Hybrid bonded chromatography materials

Abstract

The present disclosure relates to hybrid bonded siliceous materials. The materials of the present disclosure exhibit enhanced base stability and reduced baseline noise. Thus, the materials disclosed herein are suitable for use across a range of chromatography applications, including size exclusion chromatography and reverse phase chromatography.

Description

Technical Field

[0001] This technology relates to chromatographic materials having a silica-like surface. More specifically, this technology relates to chromatographic materials having a bonded phase composed of two or more silane compounds. Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 516,197, filed July 28, 2023, which is incorporated herein by reference in its entirety. Background Technology

[0003] Liquid chromatography (LC) is an analytical technique widely used throughout the pharmaceutical, biotechnology, and chemical industries. LC involves two phases: a stationary phase and a mobile phase. Typically, the stationary phase contains particles packed into the column, and the mobile phase allows the sample to flow across the stationary phase. Particulate silica is the most widely used stationary phase material due to its mechanical strength, customizable pore structure, and diversity of surface chemistry.

[0004] However, silica particles degrade over time due to the lack of stability of the base particles and their susceptibility to hydrolysis of the Si-O-Si bonds when exposed to high pH levels. These limitations significantly impact the practicality of silica-based particles in liquid chromatography applications.

[0005] A key application of liquid chromatography is size exclusion chromatography (SEC), which is frequently used to analyze the size heterogeneity of large bioanalytes, such as adeno-associated virus (AAV), mRNA, plasmids, and lipid nanoparticles. This analysis relies on the separation of analytes within the sample by size and the use of multi-angle light scattering measurements. However, due to the aforementioned limitations, it is known that these materials shed nanoscale silicate particles, which can directly and / or indirectly impede light scattering, thereby confounding the analytical results.

[0006] Therefore, there is a need in the art for silica-based chromatographic materials that have broad functionality and improved stability at high pH levels. Summary of the Invention

[0007] Generally, this technology relates to a hybrid bonded material for use in liquid chromatography applications. Therefore, in one aspect, a chromatographic material comprising particles having a silica surface, wherein the silica surface consists of a bonded phase formed by at least two silane compounds, wherein one of the at least two silane compounds is a dipoleal hybrid silane comprising two indirectly connected silica atoms, and wherein one of the at least two silane compounds is a functionalized silane. In some embodiments, the particles are polymer particles, silica particles, or inorganic-organic hybrid particles.

[0008] In some implementations, the bipedal hybrid silane is selected from the group consisting of: , and R1 is independently chlorine, methoxy, or ethoxy; R2 is independently alkyl, methoxy, or chlorine; and n and m are independently 1 to 4.

[0009] In some implementations, the functionalized silane is selected from the group consisting of: , , and R1 is independently chlorine, methoxy, or ethoxy; R2 is independently alkyl, benzyl, methoxy, or chlorine; R3 is independently alkyl, methoxy, ethoxy, or chlorine; R4 is H or benzene; R5 is hydroxyl or methoxy; n is 3, 7, or 17; m is 0 or 1; and p is an integer from 1 to 12.

[0010] In some embodiments of this technology, the bipedal hybrid silane and the functionalized silane are fed in a molar ratio of about 1:1, about 1:2, about 1:4, or about 1:10. In some embodiments, the bipedal hybrid silane and the functionalized silane are fed in a molar ratio of about 1:2 or about 1:4. In some embodiments, the surface coverage of the functionalized silane is the theoretical maximum surface coverage (μmol / m²) of the functionalized silane. 2 60% to 250%.

[0011] In one respect, functionalized silanes are Wherein R1 and R3 are ethoxy groups; R5 is a hydroxyl group; and p is 8 to 12. In some embodiments, the surface coverage of the functionalized silane is about 0.8 μmol / m². 2 Approximately 3.45 μmol / m 2 between.

[0012] In some embodiments, the chromatographic material has a pore size between about 90 Å and 5000 Å. In some embodiments, the chromatographic material has a pore size between about 100 Å and 400 Å. In some embodiments, the chromatographic material has a pore size between about 400 Å and 5000 Å. In some embodiments, the chromatographic material has a pore size between about 600 Å and 2000 Å. In some embodiments, the chromatographic material has a pore size between about 1000 Å and 2500 Å.

[0013] In some embodiments, the bipod hybrid silane and functional silane of the chromatographic material are present in a molar ratio between 0.1:1 and 3:1 (bipod hybrid silane: functional silane). In some embodiments, the bipod hybrid silane and functional silane are present in a molar ratio of about 0.1:1. In some embodiments, the bipod hybrid silane and functional silane are present in a molar ratio of about 0.4:1. In some embodiments, the bipod hybrid silane and functional silane are present in a molar ratio of about 1:1. In some embodiments, the bipod hybrid silane and functional silane are present in a molar ratio of about 1.3:1.

[0014] In some embodiments, the chromatographic material is resistant to hydrolytic corrosion, as detected by minimal change in separation performance before and after exposure to hydrolytic conditions. In some embodiments, the chromatographic material has low baseline noise as measured by a multi-angle light scattering (MALS) detector.

[0015] In one aspect, the technology includes a chromatographic column comprising the chromatographic material disclosed herein. In another aspect, the technology includes a chromatographic apparatus comprising a chromatographic column comprising the chromatographic material disclosed herein, a column injector positioned upstream of the chromatographic column, and a conduit fluidly connected to and located downstream of the chromatographic column. In some embodiments, the chromatographic apparatus further includes a detector fluidly connected to and located downstream of the chromatographic column. In some embodiments, the detector is a multi-angle light scattering (MALS) detector or an ultraviolet detector. In some embodiments, the chromatographic apparatus is suitable for size exclusion chromatography or reversed-phase chromatography. In some embodiments, the chromatographic material is resistant to hydrolytic corrosion, as detected by MALS noise and / or by separating protein and / or nucleoside samples. In some embodiments, the MALS noise is measured as peak-to-peak noise in μV, and wherein the peak-to-peak noise is ≤800 μV. Attached Figure Description

[0016] This technology will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings.

[0017] Figure 1 This is an illustration of particles having a bonded phase formed from bipedal hybrid silanes and functionalized silanes according to an embodiment of the present technology.

[0018] Figures 2A to 2D Four types of particles are depicted graphically, distinguished by the functionalized silanes used in the bonded phase. Figure 2A It contains PEG-functionalized silane. Figure 2B It contains diol-functionalized silane. Figure 2C Contains phenyl or biphenyl-functionalized silanes. Figure 2D Contains C4, C8 or C18 functionalized silanes.

[0019] Figures 3A to 3C The improved alkali stability and separation provided by the particles of this technology are demonstrated by comparing the separation performance before and after exposure to alkaline conditions. Figure 3A The method of isolating thyroglobulin, IgG, BSA, myoglobin and uracil using only PEG-functionalized silanes is demonstrated. Figure 3B and Figure 3C The hybrid-bonded materials using this technology are shown to separate thyroglobulin, IgG, BSA, myoglobin, and uracil, with the difference between these hybrid-bonded materials being their surface coverage.

[0020] Figure 4 The reduced light scattering noise provided by the particles of this technology is demonstrated, as detected by multi-angle light scattering (MALS).

[0021] Figures 5A to 5B It shows a comparison with the commercial column ( Figure 5B This technique is used to separate DNA fragments from hybrid-bonded particles. Figure 5A ).

[0022] Figures 6A to 6B It shows a comparison with the commercial column ( Figure 6B The hybrid-bonded particle separation AAV capsid using this technology ( Figure 6A ).

[0023] Figure 7 The hybridized particles used in this technique are shown to separate double-stranded and single-stranded DNA.

[0024] Figures 8A to 8B The hybrid-bonded particles used in this technique are shown to separate two lipid nanoparticle samples (LNP sample #1). Figure 8A And LNP sample #2, Figure 8B ).

[0025] Figures 9A to 9C Example proton NMR of alkaline-digested particles of this technique is provided. 1 H NMR). Figure 9A The spectra of silica particles with BTEE / PEG bonds are provided. Figure 9B The spectra of particles containing only PEG-bonded silica are provided. Figure 9C It provides potential hydrolysis products. Detailed Implementation

[0026] To facilitate understanding of this technology, certain terms are first defined. Furthermore, it should be noted that whenever values ​​or ranges of parameters are listed, the intermediate values ​​and ranges listed are also intended to be part of this disclosure. Unless otherwise defined, the word "about" means ±5%. It should also be noted that, as used herein and in the claims, the singular forms "an" and "the" include plural references unless the context clearly indicates otherwise.

[0027] definition

[0028] As used herein, the term "bipod hybrid silane" refers to a silane molecule containing two silicon atoms indirectly connected by Si-C and C-C bonds.

[0029] As used herein, the term "functionalized silane" refers to a silane molecule containing a functional group. The functional group imparts one or more chromatographic functionalities. For example, functionalized silanes may contain polyethylene glycol (PEG) groups.

[0030] As used herein, the term "bonded phase" refers to a composition bonded to the surface of particles. In a preferred embodiment, the bonded phase comprises a binary composition of a silane that imparts a bipedal hybrid region and a functional region. The term "bipedal hybrid region" refers to a portion of the bonded phase comprising a bipedal hybrid silane. The term "functional region" refers to a portion of the bonded phase comprising a functionalized silane.

[0031] As used herein, the term "hybridized" refers to a bonded phase that includes a bipedal hybrid region containing Si-C and CC bonds.

[0032] As used herein, the term "surface coverage" refers to the density of the bonded phase present on the surface of particles. In the context of this art, surface coverage may refer to the density of functional regions (i.e., functionalized silanes) within the bonded phase. Surface coverage can be calculated using weight loss from thermogravimetric analysis (TGA) and the percentage of carbon in the particles as measured by elemental analysis. Unless otherwise indicated, surface coverage is expressed per m³. 2 The particle surface area (μmol) of silane compounds was measured.

[0033] As used herein, the term "hydrolytic corrosion" refers to the hydrolysis of covalent bonds upon exposure to certain conditions. This can include, for example, the hydrolysis of Si-O-Si bonds upon exposure to alkaline conditions. In the context of silica-based particles, hydrolytic corrosion can lead to silicate shedding. These silicates can hinder downstream analysis that relies on light scattering measurements.

[0034] The term "non-porous particle" refers to particles having a pore volume of less than 0.05 cc / g. The term "porous particle" refers to particles having a pore volume of greater than 0.05 cc / g, and more preferably between 0.1 cc / g and 1.5 cc / g or 2 cc / g. The pore volume and size are determined using methods known in the art, including the nitrogen Brauner-Emmett-Teller (BET) theory or the mercury porosity method.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0036] Material

[0037] This technology relates to hybrid-bonded materials that exhibit increased stability when exposed to hydrolytic conditions such as high pH. Due to the improved stability, the hybrid-bonded materials of this disclosure result in reduced baseline noise when used in chromatographic applications. The improved stability and reduced baseline noise of the hybrid-bonded materials of this technology, compared to materials known in the art, also allow for their use in a wider range of chromatographic applications.

[0038] The material of this technology comprises a bonded phase containing at least two different silane compounds: a bipedal hybrid silane and a functionalized silane. The bipedal hybrid silane contains at least two silicon atoms indirectly connected via Si-C and C-C bonds, thereby creating a bipedal hybrid region. This bipedal hybrid region contains inherently base-stable Si-C and C-C bonds and also provides a hydrophobic microenvironment that shields the underlying Si-O-Si bonds on the particle surface. The functionalized silane provides the functional regions of the bonded phase and is connected via the bipedal hybrid region. These functional regions endow the particles with one or more advantageous chromatographic properties, thereby allowing their use in a range of chromatographic applications.

[0039] In one aspect of this technology, the bipedal hybrid silane is selected from the group consisting of: , and ;in

[0040] R1 can be chlorinated, methoxylated, or ethoxylated independently;

[0041] R2 is independently alkyl, methoxy, ethoxy, or chlorine; and

[0042] n and m are each independently 1 to 4.

[0043] In one aspect of this technology, the functionalized silane is selected from the group consisting of: , , and R1 is independently chloro, methoxy, or ethoxy;

[0044] R2 can be alkyl, benzyl, methoxy, ethoxy, or chlorine, each independently.

[0045] R3 can be alkyl, methoxy, ethoxy, or chlorine, each independently.

[0046] R4 is H or benzene;

[0047] R5 is a hydroxyl or methoxy group;

[0048] n is 3, 7, or 17;

[0049] m is 0 or 1; and

[0050] p is an integer from 1 to 12.

[0051] As will be readily understood by those skilled in the art, the functional groups of a functionalized silane determine the chromatographic properties of the resulting material. In some embodiments, the functionalized silane may impart properties favorable to size exclusion chromatography. In other embodiments, the functionalized silane may impart properties favorable to reversed-phase chromatography or ion-exchange chromatography. In some embodiments, two or more different functionalized silanes are used.

[0052] Figure 1 An exemplary particle of the present technology is illustrated. A bonded phase 110 is applied to the particle 100 below. The bonded phase 110 comprises at least one bipod hybrid silane and at least one functionalized silane. Functional groups 120 provided by the functionalized silane impart chromatographic properties to the particle. The bonded phase 110 can be applied to a range of particle types.

[0053] The particle size can range from 1 μm to 100 μm, and can be porous, surface-porous, or non-porous. The term "surface-porous" refers to particles that contain a solid core surrounded by a porous shell.

[0054] While it is necessary for the particles to have a silica surface, this silica surface can be inherent or acquired. In the former case, the particles can be porous or non-porous silica particles or inorganic-organic hybrid particles. Alternatively, particles having an acquired silica surface include polymer particles with a surface coated with a silica layer or a hybrid inorganic-organic layer.

[0055] Therefore, in some respects, particle 100 can be porous silica particles, non-porous silica particles, porous inorganic-organic hybrid particles, non-porous inorganic-organic hybrid particles, or polymer particles.

[0056] In some embodiments, the porous particles may have an average pore size between 90 Å and 5000 Å. In some embodiments, the porous particles may have an average pore size between 90 Å and 400 Å. In some embodiments, the porous particles may have an average pore size between 400 Å and 5000 Å. In some embodiments, the porous particles may have an average pore size between 600 Å and 2000 Å. In some embodiments, the porous particles may have an average pore size between 1000 Å and 2500 Å.

[0057] Figures 2A to 2D Exemplary particles of this technology are provided. Figure 2A The image shows particles in which the functionalized silane contains polyethylene glycol (PEG) groups. Figure 2B The image shows particles in which the functionalized silane contains diol groups. Figure 2C Particles in which the functionalized silane contains phenyl or biphenyl groups are shown. Figure 2D Particles containing C4, C8, or C18 groups of functionalized silane are shown. For each particle, the hybridization of the bonded phase provides a hydrophobic microenvironment protecting the underlying Si-O-Si bonds on the particle surface. Furthermore, the functionalization allows for the use of the material in various chromatographic applications. For example, Figures 2A to 2B The materials are suitable for size exclusion chromatography, while Figures 2C to 2D The materials are suitable for reversed-phase chromatography.

[0058] Since the binary composition of the bonded phase has a binary composition, the molar ratio of hybrid regions to functional regions is important for the material properties. In one aspect, particles are disclosed herein, wherein the molar ratio of hybrid regions to functional regions is between 1:1.4 and 1.3. More preferably, this ratio is between 1:2 and 1:1.6. Because the ratio of hybrid regions to functional regions depends directly on the density of the corresponding silane compound, it is necessary to determine the surface coverage of the silane compound in the bonded phase.

[0059] The resulting hybridized particles have a molar ratio of bipod hybrid silane to functionalized silane. In some embodiments, the molar ratio of bipod hybrid silane to functionalized silane is about 0.1:3 (bipod hybrid silane: functionalized silane). In some embodiments, the molar ratio of bipod hybrid silane to functionalized silane is about 0.1:1, about 0.2:1, about 0.4:1, about 0.5:1, about 0.8:1, about 1:1, about 1.3:1, about 1.5:1, about 2:1, about 2.3:1, about 2.5:1, about 3:1, or any range between said values. In some embodiments, the molar ratio of bipod hybrid silane to functionalized silane is any value between about 0.4:1 and 1.3:1. In some embodiments, the molar ratio of bipod hybrid silane to functionalized silane is any value between about 0.4:1 and 3:1. Without being bound by any particular theory, altering the ratio of bipedal hybrid silane to functionalized silane can change the surface hydrophobicity of the particles, which can impart advantages to specific chromatographic applications. For example, but not as a limitation, hybridized particles with a molar ratio in the range of 0.4:1 to 1.3:1 are suitable for use with size exclusion chromatography, and hybridized particles with a molar ratio in the range of 0.4:1 to 3:1 are suitable for use with reversed-phase chromatography.

[0060] In one aspect, the hybrid-bonded particles of this technology include:

[0061] (i)Formula Bipedal hybrid silanes, in which

[0062] R1 can be chlorinated, methoxylated, or ethoxylated independently;

[0063] R2 is independently alkyl, methoxy, ethoxy, or chlorine; and

[0064] n is 1 to 4; and

[0065] (ii) Functionalized silanes, wherein each of R1 is independently chlorinated, methoxylated, or ethoxylated;

[0066] R3 can be alkyl, methoxy, ethoxy, or chlorine, each independently.

[0067] R5 is a hydroxyl or methoxy group; and

[0068] p is an integer from 1 to 12.

[0069] In some embodiments, the hybrid-bonded particles have an average pore size of about 400 Å to 5000 Å. In some embodiments, the average pore size is about 600 Å to 2500 Å. In some embodiments, the bipedal hybrid silane and the functional silane are present in any molar ratio between about 0.1:1 and 3:1 (bipedal hybrid silane to functional silane). In some embodiments, the bipedal hybrid silane and the functional silane are present in a molar ratio between 0.4:1 and 1.3:1.

[0070] In one aspect, the hybrid-bonded particles of this technology include:

[0071] (i)Formula Bipedal hybrid silanes, in which

[0072] R1 is an ethoxy group on each of its own;

[0073] R2 is independently ethoxylated; and

[0074] n is 2; and

[0075] (ii) Functionalized silanes, wherein each R1 is an ethoxy group;

[0076] R3 is an ethoxy group on its own;

[0077] R5 is a hydroxyl group; and

[0078] p is an integer between 8 and 12.

[0079] In some embodiments, the hybrid-bonded particles have an average pore size of about 400 Å to 5000 Å. In some embodiments, the average pore size is about 600 Å to 2500 Å. In some embodiments, the bipedal hybrid silane and the functional silane are present in any molar ratio range between about 0.1:1 and 3:1 (bipedal hybrid silane to functional silane). In some embodiments, the bipedal hybrid silane and the functional silane are present in any molar ratio range between 0.4:1 and 1.3:1.

[0080] In one aspect, the hybrid-bonded particles of this technology include:

[0081] (i)Formula Bipedal hybrid silanes, in which

[0082] R1 can be chlorinated, methoxylated, or ethoxylated independently;

[0083] R2 is independently alkyl, methoxy, ethoxy, or chlorine; and

[0084] n is 1 to 4; and

[0085] (ii) Functionalized silanes, in which

[0086] R1 can be chlorinated, methoxylated, or ethoxylated independently;

[0087] R3 can be alkyl, methoxy, ethoxy, or chlorine, each independently.

[0088] R4 is H or benzene; and

[0089] m is 0 or 1.

[0090] In some embodiments, n is 2 and R4 is benzene. In some embodiments, the hybrid-bonded particles have an average pore size of about 90 Å to 400 Å. In some embodiments, the bipedal hybrid silane and the functional silane are present in any molar ratio range between about 0.1:1 and 3:1 (bipedal hybrid silane to functional silane). In some embodiments, the bipedal hybrid silane and the functional silane are present in a molar ratio between 0.4:1 and 3:1.

[0091] In one aspect, the hybrid-bonded particles of this technology include:

[0092] (i)Formula Bipedal hybrid silanes, in which

[0093] R1 can be chlorinated, methoxylated, or ethoxylated independently;

[0094] R2 is independently alkyl, methoxy, ethoxy, or chlorine; and

[0095] n is 1 to 4; and

[0096] (ii) Functionalized silanes, in which

[0097] R1 can be chlorinated, methoxylated, or ethoxylated independently;

[0098] R2 can be alkyl, benzyl, methoxy, ethoxy, or chlorine, each independently.

[0099] R3 is independently alkyl, methoxy, ethoxy, or chlorine; and

[0100] n is 3, 7, or 17.

[0101] In some embodiments, the first instance of n in the bipedal hybrid silane is 2, and the second instance of n in the functionalized silane is 3. In some embodiments, the first instance of n in the bipedal hybrid silane is 2, and the second instance of n in the functionalized silane is 7. In some embodiments, the first instance of n in the bipedal hybrid silane is 2, and the second instance of n in the functionalized silane is 17. In some embodiments, the hybrid-bonded particles have an average pore size of about 90 Å to 400 Å. In some embodiments, the bipedal hybrid silane and the functionalized silane are present in any molar ratio range between about 0.1:1 and 3:1 (bipedal hybrid silane to functionalized silane). In some embodiments, the bipedal hybrid silane and the functionalized silane are present in a molar ratio range between 0.4:1 and 3:1.

[0102] In one aspect, the hybrid-bonded particles of this technology include:

[0103] (i)Formula Bipedal hybrid silanes, in which

[0104] R1 can be chlorinated, methoxylated, or ethoxylated independently;

[0105] R2 is independently alkyl, methoxy, ethoxy, or chlorine; and

[0106] n is 1 to 4; and

[0107] (ii) Select bipod hybrid silanes from the group consisting of the following: , , and ;

[0108] Each of R1 is independently chlorinated, methoxylated, or ethoxylated;

[0109] R2 can be alkyl, benzyl, methoxy, ethoxy, or chlorine, each independently.

[0110] R3 can be alkyl, methoxy, ethoxy, or chlorine, each independently.

[0111] R4 is H or benzene;

[0112] R5 is a hydroxyl or methoxy group;

[0113] n is 3, 7, or 17;

[0114] m is 0 or 1; and

[0115] p is an integer from 1 to 12.

[0116] In some embodiments, n in the bipedal hybrid silane is firstly 2. In some embodiments, the hybrid-bonded particles have an average pore size of about 90 Å to 400 Å. In some embodiments, the bipedal hybrid silane and the functional silane are present in a molar ratio ranging from about 0.1:1 to 3:1 (bipedal hybrid silane to functional silane). In some embodiments, the bipedal hybrid silane and the functional silane are present in a molar ratio ranging from 0.4:1 to 3:1.

[0117] Material characterization

[0118] The surface coverage of silane compounds in the bonded phase has a direct impact on the improved properties of the materials disclosed herein. Therefore, it is important to first determine the theoretical maximum surface coverage of the silane compounds. The theoretical maximum surface coverage can be estimated using the hydrodynamic radius or Stokes radius of the silane compound. The hydrodynamic radius or Stokes radius can be determined using methods known in the art.

[0119] Using the hydrodynamic radius or Stokes radius of silanes, first calculate / estimate the required surface area per molecule using the following formula:

[0120]

[0121] Once the required surface area for each molecule is known, Avogadro's constant (6.022 × 10⁻⁶) can be used. 23 This determines the minimum surface area for 1 μmol. The reciprocal of this value provides the surface area per m³. 2 Maximum coverage of 1 μmol.

[0122] In some embodiments of this technology, the surface coverage of the functional areas is between 60% and 250% of the theoretical maximum coverage. In a preferred embodiment, the surface coverage of the functional areas is between 80% and 130%.

[0123] As an example of monolayer bonding, the theoretical maximum surface coverage of functionalized PEG (8 to 12 repeating units) silanes with a hydrodynamic radius of 0.61 nm is approximately 1.38 μmol / m². 2 Therefore, the desired surface coverage of functionalized silanes can be as high as 0.8 μmol / m². 2 With 3.45 μmol / m 2 Within the range between.

[0124] Once the bonded phase is applied to the particles, the actual surface coverage can be quantified by using the percentage of carbon in the product and the loss of organic components, as determined by thermogravimetric analysis (TGA). TGA quantifies the total percentage of organic matter by mass loss over a programmed temperature range of 180°C to 600°C. For example, the surface coverage of bonded silica particles can be calculated using the following formula:

[0125]

[0126] In the above calculations, C1 and M1 are the corresponding carbon percentage and molar mass of the combustible organic portion of the bonded functional silane. C2 is the carbon percentage of the combustible organic portion of the bonded bipod silane. SA is the BET surface area of ​​the particle before bonding. W is the percentage of weight loss measured by TGA (200°C to 600°C), and C is the carbon percentage found in the bonded particle.

[0127] As illustrative examples, for PEG-functionalized silanes (having 8 to 12 repeating units, with an average of 10.5 repeating units), C1 is approximately 55.3% and M is approximately 521, and for BTEE bipedal hybrid silanes, C2 is approximately 85.71%.

[0128]

[0129] If the particles below contain both carbon and combustible components, then the carbon percentage and combustible content of the base particles need to be subtracted from the calculation.

[0130] In a preferred embodiment, the bonded phase does not significantly alter the pore size of the underlying particles. The pore size of the particles can be measured using methods known in the art, including the mercury porosity method.

[0131] The molar ratio of bipod hybrid silanes and functionalized silanes in this technology can be used to digest particles. 1 To determine this, for example, ¹H NMR analysis can be used for the digestion of particles. 1 1H NMR analysis further determined the molar ratio of bridged ethylene to polyethylene oxide (BTEE / OH-PEG) bonded to silica. The particles can be digested using methods known in the art, including, for example, digestion at 64°C for 1 hour with 3 mL of 2.5 M NaOH solution containing 100:1 (v / v) D2O / H2O. Figures 9A to 9C Provided silica for PEG-only bonding obtained after alkaline digestion ( Figure 9B ), BTEE / PEG bonded silica ( Figure 9A ) and the structure of possible hydrolysis products ( Figure 9C Exemplary 1¹H NMR spectra. Chemical shifts at 3.55 ppm, 3.25 ppm, 1.35 ppm, and 0.12 ppm are associated with various methylene protons in the hydrolyzed PEG ligands. The Si-CH₂ protons of the ethylidene bridge after alkaline digestion are... 1 The 1H NMR spectrum shows chemical shifts at 0.34 ppm and 0.23 ppm.

[0132] The BTEE to PEG ratio can be calculated based on the ratio of the BTEE Si-CH2 proton integral at chemical shifts of 0.34 ppm (I1) and 0.23 ppm (I2) to the PEG proton integral at chemical shift 1.35 ppm (I3), as shown below:

[0133]

[0134] OH-PEG surface coverage can be calculated based on carbon percentage, BTEE / PEG molar ratio, and particle specific surface area (SA).

[0135] Uses of materials

[0136] The materials disclosed in this technology are suitable for a range of chromatographic applications. In some embodiments, these materials can be used in size exclusion chromatography. In some embodiments, these materials can be used in reversed-phase chromatography. In other embodiments, these materials can be used in ion-exchange chromatography.

[0137] The materials disclosed herein can be packed into chromatographic apparatuses, such as, for example, chromatographic columns. Chromatographic apparatuses include a column body formed of metal or metal alloy (e.g., titanium or stainless steel) or plastic (e.g., polyetheretherketone (PEEK)). The inner surface of the column may be coated or uncoated to minimize nonspecific interactions. Chromatographic apparatuses can be appropriately sized for use in high-performance liquid chromatography (HPLC) systems, ultra-high-performance liquid chromatography (UHPLC) systems, or rapid protein liquid chromatography (FPLC) systems. These systems can also be connected to post-column detectors, such as ultraviolet (UV) detectors, tunable UV (TUV) detectors, photodiode array (PDA) detectors, refractive index (RI) detectors, multi-angle light scattering (MALS) detectors, mass spectrometry (MS) detectors, and / or fluorescence (FL) detectors.

[0138] In one respect, the materials disclosed herein exhibit enhanced stability when exposed to alkaline conditions. This enhanced stability improves their performance in chromatographic applications and allows for repeated use with minimal loss of efficiency. Figures 3A to 3C Enhanced alkali stability of the materials disclosed herein is demonstrated when used in size exclusion chromatography. Figure 3AThe properties of unhybridized materials are shown, such as those containing only functionalized silanes without bipod hybridized silanes. After prolonged exposure to alkaline conditions (pH 8.5, 88 hours), the materials failed to achieve sufficient myoglobin separation (compare the black (post-exposure) traces and gray (pre-exposure) traces in the circled areas). Conversely, Figure 3B The hybrid-bonded materials of this technique demonstrate the ability to retain dissociated myoglobin after prolonged exposure to alkaline conditions in the same manner. However, the surface coverage of the functionalized silane and therefore the ratio of hybrid to functionalized regions in the bonded phase are important. Figure 3C (Corresponding to the material from Example 1D), which uses a material with 2.69 μmol / m 2 Materials with a PEG surface coverage of approximately 200% of the theoretical maximum surface coverage and a BTEE to PEG silane feed molar ratio of 1:1.24 are not resistant to prolonged alkaline conditions. While not wishing to be bound by any particular theory, it is believed that this is due to the self-gelling of BTEE in the bonding process, resulting in a low molar ratio of OH-PEG to BTEE, which leads to fewer hybridized regions and thus reduced hydrophobic protection. Example 11 further details the alkaline stability of the disclosed materials.

[0139] In one respect, the materials disclosed herein exhibit reduced baseline noise compared to materials known in the art. This reduced baseline noise improves the overall accuracy of analytical measurements performed using chromatographic applications. SEC-MALS is typically used to evaluate large biomolecules, determining the radius, molar mass, oligomeric state, and polydispersity of separated molecules. However, commonly used silica materials shed silicates in the 10 nm to 100 nm range, which hinders light scattering of molecules of interest. The materials disclosed herein result in a significant reduction in MALS noise, thereby allowing for more robust calculations of large biomolecules. In some embodiments of this technique, MALS noise is reduced by approximately 60 times compared to commercially available columns. Figure 4 The diagram illustrates the height-variable MALS noise produced by a commercially available column compared to a column filled with the material of this technology. Example 12 further details the reduced MALS noise of the disclosed material.

[0140] On the other hand, compared to materials known in the art, the materials disclosed herein offer superior resolution for both DNA capsids and AAV capsids. The materials of this technique are capable of resolving dsDNA ranging from 50 bp to 1500 bp. Figure 5A (gray bars), while commercially available columns have poor resolution and high baseline ( Figure 5B (gray bars). Similarly, compared to commercially available columns ( Figure 6B When separating AAV capping formulations ( Figure 6AWhen [the material is used], the material of this technology provides better resolution.

[0141] Example

[0142] Example 1: Formation of hybrid PEG-bonded phase on wide-pore silica particles

[0143] A bonded phase consisting of bipod hybrid silanes and OH-PEG silanes was formed on wide-pore silica particles. 174 mL of toluene slurry containing 15 g of silica particles (average pore size approximately 965 Å) was added to a 500 mL three-necked round-bottom flask equipped with a Dean Stark separator, condenser, thermometer, heating mantle, mechanical stirrer, and nitrogen purging system. The flask was heated to reflux at 110 °C for two hours. After two hours, 50 mL of condensed toluene was removed from the Stark separator, and 25 mL was retained until the bonding process was complete. The reaction was then cooled to below 40 °C and 360 μL of concentrated hydrogen chloride solution was added, followed by 4.46 g of [hydroxy(polyethyleneoxy)] 8-12 [Propyl]triethoxysilane (OH-PEG) and 1.19 g of 1,2-bis(triethoxysilyl)ethane (BTEE). The reaction was heated to reflux (approximately 106 °C) for 20 h, then cooled to room temperature (RT). The particles were separated by filtration and subsequently washed with toluene, ethanol, and water. After the bonding reaction, the remaining ethoxysilane was further condensed at 100 °C for 3 h using ammonium bicarbonate (100 mM). The reaction was cooled to room temperature, and the particles were separated by filtration. The particles were then washed with water to lower the pH to less than 6, washed with acetone, and vacuum dried at 70 °C for 16 h. The final particles contained approximately 1.12% (by weight) carbon and had an average pore size of approximately 980 Å. The OH-PEG coverage was approximately 1.348 μmol / m³. 2 .

[0144] The ratio of OH-PEG to BTEE can be changed by adjusting the concentration of BTEE used in the above method. Table 1 shows the parameters of the particles obtained when using different concentrations of BTEE.

[0145] Table 1: Hybridized particles generated by different BTEE concentrations

[0146]

[0147] Example 2: Delayed addition of BTEE during the formation of hybrid PEG bonded phase

[0148] The effect of delayed BTEE addition on the formation of hybrid PEG-bonded phases was evaluated. Particles were prepared as described in Example 1, except that the BTEE addition was delayed by 0, 1.5, 3, or 5 hours. As shown in Table 2, delayed BTEE addition resulted in reduced OH-PEG coverage.

[0149] Table 2: Hybridized particles resulting from delayed addition of BTEE

[0150]

[0151] Example 3: Hybrid PEG bonding on ultrawide-pore silica particles

[0152] The effect of pore size on the formation of hybrid PEG bonded phases was evaluated. Particles with different pore sizes and particle sizes were bonded as described in Example 2B and as shown in Table 3. The effect of pore size and particle size on the final particle surface coverage was negligible, and the pore size did not decrease after hybrid bonding.

[0153] Table 3: Pore size of hybrid bonded particles

[0154]

[0155] Example 4: Formation of a hybrid PEG bonded phase on hybrid coated silica particles

[0156] The ability to form a hybrid PEG-bonded phase on hybrid-coated silica particles was determined. The hybrid-coated silica particles were prepared as follows: 26 g of porous silica particles (average pore size 965 Å, pore volume 0.83 cc / g measured by a mercury porosimeter) dispersed in 480 mL of anhydrous toluene were added to a 1 L flask equipped with a Dean Stark separator, mechanical stirrer, thermocouple, condenser, and heating mantle. The reaction was heated to reflux at 110 °C for one hour, and 25 mL of toluene was retained in the Dean Stark separator until the coating process was complete. After one hour of reflux, the reaction was cooled to 40 °C. 21.3 g of pre-condensed material (denoted as PEOS) based on a 4:1 molar ratio of TEOS and BTEE was added to the reaction and stirred for 10 minutes. Then, 1.3 g of ammonium hydroxide (28% to 30% aqueous solution) was added to the reaction and stirred for another 10 minutes. The reaction was heated to 60°C and held for two hours, then cooled to below 40°C, and the particles were separated by filtration. The resulting particles were washed twice with ethanol and transferred to a 500 mL flask equipped with a mechanical stirrer, thermocouple, condenser, and heating mantle. 182 mL of purified laboratory water (i.e., Milli-Q, purchased from Millipore Sigma) was added. ® 78 mL of purified water and 26 g of ammonium hydroxide (28% to 30% aqueous solution) were added to a flask. The reaction was heated to 50 °C and maintained for two hours. The particles were then separated by filtration and washed twice with 260 mL of methanol / water (1:1, v / v), followed by two washes with 260 mL of methanol. The coated particles were dried overnight under complete vacuum at 80 °C. The resulting particles contained 0.5% carbon by weight.

[0157] The hybrid PEG-bonded phase was then added to the particles using the method described in Example 1. The resulting hybrid-bonded particles had a density of 1.26 μmol / m³. 2 The OH-PEG surface coverage, where the BTEE / OH-PEG ratio is 0.5, is shown in Table 4.

[0158] Table 4: Hybrid bonded phases on hybrid-coated silica particles

[0159]

[0160] Example 5: Formation of hybrid-bonded particles on wide-pore silica particles

[0161] A slurry of 20 g of silica particles with an average pore size of approximately 1880 Å was redispersed in 211 mL of toluene and added to a 500 mL three-necked round-bottom flask equipped with a Dean Stark separator, condenser, thermometer, heating mantle, mechanical stirrer, and nitrogen purging system. The flask was heated to reflux at 110 °C for two hours. 50 mL of condensed toluene was removed from the Stark separator, leaving 25 mL in the separator. After reflux for two hours, the reaction was cooled to below 40 °C. 600 μL of concentrated hydrogen chloride solution was added to the reaction, followed by 7.26 g of 50% (w / w) [hydroxy(polyvinyloxy)] 8-12 A solution of propyltriethoxysilane in ethanol and 0.486 g of 1,2-bis(triethoxysilyl)ethane (BTEE) dissolved in 4.11 g of toluene were used. The reaction was then heated to reflux (approximately 106 °C) for 20 hours. After cooling to room temperature, the particles were separated by filtration and washed with toluene, ethanol, and water. Further condensation of the remaining ethoxysilane was carried out at 100 °C for 3 hours using ammonium bicarbonate (20 mM) at pH 10. The reaction was cooled to room temperature, and the particles were separated by filtration. The particles were washed with water to lower the pH to below 6, washed with acetone, and dried under vacuum at 70 °C for 16 hours. The final particles (5A) contained 0.56% by weight of carbon. Particles 5B, 5C, and 5D were obtained using the same method as described above but with different concentrations of BTEE as shown in Table 5.

[0162] Table 5: Characterization of hybrid bonded phases

[0163]

[0164] Example 6: Formation of hybrid PEG-bonded phase on porous hybrid particles

[0165] Using the method described in Example 1, a hybrid PEG-bonded phase was added to hybrid particles with a pore size of about 450 Å and a C% of 6.67 wt%.

[0166] Example 7: Formation of hybrid diol-bonded phases on porous hybrid particles

[0167] A slurry containing 15 g of hybrid particles (pore size approximately 450 Å and C% 6.67) was redispersed in ethyl acetate buffer (pH 5.7) and added to a 500 mL three-necked round-bottom flask equipped with a condenser, thermometer, heating mantle, mechanical stirrer, and nitrogen purging system. The flask was heated to 70 °C, and then 5 g of (3-glycidoxypropyl)triethoxysilane and 1 g of BTEE were added. The reaction was maintained at 70 °C for 20 hours and then cooled to room temperature. The particles were separated by filtration and washed to neutral pH. The particles were transferred to a 250 mL round-bottom flask and 150 mL of 0.5 M acetic acid was added. The flask was heated to 60 °C and maintained for 20 hours. The reaction was cooled to room temperature, and the particles were separated by filtration. The particles were washed with water and methanol and then dried under vacuum at 70 °C for 16 hours.

[0168] Example 8: Formation of hybrid biphenyl bonded phase on surface hybrid particles

[0169] A slurry containing 15 g of hybrid particles (pore size approximately 130 Å and C% 6.67) redispersed in 174 mL of toluene was used. A hybrid biphenyl-bonded phase was added to the particles using the method described in Example 1, except that 4-biphenyltriethoxysilane was used instead of [hydroxyl (polyvinyloxy)]. 8-12 [Propyl]triethoxysilane.

[0170] Example 9: Formation of hybrid C18 bonded phase on hybrid-coated nonporous polymer particles

[0171] A slurry containing 15 g of nonporous polymer particles (C% 54, diameter 2.2 μm) with a hybrid coating (TEOS / BTEE molar ratio 1 / 1.5) redispersed in 174 mL of toluene was used. A hybrid C18-bonded phase was added to the particles using the method described in Example 1, except that octadecyltrimethoxysilane was used instead of [hydroxyl(polyvinyloxy)]. 8-12 [Propyl]triethoxysilane.

[0172] Example 10: Formation of hybrid C8 bonded phase on porous hybrid coated particles

[0173] A slurry containing 15 g of hybrid particles (pore size approximately 130 Å and C% 6.67) redispersed in 174 mL of toluene was used. A hybrid C8-bonded phase was added using the method described in Example 1, except that n-octyltrichlorosilane was used instead of [hydroxyl(polyvinyloxy)]. 8-12 [Propyl]triethoxysilane.

[0174] Example 11: Formation of hybrid C4-bonded phase on porous silica particles

[0175] A slurry containing 15 g of silica particles (pore size approximately 90 Å) redispersed in 174 mL of toluene was used. A hybrid C4-bonded phase was added using the method described in Example 1, except that n-butyltrichlorosilane was used instead of [hydroxyl (polyvinyloxy)]. 8-12 [Propyl]triethoxysilane.

[0176] Example 12: Alkali stability of hybrid-bonded particles

[0177] The alkali stability of the hybrid-bonded particles was evaluated. Two 4.6 × 150 mm columns were filled with materials 1A and 1D from Example 1, respectively. As a control, a third column was filled with material 2D from Example 2, which was prepared solely using OH-PEG (i.e., unhybridized).

[0178] Each column connects to Waters Acquity ™ H-class UPLC ™ A chromatographic system (purchased from Waters Technologies Corporation, Milford, MA) was used, and the column chamber was heated to 40°C. Initial isocratic separation of a protein / nucleoside mixture containing thyroglobulin (3 mg / mL), IgG (2 mg / mL), BSA (5 mg / mL), myoglobin (2 mg / mL), and uracil (0.1 mg / mL) was performed. The mobile phase was 0.02 M PBS, and the flow rate was 0.2 mL / min. The eluent was monitored using a UV detector (280 nm).

[0179] After initial separation, 100 mM ammonium acetate buffer (pH 8.5) was flowed through each column at a constant flow rate of 0.2 mL / min for 88 hours. Following this washing period, a second isocratic separation was performed. Results were obtained in... Figures 3A to 3C As shown in the image. Figure 3A A control column containing only OH-PEG is shown. As shown in the circled portion of the chromatogram, after washing in 100 mM ammonium acetate buffer (pH 8.5) for 88 hours, the column's ability to separate myoglobin was significantly reduced, as shown by comparing the gray trace (before washing) with the black trace (after washing). In contrast, Figure 3B The hybrid-bonded material 1A was shown to exhibit substantially equivalent separation before and after washing in 100 mM ammonium acetate buffer (pH 8.5). It is noteworthy that, as... Figure 3C As shown, the hybrid-bonded material 1D failed to provide any separation after washing with 100 mM ammonium acetate buffer.

[0180] Example 13: SEC-MALS noise of hybrid-bonded particles

[0181] The SEC-MALS noise of the hybrid-bonded particles was evaluated. A 4.6 × 150 mm column was filled with material 1A from Example 1. As a control, a 4.6 × 150 mm column was filled with material 2D from Example 2, which was prepared solely using OH-PEG (i.e., unhybridized).

[0182] Each column was washed with 20 column volumes (CV) of 2X PBS before being connected to the MALS detector. Baseline voltage was observed over 0.5 to 4.5 minutes (corresponding to flow rates of 54 mL to 177 mL), and peak-to-peak noise and RMS noise were calculated. RMS was calculated by dividing the peak-to-peak noise by 6.6. Columns packed with Material 1A exhibited low peak-to-peak noise after 177 mL of flow, exceeding the requirements for detailed and sensitive analysis of low-abundance large molecules. In contrast, columns packed with control Material 2D exhibited almost twice the peak-to-peak noise and RMS. Commercially available SEC columns (Sepax 1000LS from Sepax Technologies, Inc., Newark, Delaware) exhibited approximately 60 times higher peak-to-peak noise than Material 1A. Figure 4 The MALS noise generated by the commercial column (black line) relative to the column filled with material 1A (dashed line) is shown. Table 6 provides a summary of the MALS noise observed for columns filled with various materials of this technology.

[0183] Table 6: MALS noise of hybrid-bonded particles

[0184]

[0185] Example 14: Separation of DNA fragments using hybrid-bonded particles

[0186] In a column filled with hybrid bonded particles of Example 2A ( Figure 5A ) or commercial columns containing only OH-PEG bonds (i.e., unhybridized bonds) Figure 5B Fractions of the DNA ladder (0.5 mg / mL, New England Biolabs, range 1350 bp to 50 bp) were separated on a column connected to Waters Acquity. ™ H-class UPLC ™ The chromatographic system (purchased from Waters Technologies Corporation, Milford, MA) was used, with a mobile phase of 0.02 M phosphate-buffered saline and a column temperature of 30 °C to allow 5 μL of sample injection solution to flow onto the column. Figure 5AAs shown, the materials of this technique provide separation of dsDNA in the range of 766bp to 550bp, while commercial columns cannot resolve these substances and typically have a higher baseline. Figure 5B ).

[0187] Example 15: Separation of AAV capsids using hybrid-bonded particles

[0188] In a column filled with hybrid bonded particles of Example 2A ( Figure 6A ) or commercial columns containing only OH-PEG bonds (i.e., non-hybridized bonds) Figure 6B AAV2 capsid serotype formulation (15 uL, 1×10¹³ vg / mL, Virovek) was isolated using a column connected to Waters Acquity. ™ H-class UPLC ™ The chromatographic system (purchased from Waters Technologies Corporation, Milford, MA) was used, with a mobile phase of 0.02 M phosphate-buffered saline and a column temperature of 30 °C to allow 30 μL of sample injection solution to flow onto the column. Figure 6A As shown, compared to commercial columns ( Figure 6B The materials in this technology provide high monomer resolution and high molecular weight substances.

[0189] Example 16: Isolation of plasmid DNA using hybrid-bonded particles

[0190] A mixture of plasmid and viral particle DNA was separated by size exclusion chromatography on a column packed with the hybridized particles of Example 3B. Equal volumes (1 mg / mL) of plasmid dsDNA (pBR322) and viral particle ssDNA (ΦX174) were mixed, and 1 μL of the mixture was injected onto a 4.6 × 150 mm column packed with the hybridized particles of Example 3B, having an average pore size of 2000 Å. The column was connected to Waters Acquity. ™ H-Class UPLC ™ The chromatographic system (purchased from Waters Technologies Corporation, Milford, MA) was used, and samples were separated at 0.1 mL / min at 35 °C using 1X phosphate-buffered saline (1X PBS; 10.14 mM sodium phosphate pH 7.4, 137 mM sodium chloride, 2.7 mM potassium chloride). The eluent was monitored at 260 nm using a UV detector. Figure 7 As shown, the hybridized particles of Example 3B achieve the separation of double-stranded plasmid DNA from single-stranded viral particle DNA.

[0191] Example 17: Separation of lipid nanoparticles using hybrid-bonded particles

[0192] Samples containing lipid nanoparticles (LNPs) were separated using size exclusion chromatography on a column packed with hybrid bonded particles of Example 3B. 2 μL of LNP sample #1 or 4 μL of LNP sample #2 was injected onto a 4.6 × 150 mm column packed with hybrid bonded particles of Example 3B, having an average pore size of 2000 Å. The column was connected to Waters Acquity. ™ H-Class UPLC ™ The chromatographic system (purchased from Waters Technologies Corporation, Milford, MA) was used, and samples were separated at 0.1X PBS (1.014 mM sodium phosphate, pH 7.4, 13.7 mM sodium chloride, and 0.27 mM potassium chloride) at 35 °C and a flow rate of 0.1 mL / min. The eluent was monitored at 260 nm using a UV detector. Figure 8A (LNP sample #1) and Figure 8B As shown in (LNP sample #2), the hybrid-bonded particles of Example 3B were separated, demonstrating the heterogeneity of lipid nanoparticles present in the LNP sample.

Claims

1. A chromatographic material, said chromatographic material comprising: Particles having a silica surface, wherein the silica surface is composed of a bonded phase formed by at least two silane compounds, wherein: One of the at least two silane compounds is a bipod hybrid silane containing two indirectly linked silicon dioxide atoms, and One of the at least two silane compounds is a functionalized silane.

2. The chromatographic material according to claim 1, wherein the particles are polymer particles, silica particles, or inorganic-organic hybrid particles.

3. The chromatographic material according to claim 1 or 2, wherein the bipod hybrid silane is selected from the group consisting of: , and ;in R1 can be chlorinated, methoxylated, or ethoxylated independently; R2 is independently alkyl, methoxy, ethoxy, or chlorine; and n and m are each independently 1 to 4.

4. The chromatographic material according to any one of claims 1 to 3, wherein the functionalized silane is selected from the group consisting of: , , and ;in R1 can be chlorinated, methoxylated, or ethoxylated independently; R2 can be alkyl, benzyl, methoxy, ethoxy, or chlorine, each independently. R3 can be alkyl, methoxy, ethoxy, or chlorine, each independently. R4 is H or benzene; R5 is a hydroxyl or methoxy group; n is 3, 7, or 17; m is 0 or 1; and p is an integer from 1 to 12.

5. The chromatographic material according to any one of claims 1 to 4, wherein the bipod hybrid silane and the functionalized silane are fed in a molar ratio of about 1:1, about 1:2, about 1:4 or about 1:

10.

6. The chromatographic material according to claim 5, wherein the bipod hybrid silane and the functional silane are fed in a molar ratio of about 1:2 or about 1:

4.

7. The chromatographic material according to any one of claims 1 to 6, wherein the bipod hybrid silane and the functional silane are present in a molar ratio between 0.1:1 and 3:

1.

8. The chromatographic material according to any one of claims 1 to 7, wherein the surface coverage of the functionalized silane is the theoretical maximum surface coverage of the functionalized silane (μmol / m²). 2 60% to 250%.

9. The chromatographic material according to any one of claims 1 to 8, wherein the functionalized silane is ; in R1 and R3 are ethoxy groups; R5 is a hydroxyl group; and p is 8 to 12.

10. The chromatographic material according to claim 9, wherein the surface coverage of the functionalized silane is about 0.8 μmol / m². 2 Approximately 3.45 μmol / m 2 between.

11. The chromatographic material according to any one of claims 1 to 10, wherein the particles have an average pore size between about 90 Å and about 5000 Å.

12. The chromatographic material of claim 10, wherein the particles have an average pore size of about 600 Å to about 2500 Å.

13. The chromatographic material according to any one of claims 1 to 12, wherein the chromatographic material is resistant to hydrolytic corrosion, as detected by minimal change in separation performance before and after exposure to hydrolytic conditions.

14. The chromatographic material according to any one of claims 1 to 12, wherein the chromatographic material exhibits low baseline noise as measured by a multi-angle light scattering (MALS) detector.

15. A chromatographic column comprising the chromatographic material according to any one of claims 1 to 14.

16. A chromatographic apparatus, the chromatographic apparatus comprising: The chromatographic column according to claim 15; A column injector and tubing, wherein the column injector is positioned upstream of the chromatographic column, and the tubing is fluidly connected to the chromatographic column and located downstream of the chromatographic column.

17. The chromatographic apparatus of claim 16, further comprising a detector fluidly connected to and located downstream of the chromatographic column.

18. The chromatographic apparatus of claim 17, wherein the detector is a multi-angle light scattering (MALS) detector and / or an ultraviolet detector.

19. The chromatographic apparatus according to any one of claims 16 to 18, wherein the chromatographic apparatus is used in size exclusion chromatography or reversed-phase chromatography.

20. The chromatographic apparatus according to any one of claims 16 to 18, wherein the chromatographic material is resistant to hydrolytic corrosion, as detected by MALS noise and / or by separating protein and / or nucleoside samples.

21. The chromatographic apparatus of claim 20, wherein the MALS noise is measured as peak-to-peak noise in μV, and wherein the peak-to-peak noise is ≤800 μV.

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