Zwitterionic column packing

Abstract

The present invention relates to a column packing material and a method for producing the same. Specifically, the present invention relates to a column packing material used in hydrophilic interaction liquid chromatography (HILIC).

Description

Technical Field

[0001]

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 514,878, filed on July 21, 2023, the entire content of which is incorporated herein by reference.

[0002] The present invention relates to column packing materials and a method for producing the same. Specifically, the present invention relates to column packing materials used in hydrophilic interaction liquid chromatography (HILIC).

Background Art

[0002]

[0003] The hydrophilic interaction liquid chromatography (HILIC) technique is widely used for the analysis of polar compounds, which are biologically important compounds in proteomics.

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

[0003]

Chemical Formula

[0005] The zwitterionic compound sulfobetaine can be covalently bonded to silica to produce one of the most popular zwitterionic HILIC stationary phases.

[0006] Sulfobetaine stationary phases typically contain charged sulfonates and positively charged quaternary amine groups in a 1:1 ratio. These functional groups exhibit a net zero charge in the pH range of 0–14, minimizing ion exchange behavior. Evidence suggests that the zwitterionic HILIC retention mechanism in this type of phase is primarily due to hydrophilic partitioning, accompanied by weak ionic interactions from residual silanols and analysts.

[0007] The lack of ionic properties in the sulfobetaine HILIC phase presents problems when separating ionic compounds. Furthermore, the sulfobetaine HILIC phase typically experiences low reproducibility and high bleeding.

[0008] Therefore, it is desirable to develop novel types of HILIC stationary phases with improved hydrophilicity, charge properties, selectivity, and column efficiency.

[0009] The inclusion or consideration herein of any previously published literature should not necessarily be considered an endorsement that such literature is part of the latest technology or common general knowledge. [Overview of the project] [Problems that the invention aims to solve]

[0004]

[0010] This invention seeks to solve at least some of the above problems by providing a novel amphoteric ion-based HILIC phase. [Means for solving the problem]

[0005]

[0011] Therefore, the present invention relates to a method for producing chromatographic filler. The filler contains amphoteric compounds such as amino acids, aminophosphonates, and / or aminosulfonates bound to the substrate particles, and the method for forming the filler is (a)(i) Reacting functional groups on substrate particles with an organically functionalized silane compound, (ii) Process (a) (i) The product is reacted with a Michael acceptor containing a protective acid group, (iii) Remove the acid protecting group from the product of step (a) (ii), or (b)(i) Reacting a Michael acceptor containing a protective acid group with an organically functionalized silane compound, (ii) Reacting the product of step b (i) with functional groups on the substrate particles, (iii) Process (b) The present invention provides a method comprising removing an acid protecting group from the product of (ii).

[0012] In some aspects of the present invention, steps (a)(iii) or (b)(iii) may not be performed, and the column packing may be sold in a “protected form.” For example, the column packing material may be sold to the customer to be packed into the column and to have the protective groups removed in situ.

[0013] It may be preferable to remove the protecting group before the column is packed.

[0014] Alternatively, the acid-protecting group can be removed after the column packing has been added to the chromatography column.

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

[0016] As described below, amphoteric compounds are formed through a combination / reaction between an organic functional silane and a Michael acceptor containing a protective acid group, and when the protective acid group is removed, the amphoteric compound is provided.

[0017] Suitable substrate particles can include various commercially available chromatographic media such as packed beds of chromatographic particles, and many other forms including fused silica capillaries that can be used after a simple base hydrolysis treatment to derivatize the tubing and activate the surface suitably. Thus, the term "substrate particles" encompasses one or more substrates unless otherwise specified.

[0018] The substrate particles can be particulate or monolithic substrates, preferably particulate. The substrate material can be a metal oxide (this term includes in this specification semimetal oxides such as silica, and includes inorganic-organic hybrid materials (especially metal oxide-organic hybrid materials) as described, for example, in WO 00 / 45951). The substrate particles can particularly be silica (SiO2), and the term includes in this specification silica / organo hybrids, alumina (Al2O3), titania (TiO2), or zirconia (ZrO2) substrates.

[0019] Silica (this term includes in this specification silica / organo hybrids) substrate particles are most preferred.

[0020] As detailed above, the zwitterionic compound is bound to the substrate particles. In a preferred embodiment, the zwitterionic compound is bound to a functional group on the surface of the substrate particles. Typically, the bond is a covalent bond, i.e., the zwitterionic compound is covalently bound to a functional group on the surface of the substrate particles. The covalent bond can typically be formed between a functional group on the substrate particles and a silicon atom present in an organofunctional silane that generates the zwitterionic compound.

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

[0022] In step (a)(i), a functional group on the surface of the substrate particles is reacted with an organofunctional silane compound.

[0023] The functional groups on the substrate particles can be any functional groups suitable for reacting with the organic functionalized silane compound. Typically, the functional groups present on the surface of substrate particles, such as silica particles, are selected from the group consisting of epoxy, hydroxy, and amino. Preferably, the functional group may be hydroxy, i.e., the hydroxyl group is located on the surface of the substrate particles.

[0024] As used herein, the term “organofunctional silane compound” is intended to encompass hybrid compounds that combine the functionality of a reactive organic group with the inorganic functionality of an alkoxysilane in a single molecule. As used herein, organofunctional silanes may include garsil silanes.

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

[0026] In certain embodiments, the organically functionalized silane has the following structure:

[0027]

[0006] [ka] The structure has, in which X is selected from -NH2-, -NH-, -S-, or -SH-, and when X is NH2 or -SH-, only group 1 is present, i.e., group 2 is not present, and when X is -NH- or -S-, groups 1 and 2 are present.

[0028] Base 1 is formula

[0007] [ka] The formula comprises, where R1, R2, and R3 are independently selected from C1-4 alkyl or OC1-4 alkyl, as long as at least one of R1, R2, or R3 is OC1-4 alkyl.

[0029] R4 and R5 are substituted or unsubstituted C1-12 alkyl groups, provided that the combined value of R4 and R5 does not exceed that of a C14 alkyl group. For example, R4 is R5 may be C1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 alkyl, provided that the combined R4 and R5 do not exceed C14 alkyl.

[0030] A is selected from NH, CH2, or CH substituted with an aromatic group such as phenyl.

[0031] Base 2 is the already defined formula

[0008] [ka] Alternatively, it is selected from R6, where R6 is a CH2 aromatic compound.

[0032] Preferably, when X is -NH- or -S-, groups 1 and 2 are

[0009] [ka] It is possible.

[0033] Preferably, R1, R2, and R3 can be independently selected from methoxy or ethoxy.

[0034] Preferably, R4 may be a C1-4 alkyl group, and R5 may be a C1-4 alkyl group, for example, R4 may be a C1, 2, 3, or 4 alkyl group, and R5 may be a C1, 2, 3, or 4 alkyl group.

[0035] Preferably, A may be CH2 or NH, most preferably CH2.

[0036] In a particularly preferred embodiment,

[0037] X is selected from -NH2- or -SH-,

[0038] Base 1 is formula

[0010] [ka] It has, and base 2 does not exist.

[0039] R1, R2, and R3 are independently selected from methoxy or ethoxy, R4 is C1, 2, 3, or 4 alkyl, and R5 is C1, 2, 3, or 4 alkyl.

[0040] A is CH2 or NH, most preferably CH2.

[0041] In a particularly preferred embodiment,

[0042] X is -NH- or -S-, and both groups 1 and 2 are

[0011] [ka] And,

[0043] R1, R2, and R3 are independently selected from methoxy or ethoxy, R4 is C1, 2, 3, or 4 alkyl, and R5 is C1, 2, 3, or 4 alkyl.

[0044] A is CH2 or NH, most preferably CH2.

[0045] In this specification, the term "alkyl" means a linear or branched chain, either by itself or as part of another substituent, unless otherwise specified, which may be fully saturated, monounsaturated or polyunsaturated, and may include divalent and polyvalent groups with a specified number of carbon atoms (i.e., C1-10 means 1 to 10 carbon atoms).

[0046] Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl (e.g., -CH2-CH2-CH3, -CH2-CH2-CH2-), isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, and their homologs and isomers, such as n-pentyl, n-hexyl, n-heptyl, and n-octyl. An unsaturated alkyl group is a group having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, clotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl, and higher homologs and isomers. The term "alkyl" also means to include alkyl derivatives, such as "heteroalkyl," which are defined in more detail below, unless otherwise noted. Alkyl groups limited to hydrocarbon groups are called "homoalkyls." The term "alkyl" can also mean "alkylene" or "alkyldiyl," as well as alkylidene, when the alkyl group is a divalent radical.

[0047] In a preferred embodiment, alkyl as used herein may be understood to mean a substituted or unsubstituted saturated carbon chain.

[0048] Alkyl / radical substituents are generally called "alkyl substituents," and they include, but are not limited to, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl substituents. Reel, substituted or unsubstituted heterocycloalkyl, -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′″)= It can be one or more of various groups selected from NR′″, -NR-C(NR′R″)=NR′″, -S(O)R′, -S(O)2R′, -OS(O)2R′, -S(O)2NR′R″, -NRSO2R′, -CN, and -NO2, and is in the range of 0 to (2m′+1), where m′ is the total number of carbon atoms in such radical. R′, R′′, R′′′, and R′ Each of the following preferably and independently refers to hydrogen, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, for example, an aryl group substituted with 1 to 3 halogens, a substituted or unsubstituted alkyl group, an alkoxy or thioalkoxy group, or an arylalkyl group. For example, if the compound of the present invention contains two or more R groups, and two or more of the R′, R′′, R′′′, and R′′′′ groups are present, each of the R groups is independently selected as well as each of the R′, R′′, R′′′, and R′′′′ groups. If R′ and R′′ are bonded to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-membered ring, a 6-membered ring, or a 7-membered ring. For example, -NR′R″ means, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" includes groups containing carbon atoms bonded to groups other than hydrogen groups, such as haloalkyls (e.g., -CF3 and -CH2CF3) and acyls (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0049] Examples of organically functional silanes include (3-aminopropyl)trialkoxysilanes such as (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, and (3-aminopropyl)tripropoxysilane; (3-mercaptopropyl)trialkoxysilanes such as mercaptopropyl)trimethoxysilane, mercaptopropyl)triethoxysilane, and mercaptopropyl)tripropoxysilane; and bis(trimethoxysilylpropyl)amines such as bis(tri(trimethoxysilylpropyl)amine, and bis(tripropoxysilylpropyl)amine. Examples include, but are not limited to, liquidoxysilyl lupopylamine, (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.

[0050] As already mentioned, the reaction between the functional groups on the (surface) of the substrate particles and the organically functionalized silane results in the formation of bonds, such as covalent bonds, between the surface of the substrate particles and the organically functionalized silane.

[0051] In the reaction, at least one hydrolyzable alkoxy group is typically hydrolyzed to a silanol, which is then subjected to a condensation reaction with a functional group on the substrate particle (or its surface).

[0052] Therefore, method (a) as defined herein may include a step of hydrolyzing at least one hydrolyzable alkoxy group to a silanol before reacting with the functional groups on the substrate particles (or their surface). Any method suitable for hydrolyzing the alkoxy group may be used.

[0053] As those skilled in the art will understand, the type of bond formed will depend on the substrate used. For example, if the functional group present on the substrate particles (on the surface) is a hydroxyl group, then reacting the hydrolyzable / hydrolyzed alkoxy group on the organic functional silane with the hydroxyl group will result in the formation of a siloxane bond.

[0054] Therefore, in one aspect of the present invention, the method may include reacting epoxy, hydroxyl, or amino groups (preferably hydroxyl groups) present on the surface of substrate particles with hydrolyzable / hydrolyzed alkoxy groups on an organically functionalized silane compound in step (a) (i).

[0055] Typically, in step (a) (i), silica particles and organically functionalized silanes are mixed in a ratio of approximately 1: The reaction should be carried out in a ratio of approximately 1:10 to 10:1, such as 0.5 to approximately 1:2, or approximately 1:1.

[0056] Following the reaction between the functional groups on the surface of the substrate particles and the organically functionalized silane (step (a) (i)), the reaction product is then reacted with a Michael acceptor containing a protective acid functional group (step (a) (ii)).

[0057] An example of a product from step (a) (i) is shown below, where the organic functionalized silane is (3-aminopropyl)trimethoxysilane and the functional group on the substrate particles is hydroxyl.

[0012] [ka]

[0058] In steps (a) and (ii), the reaction utilizes a functional group present on the (currently bound) organofunctional silane as a Michael donor that reacts with the Michael acceptor.

[0059] The Michael acceptor can be a protected acid, such as a protected carboxylic acid, protected phosphonic acid, or protected sulfonic acid.

[0060] The protecting group used may be any group suitable for protecting a carboxylic acid, phosphonic acid, or sulfonic acid. For example, the protecting group may be methyl, ethyl, butyl, or It can be an alkyl-based protecting group such as a tert-butyl group.

[0061] Examples of Michael acceptors that can be used include α,β-unsaturated carboxylic acids such as protected methyl, ethyl, butyl, or tert-butyl acrylate, and dialkyl vinyl phosphonates such as dimethyl or diethyl ester vinyl phosphonates. Examples include, but are not limited to, vinyl sulfonate esters such as methyl, ethyl, or neo-pentyl vinyl sulfonate esters.

[0062] Those skilled in the art will understand that the product formed from the Michael addition will depend on whether the product of step (a)(i) uses a primary amine, a secondary amine, or a thiol as the Michael donor.

[0063] As a non-limiting example, the presence of a primary amine leads to the formation of a di-ester product, i.e., an amine-to-ester ratio of approximately 1:2. On the other hand, the use of a secondary amine results in the formation of a mono-ester, i.e., an amine-to-ester ratio of approximately 1:1. The ratio would be the same if the Michael donner is sulfur-based.

[0064] Typically, in step (a)(ii), the product from step (a)(i) and the Michael acceptor are reacted in a ratio of approximately 1:1 to 1:10, such as approximately 1:3.

[0065] In step (a) (iii), the acid protecting group is removed to provide an amphoteric chromatographic packing material.

[0066] Any method suitable for removing the acid protecting group may be used. For example, the treatment may use an aqueous acid, i.e., aqueous methanesulfonic acid.

[0067] Chromatography fillers may also be provided by using the method defined in process (b).

[0068] In step (b), (i), a Michael acceptor containing a protective acid group is reacted with an organofunctional silane compound.

[0069] An example of a reaction in which the organic functional silane is (3-aminopropyl)trimethoxysilane and the Michael acceptor is tert-butyl acrylate is shown below.

[0013] [ka]

[0070] In step (b) (i), the functional group of the organofunctional silane compound acts as a Michael donor.

[0071] The Michael acceptor and the organofunctional silane compound are as defined above in step (a).

[0072] Those skilled in the art will understand that the product formed by Michael addition depends on whether the organically functional silane contains a primary amine, secondary amine, or thiol as a Michael donor.

[0073] In the non-limiting examples above, a primary amine is present that leads to the formation of a di-ester product, i.e., the amine-to-ester ratio is approximately 1:2. On the other hand, the use of a secondary amine results in the formation of a mono-ester, i.e., the amine or thiol-to-ester ratio is approximately 1:1.

[0074] In step (b) (i) of the method as defined herein, the ratio of Michael donner (organofunctional silane) can be about 1:1 to about 1:5, such as about 1:1 to about 1:3.

[0075] In step (b) (ii), the product of (b) (i) is reacted with functional groups on the substrate particles.

[0076] The product of step (b)(i) contains at least one hydrolyzable alkoxy group from an organic functional silane. Similar to step (a), at least one alkoxy group may be hydrolyzed to a silanol and then subjected to a condensation reaction with a functional group (on the surface) of the substrate particles. This results in the formation of a bond, such as a covalent bond, between the functional group and the product of step (b)(i). If the functional group on the substrate particles is a hydroxyl group, the bond may be a siloxane bond.

[0077] Therefore, method (b) as defined herein may include a step of hydrolyzing at least one hydrolyzable alkoxy group to a silanol before reacting with the functional groups on the surface of the substrate particles.

[0078] Accordingly, in one aspect of the present invention, the method may include reacting epoxy, hydroxyl, or amino groups (preferably hydroxyl groups) present on the surface of the substrate particles with hydrolyzed alkoxy groups on the product of step b(i) in step (b)(ii).

[0079] The functional groups on the substrate particles and the substrate particles are as defined in step (a).

[0080] In step (b) (iii), the acid protecting group is removed to provide an amphoteric chromatographic packing material.

[0081] Similar to step (a), any method suitable for removing the acid protecting group may be used. Step (a) (iii) or (b) (iii) (removal of the acid protecting group) may be performed before or after packing the chromatographic column. If the removal is performed after packing the chromatographic column, it may be done by any means suitable for removing the acid protecting group. For example, this may be done by flushing the chromatographic column with an aqueous acid, i.e., an acid such as aqueous methanesulfonic acid.

[0082] In a more preferred embodiment, a method for preparing a chromatographic packing material, wherein the packing material comprises an amphoteric compound such as an amino acid, aminophosphonate, and / or aminosulfonate bound to a substrate particle, and the method for forming the packing material is: (a)(i) The functional groups (such as hydroxyl functional groups) on the surface of the silica substrate particles are structured as shown below:

[0083]

[0014] [ka] An organic functional silane having, in the structure, X is -NH2-, -NH- If X is selected from -S- or -SH-, and X is NH2 or -SH-, then only group 1 exists, i.e., group 2 does not exist, and if X is -NH- or -S-, then groups 1 and 2 exist.

[0084] Base 1 is formula

[0015] [ka] The formula comprises, where R1, R2, and R3 are independently selected from C1-4 alkyl or OC1-4 alkyl, as long as at least one of R1, R2, or R3 is OC1-4 alkyl.

[0085] 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 may be C1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, and R5 may be C13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0086] A is selected from NH, CH2, or CH substituted with an aromatic group such as phenyl.

[0087] Base 2 is the already defined formula

[0016] [ka] Alternatively, R6 may be selected from R6, where R6 is reacted with an organofunctional silane that is CH2-aromatic. (ii) Process (a) (i) The product of (i) is methyl, ethyl, butyl, or Reacting with at least one Michael acceptor containing a protective acid group, such as tert-butyl acrylate, dimethyl or diethyl ester vinyl sulfonate, or at least one of methyl, ethyl, or neo-pentyl sulfonate esters, (iii) Removing the protecting group from the product of step a (ii), or (b)(i) Methyl, ethyl, butyl, or tert-butyl acrylate At least one Michael acceptor containing a protective acid group, such as a rate, dimethyl or diethyl ester vinyl phosphonate, or at least one of methyl, ethyl, or neo-pentyl vinyl sulfonate esters, in the structure shown below:

[0088]

[0017] [ka] An organic functional silane having the following characteristics: In the structure, X is selected from -NH2-, -NH-, -S-, or -SH-, and when X is NH2 or -SH-, only group 1 is present, i.e., group 2 is not present, and when X is -NH- or -S-, groups 1 and 2 are present.

[0089] Base 1 is formula

[0018] [ka] The formula comprises, where R1, R2, and R3 are independently selected from C1-4 alkyl or OC1-4 alkyl, as long as at least one of R1, R2, or R3 is OC1-4 alkyl.

[0090] R4 and R5 are substituted or unsubstituted C1-12 alkyl groups, provided that the combined R4 and R5 do not exceed a C14 alkyl group. For example, R4 may be C1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, and R5 may be C13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0091] A is selected from NH, CH2, or CH substituted with an aromatic group such as phenyl.

[0092] Base 2 is the already defined formula

[0019] [ka] Alternatively, R6 is selected from R6, where R6 is reacted with an organically reactive silane that is CH2 aromatic. (ii) Step b (i) The product is reacted with functional groups (such as hydroxyl functional groups) on the surface of silica substrate particles, (iii) Step b This may include removing a protecting group from the product of (ii).

[0093] In a more preferred embodiment, the method for preparing the chromatographic packing material may be such that the packing material contains amino acids or amino acid derivatives / analogis, such as aminophosphonates and aminosulfonates, covalently bonded to silica substrate particles, and the method for forming the packing material is (a)(i) Reacting the hydroxyl functional groups on the surface of silica substrate particles with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine, (ii) Process (a) (i) The product of (i) is reacted with at least one of methyl, ethyl, butyl, or tert-butyl acrylate, dimethyl or diethyl ester vinyl sulfonate, or methyl, ethyl, or neo-pentyl sulfonate ester, (iii) Step a (ii) Removing the protecting group from the product, or (b)(i) Reacting at least one of methyl, ethyl, butyl, or tert-butyl acrylate, dimethyl or diethyl ester vinyl sulfonate, or methyl, ethyl, or neo-pentyl sulfonate ester with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine, (ii) Step b (i) The product is reacted with the hydroxyl functional group on the surface of the silica substrate particles, (iii) Step b (ii) This includes removing a protecting group from the product of (ii).

[0094] In a particularly preferred embodiment, the method for preparing a chromatographic packing material may be such that the packing material contains beta-alanine covalently bonded to silica substrate particles, and the method for forming the packing material is (a)(i) Reacting the hydroxyl functional groups on the surface of silica substrate particles with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine, (ii) Reacting the product of step (a) (i) with an acrylate alkyl ester, (iii) Removing the protecting group from the product of step a (ii), or (b)(i) Reacting an alkyl acrylate with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine, (ii) Process b (i) The product is reacted with the hydroxyl functional group on the surface of the silica substrate particles, (iii) removing a protecting group from the product of step b (ii).

[0095] The present invention provides a packing material in a form suitable for use in column chromatography. The present invention also relates to a chromatographic filler, (a) Substrate particles and (b) an amphoteric compound bonded to the substrate particles, (a)(i) Reacting functional groups on substrate particles with an organically functionalized silane compound, (ii) Process (a) (i) The product is reacted with a Michael acceptor containing a protective acid group, (iii) By removing the acid protecting group from the product of step (a) (ii), or (b)(i) Reacting a Michael acceptor containing a protective acid group with an organically functionalized silane compound, (ii) Process (b) (i) Reacting the product with functional groups on the substrate particles, (iii) A chromatographic packing material is provided, comprising an amphoteric compound formed by removing an acid protecting group from the product of step (b) (ii).

[0096] The base particles, zwitterionic compounds, functional groups on the base particles, Michael acceptors, acid protecting groups, and organic functional silane compounds are all as already defined in relation to the method of the present invention.

[0097] In a preferred embodiment, the filler is (a) Silica particles and (b) an amphoteric compound bonded to the substrate particles, (a) (i) The functional groups (such as hydroxyl functional groups) on the surface of the silica substrate particles are structured as shown below:

[0098]

[0020] [ka] An organic functional silane having, in the structure, X is -NH2-, -NH- If X is selected from -S- or -SH-, and X is NH2 or -SH-, then only group 1 exists, i.e., group 2 does not exist, and if X is -NH- or -S-, then groups 1 and 2 exist.

[0099] Base 1 is formula

[0021] [ka] The formula comprises, where R1, R2, and R3 are independently selected from C1-4 alkyl or OC1-4 alkyl, as long as at least one of R1, R2, or R3 is OC1-4 alkyl.

[0100] R4 and R5 are substituted or unsubstituted C1-12 alkyl groups, provided they are combined. Provided that R4 and R5 do not exceed C14 alkyl, for example, R4 may be C1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, and R5 may be C13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1,

[0101] A is selected from NH, CH2, or CH substituted with an aromatic group such as phenyl.

[0102] Base 2 is the already defined formula

[0022] [ka] Alternatively, R6 may be selected from R6, where R6 is reacted with an organofunctional silane that is CH2 aromatic. (ii) step (a) reacting the product of (i) with at least one Michael acceptor containing a protective acid group, such as at least one of methyl, ethyl, butyl, or tert-butyl acrylate, dimethyl or diethyl ester vinyl sulfonate, or methyl, ethyl, or neo-pentyl sulfonate ester, (iii) By removing the protecting group from the product of step a (ii), or (b)(i) At least one Michael acceptor containing a protective acid group, such as at least one of methyl, ethyl, butyl, or tert-butyl acrylate, dimethyl or diethyl ester vinyl phosphonate, or methyl, ethyl, or neo-pentyl vinyl sulfonate ester, and the structure shown below:

[0103]

[0023] [ka] An organic functional silane having the following characteristics: In the structure, X is selected from -NH2-, -NH-, -S-, or -SH-, and when X is NH2 or -SH-, only group 1 is present, i.e., group 2 is absent, and when X is -NH- or -S-, groups 1 and 2 are present.

[0104] Base 1 is formula

[0024] [ka] The formula comprises, where R1, R2, and R3 are independently selected from C1-4 alkyl or OC1-4 alkyl, as long as at least one of R1, R2, or R3 is OC1-4 alkyl.

[0105] R4 and R5 are substituted or unsubstituted C1-12 alkyl groups, provided that the combined R4 and R5 do not exceed a C14 alkyl group. For example, R4 may be C1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, and R5 may be C13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0106] A is selected from NH, CH2, or CH substituted with an aromatic group such as phenyl.

[0107] Base 2 is the already defined formula

[0025] [ka] Alternatively, R6 may be selected from R6, where R6 is reacted with an organofunctional silane that is CH2 aromatic. (ii) Step b (i) The product is reacted with the hydroxyl functional group on the surface of the silica substrate particles, (iii) Step b (ii) may include an amphoteric compound formed by removing a protecting group from the product of (ii).

[0108] In a further preferred embodiment, the filler is (a) Silica particles and (b) Amino acids or amino acid derivatives / analogis such as aminophosphonates and aminosulfonates, which are covalently bonded to silica substrate particles, (a)(i) Reacting the hydroxyl functional groups on the surface of silica substrate particles with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine, (ii) Process (a) (i) The product of (i) is methyl, ethyl, butyl, or Reacting with at least one of tert-butyl acrylate, dimethyl or diethyl ester vinyl sulfonate, or methyl, ethyl, or neo-pentyl sulfonate ester, (iii) removing the protecting group from the product of step a (ii), or (b) (i) Reacting at least one of methyl, ethyl, butyl, or tert-butyl acrylate, dimethyl or diethyl ester vinyl sulfonate, or methyl, ethyl, or neo-pentyl sulfonate ester with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine, (ii) Reacting the product of step b (i) with the hydroxyl functional groups on the surface of the silica substrate particles, (iii) Remove the protecting group from the product of step b (ii), which may include an amino acid derivative / analog formed by this process.

[0109] In a particularly preferred embodiment, the filler is (a) Silica particles and (b) Beta-alanine or a beta-alanine derivative covalently bonded to silica substrate particles, (a) (i) Reacting the hydroxyl functional groups on the surface of silica substrate particles with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine, (ii) Reacting the product of step (a) (i) with an acrylate alkyl ester, (iii) By removing the protecting group from the product of step a (ii), or (b) (i) Reacting an alkyl acrylate with (3-aminopropyl)trialkoxysilane or bis(trialkoxysilylpropyl)amine, (ii) Process b (i) The product is reacted with the hydroxyl functional group on the surface of the silica substrate particles, (iii) Step b (ii) may include beta-alanine or a beta-alanine derivative formed by removing a protecting group from the product of (ii).

[0110] The filler defined above can be obtained using the method of the present invention already defined.

[0111] As already described, the packing material defined above can be used for chromatographic separation.

[0112] To avoid ambiguity, when the term “contains” is used in this specification, the described feature must contain the listed components, but may optionally contain additional components. When the terms “consisting essentially of” or “consists essentially of” are used, the described feature must contain the listed components, but may also contain other components, provided that any component does not affect the fundamental properties of the feature. When the terms “consisting of” or “consists of” are used, this means that the described feature must contain only the listed components.

[0113] Those skilled in the art will see that the features and combinations defined for the method of the present invention also apply to fillers.

[0114] The detailed description is not limiting, but illustrates the principles of the present invention as an example. This specification makes it clear to those skilled in the art that the present invention, as well as some embodiments, modifications, variations, substitutes, and uses of the present invention described herein, can be made and used. Where used herein, the terms “about” or “approximately” with respect to any numerical value or range indicate a preferred dimensional tolerance that enables some or a group of components to function for the intended purpose described herein. [Brief explanation of the drawing]

[0026]

[0115] [Figure 1] Buffer concentration and adenosine monophosphate (AMP) retention time at pH 3 and 6.5 [Modes for carrying out the invention]

[0027]

[0116] To illustrate the present invention, the following non-limiting embodiments are shown below.

[0117] Example 1 - Column packing via method step (b)

[0118] Two beta-alanine HILIC stationary phases are synthesized through this synthetic strategy.

[0119] Scheme 1 shows a synthetic example for preparing the HILIC diacid phase. a) Organosilane synthesis: Bis(trimethoxysilylpropyl)amine (10.25 g, 30 mmol), tert-butyl acrylate (4.9 g, 39 mmol), and methanol (50 ml) were added to a 250 ml round-bottom flask equipped with a stirring bar. The resulting reaction mixture was stirred at room temperature for 18 hours. Volatile substances were then removed using a rotary evaporator. The residue was used in the next step without further purification. The yield was 99%. b) Silica substrate bonding: 10g of raw material Si particles (3μ, 120Å, SA=300m) 2 The 1 / g of the compound was transferred to a 250 ml round-bottom flask, followed by the addition of 40 ml of toluene. The mixture was sonicated for 10 minutes to prepare a homogeneous solution. Next, 7 g of the synthesized organosilane was added to the flask. A mechanical stirring system and a condenser were set up in the flask. The reaction mixture was refluxed for 24 hours. The silica resin was filtered and then washed with 150 ml of methanol. The silica was dried overnight in a conventional oven at 80°C. c) Intra-column hydrolysis: The silica obtained in the above paragraph was packed into a chromatography column. The packed column was treated with a 0.1% MSA aqueous solution at 45°C for 60 minutes to activate the HILIC phase.

[0120]

[0028] [ka] Scheme 1: Formation of a filler containing a mono-acid amphoteric compound

[0121] Example 2 - Column packing via method step (b) a) Organosilane synthesis: (3-aminopropyl)trimethoxysilane (7 g, 40 mmol), tert-butyl acrylate (12.8 g, 100 mmol), and methanol (80 ml) were added to a 250 ml round-bottom flask equipped with a stirring bar. The resulting reaction mixture was stirred at room temperature for 18 hours. Volatile substances were then removed using a rotary evaporator. The residue was used in the next step without further purification. The yield was 99%. b) Silica substrate bonding: 10g of raw material Si particles (3μ, 120Å, SA=300m) 2 The silane ( / g) was transferred to a 250 ml round-bottom flask, followed by the addition of 40 ml of toluene. The mixture was sonicated for 10 minutes to prepare a homogeneous solution. Next, 10 g of the synthesized organosilane was added to the flask. A mechanical stirring system and a condenser were set up in the flask. The reaction mixture was refluxed for 24 hours. The silica resin was filtered and then washed with 150 ml of methanol. The silica was dried overnight in a conventional oven at 80°C. c) Intra-column hydrolysis: The silica obtained in the above paragraph was packed into a chromatography column. The packed column was then treated with a 0.1% MSA aqueous solution at 45°C for 60 minutes to activate the HILIC phase.

[0122]

[0029] [ka] Scheme 2: Formation of a filler containing a diacid amphoteric compound

[0123] Example 3 - Column Retention Test

[0124] Preliminary ion exchange property tests show that the net charge of the diacid HILIC phase depends on the buffer pH. For example, at pH 3, the retention time of charged adenosine monophosphate (AMP) decreased as the buffer concentration increased. This indicates that the phase has anion exchange properties. At pH 6.5, the retention time of AMP increased as the buffer concentration increased, and therefore the net charge on the surface is negative (Figure 1). Further investigation into the separation of charged samples in the phase is needed.

[0125] Example 4 - Column packing via method (a) a) 20g of raw material Si particles (3μ, 120Å, SA=300m) 2 The (3-aminopropyl)trimethoxysilane (8g) was transferred to a 250ml round-bottom flask, followed by the addition of 60ml of toluene. The mixture was sonicated for 10 minutes to prepare a homogeneous solution. Next, 8g of (3-aminopropyl)trimethoxysilane was added to the flask. A mechanical stirring system and a condenser were set up in the flask. The reaction mixture was refluxed for 24 hours. The silica resin was filtered and then washed with 150ml of methanol. The silica was dried overnight in a conventional oven at 80°C. b) 10 g of bound Si particles obtained in the above paragraph were transferred to a 250 ml round-bottom flask, followed by the addition of 60 ml of toluene. The mixture was sonicated for 10 minutes to prepare a homogeneous solution. Then, 4 g of tert-butyl acrylate was added to the flask. A mechanical stirring system and a condenser were set up in the flask. The reaction mixture was stirred at 60°C for 24 hours. The silica resin was filtered and then washed with 150 ml of methanol. The silica was dried overnight in a conventional oven at 80°C. c) Intra-column hydrolysis: The silica obtained in the above paragraph was packed into a chromatography column. The packed column was then treated with a 0.1% MSA aqueous solution at 45°C for 60 minutes to activate the HILIC phase.

[0126] In summary, a novel type of zwitterionic HILIC stationary phase was designed and synthesized via Aza-Michael addition chemistry. High ligand yield and purity, along with an efficient hydrolytic activation step, ensure uniform distribution of amino acid functional groups on the silica surface. The resulting phase exhibits high hydrophilicity and distinctive charge properties. Furthermore, a wide variety of ligands, including beta-alanine-based ligands, can be prepared via this method, providing broad HILIC phase selection for the separation of polar compounds.

Claims

1. A method for preparing a chromatographic packing material, wherein the chromatographic packing material contains an amphoteric compound such as an amino acid or aminophosphonate, bound to a substrate particle, and the method for forming the chromatographic packing material is (a)(i) reacting the functional groups on the substrate particles with an organic functional silane compound, (ii) Reacting the product of step (a)(i) with a Michael acceptor containing a protective acid group, (iii) removing an acid protecting group from the product of step (a)(ii), or (b)(i) Reacting a Michael acceptor containing a protective acid group with an organofunctional silane compound, (ii) Reacting the product of step (b)(i) with the functional groups on the substrate particles, (iii) A method comprising removing an acid protecting group from the product of step (b)(ii).

2. The method according to claim 1, wherein the functional group in the organic functionalized silane compound is selected from amino or thio.

3. The method according to claim 1, wherein the Michael acceptor is selected from a protected α,β unsaturated acid, a dialkyl vinyl phosphonate, or a vinyl sulfonic acid ester.

4. Protected α,β-unsaturated carboxylic acid is methyl, ethyl, butyl, or The method according to claim 3, wherein the dialkyl vinyl phosphonate is selected from the group consisting of tert-butyl acrylate, the dialkyl vinyl phosphonate is selected from the group consisting of dimethyl or diethyl ester vinyl phosphonate, and the vinyl sulfonic acid ester is selected from the group consisting of methyl, ethyl, or neo-pentyl vinyl sulfonate ester.

5. The method according to claim 1, wherein the reaction between the functional group on the substrate particles and the organic functionalized silane compound in step (i), or the reaction between the functional group on the substrate particles and the product of step (b)(i) in step (b)(ii), forms a covalent bond between the substrate particles and the organic functionalized silane compound or the product of step (b)(i).

6. The method according to claim 1, wherein the base material particles are selected from the group consisting of silica or metal oxides.

7. The method according to claim 1, wherein the reaction between the functional group on the organic functionalized silane compound and the Michael acceptor is a Michael addition reaction.

8. The method according to claim 1, wherein the organic functionalized silane compound comprises at least one hydrolyzable alkoxy group.

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

10. The method according to claim 1, wherein the chromatographic filler is provided in a form suitable for use as a chromatographic filler.

11. A chromatography filler formed by the method described in any one of claims 1 to 10.

12. Chromatography filler, (a) Substrate particles and (b) an amphoteric compound bonded to the substrate particles, (a)(i) reacting the functional groups on the substrate particles with an organic functional silane compound, (ii) Reacting the product of step (a)(i) with a Michael acceptor containing a protective acid group, (iii) formed by removing the acid protecting group from the product of step (a)(ii), or (b)(i) Reacting a Michael acceptor containing a protective acid group with an organofunctional silane compound, (ii) Reacting the product of step (b)(i) with the functional groups on the substrate particles, (iii) A chromatographic packing material comprising an amphoteric compound formed by removing an acid protecting group from the product of step (b)(ii).

13. The chromatographic filler according to claim 12, wherein the functional group in the organic functional silane compound is selected from primary or secondary amines and thios.

14. The chromatographic filler according to claim 12, wherein the Michael acceptor is selected from a protective α,β unsaturated acid, a dialkyl vinyl phosphonate, or a vinyl sulfonic acid ester.

15. The chromatographic filler according to claim 14, wherein the protective α,β unsaturated carboxylic acid is selected from the group consisting of methyl, ethyl, butyl, or tert-butyl acrylate, the dialkyl vinyl phosphonate is selected from the group consisting of dimethyl or diethyl ester vinyl phosphonate, and the vinyl sulfonic acid ester is selected from the group consisting of methyl, ethyl, or neo-pentyl vinyl sulfonate ester.

16. The chromatographic filler according to claim 12, wherein the reaction between the functional group on the substrate particles and the organic functional silane compound in step (i), or the reaction between the functional group on the substrate particles and the product of step (b)(i) in step (b)(ii), forms a covalent bond between the substrate particles and the organic functional silane compound or the product of step (b)(i).

17. The chromatography filler material according to claim 12, wherein the base material particles are selected from the group consisting of silica or metal oxides.

18. The chromatography filler according to claim 12, wherein the reaction between the functional group on the organic functional silane compound and the Michael acceptor is a Michael addition reaction.

19. Chromatography filler according to any one of claims 12 to 18, wherein the filler is obtained using the method described in any one of claims 1 to 10.

20. The chromatographic filler according to any one of claims 12 to 18, wherein the chromatographic filler is suitable for use in hydrophilic interaction liquid chromatography (HILIC).

21. Use of a chromatographic packing material according to any one of claims 12 to 18 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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