Multi-component surface chemistry to eliminate retention loss in reversed phase liquid chromatography

Abstract

Provided herein is a multi-component chromatography material and its use for reversed-phase liquid chromatography. A multi-component chromatography material provided herein includes a chromatography core having an outer surface; and at least two different hydrophobic ligands covalently bound to the outer surface, the at least two different hydrophobic ligands having a total surface coverage of less than 2.0 [mu] mol / m2. Multi-component chromatography materials of the present technology are beneficial for reversed-phase liquid chromatography using highly aqueous mobile phases. For example, the chromatography materials described herein allow for mitigating or preventing significant retention loss of reversed-phase liquid chromatography columns following flow disruption.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 420,870, filed Oct. 31, 2022, the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] The present invention generally relates to compositions, methods, and devices having multi-component chromatographic materials useful in reversed-phase liquid chromatography. Specifically, the present invention relates to multi-component chromatographic materials comprising: a chromatographic core having an outer surface; and at least two different hydrophobic ligands covalently bound to the outer surface. Background Art

[0004] Reversed-phase liquid chromatography (RPLC) is one of the most widely used separation and analytical techniques, applicable to a variety of applications from separating small organic acids to separating large proteins.

[0005] Conventional reversed-phase columns employ a nonpolar stationary phase (most commonly a hydrocarbon chain chemically bonded to silica) and a polar mobile phase comprising water and at least one water-miscible organic solvent used as a modifier. RPLC columns are mainly used for separating hydrophobic compounds and moderately polar compounds. They can also be used to retain very highly polar compounds when using a highly aqueous mobile phase or a mobile phase approaching 100% water.

[0006] However, some drawbacks, including, for example, weak retention of ionic compounds and residual silanol activity that causes peak tailing of basic analytes, prevent the use of conventional reversed-phase silica columns in certain applications.

[0007] One of the most significant limitations in RPLC is the significant retention volume or time loss after column flow is stopped and then resumed when using a highly aqueous mobile phase with a particular reversed-phase LC column. Summary of the Invention

[0008] One object of the present disclosure is to improve the separation efficiency of a reversed-phase column when the highly aqueous mobile phase contains from about 98% to about 100% water.

[0009] The multi-component chromatographic material of the present invention can be used to mitigate or prevent significant retention volume or time loss after column flow is stopped and then resumed when using a highly aqueous mobile phase.

[0010] Compared to conventional reversed-phase stationary phases employed in reversed-phase liquid chromatography columns, the multi-component chromatographic material of the present invention helps to reduce the receding contact angle of water on the stationary phase used in reversed-phase liquid chromatography columns.

[0011] The technology of the present invention relates to a multi-component chromatographic material, which includes: a chromatographic core having an outer surface; and at least two different hydrophobic ligands covalently bound to the outer surface, wherein the total surface coverage of the at least two different hydrophobic ligands is less than 2.0 μmol / m 2 . In some embodiments, at least one hydrophobic ligand comprises a cycloalkyl group.

[0012] In certain embodiments, at least one hydrophobic ligand comprises an aromatic ring group, such as a phenyl group.

[0013] In one aspect, provided herein is a multi-component chromatographic material, which includes: a chromatographic core having an outer surface; and at least two different hydrophobic ligands covalently bound to the outer surface, wherein the total surface coverage of the at least two different hydrophobic ligands is less than 2.0 μmol / m 2 , wherein the at least two different hydrophobic ligands include a first hydrophobic ligand selected from C4 to C 30 phenylalkyl moiety and a second hydrophobic ligand selected from C4 to C 30 alkyl moiety.

[0014] The above aspect may include one or more of the following features. In some embodiments, the first hydrophobic ligand is a phenylhexyl moiety and the second hydrophobic ligand is a C 18 alkyl moiety. In some embodiments, the first hydrophobic ligand is a phenylhexyl moiety and the second hydrophobic ligand is a C8 alkyl moiety.

[0015] In one aspect, provided herein is a multi-component chromatographic material, which includes: a chromatographic core having an outer surface; and at least two different hydrophobic ligands covalently bound to the outer surface, wherein the total surface coverage of the at least two different hydrophobic ligands is less than 2.0 μmol / m 2 , wherein the at least two different hydrophobic ligands include a first hydrophobic ligand selected from C4 to C 42 phenylalkyl moiety and a second hydrophobic ligand selected from C4 to C 42 alkyl moiety.

[0016] In some embodiments, the first hydrophobic ligand is a phenylhexyl moiety and the second hydrophobic ligand is selected from C 23 to C 42 alkyl moiety.

[0017] In some embodiments, the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is from about 4.5:1.0 to about 1.0:4.5. In some embodiments, the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is from about 2.5:1.0 to about 1.0:2.5. In some embodiments, the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is about 1.8:1.0. In some embodiments, the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is about 1.0:1.0.

[0018] In one aspect, provided herein is a multi-component chromatographic material comprising: a chromatographic core having an outer surface; and at least two different hydrophobic ligands covalently bound to the outer surface, wherein the total surface coverage of the at least two different hydrophobic ligands is less than 2.0 μmol / m 2 , wherein the first hydrophobic ligand is a C 18 alkyl moiety and the second hydrophobic ligand is a C8 alkyl moiety, and the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is from about 2.0:1.0 to about 1.0:2.0.

[0019] In some embodiments, the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is about 1.0:1.0.

[0020] In another aspect, provided herein is a multi-component chromatographic material comprising: a chromatographic core having an outer surface; and at least two different hydrophobic ligands covalently bound to the outer surface, wherein the total surface coverage of the at least two different hydrophobic ligands is less than 2.0 μmol / m 2 , wherein the at least two different hydrophobic ligands include a first hydrophobic ligand selected from C 23 to C 42 alkyl moieties and a second hydrophobic ligand selected from C 23 to C 42 alkyl moieties, wherein the number of carbon atoms of the second hydrophobic ligand is different from the number of carbon atoms of the first hydrophobic ligand.

[0021] In some embodiments, the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is from about 2.0:1.0 to about 1.0:2.0. In one embodiment, the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is about 1:1.

[0022] The above aspects may include one or more of the following features. In some embodiments, the chromatographic core is porous and the average diameter of the pores of the chromatographic core is less than

[0023] In some embodiments, the at least two different hydrophobic ligands are modified with a capping silane (e.g., trimethylsilane) to minimize and eliminate residual silanol activity.

[0024] In some embodiments, the chromatographic core comprises a material selected from silica, alumina, titanium dioxide, zirconium oxide, and combinations thereof. In some embodiments, the chromatographic core comprises a material selected from silica monoliths, silica gels, silica / organic polymer hybrids, silica core / shell materials, and polymeric synthetic organic polymers. In one embodiment, the chromatographic core comprises an inorganic / organic hybrid material.

[0025] The technology of the present invention also relates to a reversed-phase liquid chromatography column comprising a multi-component chromatographic material according to multiple embodiments described herein.

[0026] In one aspect, provided herein is a method for selectively isolating, separating, or purifying one or more analytes from a sample, the method comprising the steps of: a) loading a sample containing the one or more analytes onto a chromatography column comprising a multi-component chromatographic material according to multiple aspects and embodiments described herein such that the one or more analytes are selectively retained on the multi-component chromatographic material; and b) eluting the retained analytes from the multi-component chromatographic material to thereby selectively isolate the one or more analytes from the sample.

[0027] In some embodiments, the retained analytes are eluted from the multi-component chromatographic material using a mobile phase comprising from about 90% to about 100% water, from about 95% to about 100% water, from about 98% to about 100% water, from about 99% to about 100% water.

[0028] In another aspect, the technology of the present invention relates to a method for reducing the receding contact angle of water on a stationary phase used in a reversed-phase liquid chromatography column. The method comprises: performing chromatographic separation of a sample using a stationary phase comprising a multi-component chromatographic material according to multiple aspects and embodiments described herein, thereby reducing the receding contact angle of water on the stationary phase to less than 90 degrees, wherein the chromatographic separation is performed using a mobile phase comprising from about 95% to about 100% water, from about 98% to about 100% water, from about 99% to about 100% water, from about 98% to about 100% water.

[0029] The materials and methods of the technology of the present invention offer many advantages.

[0030] For example, the materials and methods of the present invention technology have the advantage that retention loss is reduced when the flow rate is unexpectedly or intentionally stopped or resumed. By reducing retention loss, the materials and methods described herein improve selectivity and data reproducibility while separating polar compounds by using a highly aqueous mobile phase. That is, the materials provided herein can be used to manufacture improved columns for the reversed-phase separation of polar compounds. For example, the multi-component chromatographic materials in the stationary phases described herein provide a desired interfacial disorder that enables better peak separation and improved resolution.

[0031] The materials of the present invention technology can be effectively used at high temperatures (e.g., above 25 °C up to 80 °C) as well as at low temperatures (e.g., 25 °C or below). The conditions at high temperatures are based on the most challenging conditions for dehumidification because dehumidification is much faster at high temperatures than at low temperatures.

[0032] Another advantage of at least some of the embodiments of the present disclosure is that the materials provided herein can be used in short-trap RPLC columns that are difficult to keep wet due to their low pressure drop.

[0033] Not all of these aspects or advantages must be achieved by any particular embodiment. Accordingly, the various embodiments may be implemented in a manner that achieves or optimizes one advantage or a group of advantages taught herein without necessarily achieving other aspects or advantages taught or suggested herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present technology will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0035] Figure 1A 、 Figure 1B and Figure 1C shows an exemplary combination of two different hydrophobic ligands, the two different hydrophobic ligands including a first hydrophobic ligand and a second hydrophobic ligand. Figure 1A shows an exemplary combination of two different hydrophobic ligands, the two different hydrophobic ligands including at least one hydrophobic ligand containing an aromatic ring group (e.g., a phenyl group). Figure 1B shows an exemplary combination of two different hydrophobic ligands, the two different hydrophobic ligands containing an alkyl chain. Figure 1C shows an exemplary combination of two different hydrophobic ligands, the two different hydrophobic ligands including at least one hydrophobic ligand containing a cycloalkyl group.

[0036] Figure 2 shows an exemplary aromatic ring group that may be included in the first hydrophobic ligand of the present disclosure.

[0037] Figure 3 shows the Laplace formula that relates the pressure causing dehumidification to the contact angle of water.

[0038] Figure 4A , Figure 4B and Figure 4C The retention time of thymine is shown after stopping the flow for 64 minutes and then restarting the flow. The amount of reduction in retention time is positively correlated with the percentage of dewetting of the external surface. Figure 4A The surface concentration of pure C with 1.5 μmol / m2 is shown. 18 Dewetting of bonded phase bonded 100 angstrom silica particles. Figure 4B Dewetting of 100 angstrom silica particles bonded with a single component phenylhexyl group having a surface concentration of 1.5 micromoles per square meter is shown. Figure 4C The two-component phenylhexyl / C with a surface concentration of 1.5 μmol / m2 is shown. 18 Dewetting of bonded 100 Angstrom silica particles.

[0039] Figure 5A Phenylhexyl / C 18 Effect of the molar ratio of and temperature (24°C and 60°C) on the fraction of the surface area remaining wetted after the flow is stopped for 1 hour and then restarted. 18 The ligands were bonded to 100 angstrom silica particles with a surface concentration of 1.5 micromoles per square meter. Figure 5B The effect of the time and temperature (24°C and 60°C) at which the flow is stopped on the performance of a two-component long alkyl group (e.g., an alkyl group having at least 18 carbons, such as C 18 and C 30 )-bonded 97 angstrom silica particles, the two-component long alkyl groups having a surface concentration of 0.85 micromoles per square meter. DETAILED DESCRIPTION

[0040] Reversed phase liquid chromatography is widely known in the art for separation of water-soluble compounds, using a mobile phase as a solvent dissolving the sample to be separated, and a stationary phase as a liquid or solid carried by a packing material (ie, a support) that fills a column.

[0041] The mobile phase contains water as its main component (more than 95% water), and the stationary phase usually contains compounds with carbon chains. When a mobile phase containing water as its main component is used together with a reversed-phase stationary phase bonded with an alkyl group (usually having 8 to 18 carbons), the retention time of the sample is usually unstable, and the retention time decreases as the mobile phase flows. This phenomenon reduces the reproducibility of the retention time.

[0042] In particular, in the case where the flow of the mobile phase resumes after being temporarily stopped, it is known that the retention time is significantly reduced. In chromatography, since the separated compounds are identified based on the retention time, if the reproducibility of the retention time is low, the mobile phase cannot be used together with the stationary phase. Conventionally, as the reason for the low reproducibility of the retention time, it has been speculated that when the mobile phase containing water as its main component flows through the stationary phase, the carbon chain gradually collapses due to its hydrophobicity and the so-called "phase collapse" occurs. That is, the interaction between the stationary phase and the solute is reduced.

[0043] However, this phenomenon is now explained as a process of "pore dehumidification", in which water is expelled from the hydrophobic pore network. Recent studies have shown that this phenomenon can be basically explained by pore dehumidification, in which when the flow is suddenly stopped, the water confined in the hydrophobic mesopores is no longer in thermodynamic equilibrium with its vapor, and the column returns to atmospheric pressure. When water is confined in hydrophobic mesopores with a diameter <50 nm, the driving force for this process is the instability of the water liquid / vapor two-phase system. Due to the pressure difference between the vapor phase and the liquid phase (also known as the Laplace pressure), water is forced to leave the mesopore space in the porous reversed-phase LC material. Although the pore dehumidification phenomenon can occur in the mobile phase containing <100% water, this phenomenon becomes more significant when using a 100% water-containing mobile phase.

[0044] The kinetics of pore dehumidification depends on several parameters, including but not limited to column pressure, pore size and structure of the stationary phase material, mobile phase temperature, surface chemistry of the stationary phase material, surface concentration and molecular ordering of the bonded ligands on the surface of the stationary phase material, dissolved gases in the mobile phase, etc.

[0045] Therefore, the object of the technology of the present invention is to provide a reversed-phase liquid chromatography and a multi-component chromatographic material for use in this reversed-phase liquid chromatography, in which a mobile phase containing water as its main component (more than 95% water) can be used with any kind of column.

[0046] In the present disclosure, extensive research has been conducted to find a new chromatographic material that changes the kinetics of the dehumidification process and slows down the dehumidification of water in the case where the flow of the mobile phase is resumed or stopped.

[0047] The multi-component chromatographic material provided herein allows for the reduction of the receding contact angle, thereby contributing to slowing down the water dehumidification kinetics.

[0048] In one aspect, the present disclosure provides a multi-component chromatographic material, which includes: a chromatographic core having an outer surface; and at least two different hydrophobic ligands covalently bonded to the outer surface, wherein the total surface coverage of the at least two different hydrophobic ligands is less than 2.0 μmol / m 2 .

[0049] As used herein, the term "hydrophobic ligand" includes surface ligands that exhibit hydrophobicity on the outer surface of the chromatographic core. The hydrophobic ligands of the technology of the present invention are covalently attached to the outer surface of the chromatographic core.

[0050] As used herein, the term "covalently attached" means that the two elements either are directly covalently linked to each other (e.g., via a carbon-carbon bond) or are indirectly covalently linked to each other via an intervening chemical structure such as a bridge, spacer, linker, linking group, or any combination thereof. The term "bridge" refers to a molecular fragment that connects two different chemical elements. The terms "spacer" or "linker" are used interchangeably to refer to a single covalent bond or a series of stable covalent bonds that covalently link two or more different chemical elements, and the single covalent bond or series of stable covalent bonds are combined with 1 to 30 non-hydrogen atoms selected from the group consisting of C, N, O, S, and P. The term "linking group" is intended to denote a chemical functional group capable of covalently linking two or more chemical elements (e.g., a phosphoryl group or a sulfonyl group).

[0051] As used herein, "chromatographic surface" includes a surface that provides for chromatographic separation of a sample. In certain embodiments, the chromatographic surface is porous (e.g., having a pore volume of 0.1 cc / g to 1.5 cc / g). In some embodiments, the chromatographic surface can be the surface of a particle, superficially porous material, or monolith. In certain embodiments, the chromatographic surface is constituted by the surface of one or more particles, superficially porous materials, or monoliths used in combination during the chromatographic separation process. In certain other embodiments, the chromatographic surface is non-porous (e.g., having a pore volume of less than 0.1 cc / g, such as 0.05 cc / g).

[0052] The chromatographic surface of the technology of the present invention includes one or more functional groups that facilitate the covalent attachment of hydrophobic ligands to the outer surface of the chromatographic core.

[0053] The above aspects may include one or more of the following features. In some embodiments, at least two different hydrophobic ligands include a first hydrophobic ligand selected from a C4 to C 24 alkyl moiety and a second hydrophobic ligand selected from a C 4+n to C 24+n alkyl moiety, where n is at least 10, and the number of carbon atoms in the second hydrophobic ligand is at least 10 carbon atoms greater than the number of carbon atoms in the first hydrophobic ligand.

[0054] In some embodiments, at least two different hydrophobic ligands include a first hydrophobic ligand selected from a C 18 to C 42 alkyl moiety and a second hydrophobic ligand selected from a C 18 to C 42A second hydrophobic ligand of the alkyl moiety, wherein the number of carbon atoms of the second hydrophobic ligand is different from the number of carbon atoms of the first hydrophobic ligand.

[0055] In some embodiments, at least two different hydrophobic ligands include a first hydrophobic ligand selected from C 23 to C 42 of the alkyl moiety and a second hydrophobic ligand selected from C 23 to C 42 of the alkyl moiety, wherein the first hydrophobic ligand is different from the second hydrophobic ligand.

[0056] In some embodiments, at least two different hydrophobic ligands include a first hydrophobic ligand selected from C4 to C 30 phenylalkyl and a second hydrophobic ligand selected from C4 to C 30 of the alkyl moiety, wherein the first hydrophobic ligand is different from the second hydrophobic ligand.

[0057] In one embodiment, the first hydrophobic ligand is a phenylhexyl moiety and the second hydrophobic ligand is a C8 alkyl moiety.

[0058] In another embodiment, the first hydrophobic ligand is a phenylhexyl moiety and the second hydrophobic ligand is a C 18 alkyl moiety.

[0059] In some embodiments, at least two different hydrophobic ligands include a first hydrophobic ligand selected from C4 to C 42 phenylalkyl and a second hydrophobic ligand selected from C4 to C 42 of the alkyl moiety, wherein the first hydrophobic ligand is different from the second hydrophobic ligand.

[0060] In some embodiments, at least two different hydrophobic ligands include a first hydrophobic ligand selected from C 23 to C 42 phenylalkyl and a second hydrophobic ligand selected from C 23 to C 42 of the alkyl moiety, wherein the first hydrophobic ligand is different from the second hydrophobic ligand.

[0061] In some embodiments, at least two different hydrophobic ligands include a first hydrophobic ligand selected from C 18 to C 42 phenylalkyl and a second hydrophobic ligand selected from C 18 to C 42 of the alkyl moiety, wherein the first hydrophobic ligand is different from the second hydrophobic ligand.

[0062] In some embodiments, at least two different hydrophobic ligands include a first hydrophobic ligand selected from C4 to C 60 containing a cycloalkyl group and a second hydrophobic ligand selected from C4 to C 42A second hydrophobic ligand of the alkyl moiety, wherein the first hydrophobic ligand is different from the second hydrophobic ligand.

[0063] As used herein, "cycloalkyl" refers to a saturated aliphatic carbocyclic group. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. C4 to C containing a cycloalkyl group as used in the present disclosure 60 refers to an alkyl chain attached to the cycloalkyl group, wherein the total number of carbons in the ligand is between 4 and 60.

[0064] In some embodiments, at least two different hydrophobic ligands include a first hydrophobic ligand selected from C4 to C containing an aromatic ring 60 and a second hydrophobic ligand selected from C4 to C of the alkyl moiety, wherein the first hydrophobic ligand is different from the second hydrophobic ligand. 42 A second hydrophobic ligand of the alkyl moiety, wherein the first hydrophobic ligand is different from the second hydrophobic ligand.

[0065] As used herein, "aromatic ring" or "aryl" refers to a fully unsaturated carbocyclic ring whose planar ring has a delocalized π - electron system and contains 4n + 2π electrons, where n is an integer. The aromatic ring can be composed of six, eight, ten, or more than ten carbon atoms, and the aromatic ring can be monocyclic or polycyclic. Common aromatic rings include, but are not limited to, benzene ring, naphthalene ring, phenanthrene ring, anthracene ring, fluorene ring, and indene ring. C4 to C containing an aromatic group as used in the present disclosure 60 refers to an alkyl chain attached to the aromatic group, wherein the total number of carbons in the ligand is between 4 and 60.

[0066] In some embodiments, the aromatic group includes 5 - membered monocyclic groups and 6 - membered monocyclic groups, which may contain zero to four heteroatoms, such as furan, pyrrole, pyrroline, oxazole, thiazole, imidazole, imidazoline, pyrazole, pyrazoline, pyrazolidine, isoxazole, isothiazole, benzene, pyridine, pyridazine, pyrimidine, pyrazine, triazine, thiophene, etc. The aromatic ring can be substituted at one or more ring positions by, for example, halogen, lower alkyl, lower alkenyl, lower alkoxy, lower alkylthio, lower alkylamino, lower alkyl carboxyl, nitro, hydroxy, - CF3, - CN, etc. In other embodiments, the aromatic group includes 5 - membered polycyclic groups and 6 - membered polycyclic groups, which may contain zero to eight heteroatoms, such as indene, indolizine, indole, isoindole, dihydroindole, indazole, benzimidazole, benzothiazole, naphthalene, quinazoline, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8 - naphthyridine, quinuclidine, fluorene, carbazole, anthracene, acridine, phenazine, phenothiazine, phenoxazine, pyrene, etc. Poly - aromatic groups include fused aromatic groups.

[0067] Cycloalkyl groups and aromatic rings (e.g., phenyl) disclosed herein may contain heteroatoms. As used herein, "heteroatoms" refers to any atom other than a carbon atom that can be covalently bonded to a carbon atom. Common heteroatoms include, but are not limited to, O, S, and N.

[0068] As used herein, the term "alkyl" includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.

[0069] In addition, the term "alkyl" as used throughout this disclosure includes both "unsubstituted alkyl" and "substituted alkyl", the latter referring to an alkyl moiety having a substituent replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents may include, for example, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonate, phosphino, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), amido (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, aralkyl or aromatic or heteroaromatic moieties. Those skilled in the art will appreciate that the moieties substituted on the hydrocarbon chain may themselves be substituted, if appropriate.

[0070] Figure 1A , Figure 1B and Figure 1C An exemplary combination of two different hydrophobic ligands is shown, the two different hydrophobic ligands comprising a first hydrophobic ligand and a second hydrophobic ligand. Figure 1A As shown, in some embodiments, at least one hydrophobic ligand comprises an aromatic ring group, such as a phenyl group. Exemplary aromatic ring groups that may be included in the first hydrophobic ligand are Figure 2 Shown in.

[0071] In some embodiments, Figures 1A to 1C The number of carbon atoms (n2) of the second hydrophobic ligand shown and the number of carbon atoms (n2) of the second hydrophobic ligand are independently selected. In some embodiments, n1 is equal to n2. In other embodiments, n1 and n2 are different from each other.

[0072] In some embodiments, Figure 1A The number of carbon atoms (n1) in the alkyl chain of the first hydrophobic ligand is shown to be between 4 and 42, between 4 and 30, and between 4 and 18.

[0073] In some embodiments,Figure 1A The number of carbon atoms (n1) in the alkyl chain of the first hydrophobic ligand shown is between 23 and 42.

[0074] In some embodiments, Figure 1A The number of carbon atoms (n2) in the alkyl chain of the second hydrophobic ligand shown is between 4 and 42, between 4 and 30, between 4 and 18.

[0075] In some embodiments, Figure 1A The number of carbon atoms (n2) in the alkyl chain of the second hydrophobic ligand shown is between 23 and 42.

[0076] In some embodiments, Figure 1B The number of carbon atoms (n1) in the alkyl chain of the first hydrophobic ligand shown is between 4 and 42, between 4 and 30, between 4 and 18, between 18 and 42, between 23 and 42.

[0077] In some embodiments, Figure 1B The number of carbon atoms (n2) in the alkyl chain of the second hydrophobic ligand shown is between 4 and 42, between 4 and 30, between 4 and 18, between 18 and 42, between 23 and 42.

[0078] In some embodiments, the number of carbon atoms of the second hydrophobic ligand ( Figure 1B , n2) is at least 10 carbon atoms greater than the number of carbon atoms of the first hydrophobic ligand ( Figure 1B , n1).

[0079] As Figure 1C shown, in some embodiments, at least one hydrophobic ligand comprises a cycloalkyl group.

[0080] In some embodiments, Figure 1C The number of carbon atoms (n1) in the alkyl chain of the first hydrophobic ligand shown is between 4 and 42, between 4 and 30, between 4 and 18, between 18 and 42, between 23 and 42.

[0081] In some embodiments, Figure 1C The number of carbon atoms (n2) in the alkyl chain of the second hydrophobic ligand shown is between 4 and 42, between 4 and 30, between 4 and 18, between 18 and 42, between 23 and 42.

[0082] The molar ratio of the first hydrophobic ligand and the second hydrophobic ligand of the technology of the present invention covalently bound to the outer surface of the chromatographic core can be independently varied. In some embodiments, the number of moles of the first hydrophobic ligand and the second hydrophobic ligand present on the outer surface of the chromatographic core is the same. In some embodiments, the number of moles of the first hydrophobic ligand and the second hydrophobic ligand present on the outer surface of the chromatographic core is different.

[0083] In some embodiments, the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is from about 5.0:1.0 to about 1.0:5.0.

[0084] In some embodiments, the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is from about 2.5:1.0 to about 1.0:2.5.

[0085] In some embodiments, the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is from about 1.5:1.0 to about 1.0:1.5.

[0086] In some embodiments, the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is about 1.0:1.0.

[0087] In some embodiments, at least one hydrophobic ligand is modified with a capping silane. In other embodiments, at least two different hydrophobic ligands are modified with a capping silane.

[0088] As used herein, the term "capping" refers to placing a functional group at the end of a chain. In some embodiments, the capping silane is selected from methylhydrosilane, methyldihydrosilane, dimethylhydrosilane, and mixtures thereof.

[0089] In some embodiments, the capping silane is trimethylsilane.

[0090] In some embodiments, the chromatographic core of the technology of the present invention comprises a material selected from silica, alumina, titanium dioxide, zirconium oxide, and combinations thereof.

[0091] As used herein, the term "chromatographic core" includes chromatographic materials, including but not limited to organic materials in the form of particles, monoliths, or another suitable structure, such as silica or hybrid materials as defined herein, which form the internal portion of the materials of the present disclosure. In some aspects, the outer surface of the chromatographic core represents a chromatographic surface as defined herein, or represents a material wrapped by a chromatographic surface as defined herein. The chromatographic surface material may be disposed on the chromatographic core or bonded or annealed to the chromatographic core in a discernibly discrete or distinct transition manner, or may be bonded to the chromatographic core in a manner that blends with the surface of the chromatographic core to produce a material gradient and without a discrete internal core surface. In certain embodiments, the chromatographic surface material may be the same as or different from the material of the chromatographic core, and may exhibit physical or physicochemical properties different from those of the chromatographic core, including but not limited to pore volume, surface area, average pore diameter, carbon content, or hydrolytic pH stability.

[0092] In some embodiments, the chromatographic core includes a material selected from silica monoliths, silica gels, silica / organic polymer hybrids, silica core / shell materials, and polymeric synthetic organic polymers.

[0093] The term "core / shell material" refers to a material that includes a core portion and a shell portion surrounding the core portion. In some embodiments, the chromatographic core includes a core / shell material.

[0094] In some embodiments, the chromatographic core includes an inorganic / organic hybrid material.

[0095] As used herein, the term "inorganic / organic hybrid material" includes inorganic-like structures in which organic functional groups are integral with both the internal or "skeletal" inorganic structure and the surface of the hybrid material. The inorganic portion of the hybrid material may be, for example, alumina, silica, titanium, cerium, or zirconium or their oxides, or ceramic materials. Exemplary hybrid materials are described in US.4,017,528; 6,528,167; 6,686,035; 7,919,177 and 7,175,913.

[0096] In some embodiments, the chromatographic core is porous and may have or smaller to or larger pore diameters, for example to to to any pore diameter within the range. Porous materials are defined herein as having a pore volume of 0.2 cc / g or greater (e.g., 0.2 cc / g to about 1.5 cc / g). The pore volume is determined based on a multi-point nitrogen adsorption experiment (e.g., Micromeritics ASAP 2400, Micromeritics Instruments Inc., Norcross, GA) using methods known in the art.

[0097] In some embodiments, the average diameter of the pores of the chromatographic core is less than and has a pore volume between 0.2 cc / g and 1.5 cc / g.

[0098] In one aspect, provided herein is a reversed-phase liquid chromatography column that includes a multi-component chromatographic material according to various aspects and embodiments of the techniques of the present invention.

[0099] In another aspect, the techniques of the present invention relate to a method for selectively isolating, separating, or purifying one or more analytes from a sample using a multi-component chromatographic material according to various aspects and embodiments of the techniques of the present invention.

[0100] As used herein, the terms “purify,” “separate,” or “isolate,” which are used interchangeably herein, refer to increasing the purity of a target molecule from a composition or sample matrix (e.g., a solution containing a target molecule and one or more impurities). Generally, the purity of the target molecule is increased by removing at least one impurity from the composition (either completely or partially).

[0101] The reversed-phase liquid chromatography columns of the techniques of the present invention can be used for retaining, separating, and / or analyzing a variety of different compounds of interest from many different samples, which samples are from many different fields, such as, for example, clinical chemistry, medicine, veterinary medicine, forensic chemistry, pharmacology, the food industry, workplace safety, and environmental pollution. The plurality of samples includes, but is not limited to, small organic molecules, proteins, nucleic acids, lipids, fatty acids, carbohydrates, polymers, and the like. Similarly, the present disclosure can be used for the separation of small molecules, polar small molecules, analytes used in pharmaceuticals, biomolecules, antibodies, polymers, and oligomers, sugar, glycan analysis, petrochemical analysis, lipid analysis, peptides, phosphopeptides, oligonucleotides, DNA, RNA, polar acids, polycyclic aromatic hydrocarbons, food analysis, chemical analysis, bioanalysis, drugs of abuse, digital forensics, pesticides, agrochemicals, biosimilars, formulations.

[0102] Analytes suitable for chromatographic separation using the present disclosure can include substantially any molecule of interest, including, for example, small organic molecules, lipids, peptides, nucleic acids, synthetic polymers.

[0103] In one aspect, the techniques of the present invention relate to a method for selectively isolating, separating, or purifying one or more analytes from a sample, the method comprising the steps of: a) loading a sample containing the one or more analytes onto a chromatography column comprising a multi-component chromatographic material of the techniques of the present invention such that the one or more analytes are selectively retained on the multi-component chromatographic material; and b) eluting the retained analytes from the multi-component chromatographic material, thereby selectively isolating the one or more analytes from the sample.

[0104] In some embodiments, the retained analyte is eluted from the multi-component chromatographic material using a highly aqueous mobile phase. As used herein, a highly aqueous mobile phase refers to a mobile phase that contains from about 95% to about 100% water, from about 98% to about 100% water.

[0105] On the other hand, provided herein is a method for reducing the receding contact angle of water on a stationary phase used in a reversed-phase liquid chromatography column, the method comprising: chromatographing a sample using a stationary phase comprising a multi-component chromatographic material of the present invention, thereby reducing the receding contact angle of water on the stationary phase to less than 90 degrees.

[0106] Without wishing to be bound by theory, as the receding contact angle decreases, it is expected that the driving force for water dewetting decreases, thereby reducing retention loss.

[0107] In some embodiments, the chromatographic separation is carried out using a mobile phase comprising from about 98% to about 100% water.

[0108] Recent studies in the art have shown that retention loss observed in reversed-phase columns is essentially explained by pore dewetting, where when the flow is suddenly stopped, the water confined in the hydrophobic mesopores is no longer in thermodynamic equilibrium with its vapor and the column returns to atmospheric pressure. The equilibrium vapor pressure, Pvap, is given by the Laplace equation ( Figure 3 ). P vap depends on the receding contact angle of water on the hydrophobic surface (θ, about 93°), the liquid / vapor surface tension of water (γLV = 72 mN / m) and the mesopore radius (Rpore, about ); thus, it is typically about 10 bar to 20 bar. The pressure difference, P vap -P0 ∼ P vap is the driving force for the spontaneous extrusion of liquid water from the mesopore network. When the water has completely left the pores to reach thermodynamic equilibrium, the receding process ends. The kinetics of this process depends on several experimental parameters, including but not limited to temperature, column pressure, pore size distribution and pore size of the chromatographic material, surface coverage of the chromatographic surface, surface chemistry of the chromatographic surface, dissolved gases in the mobile phase.

[0109] Example

[0110] The following examples are included to illustrate the preferred embodiments of the present invention.

[0111] Example 1. Dewetting of a single hydrophobic ligand (C 18 , phenylhexyl) and a mixed hydrophobic ligand (C 18 , phenylhexyl)

[0112] Experiments have shown that pure C with a surface concentration of 1.5 micromoles per square meter 18 bonded phase ( Figure 4A) shows 16% dehumidification. The single-component phenylhexyl bonded phase with a surface concentration of 1.5 micromoles per square meter ( Figure 4B ) shows only 2% dehumidification. Figure 4C is obtained using a two-component phenylhexyl / C 18 bonded phase with a total surface concentration of 1.5 micromoles per square meter. The two-component phenylhexyl / C 18 bonded phase system provides 6% dehumidification.

[0113] The percentage of dehumidification is calculated using Formulas 1 and 2. The mathematical expression for % dehumidification for the outer surface is:

[0114] % dehumidification = (t R,之后 - t e ) / (t R,之前 - t e ) Formula (1)

[0115] where t R,之后 is the retention time observed after the flow rate is stopped and restored, t R,之前 is the retention time observed before the flow rate is stopped when water is in contact with the entire "outer" surface of the solid adsorbent, and t e is the interparticle elution time given by:

[0116] t e = (ε e × π × r c 2 × L) / F v Formula (2)

[0117] where ε e is the interparticle volume fraction in the chromatographic column, π is 3.1415926, r c is the inner column radius, L is the column length, and F v is the flow rate applied to the column.

[0118] Although the dehumidification of the single phenylhexyl bonded material shows better results in terms of dehumidification compared to the two-component phenylhexyl / C 18 bonded phase ( Figure 4C ), the technology of the present invention benefits from the mixed ligand technology. The advantage of the present invention of the two-component phenylhexyl / C Figure 4B is the higher retention of this stationary phase relative to pure phenylhexyl. This will provide more retention for highly polar compounds such as thymine. 18

[0119] Example 2. Dehumidification after 2.1-hour Flow Interruption

[0120] Using the same multicomponent chromatographic material and apparatus as in Example 1, the effect of flow interruption was studied.Figure 5A The Y-axis shows the fraction of the surface area that remains wetted after stopping the flow for 1 hour and then restarting. Values close to 1 indicate very little dewetting, which is desirable for RPLC. The results obtained at both 24 degrees Celsius and 60 degrees Celsius are shown in Figure 5A , where 60 degrees Celsius is the most challenging condition because dewetting is much faster at high temperatures (e.g., above 25°C up to 80°C) than at low temperatures (e.g., 25°C or below). For Figure 5A , any combination with a total surface concentration of 1.5 micromoles per square meter for phenylhexyl and C 18 groups achieves very little dewetting, with a slight improvement as the fraction of phenylhexyl groups increases.

[0121] Example 3. Influence of Flow Interruption on Dehumidification

[0122] A mixture of long alkyl groups is bonded to the outer surface at 0.85 micromoles per square meter ( Figure 5B ). The graph ( Figure 5B ) shows the relationship between the wetted surface area on the y-axis and the time the flow is stopped on the x-axis. For this material, very little dewetting was observed even after stopping the flow for 64 minutes at 60 degrees Celsius.

[0123] Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention, and thus these techniques can be considered to constitute a preferred mode for the practice of the present invention.

Claims

1. A multi-component chromatographic material, the multi-component chromatographic material comprising: A chromatographic core, the chromatographic core having an outer surface; and At least two different hydrophobic ligands, said at least two different hydrophobic ligands being covalently bound to said outer surface, wherein the total surface coverage of said at least two different hydrophobic ligands is less than 2.0 μmol / m 2 , wherein said at least two different hydrophobic ligands include a first hydrophobic ligand selected from C4 to C 30 phenylalkyl moieties and a second hydrophobic ligand selected from C4 to C 30 alkyl moieties.

2. The multi-component chromatographic material according to claim 1, wherein the first hydrophobic ligand is a phenylhexyl moiety, and the second hydrophobic ligand is a C 18 alkyl moiety.

3. The multi-component chromatographic material according to claim 1, wherein the first hydrophobic ligand is a phenylhexyl moiety, and the second hydrophobic ligand is a C8 alkyl moiety.

4. The multi-component chromatographic material according to claim 1, wherein the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is from about 2.5:1.0 to about 1.0:2.

5.

5. The multi-component chromatographic material according to claim 4, wherein the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is about 1.8:1.

0.

6. The multi-component chromatographic material according to claim 1, wherein the chromatographic core is porous, and the average diameter of the pores of the chromatographic core is less than 7. A reversed-phase liquid chromatography column, the reversed-phase liquid chromatography column comprising the multi-component chromatographic material according to any one of claims 1 to 6.

8. A method for selectively isolating, separating or purifying one or more analytes from a sample, the method comprising the steps of: a) loading a sample containing the one or more analytes onto a chromatographic column comprising a multi-component chromatographic material according to any one of claims 1 to 6 such that the one or more analytes are selectively retained on the multi-component chromatographic material; and b) eluting the retained analytes from the multi-component chromatographic material so as to selectively isolate the one or more analytes from the sample.

9. The method according to claim 8, wherein the retained analyte is eluted from the multi-component chromatographic material using a mobile phase comprising from about 98% to about 100% water.

10. A method for reducing the receding contact angle of water on a stationary phase used in a reversed-phase liquid chromatography column, the method comprising: Performing chromatographic separation of a sample using a stationary phase comprising a multi-component chromatographic material according to any one of claims 1 to 6, thereby reducing the advancing contact angle of water on the stationary phase to less than 90 degrees, wherein the chromatographic separation is carried out using a mobile phase comprising from about 98% to about 100% water.

11. A multi-component chromatographic material, the multi-component chromatographic material comprising: A chromatographic core, the chromatographic core having an outer surface; and At least two different hydrophobic ligands, said at least two different hydrophobic ligands being covalently bound to the outer surface, wherein the total surface coverage of said at least two different hydrophobic ligands is less than 2.0 μmol / m 2 , wherein the first hydrophobic ligand is a C 18 alkyl moiety and the second hydrophobic ligand is a C8 alkyl moiety, and the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is from about 2.0:1.0 to about 1.0:2.

0.

12. The multi-component chromatographic material according to claim 11, wherein the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is about 1.0:1.

0.

13. The multi-component chromatographic material according to claim 11, wherein the at least two different hydrophobic ligands are modified with a capping silane.

14. The multi-component chromatographic material according to claim 13, wherein the capping silane is trimethylsilane.

15. The multi-component chromatographic material according to claim 11, wherein the chromatographic core comprises a material selected from silica, alumina, titanium dioxide, zirconium oxide, and combinations thereof.

16. The multi-component chromatographic material according to claim 15, wherein the chromatographic core comprises a material selected from silica monoliths, silica gels, silica / organic polymer hybrids, silica core / shell materials, and polymeric synthetic organic polymers.

17. The multi-component chromatographic material according to claim 16, wherein the chromatographic core comprises an inorganic / organic hybrid material.

18. A multi-component chromatographic material, the multi-component chromatographic material comprising: A chromatographic core, the chromatographic core having an outer surface; and At least two different hydrophobic ligands, said at least two different hydrophobic ligands being covalently bound to said outer surface, wherein the total surface coverage of said at least two different hydrophobic ligands is less than 2.0 μmol / m 2 , wherein the at least two different hydrophobic ligands include a first hydrophobic ligand selected from C 23 to C 42 alkyl moieties and a second hydrophobic ligand selected from C 23 to C 42 alkyl moieties, wherein the number of carbon atoms of said second hydrophobic ligand is different from the number of carbon atoms of said first hydrophobic ligand.

19. The multi-component chromatographic material according to claim 18, wherein the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is from about 2.0:1.0 to about 1.0:2.

0.

20. The multi-component chromatographic material according to claim 19, wherein the molar ratio of the first hydrophobic ligand to the second hydrophobic ligand is about 1:1.

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