolymers of claim 64 wherein the particle diameter coefficient of variation (CV) % is less than 2.5% when measured by Coulter Counter analysis.

66. The chromatographic media for separating biopolymers of claim 47 wherein the porous substrate particles have a specific surface area of at least about 10 m2 / g.

67. The chromatographic media for separating biopolymers of claim 66 wherein the specific surface area is at least about 50 m2 / g.

68. The chromatographic media for separating biopolymers of claim 66 wherein the specific surface area is not greater than about 250 m2 / g.

69. The chromatographic media for separating biopolymers of claim 47 wherein the biopolymers include oligonucleotides, polynucleotides, peptides, proteins, or any combination thereof.

70. A method of preparing a chromatographic media having hydrophobic and ionic retention modes comprising: polymerizing a hydrophobic polymer resin monomer to form porous particles, grafting an ion exchange functional group monomer to the surface of the monodisperse, porous particles to form weakly charged porous substrate particles wherein the ion exchange functional groups monomer is in an amount of less than 5 mol%.

71. The method of claim 70 wherein the ion exchange functional groups are not greater than about 4 mol% of the porous substrate particles.

72. The method of claim 71 wherein the ion exchange functional groups are not greater than about 3 mol% of the porous substrate particles.

73. The method of claim 72 wherein the ion exchange functional groups are not greater than about 2 mol% of the porous substrate particles.

74. The method of claim 70 wherein the ion exchange functional groups are not less than about 0.01 mol% of the porous substrate particles.

75. The method of claim 70 wherein the porous substrate particles include between about 0.001 and about 0.5 wt% nitrogen.

76. The method of claim 75 wherein the porous substrate particles include between about 0.003 and about 0.4 wt% nitrogen.

77. The method of claim 76 wherein the porous substrate particles include between about 0.001 and about 0.3 wt% nitrogen.

78. The method of claim 70 wherein the hydrophobic polymer resin monomer includes vinylbenzene or derivatives thereof.

79. The method of claim 71 wherein the hydrophobic polymer resin monomer includes ethylvinylbenzene.

80. The method of claim 70 wherein the ion exchange functional group monomer include functional groups with positive charge.

81. The method of claim 80 wherein the ion exchange functional groups originate from H2C=C(CH3)CO2RINR2R3 where Ri is an alkyl group and Ri and R3 are H or alkyl groups.

82. The method of claim 81 wherein the ion exchange functional group monomer include 2-(N,N-Diethylamino)ethyl methacrylate.

83. The method of claim 70 wherein the ion exchange functional group monomer includes functional groups with negative charge.

84. The method of claim 83 wherein the ion exchange functional group monomer include methacrylic acid or acrylic acid.

85. The method of claim 70 wherein porous substrate particles are monodispcrsc having a particle diameter coefficient of variation (CV) of less than 15% when measured by Coulter Counter analysis.

86. The method of claim 85 wherein the particle diameter coefficient of variation (CV) is less than 10% when measured by Coulter Counter analysis.

87. The method of claim 86 wherein the particle diameter coefficient of variation (CV) % is less than 5% when measured by Coulter Counter analysis.

88. The method of claim 87 wherein the particle diameter coefficient of variation (CV) is less than 2.5% when measured by Coulter Counter analysis.

89. The method of claim 70 wherein the porous substrate particles have a specific surface area of at least about 10 m2 / g.

90. The method of claim 89 wherein the specific surface area is at least about 50 m2 / g.

91. The method of claim 89 wherein the specific surface area is not greater than about 250 m2 / g.

92. The method of claim 70 wherein the biopolymers include oligonucleotides, polynucleotides, peptides, proteins, or any combination thereof.

93. A method comprising: loading a biopolymer sample onto a chromatographic media having hydrophobic and ionic retention modes, the chromatographic media including weakly charged porous particles, the weakly charged porous particles including hydrophobic polymer resin subunits and ion exchange functional group subunits, the ion exchange functional group subunits being less than 5 mol% of the weakly charged porous particles, the biopolymer sample containing a plurality of biopolymer species;eluting the biopolymer sample from the chromatographic media by applying gradient of solvent conditions to the chromatographic media to cause the separation of the plurality of biopolymers species.

94. The method of claim 93 wherein the ion exchange functional groups are not greater than about 4 mol% of the porous substrate particles.

95. The method of claim 94 wherein the ion exchange functional groups are not greater than about 3 mol% of the porous substrate particles.

96. The method of claim 95 wherein the ion exchange functional groups are not greater than about 2 mol% of the porous substrate particles.

97. The method of claim 93 wherein the ion exchange functional groups are not less than about 0.01 mol% of the porous substrate particles.

98. The method of claim 93 wherein the porous substrate particles include between about 0.001 and about 0.5 wt% nitrogen.

99. The method of claim 98 wherein the porous substrate particles include between about 0.003 and about 0.4 wt% nitrogen.

100. The method of claim 99 wherein the porous substrate particles include between about 0.001 and about 0.3 wt% nitrogen.

101. The method of claim 93 wherein solvent does not include an ion pairing reagent.

102. The method of claim 93 wherein the gradient of solvent conditions includes varying the solvent, varying the ionic strength, varying the pH, or any combination thereof.

103. The method of claim 102 wherein varying the solvent includes increasing or decreasing the amount of an organic solvent.

104. The method of claim 93 wherein the biopolymers are amino acid biopolymers including peptides, proteins, or any combination thereof.

105. The method of claim 104 wherein the ionic retention modes are negatively charged.

106. The method of claim 104 the mobile phase includes an acid.

107. The method of claim 106 the acid includes formic acid, acetic acid, citric acid, other mass spec compatible acids, or any combination thereof.

108. The method of claim 104 wherein the ion exchange functional group subunits include methacrylic acid or acrylic acid.

109. The method of claim 93 wherein the biopolymers are nucleic acid biopolymers including oligonucleotides, polynucleotides, or any combination thereof.

110. The method of claim 109 wherein the ionic retention modes are positively charged.

111. The method of claim 109 wherein the mobile phase includes ammonium acetate, ammonium bicarbonate, ammonium citrate, ammonium, other mass spectrometry compatible buffers, or any combination thereof.

112. The method of claim 109 wherein the ion exchange functional groups originate from H2C=C(CH3)CO2RINR2R3 where Ri is an alkyl group and Ri and R3 are H or alkyl groups.

113. The method of claim 112 wherein the ion exchange functional group subunits include 2-(N,N-Diethylamino)ethyl methacrylate.

114. The method of claim 93 further comprising detecting one or more of the biopolymer species by mass spectroscopy.

115. The method of claim 93 wherein the hydrophobic polymer resin includes vinylbenzene or derivatives thereof.

116. The method of claim 115 wherein the hydrophobic polymer resin includes cthylvinylbcnzcnc.

117. The method of claim 93 wherein porous substrate particles are monodisperse having a particle diameter coefficient of variation (CV) % of less than 15% when measured by Coulter Counter analysis.

118. The method of claim 117 wherein the particle diameter coefficient of variation (CV) %v is less than 10% when measured by Coulter Counter analysis.

119. The method of claim 118 wherein the particle diameter coefficient of variation (CV) % is less than 5% when measured by Coulter Counter analysis.

120. The method of claim 119 wherein the particle diameter coefficient of variation (CV) % is less than 2.5% when measured by Coulter Counter analysis.

121. The method of claim 93 wherein the porous substrate particles have a specific surface area of at least about 10 m2 / g.

122. The method of claim 121 wherein the specific surface area is at least about 50 m2 / g.

123. The method of claim 121 wherein the specific surface area is not greater than about 250 m2 / g.

124. The method of claim 93 wherein the biopolymers include oligonucleotides, polynucleotides, peptides, proteins, or any combination thereof.

Images