CHROMATOGRAPHIC HARDWARE IMPROVEMENTS FOR SEPARATION OF REACTIVE MOLECULES

Abstract

The present disclosure relates to a method of reducing degradation of sample components in a liquid chromatography system. The method utilizes a masked metal frit to prevent or reduce metal surfaces from becoming catalytically active. The masked metal frit is a metal based frit that includes a coating on its exterior surfaces to mask or prevent contact between the organic solvents (and/or any analyte or other related solvent) and the underlying metal. The coating is a vapor deposited inorganic-organic hybrid coating, such as a vapor deposited C2 coating.

Claims

1. A chromatography column, comprising: a frit comprising a metal substrate and an outer coating surrounding at least a portion of a surface of the substrate, the outer coating comprising an inorganic-organic hybrid.

2. The chromatography column of claim 1, wherein the metal substrate comprises substantially pure titanium.

3. The chromatography column of claim 2, wherein the outer coating comprises C2 and/or C2C10.

4. The chromatography column of claim 2, wherein the frit further comprises an intermediate coating disposed between at least a portion of the outer coating and the surface of the metal substrate.

5. The chromatography column of claim 1, wherein the frit further comprises an intermediate coating disposed between at least a portion of the outer coating and the surface of the metal substrate.

6. The chromatography column of claim 4, wherein the intermediate coating comprises a pure metal, a metal oxide, a metal nitride, or a metal carbide.

7. The chromatography column of claim 1, wherein at least a portion of exposed metal walls housed within the chromatography column comprise a fluid-contacting coating comprising the inorganic-organic hybrid.

8. The chromatography column of claim 7, wherein the inorganic-organic hybrid comprises C2 and/or C210.

9. The chromatography column of claim 7, wherein the metal substrate comprises titanium.

10. A method of reducing metal-catalyzed reactions of sample components during liquid chromatography, the method comprising: separating a sample in a chromatography column including a masked metal frit, wherein the masked metal frit comprises a metal frit coated on exterior surfaces with an inorganic-organic hybrid coating that is non-reactive with the sample; and detecting separated sample components with a detector.

11. The method of claim 10, wherein the masked metal frit comprises a pure or substantially pure titanium frit.

12. The method of claim 10, wherein the masked metal frit comprises a titanium alloy frit.

13. The method of claim 10, wherein the masked metal frit comprises a stainless steel frit.

14. The method of claim 13, wherein the stainless steel frit includes an intermediate coating comprising titanium, a metal oxide, a metal nitride, or a metal carbide layer applied prior to the inorganic-organic hybrid coating.

15. The method of claim 13, wherein the stainless steel frit includes an intermediate layer comprising titanium, a metal oxide, a metal nitride, or a metal carbide applied simultaneously with the inorganic-organic hybrid coating.

16. The method of claim 10, wherein the inorganic-organic hybrid coating comprises an alkylsily coating.

17. The method of claim 10, wherein exposed metal walls within the chromatography column are coated with the inorganic-organic hybrid coating.

18. The method of claim 17, wherein a bioinert pump is connected to the liquid chromatography system.

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28. The chromatography column of claim 5, wherein the intermediate coating comprises a pure metal, a metal oxide, a metal nitride, or a metal carbide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The technology will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0021] FIG. 1A is a schematic of the chemical formula of Litronesib, an amine.

[0022] FIG. 1B is a schematic of the chemical formula of a nitroso impurity of Litronesib, (Z isomer).

[0023] FIG. 1C is a schematic of the chemical formula of a nitroso impurity of Litronesib, (E isomer).

[0024] FIG. 1D is a chromatogram of a Litronesib separation in which the nitroso impurities are present due to nitrosation of Litronesib during separation.

[0025] FIG. 1E is a UV chromatogram of a Litronesib separation in which the nitroso impurities are present due to nitrosation of Litronesib during separation.

[0026] FIG. 2A is a schematic of the chemical formula of Clozapine, an amine.

[0027] FIG. 2B is a schematic of the chemical formula of a n-oxide of Clozapine.

[0028] FIG. 3 is a schematic of a chromatographic flow system including a chromatography column and various other components, in accordance with an illustrative embodiment of the technology. A fluid is carried through the chromatographic flow system with a fluidic flow path extending from a fluid manager to a detector, such as a MS detector.

[0029] FIG. 4 is a flow chart showing a method of reducing degradation of sample components during liquid chromatography in accordance with an embodiment of the present technology.

[0030] FIG. 5 is a cross-sectional view schematic of an embodiment of a frit in accordance with the present technology.

[0031] FIG. 6 is a cross-sectional view schematic of an embodiment of a frit in accordance with the present technology.

[0032] FIG. 7A is a UV chromatogram of Clozapine using a stainless steel column after one injection.

[0033] FIG. 7B is a UV chromatogram of Clozapine using a stainless steel column after two injections.

[0034] FIG. 7C is a UV chromatogram of Clozapine using a stainless steel column after thirteen injections.

[0035] FIG. 8A is a TIC chromatogram of Clozapine using a stainless steel column after two injections.

[0036] FIG. 8B is a TIC chromatogram of Clozapine using a stainless steel column after thirty-four injections.

[0037] FIG. 9A is a MS spectra showing the identification of Clozapine.

[0038] FIG. 9B is a MS spectra showing the identification of the n-oxide of Clozapine.

[0039] FIG. 10A is a UV chromatogram of Clozapine using a coated (i.e., C2 coated) stainless steel column having a coated (i.e., C2 coated) Ti frit after one injection.

[0040] FIG. 10B is a UV chromatogram of Clozapine using a coated (i.e., C2 coated) stainless steel column having a coated (i.e., C2 coated) Ti frit after two injections.

[0041] FIG. 10C is a UV chromatogram of Clozapine using a coated (i.e., C2 coated) stainless steel column having a coated (i.e., C2 coated) Ti frit after thirteen injections.

[0042] FIG. 11A is a TIC chromatogram of Clozapine using a coated (i.e. C2 coated) stainless steel column having a coated (i.e., C2 coated) Ti frit after two injections.

[0043] FIG. 11B is a TIC chromatogram of Clozapine using a coated (i.e., C2 coated) stainless steel column having a coated (i.e., C2 coated) Ti frit after thirty-four injections.

[0044] FIG. 12A is a TIC chromatogram of a Clozapine separation using a C2 coated stainless steel column with uncoated stainless steel frits. The TIC chromatogram is focused on the n-oxide peak after the second injection.

[0045] FIG. 12B is a TIC chromatogram of a Clozapine separation using a C2 coated stainless steel column with C2 coated stainless steel frits. The TIC chromatogram is focused on the n-oxide peak after the second injection.

[0046] FIG. 12C is a TIC chromatogram of a Clozapine separation using a C2 coated stainless steel column with uncoated titanium frits. The TIC chromatogram is focused on the n-oxide peak after the second injection.

[0047] FIG. 12D is a TIC chromatogram of a Clozapine separation using a C2 coated stainless steel column with C2 coated titanium frits. The TIC chromatogram is focused on the n-oxide peak after the second injection.

[0048] FIG. 12E is a TIC chromatogram of a Clozapine separation using a C2 uncoated stainless steel column with uncoated stainless steel frits. The TIC chromatogram is focused on the n-oxide peak after the second injection.

[0049] FIG. 13A is a TIC chromatogram of a Clozapine separation using a C2 coated stainless steel column with uncoated stainless steel frits. The TIC chromatogram is focused on the n-oxide peak after the fifth injection.

[0050] FIG. 13B is a TIC chromatogram of a Clozapine separation using a C2 coated stainless steel column with C2 coated stainless steel frits. The TIC chromatogram is focused on the n-oxide peak after the fifth injection.

[0051] FIG. 13C is a TIC chromatogram of a Clozapine separation using a C2 coated stainless steel column with uncoated titanium frits. The TIC chromatogram is focused on the n-oxide peak after the fifth injection.

[0052] FIG. 13D is a TIC chromatogram of a Clozapine separation using a C2 coated stainless steel column with C2 coated titanium frits. The TIC chromatogram is focused on the n-oxide peak after the fifth injection.

[0053] FIG. 13E is a TIC chromatogram of a Clozapine separation using an uncoated stainless steel column with uncoated stainless steel frits. The TIC chromatogram is focused on the n-oxide peak after the fifth injection.

[0054] FIG. 14A is a TIC chromatogram of a Clozapine separation using a stainless steel column with uncoated stainless steel frits. The TIC chromatogram is focused on the z and e isomers of the Clozapine nitroso impurity peaks after the second injection.

[0055] FIG. 14B is a TIC chromatogram of a Clozapine separation using a stainless steel column with uncoated stainless steel frits. The TIC chromatogram is focused on the z and e isomers of the Clozapine nitroso impurity peaks after the thirty-fourth injection.

[0056] FIG. 15A is a TIC chromatogram of a Clozapine separation using a C2 coated stainless steel column with C2 coated Ti frits. The TIC chromatogram is focused on the location of z and e isomers of the Clozapine nitroso impurity peaks after the second injection.

[0057] FIG. 15B is a TIC chromatogram of a Clozapine separation using a C2 coated stainless steel column with C2 coated Ti frits. The TIC chromatogram is focused on the location of z and e isomers of the Clozapine nitroso impurity peaks after the thirty-fourth injection.

[0058] FIG. 16A is a MS spectra showing the identification of baicalein and baicalein oxide from the first injection of a sample on an uncoated stainless steel column.

[0059] FIG. 16B is a MS spectra showing the identification of propyl gallate and propyl gallate oxide from the first injection of the sample on an uncoated stainless steel column.

[0060] FIG. 16C is a MS spectra showing the identification of baicalein and baicalein oxide from the first injection of the sample on a C2 coated stainless steel column.

[0061] FIG. 16D is a MS spectra showing the identification of propyl gallate and propyl gallate oxide from the first injection of the sample on a C2 coated stainless steel column.

DETAILED DESCRIPTION

[0062] In general, the present disclosure is directed to devices and methods for creating an inert liquid chromatography (LC) system. Specifically, the present disclosure is directed to the application of an inorganic-organic hybrid coating applied to an underlying metal substrate to form a mask to reduce or prevent analyte-metal interactions in a LC system. In embodiments, the inorganic-organic hybrid coating is vapor deposited to create a uniform coating. In particular, the present technology relates to devices or systems including an inorganic-organic hybrid coating to mask a metal frit (e.g., a titanium frit, metal frit having a titanium layer) for use in an inert liquid chromatography system. In some instances, the present technology relates to methods of providing a coated frit to a system, and in particular a coated, inert LC system, to reduce on-column degradation, thereby increasing the strength of analytical results.

[0063] Various conditions are used in liquid chromatography (LC) to optimize the performance of analyte separations. In chromatography such as reversed-phase chromatography, an analyte is typically eluted with the use of an aqueous mobile phase and an organic solvent. Acetonitrile and methanol are common solvents used for elution but have been shown to corrode stainless steel and other metals over time. This corrosion can cause the metal surfaces of the LC system or column, particularly with regards to the frit, to become catalytically active. This can lead to analyte-metal interactions resulting in the degradation of the sample components.

[0064] Two examples of metal catalyzed analyte reactions include nitrosation and oxidation. That is, metal active surfaces exposed to organic solvents provide active sites for reactions with certain analytes, such as amines. For example, Litronesib, a kinesin inhibitor, commonly studied as a potential treatment in cancer protocols, can be transformed in a metal catalyzed reaction by nitrosation to form impurities that degrade analytic separation results. Referring to FIG. 1A shown is the chemical formula of Litronesib. In a catalytically active environment, such as an environment with exposed metal, acetonitrile and/or methanol, the polar covalent bond NH at the bottom of the structure can react in a nitrosation reaction to form either the Z isomer (FIG. 1B) or the E isomer (FIG. 1C). As a result of the nitrosation reaction, some of the original sample material is transformed to a different form, which will change the analytical findings of an investigation. For example, FIG. 1D provides a chromatogram (from Myers, et al. Journal of Chromatography A, Volume 1319, 6 Dec. 2013, pages 57-64) of a separation of a known quantity of Libronesib. Due to the nitrosation of a portion of the analytes to an impurity form (Z isomer or E isomer) impurity peaks are present in the spectra, which degrade the quality of the result. Also see FIG. 1E providing the results from a UV detector from a Litronesib separation, which was degraded by nitrosation reactions.

[0065] Oxidation catalytic reactions are also possible during chromatographic separations including metal within the flow path. For example, Clozapine, an amine and antipsychotic drug, is known to react in a separation environment having exposed metal and organic solvents. During a separation, at least a portion of Clozapine within a sample, due to the active metal sites and the organic solvents can be transformed to an n-oxide. FIGS. 2A-2B illustrate the chemical formula of Clozapine and its n-oxide.

[0066] Other analytes, besides amines, are susceptible to on-column degradation. For example, anilines have been known to degrade in a dimerization process when exposed to metal LC components in the presence of ammonium hydroxide and acetonitrile. And various additives (e.g., flavor, food additives) such as, for example, baicalin, baicalein, and propyl gallate, are known to degrade by polyphenol oxidation in a metallic LC system with formic acid and acetonitrile used in the mobile phase.

[0067] To address these degradations, several alternative surfaces have been proposed over the years including the use of titanium or titanium alloys instead of stainless steel. Titanium and its alloys is known to be less reactive than stainless steel in organic solvents. However, titanium has been shown to still be chromatographically reactive and can contribute to the adsorption of analytes. Additionally, it has been found that titanium surfaces can leach ions when used with methanol, a common LC solvent. In this way, metal-catalyzed reactions from the column, its components (e.g., frits) or LC system can still occur.

[0068] In the present technology, an application of an inorganic-organic hybrid vapor deposited coating (e.g., alkylsilyl coating, a diol coating, a phenyl coating, other ligand based coating) to stainless steel or other metal material can prevent the occurrence of degradation as caused by the column or LC system. The inorganic-organic hybrid coating can mask the metal surfaces of the column and LC system from corrosion as caused by mobile phases such as acetonitrile and methanol or even the analyte itself. The inorganic-organic hybrid coating prevents corrosion of the underlying metal and thus analyte degradation if the active sites on the metal surfaces are masked. By utilizing vapor deposition, a uniform coating on a frit masking the active site while still allowing for passage therethrough can be achieved. Reduction in degradation is realized on many different metal substrates, not just titanium and its alloys, but also stainless steel.

[0069] The present technology includes, in some embodiments, multiple layers or coatings to mask the active sites. For example, in certain embodiments, a multilayer inorganic-organic hybrid coating is applied on top of the metal frit. The multilayer coating may be a single material (e.g., C2 or C2C10) in which a base layer is applied first and then is built up in a second layer. Alternatively, the multilayer coating can comprise two different materials. For example, a base layer of C2 is vapor deposited directly onto a titanium frit followed by a growth layer or secondary layer comprising C2C10. In certain embodiments, the frit can be preprocesses prior to application of an inorganic-organic hybrid coating. For example, a stainless steel frit can be metalized with a different metal material to reduce active sites prior to the application of the inorganic-organic hybrid coating. For example, a single metal material, such as Ti can be applied as a base coating material. In other embodiments, an alloy can be applied to the frit prior to the inorganic-organic hybrid coating. In certain embodiments, a metal-oxide, metal-nitride, or in some cases, a metal-carbide base coating is applied to a stainless steel frit. In one embodiment, a titanium coating is applied (e.g., painted or vapor deposited onto) the metal frit as a base layer; next the inorganic-organic hybrid coating (e.g., C2) is vapor deposited over the Ti metalized stainless steel frit. In another embodiment, a double bilayer consisting of alumina and titania, or alumina and tantalum oxide is applied via vapor deposition to the stainless steel frit (or other component) substrate followed by the vapor application of the inorganic-organic hybrid coating on the exterior.

[0070] In some aspects, the present technology is directed to the use of a masked frit within an inert LC system. In embodiments, the present technology includes methods and systems comprising the use of a masked metal frit (e.g., inorganic-organic hybrid coated metal frit) in a LC system that has been tailored to reduce secondary interactions. For example, the present technology includes using a C2 coated titanium frit in a LC system that includes a coating along its wetted flow path. In some embodiments, the wetted flow path includes the column and connected tubing. In certain embodiments, the wetted flow path extends from the sample reservoir, through the sample injector, to connective tubing, column, and to one or more detectors downstream of the tubing. An example of one such system includes the systems described in US Patent Publication US 2020-0215457 (Jul. 9, 2020), herein incorporated by reference. In certain embodiments, the LC system also includes a specialized pump have bioinert surfaces. Examples of commercially available pumps with biocompatible pumps include, but are not limited to, bioQSM (part number 18601541), bioQSM PLUS (part number 18601581), bioQSM-XR PLUS (part number 18601584), and bioBSM (part number 18601561) all available from Waters Technologies Corporation (Milford, Mass.).

[0071] The present technology is also related to methods to reducing degradation of sample components during liquid chromatography. In general, the method aims to mask metal within the flow path to reduce possible metal-catalyzed reactions, such as nitrosation and oxidation of sample components. FIG. 3 is a representative schematic of a chromatographic flow system/device 100 that can be used to separate analytes, such as amines, in a sample. The sample may contain other analytes known to be susceptible to degradation. For example, in some embodiments, the sample can include anilines, or additives, such as baicalin, baicalein, propyl gallate, quercetin-e-rhamnoside, or rutin. Chromatographic flow system 100 includes several components including a fluid manager system 105 (e.g., controls mobile phase flow through the system), tubing 110 (which could also be replaced or used together with micro fabricated fluid conduits), fluid connectors 115 (e.g., fluidic caps), frits 120, a chromatography column 125 (which houses a frit 120 at its entrance and/or exit), a sample injector 135 including a needle (not shown) to insert or inject the sample into the mobile phase, a vial, sinker, or sample reservoir 130 for holding the sample prior to injection, a detector 150, such as a mass spectrometer, and a pressure regulator 140 for controlling pressure of the flow. Interior surfaces of the components of the chromatographic system/device form a fluidic flow path that has wetted surfaces. The fluidic flow path can have a length to diameter ratio of at least 20, at least 25, at least 30, at least 35 or at least 40. A pump for delivering fluids is part of the fluid manager 105 and the pump is not shown separately.

[0072] In the present technology, the frits 120 are positioned within the fluid flow path and has wetted surfaces. That is, the frits 120 are exposed to the organic solvents and sample passing therethrough. In general, metal frits are preferred for several reasons. For example, metal frits can be formed and shaped according to a desired need. Metal frits have well-understood permeability and particle retention capability. In addition, metal frits maintain their shape and structure even after extended periods of use. Metal frits do however contribute to analyte losses by providing active sites for metal-catalytic reactions. To reduce or prevent those reactions, but to maintain the structural integrity provided by metal frits, methods in accordance with the present disclosure separate samples with masked or coated metal frits to prevent analyte-metal interactions in the liquid chromatography column 125.

[0073] FIG. 4 provides a flow chart illustrating a method of the present technology. The method 400 of reducing degradation of sample components during analysis (e.g., a liquid chromatographic separation) includes step 405 separating a sample using a column having a masked metal frit; and step 410 detecting the separated sample components with a detector. The masked metal frit of step 405 is a metal frit coated on exterior surfaces with a vapor deposited inorganic-organic hybrid coating. The coating on the masked or coated metal frit forms a barrier to prevent interaction between the underlying metal and the organic solvents/sample flowing through and about the frit. That is, the coating prevents analyte-metal interactions and thus reduces degradation of the sample components during liquid chromatography.

[0074] Some embodiments of the method, further include separating the sample in an inert liquid chromatography system that not only includes masked metal frits, but also coated wetted surfaces, and components (e.g., sample injectors and tubing connecting column to other components) and in some instances a bioinert pump for delivery of fluids.

[0075] The masked frit 500 shown in FIG. 5 includes a metal substrate 505 which provides the structure and integrity to the frit and at least one coating layer 510. The at least one coating layer 510 is typically vapor deposited, such that the entire exterior of the masked frit 500 is protected by the coating—such that there are no active metal sites available for solvent/analyte interaction.

[0076] The inorganic-organic hybrid coatings protect the underlying metal material from interaction with organic solvents/metal reactive analytes. In one embodiment, the inorganic-organic hybrid coating is an alkylsilyl coating. The alkylsilyl coating is inert to at least one of the analytes in the sample. In some embodiments, the alkylsilyl coating is a organosilica coating. In certain embodiments, the alkylsilyl coating is an inorganic-organic hybrid material that forms the wetted surface or that coats the wetted surfaces (e.g., almost the entirety of the wetted surface, more than 95% of exposed surface, more than 97% of exposed surface, more than 99% of the exposed surface).

[0077] The inorganic-organic coating can have a contact angle of at least about 15°. In some embodiments, the coating can have a contact angle of less than or equal to 30°. The contact angle can be less than or equal to about 115°. In some embodiments, the contact angle of the coating is between about 15° to about 90°, in some embodiments about 15° to about 105°, and in some embodiments about 15° to about 115°. For example, the contact angle of the coating can be about 0°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, 90°, 95°, 100°, 105°, 110°, or 115°.

[0078] The thickness of the inorganic-organic hybrid coating, e.g., the alkylsilyl coating, can be at least about 100 Å. For example, the thickness can be between about 100 Å to about 1600 Å. The thickness of the coating can be about 100 Å, 200 Å, 300 Å, 400 Å, 500 Å, 600 Å, 700 Å, 800 Å, 900 Å, 1000 Å, 1100 Å, 1200 Å, 1300 Å, 1400 Å, 1500 Å or 1600 Å. The thickness of the coating (e.g., a vapor deposited alkylsilyl coating) can be detected optically by the naked eye. For example, more opaqueness and coloration is indicative of a thicker coating. From thin to thick, the color changes from yellow, to violet, to blue, to slightly greenish and then back to yellow when coated parts are observed under full-spectrum light, such as sunlight. For example, when the alkylsilyl coating is 300 Å thick, the coating can appear yellow and reflect light with a peak wavelength between 560 and 590 nm. When the alkylsilyl coating is 600 Å thick, the coating can appear violet and reflect light with a peak wavelength between 400 and 450 nm. When the alkylsilyl coating is 1000 Å thick, the coating can appear blue and reflect light with a peak wavelength between 450 and 490 nm. See, e.g., Faucheu et al., Relating Gloss Loss to Topographical Features of a PVDF Coating, Published Oct. 6, 2004; Bohlin, Erik, Surface and Porous Structure of Pigment Coatings, Interactions with flexographic ink and effects of print quality, Dissertation, Karlstad University Studies, 2013:49.

[0079] The inorganic-organic hybrid coating can be the product of vapor deposited bis(trichlorosilyl)ethane, bis(trimethoxysilyl)ethane, bis(trichlorosilyl)octane, bis(trimethoxysilyl)octane, bis(trimethoxysilyl)hexane, and bis(trichlorosilyl)hexane.

[0080] In some aspects, at least a portion of the wetted surfaces are coated with multiple layers of the same or different alkylsilyls, where the thickness of the alkylsilyl coatings correlate with the number of layering steps performed (e.g., the number of deposited layers of alkylsilyl coating on wetted surfaces of the frits (or in the case of an inert LC system along wetted surfaces such as column walls, fittings, injectors, etc).

[0081] The metal frits can have multiple coatings, such as multiple alkylsilyl coatings. For example, a second alkylsilyl coating can be in direct contact with a first or base alkylsilyl coating. In one embodiment, a titanium metal frit is coated with a base coating of C2 and a second coating of C2C10.

[0082] In one aspect, the inorganic-organic hybrid coating is n-decyltrichlorosilane, (3-glycidyloxypropyl)trimethoxysilane (GPTMS), (3-glycidyloxypropyl)trimethoxysilane (GPTMS) followed by hydrolysis, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, trimethylchlorosilane, trimethyldimethyaminosilane, methoxy-polyethyleneoxy(3)silane propyltrichlorosilane, propyltrimethoxysilane, (heptadecafluoro-1,1,2,2-tetrahydrodecyl)tris(dimethylamino)silane, (heptadecafluoro-1,1,2,2-tetrahydrodecyl)trischlorosilane, (heptadecafluoro-1,1,2,2-tetrahydrodecyl)trimethoxysilane vinyltrichlorosilane, vinyltrimethoxysilane, allyltrichlorosilane, 2-[methoxy(polyethyleneoxy)3propyl]trichlorosilane, 2-[methoxy(polyethyleneoxy)3propyl]trimethoxysilane, or 2-[methoxy(polyethyleneoxy)3propyl]tris(dimethylamino)silane.

[0083] Other coating materials are possible besides alkylsilyl coatings. For example, other inorganic-organic hybrid coatings including diol, phenyl, or other ligands are available for use to protect the metal frit from undesired interactions.

[0084] In addition to applying an inorganic-organic hybrid coating to the metal frit, other processing can be used to reduce the undesired degradation of sample components. For example, if a stainless steel frit is desired due to its structural integrity, a metal containing exterior coating of titanium can be applied to its exterior surface either prior to or simultaneously with the vapor deposition of the inorganic-organic hybrid coating. Other types of metallization or metal containing coatings (prior to the deposition of the exterior inorganic-organic hybrid coating) of the stainless steel frit (or other metal LC component) are also possible. Instead of metallizing with titanium, the stainless steel frit could be first coated with iron, silicon, manganese, nickel, molybdenum, tin, cobalt, aluminum, copper, vanadium, chromium or boron. In certain embodiments, the metal frit can be coated with gold, platinum, silver, tungsten, tantalum, or iridium. In some embodiments, the stainless steel frit could first be coated or treated with carbon (e.g., diamond film), phosphorous, or sulfur prior to the exterior inorganic-organic hybrid being deposited to prevent analyte interactions.

[0085] The masked frit can include multiple coating layers. For example, referring to FIG. 6, shown is a masked frit 600 including a metal containing layer 607 (e.g., layer containing a metal, alloy, metal-oxide, metal-nitride, or metal-carbide) covering the metal substrate 605, followed by one or more vapor deposited inorganic-organic hybrid coatings 610.

[0086] In some embodiments, the metal containing layer 607 is a single layer consisting of a single material (e.g., a Ti layer, a Ti.sub.2O.sub.3 layer, etc.). The metal containing layer 607 can also be a bilayer consisting of two different materials (e.g., layer of alumina followed by layer of titania). The metal containing layer 607 can comprise a pure or substantially pure elemental metal (e.g., Ti, Au, Pt). In other embodiments, the metal containing layer is an alloy, such as a Ti 6 Al-4V. In certain embodiments, the metal containing layer is an oxide, a nitride, or a carbide. For example, the metal containing layer can be alumina, silicon nitride, or titanium carbide. In embodiments, the metal containing layer is vapor deposited and in the case of an oxide, nitride, or carbide, the vapor deposition utilizes oxygen, nitrogen or carbon precursors in addition to the metal precursors. Table 1 provides a list of ligand types of interest which form the precursors used in the formation of the metal containing layer. Table 2 provides the precursors for forming oxides, nitrides, and carbides. The precursors listed in table 2 can be provided in an unactivated or plasma activated state. In some embodiments, the carbon precursors provided in Table 2 can be used in conjunction with metal oxides and nitrides to add organic bridges to these films.

TABLE-US-00001 TABLE 1 Ligand types of interest which form the precursor used for deposition Metal Ligand Types Scandium Cyclopentadienyl, amide, imide, amidinate Yttrium Cyclopentadienyl, amide, imide, amidinate Titanium halide, Cyclopentadienyl, alkoxide, amide, imide, amidinate Zirconium halide, Cyclopentadienyl, alkoxide, amide, imide, amidinate Hafnium halide, Cyclopentadienyl, alkoxide, amide, imide, amidinate Vanadium halide, alkoxide, amide, imide, amidinate Niobium halide, alkoxide, amide, imide Tantalum halide, alkoxide, amide, imide Chromium halide, alkoxide, amide, imide Molybdenum halide, amide, imide Tungsten halide, amide, imide Aluminum halide, alkyl, alkoxide, amide, imide, amidinate Boron halide, alkyl, alkoxide Silicon halide, alkyl, alkoxide, amide Germanium halide, alkyl, alkoxide, amidinate

TABLE-US-00002 TABLE 2 Oxide, Nitride and Carbide Precursors Oxygen precursors Water, hydrogen peroxide, oxygen, ozone, alcohols Nitrogen precursors Nitrogen, ammonia, hydrazine Carbon precursors Acetylene, formic acid, carbon (see ligands from Table 1), alcohols, acids, anhydrides

[0087] The masked frits of the present technology provide a major advance over uncoated frits. In the Example section below, evaluations of different frit materials in combination with different LC systems illustrate the technology reduces on-column catalytic reactions, such as, for example, oxidation and nitrosation, to a great extent over non-coated hardware. As a result, more robust analysis with strengthen results are provided over the conventional routes of separation.

EXAMPLES

Example 1: On-Column Oxidation of Different Column Technologies

[0088] Clozapine was prepared in 0.1% (w/v) 20/80/0.08 (acetonitrile/water/acetic acid). Analyses of these samples were performed using a Waters ACQUITY UPLC I-Class LC system and the separation method outlined below. FIGS. 7A-7C and FIGS. 8A-8B present the on-column oxidation results of the separations on an uncoated stainless steel column (Column A) from multiple injections. FIGS. 10A-10C and FIGS. 11A-11B present the on-column oxidation results of the separations on a C2 coated stainless steel column utilizing a C2 coated Ti frit (Column B) from multiple injections. FIG. 9A presents a mass spectra of Clozapine having a m/z=327, whereas FIG. 9B presents a mass spectra of n-oxide Clozapine having a m/z=343.

TABLE-US-00003 TABLE 3 Separation details for Example 1 Test Conditions Column A Hybrid Silica C18, 130 angstroms, 1.7 μm (Conventional) packed within a stainless steel column 2.1 × 50 mm (with stainless steel frit) Column B (Coated Hybrid Silica C18, 130 angstroms, 1.7 μm Column) packed within a C2 coated stainless steel column 2.1 × 50 mm (with C2 coated titanium frit) Sample 6 mg/mL Clozapine in acetonitrile/water/acetic acid Solvent Conditions Solvent Line A 0.05% (w/v) ammonium hydroxide in water Solvent Line B Acetonitrile Column Temperature 30° C. Injection Volume 0.5 μL (first injection), 0.25 μL (successive injections) Diluent 20/80/0.8 (w/v) acetonitrile/water/acetic acid UV Detection 290 nm MS Conditions Mode ESI positive, sensitivity Mass Range 50-1500 m/z Capillary 3.5 kV Sample Cone 40 V Source Offset 80 V Source Temperature 100° C. Desolvation Temperature 250° C. Desolvation Gas 600 L/h Quadrupole Option Automatic Gradient Table: Time (min) Flow Rate (mL/min) % A % B Curve Initial 0.310 75.0 25.0 Initial 0.45 0.310 75.0 25.0 6 10.31 0.310 20.0 80.0 6 11.20 0.310 20.0 80.0 6 11.25 0.310 20.0 80.0 6 12.55 0.310 20.0 80.0 6 13.00 0.310 75.0 25.0 6 15.00 0.310 75.0 25.0 6

[0089] The choice of column technology has an effect on metal-catalyzing reactions. In this example, two types of columns were investigated. Column A is an uncoated stainless steel column and Column B is a C2 coated stainless steel column.

[0090] FIGS. 7A-7C illustrate the increasing effect of metal-catalyzing reaction for the separation of Clozapine on an uncoated column (Column A). In FIG. 7A after the first injection a clean Clozapine peak is observed. However, after more sample is provided to the system (2 injections shown in FIG. 7B and 13 in FIG. 7C), the oxide form of Clozapine appears, degrading the results. FIGS. 8A and 8B illustrate that the increase in the oxide form continues to increase and greatly effect results—compare oxide peak after two injections in FIG. 8A to the oxide peak after 34 injections in FIG. 8B.

[0091] The degradation of results can be minimized by the selection of column body material. The spectra shown in FIGS. 7A-8B present results from an uncoated stainless steel column, and present the growth of the n-oxide peak with increasing exposure to the sample and mobile phase. However, by selecting a different column technology, a stainless steel column coated with an inorganic-organic hybrid material (i.e., C2), degradation of results is minimized as the oxide peak (while present) does not increase to the same extent as shown in FIG. 7A-8B. FIGS. 10A-10C provide spectra after the first, second and thirteen (respectively) on a C2 coated column. FIGS. 11A-11B illustrate the change in the n-oxide peak between the second and thirty-fourth injections.

Example 2: On-Column Oxidation of Different Frit Materials

[0092] Clozapine was prepared in 0.1% (w/v) 20/80/0.08 acetonitrile/water/acetic acid. Analysis of these samples were performed using a coated inert system (ACQUITY UPLC PREMIER LC system, commercially available from Waters Corporation, Milford Mass.) and the separation method outline below. FIGS. 12A-12D (after second injection) and FIGS. 13A-13D (after fifth injection) present the on-column oxidation results of the separations from multiple injections (i.e., 2.sup.nd, and 5.sup.th) in columns using different frit materials (e.g., uncoated metals such as stainless steel and titanium, and coated metals including C2 coated stainless steel frits and C2 coated titanium frits).

TABLE-US-00004 TABLE 4 Separation details for Example 2 Test Conditions Column Hybrid Silica C18, 130 angstroms, 1.7 μm packed within a C2 coated stainless steel 2.1 × 50 mm column; frits (A-D) evaluated by use of a single type in the column Frit A Stainless steel Frit B C2 coated stainless steel Frit C Titanium Frit D C2 coated titanium Sample 6 mg/mL Clozapine in acetonitrile/water/acetic acid Solvent Conditions Solvent Line A 0.05% (w/v) ammonium hydroxide in water Solvent Line B Acetonitrile Column Temper 30° C. Injection Volume 0.5 μL (first injection), 0.25 μL (successive injections) Diluent 20/80/0.8 (w/v) acetonitrile/water/acetic acid UV Detection 290 nm MS Conditions Mode ESI positive, sensitivity Mass Range 50-1500 m/z Capillary 3.5 kV Sample Cone 40 V Source Offset 80 V Source Temperature 100° C. Desolvation 250° C. Temperature Desolvation Gas 600 L/h Quadrupole Option Automatic Gradient Table: Time (min) Flow Rate (mL/min) % A % B Curve Initial 0.310 75.0 25.0 Initial 0.45 0.310 75.0 25.0 6 10.31 0.310 20.0 80.0 6 11.20 0.310 20.0 80.0 6 11.25 0.310 20.0 80.0 6 12.55 0.310 20.0 80.0 6 13.00 0.310 75.0 25.0 6 15.00 0.310 75.0 25.0 6

[0093] As shown in an evaluation of different frit materials used in conjunction with hybrid organic-inorganic coating (in this example C2 coating), both stainless steel and titanium frits with C2 coating technology provide reductions in on-column degradation of amine versus their corresponding non-coated fits. The separation of Clozapine, a compound known to undergo on-column oxidation with ammonium hydroxide and acetonitrile mobile phases, resulted in 50% less clozapine N-oxide using C2 coated stainless steel frits over stainless steel frits (uncoated). An 80% reduction was seen using C2 on titanium fits over titanium frits without a coating.

TABLE-US-00005 TABLE 5 Percentage of Oxidized Clozapine from Total Peak Area Tube Material SS C2SS C2SS C2SS C2SS Frit Material SS SS C2SS Ti C2Ti % after 2.sup.nd Injection 1.03 1.11 0.53 0.04 0.01 % after 5.sup.th Injection 2.08 1.83 0.9 0.21 0.41 SS = Stainless Steel; Ti = Titanium; C2SS = C2 coating on Stainless Steel; C2Ti = C2 coating on Titanium. See FIGS. 12A-12E and 13A-13E.

[0094] Interestingly, when comparing the performance of the frits studied, the C2 coated titanium frits produced the least amount of oxidative species followed by the column packed with titanium frits. The C2 coated stainless steel frits, while providing benefits over standard stainless steel frits, was still 77% less effective at preventing on-column oxidation than when titanium frits were used. It is reasoned that the C2 coating may not be as effectively applied to stainless steel frits and that an improved process or the addition of a metallic layer could prove more effective. Nevertheless, C2 coating yields significantly lower oxidative species and, in the case of C2 coated titanium, could even potentially prevent the formation of oxidative species, as demonstrated throughout 34 injections on a C2 stainless steel coated column with C2 coated Ti frit shown in FIG. 11B. For example, compare FIGS. 11A and 11B showing little to no growth of the oxide peak.

[0095] The results of using a masked or coated frit also show a decrease in nitrosation during the separation of Clozapine with ammonium hydroxide and acetonitrile mobile phases. While nitrosation of Clozapine was not as prominent as the oxidized form, reductions in nitrosation were also recorded. FIG. 14 A and FIG. 14 B provide the spectra at the known Clozapine nitroso impurities peaks (peaks for z and e isomers) after the second injection and thirty-fourth injection of Clozapine in an uncoated column using an uncoated stainless steel frit. As more Clozapine is injected into the system, additional nitrosation occurs. However, nitrosation is stunted in the C2 coated stainless steel column including a C2 coated titanium frit. Compare the lack of growth of the nitrosation peaks (e.g., peaks at retention times 8.52 and 8.66) in FIGS. 14A and 14B, with the lack of peaks in FIGS. 15A and 15B.

[0096] Accordingly, in other embodiments of this invention, the hybrid organic-inorganic is not restricted to C2 coatings but to any coating or ligand that can mask metal-analyte interactions. Thus, different modes of chromatography as well as various analytes (i.e., not just amines) can benefit from this protection. For example, the present technology can be used in the separation or study of any metal reactive analyte—such as for example, anilines). Alternative byproducts of degradation or metal-catalyzed reactions can also be prevented or reduced. It can be foreseen that this technology can be applied to the entire LC system or MS instrument or even different surfaces, such as sample vials or mobile phase containers, to prevent surface-catalyzed reactions that could occur within these instruments or containers.

Example 3: On-Column Oxidation of Different Column Technologies

[0097] Baicalein, baicalein oxide, propyl gallate, and propyl gallate oxide were prepared in methanol/water (1:1, v/v) to create a single sample. Analyses of the sample (i.e., the analytes in the sample) were performed using a Waters ACQUITY UPLC H-Class Bio Binary system and the separation conditions outlined below using a stainless steel, uncoated column with uncoated frit (Column A) and a coated stainless steel column with coated frit (Column B). The mobile phase included acetonitrile as well as formic acid, which are known to cause polyphenol oxidation of these analytes in the presence of metals. FIGS. 16A-16B present the on-column oxidation results of the separation from the first injection on an uncoated stainless steel column (Column A); and FIGS. 16C-16D present the results on a C2 coated stainless steel column (Column B). Mass spectra are presented, with the compounds and their oxidized forms labeled in the mass spectra. These spectra show that the oxide peaks (i.e., baicalein oxide and propyl gallate oxide) are significantly greater (i.e., contain greater amount of the oxide forms) in the uncoated Column A, than in coated Column B (which included a titanium frit having a coating covering its wetted surfaces).

TABLE-US-00006 TABLE 6 Separation details for Example 3 Test Conditions Column A Hybrid Silica C18, 130 angstroms, 1.7 μm (Conventional) packed within a stainless steel 2.1 × 50 mm column with stainless steel frit Column B (Coated Hybrid Silica C18, 130 angstroms, 1.7 μm Column) packed within a C2 coated stainless steel column 2.1 × 50 mm; with coated titanium frit Sample 0.1 mg/mL solutions of baicalein, baicalein oxide, propyl gallate, and propyl gallate oxide Solvent Conditions Solvent Line A 0.1% formic acid in water Solvent Line B Acetonitrile Column Temperature 30° C. Injection Volume 0.5 μL (first injection) Diluent Methanol/water (1:1, v/v) UV Detection 280 nm Gradient Table: Time (min) Flow Rate (mL/min) % A % B Curve Initial 0.400 95.0 5.0 Initial 10.00 0.400 30.0 70.0 6 10.10 0.400 95.0 5.0 6 12.00 0.400 95.0 5.0 6