阅读说明：本技术 用于捕集-洗脱混合模式色谱法的色谱系统和方法 (Chromatography system and method for trap-elute mixed mode chromatography ) 是由 M·A·劳伯 B·O·奥肯德吉 于 2019-07-09 设计创作，主要内容包括：在各个方面,本公开涉及用于执行液相色谱的材料(例如,套件、柱组件、液相色谱系统等)方法,其采用第一柱(例如,捕集柱)和第二柱(例如,分析柱)。第一柱包括具有第一色谱表面的第一色谱材料,该第一色谱表面包含第一疏水表面基团和具有第一pKa值的第一可离子化表面基团。第二柱包括具有第二色谱表面的第二色谱材料,该第二色谱表面包含第二疏水表面基团和(a)永久性离子化的表面基团或(b)具有第二pKa值的第二可离子化表面基团。第一疏水表面基团的疏水性小于第二疏水表面基团的疏水性。此外,在第二色谱表面包含第二可离子化P表面基团的情况下,第一pKa值可以与第二pKa值相差1个单位至12个单位。(In various aspects, the present disclosure relates to methods of materials (e.g., kits, column assemblies, liquid chromatography systems, etc.) for performing liquid chromatography that employ a first column (e.g., a trapping column) and a second column (e.g., an analytical column). The first column includes a first chromatographic material having a first chromatographic surface comprising a first hydrophobic surface group and a first ionizable surface group having a first pKa value. The second column includes a second chromatographic material having a second chromatographic surface comprising a second hydrophobic surface group and either (a) a permanently ionized surface group or (b) a second ionizable surface group having a second pKa value. The hydrophobicity of the first hydrophobic surface group is less than the hydrophobicity of the second hydrophobic surface group. Further, where the second chromatographic surface comprises a second ionizable P surface group, the first pKa value may differ from the second pKa value by from 1 unit to 12 units.)

1. a liquid chromatography system, comprising:

(a) a trapping column comprising a first chromatographic material having a first chromatographic surface comprising a first hydrophobic surface group and a first ionizable surface group having a first pKa value,

(b) an analytical column comprising a second chromatographic material having a second chromatographic surface comprising a second hydrophobic surface group having a hydrophobicity greater than that of the first hydrophobic surface group and (i) a permanently ionized surface group or (ii) a second ionizable surface group having a second pKa value that differs from the first pKa value by 1 unit to 12 units,

(c) a sample injector for introducing a liquid sample into the system,

(d) a detector capable of detecting a characteristic of the component,

(e) a first flow path including the injector and the trap column, but not the analytical column,

(f) a second flow path comprising the trapping column and the analysis column, an

(g) One or more mobile phase delivery sources configured to pump a first mobile phase along the first flow path and a second mobile phase along the second flow path.

2. The liquid chromatography system of claim 1, wherein the one or more mobile phase delivery sources comprise a first pump configured to pump the first mobile phase along the first flow path and a second pump, which may be the same or different from the first pump, configured to pump the second mobile phase along the second flow path.

3. The liquid chromatography system of any of claims 1-2, wherein the first chromatographic material is in the form of first particles, and wherein the second chromatographic material is in the form of second particles.

4. The liquid chromatography system of claim 3, wherein a first diameter of the first particles is equal to or greater than a second diameter of the second particles.

5. The liquid chromatography system of claim 4, wherein the ratio of the first particle diameter to the second particle diameter is in the range from 1 to 10.

6. The liquid chromatography system of any of claims 4-5, wherein the first diameter is in a range from 2 microns to 10 microns.

7. The liquid chromatography system of any of claims 1-6, wherein the inner diameter of the trap column is greater than or equal to the inner diameter of the analytical column, and wherein the length of the trap column is shorter than the length of the analytical column.

8. The liquid chromatography system of any of claims 1-7, wherein the inner diameter of the trapping column is 1.5 to 5 times the inner diameter of the analytical column.

9. The liquid chromatography system of any of claims 1-8, wherein the volume of the trapping column is in a range from 0.05 to 0.5 times the volume of the analytical column.

10. The liquid chromatography system of any one of claims 1-9, wherein the first hydrophobic surface group and the second hydrophobic surface group are hydrocarbon groups, and wherein the second hydrophobic surface group contains more carbon atoms than the first hydrophobic surface group.

11. The liquid chromatography system of claim 10, wherein the second hydrocarbon group contains 2 to 20 more carbon atoms than the first hydrocarbon group.

12. The liquid chromatography system of any of claims 10-11, wherein the first hydrocarbon group is a first alkyl group containing from 3 to 8 carbon atoms, and wherein the second hydrocarbon group is a second alkyl group containing from 10 to 24 carbon atoms.

13. The liquid chromatography system of claim 12, wherein the first alkyl group contains 4 carbon atoms, and wherein the second group contains 18 carbon atoms.

14. The liquid chromatography system of any one of claims 1-13, wherein the first ionizable group is present at a surface concentration that is less than or equal to a surface concentration of the permanently ionized group or second ionizable group.

15. The liquid chromatography system of any one of claims 1-14, wherein the first ionizable group is present at a surface concentration in a range from 0.03 to 0.3 micromoles per square meter.

16. The liquid chromatography system of any one of claims 1-15, wherein the first ionizable group and the permanently ionized group or second ionizable group are positively charged upon ionization.

17. The liquid chromatography system of claim 16, wherein the second chromatographic surface comprises the second ionizable surface group, wherein the first pKa value and the second pKa value are greater than 3, and wherein the second pKa value is from 1 unit to 7 units greater than the first pKa value.

18. The liquid chromatography system of any one of claims 1-17, wherein (a) the first ionizable group and (b) the permanently ionized group or second ionizable group comprises an amine group.

19. The liquid chromatography system of any one of claims 18, wherein the first ionizable group is selected from a primary amine group, a secondary amine group, and a tertiary amine group, and the permanently ionized group or second ionizable group is selected from a secondary amine group, a tertiary amine group, and a quaternary ammonium group.

20. The liquid chromatography system of any one of claims 1-17, wherein the first ionizable group is selected from a 4-pyridylethyl, 2-imidazolinylpropyl, 3-propylaniline, or an imidazole group.

21. The liquid chromatography system of any one of claims 1-18, wherein the second ionizable group is selected from the group consisting of a diethylaminopropyl, ethylaminopropyl, dimethylaminopropyl, methylaminopropyl, aminopropyl, diethylaminomethyl, 3- [ bis (2-hydroxyethyl) amino ] propyl, N-butyl-aza-silacyclopentane, N-methyl-aza-silacyclopentane, or a bis-3-methylaminopropylsilyl group.

22. The liquid chromatography system of any one of claims 1 to 21, wherein the molar ratio of the first hydrophobic surface group to the first ionizable group is in a range from 5:1 to 200: 1.

23. The liquid chromatography system of any one of claims 1-15, wherein the first ionizable group and the second ionizable group are negatively charged upon ionization.

24. The liquid chromatography system of claim 23, wherein the first ionizable group is a carboxylic acid group and the second ionizable group is selected from the group consisting of a sulfonic acid group and a carboxylic acid group.

25. The liquid chromatography system of any one of claims 23-24, wherein the second pKa value is 1 to 4 units less than the first pKa value.

26. The liquid chromatography system of any one of claims 1-25, wherein the first chromatographic material is in the form of first particles having a core of first material, and wherein the second chromatographic material is in the form of second particles having a core of second material.

27. The liquid chromatography system of claim 26, wherein the first material and the second material are organic materials, inorganic materials, or hybrid organic-inorganic materials.

28. The liquid chromatography system of claim 26, wherein the first material and the second material are selected from the group consisting of silica-based materials, alumina-based materials, titania-based materials, zirconia-based materials, and carbon-based materials.

29. The liquid chromatography system of claim 26, wherein the first and second materials are silica-based materials formed by hydrolytic condensation of one or more organosilane compounds.

30. The liquid chromatography system of claim 29, wherein the organosilane compound comprises one or more alkoxysilane compounds.

31. The liquid chromatography system of claim 29, wherein the organosilane compound is prepared from a tetraalkoxysilane and an alkylalkoxysilane.

32. The liquid chromatography system of claim 26, wherein the first material and the second material comprise organic polymers.

33. The liquid chromatography system of any of claims 1-32, in which the sample injector comprises a sample loop or a flow-through needle.

34. A method of performing liquid chromatography analysis on a liquid sample comprising a plurality of components using the liquid chromatography system of any of claims 1-33, the method comprising:

introducing the liquid sample into the system via the injector;

flowing the first mobile phase through the first flow path using the one or more mobile phase delivery sources such that the liquid sample is directed through the trapping column and such that the trapping column traps at least a portion of the components of the liquid sample as trapped components;

flowing the second mobile phase through the second flow path using the one or more mobile phase delivery sources, wherein flowing through the second flow path comprises flowing the second mobile phase (a) through the trapping column such that at least some of the trapped components elute from the trapping column as eluted components, and (b) through the analysis column such that at least some of the eluted components are separated as separated components; and is

Passing the separated eluate component to the detector.

35. The method of claim 34, wherein the component comprises a glycan.

36. The method of claim 34, wherein the component comprises labeled glycans.

37. The method of claim 36, wherein the labeled glycan is labeled with a labeling reagent selected from the group consisting of an MS-active rapid fluorescent labeling compound, a procainamide reagent, or a procaine reagent.

38. The method of any one of claims 36 to 37, wherein the labeled glycans are labeled with a glycan labeling reagent that provides an amphiphilic strongly basic moiety with a pKa value greater than 6.

39. The method of any one of claims 34 to 38, wherein the first mobile phase comprises (i) water or (ii) an aqueous solution of a first organic acid and/or a first organic acid salt.

40. The method of claim 39, wherein the second mobile phase comprises a solution of a second organic acid, which may be the same or different from the first organic acid, and/or a second organic acid salt, which may be the same or different from the first organic acid salt.

41. The method of any one of claims 34 to 38, wherein the first mobile phase comprises a solution of an organic acid and an organic acid salt in a solvent comprising water and an organic solvent, and wherein the second mobile phase comprises an elution process during which the concentration of the organic acid increases and the concentration of the organic acid salt increases.

Technical Field

The present disclosure relates to the field of liquid chromatography.

Background

Mixed mode chromatography refers to a chromatographic method that utilizes more than one form of interaction between a stationary phase and an analyte to effect separation of the analyte. Mixed mode chromatography is a promising technique for obtaining new separation selectivities. However, mixed mode chromatography can be difficult to implement, as it inherently relies on more than one retention mechanism.

WO 2017/189357 to Lauber et al, entitled "Charged Surface Reversed Phase Chromatographic Materials Method for Analysis of polysaccharides with Modified Amphipathic residues, Strongly Basic Moites", which is hereby incorporated by reference in its entirety, describes the use of so-called Charged Surface Reversed Phase Chromatographic Materials for mixed mode pattern separation of Glycans Modified with Amphipathic Strongly Basic residues, wherein separation is achieved by exploiting the differences in hydrophobicity and charge properties that exist between Glycans. Charged surface-reversed phase adsorbents suitable for these separations have been described in detail. Briefly, the charged surface reversed phase material may be formed from a High Purity Chromatographic Material (HPCM) having a chromatographic surface comprised of hydrophobic surface groups and one or more ionizable modifiers. In certain aspects, such charged surface inverse materials have been described: hydrophobic surface groups in HPCM: ionizable modificationRatio of the modifying agent is in the range of from about 5:1 to about 22:1, and concentration of the ionizable modifying agent is less than about 0.5 micromole/m²And has a C4 to C30 hydrophobic surface group. The use of such materials, where the chromatographic surface results from Diethylaminopropyl (DEAP) ionizable modifiers, C18 hydrophobic groups, and end capping on the bridged ethylene hybrid particles, has been demonstrated to be an exemplary embodiment for the isolation of glycans labeled with amphiphilic strongly basic moieties, such as those labeled with rapidfluor-MS reagents available from Waters Corporation (Milford, MA, USA). This so-called diethylaminopropyl charged surface hybrid stationary phase material (DEAP HPCM) allows for highly efficient separation of Rapi Fluor-MS reagent-labeled glycans, since they have been modified with an ionizable modifier of relatively high pKa (about 10), which results in a unique, distinct anion retention.

Nanoscale liquid chromatography (nanoLC) enables analysis of the smallest sample volume with relatively high mass spectral sensitivity relative to higher flow rate chromatographic separations, such as those achieved with narrow bore (e.g., 2.1mm ID) columns. However, in the case of using nanoLC, it is often not reasonable to use only a single column to analyze a large volume sample. Instead, it is preferred to perform on-line capture of analytes at micro-scale flow rates and then elute and separate those analytes on an analytical column, with significantly lower nano-scale flow rates being employed in the analytical column. Typically, samples of tens of microliters must be analyzed, and in these samples, the analyte may be present in concentrations that differ by more than several orders of magnitude. Trapping on a trapping column amenable to high flow rates allows for higher flux efficiencies and desalting in the step of transferring unwanted solutes out of the downstream mass spectrometer without adding additional connections and tubing after the analytical column. The trap column can also be used as a guard column to maintain the performance of the analytical column. In addition to its role as a guard column, an effective trapping column can also be packed with larger diameter particles (typically about 5 μm) to enable higher speed loading and retention of large, potentially dilutable sample volumes.

Disclosure of Invention

In various aspects, the present disclosure relates to methods of materials (e.g., kits, column assemblies, liquid chromatography systems, etc.) for performing liquid chromatography that employ a first column (e.g., a trapping column) and a second column (e.g., an analytical column). The first column includes a first chromatographic material having a first chromatographic surface comprising a first hydrophobic surface group and a first ionizable surface group having a first pKa value. The second column includes a second chromatographic material having a second chromatographic surface comprising a second hydrophobic surface group and either (a) a permanently ionized surface group or (b) a second ionizable surface group having a second pKa value. The hydrophobicity of the first hydrophobic surface group is less than the hydrophobicity of the second hydrophobic surface group. Further, where the second chromatographic surface comprises a second ionizable surface group, the first pKa value may differ from the second pKa value by from 1 unit to 12 units, more typically from 2 units to 6 units.

In various embodiments that may be used in combination with any of the preceding aspects, the first chromatographic material may be in the form of first particles and the second chromatographic material may be in the form of second particles. In these embodiments, the first diameter of the first particle may be greater than or equal to the second diameter of the second particle. For example, the ratio of the first particle diameter to the second particle diameter may range from 1 to 10 (typically from 1.5 to 3), among other values. In these embodiments, the first diameter may range from 2 microns to 10 microns (typically from 2.5 microns to 5 microns), and the second diameter may range from 1 micron to 5 microns (typically from 1.5 microns to 3 microns), among other values.

In various embodiments that may be used in combination with any of the above aspects and embodiments, the first chromatographic material may be in the form of first particles having a core of the first material, and the second chromatographic material may be in the form of second particles having a core of the second material.

The first material and the second material may be organic materials, inorganic materials, or organic-inorganic hybrid materials. The first and second materials may be selected from, for example, silica-based materials, alumina-based materials, titania-based materials, zirconia-based materials, carbon-based materials, and polymeric materials.

Where the first and second materials are silica-based materials, such materials are formed by hydrolytic condensation of one or more organosilane compounds. Examples of the organosilane compound include alkoxysilane compounds such as tetraalkoxysilanes (e.g., Tetramethoxysilane (TMOS), Tetraethoxysilane (TEOS), etc.), alkylalkoxysilanes such as alkyltrialkoxysilanes (e.g., methyltrimethoxysilane, Methyltriethoxysilane (MTOS), ethyltriethoxysilane, etc.) and bis (trialkoxysilyl) alkanes (e.g., bis (trimethoxysilyl) methane, bis (trimethoxysilyl) ethane, bis (triethoxysilyl) methane, bis (triethoxysilyl) ethane (BTEE), etc.), and combinations of the foregoing. In particular embodiments, the silica-based material may be formed by hydrolytically condensing a tetraalkoxysilane (e.g., TMOS or TEOS) and an alkylalkoxysilane (e.g., MTOS or BTEE). Where the first and second materials are polymeric materials, the first and second materials may include copolymers including hydrophilic monomers (e.g., N-vinyl pyrrolidone, N-vinyl caprolactam, etc.) and hydrophobic monomers (e.g., divinylbenzene, styrene, etc.).

In various embodiments that may be used in combination with any of the above aspects and embodiments, the first column may have an inner diameter greater than or equal to an inner diameter of the second column. For example, the inner diameter of the first column may be 1 to 5 times (typically 1.5 to 3 times, more typically about 2 times) the inner diameter of the second column, among other values. Further, the length of the first column may be shorter than the length of the second column. Further, the volume of the first column may be in the range of from 0.05 to 0.5 (typically 0.1 to 0.3) times the volume of the second column, among other values.

In various embodiments that may be used in combination with any of the above aspects and embodiments, the first hydrophobic surface group is a first hydrocarbon group consisting of carbon atoms and hydrogen atoms, and the second hydrophobic surface group is a second hydrocarbon group consisting of carbon atoms and hydrogen atoms. Examples of the first hydrocarbon group and the second hydrocarbon group include (a) an alkyl group, (b) an alkenyl group, (c) an alkynyl group, (d) an aromatic group, or a group formed from any combination of groups (a), (b), (c), and (d). Generally, the second hydrocarbon group contains more carbon atoms than the first hydrocarbon surface group. For example, the second hydrocarbon group can contain from 2 to 22 (more typically 4 to 14) more carbon atoms than the first hydrocarbon group. In certain embodiments, the second hydrocarbon group contains from 10 carbon atoms to 24 carbon atoms (in particular embodiments, 16 carbon atoms to 20 carbon atoms) and the first hydrocarbon group contains from 3 carbon atoms to 8 carbon atoms (in particular embodiments, 4 carbon atoms).

In various embodiments that may be used in combination with any of the above aspects and embodiments, the first ionizable group is present at a surface concentration that is less than or equal to the surface concentration of the permanently ionized group or the second ionizable group (e.g., the first ionizable group may be present at a surface concentration that is equal to 1% to 100%, more typically 15% to 50%, even more typically 20% to 30% of the surface concentration of the second ionizable group). The first ionizable group can be present at a surface concentration in a range from 0.03 to 0.3 micromoles per square meter (typically from 0.05 to 0.1 micromoles per square meter, more typically 0.06 to 0.08 micromoles per square meter, among other values).

In various embodiments that may be used in combination with any of the above aspects and embodiments, the first ionizable group and the permanently ionized group or the second ionizable group are positively charged upon ionization, in which case the ionizable group or the permanently ionized group may comprise, for example, a positively charged nitrogen atom. In these embodiments, the first ionizable group and the permanently ionized group or the second ionizable group can comprise an amine group (including, for example, alkylamine groups, arylamine groups, imidazole groups, guanidino groups, amidino groups, quinolinyl groups, imine groups, and indole groups, and the like). In these embodiments, the first ionizable group can include an amine group selected from a primary amine group, a secondary amine group, and a tertiary amine group, and the permanently ionizable group or the second ionizable group can include an amine group selected from a primary amine group, a secondary amine group, a tertiary amine group, and a quaternary ammonium group. In embodiments in which the second chromatographic surface comprises a second ionizable surface group, the first pKa value and the second pKa value may be greater than 3, and the second pKa value may be from 1 unit to 11 units (and typically from 2 units to 10 units) greater than the first pKa value. In certain embodiments, the first ionizable group can be a 4-pyridylethyl (4-PE), 2-pyridylethyl, 2-imidazolinylpropyl, 3-propylaniline, or an imidazole group. In certain embodiments, the second ionizable group is a Diethylaminopropyl (DEAP), ethylaminopropyl, dimethylaminopropyl, methylaminopropyl, aminopropyl, diethylaminomethyl, 3- [ bis (2-hydroxyethyl) amino ] propyl, N-butyl-aza-silacyclopentane, N-methyl-aza-silacyclopentane, or bis-3-methylaminopropylsilyl group.

Alternatively, in various embodiments that may be used in combination with any of the above aspects and embodiments, the first ionizable group and the second ionizable group are negatively charged upon ionization. For example, the first ionizable group can be a carboxylic acid group, and the second ionizable group can be selected from a sulfonic acid group and a carboxylic acid group, among other possible groups. In embodiments, the second pKa value may be 1 to 4 units less than the first pKa value.

In various embodiments that may be used in combination with any of the above aspects and embodiments, the molar ratio of the first hydrophobic surface group to the first ionizable group may be in a range from 5:1 to 200: 1.

In various aspects, the present disclosure relates to a kit comprising a first column (e.g., a trapping column) according to any of the above aspects and embodiments and a second column (e.g., an analytical column) according to any of the above aspects and embodiments.

In some embodiments, the kit may further comprise a glycan-labeling reagent. In certain of these embodiments, the glycan-labeling reagent may be selected from the group consisting of an MS-active rapid fluorescent labeling compound, a procainamide reagent, and a procaine reagent. In certain of these embodiments, glycans can be labeled using a glycan labeling reagent that provides an amphiphilic basic moiety that: (a) has a pKa value greater than 7, greater than 8, or even greater than 9, and/or (b) has a Log P value between 0 and 5, typically a Log P value between 1 and 5, more typically a Log P value between 1 and 3. Specific examples of glycan-labeling reagents used in the present disclosure include RapidFluor-MS reagent, wherein the glycan-labeling reagent is

Available from Waters Corporation (Milford, MA, USA), or InstantPC reagent, available from Prozyme Inc. (Hayward, Calif., USA). Additional glycan-labeling reagents are described in WO 2013/049622 to broussiche et al, which is hereby incorporated by reference in its entirety.

In various embodiments that may be used in conjunction with any of the above aspects and embodiments, the kit may include one or more mobile phases for use with the first column and/or the second column. The one or more mobile phases may comprise an aqueous solution of an organic acid and/or an organic acid salt. Examples of the organic acid include formic acid, difluoroacetic acid, trifluoroacetic acid, acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, maleic acid, and the like, and include organic hydroxy acids such as glycolic acid, lactic acid, tartaric acid, malic acid, citric acid, gluconic acid, and the like. Examples of the organic acid salt include salts having an organic acid anion (for example, selected from formate, difluoroacetate, trifluoroacetate, acetate, propionate, butyrate, carbonate, bicarbonate, oxalate, malonate, succinate, maleate, glutarate, glycolate, lactate, malate, citrate, gluconate, and the like), and salts having a cation selected from group I metal cations, group II metal cations, ammonium cations, and amine cations). In particular embodiments, the one or more mobile phases comprise an aqueous solution of formic acid and ammonium formate.

In various aspects, the present disclosure relates to a column assembly comprising a first column (e.g., a trapping column) according to any of the above aspects and embodiments and a second column (e.g., an analytical column) according to any of the above aspects and embodiments. Additionally, the column assembly may further include a multi-way valve having a first port, a second port, and a third port, wherein the first column is connected to the multi-way valve via the first port, wherein the second column is connected to the multi-way valve via the second port. In the first multi-way valve position, the first port is in fluid communication with the second port (i.e., the first column is in fluid communication with the second column), but not the third port. In the second multi-way valve position, the first port is in fluid communication with the third port, but not the second port (i.e., the first column is not in fluid communication with the second column). In certain embodiments, the waste line is connected to the multi-way valve via a third port. It is also known in the art to provide a vent column in which an on-off valve is placed outside of the sample stream and the on-off valve defines the flow path.

In various aspects, the present disclosure relates to a liquid chromatography system comprising: (a) a first column (e.g., a trapping column) according to any of the above aspects and embodiments, and (b) a second column (e.g., an analytical column) according to any of the above aspects and embodiments. In addition, the liquid chromatography system may further include: (c) a sample injector configured to introduce a sample liquid into the system, (d) a detector configured to detect a component (e.g., an analyte) of the sample liquid, (e) a first flow path including a sample loop or a flow needle and a first column but not a second column, (f) a second flow path including a first column and a second column, (g) a first pump configured to pump a first mobile phase along the first flow path, and (h) a second pump, which may be the same or different from the first pump, configured to pump a second mobile phase along the second flow path. In some embodiments, the sample injector may comprise a sample loop. In some embodiments, the sample injector may include a flow-through needle.

In various aspects, the present disclosure relates to methods of performing liquid chromatography analysis on a liquid sample comprising a plurality of components using a liquid chromatography system, such as those described in the preceding paragraphs. In some embodiments, the method comprises (a) introducing a liquid sample into the system via a sample injector; (b) flowing the first mobile phase through a first flow path using a first pump such that the liquid sample is directed through a first column and such that the first column traps at least a portion of a component of the liquid sample as a trapped component; (c) flowing the second mobile phase through a second flow path using a second pump, wherein flowing through the second flow path comprises flowing the second mobile phase (i) through the first column such that at least some of the captured component elutes from the first column as an eluted component, and (ii) through the second column such that at least some of the eluted component is separated as a separated component; and flowing the separated components to a detector.

In various aspects, the present disclosure relates to methods for performing liquid chromatography analysis on a liquid sample comprising a plurality of components (e.g., analytes), the methods comprising (a) loading the liquid sample into a sample injector (e.g., a sample injector comprising a sample loop or a flow-through needle) of a liquid chromatography system; (b) flowing a first mobile phase through a first flow path in a liquid chromatography system, wherein flowing through the first flow path comprises flowing the first mobile phase through a sample injector (thereby picking up the sample) and a first column (e.g., a trapping column) according to any of the above aspects and embodiments, wherein the first column traps at least some of the components of the liquid sample as trapped components; (c) flowing the second mobile phase through a second flow path in the liquid chromatography system, wherein flowing through the second flow path comprises flowing the second mobile phase through a first column to elute at least some of the captured components as eluted components from the first column, followed by flowing the second mobile phase and the eluted components through a second column (e.g., an analytical column) according to any of the above aspects and embodiments, wherein the second column separates at least some of the eluted components as separated components. In various embodiments, the method further comprises flowing the separated component to a detector configured to detect a characteristic of the separated component.

In various embodiments that may be used in conjunction with any of the above aspects and embodiments, the plurality of components in the sample fluid include glycans (which may vary in charge, including neutral glycans and positively charged glycans).

In various embodiments that may be used in combination with any of the above aspects and embodiments, the plurality of components in the sample liquid comprise labeled glycans. In some of these methods, the glycans can be labeled using a glycan labeling reagent, which can be selected from a Mass Spectrometry (MS) active fast fluorescent labeling compound, a procainamide reagent, and a procaine reagent. In some of these methods, glycans can be labeled using a glycan labeling reagent that provides an amphiphilic basic moiety that: (a) has a pKa value greater than 7, greater than 8, or even greater than 9, and/or (b) has a Log P value between 0 and 5, typically a Log P value between 1 and 5, more typically a Log P value between 1 and 3. Specific examples of glycan-labeling reagents used in the present disclosure include rapid fluor-MS reagent available from Waters Corporation (Milford, MA, USA) or InstantPC reagent available from ProZyme Inc (Hayward, CA USA).

In various embodiments that may be used in conjunction with any of the above aspects and embodiments, the first mobile phase may comprise water or an aqueous solution of an organic acid and/or organic acid salt, examples of which are provided above.

In various embodiments that may be used in conjunction with any of the above aspects and embodiments, the second mobile phase may include a solution of an organic acid and/or an organic acid salt, examples of which are provided above. In some of these embodiments, the second mobile phase comprises a solution of an organic acid and/or organic acid salt in a solvent comprising water and an organic solvent (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, acetone, ethyl acetate, methyl ethyl ketone, tetrahydrofuran, and blends thereof). In some of these embodiments, the second mobile phase comprises an organic acid and an organic acid salt. In some of these embodiments, an elution process of the second mobile phase is provided during which the concentration of the organic acid increases and the concentration of the organic acid salt increases.

Drawings

Fig. 1A is a schematic diagram of a liquid chromatography system according to the present disclosure.

Fig. 1B-1C illustrate two flow paths of a trap-elute chromatography system including a trap column and an analytical column according to the present disclosure. Fig. 1B is a schematic view of the fluid path for loading the trapping column. Figure 1C is a schematic of the fluid path used to elute from the capture column and create a gradient across the analytical column.

Figure 2A is a schematic of a trap-elute chromatography system including a trap column and an analytical column according to one embodiment of the present disclosure.

Figure 2B is a schematic diagram of a trap-elute chromatography system including a trap column and an analytical column according to another embodiment of the present disclosure.

FIG. 3 shows the stationary phase (DEAP HPCM)1.7 μm) of the mixed mode retention. The approximate net charge of each species in the presence of mobile phase conditions was recorded.

Fig. 4 shows the mixed-mode retention of four phases with different surface charges and alkyl chain lengths. The retention times of neutral, mono-and di-sialylated RapiFluor-MS reagent labelled glycans are shown (DEAP ═ diethylaminopropyl, tC18 ═ trifunctional C18, 4-PE ═ 4-pyridylethyl, tC8 ═ trifunctional C8).

Fig. 5A to 5D show a trap-elution chromatogram combining the trap using phase 2 and the analysis chromatogram using phase 1. Fluorescence chromatograms of RapiFluor-MS reagent-labeled human IgG glycans obtained with different concentrations of ammonium formate under initial mobile phase conditions are shown.

Figure 6 illustrates the mixed mode retention of the four phases that differ in alkyl chain length, ionizable modifier, and coverage of ionizable modifier. The retention times of neutral, mono-and di-sialylated RapiFluor-MS reagent labelled glycans are shown (DEAP ═ diethylaminopropyl, tC18 ═ trifunctional C18, 4-PE ═ 4-pyridylethyl).

Fig. 7A to 7C show capture-elution chromatography combined with the analysis chromatography using the capture of three different phases and phase 1. Fluorescence chromatograms of RapiFluor-MS reagent-labeled human IgG glycans obtained with different concentrations of ammonium formate under initial mobile phase conditions are shown.

Fig. 8A to 8C show the recovery from the capture-elution chromatography as a function of the ammonium formate concentration used for the initial mobile phase/capture conditions. Fig. 8A to 8C show the recovery of three different classes of rapiFluor-MS reagent labelled neutral N-glycans provided as observed fluorescence peak areas. In fig. 8A to 8C, the maximum value of the fluorescence peak area is marked with a horizontal dotted line as estimated from the fluorescence peak area observed by direct injection (no trapping) analysis as shown in fig. 8D.

Fig. 9A to 9C show trap-elution chromatograms optimized for initial and final ammonium formate mobile phase concentrations. Separation of rapiFluor-MS reagent labeled glycans from pooled human IgG and bovine fetuin is shown. FIG. 9A is a fluorescence chromatogram obtained by direct injection (without capture) of initial buffer concentration and final buffer concentration with 0mM ammonium formate/22 mM formic acid and 22mM ammonium formate/22 mM formic acid, respectively. FIG. 9B is a fluorescence chromatogram obtained by direct injection (without capture) of initial buffer concentration and final buffer concentration with 14mM ammonium formate/14 mM formic acid and 22mM ammonium formate/22 mM formic acid, respectively. Fig. 9C is a fluorescence chromatogram obtained from capture-elution chromatography using initial buffer concentrations and final buffer concentrations of 14mM ammonium formate/14 mM formic acid and 22mM ammonium formate/22 mM formic acid, respectively.

Detailed Description

Systems and methods for performing trap-elute liquid chromatography on a liquid sample are schematically illustrated in fig. 1A-1C. Turning to fig. 1A, a liquid chromatography system 100 is shown, comprising: (a) a trapping column 106 comprising a stationary phase material (described in further detail below), also referred to herein as "trapping phase material", "trapping phase" or "first chromatographic material", (b) an analytical column 107 comprising a stationary phase material (described in further detail below), also referred to herein as "analytical phase material", "analytical phase" or "second chromatographic material", (c) a sample injector comprising a sample loop 112 and a six-port sample valve 102 for receiving a liquid sample into the sample loop 112 (e.g., via a sampling accessory such as a sample needle 108), (d) a first flow path F1 comprising the trapping column 106 but not the analytical column 107, (e) a second flow path F2 comprising the trapping column 106 and the analytical column 107, (F) a first mobile phase delivery source (e.g., a first pump 104a is shown) configured to pump a first mobile phase along a first flow path, and (g) a second mobile phase delivery source (e.g., a second pump 104b is shown) configured to pump a second mobile phase along a second flow path. A multi-port trap-and-elute valve (three-port valve 103 is shown) is used to establish the first flow path and the second flow path. (in other embodiments, a vented column arrangement may be employed.) although not shown, the liquid chromatography system 100 may also include one or more detectors configured to analyze separated sample components emerging from the analytical column, including detectors for performing Mass Spectrometry (MS). These include electrospray ionization mass spectrometry (ESI-MS), matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), and time-of-flight mass spectrometry (TOFMS), nuclear magnetic resonance, infrared analysis, ultraviolet analysis, or combinations thereof, and the like.

In a first step, and referring in particular also to fig. 1B, the method comprises introducing a liquid sample via a sample injector, flowing the first mobile phase through a first flow path F1 (indicated with a dashed arrow) using a first pump 104a such that the liquid sample is directed through the trapping column 106 and such that the trapping column 106 traps at least a portion of the components of the liquid sample as trapped components. During this first step, multiport trap-elute valve 103 can be set to a first position such that the first mobile phase emerging from trap column 106 is directed to waste line 111. After this first step, and with particular reference also to fig. 1C, the second mobile phase is flowed through a second flow path F2 (indicated with dashed arrows) using a second pump 104b, wherein flowing through the second flow path F2 comprises flowing the second mobile phase (a) through the trapping column 106 such that at least some of the trapped component elutes from the trapping column 106 as an eluted component, and (b) through the analysis column 107 such that at least some of the eluted component is separated as a separated component. The separated eluted components may then be subjected to further analysis as required. During this first step, the multi-port trap-and-elute valve 103 can be set to a second position such that the second mobile phase emerging from the trap column 106 is directed to the analytical column 107.

Although the first pump 104a and the second pump 104b are described in conjunction with fig. 1A-1C, in other embodiments, a single pump may be used to pump the mobile phase.

Additional details will now be described in connection with the liquid chromatography system 100 shown in FIG. 2A. The illustrated system 100 includes a six-port sample valve 102 connected to a mobile phase delivery source 104. In the embodiment shown, mobile phase delivery source 104 is a miniature binary solvent manager (μ BSM) system available from Waters Corporation that is capable of delivering up to four solvents in various combinations of two solvents. The mobile phase delivery source 104 delivers the mobile phase liquid through the selected internal fluid pathway of the six-port injection valve 102. A trap column 106 and an analytical column 107 separated by a trap-elute valve 103 are also connected to the six-port sample injection valve 102. As previously indicated, the capture column 106 and analytical column 107 act in concert with the mobile phase to effect capture and separation of the various sample components introduced into the system.

A sampling accessory (in this embodiment, a sample needle 108) is also connected to the six-port sample injection valve 102. The sample needle 108 may be inserted into a sample container (not shown) for obtaining an aliquot of a sample solution contained within the sample container. The sample needle 108 is the entry point for the sample solution into the sample intake or aspiration path to the sample loop 112 of the injection valve 102. The syringe 110 is connected to the six-port injection valve 102. The injector 110 cooperatively interacts with the injection valve 102 to control the uptake or aspiration of a sample through the sample needle 108, typically by causing a prescribed volume displacement of the sample fluid within the sample needle 108. The sample loop 112 in fluid communication with the six-port sample valve 102 has an upstream loop end 112u and a downstream loop end 112 d. When the six-port sample valve 102 is in the sample intake configuration, the syringe 110 is in fluid communication with the upstream loop end 112u of the outer sample loop 112, while the sample needle 108 is in fluid communication with the downstream loop end 112 d. In this exemplary configuration, a sample may be drawn from a sample container into the sample loop 112 by the action of the syringe 110.

On the other hand, when the six-port sample valve 102 is switched to the trapping/eluting configuration, the fluid communication pattern inside the valve 112 changes, thereby redirecting fluid flow. With the sample valve 102 in the trapping/elution configuration, the mobile phase delivery source 104 is in fluid communication with the upstream loop end 112u of the sample loop 112, while the trapping column 106 is in fluid communication with the downstream end 112d of the sample loop 112. In addition, when the sample valve 102 is in the trap/elute configuration, the downstream end 112d of the sample loop 112 is in fluid communication with the upstream end 106u of the trap column 106 via port 6 and line 113. Thus, when sample valve 102 is in the trapping/elution configuration, the mobile phase flow from mobile phase delivery source 104 pushes any sample solution contained within sample loop 112 through line 113 and into trapping column 106.

Downstream of the trap column 106 is a trap-elute valve 103. Although the illustrated valve 103 is a six-port valve, in the illustrated embodiment, the six-port valve is actually a three-port valve in that three of the ports are plugged with the pin plugs 109. Thus, a simple three-way valve with three ports and two flow modes (e.g., at a T-valve as shown in fig. 1A) may also be used as the trap-elute valve 103. The downstream end of the trap column is connected to the port of the trap-elute valve 103 at port 1 as shown. Port 2 of trap-and-elute valve 103 is attached to waste line 111 and port 6 is connected to trap column 107. In the trapping position of the trap-and-elute valve 103, the downstream end 106d of the trap column 106 is in fluid communication with the waste line 111, thereby providing a first flow path for the mobile phase.

On the other hand, with trap-elution valve 103 in the elution position, downstream end portion 106d of trap column 106 is in fluid communication with upstream end portion 107u of analytical column 107, thereby providing a second flow path for the mobile phase. In this position, the mobile phase flow from the mobile phase delivery source 104 pushes the sample contained within the capture column 106 from the capture column 106 through the analytical column 107.

Fig. 2B illustrates an alternative embodiment of a liquid chromatography system 100 shown in accordance with the present disclosure. As can be seen by comparing fig. 2A and 2B, system 100 differs in that it includes an additional mobile phase delivery source 104a in fluid communication with port 5, trap-and-elute valve 103 is a six-port valve with no ports plugged, line 113 is in fluid communication with port 2 of trap-and-elute valve 103 (rather than upstream end 106u of trap-and-elute valve 106), waste line 111 is in fluid communication with port 3 of trap-and-elute valve 103, and the trap-and-elute valve 103 is in fluid connection with ports 1 and 4.

As shown in fig. 2A, when the six-port sample valve 102 is in the sample intake configuration, the syringe 110 is in fluid communication with the upstream loop end 112u of the outer sample loop 112, while the sample needle 108 is in fluid communication with the downstream loop end 112d of the outer sample loop 112, thereby allowing sample to be drawn from the sample container into the sample loop 112 by action of the syringe 110. Also similar to fig. 2A, when the six-port sample valve 102 is switched to the trapping configuration, the mobile phase flow from the mobile phase delivery source 104 pushes any sample solution contained within the sample loop 112 into line 113.

Six-port trap-and-elute valve 103 can be placed in a variety of configurations, including a trapping configuration and an eluting configuration. When the six-port trap-and-elute valve 103 is in the trapping configuration, the upstream end 106u of the trap column 106 is in fluid communication with line 113, allowing the sample solution to be received from the six-port sample valve 102 (when the sample valve 102 is in the trapping configuration) while the downstream end 106d of the trap column 106 is in fluid communication with the waste line 111. Thus, in the capture position of capture-elution valve 103, a first flow path for the mobile phase is created that extends from mobile phase delivery source 104, through sample loop 112, through line 113, through capture column 106, and into waste line 111.

On the other hand, when the six-port trap-and-elute valve 103 is in the elution configuration, the upstream end 106u of the trap column 106 is in fluid communication with the additional mobile phase delivery source 104a, while the downstream end 106d of the trap column 106 is in fluid communication with the upstream end 107u of the analytical column 107. Thus, in the elution position of trap-elution valve 103, a second flow path for mobile phase is created that extends from additional mobile phase delivery source 104a, through trap column 106, and then through analysis column 107.

Having described embodiments of capture-eluent phase chromatography system 100 and its operation, the use of such a system for mixed mode capture-elution chromatography will now be described. To the best of the inventors' knowledge, systems and techniques for trap-elute chromatography with mixed mode separation have not been developed. It will be appreciated that the development of a given trap-elute mixed mode chromatography system is not trivial, as multiple retention mechanisms are considered when seeking to match the characteristics of the trap phase with those of the selected mixed mode analysis phase. In the following disclosure, the best chromatographic materials and methods for use with mixed mode capture-elution chromatography are described.

Generally, the trapping column employed in the present disclosure is selected to have a lower retention when compared to the analytical column. This relationship between capture column retention and analytical column retention during gradient elution ensures that the analyte refocuses onto the analytical column, delivering a high peak volume separation to any downstream detector.

In developing suitable trap phase materials and analysis phase materials, the present inventors have analyzed the relative retention of the trap phase relative to the analysis phase to establish an optimized trap-elute chromatography system. Thus, screening experiments involving one-dimensional separations on various stationary phases can be used to assess the potential utility of a capture phase for use with a given analysis phase. That is, the suitability of a capture phase can be assessed by comparing its retention curve to that of the expected analysis phase. In the following specific embodiment, the analytical phase employed is DEAP HPCM material as described in WO 2017/189357 to Lauber et al.

As mentioned above, WO 2017/189357 to Lauber et al describes the use of the so-called diethylaminopropyl charged surface hybrid stationary phase material (DEAP HPCM) described therein for the separation of Glycans labeled with strongly basic amphiphilic moieties such as the Labeling Reagent Rapi Fluor-MS Reagent, available from Waters Corporation (Milford, MA, USA) and described, for example, in WO 2013/049622 and Matthew A. Lauber et al, "Rapid Preparation of recovered N polysaccharides for HILIC Analysis Using a laboratory Reagent and Sensitive Fluorescence and ESI-MS Detection," anal. chem.2015,87, 5401-. DEAP HPCM previously has been shown to be highly effective in isolating Rapi Fluor-MS labeled glycans because such glycans have been modified with an ionizable modifier of relatively high pKa (about 10), which results in a unique apparent anion retention.

The procedures associated with the generation of fig. 3 to 9C are described in detail in the following embodiments. Turning to fig. 3, a representation of the retention curve of the DEAP HPCM stationary phase is shown in the form of an extracted ion chromatogram of neutral (net charge ═ 1) (named FA2G2), monosialylated (net charge ═ 0) (named FA2G2S1), and disialylated (net charge ═ 1) (named FA2G2) RapiFluor-MS labeled N-glycan species. The retention times of the three indicated species provide an indication of the retention and selectivity of the stationary phase. As shown in the graph of fig. 4, it was found that DEAP HPCM (phase 1) has a relatively high average retention due to its long C18 hydrophobic linkage, but also exhibits high selectivity (i.e., difference in retention between different charged species) due to its high pKa ionizable modifier (i.e., diethylaminopropyl ionizable modifier) and relatively strong anion exchange characteristics.

The ideal trapping phase for DEAP HPCM will be one that has a very low degree of reduced retention for each type (charge state) of analyte. To do this, the inventors examined the retention curves for three potential trapping phases, each with a unique combination of alkyl linkages and ionizable modifiers, specifically, a combination of C4 alkyl linkages and 4-pyridylethyl (4-PE) ionizable groups (phase 2 in fig. 3), a combination of C8 alkyl linkages and 4-PE ionizable groups (phase 3 in fig. 3), and a combination of C4 alkyl linkages and no ionizable groups (phase 4 in fig. 3). It has been hypothesized that shorter alkyl linkages would be a viable route to find suitable capture. However, it can be seen from the phase 4 results that alkyl C4 linkages without any ionizable modifier produce a suboptimal retention profile, especially for species with a positive net charge in solution (FA2G 2). Therefore, the inventors also investigated to change the surface potential of the trapping phase. In order not to produce any mixed mode selectivity that would be greater than the DEAP HPCM analytical phase, an ionizable modifier with a lower pKa (4-pyridylethyl, 4-PE) was then investigated. Interestingly, the retention curve for phase (phase 3) combining C8 alkyl linkages with a 4-PE ionizable modifier proved to be very similar to that of DEAP HPCM, yet phase 3 still exhibited higher retention than expected for certain species with a positive net charge in solution, such as FA2G 2. In contrast, the phase paired with the weaker pKa 4-PE modifier based on C4 alkyl linkages (phase 2) gave a near ideal retention curve for each analyte class. These data demonstrate that for a given mixed mode analysis stationary phase, the optimum material for trapping can be generated by reducing the retention and mixed mode selectivity.

Based on the screening experiments described above, a phase 2 trap column was selected for use in the trap-elute configuration along with a DEAP HPCM (phase 1) analytical column (fig. 5A to 5D). Methods were explored for optimizing the isolation of RapiFluor-MS labeled human IgG N-glycans, starting with the use of previously discovered conditions, with an exemplary direct injection separation using a DEAP HPCM column. This particular process requires the use of an initial mobile phase composition of 100% water, which is also a definition of the trapped mobile phase composition of the trap-elute configuration. FIG. 5D shows the chromatogram obtained using this method for trapping onto phase 2. Notably, this chromatogram was found to lack a positively net charged glycan structure (RapiFluor-MS reagent labeled neutral N-glycans). The present inventors have previously found that a significant effect on the retention of these species is associated with charge repulsion. Thus, the ionic strength of the initial mobile phase composition is increased, followed by a significant increase in recovery of species with a positive net charge state (fig. 5A-5C). However, it was found that the initial mobile phase composition using ≧ 15mM ammonium formate/15 mM formic acid was insufficient to effectively retain the least hydrophobic glycan species (e.g., mannose 5). Furthermore, the use of higher buffer concentrations at the initial conditions of the gradient results in an undesirable decrease in anion exchange selectivity.

To further adjust the retentionCoverage of 4-PE from about 0.3. mu. mol/m²(phase 2) Change was 0.2. mu. mol/m²(phase 5) and 0.1. mu. mol/m²(phase 6). The retention of each phase as well as the retention of phase 1 is shown in fig. 6. Phase 5 and phase 6 each show a favorable increase in retention of the probe to the rapiFluor-MS reagent labeled neutral glycan species (FA2G 2). Furthermore, their performance in the trap-elute configuration was found to be quite promising, as shown in fig. 7. In particular, phase 6 appears to significantly improve the recovery of the least hydrophobic neutral N-glycans and is less dependent on the initial mobile phase composition. To observe this quantitatively, we analyzed the peak area of the weakest retained glycan species as a function of the composition of the trapping phase and the initial mobile phase. Figures 8A to 8C show these data together with the values labeled for the peak areas for the indicated glycan species as observed with the direct injection experiments. In fact, phase 6 proved to be the most suitable capture phase, considering that even with the method of using low buffer concentrations for the initial mobile phase composition, phase 6 showed a recovery rate in the spectrum that retained the weakest species.

The combination of the phase 6 trap column and the DEAP HPCM analytical column was further optimized. By adjusting the ionic strength gradient, it was found that this trap-elute method can produce resolution comparable to direct injection separation. Fig. 9A-9C show exemplary results of this trap-elute mixed mode chromatography along with a comparative example of separation as obtained with a direct injection LC configuration.

There is a continuing need in the art for methods of trap-elution chromatography that can facilitate mixed mode separations. The present disclosure addresses the challenge of creating a trapping phase having properties that best match those of a selected mixed mode analysis phase, i.e., with reduced retention, but similar mixed mode selectivity, for different classes of related analyte types. Selected ones of these analytes include acidic metabolites of the Kreb's cycle (including but not limited to citrate, isocitrate, ketoglutarate, succinate, fumarate, malate, and oxaloacetate), oligonucleotides, glyphosate and its related derivative analogs, as well as peptides and glycans.

It has also been found that by using a weaker (i.e., lower pKa base/higher pKa acid) ionizable modifier and a shorter hydrophobic alkyl group than the intended analytical phase, capture materials with suitably reduced retention and mixed mode selectivity can be prepared. In one embodiment, the ionizable modifier for the trapping phase is selected to have a pKa that differs from the pKa of the ionizable modifier for the analytical phase by at least 1 unit, more desirably by at least 2 units. In this same embodiment, hydrophobic surface groups are selected for the trapping phase that contain fewer carbon atoms than the analysis phase, typically at least 2 carbons less, more desirably at least 4 carbons less, and preferably at least 6 carbons less.

Furthermore, it has been found that by adjusting the coverage of its ionizable modifier, the material can be trapped more efficiently. In one embodiment, the coverage of ionizable modifier is less than 0.3 μmol/m²And still more desirably less than 0.2. mu. mol/m²And further less than or equal to 0.1. mu. mol/m²。

In addition, the present disclosure provides for the use of solutions having ionic strength as a means of increasing the capture recovery of analytes where charge repulsion is encountered. Thus, in yet another embodiment of the present disclosure, the indicated capture materials may be used with initial mobile phase total buffer concentrations greater than 0mM, more desirably greater than 1mM, but typically less than 40mM, so as not to unduly significantly limit the available mixed mode selectivity.

Additional aspects of the disclosure will now be described in the following enumerated aspects:

aspect a 1. A liquid chromatography system comprising: a trapping column comprising a first chromatographic material having a first chromatographic surface comprising a first hydrophobic surface group and a first ionizable surface group having a first pKa value; an analytical column comprising a second chromatographic material having a second chromatographic surface comprising a second hydrophobic surface group having a hydrophobicity greater than the hydrophobicity of the first hydrophobic surface group and (i) a permanently ionized surface group or (ii) a second ionizable surface group having a second pKa value that differs from the first pKa value by 1 unit to 12 units; a sample injector for introducing a liquid sample into the system; a detector capable of detecting a characteristic of the constituent; a first flow path including a sample injector and a trapping column, but not including an analytical column; a second flow path comprising a trapping column and an analysis column; and one or more mobile phase delivery sources configured to pump a first mobile phase along a first flow path and a second mobile phase along a second flow path.

Aspect a 2. The liquid chromatography system of aspect a1, wherein the one or more mobile phase delivery sources comprise a first pump configured to pump the first mobile phase along the first flow path and a second pump, which may be the same or different from the first pump, configured to pump the second mobile phase along the second flow path.

Aspect a 3. The liquid chromatography system of any of aspects a1 to a2, wherein the first chromatographic material is in the form of first particles, and wherein the second chromatographic material is in the form of second particles.

Aspect a 4. The liquid chromatography system of aspect a3, wherein the first diameter of the first particles is equal to or greater than the second diameter of the second particles.

Aspect a 5. The liquid chromatography system of aspect a4, wherein the ratio of the first particle diameter to the second particle diameter is in the range of from 1 to 10.

Aspect a 6. The liquid chromatography system of any of aspects a4 to a5, wherein the first diameter is in a range from 2 microns to 10 microns.

Aspect a 7. The liquid chromatography system of any of aspects a1 to a6, wherein the inner diameter of the trapping column is greater than or equal to the inner diameter of the analytical column, and wherein the length of the trapping column is shorter than the length of the analytical column.

Aspect A8. The liquid chromatography system of any of aspects a1 to a7, wherein the inner diameter of the trapping column is 1.5 to 5 times the inner diameter of the analytical column.

Aspect a 9. The liquid chromatography system of any of aspects a1 to A8, wherein the volume of the trapping column is in the range of from 0.05 to 0.5 times the volume of the analytical column.

Aspect a 10. The liquid chromatography system of any of aspects a1 to a9, wherein the first hydrophobic surface group and the second hydrophobic surface group are hydrocarbon groups, and wherein the second hydrophobic surface group contains more carbon atoms than the first hydrophobic surface group.

Aspect a 11. The liquid chromatography system of aspect a10, wherein the second hydrocarbon group contains 2 to 20 more carbon atoms than the first hydrocarbon group.

Aspect a 12. The liquid chromatography system of any of aspects a10 to a11, wherein the first hydrocarbon group is a first alkyl group containing from 3 to 8 carbon atoms, and wherein the second hydrocarbon group is a second alkyl group containing from 10 to 24 carbon atoms.

Aspect a 13. The liquid chromatography system of aspect a12, wherein the first alkyl group contains 4 carbon atoms, and wherein the second group contains 18 carbon atoms.

Aspect a 14. The liquid chromatography system of any of aspects a1 to a13, wherein the first ionizable group is present at a surface concentration that is less than or equal to the surface concentration of the permanently ionized group or the second ionizable group.

Aspect a 15. The liquid chromatography system of any of aspects a1 to a14, wherein the first ionizable group is present at a surface concentration in a range from 0.03 micromoles per square meter to 0.3 micromoles per square meter.

Aspect a 16. The liquid chromatography system of any of aspects a1 to a15, wherein the first ionizable group and the permanently ionized group or the second ionizable group are positively charged upon ionization.

Aspect a 17. The liquid chromatography system of aspect a16, wherein the second chromatographic surface comprises a second ionizable surface group, wherein the first pKa value and the second pKa value are greater than 3, and wherein the second pKa value is 1 unit to 7 units greater than the first pKa value.

Aspect a 18. The liquid chromatography system of any of aspects a1 to a17, wherein (a) the first ionizable group and (b) the permanently ionized group or the second ionizable group comprises an amine group.

Aspect a 19. The liquid chromatography system of any of aspects a18, wherein the first ionizable group is selected from the group consisting of a primary amine group, a secondary amine group, and a tertiary amine group, and the permanently ionizable group or the second ionizable group is selected from the group consisting of a secondary amine group, a tertiary amine group, and a quaternary ammonium group.

Aspect a 20. The liquid chromatography system of any of aspects a1 to a17, wherein the first ionizable group is selected from a 4-pyridylethyl, 2-imidazolinylpropyl, 3-propylaniline, or an imidazole group.

Aspect a 21. The liquid chromatography system of any of aspects a1 to a18, wherein the second ionizable group is selected from the group consisting of a diethylaminopropyl, ethylaminopropyl, dimethylaminopropyl, methylaminopropyl, aminopropyl, diethylaminomethyl, 3- [ bis (2-hydroxyethyl) amino ] propyl, N-butyl-aza-silacyclopentane, N-methyl-aza-silacyclopentane, or a bis-3-methylaminopropylsilyl group.

Aspect a 22. The liquid chromatography system of any of aspects a1 to a21, wherein the molar ratio of the first hydrophobic surface group to the first ionizable group is in a range from 5:1 to 200: 1.

Aspect a 23. The liquid chromatography system of any of aspects a1 to a15, wherein the first ionizable group and the second ionizable group are negatively charged upon ionization.

Aspect a 24. The liquid chromatography system of aspect a23, wherein the first ionizable group is a carboxylic acid group and the second ionizable group is selected from the group consisting of a sulfonic acid group and a carboxylic acid group.

Aspect a 25. The liquid chromatography system of any of aspects a23 to a24, wherein the second pKa value is 1 to 4 units less than the first pKa value.

Aspect a 26. The liquid chromatography system of any of aspects a1 to a25, wherein the first chromatographic material is in the form of first particles having a core of the first material, and wherein the second chromatographic material is in the form of second particles having a core of the second material.

Aspect a 27. The liquid chromatography system of aspect a26, wherein the first material and the second material are organic materials, inorganic materials, or organic-inorganic hybrid materials.

Aspect a 28. The liquid chromatography system of aspect a26, wherein the first material and the second material are selected from the group consisting of a silica-based material, an alumina-based material, a titania-based material, a zirconia-based material, and a carbon-based material.

Aspect a 29. The liquid chromatography system of aspect a26, wherein the first material and the second material are silica-based materials formed by hydrolytic condensation of one or more organosilane compounds.

Aspect a 30. The liquid chromatography system of aspect a29, wherein the organosilane compound comprises one or more alkoxysilane compounds.

Aspect a 31. The liquid chromatography system of aspect a29, wherein the organosilane compound is prepared from a tetraalkoxysilane and an alkylalkoxysilane.

Aspect a 32. The liquid chromatography system of aspect a26, wherein the first material and the second material comprise organic polymers.

Aspect a 33. The liquid chromatography system of any of aspects a1 to a32, wherein the sample injector comprises a sample loop or a flow-through needle.

Aspect a 34. A method of performing liquid chromatography analysis on a liquid sample comprising a plurality of components using the liquid chromatography system of any of aspects a1 through a33, the method comprising: introducing a liquid sample into the system via an injector; flowing a first mobile phase through a first flow path using one or more mobile phase delivery sources such that a liquid sample is directed through a trapping column and such that the trapping column traps at least a portion of a component of the liquid sample as a trapped component; flowing a second mobile phase through a second flow path using one or more mobile phase delivery sources the one or more mobile phase delivery sources, wherein flowing through the second flow path comprises flowing the second mobile phase (a) through a trapping column such that at least some of the trapped component elutes from the trapping column as an eluted component, and (b) through an analysis column such that at least some of the eluted component is separated as a separated component; and passing the separated eluate component to a detector.

Aspect a 35. The method of aspect a34, wherein the component comprises a glycan.

Aspect a 36. The method of aspect a34, wherein the component comprises a labeled glycan.

Aspect a 37. The method of aspect a36, wherein the labeled glycan is labeled with a labeling reagent selected from the group consisting of an MS-active rapid fluorescent labeling compound, a procainamide reagent, or a procaine reagent.

Aspect a 38. The method of any one of aspects a36 to a37, wherein the labeled glycan is labeled with a glycan labeling reagent that provides an amphiphilic strongly basic moiety having a pKa value greater than 6.

Aspect a 39. The method of any of aspects a34 to a38, wherein the first mobile phase comprises (i) water or (ii) an aqueous solution of the first organic acid and/or the first organic acid salt.

Aspect a 40. The method of aspect a39, wherein the second mobile phase includes a solution of a second organic acid, which may be the same as or different from the first organic acid, and/or a second organic acid salt, which may be the same as or different from the first organic acid salt.

Aspect a 41. The method of any one of aspects a34 to a38, wherein the first mobile phase comprises a solution of an organic acid and an organic acid salt in a solvent comprising water and an organic solvent, and wherein the second mobile phase comprises an elution process during which a concentration of the organic acid increases and a concentration of the organic acid salt increases.

Aspect B1. A chromatography column comprising chromatography particles comprising a core and a chromatography surface comprising hydrophobic surface groups having from 3 to 8 carbon atoms and ionizable surface groups having a pKa value in the range of from 3 to 8, wherein the ionizable groups are present at a surface concentration in the range of from 0.03 micromoles per square meter to 0.3 micromoles per square meter.

Aspect B2. The chromatography column of aspect B1, wherein the ionizable group is selected from the group consisting of a 4-pyridylethyl, 2-imidazolinylpropyl, 3-propylaniline, or an imidazole group.

Aspect B3. The chromatography column of any of aspects B1 to B2, wherein the molar ratio of the first hydrophobic surface group to the ionizable group is in the range of from 5:1 to 200: 1.

Aspect B4. The chromatography column of any of aspects B1 to B3, wherein the core is selected from an organic material, an inorganic material, or an organic-inorganic hybrid material.

Aspect B5. The chromatography column of any of aspects B1 to B3, wherein the core is selected from the group consisting of silica-based materials, alumina-based materials, titania-based materials, zirconia-based materials, and carbon-based materials.

Aspect B6. The chromatography column of any of aspects B1 to B3, wherein the core is a silica-based material formed by hydrolytic condensation of one or more organosilane compounds.

Aspect B7. The chromatography column of aspect B6, wherein the organosilane compound comprises one or more alkoxysilane compounds.

Aspect B8. The chromatography column of aspect B6, wherein the organosilane compound is prepared from a tetraalkoxysilane and an alkylalkoxysilane.

Aspect C1. A kit, comprising: (a) a trap column comprising a first chromatographic material having a first chromatographic surface comprising a first hydrophobic surface group and a first ionizable surface group having a first pKa value, and (b) an analytical column comprising a second chromatographic material having a second chromatographic surface comprising a second hydrophobic surface group that is more hydrophobic than the first hydrophobic surface group and either (i) a permanently ionized surface group or (ii) a second ionizable surface group having a second pKa value that differs from the first pKa value by 1 unit to 12 units.

Aspect C2. The kit of aspect C1, wherein the first chromatographic material is in the form of first particles, and wherein the second chromatographic material is in the form of second particles.

Aspect C3. The kit of aspect C2, wherein the first diameter of the first particle is equal to or greater than the second diameter of the second particle.

Aspect C4. The kit of aspect C3, wherein the ratio of the first particle diameter to the second particle diameter is in the range from 1 to 10.

Aspect C5. The kit of any one of aspects C3 to C4, wherein the first diameter is in a range from 2 microns to 10 microns.

Aspect C6. The kit of any one of aspects C1 to C5, wherein an inner diameter of the trapping column is greater than or equal to an inner diameter of the analytical column, and wherein a length of the trapping column is shorter than a length of the analytical column.

Aspect C7. The kit of any one of aspects C1 to C6, wherein the inner diameter of the trapping column is 1.5 to 5 times the inner diameter of the analytical column.

Aspect C8. The kit of any one of aspects C1 to C7, wherein the volume of the trapping column is in the range of from 0.05 to 0.5 times the volume of the analytical column.

Aspect C9. The kit of any one of aspects C1 to C8, wherein the first hydrophobic surface group and the second hydrophobic surface group are hydrocarbon groups, and wherein the second hydrophobic surface group contains more carbon atoms than the first hydrophobic surface group.

Aspect C10. The kit of aspect C9, wherein the second hydrocarbon group contains 2 to 20 more carbon atoms than the first hydrocarbon group.

Aspect C11. The kit of any of aspects C9 to C10, wherein the first hydrocarbon group is a first alkyl group containing from 3 to 8 carbon atoms, and wherein the second hydrocarbon group is a second alkyl group containing from 10 to 24 carbon atoms.

Aspect C12. The kit of aspect C11, wherein the first alkyl group contains 4 carbon atoms, and wherein the second group contains 18 carbon atoms.

Aspect C13. The kit of any one of aspects C1 to C12, wherein the first ionizable group is present at a surface concentration that is less than or equal to the surface concentration of the permanently ionized group or the second ionizable group.

Aspect C14. The kit of any one of aspects C1 to C13, wherein the first ionizable group is present at a surface concentration ranging from 0.03 micromoles per square meter to 0.3 micromoles per square meter.

Aspect C15. The kit of any one of aspects C1 to C14, wherein the first ionizable group and the permanently ionized group or the second ionizable group are positively charged upon ionization.

Aspect C16. The kit of aspect C15, wherein the second chromatographic surface comprises a second ionizable surface group, wherein the first pKa value and the second pKa value are greater than 3, and wherein the second pKa value is 1 unit to 7 units greater than the first pKa value.

Aspect C17. The kit of any one of aspects C1 to C16, wherein (a) the first ionizable group and (b) the permanently ionized group or the second ionizable group comprises an amine group.

Aspect C18. The kit of any of aspects C17, wherein the first ionizable group is selected from the group consisting of a primary amine group, a secondary amine group, and a tertiary amine group, and the permanently ionizable group or the second ionizable group is selected from the group consisting of a primary amine group, a secondary amine group, a tertiary amine group, and a quaternary ammonium group.

Aspect C19. The kit of any one of aspects C1 to C16, wherein the first ionizable group is selected from a 4-pyridylethyl, 2-imidazolinylpropyl, 3-propylaniline, or an imidazole group.

Aspect C20. The kit of any one of aspects C1 to C17, wherein the second ionizable group is selected from a diethylaminopropyl, ethylaminopropyl, dimethylaminopropyl, methylaminopropyl, aminopropyl, diethylaminomethyl, 3- [ bis (2-hydroxyethyl) amino ] propyl, N-butyl-aza-silacyclopentane, N-methyl-aza-silacyclopentane, or a bis-3-methylaminopropylsilyl group.

Aspect C21. The kit of any one of aspects C1 to C20, wherein the molar ratio of the first hydrophobic surface group to the first ionizable group is in a range from 5:1 to 200: 1.

Aspect C22. The kit of any one of aspects C15 to C21, wherein the kit comprises a glycan-labeling reagent

Aspect C23. The kit of aspect C22, wherein the glycan labeling reagent is selected from the group consisting of an MS-active rapid fluorescent labeling compound, a procainamide reagent, and a procaine reagent.

Aspect C24. The kit of any one of aspects C22 to C23, wherein the glycan labeling reagent provides an amphiphilic strongly basic moiety with a pKa value greater than 6.

Aspect C25. The kit of any one of aspects C1 to C14, wherein the first ionizable group and the second ionizable group are negatively charged upon ionization.

Aspect C26. The kit of aspect C25, wherein the first ionizable group is a carboxylic acid group and the second ionizable group is selected from the group consisting of a sulfonic acid group and a carboxylic acid group.

Aspect C27. The kit of any one of aspects C25 to C26, wherein the second pKa value is 1 to 4 units less than the first pKa value.

Aspect C28. The kit of any one of aspects C1 to C27, wherein the kit further comprises an eluent.

Aspect C29. The kit of any one of aspects C1 to C28, wherein the first chromatographic material is in the form of first particles having a core of the first material, and wherein the second chromatographic material is in the form of second particles having a core of the second material.

Aspect C30. The kit of aspect C29, wherein the first material and the second material are organic materials, inorganic materials, or hybrid organic-inorganic materials.

Aspect C31. The kit of aspect C29, wherein the first material and the second material are selected from the group consisting of a silica-based material, an alumina-based material, a titania-based material, a zirconia-based material, and a carbon-based material.

Aspect C32. The kit of aspect C29, wherein the first material and the second material are silica-based materials formed by hydrolytic condensation of one or more organosilane compounds.

Aspect C33. The kit of aspect C32, wherein the organosilane compound comprises one or more alkoxysilane compounds.

Aspect C34. The kit of aspect C32, wherein the organosilane compound is prepared from a tetraalkoxysilane and an alkylalkoxysilane.

Aspect C35. The kit of aspect C29, wherein the first material and the second material comprise organic polymers.

Aspect D1. A method for performing liquid chromatography analysis on a liquid sample, the method comprising: loading a liquid sample comprising a plurality of components into a first flow path in a liquid chromatography system; flowing the first mobile phase through a first flow path such that the liquid sample is directed through a trapping column, wherein the trapping column comprises a first chromatographic material having a first chromatographic surface comprising a first hydrophobic surface group and a first ionizable surface group having a first pKa value, and wherein the trapping column traps at least some of the components of the liquid sample as trapped components; and flowing the second mobile phase through a second flow path in the liquid chromatography system, wherein flowing through the second flow path comprises flowing the second mobile phase through a trapping column to elute at least some of the trapped components as eluted components from the trapping column, and flowing the second mobile phase and the eluted components through an analytical column capable of separating at least some of the eluted components as separated components, wherein the analytical column comprises a second chromatographic material having a second chromatographic surface comprising a second hydrophobic surface group having a hydrophobicity greater than the hydrophobicity of the first hydrophobic surface group and either (i) a permanently ionized surface group or (ii) a second ionizable surface group having a second pKa value that differs from the first pKa value by 1 unit to 12 units.

Aspect D2. The method of aspect D1, further comprising flowing the separated component to a detector capable of detecting a characteristic of the separated component.

Aspect D3. The method of any one of aspects D1 to D2, wherein the first chromatographic material is in the form of first particles, and wherein the second chromatographic material is in the form of second particles.

Aspect D4. The method of aspect D3, wherein the first diameter of the first particle is equal to or greater than the second diameter of the second particle.

Aspect D5. The method of aspect D4, wherein the ratio of the first particle diameter to the second particle diameter is in the range of from 1 to 10.

Aspect D6. The method of any one of aspects D4 to D5, wherein the first diameter is in a range from 2 microns to 10 microns.

Aspect D7. The method of any one of aspects D1 to D6, wherein the inner diameter of the trapping column is greater than or equal to the inner diameter of the analytical column, and wherein the length of the trapping column is shorter than the length of the analytical column.

Aspect D8. The method of any one of aspects D1 to D7, wherein the inner diameter of the trapping column is 1.5 to 5 times the inner diameter of the analytical column.

Aspect D9. The method of any one of aspects D1 to D8, wherein the volume of the trapping column is in the range of from 0.05 to 0.5 times the volume of the analytical column.

Aspect D10. The method of any one of aspects D1 to D9, wherein the first hydrophobic surface group and the second hydrophobic surface group are hydrocarbon groups, and wherein the second hydrophobic surface group contains more carbon atoms than the first hydrophobic surface group.

Aspect D11. The method of aspect D10, wherein the second hydrocarbon group contains 2 to 20 more carbon atoms than the first hydrocarbon group.

Aspect D12. The method of any one of aspects D10 to D11, wherein the first hydrocarbon group is a first alkyl group containing from 3 to 8 carbon atoms, and wherein the second hydrocarbon group is a second alkyl group containing from 10 to 24 carbon atoms.

Aspect D13. The method of aspect D12, wherein the first alkyl group contains 4 carbon atoms, and wherein the second group contains 18 carbon atoms.

Aspect D14. The method of any one of aspects D1 to D13, wherein the first ionizable group is present at a surface concentration that is less than or equal to the surface concentration of the permanently ionized group or the second ionizable group.

Aspect D15. The method of any one of aspects D1 to D14, wherein the first ionizable group is present at a surface concentration in a range from 0.03 micromoles per square meter to 0.3 micromoles per square meter.

Aspect D16. The method of any one of aspects D1 to D15, wherein the first ionizable group and the permanently ionized group or the second ionizable group are positively charged upon ionization.

Aspect D17. The method of aspect D16, wherein the second chromatographic surface comprises a second ionizable surface group, wherein the first pKa value and the second pKa value are greater than 3, and wherein the second pKa value is 1 unit to 7 units greater than the first pKa value.

Aspect D18. The method of any one of aspects D1 to D17, wherein (a) the first ionizable group and (b) the permanently ionized group or the second ionizable group comprises an amine group.

Aspect D19. The method of any of aspects D18, wherein the first ionizable group is selected from a primary amine group, a secondary amine group, and a tertiary amine group, and the permanently ionized group or the second ionizable group is selected from a secondary amine group, a tertiary amine group, and a quaternary ammonium group.

Aspect D20. The method of any one of aspects D1 to D17, wherein the first ionizable group is selected from a 4-pyridylethyl, 2-imidazolinylpropyl, 3-propylaniline, or an imidazole group.

Aspect D21. The method of any one of aspects D1 to D18, wherein the second ionizable group is selected from the group consisting of a diethylaminopropyl, ethylaminopropyl, dimethylaminopropyl, methylaminopropyl, aminopropyl, diethylaminomethyl, 3- [ bis (2-hydroxyethyl) amino ] propyl, N-butyl-aza-silacyclopentane, N-methyl-aza-silacyclopentane, or a bis-3-methylaminopropylsilyl group.

Aspect D22. The method of any one of aspects D1 to D21, wherein the molar ratio of the first hydrophobic surface group to the first ionizable group is in the range of from 5:1 to 200: 1.

Aspect D23. The method of any one of aspects D1 to D15, wherein the first ionizable group and the second ionizable group are negatively charged upon ionization.

Aspect D24. The method of aspect D23, wherein the first ionizable group is a carboxylic acid group and the second ionizable group is selected from the group consisting of a sulfonic acid group and a carboxylic acid group.

Aspect D25. The method of any one of aspects D23 to D24, wherein the second pKa value is 1 to 4 units less than the first pKa value.

Aspect D26. The method of any one of aspects D1 to D25, wherein the first chromatographic material is in the form of first particles having a core of the first material, and wherein the second chromatographic material is in the form of second particles having a core of the second material.

Aspect D27. The method of aspect D26, wherein the first material and the second material are organic materials, inorganic materials, or organic-inorganic hybrid materials.

Aspect D28. The method of aspect D26, wherein the first material and the second material are selected from the group consisting of a silica-based material, an alumina-based material, a titania-based material, a zirconia-based material, and a carbon-based material.

Aspect D29. The method of aspect D26, wherein the first material and the second material are silica-based materials formed by hydrolytic condensation of one or more organosilane compounds.

Aspect D30. The method of aspect D29, wherein the organosilane compound comprises one or more alkoxysilane compounds.

Aspect D31. The method of aspect D29, wherein the organosilane compound is prepared from a tetraalkoxysilane and an alkylalkoxysilane.

Aspect D32. The method of aspect D26, wherein the first material and the second material comprise organic polymers.

Aspect D33. The method of any one of aspects D1 to D32, wherein the component comprises a glycan.

Aspect D34. The method of any one of aspects D1 to D32, wherein the component comprises a labeled glycan.

Aspect D35. The method of aspect D34, wherein the glycan is labeled with a labeling reagent selected from the group consisting of an MS-active rapid fluorescent labeling compound, a procainamide reagent, or a procaine reagent.

Aspect D36. The method of any one of aspects D34 to D35, wherein the glycan is labeled with a glycan-labeling reagent that provides an amphiphilic strongly basic moiety having a pKa value greater than 6.

Aspect D37. The method of any one of aspects D1 to D36, wherein the first mobile phase comprises (i) water or (ii) an aqueous solution of the first organic acid and/or the first organic acid salt.

Aspect D38. The method of aspect D37, wherein the second mobile phase comprises a solution of a second organic acid, which may be the same as or different from the first organic acid, and/or a second organic acid salt, which may be the same as or different from the first organic acid salt.

Aspect D39. The method of any one of aspects D1 to D36, wherein the first mobile phase comprises a solution of an organic acid and an organic acid salt in a solvent comprising water and an organic solvent, and wherein the second mobile phase comprises an elution process during which a concentration of the organic acid increases and a concentration of the organic acid salt increases.

Examples

Material

All reagents described in the procedures below were used as received unless otherwise indicated. Those skilled in the art will recognize that there are equivalents, and thus, although supplies and suppliers are listed, the listed supplies/suppliers should in no way be construed as limiting.

Characterization of

The stationary phase produced by the above procedure was characterized in the following manner. The% C value was measured by coulometric carbon analyser (model CM5300, CM5014, UIC inc., Joliet, IL). Specific Surface Area (SSA), Specific Pore Volume (SPV), and average pore size (APD) of these materials use multipoint N₂Adsorption measurements (Micromeritics ASAP 2400; Micromeritics Instruments Inc., Norcross, GA, Nocroms, Georgia). Calculation of SSA Using the BET method, SPV is for P/P₀>0.98, and calculating APD from the desorption segment of the isotherm using the BJH method. Particle size was measured using a Beckman Coulter Multisizer 3 analyser (30 μm wells, 70000 counts; Miami, FL). The particle size (dp) is measured as the 50% cumulative diameter of the particle size distribution on a volume basis. The total surface coverage of the alkyl hydrophobic groups was determined by the difference in% C of the particles before and after surface modification as measured by elemental analysis. Those skilled in the art will recognize that there are instruments listed above, and so onThe same, therefore, should not be construed as limiting.

Example 1

Synthesis of DEAP HPCM stationary phase

The DEAP HPCM stationary phase (phase 1) was synthesized according to the following procedure:

step 1: using a Dean-Stark separator, the compound of formula (O)_1.5SiCH₂CH₂SiO_1.5)(SiO₂)₄BEH porous particles (Waters Corporation, Milford, MA; 6.5% C; SSA 75-200 m)²/g；SPV＝0.60-0.75cc/g；) (prepared according to the method described in U.S. Pat. No. 6,686,035) was refluxed in toluene (5mL/g, Fisher Scientific, Fairlawn, NJ) for 1 hour. After cooling, redistilled (N, N-diethylaminopropyl) trimethoxysilane (DEAP, Silar Laboratories, Wilmington, N.C.) at 0.3. mu. mol/m²Added and the reaction heated to reflux for 2 hours. The reaction was then cooled and the product was filtered and washed sequentially with toluene, 1:1v/v acetone/water and acetone (all solvents from Fisher Scientific, Fairlawn, NJ). The product was then dried at 80 ℃ under reduced pressure for 16 hours.

Step 2: the material from step 1 was refluxed in toluene (5mL/g, Fisher Scientific, Fairlawn, NJ) for 1 hour using a Dean-Stark separator. After cooling, imidazole (Aldrich, Milwaukee, Wis.) and octadecyltrichlorosilane (Gelest Inc., Morrisville, Pa.) were added at 2.3. mu. mol/m²Added and the reaction heated to reflux for 16 hours. The reaction was then cooled and the product was filtered and washed sequentially with toluene, 1:1v/v acetone/water and acetone (all solvents from Fisher Scientific, Fairlawn, NJ). The material was then refluxed in an acetone/aqueous 0.1M ammonium bicarbonate solution (pH 10) at 50 ℃ for 20 hours (hydrolysis). After hydrolysis, the material was washed sequentially with 1:1v/v acetone/water and acetone (all solvents were from the Silmer Feishell science, Fisher, Phili, N.J.)Scientific, Fairlawn, NJ)). The product was then dried at 80 ℃ under reduced pressure for 16 hours.

And step 3: the material from step 2 was refluxed in toluene (5mL/g, Fisher Scientific, Fairlawn, NJ) for 1 hour using a Dean-Stark separator. After cooling, imidazole (Aldrich, Milwaukee, WI) and triethylchlorosilane (TECS, Gelest inc. of molysill, PA) were added and the reaction was heated to reflux for 4 hours. The reaction was then allowed to cool, imidazole and chlorotrimethylsilane (Aldrich, Milwaukee, WI) were added to the reaction, and the reaction was heated to reflux for a further 16 hours. The reaction was then allowed to cool, the product was filtered, and washed with toluene, 1:1v/v acetone/water, and acetone in that order (all solvents were from Fisher Scientific, Fairlawn, NJ). The product was then dried at 80 ℃ under reduced pressure for 16 hours.

Information related to the DEAP HPCM phase (phase 1) can be found below:

¹as described in US7919177, US 7223473, US 6686035

Example 2

Synthesis of HPCM with ionizable modifier and alkyl hydrophobe

Step 1: using a Dean-Stark separator, the compound of formula (O)_1.5SiCH₂CH₂SiO_1.5)(SiO₂)₄BEH porous particles (Waters Corporation, Milford, MA; 6.5% C; SSA 75 m)²/g-200m²/g；SPV＝0.60cc/g-0.75cc/g；) (prepared according to the method described in U.S. Pat. No. 6,686,035) was refluxed in toluene (5mL/g, Fisher Scientific, Fairlawn, NJ) for 1 hour. While cooling, at 0.1. mu. mol/m²To 0.3. mu. mol/m²2- (4-pyridylethyl) triethoxysilane (4PE, Gelest Incorporated, Morrisville, Pa.) was added and the reaction was heated to reflux for 1 hour. The reaction was then allowed to cool and imidazole and component a silane additives including t-butyldimethylsilyl chloride or octyltrichlorosilane (C8, Aldrich, st. The reaction was then heated to reflux for 20 hours. The reaction was allowed to cool and the product was filtered and washed successively with toluene, 1:1v/v acetone/water and acetone (all solvents were obtained from Fisher Scientific, fairlaw, NJ). The material was then refluxed in an acetone/aqueous 0.1M ammonium bicarbonate solution (pH 10) at 50 ℃ for 20 hours (hydrolysis). After hydrolysis, the material was washed with 1:1v/v acetone/water and acetone in that order (all solvents were from Fisher Scientific, Fairlawn, NJ). The product was then dried at 80 ℃ under reduced pressure for 16 hours.

Step 2: for phase 3, the material from step 1 was refluxed in toluene (5mL/g, Fisher Scientific, Fairlawn, NJ) for 1 hour using a Dean-Stark trap. On cooling, imidazole (Aldrich, Milwaukee, WI) and triethylchlorosilane (TECS, Gelest inc., Morrisville, PA) were added and the reaction was heated to reflux for 4 hours. The reaction was then allowed to cool and imidazole and chlorotrimethylsilane (TMCS, Aldrich, Milwaukee, WI) were added to the reaction. The reaction was then heated to reflux for an additional 16 hours after which the reaction was allowed to cool and the product was filtered and washed successively with toluene, 1:1v/v acetone/water and acetone (all solvents were obtained from Fisher Scientific, fairlaw, NJ). The product was then dried at 80 ℃ under reduced pressure for 16 hours.

Information regarding the HPCM phase with ionizable modifier and alkyl hydrophobic group can be found below:

¹as described in US7919177, US 7223473, US 6686035

Example 3

Screening for Mixed mode Retention of stationary phase

Various high purity chromatographic materials (HCPM) were screened using RapiFluor-MS reagent labeled glycans from human igg (higg) and simple linear gradient one-dimensional separation.

N-glycans from human IgG (Sigma I4506) and bovine fetuin (Sigma F3004) were labeled with rapidfluor-MS reagent according to previously published conditions. (Lauber, M.A.; Yu, Y.Q.; Broussiche, D.W.; Hua, Z.; Koza, S.M.; Magnelli, P.; Guthrie, E.; Taron, C.H.; Fountain, K.J., Rapid prediction of Released N-Glycans for HILIC Analysis Using a laboratory reaction that is Sensitive to heat environments and ESI-MS detection end. anal m.2015 87(10), 5401-9). LC-MS analysis was performed using the conditions indicated below to generate the data shown in fig. 3, 4 and 6.

LC conditions used with Waters ACQUITY UPLC H-Class Bio:

gradiometer:

MS conditions used with the Waters Xevo G2-XS QTof system:

example 4

Trap-elute mixed mode chromatography

The trap-elute chromatography was performed in the configuration depicted in fig. 3 according to the LC-MS conditions listed below. Data corresponding to these experimental conditions are shown in fig. 5, 7, 8 and 9.

LC conditions used with Waters ACQUITY UPLC H-Class Bio:

a capturing gradiometer:

analyzing a gradiometer:

MS conditions used with the Waters Xevo G2-XS QTof system: