WO2025024069A2

WO2025024069A2 - Zwitterionic column packing

Info

Publication number: WO2025024069A2
Application number: PCT/US2024/034611
Authority: WO
Inventors: Richard Thomas Williams; Xiao CUI
Original assignee: Thermo Electron Manufacturing Ltd; Dionex Corp
Current assignee: Thermo Electron Manufacturing Ltd; Dionex Corp
Priority date: 2023-07-21
Filing date: 2024-06-19
Publication date: 2025-01-30
Anticipated expiration: 2026-01-21
Also published as: WO2025024069A3

Abstract

The present invention relates to column packing material and methods of making thereof. In particular, the present invention relates to column packing material that is used in Hydrophilic Interaction Liquid Chromatography (HILIC).

Description

ZWITTERIONIC COLUMN PACKING

Cross-Reference to Related Application

[0001] This application claims priority to US Provisional Application S/N 63/514,878, Filed July 21, 2023, the entire contents of which is incorporated herein by reference.

Field of the Invention

[0002] The present invention relates to column packing material and methods of making thereof. In particular, the present invention relates to column packing material that is used in Hydrophilic Interaction Liquid Chromatography (HILIC).

Background of the Invention

[0003] The Hydrophilic Interaction Liquid Chromatography (HILIC) technique has been extensively utilized in the analysis of polar compounds, biologically important compounds in proteomics.

[0004] Several columns have been commercially available such as Thermo Amide HILIC, ZIC- HILIC and so on. It has been reported that nearly 30% of HILIC applications used zwitterionic HILIC phase. However, the selection of zwitterionic separation material is limited. At present, one of the most used zwitterionic compound for HILIC phase is sulfobetaine as shown below.

Sulfobetaine (SB)

[0005] Sulfobetaine, a zwitterionic compound, can be covalently bonded to silica to make one of the most popular zwitterionic HILIC stationary phases.

[0006] Sulfobetaine stationary phases typically contain negatively-charged sulfonate and

SUBSTITUTE SHEET (RULE 26) positively- charged quaternary amine groups in a 1 to 1 ratio. These functional groups exhibit a net zero charge in the pH range from 0-14, which minimizes the ion-exchange behavior. Evidence suggests the zwitterionic HILIC retention mechanism for this type of phase is mainly from hydrophilic partitioning, with weak ionic interactions from residual silanols and analysts.

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

[0008] It is therefore desirable to develop a new type of HILIC stationary phase with improved hydrophilicity, charge properties, selectively and column efficiency.

[0009] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Description of the Invention

[0010] The present invention seeks to solve at least some of the above issues by providing new zwitterionic based HILIC phases.

[0011] Thus, the present invention provides a method for making chromatographic packing material, wherein the packing material comprises a zwitterionic compound, such as amino acid, amino phosphonate and/or amino sulfonate, bonded to substrate particles and the method for forming the packing material comprises:

(a) (i) reacting functional groups on the substrate particles with an organofunctional silane compound;

(ii) reacting the product of step (a) (i) with a Michael acceptor containing a protected acid group; and

(iii) removing the acid protecting group from the product of step (a) (ii); or

(b) (i) reacting a Michael acceptor containing a protected acid group

2

SUBSTITUTE SHEET (RULE 26) with an organofunctional silane compound;

(ii) reacting the product of step b (i) with functional groups on the substrate particles; and

(iii)removing the acid protecting group from the product of step (b) (ii).

[0012] In an aspect of the invention, step (a) (iii) or step (b) (iii) may not be performed and the column packing may be sold in “protected form”. For example, the column packing material may be packed into columns and sold for the customer to remove the protecting groups in-situ.

[0013] It may be preferred that the protecting groups are removed before being packed in columns.

[0014] Alternatively, the acid protecting groups may be removed after the column packing has been added to a chromatography column,

[0015] As used herein, the term “zwitterionic” is intended to encompass negatively charged compounds that intramolecularly comprise both a positive and a negative charge. Examples of zwitterionic compounds that may be used in the present invention include amino acids and amino acid derivatives/analogues, such as amino phosphonates and amino sulfonates.

[0016] As described below, the zwitterionic compound is formed through a combination of/reaction between an organofunctional silane and Michael acceptor containing a protected acid group, which when removed provides a zwitterionic compound.

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

[0018] The substrate particles may be a particulate or monolithic substrate, preferably particulate. The substrate particle material may be a metal oxide (which term herein includes

3

SUBSTITUTE SHEET (RULE 26) a metalloid oxide, such as silica for example, and includes an inorganic-organic hybrid material (especially a metal oxide-organic hybrid material), as described in WO 00/45951 for example). The substrate particles may, in particular, be silica (SiO2), which term herein includes a silica/organo hybrid, alumina (A12O3), titania (TiO2), or zirconia (ZrO2) substrate.

[0019] Silica (which term herein includes a silica/organo hybrid) substrate particles are most preferred.

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

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

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

[0023] The functional groups on the substrate particles may be any functional group suitable for reacting with an organofunctional silane compound. Typically, the functional groups present on the surface of the 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. hydroxy groups located on the surface of the substrate particles.

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

[0025] The organofunctional silane compound comprises at least one hydrolysable alkoxy

4

SUBSTITUTE SHEET (RULE 26) group, and at least one reactive organic group, such as a primary amine, a secondary amine and/or thiol.

[0026] In a particular aspect, the organofunctional silane has the structure shown below: Group 1 - X - Group 2

[0027] Wherein X is selected from -NH2-, -NH-, -S- or -SH-, wherein when X is NH2 or - SH- only Group 1 is present, i.e. group 2 is absent, when X is -NH- or -S- Group 1 and 2 are present;

[0028] Group 1 has the formula

wherein Rl, R2 and R3 are independently selected from Cl-4 alkyl or OC1-4 alkyl, so long as at least one of Rl, R2 or R3 is OC1-4 alkyl;

[0029] R4 and R5 are substituted or unsubstituted Cl- 12 alkyl, with the proviso that R4 and R5 combined do not exceed C14 alkyl, for example R4 may be Cl, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 alkyl and R5 may be C13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 alkyl, with the proviso that R4 and R5 combined do not exceed C14 alkyl;

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

[0031] Group 2 is selected from the formula

previously defined, or R6, wherein R6 is CH2aromatic.

[0032] Preferably, where X is -NH- or -S-, Group 1 and 2 may

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

[0034] Preferably, R4 may be Cl-4 alkyl and R5 may be Cl-4 alkyl, for example, R4 may be Cl,

SUBSTITUTE SHEET (RULE 26) 2, 3 or 4 alkyl and R5 may be Cl, 2, 3 or 4 alkyl.

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

[0036] In a particularly preferred aspect,

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

[0038] Group 1 has the formula

Group 2 is absent:

[0039] Rl, R2 and R3 are independently selected from methoxy or ethoxy; R4 is Cl, 2, 3 or 4 alkyl and R5 is Cl, 2, 3 or 4 alkyl; and

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

[0041] In a further particularly preferred aspect,

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

[0043] Rl, R2 and R3 are independently selected from methoxy or ethoxy; R4 is Cl, 2, 3 or 4 alkyl and R5 is Cl, 2, 3 or 4 alkyl; and

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

[0045] Herein the term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e., Cl- 10 means one to ten carbons).

[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, homologs and isomers of, for example, n-pentyl, n-hexyl, n-

6

SUBSTITUTE SHEET (RULE 26) heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2- isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3- propynyl, 3-butynyl, and the higher homologs and isomers. The term “alkyl,” unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as “hetero alkyl”. Alkyl groups that are limited to hydrocarbon groups are termed “homoalkyl”. The term “alkyl” can also mean “alkylene” or “alkyldiyl” as well as alkylidene in those cases where the alkyl group is a divalent radical.

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

[0048] Substituents for the alkyl groups/radicals are generically referred to as “alkyl group substituents,” and they can be one or more of a variety of groups selected from, but not limited to: substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, — OR', =0, =NR', =N — OR', — NR'R'', — SR', -halogen, — SiR'R' R'", — OC(O)R', — C(O)R', — CO2 R', — CONR'R'', — OC(O)NR'R", — NR''C(O)R', — NR'— C(O)NR''R"', — NR"C(O)2 R', — NR— C(NR'R''R"')=NR'", — NR— C(NR'R'')=NR'", — S(O)R', — S(O)2 R', — OS(O)2 R', — S(O)2 NR’R'', — NRSO2 R', — CN and — NO2 in a number ranging from zero to (2m'+l), where m' is the total number of carbon atoms in such radical. 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 groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R'', R'" and R'"' groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, — NR'R'' is meant to include, but not be limited to, 1 -pyrrolidinyl and 4- morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., — CF3 and — CH2 CF3 ) and acyl (e.g., — C(O)CH3 , — C(O)CF3 , — C(O)CH2 OCH3 , and the like).

7

SUBSTITUTE SHEET (RULE 26) [0049] Examples organofunctional silanes include, but are not limited to, (3- aminopropyl)trialkoxylsilanes, such as (3-aminopropyl)trimethoxysilane, (3- aminopropyl)triethoxysilane and (3-aminopropyl)tripropoxysilane; (3- mercaptopropyl)trialkoxysilanes, such as mercaptopropyl)trimethoxysilane, mercaptopropyljtriethoxysilane and mercaptopropyl)tripropoxysilane; bis(trialkoxysilylrpopyl)amines, such as bis(trimethoxysilylpropyl)amine. bis(triethoxypropyl)amine and bis(tripropoxysilylpropyl)amine,

(AMINOETHYL AMINOMETHYL)PHENETHYLTRIMETHOXYSILANE,N-(2-N-BENZYLAMINOETHYL)-3-

AMINOPROPYLTRIMETHOXYSILANE, N-(2-AMINOETHYL)-3-

AMINOPROPYLMETHYLDIETHOXYSILANE, N-(6-

AMINOHEXYL) AMINOPROPYLTRIMETHOXYSILANE, N-(2-AMINOETHYL)-l 1-

AMINOUNDECYLTRIMETHOXYSILANE, N-(2-AMINOETHYL)-3-

AMINOPROPYLTRIMETHOXYSILANE, and N-(6-

AMINOHEXYL) AMINOMETH YLTRIETHOXYSILANE.

[0050] As noted previously, reaction between the functional groups on (the surface of) the substrate particles and the organofunctional silane forms a bond, such as a covalent bond, between the surface of the substrate particles and the organofunctional silane.

[0051] In the reaction, the at least one hydrolysable alkoxy group(s) may typically be hydrolyzed to silanols, and then subject to a condensation reaction with the functional group on (the surface of) the substrate particles.

[0052] Thus, the method (a) defined herein may comprise the step of hydrolyzing the at least one hydrolysable alkoxy group(s) to silanols before reacting with the functional groups on (the surface of) the substrate particles. Any method suitable for the hydrolysis of alkoxy groups may be used.

[0053] As the skilled person would appreciate, the bond formed will depend on the substrates used. For example, where the functional groups present on (the surface of) the substrate particles are hydroxyl groups, reacting a hydrolysable/hydrolyzed alkoxy group on an organofunctional silane with a hydroxyl group will result in the formation of a siloxane bond.

[0054] Thus, in an aspect of the invention, the method may include reacting epoxy, hydroxyl, or

SUBSTITUTE SHEET (RULE 26) amino groups (preferably hydroxyl groups) present on the surface of the substrate particles with hydrolysable/hydrolyzed alkoxy groups on the org anofunctional silane compound in step (a) (i).

[0055] Typically, in step (a) (i), the silica particles and organofunctional silane are reacted in a ratio of from about 1:10 to about 10:1, such as from about 1:0.5 to about 1:2, or about 1:1.

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

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

[0058] In step (a) (ii), the reaction utilizes the functional group present on the (now bonded) organofunctional silane as a Michael donor, which reacts with a Michael acceptor.

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

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

[0061] Examples of Michael acceptors that may be used include, but are not limited to, protected a,0- unsaturated carboxylic acids, such as methyl, ethyl, butyl or tert-butyl acrylate, di-alkyl vinyl phosphonates, such as di-methyl or di-ethyl ester vinyl phosphonates and vinyl sulfonate esters, such as methyl, ethyl or neo-pentyl vinyl sulfonate ester.

[0062] It will be appreciated by the skilled person that the product formed from the Michael

9

SUBSTITUTE SHEET (RULE 26) addition will depend on whether the product of step (a) (i) uses a primary amine, a secondary amine or thiol as the Michael donor.

[0063] As a non-limiting example, if a primary amine is present this will lead to the formation of a di-ester product, i.e. the ratio of amine to ester is about 1:2. Whereas, the use of a secondary amine would result in the formation of a mono-ester, i.e. the ratio of amine to ester is about 1:1. The ratios would be the same where the Michael donor is sulfur based.

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

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

[0066] Any method suitable for the removal of acid protecting groups may be used. For example, treatment using an acid, such as an aqueous acid, i.e. aqueous methanesulfonic acid.

[0067] The chromatographic packing material may also be provided by using a method as defined in step (b).

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

[0069] An example of the reaction, where the organofunctional silane is (3- aminopropyl)trimethoxysilane and Michael acceptor is tert-butyl acrylate is shown below.

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

SUBSTITUTE SHEET (RULE 26) [0071] The Michael acceptor and organofunctional silane compound are as defined above for step (a).

[0072] It will be appreciated by the skilled person that the product formed from the Michael addition will depend on whether the organofunctional silane comprises a primary amine, a secondary amine or thiol as the Michael donor.

[0073] In the above non-limiting example, a primary amine is present leading to the formation of a di- ester product, i.e. the ratio of amine to ester is about 1 :2. Whereas, the use of a secondary amine would result in the formation of a mono-ester, i.e. the ratio of amine or thiol to ester is about 1:1.

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

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

[0076] The product of step (b) (i) comprises at least one hydrolysable alkoxy group from the organofunctional silane. As in step (a), the at least one alkoxy group may be hydrolyzed to a silanol, and then subject to a condensation reaction with the functional groups (on the surface of) the substrate particles. Resulting in the formation of a bond, such as a covalent bond, between the functional group and the product of step (b) (i). Where the functional group on the substrate particles is a hydroxy group, the bond may be a siloxane bond.

[0077] Thus, the method (b) defined herein may comprise the step of hydrolyzing the at least one hydrolysable alkoxy group(s) to silanols before reacting with the functional groups on (the surface of) the substrate particles.

[0078] Thus, in an aspect of the 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 for step (a). n

SUBSTITUTE SHEET (RULE 26) [0080] In step (b) (iii), the acid protecting group is removed to provide the zwitterionic chromatographic packing material.

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

[0082] In a further preferred aspect, the method for making chromatographic packing material, wherein the packing material comprises a zwitterionic compound, such as amino acid, amino phosphonate and/or amino sulfonate, bonded to substrate particles and the method for forming the packing material may comprise:

(a) (i) reacting functional groups (such as hydroxyl functional groups) on the surface of the silica substrate particles with an organofunctional silane having the structure shown below:

Group 1 - X - Group 2

[0083] Wherein X is selected from -NH2-, -NH-, -S- or -SH-, wherein when X is NH2 or -SH- only Group 1 is present, i.e. group 2 is absent, when X is -NH- or -S- Group 1 and 2 are present;

[0084] Group 1 has the formula

wherein Rl, R2 and R3 are independently selected from Cl -4 alkyl or OC1-4 alkyl, so long as at least one of Rl, R2 or R3 is OC1-4 alkyl;

[0085] R4 and R5 are substituted or unsubstituted Cl- 12 alkyl, with the proviso that R4 and R5 combined do not exceed C 14 alkyl, for example R4 may be Cl, 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;

SUBSTITUTE SHEET (RULE 26) [0087] Group 2 is selected from the formula

as previously defined, or R6, wherein R6 is CH2-aromatic;

(ii) reacting the product of step (a) (i) with at least one Michael acceptor containing a protected acid group, such as at least one of methyl, ethyl, butyl or tert-butyl acrylate, di- methyl or di-ethyl ester vinyl phosphonate or methyl, ethyl or neo-pentyl vinyl sulfonate ester; and

(iii) removing the protecting group from the product of step a (ii); or

(b) (i) reacting at least one Michael acceptor containing a protected acid group, such as at least one of methyl, ethyl, butyl or tert-butyl acrylate, di-methyl or di-ethyl ester vinyl phosphonate or methyl, ethyl or neo-pentyl vinyl sulfonate ester with an organofunctional silane having the structure shown below:

Group 1 - x - Group 2

[0088] Wherein X is selected from -NH2-, -NH-, -S- or -SH-, wherein when X is NH2 or -SH- only Group 1 is present, i.e. Group 2 is absent, and when X is -NH- or -S- Group 1 and 2 are present;

[0089] Group 1 has the formula

[0090] R4 and R5 are substituted or unsubstituted C1-12 alkyl, with the proviso that R4 and R5 combined do not exceed C14 alkyl, for example R4 may be Ci,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] Group 2 is selected from the formula

as previously defined, or R6,

SUBSTITUTE SHEET (RULE 26) wherein R6 is CH2aromatic;

(ii) reacting the product of step b (i) with functional groups (such as hydroxyl functional groups) on the surface of the silica substrate particles.

(iii)removing the protecting group from the product of step b (ii).

[0093] In a further preferred aspect, the method for making chromatographic packing material, may be wherein the packing material comprises amino acid or amino acid derivatives/analogues, such as amino phosphonates and amino sulfonates, covalently bonded to silica substrate particles and the method for forming the packing material comprises:

(a) (i) reacting hydroxyl functional groups on the surface of the silica substrate particles with (3-aminopropyl) trialkoxysilane or bis(trialkoxysilylpropyl)amine;

(ii) reacting the product of step (a) (i) with at least one of methyl, ethyl, butyl or tertbutyl acrylate, di-methyl or di-ethyl ester vinyl phosphonate or methyl, ethyl or neopentyl vinyl sulfonate ester; and

(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, di-methyl or di-ethyl ester vinyl phosphonate or methyl, ethyl or neo-pentyl vinyl sulfonate ester with (3- aminopropyl) trialkoxysilane or bis(trialkoxysilylpropyl)amine;

(ii) reacting the product of step b (i) with hydroxyl functional groups on the surface of the silica substrate particles.

(iii) removing the protecting group from the product of step b (ii).

[0094] In a particularly preferred aspect, the method for making chromatographic packing material, may be wherein the packing material comprises beta-alanine covalently bonded to silica substrate particles and the method for forming the packing material comprises:

(a) (i) reacting hydroxyl functional groups on the surface of the silica substrate particles with a (3-aminopropyl) trialkoxysilane or bis(trialkoxysilylpropyl)amine;

(ii) reacting the product of step (a) (i) with an acrylate alkyl ester; and

(iii) removing the protecting group from the product of step a (ii); or

14

SUBSTITUTE SHEET (RULE 26) (b) (i) reacting an acrylate alkyl ester with a (3-aminopropyl) trialkoxysilane or bis(trialkoxysilylpropyl)amine;

(ii)reacting the product of step b (i) with hydroxyl functional groups on the surface of the silica substrate particles.

(iii) removing the protecting group from the product of step b (ii).

[0095] The method of the invention provides the packing material in a form suitable to be used for column chromatography.

The present invention also provides chromatographic packing material comprising:

(a) Substrate particles; and

(b) A zwitterionic compound, bonded to the substrate particles, formed by:

(iii) removing the acid protecting group from the product of step (a) (ii); or

(b) (i) reacting a Michael acceptor containing a protected acid group with an organofunctional silane compound;

(iii) removing the acid protecting group from the product of step (b) (ii).

[0096] The substate particles, zwitterionic compound, functional groups on the substrate particles, Michael acceptor, acid protecting group and organofunctional silane compound are all as previously defined with respect to the method of the invention.

[0097] In a preferred aspect, the packing material may comprise:

(a) Silica particles; and

(b) A zwitterionic compound, bonded to the substrate particles formed by:

(i) reacting functional groups (such as hydroxyl functional groups) on the surface of the silica substrate particles with an organofunctional silane having the structure

15

SUBSTITUTE SHEET (RULE 26) shown below:

Group 1 - X - Group 2

[0098] Wherein X is selected from -NH2-, -NH-, -S- or -SH-, wherein when X is NH2 or -SH- only Group 1 is present, i.e Group 2 is absent, and when X is -NH- or -S- Group 1 and 2 are present;

[0099] Group 1 has the formula

, wherein Rl, R2 and R3 are independently selected from Cl-4 alkyl or OC1-4 alkyl, so long as at least one of Rl, R2 or R3 is OC1-4 alkyl;

[0100] R4 and R5 are substituted or unsubstituted Cl- 12 alkyl, with the proviso that R4 and R5 combined do not exceed C14 alkyl, for example R4 may be Cl, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 and R5 may be C 13, 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] Group 2 is selected from the formula

as previously defined, or R6, wherein R6 is CH2aromatic;

(iii) removing the protecting group from the product of step a (ii) ; or

(b) (i) reacting with at least one Michael acceptor containing a protected acid group, such as at least one of methyl, ethyl, butyl or tert-butyl acrylate, di-methyl or di-ethyl ester vinyl phosphonate or methyl, ethyl or neo-pentyl vinyl sulfonate ester with an organofunctional silane having the structure shown below:

SUBSTITUTE SHEET (RULE 26) Group 1 - x - Group 2

[0103] Wherein X is selected from -NH2-, -NH-, -S- or -SH-, wherein when X is NH2 or -SH- only Group 1 is present, i.e. Group 2 is absent, and when X is -NH- or -S- Group 1 and 2 are present;

[0104] Group 1 has the formula

[0105] R4 and R5 are substituted or unsubstituted C1-12 alkyl, with the proviso that R4 and R5 combined do not exceed C14 alkyl, for example R4 may be Ci, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 a nd 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] Group 2 is selected from the formula

as previously defined, or R6, wherein R6 is CH2aromatic;

(iii)removing the protecting group from the product of step b (ii).

[0108] In a further preferred aspect, the packing material may comprise:

(a) Silica particles; and

(b) An amino acid or amino acid derivative/analogue, such as amino phosphonates and amino sulfonates, covalently bonded to the silica substrate particles formed by:

(a) (i) reacting hydroxyl functional groups on the surface of the silica substrate

SUBSTITUTE SHEET (RULE 26) particles with (3-aminopropyl) trialkoxysilane or bis(trialkoxysilylpropyl)amine;

(ii) reacting the product of step (a) (i) with at least one of methyl, ethyl, butyl or tert-butyl acrylate, di-methyl or di-ethyl ester vinyl phosphonate or methyl, ethyl or neo-pentyl vinyl sulfonate ester; and

(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 di-ethyl ester vinyl phosphonate or methyl, ethyl or neo-pentyl vinyl sulfonate ester with (3-aminopropyl) trialkoxysilane or bis(trialkoxysilylpropyl)amine;

(iii) removing the protecting group from the product of step b (ii).

[0109] In a particularly preferred aspect, the packing material may comprise:

(a) Silica particles; and

(b) Beta-alanine or beta-alanine derivative covalently bonded to the silica substrate particles formed by:

(ii) reacting the product of step (a) (i) with an acrylate alkyl ester; and

(iii) removing the protecting group from the product of step a (ii); or

(b)(i) reacting an acrylate alkyl ester with a (3-aminopropyl) trialkoxysilane or bis(trialkoxysilylpropyl)amine;

(iii)removing the protecting group from the product of step b (ii).

[0110] The packing material defined above may be obtained using a method of the invention as previously defined.

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SUBSTITUTE SHEET (RULE 26) [0111] As noted previously, the packing material defined above maybe used in chromatographic separation.

[0112] For the avoidance of doubt, in this specification when we use the term “comprising” or “comprises” we mean that the feature being described must contain the listed component(s) but may optionally contain additional components. When we use the term “consisting essentially of’ or “consists essentially of’ we mean that the feature being described must contain the listed component(s) and may also contain other components provided that any components do not affect the essential properties of the feature. When we use the term “consisting of’ or “consists of’ we mean that the feature being described must contain the listed component(s) only.

[0113] It would be clear to the person skilled in the art that the features and combinations defined with respect to the method of the invention, apply equally to the packing material.

[0114] The detailed description illustrates, by way of example, not by way of limitation, the principles of the invention. The description will clearly enable one skilled in the art to make and use the invention, and described several embodiments, adaptions, variations, alternatives and uses of the invention. As used herein, the terms “about” or “approximately” for any numerical values or ranges indicate a suitable dimensional tolerance that allows the part or collection of components to functions for its intended purpose as described.

Brief Description of the Drawings

[0115] Figure 1 : Buffer concentration and adenosine monophosphate (AMP) retention time at pH 3 and 6.5

[0116] In order to illustrate the present invention, the following non-limited examples of its practice are given below.

[0117] Example 1 - Column Packing via Method Step (b)

[0118] Two beta-alanine HILIC stationary phases have been synthesized via this synthetic strategy.

[0119] Scheme 1 shows a synthesis example of preparing di-acid HILIC phase. a) Organosilane synthesis: In a 250 ml round bottom flask equipped with a stirring bar was

19

SUBSTITUTE SHEET (RULE 26) added Bis(trimethoxysilylpropyl)amine (10.25 g, 30mmol), tert-butyl acrylate (4.9g, 39 mmol) and methanol (50 ml). The resulting reaction mixture was stirred at room temperature for 18 hrs. Then volatiles were removed by rotavapor. Residue was used for the next step without further purification. Yield was 99%. b) Silica substrate bonding: 10 g of raw Si particle (3p, 120A, S A=300m²/g) was transferred into a 250ml round bottom flask followed by the addition of 40 ml of toluene. The mixture was sonicated for 10 min to make a homogeneous solution. Then 7g of synthesized organosilane was added into the flask. Set up the mechanical stirring system and condenser on the flask. The reaction mixture was refluxed for 24 hours. Filtered out the silica resin followed by washing with 150 ml methanol. The silica was dried on conventional oven at 80°C for overnight. c)In column hydrolysis: Silica as obtained in the above paragraph was packed into a chromatography column. The packed column was treated with 0.1% MSA aqueous solution at 45 °C for 60min to activate the HILIC phase.

Mono-ester silane Mono-ester Mono-acid

[0120] Scheme 1: Formation of packing material comprising mono-acid zwitterionic compound

[0121] Example 2 - Column Packing via Method Step (b) a) Organosilane synthesis: In a 250 ml round bottom flask equipped with a stirring bar was added (3-aminopropyl)trimethoxysilane (7 g, 40mmol), tert-butyl acrylate (12.8g, 100 mmol) and methanol (80 ml). The resulting reaction mixture was stirred at room temperature for 18 hrs. Then volatiles were removed by rotavapor. Residue was used for the next step without further purification. Yield was 99%. b) Silica substrate bonding: 10 g of raw Si particle (3p, 120A, SA=300m²/g) was transferred into a 250ml round bottom flask followed by the addition of 40 ml of toluene. The mixture was sonicated for 10 min to make a homogeneous solution. Then 10g of synthesized organosilane was added into the flask. Set up the mechanical stirring system and condenser on the flask. The reaction mixture was refluxed for 24 hours. Filtered out the silica resin followed by

SUBSTITUTE SHEET (RULE 26) washing with 150 ml methanol. The silica was dried on conventional oven at 80°C for overnight. c) In column hydrolysis: Silica as obtained in the above paragraph was packed into a chromatography column. Then the packed column was treated with 0.1% MSA aqueous solution at 45 °C for 60min to activate the HILIC phase.

Di-ester silane Di-ester Di-acid

[0122] Scheme 2: Formation of packing material comprising di-acid zwitterionic compound

[0123] Example 3 - column retention study

[0124] Preliminary ion exchange property study shows that the net charge on the di-acid HILIC phase depends on the buffer pH. For example, at pH3, the retention time of negatively chargedadenosine monophosphate (AMP) decreased when the buffer concentration increases. This indicates the phase has anion exchange properties. At pH 6.5, the retention time of AMP is getting longer when the buffer concentration increases, therefore the net charge on the surface is negative (Fig. 1). In future, the investigation of charged sample separation on the phases is needed.

[0125] Example 4 - Column Packing via Method Step (a). a) 20 g of raw Si particle (3p, 120A, SA=300m²/g) was transferred into a 250ml round bottom flask followed by the addition of 60 ml of toluene. The mixture was sonicated for 10 min to make a homogeneous solution. Then 8g of (3-aminopropyl)trimethoxysilane was added into the flask. Set up the mechanical stirring system and condenser on the flask. The reaction mixture was refluxed for 24 hours. Filtered out the silica resin followed by washing with 150 ml methanol. The silica was dried on conventional oven at 80°C for overnight. b) 10 g of bonded Si particle obtained in paragraph above was transferred into a 250ml round bottom flask followed by the addition of 60 ml of toluene. The mixture was

21

SUBSTITUTE SHEET (RULE 26) sonicated for 10 min to make a homogeneous solution. Then 4g of tert-butyl acrylate was added into the flask. Set up the mechanical stirring system and condenser on the flask. The reaction mixture was heated at 60°C for 24 hours. Filtered out the silica resin followed by washing with 150 ml methanol. The silica was dried on conventional oven at 80°C for overnight. c) In column hydrolysis: Silica as obtained in the above paragraph was packed into a chromatography column. Then the packed column was treated with 0.1% MSA aqueous solution at 45 °C for 60min to activate the HILIC phase.

[0126] In summary, a new type of zwitterionic HILIC stationary phase was designed and synthesized via Aza-Michael addition chemistry. The high yield and purity of the ligand, and efficient hydrolysis activation step ensure the amino acid functional groups uniformly distributed on the silica surface. The results of the phase exhibits high hydrophilicity and unique charge properties. In addition, a large variety of ligands, such as beta-alanine based ligands, can be prepared via this method, which gives a large HILIC phase selection in the separation of polar compounds.

SUBSTITUTE SHEET (RULE 26)

Claims

1. A method for making chromatographic packing material, wherein the packing material comprises a zwitterionic compound, such as amino acid or amino phosphonate, compound bonded to substrate particles and the method for forming the packing material comprises:

(ii) reacting the product of step (a) (i) with a with a Michael acceptor containing a protected acid group; and

(iii) removing the acid protecting group from the product of step (a) ii); or

(ii) reacting the product of step b (i) with functional groups on the substrate particles.; and

(iii) removing the acid protecting group 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 thio.

3. The method according to claim 1 or 2, wherein the Michael acceptor is selected from a protected a,P-unsaturated acid, a di-alkyl vinyl phosphonate or a vinyl sulfonic acid ester.

4. The method according to claim 3, wherein the protected a,p-unsaturated carboxylic acid is selected from the group consisting of methyl, ethyl, butyl or tert-butyl acrylate, the di-alkyl vinyl phosphonate is selected from the group consisting of di-methyl or di-ethyl ester vinyl phosphonates and the vinyl sulfonic acid ester is selected from the group consisting of methyl, ethyl or nco-pcntyl vinyl sulfonate ester.

5. A method according to any one of the preceding claims, wherein the reaction between functional groups on the substrate particles and the organofunctional silane compound in step (i) or the functional groups on the substrate particles and the product of step h (i) in step (b) (ii) forms covalent bonds between the substrate particles and the organofunctional silane compound or product of step (b) (i).

6. The method according to any one of the preceding claims, wherein the substrate particles are selected from the group consisting of silica 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 Michael acceptor is a Michael addition type reaction.

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

9. A method according to claim 8, wherein the at least one hydrolysable group is hydrolysed before being reacted in step (a) (i) or step (b) (ii)

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

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

12. Chromatographic packing material comprising:

(a) Substrate particles; and

(b) A zwitterionic compound, bonded to the substrate particles, formed by:

(iii) removing the acid protecting group from the product of step (a) (ii); or

(b) (i) reacting a Michael acceptor containing a protected acid group with an organofunctional silane compound; (ii) reacting the product of step b (i) with functional groups on the substrate particles; and

(iii) removing the acid protecting group from the product of step (b) (ii).

13. The packing material according to claim 12, wherein the functional group in the organofunctional silane compound is selected from primary or secondary amine, thio.

14. The packing material according to claim 12 or 13, wherein the Michael acceptor is selected from a protected a,P-unsaturated acid, a di-alkyl vinyl phosphonate or a vinyl sulfonic acid ester.

15. The packing material according to claim 14, wherein the protected a,P- unsaturated carboxylic acid is selected from the group consisting of methyl, ethyl, butyl or tert-butyl acrylate, the di-alkyl vinyl phosphonate is selected from the group consisting of di-methyl or di-ethyl ester vinyl phosphonates and the vinyl sulfonic acid ester is selected from the group consisting of methyl, ethyl or neo-pentyl vinyl sulfonate ester.

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

17. The packing material according to any one of claim 12 to 16, wherein the substrate particles are selected from the group consisting of silica or metal oxides.

18. The packing material 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 type reaction.

19. The packing material according to any one of claims 12 to 18, wherein the packing is obtained using a method as 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 use in in Hydrophilic Interaction Liquid Chromatography (HILIC).

21. The use of 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 Chromatographic (HILIC) separation.