Mixed mode chromatographic packing material

Abstract

To provide a method for producing a chromatographic packing material.SOLUTION: This packing material comprises a functionalized silicone-based polymer layer bonded to substrate particles. This method comprises: (a) (i) forming silicone-based polymer encapsulated substrate particles comprising pendant functional groups by reacting functional groups on the substrate particles with a silicone-based polymer product comprising pendant functional groups; and (ii) functionalizing the silicone-based polymer encapsulated substrate particles by reacting the pendant functional groups on the silicone-based polymer encapsulated substrate particles with a hydrophobic compound and an amine-containing compound; or comprises (b) (i) forming a functionalized silicone-based polymer by reacting pendant functional groups on the silicone-based polymer product with a hydrophobic compound and an amine-containing compound; and (ii) reacting the product of step b (i) with functional groups on the substrate particles.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of chromatographic sample separation, including liquid chromatography and solid-phase extraction, and in particular to materials and the synthesis of materials for use as stationary phases in chromatographic sample separation. The invention further relates to the use of the materials, in particular in the separation of hydrophilic and hydrophobic peptides, non-glycosylated and N-linked glycosylated peptides, deamidated peptides, and oxidized peptides. The invention also relates to chromatography columns and solid-phase extraction columns containing the materials as stationary phases. [Background technology]

[0002] Liquid chromatography (LC), e.g., HPLC and UHPLC, as well as solid-phase extraction (SPE), are routinely used in both analytical and preparative chromatographic applications to determine the quality and quantity of analytes in various samples. In these chromatographic techniques, separation of a sample containing a mixture of components (also called analytes) is achieved by conveying the sample in a liquid mobile phase through a stationary phase within a column, thereby separating the sample into its components, since each component partitions differently between the mobile and stationary phases (i.e., the components have different partition coefficients). The stationary phase is typically in the form of a particle bed packed within the column or a monolithic material retained within the column. Silica particles are commonly used for stationary phases, either as nonporous, fully porous, or superficially porous particles. Silica-based HPLC columns are used for a wide range of applications due to their excellent physical strength, high efficiency, and mature surface-bonded chemistry.

[0003] The choice of stationary phase for an analyte is primarily driven by the column chemistry, which is important in LC separations. Column chemistry is typically controlled by modifying the stationary phase surface, typically by attaching ligands to the surface. Introducing various functional groups onto the stationary phase surface allows for the implementation of mixed-mode chromatography, where multiple separation mechanisms can be used simultaneously.

[0004] However, silica-based columns have pH limitations. Under acidic conditions, the bound ligand can be cleaved at the siloxane (Si-O-Si) bond between the silica surface and the ligand, resulting in a loss of hydrophobic retention in the case of C18-bonded columns. Furthermore, silica particles tend to dissolve at low pH, resulting in poor chromatographic performance and reduced column life. Under alkaline conditions, hydroxide ions can erode the silica substrate by breaking the siloxane bonds in the silica backbone, causing the collapse of the packing layer or headspace in the column.

[0005] Stationary phase media for HPLC separations are generally produced by modifying silica surfaces with silylating agents. Monofunctional silylating agents are often used to form monolayer surface coatings, while bifunctional and trifunctional silylating agents are used to form polymer coatings on silica surfaces, which generally improve chemical stability (UK Patent No. 2,549,417). However, the use of some silylating agents results in coatings with undesirable properties, including hydrolytic instability and inadequate masking of acidic silanols on the silica surface.

[0006] The presence of acidic silanols on the surface of silica particles has been a known challenge in LC. This is because acidic silanols can cause irreversible absorption of basic solutions, resulting in strong peak imbalances and retention times highly dependent on the solution concentration. To mitigate this issue, end-capping techniques using activated silanes such as hexaethyldisilane and bis-trimethylsilylacetamide have been used to improve bonded phase behavior (U.S. Patent No. 5,134,110, U.S. Patent No. 7,534,352). However, this approach is primarily applicable to RP materials and is limited by the introduction of polar functional groups, which can lead to different selectivities in mixed-mode stationary phases. These groups can react with the end-capping silane, potentially compromising the stability and performance of the stationary phase.

[0007] C18 HPLC columns are primarily used in RP HPLC, where the primary interaction between the stationary phase and the analyte is a hydrophobic interaction caused by partitioning forces.

[0008] Secondary interactions in the unendcapped C18 phase include hydrophilic interactions between the remaining silanol groups and the analyte.

[0009] Traditional C18 columns are well known in the art and are designed to achieve separations based on specific requirements, resulting in varying product characteristics: 1) Particle pore size: Smaller pore sizes (e.g., 80 Å) are suitable for smaller molecules, while larger pore sizes (e.g., 300 Å) are suitable for larger molecules. 2) Particle surface area: The greater the surface area, the higher the retention. 3) Particle size: Smaller particles (e.g., 1.5 μm) provide higher separation efficiency, while larger particles (e.g., 5 μm) reduce column backpressure. 4) Column dimensions: Longer columns (e.g., 20 cm long) provide better separation and resolution, while shorter columns (e.g., 5 cm long) allow for faster separation and analysis. 5) Certain end caps are used to reduce silanol activity and increase particle stability.

[0010] Traditional C18 columns are widely used in various industries, including pharmaceuticals, environmental analysis, food analysis, etc. C18 phases are commonly used for the analysis of small molecules, peptides, proteins, and other compounds of interest.

[0011] However, while traditional C18 columns are effective for separating nonpolar and moderately polar compounds, they offer limited selectivity for highly polar or ionic compounds and their sample mixtures. This limitation necessitates the consideration of new approaches to enhance the separation and resolution of analytes of interest, such as the implementation of alternative chemistries, mixed-mode methods, or multidimensional LC techniques. One particular area is proteomics and peptide analysis, where a combination of hydrophobic and ion-exchange interactions can be utilized to improve separation and resolution.

[0012] Hybrid materials can address chromatographic challenges, such as improved stability at both high and low pH levels (U.S. Patent No. 9,120,083). However, when the test environment changes, hybrid materials tend to exhibit retention time drain, resulting in poor baseline peak shape at low pH, which negatively impacts loading rates and peak capacity. Furthermore, the presence of surface biogroups, especially methyl groups, can reduce the concentration of the surface bonded phase, thereby compromising the efficiency of chromatographic separations.

[0013] Therefore, although end-capping techniques and hybrid materials have proven beneficial in improving the properties of LC stationary phases, their application in mixed-mode chromatographic separations remains limited. Innovative approaches are needed to overcome these limitations and develop pH-stable and efficient mixed-mode stationary phases that offer improved selectivity, good peak shape, and reversibility [for charged analytes] while maintaining the integrity of the surface modification.

[0014] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Summary of the Invention

[0015] The present invention provides a method for producing a chromatographic packing material, the packing material comprising a functionalized silicone-based polymer layer bonded to a substrate particle, the method for forming the packing material comprising the steps of: (a) (i) reacting functional groups on the substrate particles with a silicone-based polymer product containing pendant functional groups to form silicone-based polymer-encapsulated substrate particles containing pendant functional groups; (ii) functionalizing the silicone-based polymer-encapsulated substrate particles by reacting pendant functional groups on the silicone-based polymer-encapsulated substrate particles with at least one hydrophobic compound and at least one amine-containing compound (and forming functionalized silicone-based polymers attached to the substrate particles); or (b) (i) reacting pendant functional groups on the silicone-based polymer product with at least one hydrophobic compound and at least one amine-containing compound to form a functionalized silicone-based polymer; (ii) reacting the product of step b(i) with functional groups on said substrate particles.

[0016] Substrate particles, as defined herein, may be particulate substrates or monolithic substrates, preferably particulate substrates.

[0017] The substrate material may be a metal oxide (this term is used herein to include semi-metal oxides such as silica, including inorganic-organic hybrid materials (especially metal oxide-organic hybrid materials), such as those described in WO 00 / 45951). The substrate may in particular be silica (SiO), and this term is used herein to include silica / organohybrid, alumina (AlO), titania (TiO), or zirconia (ZrO) substrates.

[0018] Silica (as this term is used herein to include silica / organohybrid) substrates are most preferred.

[0019] As used herein, the term "silicone-based" is intended to encompass polymers and / or compounds that include silicone.

[0020] As used herein, the term "functionalized silicone-based polymer layer" is intended to cover a silicone-based polymer product to which compounds have been added that alter the functionality of the polymer, specifically compounds such as hydrophobic compounds and amine-containing compounds, as well as compounds that result in further polymerization.

[0021] As used herein, the term "pendant" is as defined by the IUPAC chemical classification system: a pendant group (sometimes written as pendant) is a group of atoms attached to the backbone chain of a longer molecule (usually a polymer). A pendant group differs from a pendant chain because it is neither an oligomer nor a polymer.

[0022] The additional compound may be a single compound or may itself be a monomer that forms an additional polymer layer attached to the silicone-based polymer either before or after addition to the silicone-based polymer layer, as described in more detail below.

[0023] The functionalized silicone-based polymer layer is bonded to the substrate particle. Typically, the functionalized silicone-based polymer layer is bonded to the surface of the substrate particle, preferably via a covalent bond. That is, the functionalized silicone-based polymer layer is covalently bonded to the substrate particle.

[0024] The functionalized silicone-based polymer attached to the substrate particle can be formed by either step (a) or step (b) of the present method.

[0025] In step (a)(i), a silicone-based polymer-encapsulated substrate particle containing pendant functional groups is formed by reacting the functional groups (as defined above) of the substrate particle with a silicone-based polymer product containing pendant functional groups.

[0026] Typically, the reaction of the functional groups on the substrate particles with the silicone-based polymer product containing the pendant functional groups forms a covalent bond.

[0027] The functional groups of the substrate particles can be selected from hydroxyl, epoxy, and thiol. In the most preferred embodiment, the functional groups are hydroxyl groups.

[0028] The functional groups of the substrate particles (those that react with the silicone-based polymer product containing pendant functional groups) are typically located on the surface of the substrate particles. For example, the silicone-based polymer product containing pendant functional groups can react with functional groups, i.e., hydroxyl groups, on the surface of the substrate particles.

[0029] The silicone-based polymer product preferably contains and utilizes a silyl group or other group for attaching the polymer to a substrate (especially a silica substrate). Therefore, the silyl group is preferably an activated silyl group, i.e., it preferably has a group (leaving group) that can react with the substrate (especially silica) surface and allows the polymer to bond to the substrate surface. In this way, the silyl group covalently bonds the polymer to the silica substrate via a siloxane bond (Si-O-Si). The first Si atom in the siloxane bond originates from the silyl group. The second Si atom in the siloxane bond originates from silica, i.e., the silica surface.

[0030] The silyl or other groups of the polymer molecules are preferably represented by the formula: [ka] In the formula, R 1 , R 2 , R 3 At least one of R is a leaving group.1 , R 2 , R 3 are independently selected from an oxygen atom (e.g., attached to a substrate (silicon) atom in the substrate or attached to another silicon atom of the polymer), a hydroxyl group, a halogen atom, an alkoxy group (i.e., methoxy, ethoxy, etc.), a dialkylamino group, an acyl group, an alkyl group (optionally a heteroalkyl or heterocycloalkyl group), an aryl group (optionally a heteroaryl group), or a pendant functional group (reactive group) (i.e., a first reactive group as described herein, e.g., an olefinic group such as vinyl, allyl, etc.).

[0031] base R 1 , R 2 , and R 3 may be the same or all different. Preferably, R 1 , R 2 , R 3 At least one, and optionally two, of the groups is a leaving group. More preferably, R 1 , R 2 , R 3 At least one of the groups is an alkoxy group (preferably methoxy, ethoxy or propoxy, especially methoxy), a dialkylamino group, or a halogen atom.

[0032] The silicone-based polymer product also includes a plurality of pendant functional groups (hereinafter referred to as first reactive groups).

[0033] The pendant functional group (first reactive group) may preferably be a reactive olefin group or a reactive thiol group, particularly an olefin group. The reactive olefin group of the polymer is preferably a vinyl or allyl group. The multiple reactive groups may preferably all be of the same type, for example, all vinyl.

[0034] The pendant functional group can be a functional group capable of participating in a radical polymerization reaction, for example, the pendant functional group capable of participating in a radical polymerization reaction can be a vinyl, allyl, methacrylic, acrylate, acrylamide, methacrylamide, and styrene group, preferably a vinyl group.

[0035] The polymer preferably has a "constant" distance between adjacent first reactive groups (e.g., adjacent vinyl groups), i.e., the distance between adjacent first reactive groups is substantially uniform for all reactive groups in the polymer molecule. For example, homopolymer 1 in Scheme 1 of Figure 1 has a repeating 3-atom spacing between first reactive groups (e.g., vinyl groups). The silicone-based polymer product preferably comprises a vinyl siloxane polymer (i.e., a siloxane polymer having reactive vinyl groups). The size (i.e., number average molecular weight, MWn) of the siloxane polymer (optionally vinyl) is preferably 500 to 10,000 Daltons (Da). The vinyl siloxane polymer is preferably a vinyl alkoxy siloxane polymer.

[0036] In the case of vinyl siloxane polymers, the silyl groups typically have a leaving group (a terminal silyl group may have two leaving groups), a reactive group, and two bonds (a terminal silyl group has one such bond) at each oxygen atom (attached to adjacent silicon atoms in the polymer). The leaving group allows the polymer to form a siloxane bond to the substrate silica surface.

[0037] The vinyl siloxane polymer can have the formula I: [ka] wherein n is an integer from 3 to 100, and R1 and R2 are independently selected from alkoxy, particularly methoxy and ethoxy, hydroxyl, and halo (particularly Cl). R1 ​​and R2 are preferably independently selected from alkoxy, particularly methoxy and ethoxy, and hydroxyl. R1 and R2 are more preferably independently selected from alkoxy, particularly methoxy and ethoxy. R1 and R2 are particularly preferably the same, and preferably both are either methoxy or ethoxy.

[0038] Alternatively, the vinyl siloxane polymer may have the formula II: [ka]

[0039] Alternatively, the silicone-based polymer product may have ethoxy groups, or hydroxyl groups, in place of the methoxy groups of Formula II.

[0040] The silicone-based polymer can be a copolymer, for example, a copolymer containing a mixture of vinyl siloxane units and alkyl siloxane units (especially C1-C4 alkyl). The copolymer can be, for example, a copolymer of the nominal formula III [ka] wherein R1 and R2 are as defined for formulae I and II, n is an integer from 3 to 100, and m is an integer from 1 to 70 (preferably 1 to 20), or, for example, formula IV [ka] may have:

[0041] The vinylsiloxane and alkylsiloxane units in the above formula may be present in the polymer as blocks, or randomly distributed, or in alternating positions. Alternatively, the polymer may have methoxy or hydroxyl groups in place of the ethoxy groups of Formula IV.

[0042] The silicone-based polymer product includes at least one silyl-group-containing polymer (i.e., at least one polymer containing multiple silyl groups), preferably having a MWn of 500 to 10,000 Daltons. Specifically, such a polymer may be a modified polybutadiene, particularly a silyl-modified polybutadiene, preferably having a MWn of 500 to 10,000 Daltons.

[0043] The silicone-based polymer product preferably contains a trialkoxysilyl group (e.g., trimethoxysilyl or triethoxysilyl) as the silyl group. The at least one silyl-modified polybutadiene preferably contains a trialkoxysilyl-modified polybutadiene (the size (i.e., molecular weight, MWn) of the trialkoxysilyl-modified polybutadiene polymer is preferably 500 to 10,000 daltons). For example, the polybutadiene may be a trimethoxysilyl-modified polybutadiene or a triethoxysilyl-modified polybutadiene.

[0044] The silyl-modified polybutadiene may have a nominal repeat unit of formula V: [ka] In the formula, R 1 , R 2 and R 3 are independently selected from alkoxy, particularly methoxy and ethoxy, hydroxyl, halo (particularly Cl), and alkyl (particularly C1-C3 alkyl, more particularly methyl), with the proviso that R 1 , R 2 and R 3 At least one of R is a leaving group (especially methoxy or ethoxy). 1 , R 2 and R 3 is preferably independently selected from alkoxy, especially methoxy and ethoxy, and hydroxyl. 1 , R 2 and R 3are more preferably independently selected from alkoxy, especially methoxy and ethoxy; R 1 , R 2 and R 3 are particularly preferably the same, and preferably both are either methoxy or ethoxy.

[0045] The silyl-modified polybutadiene may be an alkoxysilyl-modified polybutadiene having a nominal repeat unit of formula VI. [ka] In the formula, each R 1 are independently either methoxy or ethoxy. Preferably, all R 1 are the same group.

[0046] for example: [ka] is.

[0047] Alternatively, the polymer may have methoxy or hydroxyl groups in place of the ethoxy groups of Formula VII.

[0048] In another preferred embodiment, the silyl-modified polybutadiene can be an alkylalkoxysilyl-modified polybutadiene. For example, the alkylalkoxysilyl-modified polybutadiene can have a nominal repeat unit of Formula VIII: [ka]

[0049] Alternatively, the polymer may have methoxy or hydroxyl groups in place of the ethoxy groups of formula VIII.

[0050] After reacting with the functional groups on the substrate particle, such as on the surface of the substrate particle (i.e., step (a)(i)), the silicone-based polymer product, in turn, encapsulates the substrate particle to form a silicone-based polymer-encapsulated substrate particle comprising pendant functional groups.

[0051] Examples of surface-bonded silicone-based polymer layers formed from polymers of Formula I or II (containing methoxy or ethoxy groups) in which vinyl reactive groups (vinyl pendant functional groups) are present are shown below: [ka]

[0052] In Formula I or II, any number of vinyl groups can be replaced with alkyl groups, for example, C1-C4 alkyl, particularly C1-C3 alkylation can be used.

[0053] Covalent attachment of polymers of Formula V, VI, or VII to silica can result in silicone-based polymer encapsulated substrate particles in which the silicone-based polymer product contains pendant functional groups attached to the surface as follows: [ka]

[0054] Covalent attachment of the polymer of Formula VIII to silica can result in silicone-based polymer encapsulated substrate particles in which the silicone-based polymer product contains pendant functional groups attached to the surface as follows: [ka]

[0055] The use of the polymers described herein offers numerous benefits, including controllability compared to very high MW polymers, which may tend to clog pores in the substrate, while allowing for multiple attachment to the substrate for stability, allowing for subsequent surface modification using copolymerization of compounds containing vinyl functional groups, and the "constant" distance between adjacent reactive (e.g., vinyl) groups favors the formation of a more uniform protective layer on the substrate surface, allowing for flexibility in the stationary phase or column chemistry through the choice of functional groups to attach to the polymer.

[0056] After forming the silicone-based polymer encapsulated substrate particles containing pendant functional groups as described above in step (a)(i), the encapsulated particles were further reacted in step (a)(ii) to functionalize the silicone-based polymer layer.

[0057] In step (a)(ii), this is accomplished by reacting pendant functional groups on the silicone-based polymer product with at least one hydrophobic compound and at least one amine-containing compound.

[0058] After step (a)(i), the pendant functional groups are located on the surface of the silicone-based polymer encapsulated substrate particles, i.e., in step (a)(ii), functionalizing the silicone-based polymer encapsulated substrate particles may comprise reacting the pendant functional groups located on the surface of the silicone-based polymer encapsulated substrate particles with at least one hydrophobic compound and at least one amine-containing compound.

[0059] This allows the surface of the base particle to be completely covered with reactive groups.

[0060] The at least one hydrophobic compound typically comprises at least one group reactive with the pendant functional groups on the silicone-based polymer-encapsulated substrate particle. For example, the at least one hydrophobic compound may comprise one, two, or three groups reactive with the pendant functional groups located on the silicone-based polymer-encapsulated substrate particle. The groups may be selected from the group consisting of vinyl groups, allyl groups, and / or thiol groups.

[0061] In a preferred embodiment, at least one hydrophobic compound is a branched or unbranched C4-C 30 In one embodiment of the present invention, at least one branched or unbranched C4-C 30 The alkyl may be monounsaturated, i.e., contain a double bond. For example, at least one branched or unbranched C4-C6 alkyl may contain at least one double bond. 30 The alkyl may preferably contain a terminal double bond (vinyl group). In a preferred embodiment, C4 to C 30 The alkyl is unbranched and monounsaturated, particularly where the monounsaturation is terminal (ie, vinyl).

[0062] Examples of the at least one hydrophobic compound include, for example, 1-butene, 1-pentene, 1-hexene, 1-heptene, 1-octene, 1-nonene, 1-decene, 1-undecene, 1-dodecene, 1-tridecene, 1-tetradecene, 1-pentadecene, 1-hexadecene, 1-heptadecene, 1-octadecene, 1-nonadecene, 1-eicosene, 1-heneicosene, 1-docosene, 1-tricosene, 1-tetracosene, 1-pentacosene, 1-hexacosene, and 1-heptacosene. and positional isomers of butene, pentene, hexene, octene, nonene, decene, undecene, dodecene, tridecene, tetradecene, pentadecene, hexadecene, heptadecene, octadecene, nonadecene, eicosene, heneicosene, docosene, tricosene, tetracosene, pentacosene, hexacosene, heptacosene, octocosene, nonacosene, and tricontene, such as 1-octocosene, 1-nonacosene, and 1-trichotene.

[0063] The at least one amine-containing compound can include at least one amino group selected from an ammonium group, a primary amine group, a secondary amine group, a tertiary amine group, and a quaternary amine group.

[0064] In one aspect of the invention, the at least one amine-containing compound is a branched or unbranched C4-C amine substituted with at least one amino group selected from ammonia, primary amine, secondary amine, tertiary amine, and quaternary amine groups. 30 The at least one amine-containing compound may also be monounsaturated, i.e., the at least one amine-containing compound may be a monounsaturated, branched or unbranched C4-C alkyl substituted with at least one amino group selected from ammonia, primary amine, secondary amine, tertiary amine, and quaternary amine groups. 30 In certain embodiments, the at least one amine-containing compound is a monounsaturated, unbranched C4-C alkyl substituted with at least one amino group selected from ammonia, primary amine, secondary amine, tertiary amine, and quaternary amine groups. 30 It can be alkyl.

[0065] The amine-containing compound may preferably have the formula: [ka] wherein each occurrence of R1 and R2 independently represents hydrogen, C1-C 18 Alkylation, C2-C 18 Alkenyl, C2-C 18 Alkynyl, C3-C 18 Cycloalkyl, C1-C 18 Heterocycloalkyl, C5-C 18 Allyl, C5-C 18 Aryloxy or C1-C 18 R3 represents heteroaryl. R3 is C1 to C 18 Alkyl, C2-C 18 Alkenyl, C2-C 18 Alkynyl, C3-C 18 Cycloalkyl, C1-C 18Heterocycloalkyl, C5-C 18 Allyl, C5-C 18 R4 represents hydrogen, C1-C 18 Alkyl, C2-C 18 Alkenyl, C2-C 18 Alkynyl, C3-C 18 Cycloalkyl, C1-C 18 Heterocycloalkyl, C5-C 18 Allyl, C5-C 18 Aryloxy, or C1-C 18 Represents heteroaryl.

[0066] In a preferred embodiment, the amine-containing compound may have the formula: [ka] Here, R1 and R2 are hydrogen, and R3 is C1 to C 18 Alkyl, R4 is C1-C 18 Represents alkyl.

[0067] For example, the amine-containing compound may preferably be oleylamine.

[0068] At least one hydrophobic compound and at least one amine-containing compound can react with the pendant functional groups in a radical polymerization reaction to form an additional polymer layer attached to the silicone-based polymer layer.

[0069] As previously mentioned, reaction of the pendant functional groups on the silicone-based polymer-encapsulated substrate particles formed in step (a)(i) with at least one hydrophobic compound and at least one amine-containing compound can form an additional polymer layer attached to the silicone-based polymer-encapsulated substrate particles, which may hereinafter also be referred to as an additional or further polymer layer.

[0070] Thus, step (a)(ii) may involve functionalizing the silicone-based polymer-encapsulated substrate particle by reacting pendant functional groups on the silicone-based polymer-encapsulated substrate particle with at least one hydrophobic compound and at least one amine-containing compound in a radical polymerization reaction to form an additional (or further) polymer layer.

[0071] Alternatively, step (a)(ii) may comprise encapsulating the silicone-based polymer-encapsulated substrate particles by reacting pendant functional groups on the silicone-based polymer-encapsulated substrate particles with at least one hydrophobic compound and at least one amine-containing compound in a radical polymerization reaction.

[0072] Step (a)(i) of the method of the present invention can typically be carried out at a temperature of about 20°C to about 250°C, preferably about 100°C to about 225°C, for example about 200°C.

[0073] Typically, step (a)(i) of the present method can be carried out for about 2 hours to about 72 hours, preferably about 24 hours to about 64 hours, for example, about 48 hours.

[0074] Step (a)(i) can be carried out in the presence of a solvent. Preferred solvents are water-immiscible organic solvents such as toluene, ethylbenzene, dimethylbenzene, n-hexane, n-heptane, n-pentane, n-octane, cyclohexane, etc. Preferably, the solvent is toluene.

[0075] Alternatively, step (a)(i) of the process may be carried out under reduced pressure, such as 500 mbar or less.

[0076] When step (a)(i) is carried out under reduced pressure, it may be preferred to carry out the step in the absence of a solvent.

[0077] Step (a)(ii) of the process of the present invention may also be carried out preferably in the absence of a solvent, at elevated temperature and / or reduced pressure.

[0078] It may be preferable to carry out step (a)(ii) in the presence of an initiator to facilitate the free radical polymerization reaction.

[0079] Desirably, the elevated temperature in step (a)(ii) is at least about 100° C., or at least about 110° C., or at least about 120° C., or at least about 140° C. For example, the elevated temperature in step (a)(ii) can be up to about 300° C., up to about 200° C., up to about 190° C., or up to about 180° C., or up to about 160° C.

[0080] More preferably, the elevated temperature in step (a)(ii) can be about 100°C to about 300°C, about 100°C to about 200°C, about 110°C to about 190°C, about 120°C to about 180°C, or about 130°C to about 170°C.

[0081] The reduced pressure for step (a)(i) or (ii) may preferably be less than 500 mbar, more preferably less than 400 mbar, even more preferably less than 300 mbar, even more preferably less than 200 mbar, and most preferably less than 100 mbar.

[0082] For example, the pressure is preferably at least 0.01 mbar, more preferably at least 0.1 mbar, or at least 1 mbar.

[0083] The reduced pressure and elevated temperature for step (a)(i) or (ii) may preferably be applied simultaneously for at least a certain period (first reaction period).

[0084] This period of time may be at least 1 hour, or at least 2 hours, or at least 4 hours, or at least 8 hours, or at least 12 hours. This period of time may be up to 20 hours, or up to 30 hours.

[0085] The reduced pressure is preferably applied for substantially the same period of time as the elevated temperature.

[0086] Both steps (a)(i) and (ii) can be carried out under an inert environment, i.e., under an inert gas (e.g., nitrogen or argon). The reactants are preferably purged with an inert gas before applying elevated temperatures, if reacted under reduced pressure, prior to the step.

[0087] As detailed above, the functionalized silicone-based polymer attached to the substrate particles can be formed by either step (a) or step (b) of the present method.

[0088] In step (b)(i), pendant functional groups on the silicone-based polymer product are reacted with at least one hydrophobic ligand and at least one amine-containing ligand to form a functionalized silicone-based polymer.

[0089] Step (b)(i) is carried out to form a functionalized silicone-based polymer before the substrate particles are encapsulated.

[0090] Similar to step (a)(ii) above, the reaction of pendant functional groups on the silicone-based polymer product with at least one hydrophobic compound and at least one amine-containing compound in step (b)(i) can form an additional polymer layer attached to the silicone-based polymer product, which may hereinafter be referred to as the additional or further polymer layer.

[0091] That is, step (b)(i) can include reacting pendant functional groups on the silicone-based polymer product with at least one hydrophobic compound and at least one amine-containing compound in a radical polymerization reaction.

[0092] Thus, step (b)(i) may involve functionalizing the silicone-based polymer product by reacting pendant functional groups on the silicone-based polymer-encapsulated substrate particles with at least one hydrophobic compound and at least one amine-containing compound in a radical polymerization reaction to form an additional (or further) polymer layer.

[0093] Encapsulation of the substrate particles occurs in step (b)(ii) where the functionalized silicone-based polymer (ie, the product of step (b)(i)) reacts with the functional groups on the substrate particles.

[0094] The functional group on the substrate particle, the substrate particle, the silicone-based polymer product, the pendant functional group on the silicone-based polymer product, the at least one hydrophobic ligand, and the at least one amine-containing ligand are as defined in step (a) above.

[0095] As with steps (a)(i) and (ii) above, step (b)(i) or (ii) may be carried out at elevated temperature and / or reduced pressure as previously defined.

[0096] Typically, in either step (a)(ii) or step (b)(i), the ratio of the at least one hydrophobic compound to the at least one amine-containing compound is from about 0:1 to about 50:1, such as from about 2:1 to about 8:1, for example, about 4:1.

[0097] It should be noted that in any polymerization reaction, suitable polymerization reagents can be used, which will depend on the compounds present, and the expert will be able to determine which is most suitable.

[0098] In a preferred embodiment, in a method for producing a chromatographic packing material, the packing material comprises a functionalized silicone-based polymer layer bonded to a substrate particle, the method for forming the packing material comprises: (a) (i) forming a silicone-based polymer encapsulated substrate particle comprising pendant functional groups by reacting hydroxyl, epoxy and / or thiol functional groups on the substrate particle with a silicone-based polymer product comprising pendant functional groups, wherein the silicone-based polymer product is a vinyl silxane having the formula: [ka] wherein n is an integer from 3 to 100, and R1 and R2 are independently selected from the group consisting of alkoxy, hydroxyl, and halo; (ii) converting the pendant vinyl functional groups on the silicone-based polymer encapsulated substrate particles into at least one monounsaturated, unbranched C4-C6 30 alkyl compounds and at least one monounsaturated, unbranched C4-C substituted with at least one amino group selected from ammonia, primary amine, secondary amine, tertiary amine, and quaternary amine groups. 30 functionalizing the silicone-based polymer encapsulated substrate particles by reacting them with an alkyl; or (b) (i) forming a functionalized silicone-based polymer by reacting pendant vinyl functional groups on a silicone-based polymer product, wherein the silicone-based polymer product is a vinyl siloxane having the formula: [ka] wherein n is an integer from 3 to 100, and R1 and R2 are independently at least one monounsaturated, unbranched C4-C 30 alkyl compounds and at least one monounsaturated, unbranched C4-C substituted with at least one amino group selected from ammonia, primary amine, secondary amine, tertiary amine, and quaternary amine groups. 30 wherein the alkyl is selected from the group consisting of alkoxy, hydroxyl, and halo; (ii) reacting the product of step b(i) with hydroxyl, epoxy and / or thiol functional groups on the substrate particles.

[0099] The resulting chromatography packing is provided in a form suitable for use as a chromatographic packing.

[0100] Thus, the present invention also provides a chromatographic packing material formed by the method defined above.

[0101] The present invention also provides (i) substrate particles; (ii) a functionalized silicone-based polymer layer bonded to a substrate particle, wherein the functionalized silicone-based polymer layer bonded to the substrate particle comprises: (a) (i) reacting functional groups on the substrate particles with a silicone-based polymer product containing pendant functional groups to form silicone-based polymer-encapsulated substrate particles containing pendant functional groups; and (ii) functionalizing the silicone-based polymer-encapsulated substrate particles by reacting the pendant functional groups on the silicone-based polymer-encapsulated substrate particles with at least one hydrophobic compound and at least one amine-containing compound; or (b) (i) reacting pendant functional groups on the silicone-based polymer product with at least one hydrophobic compound and at least one amine-containing compound to form a functionalized silicone-based polymer; and (ii) reacting the product of step b(i) with functional groups on said substrate particles.

[0102] Hereinafter, this may be referred to as the chromatography packing material of the present invention or the packing material of the present invention.

[0103] In the filler of the present invention, the bond between the functionalized silicone-based polymer and the substrate particle can be a covalent bond.

[0104] As detailed above with respect to the methods of the present invention, this is because bonds can typically be formed by reaction between hydroxyl, epoxy, and / or thiol functional groups on the substrate particle (e.g., the surface of the substrate particle) and leaving groups, such as alkoxy groups, present on the silicone-based polymer product.

[0105] The functional group on the substrate particle, the substrate particle, the silicone-based polymer product, the pendant functional group on the silicone-based polymer product, the at least one hydrophobic ligand, and the at least one amine-containing ligand are as defined above in the inventive content.

[0106] In one embodiment of the filler of the present invention, the ratio of the at least one hydrophobic compound to the at least one amine-containing compound present in the filler can be from about 0:1 to about 50:1, for example, from about 2:1 to about 8:1, for example, about 4:1.

[0107] In a preferred embodiment, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis. (i) metal oxide or organic polymer substrate particles, preferably silica substrate particles, containing hydroxyl, epoxy and / or thiol functional groups; (ii) a functionalized silicone-based polymer layer covalently bonded to a substrate particle, wherein the functionalized silicone-based polymer layer covalently bonded to the substrate particle comprises: (c) (i) forming a silicone-based polymer encapsulated substrate particle comprising pendant functional groups by reacting hydroxyl, epoxy and / or thiol functional groups on the substrate particle with a silicone-based polymer product comprising pendant functional groups, wherein the silicone-based polymer product is a vinyl silxane having the formula: [ka] wherein n is an integer from 3 to 100, and R1 and R2 are independently selected from the group consisting of alkoxy, hydroxyl, and halo; and (ii) converting the pendant vinyl functional groups on the silicone-based polymer encapsulated substrate particles into at least one monounsaturated, unbranched C4-C6 30 alkyl compounds and at least one monounsaturated, unbranched C4-C substituted with at least one amino group selected from ammonia, primary amine, secondary amine, tertiary amine, and quaternary amine groups. 30 or by functionalizing the silicone-based polymer encapsulated substrate particles by reacting them with an alkyl; (d) (i) forming a functionalized silicone-based polymer by reacting pendant vinyl functional groups on a silicone-based polymer product, wherein the silicone-based polymer product is a vinyl siloxane having the formula: [ka] wherein n is an integer from 3 to 100, and R1 and R2 are independently at least one monounsaturated, unbranched C4-C 30 alkyl compounds and at least one monounsaturated, unbranched C4-C substituted with at least one amino group selected from ammonia, primary amine, secondary amine, tertiary amine, and quaternary amine groups. 30 wherein the alkyl is selected from the group consisting of alkoxy, hydroxyl, and halo; and (ii) reacting the product of step d(i) with hydroxyl, epoxy and / or thiol functional groups on the substrate particles.

[0108] In certain embodiments, the filler material may be obtained using a method as defined herein.

[0109] The packing materials of the present invention can be used in chromatographic separations.

[0110] The specific combination of at least one hydrophilic compound and at least one amine-containing compound in the additional polymer layer has been shown to unexpectedly and unexpectedly provide particularly effective water retention and ion exchange selectivity, leading to excellent selectivity for glycosylated and deaminated peptides, and also provides protection from low pH and acidic aqueous mobile phases.

[0111] The present invention therefore also provides the use of a packing material as defined herein in chromatographic separations, in particular in the separation of glycopeptides.

[0112] In one aspect of the invention, use in chromatographic separations is preferred when carried out using mobile phases with a pH of 1.0 or less, or when carried out using mobile phases with a pH of 13.0 or more.

[0113] For example, it is used to separate glycopeptides using a mobile phase of pH 1.0 or less, or to separate glycopeptides using a mobile phase of pH 13.0 or more.

[0114] In view of the above, the present invention also provides a chromatographic separation device comprising a packing material as defined above, preferably the packing material being obtained using a method also defined above.

[0115] For the avoidance of doubt, when the term "comprising" or "comprises" is used herein, the described feature must include the recited elements, but may optionally include additional elements. When the term "consisting essentially of" or "consists essentially of" is used, the described feature must include the recited elements, but may also include other elements, provided that any element does not affect the basic characteristics of the feature. When the term "consisting of" or "consists of" is used, this means that the described feature must contain only the recited elements.

[0116] As used herein, the term "at least one" is intended to mean that the compound contains one or more of the recited characteristics, specifically one, two, three or four, one to four, or one to three of the recited characteristics.

[0117] It will be clear to those skilled in the art that the features and combinations defined for the method of the present invention apply equally to the filler material.

[0118] The detailed description illustrates, by way of example, and not by way of limitation, the principles of the present invention. This specification clearly enables one skilled in the art to make and use the present invention, as well as several embodiments, adaptations, variations, alternatives, and uses of the invention described. As used herein, the terms "about" or "approximately" in connection with any numerical value or range indicate a suitable dimensional tolerance that allows a portion or group of components to function for its intended purpose as described. [Brief explanation of the drawings]

[0119] [Figure 1]Scheme 1: shows the formation of silicone-based polymer-encapsulated silica-based particles containing pendant vinyl functional groups. Scheme 2: shows the formation of functionalized silicone-based polymer-encapsulated silica-based particles. [Figure 2] 1 shows the results of ion exchange tests obtained using a CAD detector on an ammonium chloride sample: (A) packing material according to the invention, (B) a well-known C18 column (Brand A). [Figure 3] 1 shows the results of hydrolytic stability testing at pH 2 (0.1 M TFA) and a temperature of 50° C.: (A) performance of the filler of the present invention at 40 hours of exposure time; (B) percent retention remaining. [Figure 4] LC-MS patterns of a peptide derived from the amino acid sequence DIQMTQSPSTLSASVGD characterized by oxidation are shown: (A) packing material of the present invention, (B) conventional C4 column (Brand A), (C) conventional C18 column (Brand A), (D) charged surface hybrid (Brand B). [Figure 5] LC-MS patterns of a peptide derived from the amino acid sequence GFYPSDIAVEWESNGQPENNYK characterized by deamidation are shown: (A) the packing material of the present invention, (B) a conventional C4 column (Brand A), (C) a conventional C18 column (Brand A), and (D) a charged surface hybrid (Brand B). [Figure 6] LC-MS patterns of a peptide derived from the amino acid sequence EEQYNSTYR characterized by N-glycanation are shown: (A) the packing material of the present invention, (B) a conventional C4 column (Brand A), (C) a conventional C18 column (Brand A), and (D) a charged surface hybrid (Brand B). DETAILED DESCRIPTION OF THE INVENTION

[0120] To illustrate the invention, non-limiting examples of its implementation are given below.

[0121] Example 1 Synthesis of Materials for Use as Chromatographic Stationary Phase Materials (Phase 1) 1.1 Preparation of vinyl-functionalized silica (step (a)(i) - Figure 1, Scheme 1) 20 g of dried solid-core porous spherical silica particles (dp, 2 μm, surface area, 75 m 2 The silica particles were transferred to a 250 mL round-bottom flask (160 oz, pore size) and a mixture of 7 g of vinylethoxysiloxane homopolymer (e.g., Gelest) and 0.44 g of tetramethylethylenediamine (e.g., Sigma-Aldrich) in toluene (60 mL) was added. After carefully dispersing the top of the slurry, the reaction mixture was brought to a steady reflux and stirred for 48 hours. The silica particles were filtered and washed thoroughly with acetone, an acetone:water solution (1:1, v / v), an acetonitrile:water solution (1:1, v / v), and then a mixture of 5% formic acid and acetonitrile:water solution (1:1, v / v). After filtration, washing with acetonitrile:water solution (1:1, v / v), an acetone:water solution (1:1, v / v), and acetone, the resulting silica was dried overnight at 140 °C under vacuum. The dried silica was redissolved in 60 mL of toluene, followed by the addition of 7 g of vinyldimethylethoxysilane (e.g., Gelest) and 0.56 g of tetramethylethylenediamine (e.g., Sigma-Aldrich). The resulting mixture was refluxed for 16 hours. The functionalized silica particles were filtered and washed thoroughly with toluene, dioxane, methanol, and acetone to yield silicone-based polymer-encapsulated substrate particles containing pendant vinyl functional groups.

[0122] 1.2 Preparation of polymer-encapsulated silica phase (Phase 1) using free radical polymerization reaction (step (a)(ii) - Figure 1, Scheme 2): 12 mL of dichloromethane was added to 10 g of vinyl-functionalized silica, 2 g of 1-octadecene (e.g., Sigma-Aldrich), 0.5 g of oleylamine (e.g., Thermo Scientific), and 0.5 g of dicumyl peroxide (e.g., Sigma-Aldrich). The resulting mixture was sonicated until homogeneous, and then all volatiles were removed under reduced pressure using a rotary evaporator. The resulting solvent-free mixture was then transferred to a reactor, which was sealed, flushed with an inert gas (e.g., nitrogen or argon) for 15 minutes, and heated to 160 °C. After holding at the same temperature for 16 hours, the reaction was cooled, and the reaction mixture was dispersed in heptane and sonicated for 15 minutes. After filtration, the cake was thoroughly washed with toluene, dioxane, methanol, and acetone to obtain the polymer-encapsulated silica (Phase 1).

[0123] Example 2: Synthesis of materials for use as chromatographic stationary phase materials (Phase 2) 1.1 Preparation of vinyl-functionalized silica via solvent-free conditions at elevated temperatures Dry solid core porous spherical silica particles (dp, 2 μm; surface area, 75 m 220 g of silica (e.g., 160 Å / g; pore size) was transferred to a 250 mL round-bottom flask, followed by the addition of a mixture of 7 g of vinylethoxysiloxane homopolymer (e.g., Gelest) in a suitable solvent (e.g., dichloromethane). The resulting mixture was sonicated until homogeneous, and then all volatiles were completely removed under reduced pressure. The dried mixture was placed in a reactor equipped with heating and vacuum capabilities. After placing a catalyst (e.g., 0.5 g of tetramethylethylenediamine) in the reactor, the reactor was sealed and then flushed with an inert gas (e.g., nitrogen or argon) for 30 minutes. The reactor was then evacuated to a certain value (e.g., less than 100 mbar) using a vacuum pump. The reactor was then heated to the desired temperature (100 °C) and maintained at the same temperature for at least 16 hours. After cooling, the silica particles were dispersed in toluene (100 mL) and sonicated for 30 minutes. After filtration, the cake was washed with toluene and acetone. The resulting silica was dispersed in a mixture of 5% acetic acid solution (CHCN:HO = 1:1, v / v) and left for 12 hours. After filtration and washing with acetone, the resulting silica was dried under vacuum at 105 °C for 12 hours. The dried silica was again placed in a reactor equipped with heating and vacuum capabilities. After placing a catalyst (e.g., 0.5 g of tetramethylethylenediamine) and 7 g of vinyldimethylethoxysilane in the reactor, the reactor was sealed and then flushed with an inert gas (e.g., nitrogen or argon) for 30 minutes. The reactor was then evacuated to the desired pressure (e.g., less than 100 mbar) using a vacuum pump. The reactor was then heated to the desired temperature (100 °C) and maintained at the same temperature for at least 16 hours. After cooling, the silica particles were dispersed in toluene (100 mL) and sonicated for 30 minutes. After filtration, the cake was washed with toluene and acetone to obtain silicone-based polymer-encapsulated substrate particles containing pendant vinyl functional groups.

[0124] 1.2 Preparation of polymer-encapsulated silica phase (second phase) using free radical polymerization reaction It was synthesized similarly to the method described in Example 1, section 1.2.

[0125] Example 3 Separation of a negatively charged analyte (chloride ion) under different buffer concentrations. The mixed-mode stationary phase described in Example 1 was packed into a chromatography column (150 mm i.d. x 2.1 mm) and applied to the separation of negatively charged analytes (chloride ions) under different buffer concentrations. As shown in Figure 2, the effect of ammonium formate concentration (counterion) on the retention of negatively charged analytes was observed for the first-phase column (A) and the conventional C18 column (B) while the ACN content and pH of the mobile phase were kept constant. As expected, the retention status of the conventional C18 column did not change with the counterion concentration. Retention is affected when a mixed-mode retention mechanism is present (first phase). The retention of negatively charged analytes increased with increasing counterion buffer concentration. This indicates that the synthetic approach of this invention can yield a mixed-mode phase with ion-pairing (anion-exchange) properties. Column: 150mm x 2.1mm inner diameter Mobile phase: 100mM ammonium formate pH 3.0, acetonitrile, deionized water Flow rate: 0.3mL / min Column temperature: 30°C Detector: Charged Aerosol Detector (CAD)

[0126] Example 4 Hydrolytic stability test The mixed-mode stationary phase described in Example 1 was packed into a chromatography column (50 mm x 4.6 mm internal diameter) and subjected to a hydrolytic stability test. The performance test and low-pH mobile phase treatment were repeated 11 times (for a total of 40 hours). At 4-hour intervals, a sample containing acenaphthene (uracil as a void marker) was applied to the performance test. As shown in Figure 3, the retention of acenaphthene during the performance test decreased by only 2.3% after 40 hours of acidic treatment, indicating that mixed-mode stationary phase 1 is highly stable under acidic conditions. Column: 50mm x 4.6mm inner diameter Mobile phase: Hydrolysis stability test 0.1% TFA (wettable), column performance test 50% ACN, 50% 0.01M ammonium acetate pH 5.0 Flow rate: 0.4mL / min Column temperature: 50℃ Detector: UV, 254 nm,

[0127] Example 5 Isolation of oxidized peptides. The mixed-mode stationary phase 1 described in Example 1 was packed into a chromatography column (150 mm x 2.1 mm internal diameter) for analysis of post-translationally modified peptides. Figure 4 shows the LC-MS patterns of a peptide derived from the amino acid sequence DIQMTQSPSTLSASVGD, characterized by oxidation, on: (A) the packing material of the present invention, (B) a conventional C4 column (Brand A), (C) a conventional C18 column (Brand A), and (D) a charged surface hybrid (Brand B). The same sample of a NISTmAb tryptic digest containing the oxidized peptide (amino acid sequence: DIQMTQSPSTLSASVGDR) was analyzed.

[0128] The packing of the present invention showed peak shape and resolution of oxidized peptides from interfering peaks comparable to non-mixed-mode stationary phases, which is beneficial since mixed-mode stationary phases typically produce poor peak shape for oxidized peptides.

[0129] The test conditions were as follows: column, packing material of the present disclosure: 150 mm x 2.1 mm inner diameter; mobile phase: ultrapure water containing 0.1% formalin acid and acetonitrile containing 0.1% formalin acid; flow rate: 0.4 mL / min; column temperature: 50°C; detector: high-resolution mass spectrometer; sample: NISTmAb digestion; injection volume: 8 μL.

[0130] Example 6 Isolation of deamidated peptides. The mixed-mode stationary phase 1 described in Example 1 was packed into a chromatography column (150 mm x 2.1 mm internal diameter) for analysis of post-translationally modified peptides. Figure 5 shows the LC-MS patterns of a peptide derived from the amino acid sequence GFYPSDIAVEWESNGQPENNYK, characterized by deamidation, on (A) the packing material of the present invention, (B) a conventional C4 column (Brand A), (C) a conventional C18 column (Brand A), and (D) a charged surface hybrid (Brand B). The same sample of NISTmAb tryptic digest containing the deamidated peptide (amino acid sequence: GFYPSDIAVEWESNGQPENNYK) was analyzed.

[0131] Since glutamine and multiple asparagine amino acids in a peptide can be deamidated, multiple deamidated peaks are expected. The packing material of the present invention shows the separation of multiple peptide peaks with single deamidation, which is useful for detailed analysis of post-translationally modified peptides.

[0132] The test conditions were as follows: column, packing material of the present disclosure: 150 mm x 2.1 mm inner diameter; mobile phase: ultrapure water containing 0.1% formalin acid and acetonitrile containing 0.1% formalin acid; flow rate: 0.4 mL / min; column temperature: 50°C; detector: high-resolution mass spectrometer; sample: NISTmAb digestion; injection volume: 8 μL.

[0133] Example 7 Separation of aminoglycoside peptides. The mixed-mode stationary phase 1 described in Example 1 was packed into a chromatography column (150 mm x 2.1 mm internal diameter) for analysis of post-translationally modified peptides. Figure 6 shows the LC-MS patterns of a peptide derived from the amino acid sequence EEQYNSTYR, characterized by N-glycanation, using: (A) the packing material of the present invention, (B) a conventional C4 column (Brand A), (C) a conventional C18 column (Brand A), and (D) a charged surface hybrid (Brand B). The same sample of NISTmAb tryptic digest containing oxidized peptides (amino acid sequence: EEQYNSTYR) was analyzed.

[0134] Due to the different types of glycans attached to peptides, multiple N-linked glycopeptide peaks are expected. The packing materials of the present invention are excellent at separating multiple glycopeptide peaks, making them useful for detailed analysis of glycopeptides. Chromatographic resolution of these peaks is important for successful identification by fragmentation spectroscopy, because multiple co-eluting glycan species can cause unintended fragmentation in the ion source, reducing the intensity of spectral peaks and potentially interfering with each other. Therefore, the packing materials of the present invention are useful for reducing interference between peptide glycoforms.

[0135] The test conditions were as follows: column, packing material of the present disclosure: 150 mm x 2.1 mm inner diameter; mobile phase: ultrapure water containing 0.1% formalin acid and acetonitrile containing 0.1% formalin acid; flow rate: 0.4 mL / min; column temperature: 50°C; detector: high-resolution mass spectrometer; sample: NISTmAb digestion; injection volume: 8 μL.

Claims

1. 1. A method for producing a chromatography packing, the packing comprising a functionalized silicone-based polymer layer bonded to a substrate particle, the method comprising: (a) (i) reacting functional groups on the substrate particles with a silicone-based polymer product containing pendant functional groups to form silicone-based polymer-encapsulated substrate particles containing pendant functional groups; (ii) functionalizing the silicone-based polymer-encapsulated substrate particles by reacting the pendant functional groups on the silicone-based polymer-encapsulated substrate particles with at least one hydrophobic compound and at least one amine-containing compound; or (b) (i) forming a functionalized silicone-based polymer by reacting pendant functional groups on the silicone-based polymer product with at least one hydrophobic compound and at least one amine-containing compound; (ii) reacting the functionalized silicone-based polymer with functional groups on the substrate particles.

2. The method of claim 1 , wherein the functional groups on the substrate particles are selected from hydroxyl, epoxy, and thiol.

3. The method of claim 1 , wherein the substrate particles are selected from the group consisting of metal oxides and organic polymers.

4. 2. The method of claim 1, wherein the pendant functional groups on the silicone-based polymer encapsulated substrate particles or the silicone-based polymer product are reactive olefinic or reactive thiol groups, preferably olefinic groups.

5. The method of claim 1 , wherein the silicone-based polymer product comprises at least one leaving group, preferably an alkoxy leaving group.

6. The method of claim 1 , wherein the silicone-based polymer product comprises a vinyl siloxane.

7. The at least one hydrophobic compound is ４ ~C ３０ The method of claim 1 , wherein the alkyl is alkyl.

8. The at least one amine-containing compound is a C substituted with at least one amino group selected from ammonia, primary amine, secondary amine, tertiary amine, and quaternary amine groups. ４ ~C ３０ The method of claim 1 , wherein the alkyl is alkyl.

9. 10. The method of claim 1, wherein the reaction of the pendant functional groups on the silicone-based polymer-encapsulated substrate particles with the at least one hydrophobic compound and at least one amine-containing compound in step (a)(ii) forms an additional polymer layer attached to the silicone-based polymer-encapsulated substrate particles.

10. 10. The method of claim 1, wherein the reaction of the pendant functional groups on the silicone-based polymer product with the at least one hydrophobic compound and at least one amine-containing compound in step (b)(i) forms an additional polymer layer attached to the silicone-based polymer product.

11. 10. The method of claim 1, wherein in step (a)(ii) or step (b)(i), the ratio of the at least one hydrophobic compound to the at least one amine-containing compound is from about 0:1 to about 50:

1.

12. The method of claim 1 , wherein the packing material is provided in a form suitable for use as a chromatographic packing.

13. A chromatography packing material formed by the method of any one of claims 1 to 12.

14. Base particles; a functionalized silicone-based polymer layer bonded to the substrate particle, The functionalized silicone-based polymer layer comprises: (a) (i) reacting functional groups on the substrate particles with a silicone-based polymer product containing pendant functional groups to form silicone-based polymer-encapsulated substrate particles containing pendant functional groups; and (ii) functionalizing the silicone-based polymer-encapsulated substrate particles by reacting the pendant functional groups on the silicone-based polymer-encapsulated substrate particles with at least one hydrophobic compound and at least one amine-containing compound; or (b) (i) forming a functionalized silicone-based polymer by reacting pendant functional groups on the silicone-based polymer product with at least one hydrophobic compound and at least one amine-containing compound; and (ii) A chromatography packing prepared by reacting said functionalized silicone-based polymer with functional groups on said substrate particles.

15. 15. The chromatography packing of claim 14, wherein the functional groups on the substrate particles are selected from hydroxyl, epoxy, and thiol.

16. 15. The chromatography packing of claim 14, wherein the substrate particles are selected from the group consisting of metal oxides and organic polymers.

17. 15. The chromatography packing of claim 14, wherein the pendant functional groups on the silicone-based polymer encapsulated substrate particle or the silicone-based polymer product are reactive olefinic or reactive thiol groups, preferably olefinic groups.

18. 15. The chromatography packing of claim 14, wherein the silicone-based polymer product comprises at least one leaving group, preferably an alkoxy group.

19. 15. The chromatography packing of claim 14, wherein the silicone-based polymer product comprises a vinyl siloxane.

20. The at least one hydrophobic compound is C ４ ~C ３０ 15. The chromatography packing of claim 14, which is alkyl.

21. The at least one amine-containing compound is a C substituted with at least one amino group selected from ammonia, primary amine, secondary amine, tertiary amine, and quaternary amine groups. ４ ~C ３０ 15. The chromatography packing of claim 14, which is alkyl.

22. 15. The chromatography packing of claim 14, wherein the reaction of the pendant functional groups on the silicone-based polymer-encapsulated substrate particles with the at least one hydrophobic compound and the at least one amine-containing compound in step (a)(ii) forms an additional polymer layer attached to the silicone-based polymer-encapsulated substrate particles.

23. 15. The chromatography packing of claim 14, wherein the reaction of the pendant functional groups on the silicone-based polymer product with the at least one hydrophobic compound and the at least one amine-containing compound in step (b)(i) forms an additional polymer layer attached to the silicone-based polymer product.

24. 15. The chromatography packing of claim 14, wherein the ratio of said at least one hydrophobic compound to said at least one amine-containing compound is from about 0:1 to about 50:

1.

25. Chromatography packing material according to any one of claims 14 to 24, wherein the chromatography packing material is obtainable using a method according to any one of claims 1 to 12.

26. Use of a chromatographic packing material according to any one of claims 14 to 24 in chromatographic separations, preferably in the separation of glycopeptides.

27. 27. The use according to claim 26, wherein the chromatographic separation is carried out using a mobile phase with a pH of 1.0 or less or a pH of 13.0 or more.

28. A chromatographic separation device comprising the chromatographic packing material according to any one of claims 14 to 24.

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