Zwitterionic column filler

Abstract

The invention relates to column packing and a preparation method thereof. In particular, the present invention relates to a column packing for hydrophilic interaction liquid chromatography (HILIC).

Description

[0001] Cross-reference to related applications This application claims priority to U.S. Provisional Application No. 63 / 514,878, filed July 21, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to column packing materials and their preparation methods. Specifically, this invention relates to column packing materials for hydrophilic interaction liquid chromatography (HILIC). Background Technology

[0003] Hydrophilic interaction liquid chromatography (HILIC) has been widely used in the analysis of polar compounds and compounds of important biological significance in proteomics.

[0004] Several commercially available chromatography columns are available, such as Thermo Amide HILIC and ZIC-HILIC. It has been reported that nearly 30% of HILIC applications use zwitterionic HILIC stationary phases. However, the selection of zwitterionic separation materials is limited. Currently, one of the most commonly used zwitterionic compounds for HILIC stationary phases is sulfobetaine, as shown below.

[0005] Sulfobetaine is an amphoteric compound that can be covalently bonded to silica to prepare one of the most commonly used zwitterionic HILIC stationary phases.

[0006] Sulfobetaine stationary phases typically contain a 1:1 ratio of negatively charged sulfonate groups to positively charged quaternary ammonium groups. These functional groups exhibit a net zero charge in the pH range of 0–14, thereby minimizing ion exchange. Evidence suggests that the zwitterionic HILIC retention mechanism of such stationary phases primarily stems from hydrophilic partitioning, accompanied by weak ion interactions between residual silanol groups and the analyte.

[0007] The lack of ionic properties in the sulfobetaine HILIC stationary phase leads to problems in the separation of ionic compounds. Furthermore, sulfobetaine HILIC stationary phases typically suffer from low reproducibility and significant stationary phase loss.

[0008] Therefore, it is essential to develop a novel HILIC stationary phase that improves upon hydrophilicity, charge properties, selectivity, and column efficiency.

[0009] The listing or discussion of prior disclosures in this specification should not necessarily be construed as an admission that such documents constitute part of the prior art or are common general knowledge. Summary of the Invention

[0010] The present invention aims to solve at least some of the above-mentioned problems by providing a novel zwitterionic HILIC stationary phase.

[0011] Therefore, the present invention provides a method for preparing chromatographic packing material, wherein the packing material comprises a zwitterionic compound (e.g., amino acid, aminophosphonate, and / or aminosulfonate) bonded to matrix particles, and the method for preparing the packing material includes: (a) (i) Reacting the functional groups on the matrix particles with an organofunctional silane compound; (ii) Reacting the product of steps (a) and (i) with a Michael acceptor containing a protected acid group; and (iii) Remove the acid protecting group from the product of steps (a) and (ii); or (b) (i) React Michael acceptors containing protected acid groups with organofunctional silane compounds; (ii) React the product of step (b) (i) with the functional groups on the matrix particles; and (iii) Remove the acid protecting groups from the product of step (b) (ii).

[0012] In one aspect of the invention, steps (a) and (iii) or (b) and (iii) may be omitted, and the column packing material may be sold in a "protected form." For example, the column packing material may be packed into a column and sold for in-situ deprotection by the customer.

[0013] Preferably, the protecting group can be removed before being packed into the chromatographic column.

[0014] Alternatively, the acid protecting group can be removed after the column packing material is loaded into the column.

[0015] As used herein, the term "zwitterionic" is intended to encompass negatively charged compounds that contain both positive and negative charges within their molecules. Examples of zwitterionic compounds that can be used in this invention include amino acids and their derivatives / analogs, such as aminophosphonates and aminosulfonates.

[0016] As described below, the zwitterionic compound is formed by the binding / reaction between an organofunctional silane and a Michael acceptor containing a protected acid group, the protected acid group being removed to provide the zwitterionic compound.

[0017] Suitable matrix particles may include a variety of commercially available chromatographic media (such as packed beds of chromatographic particles), as well as many other specifications, including appropriately derivatized tubing and fused silica capillaries that can be used after a simple alkaline hydrolysis treatment to activate the surface. Therefore, unless otherwise stated, the term "matrix particles" encompasses one or more matrices.

[0018] The matrix particles can be granular or monolithic, preferably granular. The matrix particle material can be a metal oxide (this term includes metal-like oxides, such as silicon dioxide; and includes inorganic-organic hybrid materials (especially metal oxide-organic hybrid materials), such as those described in WO 00 / 45951). The matrix particles can particularly be silicon dioxide (SiO2, this term includes silicon dioxide / organic hybrid materials), alumina (Al2O3), titanium dioxide (TiO2), or zirconium oxide (ZrO2) matrices.

[0019] The preferred matrix particles are silica (the term includes silica / organic hybrid materials in this document).

[0020] As detailed above, the zwitterionic compound is bonded to the matrix particles. In a preferred aspect, the zwitterionic compound is bonded to functional groups on the surface of the matrix particles. Typically, the bond is a covalent bond, meaning the zwitterionic compound is covalently bonded to the functional groups on the surface of the matrix particles. The covalent bond can typically be formed between the functional groups on the matrix particles and the silicon atoms contained in the organofunctional silane that forms the zwitterionic compound.

[0021] In the method of the present invention, the filler may be prepared using step (a) or step (b).

[0022] In step (a) (i), the functional groups on the matrix particles (surface) are reacted with an organofunctional silane compound.

[0023] The functional groups on the matrix particles can be any functional groups suitable for reacting with organofunctional silane compounds. Typically, the functional groups present on the surface of the matrix particles (e.g., silica particles) are selected from the group consisting of epoxy groups, hydroxyl groups, and amino groups. Preferably, the functional group can be a hydroxyl group, i.e., a hydroxyl group located on the surface of the matrix particles.

[0024] As used herein, the term "organofunctional silane compound" is intended to encompass hybrid compounds that combine the functionality of reactive organic groups with the inorganic functionality of alkoxysilanes in a single molecule. Organofunctional silanes as used herein may include Garsil silanes.

[0025] The organofunctional silane compound contains at least one hydrolyzable alkoxy group and at least one reactive organic group, such as a primary amino group, a secondary amino group, and / or a thiol group.

[0026] In certain respects, the organofunctional silane has the following structure: X is selected from -NH2, -NH-, -S- or -SH-. When X is NH2 or -SH-, only group 1 exists, that is, group 2 does not exist. When X is -NH- or -S-, both group 1 and group 2 exist.

[0027] Group 1 has the formula R1, R2 and R3 are independently selected from C1-4 alkyl or OC1-4 alkyl, provided that at least one of R1, R2 or R3 is OC1-4 alkyl; R4 and R5 are substituted or unsubstituted C1-12 alkyl groups, provided that the combined number of R4 and R5 does not exceed C14 alkyl groups; for example, R4 can be C1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 alkyl groups, and R5 can be C13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 alkyl groups, provided that the combined number of R4 and R5 does not exceed C14 alkyl groups; A is selected from NH, CH2, or CH substituted with an aryl group (e.g., phenyl); Group 2 is selected from the formula as defined above. Or R6, where R6 is -CH2-aryl.

[0028] Preferably, when X is -NH- or -S-, group 1 and group 2 can be .

[0029] Preferably, R1, R2 and R3 can be independently selected from methoxy or ethoxy.

[0030] Preferably, R4 can be a C1-4 alkyl group and R5 can be a C1-4 alkyl group; for example, R4 can be a C1, 2, 3 or 4 alkyl group and R5 can be a C1, 2, 3 or 4 alkyl group.

[0031] Preferably, A can be CH2 or NH, and most preferably CH2.

[0032] In a particularly preferred aspect, X is selected from -NH2- or -SH-; Group 1 has the formula Furthermore, group 2 is absent; R1, R2, and R3 are independently selected from methoxy or ethoxy; R4 is a C1, 2, 3, or 4 alkyl group; and R5 is a C1, 2, 3, or 4 alkyl group; and A can be CH2 or NH, with CH2 being the most preferred.

[0033] In yet another particularly preferred aspect X is -NH- or -S-, and both group 1 and group 2 are... ; R1, R2, and R3 are independently selected from methoxy or ethoxy; R4 is a C1, 2, 3, or 4 alkyl group; and R5 is a C1, 2, 3, or 4 alkyl group; and A can be CH2 or NH, with CH2 being the most preferred.

[0034] In this document, unless otherwise stated, the term "alkyl" itself or as part of another substituent refers to a straight or branched chain that may be fully saturated, monounsaturated or polyunsaturated, and may include divalent and polyvalent groups having a specified number of carbon atoms (i.e., C1-10 means one to ten carbons).

[0035] Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl (e.g., —CH2 —CH2 —CH3, —CH2 —CH2 —CH2 —), isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, and homologues and isomers such as n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl groups are alkyl groups having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propynyl, crotonyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl, and higher homologues and isomers. Unless otherwise stated, the term "alkyl" is also intended to include those alkyl derivatives, such as "heteroalkyl," as defined in more detail below. Alkyl groups limited to hydrocarbon groups are collectively referred to as "homoalkyl." When the alkyl group is a divalent group, the term "alkyl" may also refer to "alkylene", "alkyldiyl", or "alkylene".

[0036] In a preferred aspect, the alkyl group used herein may be considered to mean a substituted or unsubstituted saturated carbon chain.

[0037] Substituents in alkyl groups are collectively referred to as "alkyl substituents," and can be selected from, but not limited to, one or more of the following groups: substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, substituted or unsubstituted heterocyclic alkyl groups, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, —halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO2R′ , —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)2R′, —NR—C(NR′R″R′″ )═NR′″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)2R′, —OS(O)2R′, —S(O)2NR′R″, —NRSO2R′, —CN And —NO2, the number of which ranges from zero to (2m′+1), where m′ is the total number of carbon atoms in such groups. R′, R″, R′″ and R″″ each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or aralkyl. For example, when the compounds of the present invention contain more than one R group, each R group is independently selected; when more than one such group is present, each R′, R″, R′″ and R″″ group is also independently selected. When R′ and R″ are attached to the same nitrogen atom, they can combine with that nitrogen atom to form a 5, 6 or 7-membered ring. For example, —NR′R″ is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. Based on the discussion of substituents above, those skilled in the art should understand that the term "alkyl" is intended to include groups comprising a carbon atom bonded to a group other than a hydrogen group, such as haloalkyl (e.g., —CF3 and —CH2 CF3) and acyl (e.g., —C(O)CH3, —C(O)CF3, —C(O)CH2 OCH3, etc.).

[0038] Examples of organofunctional silanes include, but are not limited to: (3-aminopropyl)trialkoxysilanes, such as (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, and (3-aminopropyl)tripropoxysilane; (3-mercaptopropyl)trialkoxysilanes, such as (3-mercaptopropyl)trimethoxysilane, (3-mercaptopropyl)triethoxysilane, and (3-mercaptopropyl)tripropoxysilane; and bis(trialkoxysilylpropyl)amines, such as bis(trimethoxysilylpropyl)amine, bis(triethoxysilylpropyl)amine, and bis(triethoxysilylpropyl)amine. Alkylpropyl)amine and bis(tripropoxysilylpropyl)amine; (aminoethylaminomethyl)phenethyltrimethoxysilane, N-(2-N-benzylaminoethyl)-3-aminopropyltrimethoxysilane, N-(2-aminoethyl)-3-aminopropylmethyldiethoxysilane, N-(6-aminohexyl)aminopropyltrimethoxysilane, N-(2-aminoethyl)-11-aminoundecyltrimethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and N-(6-aminohexyl)aminomethyltriethoxysilane.

[0039] As mentioned earlier, the reaction between the functional groups on the matrix particles (surface) and the organofunctional silanes forms bonds (e.g., covalent bonds) between the matrix particle surface and the organofunctional silanes.

[0040] In this reaction, the at least one hydrolyzable alkoxy group is typically hydrolyzed to a silanol group, which then undergoes a condensation reaction with functional groups on the matrix particles (surface).

[0041] Therefore, method (a) as defined herein may include the step of hydrolyzing at least one hydrolyzable alkoxy group to a silanol group prior to reaction with functional groups on the matrix particles (surface). Any method suitable for alkoxy hydrolysis may be used.

[0042] As those skilled in the art will understand, the bonds formed will depend on the matrix used. For example, in the case where the functional group present on the matrix particles (surface) is a hydroxyl group, the reaction of the hydrolyzable / hydrolyzable alkoxy group on the organofunctional silane with the hydroxyl group will lead to the formation of siloxane bonds.

[0043] Therefore, in one aspect of the invention, the method may include: reacting epoxy, hydroxyl, or amino (preferably hydroxyl) groups present on the surface of the matrix particles with hydrolyzable / hydrolyzable alkoxy groups on the organofunctional silane compound in step (a) (i).

[0044] Typically, in step (a) (i), silica particles react with organofunctional silanes in a ratio of about 1:10 to about 10:1, for example about 1:0.5 to about 1:2, or about 1:1.

[0045] After the functional groups on the surface of the matrix particles react with organofunctional silanes (step (a)(i)), the product of the reaction is then reacted with a Michael acceptor containing protected acid functional groups (step (a)(ii)).

[0046] The following shows an example of the product of step (a) (i), wherein the organofunctional silane is (3-aminopropyl)trimethoxysilane and the functional group on the matrix particles is hydroxyl.

[0047] In steps (a) and (ii), the reaction utilizes the functional groups present on the (now bonded) organofunctional silane as Michael donors, which react with Michael acceptors.

[0048] Michael receptors can be protected acids, such as protected carboxylic acids, protected phosphonic acids, or protected sulfonic acids.

[0049] The protecting group used can be any group suitable for protecting carboxylic acids, phosphonic acids, or sulfonic acids. For example, the protecting group can be an alkyl protecting group, such as methyl, ethyl, butyl, or tert-butyl.

[0050] Examples of usable Michael receptors include, but are not limited to: protected α,β-unsaturated carboxylic acids, such as methyl acrylate, ethyl acrylate, butyl acrylate, or tert-butyl acrylate; dialkyl vinylphosphonates, such as dimethyl vinylphosphonate or diethyl vinylphosphonate; and vinyl sulfonates, such as methyl vinylsulfonate, ethyl vinylsulfonate, or neopentyl vinylsulfonate.

[0051] Those skilled in the art will understand that the product formed by the Michael addition reaction will depend on whether the product of step (a)(i) uses a primary amino group, a secondary amino group, or a thiol group as the Michael donor.

[0052] As a non-limiting example, the presence of a primary amine will result in the formation of a diester product, with an amino to ester ratio of approximately 1:2. The use of a secondary amine will result in the formation of a monoester, with an amino to ester ratio of approximately 1:1. The same ratio will also apply when the Michael donor is a thiodont.

[0053] Typically, in step (a) and (ii), the product of step (a) and (i) is reacted with the Michael receptor in a ratio of about 1:1 to about 1:10, for example, about 1:3.

[0054] In steps (a) and (iii), the acid protecting groups are removed to obtain zwitterionic chromatography packing material.

[0055] Any suitable method for removing acid protecting groups can be used. For example, treatment can be performed using an acid, such as an aqueous solution of methanesulfonic acid.

[0056] The chromatographic packing material can also be prepared by the method defined in step (b).

[0057] In step (b)(i), the Michael acceptor containing protected acid groups is reacted with an organofunctional silane compound.

[0058] The following shows an example of the reaction in which the organofunctional silane is (3-aminopropyl)trimethoxysilane and the Michael acceptor is tert-butyl acrylate.

[0059] In step (b)(i), the functional group of the organofunctional silane compound is used as a Michael donor.

[0060] Michael receptors and organofunctional silane compounds are as defined above for step (a).

[0061] Those skilled in the art will understand that the product formed by the Michael addition reaction will depend on whether the organofunctional silane contains a primary amino group, a secondary amino group, or a thiol group as a Michael donor.

[0062] In the above non-limiting examples, the presence of a primary amine leads to the formation of a diester product, i.e., the ratio of amine to ester is approximately 1:2. The use of a secondary amine, however, results in the formation of a monoester, i.e., the ratio of amine or thiol group to ester is approximately 1:1.

[0063] In method step (b)(i) as defined herein, the ratio of Michael donor (organofunctional silane) to Michael acceptor may be from about 1:1 to about 1:5, for example from about 1:1 to about 1:3.

[0064] In step (b)(ii), the product of step (b)(i) is reacted with the functional groups on the matrix particles.

[0065] The product of step (b)(i) contains at least one hydrolyzable alkoxy group derived from the said organofunctional silane. Similar to step (a), the at least one alkoxy group is hydrolyzed to a silanol group, which then undergoes a condensation reaction with a functional group on the matrix particles (surface). This forms a bond, such as a covalent bond, between the functional group and the product of step (b)(i). If the functional group on the matrix particles is a hydroxyl group, the bond can be a siloxane bond.

[0066] Therefore, the method (b) defined herein may include the step of hydrolyzing at least one hydrolyzable alkoxy group to a silanol group before reacting with the functional groups on the matrix particles (surface).

[0067] Therefore, in one aspect of the invention, the method may include: in step (b)(ii), reacting an epoxy, hydroxyl, or amino group (preferably hydroxyl) present on the surface of the matrix particles with a hydrolyzed alkoxy group on the product of step (b)(i).

[0068] The functional groups on the matrix particles and the matrix particles are as defined for step (a).

[0069] In steps (b) and (iii), the acid protecting groups are removed to obtain zwitterionic chromatography packing material.

[0070] Similar to step (a), any suitable method for removing acid protecting groups can be used. Steps (a) (iii) or (b) (iii) (removal of acid protecting groups) can be performed before or after packing the column. If the removal is performed after packing, it can be carried out by any means suitable for removing acid protecting groups. For example, the removal can be performed by rinsing the column with an acid (e.g., an aqueous solution of an acid, i.e., an aqueous solution of methanesulfonic acid).

[0071] In another preferred aspect, the present invention provides a method for preparing chromatographic packing material, wherein the packing material comprises a zwitterionic compound (e.g., amino acid, aminophosphonate, and / or aminosulfonate) bonded to matrix particles, and the method for preparing the packing material may include: (a) (i) Reacting functional groups (e.g., hydroxyl functional groups) on the surface of silica matrix particles with an organofunctional silane having the following structure: X is selected from -NH2, -NH-, -S- or -SH-. When X is NH2 or -SH-, only group 1 exists, that is, group 2 does not exist. When X is -NH- or -S-, both group 1 and group 2 exist.

[0072] Group 1 has the formula R1, R2 and R3 are independently selected from C1-4 alkyl or OC1-4 alkyl, provided that at least one of R1, R2 or R3 is OC1-4 alkyl; R4 and R5 are substituted or unsubstituted C1-12 alkyl groups, provided that the combined number of R4 and R5 does not exceed C14 alkyl groups; for example, R4 can be C1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, and R5 can be C13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1. A is selected from NH, CH2, or CH substituted with an aryl group (such as phenyl); Group 2 is selected from the formula as defined above. Or R6, where R6 is -CH2-aryl; (ii) Reacting the product of step (a) (i) with at least one Michael acceptor containing a protected acid group (e.g., at least one compound selected from: methyl acrylate, ethyl acrylate, butyl acrylate or tert-butyl acrylate, dimethyl vinylphosphonate or diethyl vinylphosphonate, or methyl vinylsulfonate, ethyl vinylsulfonate or neopentyl vinylsulfonate); and (iii) Remove the protecting group from the product of step a (ii); or (b) (i) Reacting at least one Michael acceptor containing a protected acid group (e.g., at least one compound selected from: methyl acrylate, ethyl acrylate, butyl acrylate or tert-butyl acrylate, dimethyl vinylphosphonate or diethyl vinylphosphonate, or methyl vinylsulfonate, ethyl vinylsulfonate or neopentyl vinylsulfonate) with an organofunctional silane having the structure shown below: X is selected from -NH2-, -NH-, -S- or -SH-. When X is NH2 or -SH-, only group 1 exists, that is, group 2 does not exist; when X is -NH- or -S-, both group 1 and group 2 exist. Group 1 has the formula R1, R2 and R3 are independently selected from C1-4 alkyl or OC1-4 alkyl, provided that at least one of R1, R2 or R3 is OC1-4 alkyl; R4 and R5 are substituted or unsubstituted C1-12 alkyl groups, provided that the combined R4 and R5 do not exceed C14 alkyl groups. For example, R4 can be C1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, and R5 can be C13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1. A is selected from NH, CH2, or CH substituted with an aryl group (e.g., phenyl); Group 2 is selected from the formula as defined above. Or R6, where R6 is -CH2-aryl; (ii) React the product of step b (i) with functional groups (e.g., hydroxyl functional groups) on the surface of the silica matrix particles. (iii) Remove protecting groups from the product of step (b) (ii).

[0073] In another preferred aspect, the method for preparing the chromatographic packing material may be as follows: wherein the packing material comprises an amino acid or an amino acid derivative / analogue (e.g., aminophosphonates and aminosulfonates) covalently bonded to silica matrix particles, and the method for preparing the packing material includes: (a) (i) Reacting the hydroxyl functional groups on the surface of the silica matrix particles with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine; (ii) Reacting the product of step (a) (i) with at least one of methyl acrylate, ethyl acrylate, butyl acrylate or tert-butyl acrylate, dimethyl vinylphosphonate or diethyl vinylphosphonate, or methyl vinylsulfonate, ethyl acrylate or neopentyl acrylate; and (iii) Remove the protecting group from the product of step a (ii); or (b) (i) Reaction of at least one of methyl acrylate, ethyl acrylate, butyl acrylate or tert-butyl acrylate, dimethyl vinylphosphonate or diethyl vinylphosphonate, or methyl vinylsulfonate, ethyl vinylsulfonate or neopentyl vinylsulfonate with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine. (ii) React the product of step b (i) with the hydroxyl functional groups on the surface of the silica matrix particles; (iii) Remove protecting groups from the product of step (b) (ii).

[0074] In a particularly preferred aspect, the method for preparing the chromatographic packing material may be as follows: wherein the packing material comprises β-alanine covalently bonded to silica matrix particles, and the method for preparing the packing material includes: (a) (i) Reacting the hydroxyl functional groups on the surface of the silica matrix particles with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine; (ii) React the product of steps (a) and (i) with an alkyl acrylate; and (iii) Remove the protecting group from the product of step a (ii); or (b) (i) Reacting alkyl acrylates with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine; (ii) React the product of step b (i) with the hydroxyl functional groups on the surface of the silica matrix particles; (iii) Remove protecting groups from the product of step (b) (ii).

[0075] The method of the present invention provides packing material suitable for column chromatography.

[0076] The present invention also provides a chromatographic packing material comprising: (a) matrix particles; and (b) A zwitterionic compound bonded to the matrix particles, which is prepared by the following manner: (a) (i) Reacting the functional groups on the matrix particles with an organofunctional silane compound; (ii) Reacting the product of steps (a) and (i) with a Michael acceptor containing a protected acid group; and (iii) Remove the acid protecting group from the product of steps (a) and (ii); or (b) (i) React Michael acceptors containing protected acid groups with organofunctional silane compounds; (ii) React the product of step (b) (i) with the functional groups on the matrix particles; and (iii) Remove the acid protecting groups from the product of step (b) (ii).

[0077] The matrix particles, the zwitterionic compound, the functional groups on the matrix particles, the Michael acceptor, the acid protecting group, and the organofunctional silane compound are all as defined above in relation to the method of the present invention.

[0078] In a preferred aspect, the packing may comprise: (a) silica particles; and (b) A zwitterionic compound bonded to the matrix particles, which is prepared by the following manner: (i) Reacting functional groups (e.g., hydroxyl functional groups) on the surface of silica matrix particles with an organofunctional silane having the following structure: X is selected from -NH2-, -NH-, -S- or -SH-. When X is NH2 or -SH-, only group 1 exists, that is, group 2 does not exist; and when X is -NH- or -S-, group 1 and group 2 exist simultaneously. Group 1 has the formula R1, R2 and R3 are independently selected from C1-4 alkyl or OC1-4 alkyl, provided that at least one of R1, R2 or R3 is OC1-4 alkyl; R4 and R5 are substituted or unsubstituted C1-12 alkyl groups, provided that the combined number of R4 and R5 does not exceed C14 alkyl groups; for example, R4 can be C1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, and R5 can be C13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1. A is selected from NH, CH2, or CH substituted with an aryl group (such as phenyl); Group 2 is selected from the formula as defined above. Or R6, where R6 is -CH2-aryl; (ii) Reacting the product of step (a) (i) with at least one Michael acceptor containing a protected acid group (e.g., at least one compound selected from: methyl acrylate, ethyl acrylate, butyl acrylate or tert-butyl acrylate, dimethyl vinylphosphonate or diethyl vinylphosphonate, or methyl vinylsulfonate, ethyl vinylsulfonate or neopentyl vinylsulfonate); and (iii) Remove the protecting group from the product of step a (ii); or (b) (i) Reacting at least one Michael acceptor containing a protected acid group (e.g., at least one compound selected from: methyl acrylate, ethyl acrylate, butyl acrylate or tert-butyl acrylate, dimethyl vinylphosphonate or diethyl vinylphosphonate, or methyl vinylsulfonate, ethyl vinylsulfonate or neopentyl vinylsulfonate) with an organofunctional silane having the structure shown below: X is selected from -NH2-, -NH-, -S- or -SH-. When X is NH2 or -SH-, only group 1 exists, that is, group 2 does not exist; when X is -NH- or -S-, both group 1 and group 2 exist. Group 1 has the formula R1, R2 and R3 are independently selected from C1-4 alkyl or OC1-4 alkyl, provided that at least one of R1, R2 or R3 is OC1-4 alkyl; R4 and R5 are substituted or unsubstituted C1-12 alkyl groups, provided that the combined R4 and R5 do not exceed C14 alkyl groups. For example, R4 can be C1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, and R5 can be C13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1. A is selected from NH, CH2, or CH substituted with an aryl group (e.g., phenyl); Group 2 is selected from the formula as defined above. Or R6, where R6 is -CH2-aryl; (ii) React the product of step b (i) with the hydroxyl functional groups on the surface of the silica matrix particles; (iii) Remove protecting groups from the product of step (b) (ii).

[0079] In yet another preferred aspect, the packing may comprise: (a) Silica particles; and (b) An amino acid or an amino acid derivative / analyte (e.g., aminophosphonate and aminosulfonate) covalently bonded to the silica matrix particles, prepared by: (a) (i) Reacting the hydroxyl functional groups on the surface of the silica matrix particles with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine; (ii) Reacting the product of step (a) (i) with at least one of methyl acrylate, ethyl acrylate, butyl acrylate or tert-butyl acrylate, dimethyl vinylphosphonate or diethyl vinylphosphonate, or methyl vinylsulfonate, ethyl vinylsulfonate or neopentyl vinylsulfonate; and (iii) Remove the protecting group from the product of step a (ii); or (b) (i) Reaction of at least one of methyl acrylate, ethyl acrylate, butyl acrylate or tert-butyl acrylate, dimethyl vinylphosphonate or diethyl vinylphosphonate, or methyl vinylsulfonate, ethyl vinylsulfonate or neopentyl vinylsulfonate with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine. (ii) React the product of step b (i) with the hydroxyl functional groups on the surface of the silica matrix particles; (iii) Remove protecting groups from the product of step (b) (ii).

[0080] In a particularly preferred aspect, the packing may comprise: (a) Silica particles; and (b) β-alanine or a β-alanine derivative covalently bonded to the matrix particles, which is prepared by the following method: (a) (i) Reacting the hydroxyl functional groups on the surface of the silica matrix particles with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine; (ii) React the product of steps (a) and (i) with an alkyl acrylate; and (iii) Remove the protecting group from the product of step a (ii); or (b) (i) Reacting alkyl acrylates with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine; (ii) React the product of step b (i) with the hydroxyl functional groups on the surface of the silica matrix particles; (iii) Remove protecting groups from the product of step (b) (ii).

[0081] The filler defined above can be obtained by using the method of the present invention as described above.

[0082] As previously stated, the packing material defined above can be used for chromatographic separation.

[0083] For the avoidance of doubt, when the terms "comprising" or "including" are used in this specification, it means that the described feature must contain the listed components, but may optionally contain additional components. When the terms "consistently composed of" or "essentially composed of" are used, it means that the described feature must contain the listed components, and may also contain other components, provided that such components do not affect the essential properties of the feature. When the terms "composed of" or "consisting of" are used, it means that the described feature must contain only the listed components.

[0084] It will be apparent to those skilled in the art that the features and combinations defined for the method of the present invention are equally applicable to the packing.

[0085] The specific embodiments described herein illustrate the principles of the invention by way of example rather than limitation. This specification will clearly enable those skilled in the art to make and use the invention, and describes several embodiments, modifications, variations, alternatives, and uses of the invention. As used herein, the terms “about” or “approximately” for any numerical value or range indicate a suitable dimensional tolerance that allows a group of parts or components to achieve the intended purpose as described above. Brief description of the attached diagram Figure 1 Buffer concentration and adenosine monophosphate (AMP) retention time at pH 3 and 6.5 To illustrate the present invention, the following non-limiting embodiments of the present invention are given below.

[0087] Example 1 – Column packing via method step (b) Using this synthetic strategy, two β-alanine HILIC stationary phases have been synthesized.

[0088] Scheme 1 shows a synthetic example of preparing a diacid-type HILIC stationary phase.

[0089] a) Organosilanes synthesis: In a 250 ml round-bottom flask equipped with a magnetic stirrer, bis(trimethoxysilylpropyl)amine (10.25 g, 30 mmol), tert-butyl acrylate (4.9 g, 39 mmol), and methanol (50 ml) were added. The resulting reaction mixture was stirred at room temperature for 18 hours. Volatile substances were then removed by rotary evaporation. The residue was used directly in the next step without further purification. The yield was 99%.

[0090] b) Silica matrix bonding: 10 g of silica raw material particles (particle size 3 µm, pore size 120 Å, specific surface area = 300 m²) were bonded together. 2 Transfer 7 g of the synthesized organosilane to a 250 ml round-bottom flask, followed by the addition of 40 ml of toluene. Sonicate the mixture for 10 minutes to form a homogeneous suspension. Then add 7 g of the synthesized organosilane to the flask. Attach a mechanical stirrer and a condenser to the flask. Reflux the reaction mixture for 24 hours. Filter out the silica resin and wash with 150 ml of methanol. Dry the silica overnight in a standard oven at 80°C.

[0091] c) In-column hydrolysis: The silica prepared in the above section is packed into the chromatographic column. The packed column is then treated with 0.1% MSA aqueous solution at 45°C for 60 minutes to activate the HILIC stationary phase.

[0092] Option 1: Preparation of fillers containing monoacid zwitterionic compounds Example 2 - Column filling via method step (b) a) Organosilanes synthesis: In a 250 ml round-bottom flask equipped with a magnetic stirrer, (3-aminopropyl)trimethoxysilane (7 g, 40 mmol), tert-butyl acrylate (12.8 g, 100 mmol), and methanol (80 ml) were added. The resulting reaction mixture was stirred at room temperature for 18 hours. Volatile substances were then removed by rotary evaporation. The residue was used directly in the next step without further purification. The yield was 99%.

[0093] b) Silica matrix bonding: 10 g of silica raw material particles (particle size 3 µm, pore size 120 Å, specific surface area = 300 m²) were bonded together. 2 Transfer the 10 g / g of synthesized organosilane to a 250 ml round-bottom flask, followed by the addition of 40 ml of toluene. Sonicate the mixture for 10 minutes to form a homogeneous suspension. Then add 10 g of the synthesized organosilane to the flask. Attach a mechanical stirrer and a condenser to the flask. Reflux the reaction mixture for 24 hours. Filter out the silica resin and wash with 150 ml of methanol. Dry the silica in a conventional oven at 80°C overnight.

[0094] c) In-column hydrolysis: The silica prepared in the above section is packed into the chromatographic column. Then, the packed column is treated with 0.1% MSA aqueous solution at 45°C for 60 minutes to activate the HILIC stationary phase.

[0095] Option 2: Preparation of fillers containing zwitterionic compounds of diacids Example 3 – Study on column retention behavior Preliminary ion exchange characteristics studies show that the net charge of the diacid-type HILIC stationary phase depends on the pH of the buffer solution. For example, at pH 3, the retention time of negatively charged adenosine monophosphate (AMP) decreases with increasing buffer concentration. This indicates that the stationary phase has anion exchange properties. At pH 6.5, the retention time of AMP increases with increasing buffer concentration, therefore the net surface charge is negative. Figure 1 Further in-depth research is needed on the separation behavior of charged samples on this stationary phase in the future.

[0096] Example 4 – Column filling via method step (a).

[0097] a) 20 g of silica raw material particles (particle size 3 µm, pore size 120 Å, specific surface area = 300 m²) 2 Transfer 8 g of (3-aminopropyl)trimethoxysilane to a 250 ml round-bottom flask, followed by the addition of 60 ml of toluene. Sonicate the mixture for 10 minutes to form a homogeneous suspension. Then add 8 g of (3-aminopropyl)trimethoxysilane to the flask. Attach a mechanical stirrer and condenser to the flask. Reflux the reaction mixture for 24 hours. Filter out the silica resin and wash with 150 ml of methanol. Dry the silica in a standard oven at 80°C overnight.

[0098] b) Transfer 10 g of the bonded silica particles obtained in the above paragraph to a 250 ml round-bottom flask. Then add 60 ml of toluene. Sonicate the mixture for 10 minutes to form a homogeneous suspension. Then add 4 g of tert-butyl acrylate to the flask. Attach a mechanical stirrer and condenser to the flask. Heat the reaction mixture at 60°C for 24 hours. Filter out the silica resin and wash with 150 ml of methanol. Dry the silica overnight in a standard oven at 80°C.

[0099] c) In-column hydrolysis: The silica prepared in the above section is packed into the chromatographic column. Then, the packed column is treated with 0.1% MSA aqueous solution at 45°C for 60 minutes to activate the HILIC stationary phase.

[0100] In summary, this paper presents a novel zwitterionic HILIC stationary phase designed and synthesized via aza-Michael addition chemistry. The high yield and purity of the ligands, along with an efficient hydrolysis activation step, ensured the uniform distribution of amino acid functional groups on the silica surface. The stationary phase exhibits high hydrophilicity and unique charge properties. Furthermore, this method allows for the preparation of a wide variety of ligands (such as β-alanine-based ligands), providing a broad selection of HILIC stationary phases for the separation of polar compounds.

Claims

1. A method for preparing a chromatographic packing material, wherein the packing material comprises a zwitterionic compound (e.g., an amino acid or an aminophosphonate) bonded to matrix particles, and the method for preparing the packing material comprises: (a) (i) Reacting the functional groups on the matrix particles with an organofunctional silane compound; (ii) Reacting the product of steps (a) and (i) with a Michael acceptor containing a protected acid group; and ； (iii) Remove the acid protecting groups from the products of steps (a) and (ii); or (b) (i) React Michael acceptors containing protected acid groups with organofunctional silane compounds; (ii) React the product of step (b) (i) with the functional groups on the matrix particles; as well as (iii) Remove the acid protecting groups from the product of step (b) (ii).

2. The method according to claim 1, wherein the functional group in the organofunctional silane compound is selected from amino or thiol groups.

3. The method according to claim 1 or 2, wherein the Michael receptor is selected from protected α,β-unsaturated acids, dialkyl vinyl phosphonates, or vinyl sulfonates.

4. The method according to claim 3, wherein the protected α,β-unsaturated carboxylic acid is selected from methyl acrylate, ethyl acrylate, butyl acrylate or tert-butyl acrylate; the dialkyl vinylphosphonate is selected from dimethyl vinylphosphonate or diethyl vinylphosphonate; and the vinyl sulfonate is selected from methyl vinylsulfonate, ethyl vinylsulfonate or neopentyl vinylsulfonate.

5. The method according to any one of the preceding claims, wherein The reaction between the functional group on the matrix particles in step (i) and the organofunctional silane compound, or the reaction between the functional group on the matrix particles in step (b) (ii) and the product of step (b) (i), forms a covalent bond between the matrix particles and the organofunctional silane compound or the product of step (b) (i).

6. The method according to any one of the preceding claims, wherein the matrix particles are selected from the group consisting of silicon dioxide or metal oxides.

7. The method according to any one of the preceding claims, wherein the reaction between the functional group on the organofunctional silane compound and the Michael acceptor is a Michael addition reaction.

8. The method according to any one of the preceding claims, wherein the organofunctional silane compound comprises at least one hydrolyzable alkoxy group.

9. The method according to claim 8, wherein the at least one hydrolyzable group is hydrolyzed before the reaction is carried out in step (a)(i) or step (b)(ii).

10. The method according to any one of the preceding claims, wherein the packing material is provided in a form suitable for use as a chromatographic packing material.

11. A chromatographic packing material prepared by any one of the preceding claims.

12. Chromatographic packing material, comprising: (a) matrix particles; and (b) A zwitterionic compound bonded to the matrix particles, which is prepared by the following manner: (a) (i) Reacting the functional groups on the matrix particles with an organofunctional silane compound; (ii) React the product of step (a) (i) with a Michael acceptor containing a protected acid group; as well as (iii) Remove the acid protecting groups from the products of steps (a) and (ii); or (b) (i) React Michael acceptors containing protected acid groups with organofunctional silane compounds; (ii) React the product of step (b) (i) with the functional groups on the matrix particles; as well as (iii) Remove the acid protecting groups from the product of step (b) (ii).

13. The filler according to claim 12, wherein the functional group in the organofunctional silane compound is selected from primary amino, secondary amino, and thiol groups.

14. The packing material according to claim 12 or 13, wherein the Michael acceptor is selected from protected α,β-unsaturated acids, dialkyl vinyl phosphonates, or vinyl sulfonates.

15. The filler according to claim 14, wherein the protected α,β-unsaturated carboxylic acid is selected from methyl acrylate, ethyl acrylate, butyl acrylate or tert-butyl acrylate; the dialkyl vinylphosphonate is selected from dimethyl vinylphosphonate or diethyl vinylphosphonate; and the vinyl sulfonate is selected from methyl vinylsulfonate, ethyl vinylsulfonate or neopentyl vinylsulfonate.

16. The filler according to any one of claims 12 to 15, wherein the reaction between the functional groups on the matrix particles in step (i) and the organofunctional silane compound, or the reaction between the functional groups on the matrix particles in step (b) (ii) and the product of step (b) (i), forms a covalent bond between the matrix particles and the organofunctional silane compound or the product of step (b) (i).

17. The filler according to any one of claims 12 to 16, wherein the matrix particles are selected from the group consisting of silica or metal oxides.

18. The filler according to any one of claims 12 to 17, wherein the reaction between the functional group on the organofunctional silane compound and the Michael acceptor is a Michael addition reaction.

19. The packing material according to any one of claims 12 to 18, wherein the packing material is obtained using the method defined in any one of claims 1 to 10.

20. The packing material according to any one of claims 12 to 19, wherein the packing material is suitable for hydrophilic interaction liquid chromatography (HILIC).

21. Use of the packing material according to any one of claims 12 to 20 in chromatographic separation.

22. The use according to claim 21, wherein the chromatographic separation is hydrophilic interaction liquid chromatography (HILIC) separation.

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