Patent Application
20200215457
This technology relates to the use of vapor deposition coated flow paths for improved chemical separation (e.g., chromatography) and other analytical or preparative processes (e.g., extraction, filtration, sample transfer, fluid handlers and multi-channel processing). More specifically, this technology relates to devices used in the analysis or preparation of fluid samples having coated flow paths, methods of analyzing or preparing a sample (for example, glycans, peptides, pesticides, and citric acid cycle metabolites) using a fluidic system that includes coated flow paths, and methods of tailoring a fluidic flow path for an improved processing, analysis or preparation of a sample.
Analytes that interact with metal have often proven to be very challenging to separate. The desire to have high pressure capable chromatographic systems with minimal dispersion has required that flow paths decrease in diameter and be able to withstand increasingly high pressures at increasingly fast flow rates. As a result, the material of choice for chromatographic flow paths is often metallic in nature. This is despite the fact that characteristics of certain analytes, for example, biomolecules, proteins, glycans, peptides, oligonucleotides, pesticides, bisphosphonic acids, anionic metabolites, and zwitterions like amino acids and neurotransmitters, are known to have unfavorable interactions, so called chromatographic secondary interactions, with metallic surfaces.
The proposed mechanism for metal specific binding interactions requires an understanding of the Lewis theory of acid-base chemistry. Pure metals and metal alloys (along with their corresponding oxide layers) have terminal metal atoms that have characteristics of a Lewis acid. More simply, these metal atoms show a propensity to accept donor electrons. This propensity is even more pronounced with any surface metal ions bearing a positive charge. Analytes with sufficient Lewis base characteristics (any substance that can donate non-bonding electrons) can potentially adsorb to these sites and thus form problematic non-covalent complexes. It is these substances that are defined as metal-interacting analytes.
For example, analytes having phosphate groups are excellent polydentate ligands capable of high affinity metal chelation. This interaction causes phosphorylated species to bind to the flow path metals thus reducing the detected amounts of such species, a particularly troublesome effect given that phosphorylated species are frequently the most important analytes of an assay.
Other characteristics of analytes can likewise pose problems. For example, carboxylate groups also have the ability to chelate to metals, albeit with lower affinities than phosphate groups. Yet, carboxylate functional groups are ubiquitous in, for example, biomolecules, giving the opportunity for cumulative polydentate-based adsorptive losses. These complications can exist not only on peptides and proteins, but also glycans. For example, N-glycan species can at times contain one or more phosphate groups as well as one or more carboxylate containing sialic acid residues. Additionally, smaller biomolecules such as nucleotides and saccharides, like sugar phosphates, can exhibit similar behavior to the previously mentioned N-glycan molecules. Moreover, chromatographic secondary interactions can be especially problematic with biomolecules, particularly larger structures, because they have a capacity (via their size and structural order) to form microenvironments that can adversely interact with separation components and flow path surfaces. In this case, a biomolecule or analyte having larger structures, can present structural regions with chemical properties that amplify a secondary interaction to the material of a flow path. This, combined with the cumulative metal chelation effects curtails the overall effective separation of biomolecules, pesticides, bisphosphonic acids, anionic metabolites, and zwitterions like amino acids and neurotransmitters.
An alternative to using metal flow paths is to use flow paths constructed from polymeric materials, such as polyether ether ketone (PEEK). PEEK tubing, like most polymeric materials, is formed by means of an extrusion process. With polymeric resin, this manufacturing process can lead to highly variable internal diameters. Accordingly, PEEK column hardware yields unfavorable differences in the retention times as can be observed from switching between one column and the next. Often, this variation can be a factor of three higher than a metal constructed column. In addition, the techniques for fabricating polymer based frits are not yet sufficiently optimized to afford suitably rugged components for commercial HPLC columns. For example, commercially available PEEK frits tend to exhibit unacceptably low permeability.
Other analytical or preparative devices that include fluidic flow paths experience similar challenges. These devices can be made from metals, polymeric materials (e.g., PEEK, polypropylene), plastics or glass. A common example includes any and all labware. It is a common occurrence for analytes to adsorb and be lost to labware during the manipulation of samples prior to and during analysis. Labware prone to these issues can include, but is not limited to, beakers, centrifuge tubes, pipette tips, solid phase extraction devices, molecular weight cutoff apparatus, dialysis chambers, and LC autosampler vials and well plates. Adsorptive losses to the labware decreases the strength of analytical results or amount of preparative sample.
For example, most pipette tips are made of polypropylene, as it is preferred for the sake of chemical resistance to common acids, bases and organic solvents. However, the hydrophobicity of polypropylene is known to cause high levels of adsorptive losses when used with biological analytes, like proteins and peptides. As a result, polypropylene can be identified as a major contributor to undesired sample loss Likewise, the frits that are commonly used in extraction devices can also cause issues with adsorptive losses. In general, frits for extraction devices are commonly made from a breathable high density polyethylene or from polypropylene, such as for example Vyon® F material (available from PAR Group Limited, UK). These materials are also sufficiently hydrophobic to cause adsorptive analyte loss.
Ongoing efforts to reduce interaction between wetted surfaces and fluidic samples to provide improved outcomes are therefore needed.
One advantage of the alkylsilyl coatings of the present technology is that metal chromatographic flow paths can be used while minimizing the interactions between analytes and the metal flow paths. Coating the flow path of instrumentation and chromatographic devices with certain alklysilyl compositions improves multiple aspects of liquid chromatography separations where the analyte of interest is a metal-interacting analyte. The use of alkylsilyl coatings on metal flow paths allow the use of metal chromatographic flow paths, which are able to withstand high pressures at fast flow rates, while minimizing the secondary chromatographic interactions between the analyte and the metal. Therefore, high pressure components can be manufactured out of stainless steel or other metallic or high pressure material. These components made of high pressure material can then be tailored in that the internal flow paths can be modified with a coating to address the hydrophobicity of the flow path and reduce secondary chromatographic interactions. The entire summary of the technology will be rewritten after claims are finalized
Provided herein, therefore, are methods for isolating analytes comprising the use of vapor depositing one or more alklysilyl derivatives to at least one component of a fluidic system to form a bioinert or low-bind coating, and eluting the analyte through the system. Unlike ambient, liquid phase silanization, coatings which are vapor deposited tend to produce, more resilient modifications of substrates with precisely controlled thicknesses. Also, because vapor deposition is a non-line-of-sight process, this leads to a more uniform coating over substrate contours and complex surfaces. This advantage allows for coatings to be applied to flow paths with narrow internal diameters and curved surfaces, therefore addressing the need for increasingly high pressures at increasingly fast flow rates.
Also provided herein are methods of tailoring a fluidic flow path for separation of a sample comprising an analyte that includes infiltrating a vaporized source of one or more alkylsilyl derivatives through the fluidic flow path to form a bioinert (or low-bind) coating and controlling temperature and pressure to deposit a first coating on wetted surfaces of the flow path.
Also provided are methods of tailoring a fluidic flow path for separation of a sample including an analyte comprising assessing the polarity of the analyte, selecting an appropriate alkylsilyl derivative, and adjusting the hydrophobicity of wetted surfaces of the flow path by vapor depositing the appropriate alkylsilyl derivative to form a bioinert, low-bind coating.
Further provided herein are methods of improving baseline returns in a chromatographic system comprising introducing a sample including an analyte into a fluidic system comprising at least one vapor deposited alkylsilyl derivative to form a bioinert, low-bind coating, and eluting the sample through the system.
The disclosed methods can be applied to stainless steel or other metallic flow path components and provides a manufacturing advantage over alternative non-metallic or non-metallic lined components.
In one aspect, the technology includes a chromatographic device for separating analytes in a sample. The device includes a sample injector having a sample injection needle for injecting the sample into the mobile phase, a sample reservoir container in fluid communication with the sample injector, a chromatography column downstream of the sample injector, the chromatography column having fluid connectors, and fluid conduits connecting the sample injector and the chromatography column. Interior surfaces of the fluid conduits, sample injector, sample reservoir container, and chromatography column form a fluidic flow path having wetted surfaces. At least a portion of the wetted surfaces of the fluidic flow path are coated with a alkylsilyl coating. The alkylsilyl coating is inert to at least one of the analytes in the sample. The alkylsilyl coating is deposited by a thermal decomposition of a carbosilane followed by an oxidation to completely cover the at least a portion of the wetted surfaces with the alkylsilyl coating.
The device can include one or more of the following embodiments in any combination thereof.
The alkylsilyl coating can have a contact angle of at least 15°. In some embodiments, the alkylsilyl coating has a contact angle less than or equal to 30° or less than or equal to 115°. In some embodiments, the alkylsilyl coating has a contact angle of between 5 and 115 degrees. In some embodiments, the alkylsilyl coating has a contact angle of between 15 and 85 degrees.
The chromatographic device can also include a detector downstream of the chromatography column. The fluidic flow path can also include the detector. In some embodiments the detector is a mass spectrometer and the fluidic flow path includes wetted surfaces of an electrospray needle or at least the flow path includes tubing and intake to MS device.
In some embodiments, the fluidic flow path has a length to diameter ratio of at least 20. The alkylsilyl coating can have a thickness of at least 100 Å uniformly coated.
In some embodiments, the wetted surface of the fluidic flow path is defined at least in part by an interior surface of a column or an interior surface of a sample injection needle. The wetted surface of the fluidic flow path can extend from an interior surface of a sample injection needle through the interior surface of a column. In some embodiments, the wetted surface of the fluidic flow path extends from a sample reservoir container disposed upstream of an in fluidic communication with an interior surface of a sample injection needle throughout the fluidic system to a connector or port of a detector.
In some embodiments, the alkylsilyl coating is deposited by a thermal decomposition of a carbosilane followed by an oxidation and a functionalization with silane to completely cover the at least a portion of the wetted surfaces with the alkylsilyl coating.
In some embodiments, the functionalization with silane includes treating with an organosilane reagent.
In some embodiments, the carbosilane is selected from the group consisting of: dimethylsilane, trimethylsilane, dialkylsilyl dihydride, alkylsilyl trihydride, bis(trichlorosilyl)ethane, bis(trimethoxysilyl)ethane, (3-glycidyloxypropyl) trimethoxysilane, n-decyltrichlorosilane, trimethylchlorosilane, trimethyldimethyaminosilane, methoxy-polyethyleneoxy(1-10) propyl trichlorosilane, or methoxypolyethyleneoxy(1-10) propyl trimethoxysilane, and combinations thereof.
In some embodiments, one or more of the following reagents are used in the oxidation of the thermally decomposed carbosilane: water, oxygen, air, nitrous oxide, ozone, or peroxide.
In some embodiments, the alkylsilyl coating does not affect retentivity of the sample.
In some embodiments, the alkylsilyl coating includes one or more of the following groups: N—OH, Si—OH or C—OH.
In one aspect, the technology includes method of separating a sample. The method includes introducing the sample to a fluidic system including a flow path disposed in an interior of the fluidic system, the flow path includes an alkylsilyl coating covering wetted surfaces and deposited on the wetted surfaces by thermal decomposing a carbosilane followed by oxidizing the wetted surface. The alkylsilyl coating is inert to at least one analyte in the sample.
The method can include one or more of the following embodiments in any combination thereof.
In one embodiment, the method includes functionalizing after oxidizing the decomposed carbosilane.
In one embodiment, the method includes controlling an amount of oxidation after decomposing the carbosilane to adjust the percentage of Si—C bonds in the alkylsilyl coating.
In one embodiment, the method includes tuning the oxidizing by controlling the amount of one or more of the following groups: N—OH, Si—OH or C—OH.
In one embodiment, the method includes tuning the oxidized surface by controlling the ratio of Si—OH and C—OH groups to C—H and Si—C groups.
In one aspect, the technology includes a method of improving separation of a sample including at least one analyte. The method includes creating an alkylsilyl coating covering at least a portion of a fluidic flow path in a separation device, wherein the alkylsilyl coating is inert to the at least one analyte and is deposited by: (i) decomposing a carbosilane vapor within the fluidic flow path; (ii) followed by oxidizing the coating to create an oxidized surface; and (iii) tuning the oxidized surface by controlling the ratio of Si—OH and C—OH groups to C—H and Si—C groups; and injecting the sample into the separation device to flow along the coated fluidic flow path for separation.
In another aspect, the technology includes a method of improving separation of a sample including at least one analyte. The method includes creating an alkylsilyl coating covering at least a portion of a fluidic flow path in a separation device, wherein the alkylsilyl coating is inert to the at least one analyte and is deposited by: (i) decomposing a carbosilane vapor within the fluidic flow path; (ii) followed by oxidizing the coating to create an oxidized surface; and (iii) tuning the oxidized surface by controlling the amount of N—OH, Si—OH and C—OH groups; and injecting the sample into the separation device to flow along the coated fluidic flow path for separation.
The above two aspects can include one or more of the following embodiments in any combination thereof.
In one embodiment, the method can include functionalizing after oxidizing the decomposed carbosilane.
In one embodiment, the method can include controlling an amount of oxidation after decomposing the carbosilane to adjust the percentage of Si—C bonds in the alkylsilyl coating.
In one embodiment, the method can include controlling deposition of the alkylsilyl coating to create a contact angle of between 5 degrees and 115 degrees.
In another embodiment, the method can include controlling deposition of the alkylsilyl coating to create a contact angle of between 15 and 85 degrees.
In general, a number of aspects of the technology are directed to (1) devices having an alkylsilyl coating; (2) methods of tailoring or tuning a flow path for isolation of an analyte or processing a sample; (3) method of isolating an analyte in a sample, in particular a metal-interacting analyte; and (4) kits comprising various labware or chromatographic components coated with an alkylsilyl coating and instructions for use. In some aspects, a bioinert, low-bind coating is used to modify a flow path to address flow path interactions with an analyte or sample to be processed. That is, the bioinert, low-bind coating minimizes surface reactions with the interacting analyte and allows the analyte to pass along a flow path without clogging, attaching to surfaces, or change in analyte properties. The reduction/elimination of these interactions is advantageous because it allows for accurate quantification and analysis of a sample containing an interacting analyte, for example biomolecules, proteins, glycans, peptides, oligonucleotides, pesticides (e.g., glyphosate), bisphosphonic acids (e.g., bisphosphonates), anionic metabolites, and zwitterions like amino acids and neurotransmitters. The biomolecule can be selected from a peptide or peptide fragment, an oligopeptide, a protein, a glycan, a nucleic acid or nucleic acid fragment, a growth factor, a carbohydrate, a fatty acid, and a lipid. In one aspect, the biomolecule is a protein, a peptide, or a glycan. The biomolecule can be a phosphoglycan or a phosphopeptide.
In the present technology, vapor deposited alkylsilyl coatings on wetted surfaces of fluidic systems (e.g., liquid chromatography systems, extraction devices, pipettes, etc) to modify the fluidic path and decrease secondary interactions. As such, they are bioinert or low-bind (meaning that analytes of a sample do not interact, or have minimal interaction, with the alkylsilyl coating). In addition, the deposited coatings are highly tunable to provide a range of desirable contact angles (e.g., make the wetted surfaces hydrophilic or hydrophobic), chemistries, and properties to achieve a desired effect on the flow path and ultimately the sample passing through the flow path.
The detector 150, can be a mass spectrometer. The fluidic flow path can include wetted surfaces of an electrospray needle (not shown).
At least a portion of the wetted surfaces can be coated with an alkyl silyl coating, described in detail herein, to tailor its hydrophobicity. The coating can be applied by vapor deposition. As such, methods and devices of the present technology provide the advantage of being able to use high pressure resistant materials (e.g., stainless steel) for the creation of the flow system, but also being able to tailor the wetted surfaces of the fluidic flow path to provide the appropriate hydrophobicity so deleterious interactions or undesirable chemical effects on the sample can be minimized.
The alkylsilyl coating can be provided throughout the system from the tubing or fluid conduits 110 extending from the fluid manager system 105 all the way through to the detector 150. The coatings can also be applied to portions of the fluidic fluid path. That is, one may choose to coat one or more components or portions of a component and not the entire fluidic path. For example, the internal portions of the column 125 and its frits 120 and end caps 115 can be coated whereas the remainder of the flow path can be left unmodified. Further, removable/replaceable components can be coated. For example, the vial or sinker 130 containing the sample reservoir can be coated as well as frits 120.
In one aspect, the flow path of the fluidic systems described herein is defined at least in part by an interior surface of tubing. In another aspect, the flow path of the fluidic systems described herein is defined at least in part by an interior surface of microfabricated fluid conduits. In another aspect, the flow path of the fluidic systems described herein is defined at least in part by an interior surface of a column. In another aspect, the flow path of the fluidic systems described herein is defined at least in part by passageways through a frit. In another aspect, the flow path of the fluidic systems described herein is defined at least in part by an interior surface of a sample injection needle. In another aspect, the flow path of the fluidic systems described herein extends from the interior surface of a sample injection needle throughout the interior surface of a column. In another aspect, the flow path extends from a sample reservoir container (e.g. sinker) disposed upstream of and in fluidic communication with the interior surface of a sample injection needle throughout the fluidic system to a connector/port to a detector.
In some embodiments, only the wetted surfaces of the chromatographic column and the components located upstream of the chromatographic column are coated with the alkylsilyl coatings described herein while wetted surfaces located downstream of the column are not coated. The coating can be applied to the wetted surfaces via vapor deposition. Similarly, the “wetted surfaces” of labware or other fluid processing devices may benefit from alkylsiyl coatings described herein. The “wetted surfaces” of these devices not only include the fluidic flow path, but also elements that reside within the fluidic flow path. For example, frits and/or membranes within a solid phase extraction device come in contact with fluidic samples. As a result, not only the internal walls within a solid phase extraction device, but also any frits/membranes are included within the scope of “wetted surfaces.” All “wetted surfaces” or at least some portion of the “wetted surfaces” can be improved or tailored for a particular analysis or procedure by including one or more of the coatings described herein. The term “wetted surfaces” refers to all surfaces within a device (e.g., chromatography column, chromatography injection system, chromatography fluid handling system, labware, solid phase extraction device, pipette tips, centrifuge tubes, beakers, dialysis chambers, etc.) that come into contact with a fluid, especially a fluid containing an analyte of interest.
At least a portion of the wetted surfaces are coated with an alkylsilyl coating. The alkylsilyl coating is inert to at least one of the analytes in the sample. The alkylsilyl coating can have the
Formula I:
R1, R2, R3, R4, R5, and R6 are each independently selected from (C1-C6)alkoxy, —NH(C1-C6)alkyl, —N((C1-C6)alkyl)2, OH, ORA, and halo (i.e., a halogen, for example chloro). RA represents a point of attachment to the interior surfaces of the fluidic system. At least one of R1, R2, R3, R4, R5, and R6 is ORA. X is (C1-C20)alkyl, —O[(CH2)2O]1-20—, —(C1-C10)[NH(CO)NH(C1-C10)]1-20—, or —(C1-C10)[alkylphenyl(C1-C10)alkyl]1-20—.
When used in the context of a chemical formula, a hyphen (“-”) indicates the point of attachment. For example, when X is —[(C1-C10)alkylphenyl(C1-C10)alkyl]1-20—, that means that X is connected to SiR1R2R3 via the (C1-C10)alkyl and connected to SiR4R5R6 via the other (C1-C10)alkyl. This applies to the remaining variables.
In one aspect, X in Formula I is (C1-C15)alkyl, (C1-C12)alkyl, or (C1-C10)alkyl. In some aspects, X in Formula I is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, iso-butyl, t-butyl, pentyl, hexyl, heptyl, nonyl, or decanyl. In other aspect, X in Formula I is ethyl or decanyl.
In one aspect, at least one of R1, R2, R3, R4, R5, and R6 is (C1-C6)alkoxy, e.g., ethoxy, wherein the values for X are described in Formula I or the preceding paragraph. In another aspect, at least two of R1, R2, R3, R4, R5, and R6 is (C1-C6)alkoxy, e.g., ethoxy, wherein the values for X are described in Formula I or the preceding paragraph. In another aspect, at least three of R1, R2, R3, R4, R5, and R6 is (C1-C6)alkoxy, e.g., ethoxy, wherein the values for X are described in Formula I or the preceding paragraph. In another aspect, at least four of R1, R2, R3, R4, R5, and R6 is (C1-C6)alkoxy, e.g., ethoxy, wherein the values for X are described in Formula I or the preceding paragraph. In another aspect, at least five of R1, R2, R3, R4, R5, and R6 is (C1-C6)alkoxy, e.g., ethoxy, wherein the values for X are described in Formula I or the preceding paragraph.
In one aspect, at least one of R1, R2, R3, R4, R5, and R6 is halo, e.g., chloro, wherein the values for X are described in Formula I or the preceding paragraphs above. In another aspect, at least two of R1, R2, R3, R4, R5, and R6 is halo, e.g., chloro, wherein the values for X are described in Formula I or the preceding paragraphs above. In another aspect, at least three of R1, R2, R3, R4, R5, and R6 is halo, e.g., chloro, wherein the values for X are described in Formula I or the preceding paragraphs above. In another aspect, at least four of R1, R2, R3, R4, R5, and R6 is halo, e.g., chloro, wherein the values for X are described in Formula I or the preceding paragraphs above. In another aspect, at least five of R1, R2, R3, R4, R5, and R6 is halo, e.g., chloro, wherein the values for X are described in Formula I or the preceding paragraphs above.
In another aspect, R1, R2, R3, R4, R5, and R6 are each methoxy or chloro.
In some embodiments, the alkylsilyl coating of Formula I is a organosilica coating. In certain embodiments, the alkylsilyl coating of Formula I is a hybrid inorganic/organic material that forms the wetted surface or that coats the wetted surfaces.
The alkylsilyl coating of Formula I can have a contact angle of at least about 15°. In some embodiments, the alkylsilyl coating of Formula I 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 alkylsilyl coating of Formula I 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 alkylsilyl coating of Formula I 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°.
The thickness of 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 alkylsilyl coating for Formal I 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 alkylsilyl 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. Thus, coatings with pronounced visual distinction are an embodiment of this technology. 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.
In one aspect, the vapor deposited coating of Formula I is the product of vapor deposited bis(trichlorosilyl)ethane, bis(trimethoxysilyl)ethane, bis(trichlorosilyl)octane, bis(trimethoxysilyl)octane, bis(trimethoxysilyl)hexane, and bis(trichlorosilyl)hexane.
In some aspects, at least a portion of the wetted surfaces are coated with multiple layers of the same or different alkyslilyls, 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 (e.g., internal surfaces of the fluidic flow path of the chromatographic system/device or internal surfaces or fluid interfacing/contacting surfaces of labware or other analytical devices, such as frits within a solid phase extraction device together with interior walls of the solid phase extraction device). In this manner, increasingly thick bioinert coatings can be produced and tailored to achieve desirable separations.
The chromatographic device can have a second alkylsilyl coating in direct contact with the alkylsilyl coating of Formula I. The second alkylsilyl coating has the Formula II
wherein R7, R8, and R9 are each independently selected from —NH(C1-C6)alkyl, —N[(C1-C6)alkyl]2, (C1-C6)alkoxy, (C1-C6)alkyl, (C1-C6)alkenyl, OH, and halo; R10 is selected from (C1-C6)alkyl, —ORB, —[O(C1-C3)alkyl]1-100(C1-C6)alkyl, —[O(C1-C3)alkyl]1-10OH and phenyl. (C1-C6)alkyl is optionally substituted with one or more halo. The phenyl is optionally substituted with one or more groups selected from (C1-C3)alkyl, hydroxyl, fluorine, chlorine, bromine, cyano, —C(O)NH2, and carboxyl. RB is —(C1-C3)alkyloxirane, —(C1-C3)alkyl-3,4-epoxycyclohexyl, or —(C1-C4)alkylOH. The hashed bond to R10 represents an optional additional covalent bond between R10 and the carbon bridging the silyl group to form an alkene, provided y is not 0. y is an integer from 0 to 20.
In one aspect, y in Formula II is an integer from 1 to 15. In another aspect, y in Formula II is an integer from 1 to 12. In another aspect, y in Formula II is an integer from 1 to 10. In another aspect, y in Formula II is an integer from 2 to 9.
In one aspect R10 in Formula II is methyl and y is as described above for Formula II or the preceding paragraph.
In one aspect, R7, R8, and R9 in Formula II are each the same, wherein R10 and y are as described above. In one aspect, R7, R8, and R9 are each halo (e.g., chloro) or (C1-C6)alkoxy such as methoxy, wherein R10 and y are as described above.
In one aspect, y in Formula II is 9, R10 is methyl, and R7, R8, and R9 are each ethoxy or chloro.
In one aspect, the coating of the formula II 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.
The alkylsilyl coating of Formula I and II can have a contact angle of at least about 15°. In some embodiments, the alkylsilyl coating of Formula I and II can have a contact angle of less than or equal to 105°. The contact angle can be less than or equal to about 115°. In other embodiments, the contact angle can be less than or equal to about 90°. In some embodiments, the contact angle of the alkylsilyl coating of Formula I and II is between about 15° to about 115°. For example, the contact angle of the alkylsilyl coating of Formula I and II 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°.
The thickness of the multi-layered 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 multi-layered alkylsilyl coating for Formal I 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.
In one aspect, the alkylsilyl coating of Formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula II is (3-glycidyloxypropyl)trimethoxysilane. In another aspect, the alkylsilyl coating of Formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula II is (3-glycidyloxypropyl)trimethoxysilane followed by hydrolysis. In one aspect, the alkylsilyl coating of Formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula II is n-decyltrichlorosilane. The alkylsilyl coating of Formula I can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula II can be trimethylchlorosilane or trimethyldimethyaminosilane. In one aspect, the alkylsilyl coating of Formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula II is methoxy-polyethyleneoxy(3) propyl tricholorosilane or methoxy-polyethyleneoxy(3) propyl trimethoxysilane.
The chromatographic device can have an alkylsilyl coating in direct contact with the alkylsilyl coating of Formula III in direct contact with the alkylsilyl coating of Formual I.
R11, R12, R13, R14, R15, and R16 are each independently selected from (C1-C6)alkoxy, —NH(C1-C6)alkyl, —N((C1-C6)alkyl)2, OH, and halo (i.e., a halogen, for example, chloro). Z is (C1-C20)alkyl, —O[(CH2)2O]1-20—, —(C1-C10)[NH(CO)NH(C1-C10)]1-20—, or —(C1-C10)[alkylphenyl(C1-C10)alkyl]1-20—.
In some aspects, Z in Formula III is (C1-C10)alkyl; and R1, R2, R3, R4, R5, and R6 are each methoxy or chloro. In other aspects, Z in Formula III is (C2-C10)alkyl. In other aspects, Z in Formula III is ethyl.
In the layered alkylsilyl coating of Formula I and Formula III, Formula I and Formula III can be the same (for example, C2C2) or Formula I and Formula III can be different. Formula III is attached directly to the coating of Formula I, i.e., in Formula III, there is no point of attachment to the interior of the fluidic system; instead Formula III is deposited directly on Formula I.
The alkylsilyl coating of Formula III can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane. The alkylsilyl coating of Formula I can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula III can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.
The alkylsilyl coating of Formula I and III can have a contact angle of at least about 15°. In some embodiments, the alkylsilyl coating of Formula I and III can have a contact angle of less than or equal to 115°. The contact angle can be less than or equal to about 115°. In some embodiments, the contact angle of the alkylsilyl coating of Formula I and III is between about 15° to about 115°. For example, the contact angle of the alkylsilyl coating of Formula I and III 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°.
The thickness of the multi-layered 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 multi-layered alkylsilyl coating for Formula I 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.
In one aspect, the alkylsilyl coating of Formula II is applied directly to wetted surfaces of the fluidic flow path. Therefore, in some embodiments, one of R7, R8, and R9 of Formula II can also include ORA, where RA represents a point of attachment to the interior surfaces (e.g., wetted surfaces) of the fluidic system. In other embodiments, R7, R8, and R9 of the alkylsilyl coating of Formula II does not include ORA, by the alkylsilyl coating of Formula II is deposited directly onto wetted surfaces of the fluidic flow path that have been pre-treated with, for example, a plasma.
In one aspect, stainless steel flow path components, including but not limited to tubing, microfabricated fluid conduits, column frits, column inlet tubing, and sample injection needles, are coated via vapor deposition with one or more of the disclosed alkylsilyls. In one aspect, these coated components are annealed to alter their chemical or physical properties.
In another aspect, flow path components that are made of other materials than stainless steel or other metallics, (e.g., polymers, glass, etc.) are coated via vapor deposition with one or more of the disclosed alkylsilyls. In particular, frits for use within the chromatography or fluid injection system or sample vials connectable to the injection needle are coated.
In another aspect, wetted surfaces of labware or at least some portion of wetted surfaces of labware are coated via vapor deposition with one or more of the disclosed alkylsilyls. In certain embodiments, the vapor deposited coatings are used to minimize adsorptive losses of the sample. In some embodiments, the vapor deposited coating is both neutral (low in ionic properties) and hydrophilic (exhibiting a contact angle less than)60°. The coating can be used to mitigate issues with many different types of materials, including glass and polymeric compositions, such as polypropylene or polyethylene.
Exemplary coatings with their respective approximate thickness and contact angle are provided in Table 1.
Referring to VPD #1 (C2-GPTMS-OH), the first coating layer, C2 shown below, is a layer according to Formula I, described above.
structure of bis(trichlorosilyl)ethane or bis(trismethoxysilyl)ethane (C2)
The second layer of VPD #1, GPTMS-OH, shown below, is a layer according to Formula II.
VPD #3 (C2-C2) is an example of a coating of Formula I and then a coating for Formula III.
VPD #7 (C2C10) is another example of a coating of Formula I and a second layer of Formula II. The structure of bis(trichlorosilyl)ethane or bis(trismethoxysilyl)ethane (C2) is shown above. The structure of C10 is shown below.
VPD #11 (C2C3) is another example of a coating of Formula I and a second layer of Formula II. The structure of bis(trichlorosilyl)ethane or bis(trismethoxysilyl)ethane (C2) is shown above. The structure of C3 is shown below.
VPD #13 is another example of a coating of Formula I and a second layer of Formula II. The structure of bis(trichlorosilyl)ethane or bis(trismethoxysilyl)ethane (C2) is shown above. The structure of methoxy-polyethyleneoxy(3)propyl trichlorosilane (PEO) is shown below.
Alternatively, commercially available vapor deposition coatings can be used in the disclosed systems, devices, and methods, including but not limited to Dursan® and Dursox® (both commercially available from SilcoTek Corporation, Bellefonte, Pa.). The process for making is described in U.S. application Ser. No. 14/680,669, filed on Apr. 7, 2015, and entitled “Thermal Chemical Vapor Deposition Coated Article and Process,” which claims priority to and benefit of U.S. Provisional Application No. 61/976,789 filed Apr. 8, 2014. The contents of each application are incorporated herein by reference in their entirety.
A coating process 400 (see
The article 503 includes a surface 505, which is or includes the interior surface, an exterior surface, or a combination thereof. The surface 505 has surface properties achieved through the coating process 400 controllably depositing a layer 502. The layer 502 imparts a surface effect to the substrate 500, the coating 501, the article 503, or combinations thereof. The substrate 500 is any suitable substrate, such as, a metallic substrate (ferrous or non-ferrous), stainless steel, titanium, a glass substrate, a ceramic substrate, ceramic matrix composite substrate, a composite metal substrate, a coated substrate, a fiber substrate, a foil substrate, a film, or a combination thereof.
The pretreatment (step 402) is or includes any suitable techniques taken to prepare a chamber, the surface 505, the substrate 500, or a combination thereof. In one embodiment, the chamber is a chemical vapor deposition chamber, for example, with tubing connections to allow gas flow in and out of the chemical vapor deposition chamber. In a further embodiment, the chamber includes multiple controlled inlets and outlets configured for providing and removing multiple gas streams and/or a vacuum connected to one or more outlet tubes.
Suitable techniques for the pretreatment (step 402) include, but are not limited to, cleaning, pre-heating, isolating the substrate 500 and/or the surface 505, surface treatment techniques, evacuating the chamber (for example, with the flow of gas and/or maintenance of a vacuum in the chamber providing a controlled atmosphere), flushing/purging the chamber (for example, with an inert gas such as nitrogen, helium, and/or argon), or a combination thereof. In one embodiment, a heat source controls the temperature in the chamber, for example, to desorb water and remove contaminants from the surface 505. In one embodiment, the heating is at a temperature above about 100° C. (for example, about 450° C.) and/or at a pressure (for example, between about 1 atmosphere and about 3 atmospheres, between about 1 atmosphere and about 2 atmospheres, between about 2 atmospheres and about 3 atmospheres, about 1 atmosphere, about 2 atmospheres, about 3 atmospheres, or any suitable combination, Sub-combination, range, or Sub-range therein). In one embodiment, the heating is for a period of time (for example, between about 3 minutes and about 15 hours, between about 0.5 hours and about 15 hours, for about 3 minutes, for about 0.5 hours, for about 2 hours, for about 15 hours, or any suitable combination, sub-combination, range, or sub-range therein).
In one embodiment, the pretreatment (step 402) includes pre-exposure of the substrate 500 to a thermal oxidative environment. Pre-exposure of the substrate 500 to the thermal oxidative environment pre-oxidizes the surface 505 of the substrate 500, increasing stability of both the surface 505 and the substrate 500. The increased stability of the substrate 500 increases the stability of the coating 501 formed over the substrate 500.
The thermal oxidative environment is at any suitable temperature(s) allowing oxidation. Suitable temperatures include, but are not limited to between about 100° C. and about 700° C., between about 100° C. and about 450° C., between about 100° C. and about 300° C., between about 200° C. and about 500° C., between about 300° C. and about 600° C., between about 450° C. and about 700° C., about 700° C., about 450° C., about 100° C., or any suitable combination, sub-combination, range, or sub-range thereof.
The substrate 500 is pre-exposed to the thermal oxidative environment for any suitable duration allowing oxidation. Suitable duration including, but are not limited to, between about 30 minutes and 6 hours, between about 30 minutes and about 4 hours, between about 1 hour and about 4 hours, up to about 10 hours, up to about 4 hours, up to about 2 hours, up to about 30 minutes, or any combination, sub combination, range or sub-range thereof.
The increased stability of the coating 501 is detectable by contact angle measurements for both water and hexadecane, for example, after exposure of the substrate 500 to room air at 450° C. for 30 minutes. In one embodiment, the substrate 500 is X40CrMoV5-1 having a composition including by weight percent between about 0.37% and about 0.42% carbon, between about 0.90% and about 1.20% silicon, between about 0.30% and about 0.50% manganese, up to about 0.030% phosphorous, up to about 0.030% sulfur, between about 4.80% and about 5.50% chromium, between about 1.20% and about 1.50% molybdenum, between about 0.90% and about 1.10% vanadium, the rest being substantially iron.
In another embodiment, without pre-oxidation of the substrate 500, the contact angle of water on X40CrMoV5-1 after 30 minutes of exposure to 450° C. in room air drops to 28.8° from an initial value of 146.9, a 118.1 change. However, with pre-oxidation of the substrate 500, the contact angle of water on X40CrMoV5-1 after 30 minutes of exposure to 450° C. in room air increases to 127.4° from an initial value of 126.2°, a 1.2° change. In another example, without pre-oxidation the contact angle of hexadecane on X40CrMoV5-1 after 30 minutes of exposure to 450° C. in room air drops to approximately 0° from an initial value of 92.3°, a 92.3° change. However, with pre-oxidation, the contact angle of hexadecane on X40CrMoV5-1 after 30 minutes of exposure to 450° C. in room air increases to 72.1° from an initial value of 66.5°, a 5.6° change.
The thermal decomposition (step 404) is or includes thermal decomposition of one or more precursor materials. In one embodiment, the precursor material is or includes dimethylsilane, for example, in gaseous form. In general, dimethylsilane is not readily obtainable due to the low demand for it. Dimethylsilane has been regarded as undesirable in some chemical vapor deposition applications because it includes carbon and is much more expensive than silane. Silane and the monomethyl analogue to dimethylsilane, methylsilane, are both pyrophoric and may explode in air. Dimethylsilane, although flammable, is not pyrophoric. Thus, use of dimethylsilane decreases safety risks. In addition, use of dimethyl silane results in inertness of a coating and/or chemical resistance, thereby protecting the surface 505 of the substrate 500. Other suitable precursor materials include, but are not limited to, trimethylsilane, dialkylsilyl dihydride, alkylsilyl trihydride, and combinations thereof. In one embodiment, the materials are non-pyrophoric, for example, dialkylsilyldihydride and/or alkylsilyl trihydride.
The thermal decomposition (step 404) includes any suitable thermal decomposition parameters corresponding to the precursor material, for example, as is described in U.S. Pat. No. 6,444,326, which is incorporated herein by reference in its entirety, to apply material through deposition. If a thicker deposition of the layer 502 is desired, the deposition temperature, the deposition pressure, the deposition time, or a combination thereof are increased or decreased. Suitable thicknesses of the coating 501 include, but are not limited to, between about 100 nm and about 10,000 nm, between about 200 nm and about 5,000 nm, between about 300 nm and about 1,500 nm, or any suitable combination, sub-combination, range, or sub-range therein.
Additionally or alternatively, in one embodiment, a plurality of the layers 502 are applied by repeating the deposition. In one embodiment, the thermal decomposition (step 404) pressure is between about 0.01 psia and about 200 psia, 1.0 psia and about 100 psia, 5 psia and about 40 psia, about 1.0 psia, about 5 psia, about 40 psia, about 100 psia, 200 psia, or any suitable combination, sub-combination, range, or sub-range therein. In one embodiment, the thermal decomposition (step 404) temperature is between about 200° C. and 600° C., between about 300° C. and 600° C., between about 400° C. and about 500° C., about 300° C., about 400° C., about 500° C., about 600° C., or any suitable combination, sub-combination, range, or sub-range therein. In one embodiment, the thermal decomposition (step 404) period is for a duration of about 10 minutes to about 24 hours, about 30 minutes to about 24 hours, about 10 minutes, about 30 minutes, about 15 hours, about 24 hours, or any suitable combination, sub-combination, range, or sub-range therein.
The thermal decomposition (step 404) forms the layer 502, for example, having improved chemical resistance, improved inertness, and/or improved adhesion over non-diffusion coatings and/or coatings not having the thermally decomposed material. The layer 502 includes any suitable thermally decomposed material corresponding to the precursor material. The thermally decomposed material is formed by the thermal decomposition (step 404) at a pressure and a temperature sufficient to decompose the precursor material, thereby depositing constituents from the thermally decomposed material onto the substrate 500, for example, with an inert gas such as nitrogen, helium, and/or argon, as a partial pressure dilutant.
In one embodiment, the thermally decomposed material is or includes carbosilane (for example, amorphous carbosilane), corresponding to the precursor including the dimethylsilane, which, although not intending to be bound by theory, is believed to be a recombination of carbosilyl (disilyl or trisilyl fragments) formed from the carbosilane. In one embodiment, the thermally decomposed material includes molecules, such as, silicon, carbon, and hydrogenatoms, that serve as active sites. The molecules are positioned within the layer 502 and include a first portion 504 and a second portion 506. Generally, the first portion 504 and the second portion 506 of the layer 502 are not spatially resolvable (for example, the first portion 504 and the second portion 506 are defined by the molecules deposited on the layer 502 and the molecules are capable of being interspersed throughout the layer 502). Furthermore, use of the terms “first and “second is not intended to imply any sequentiality, difference in quantity, difference in size, or other distinction between the two portions. To the contrary, the terms “first and “second are used for distinguishing molecular composition of the two portions. For example, in one embodiment, as is shown in
In one embodiment, the composition of the layer 502 is about 1:0.95:0.12 ratio of C:Si:O. In contrast, the composition of the dimethylsilane introduced into the chemical vapor deposition chamber according to an embodiment has about a 2:1 ratio of C:Si. Although not intending to be bound by theory, it is believed that CHx (x=0-3) moieties are retained and Si—C bonds are broken thus indicating that layer 502 includes an amorphous array of Si—C bonding. The amorphous array provides additional benefits such as decreased cracking or flaking, for example, upon tensile or compressive forces acting on the substrate 500, increased adhesion, or a combination thereof. In one embodiment, multiple layers of the coating 501, or similar coatings, are deposited for thicker layers or for desired properties.
In one embodiment, upon the thermally decomposed materials forming the layer 502 through the thermal decomposition (step 404), the chamber is purged. The purging removes remaining decomposition materials, unbound thermally decomposed materials, and/or other materials or constituents present within the chamber.
The oxidation (step 408) is or includes exposure to any suitable chemical species or oxidation reagent capable of donating a reactive oxygen species under oxidation conditions to form the oxidized layer 507. The oxidation (step 408) is of the layer 502 and forms the oxidized layer 507. In an embodiment with the layer 502 being amorphous carbosilane, the oxidized layer 507 formed by the oxidation (step 408) is or includes amorphous carboxysilane. In general, the oxidation (step 408) are bulk reactions that affect the bulk of the coating 501. In one embodiment, the degree of oxidization is controlled by increasing or decreasing the temperature within the chamber, the exposure time within the chamber, the type and/or amount of diluent gases, pressure, and/or other suitable process conditions. Control of the degree of the oxidization increases or decreases the amount and/or depth of the oxidized layer 507 and, thus, the wear resistance and/or hardness of the coating 501.
Suitable oxidation reagents for the oxidation (step 408) include, but are not limited to, water (alone, with zero air, or with an inert gas), oxygen, air (alone, not alone, and/or as zero air), nitrous oxide, ozone, peroxide, or a combination thereof. As used herein, the term “Zero air refers to atmospheric air having less than about 0.1 ppm total hydrocarbons. In one embodiment, the oxidation reagent consists of gaseous reagents. Due to the gaseous processing agents (for example, dimethylsilane and/or nitrogen) being in the gas phase, use of the gaseous oxidation reagent results in simpler scale-up for manufacturing, a more transferable process, and a more economical process.
The oxidation reagent used for the oxidation (step 408) is introduced at any suitable operational conditions permitting the formation of the oxidized layer 507. Suitable operational conditions include, but are not limited to, being in the presence of an inert gas, being at a pressure (for example, between about 1 to 200 psia), being subjected to a temperature (for example, about 450° C.), being for a period of time (for example, for about two hours), other parameters as are described above with reference to the thermal decomposition (step 404), or a combination thereof.
In one embodiment, depending upon the selected species of the oxidation reagent, additional features are present, for example, for safety purposes. Such features include the chamber having a size, weight, and/or corrosion-resistance permitting reactions to occur safely. In one embodiment, to safely inject water into the chamber as the oxidation reagent, substantial cooling is used. For example, in embodiments with the chamber operating at temperature of greater than about 300° C., the chamber is first cooled below about 100° C., which is capable of resulting in a drain on energy and/or time of manufacturing resources.
The oxidized layer 507 formed by the oxidation (step 408) includes properties corresponding to the oxidation reagent used and the operational parameters. In one embodiment, in comparison to the layer 502, the oxidized layer 507 is over-oxidized and/or has a contact angle on a Si wafer of about 60° has an increased amount of N—H, Si—OH, and/or C—OH groups, has fragile scratch resistance, has increased acid resistance, has increased corrosion resistance, or a combination thereof.
The oxidized layer 507 includes various comparative properties relative to the layer 502, and/or embodiments with the oxidized layer 507 being formed by different oxidation reagents. For example, the oxidized layer 507 has decreased chemical resistance, has decreased scratch resistance, has decreased hardness, or a combination thereof. In one embodiment, the oxidized layer 507 is oxidized and/or has a contact angle on a Si wafer of about 86.6° has decreased friction (for example, in comparison to embodiments with the oxidizing reagent being zero air and water), has decreased wear resistance (for example, in comparison to embodiments with the oxidizing reagent being zero air and water), includes Si—O—Si groups (for example, capable of being shown by FT-IR data having a growth of the Si—O—Si peak at 1026.9 cm−1 compared to the non-water functionalized peak at 995.2 cm−1), or a combination thereof. In one embodiment, the oxidized layer 507 is over-oxidized, has a decreased amount of C—H groups (for example, in comparison to embodiments with the oxidizing reagent being water alone), has a decreased amount of Si—C groups (for example, in comparison to embodiments with the oxidizing reagent being water alone), has an increased amount of Si—OH/C—OH groups (for example, in comparison to embodiments with the oxidizing reagent being water alone), or a combination thereof. In one embodiment, the oxidized layer 507 has lower coefficient of friction (for example, in comparison to embodiments with the oxidization agent being zero air and water), has increased wear resistance (for example, in comparison to embodiments with the oxidization agent being zero air and water), includes Si—O—Si groups, or a combination thereof.
In one embodiment, the coefficient of friction is decreased by the oxidation (step 408). For example, in an embodiment with the oxidation (step 408) of the layer 502, the layer 502 includes a first coefficient of friction (for example, about 0.97) prior to the oxidation (step 408) and a second coefficient of friction (for example, about 0.84) after the oxidation (step 408).
In one embodiment, the wear rate is decreased by the oxidation (step 408). For example, in an embodiment with the oxidation (step 408) of the layer 502, the layer 502 includes a first wear rate (for example, 4.73×10-4 mm3/N/m) prior to the oxidation (step 408) and a second wear rate (for example, about 6. 75×10-5 mm3 /N/m) after the oxidation (step 408).
The post-oxidation functionalization (step 210) is or includes thermal coupling of one or more materials.
In one embodiment, the post-oxidation functionalization (step 210) modifies the oxidized layer 507, for example, by heating and/or modifying the surface, to form the oxidized then functionalized layer 509 shown in
In one embodiment, the oxidized-then-functionalized layer 509 has a contact angle for deionized water on a mirror surface of greater than about 105°, greater than about 110°, greater than about 112°, greater than about 115°, between about 100° and about 114°, about 110.3°, about 112.1°, about 113.7°, or any suitable range, sub-range, combination, or sub-combination thereof. Additionally or alternatively, in one embodiment, the oxidized-then-functionalized layer 509 has a contact angle for deionized water on a mirror surface that is less than polytetrafluoroethylene, for example, by about 1°, about 2°, between about 1° and about 2°, or any suitable range, sub-range, combination, or sub-combination thereof.
In one embodiment, the oxidized-then-functionalized layer 509 has a contact angle for deionized water on a rough surface of greater than about 140°, greater than about 145°, between about 140° and about 150°, about 142.7°, about 145.7°, about 148.1°, or any suitable range, subrange, combination, or sub-combination thereof. Additionally or alternatively, in one embodiment, the oxidized-then-functionalized layer 509 has a contact angle for deionized water on a rough surface that is greater than polytetrafluoroethylene, for example, by about 25°, about 30°, between about 20° and about 35°, or any suitable range, sub-range, combination, or subcombination thereof.
In one embodiment, the oxidized-then-functionalized layer 509 has greater anti-stiction properties than the oxidized layer 507, for example, formed with zero air as the binding reagent. As such, in one embodiment of the coating process 400, the oxidized-then-functionalized layer 509 has increased anti-stiction.
By modifying and varying the R-groups, or by using other molecules capable of hydroxyl reactivity, surface properties of the oxidized-then-functionalized layer 509 are adjusted. For example, in one embodiment, the adjustments increase or decrease hardness and anti-stiction, wear resistance, inertness, electrochemical impedance, contact angle, or a combination thereof, thereby providing physical performance characteristics expanding the applicability and durability for use in biomedical and marine fields.
In one aspect, the alkylsilyl coatings described herein enhance the corrosion performance of metals, e.g., as in metallic chromatography columns. Depending on the denseness and thickness, the coatings act as a barrier, thereby preventing water and corrosive molecules from reacting with the base metal. While increasing the hydrophobicity and density improves the corrosion performance, even coatings derived from C2 and GPTMS (C2-GPTMS) followed by hydrolysis to form C2-GPTMS-OH shows a 10× improvement in the ASTM G48 Method A pitting corrosion, see e.g., Example 4 below. In terms of most corrosion resistant to least, the ranking is the material formed from VPD #7>2>1 (bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane as a first layer followed by GPTMS then hydrolysis to form GPTMS-OH as a second layer). This also correlates to hydrophobicity rankings.
The coatings described above can be used to tailor a fluidic flow path of a chromatography system for the separation of a sample. The coatings can be vapor deposited. In general, the deposited coatings can be used to adjust the hydrophobicity of internal surfaces of the fluidic flow path that come into contact with a fluid (i.e. wetted surfaces or surfaces coming into contact with the mobile phase and/or sample/analyte). By coating wetted surfaces of one or more components of a flow path within a chromatography system, a user can tailor the wetted surfaces to provide a desired interaction (or lack of interaction) between the flow path and fluids therein (including any sample, such as biomolecules, proteins, glycans, peptides, oligonucleotides, pesticides, bisphosphonic acids, anionic metabolites, and zwitterions like amino acids and neurotransmitters, within the fluid). The wetted surfaces need not be within a chromatography system. Other devices or labware can also be tailored. That is, any fluid contacting surface, such as frits within an extraction device, or the interior of a pipette tip, can be tailored to provide the desired interaction or lack of interaction between the wetted surfaces and fluids therein.
In one aspect, an effective coating is produced from a low temperature, vacuum-assisted vapor deposition process. In one aspect, an oxygen plasma pretreatment step precedes the coating deposition. The oxygen plasma removes organic compounds and improves surface wettability for the coatings. Time, temperature, and pressure are controlled for each processing step. Each coating run can use a silicon wafer to monitor the thickness and contact angle of the resultant coating. Ellipsometry can be used to measure the coating thickness, and an optical goniometer can be used to measure the contact angle of the coating. A post coating annealing step can be utilized to increase coating cross-linking and increase coating hydrophobicity.
The method has certain steps which are optional as indicated by the dashed outline surrounding a particular step. Method 200 can start with a pretreatment step (205) for cleaning and/or preparing a flow path within a component for tailoring. Pretreatment step 205 can include cleaning the flow path with plasma, such as oxygen plasma. This pretreatment step is optional.
Next, an infiltration step (210) is initiated. A vaporized source of an alkylsilyl compound (e.g., the alkylsilyl compounds of Formulas I, II and/or III) is infiltrated into the flow path to coat the wetted surfaces. The vaporized source is free to travel throughout and along the internal surfaces of the flow path. Temperature and/or pressure is controlled during infiltration such that the vaporized source is allowed to permeate throughout the internal flow path and to deposit a coating from the vaporized source on the exposed surface (e.g., wetted surfaces) of the flow path as shown in step 215. Additional steps can be taken to further tailor the flow path. For example, after the coating is deposited, it can be heat treated or annealed (step 220) to create cross linking within the deposited coating and/or to adjust the contact angle or hydrophobicity of the coating. Additionally or alternatively, a second coating of alkylsilyl compound (having the same or different form) can be deposited by infiltrating a vaporized source into the flow path and depositing a second or additional layers in contact with the first deposited layer as shown in step 225. After the deposition of each coating layer, an annealing step can occur. Numerous infiltration and annealing steps can be provided to tailor the flow path accordingly (step 230).
A method of tailoring a fluidic flow path for separation of a sample is provided that includes assessing a polarity of an analyte in the sample and selecting a chromatographic media based on the analyte in the sample. An alkylsilyl coating is selected based on the polarity of the analyte in the sample. The alkylsilyl coating is selected so that the coating is inert to the analyte(s) being separated. In other words, the alkylsilyl coating does not produce any secondary chromatographic effects that are attributable to the alkylsilyl coating. In some embodiments, the analyte is a biomolecule. The biomolecule can be a peptide or peptide fragment, an oligopeptide, a protein, a glycan, a nucleic acid or nucleic acid fragment, a growth factor, a carbohydrate, a fatty acid or a lipid. The analyte can be a citric acid cycle metabolite. The analyte can be a pesticide.
A method of tailoring a fluidic flow path within a processing device includes assessing the hydrophobicity of an analyte within a fluid to be processed and of wetted surfaces of the processing device. An alkylsily coating is selected to minimize adsorptive losses to the wetted surfaces based on a difference in hydrophobicity of the analyte and the wetted surfaces of the processing device.
The alkylsilyl coating can have the Formula I, II, or III as described above. In one embodiment, the alkylsilyl coating has the Formula I as a first layer and Formula II as a second layer. In some embodiments, there is only a single layer coating having Formula I (e.g., bis(trichlorosilyl)ethane or bis(trimethoxysilyl)eithane). In some embodiments, there is only a single layer coating having Formula II (e.g., (3-glycidyloxypropyl)trimethoxysilane, n-decyltrichlorosilane, trimethylchlorosilane, trimethyldimethyaminosilane, or methoxy-polyethyleneoxy(3)silane). In some embodiments, there is only a single layer coating having Formula III (e.g., bis(trichlorosilyl)ethane or bis(trimethoxysilyl)eithane).
The method also includes adjusting a hydrophobicity of the wetted surfaces of the fluidic flow path by vapor depositing the alkylsilyl coating onto the wetted surfaces of the fluidic flow path. In some embodiments, the hydrophobicity of the wetted surfaces is adjusted by adjusting the contact angle of the alkylsilyl coating. For example, the contact angle of the alkylsilyl coating can be between about 0° to about 115°. In cases where the underlying material of the wetted surfaces is hydrophobic, alkylsilyl coatings that are hydrophilic (exhibiting a contact angle of less than about) 60° are preferred. In embodiments where the underlying material of the wetted surfaces is hydrophilic, and there is a preference to increase hydrophobicity, then a alkylsilyl coating exhibiting a contact angle of greater than 60° is vapor deposited.
The analyte in the sample can be retained with a retentivity within 10% of the retentivity attributable to the chromatography media. In some embodiments, the sample can be retained with a retentivity within 5% or within 1% of the retentivity attributable to the chromatography media. Therefore, the alkylsilyl coating solves the problem of metal interaction between the analyte and the metal chromatographic materials without introducing any secondary reactions that would have a negative effect on the quality of the separation. The alkylsilyl coating does not impart any retention mechanism on the analyte of interest, making the coating inert to the analyte of interest and low-bind.
In addition, the alkylsilyl coating does not produce any changes to peak width. The analyte in the sample has a peak width that is within 10%, 5%, or 1% of the peak width attributable to the chromatographic media.
The wetted surfaces of the fluidic flow path can be any of those described above with respect to aspects and embodiments of the chromatographic device.
The method can also include annealing the alkylsilyl coating after vapor depositing the alkylsilyl coating on the wetted surfaces of the fluidic flow path. Typically, the annealing cycle involves subjecting the coating to 200° C. for 3 hours under vacuum.
The method can also include assessing the polarity of the chromatographic media and selecting the alkylsilyl coating based on the polarity of the analyte and the chromatographic media. The method can also include eluting the sample through the fluidic flow path, thereby isolating the analyte.
In some embodiments, the alkylsilyl coating is modified with a silanizing reagent to obtain a desired thickness of the alkylsilyl coating. The silanizing reagent can be a non-volatile zwitterion. The non-volatile zwitterion can be sulfobetaine or carboxybetaine. In some embodiments, the silanizing reagent is an acidic or basic silane. The silanizing reagent can introduce polyethylene oxide moieties, such as methoxy-polyethyleneoxy(6-9)silane, the structure of which is shown below.
In some aspects, the method of tailoring a fluidic flow path for processing a sample including a biomolecule further comprises: pretreating the wetted surfaces of the flow path with a plasma prior to depositing the first coating. In other aspects, the method of tailoring a fluidic flow path for processing a sample including a metal interacting analyte further comprises annealing the first coating at a temperature to increase cross-linking in the first coating. In yet another aspect, the method of tailoring a fluidic flow path for separation of a sample including a metal interacting analyte further comprises annealing the first coating at a temperature to alter hydrophobicity.
In one aspect, the method of tailoring a fluidic flow path for separation of a sample including a metal interacting analyte further comprises performing a second infiltration with a vaporized source having the Formula II, wherein the features for Formula II are as described above; along and throughout the interior flow path of the fluidic system to form a second coating deposited in direct contact with the first coating. In one aspect, the step of performing a second infiltration in the preceding method further comprises performing an annealing step after depositing the second coating. In another aspect, the preceding method further comprises connecting in fluid communication with the flow path at least one coated component selected from the group consisting of a sample reservoir container and a frit.
Also provided herein is a method of tailoring a fluidic flow path for separation of a sample including a metal interacting analyte, the method comprising: assessing polarity of the analyte in the sample; selecting an alkylsilyl coating having the Formula I, wherein the features for Formula I are as described above, and desired contact angle based on polarity assessment; and adjusting the hydrophobicity of wetted surfaces of the flow path by vapor depositing an alkylsilyl having the Formula III, wherein the features for Formula III are as described above, and providing the desired contact angle. In some embodiments of the above method, in addition to assessing polarity of the analyte in the sample, the polarity of a stationary phase disposed within at least a portion of the flow path is also assessed and the polarity assessment is obtained from both the polarity of the biomolecule in the sample and the stationary phase.
In one aspect, provided herein are methods of isolating an analyte. The method includes introducing a sample including a glycan, a peptide, a pesticide, or a citric acid cycle metabolite into a fluidic system including a flow path disposed in an interior of the fluidic system. The flow path includes a first vapor deposited alkylsilyl inert coating having the Formula I described above and a second vapor deposited coating of the Formula II described above. The sample is eluted through the fluidic system, thereby isolating the glycan, peptide, pesticide, or citric acid cycle metabolite.
The glycan can be a phosphoglycan. The peptide can be a phosphopeptide and the pesticide can be glyphosate. The citric acid cycle metabolite can be citric acid or malic acid.
When the analyte is a glycan, peptide or pesticide, the alkylsilyl coating of Formula I can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula II can be n-decyltrichlorosilane. When the analyte is a citric acid cycle metabolite, the alkylsilyl coating of Formula I can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula II can be trimethylchlorosilane or trimethyldimethyaminosilane.
The flow path can be defined at least in part by the interior surface of a chromatographic system. The flow path can be further defined at least in part by passageways through a frit of the chromatographic column. The flow path can be defined at least in part by interior surfaces of tubing. The flow path can be any flow path described herein, for example, the flow paths described with respect to the chromatographic device.
Also provided herein is a method of improving baseline returns in a chromatographic system, the method comprising: introducing a sample including an analyte into a fluidic system including a flow path disposed in an interior of the fluidic system, the flow path having a length to diameter ratio of at least 20 and comprising a vapor deposited alkylsilyl coating having the Formula I, wherein the features for Formula I are as described above, a thickness of at least 100 angstroms and a contact angle of about 30 degrees to 115 degrees; and eluting the sample through the fluidic system, thereby isolating the biomolecule. In some embodiments, the method includes a second layer of Formula II or Formula III, wherein the features of Formula II and II are described above.
Also provided herein is a method of minimizing adsorptive losses, the method comprising: introducing a sample including an analyte into a processing device including a flow path disposed in an interior of the processing device, the flow path comprising a vapor deposited alkylsilyl coating having the Formula I, wherein the features for Formula I are as described above, a thickness of at least 100 angstroms and a contact angle of about 5 to 60°; and flowing the sample through the processing device. In some embodiments, the method includes a second layer of Formula II or Formula III, wherein the features of Formula II and II are described above.
Also provided here are kits. The kits include chromatographic components, for example, a chromatographic column, that has been coated with an alkylsilyl coating of Formulas I, II, and/or III, as described above. Other components can be provided in the kit that can also include the coatings described herein, for example, the tubing, frits, and/or connectors. The kit can also include instructions for separating analytes, for example, biomolecules, proteins, glycans, peptides, oligonucleotides, pesticides, bisphosphonic acids, anionic metabolites, and zwitterions like amino acids and neurotransmitters.
Embodiments may be directed to labware instead of chromatographic components. For example, the kit could include one or more of a beaker, extraction device, pipette tip, dialysis chamber, autosampler vial or plates that have been coated with an alkylsilyl coating of Formulas I, II, and/or III, as described above.
Separation of Phosphoglygans
The disclosed coatings, which can be vapor deposited, have been found to dramatically improve separations of phosphoglycans by hydrophilic interaction chromatography (HILIC). To demonstrate the significance of this, the released N-glycans from a recombinant alpha-galactosidase which can be used as an enzyme replacement therapy for Fabry's disease were evaluated. This particular type of enzyme is taken up from circulation and delivered intercellularly to lysosomes through the mannose-6-phosphate pathway, making it important to identify and monitor the levels of phosphorylated glycans that are present on its surface. Vapor deposition coated stainless steel column tubes along with matching coated stainless steel frits were first tested against corresponding untreated stainless steel hardware. In this instance, two different types of coating chemistries were used. The coating chemistries used to coat the frits and tubing were VPD #2 and VPD #7.
The increased recovery of phosphorylated glycans using coated column hardware and fits shows that adsorption to metallic column hardware surfaces is detrimental to recovery. With this in mind, separations were also performed with vapor deposition coated stainless steel sample flow path components (
Separation of Other Phosphorylated Molecules
The principles learned from using vapor deposition coatings for phosphoglycan analysis were extended to facilitate the analysis of other types of phosphorylated biomolecules. In which case, the coatings have been found to be beneficial to improving the recovery of phosphorylated peptides under reversed phase chromatography conditions. To demonstrate these recovery advantages, we evaluated a mixture containing phosphopeptides. This particular sample contains three peptides that are singly phosphorylated and one that is doubly phosphorylated. Vapor deposition coated stainless steel column tubes along with matching coated stainless steel frits were first tested against corresponding untreated stainless steel hardware.
As such, in one aspect, the vapor deposition coated column hardware is used to improve the recovery of phosphorylated biomolecules during analyses by liquid chromatography. In yet another embodiment of this invention, vapor deposition coated flow path components are used in conjunction with vapor deposition coated column hardware to improve the recovery of phosphorylated biomolecules during analyses by liquid chromatography.
The effects of this finding have been demonstrated for two examples of phosphorylated biomolecules, phosphorylated glycans and phosphorylated peptides. Phosphorylated biomolecules refer to any molecule naturally produced by an organism that contains a phospho group, including but not limited to phosphorylated proteins and polynucleotides. Furthermore, it is reasonable to envision this disclosure being used to improve liquid chromatographic analyses of smaller biomolecules, including but not limited to phospholipids, nucleotides and sugar phosphates. Indeed, vapor deposition coated column hardware has been found to be useful in improving the recovery and peak shape of sugar phosphates and nucleotides. The effects of employing vapor deposition coated versus untreated column hardware for the reversed phase LC analyses of glucose-6-phosphate, fructose-6-phosphate, adenosine triphosphate, and adenosine monophosphate are captured in
Separation of Sialylated Glycans and Molecules Having Carboxylic Acid Moieties
It has additionally been discovered that vapor deposition coated hardware can be of benefit to mixed mode separations of sialylated glycans. In such a technique, sialylated glycans can be resolved using a stationary phase that exhibits anion exchange and reversed phase retention mechanisms. It was just recently discovered that a unique class of stationary phase, referred to as charged surface reversed phase chromatographic materials and described in International Application No. PCT/US2017/028856, entitled “CHARGED SURFACE REVERSED PHASE CHROMATOGRAPHIC MATERIALS METHOD FOR ANALYSIS OF GLYCANS MODIFIED WITH AMPHIPATHIC, STRONGLY BASED MOIETIES” and published as WO2017/189357 (and incorporated herein by reference in its entirety), is ideally suited to producing these types of separations. The use of a high purity chromatographic material (HPCM) with a chromatographic surface comprised of a diethylaminopropyl (DEAP) ionizable modifier, a C18 hydrophobic group and endcapping on a bridged ethylene hybrid particle has proven to be an exemplary embodiment for the separation of glycans labeled with amphipathic, strongly basic moieties, like that imparted by the novel labeling reagent described in International Application No. PCT/US2017/028856 (WO2017/189357). This so-called diethylaminopropyl high purity chromatographic material (DEAP HPCM) stationary phase is effective in separating acidic glycans as a result of it being modified with a relatively high pKa (˜10) ionizable modifier that yields uniquely pronounced anionic retention.
In an application to DEAP HPCM mixed mode separations of sialylated glycans, vapor deposition coated hardware has been shown to yield improved chromatographic recoveries and peak shapes of glycans containing greater than three sialic acid residues. A comparison of fluorescence chromatograms for fetuin N-glycans obtained with untreated stainless steel versus VPD #7 coated hardware is provided in
As with phosphorylated species, this effect on the chromatography of sialylated glycans is believed to result from masking the metallic surface of the hardware and minimizing adsorptive sample losses that can occur with analytes that exhibit a propensity for metal chelation. However, the origin of the metal chelation is different in that the effect is a consequence of a glycan carrying multiple carboxylate residues versus one or two phosphorylated residues. Carboxylate containing compounds generally have a weak affinity for metals. Yet, when there are multiple carboxylate moieties present in one molecule, an opportunity for polydentate chelation is created, as is the case with tetra- and penta-sialylated glycans.
Accordingly, in an embodiment of this invention, vapor deposition coated column hardware is used during liquid chromatography of biomolecules containing greater than three carboxylic acid residues as a means to improve their peak shape and recovery. In yet another embodiment of this invention, vapor deposition coated flow path components are used in conjunction with vapor deposition coated column hardware to improve the peak shape and recovery of biomolecules containing greater than three carboxylic acid residues.
Separation of Proteins
Certain vapor deposition coatings have also been found to beneficially impact protein reversed phase chromatography. To demonstrate such, we evaluated a paradigmatic protein separation that is very important to the analysis of biopharmaceuticals, a monoclonal (mAb) subunit separation with MS-friendly, formic acid modified mobile phase. Using such a test, numerous combinations of column hardware materials have been examined. Vapor deposition coated stainless steel column tubes along with matching coated stainless steel frits were first tested against corresponding untreated stainless steel hardware.
An effect such as this can be very significant to protein reversed phase separations, particularly those intended to facilitate detection by online electrospray ionization (ESI)—mass spectrometry (MS). Often, it is critical to have quick returns to baseline in ESI-MS data given that it will make the assignment of chromatographic peaks less ambiguous. Signal from previously eluted species will be less abundant and therefore less confounding in data accumulated for later eluting peaks. With this in mind, 11 additional combinations of column hardware materials were screened, using ESI-MS detection as the means to assessing the quality of the data.
As such, in an embodiment of this invention, vapor deposition coated column hardware is used to improve the chromatographic performance of protein reversed phase separations. In yet another embodiment of this invention, a vapor deposition coating with a contact angle of >90°, more preferably greater than 100 Å, is used to coat the tubing and frits of a column, or chromatographic device, as a means to improve the baseline and/or tailing factors of protein separations.
In a separate embodiment, this invention may utilize a frit material that is constructed of a specific polymer, such that an equivalently hydrophobic surface is achieved, specifically one with a contact angle greater than 90°, more preferably greater than 100 Å. Polytetrafluoroethylene (PTFE), polymethylpentene (PMP), high density polyethylene (HDPE), low density polyethylene (LDPE) and ultra high molecular weight polyethylene (UHMWPE) are examples of hydrophobic polymers that could be suitable for use as the frit or column material in other embodiments of this invention. In fact, an inlet frit constructed of porous PTFE (1.5 mm thick, Porex PM0515) was found to favorably affect protein reversed phase baselines, in a manner similar to that of the previously mentioned VPD #7 vapor deposition coated inlet frit (
Finally, vapor deposition coated hardware has been found to be of benefit to aqueous biomolecule separations, such as protein ion exchange chromatography. When looking to understand the charge heterogeneity of a sample, an analyst will often choose to resolve the components of a sample by ion exchange. In the case of protein therapeutics, this type of analysis is performed as a means to interrogate so-called charge variants, such as deamidiation variants, that can have a detrimental effect on the efficacy of the corresponding drug product. Charge variant separations by way of ion exchange can therefore be critical to the effectiveness of a characterization approach for a protein therapeutic, most particularly a monoclonal antibody. Being such an important analytical approach, protein ion exchange must be robust and able to quickly and reliably yield accurate information.
To this end, ion exchange separations of a monoclonal antibody were evaluated, and the effects of using uncoated versus vapor deposition coated column hardware were contrasted.
Prior to coating, all metal components are passivated according to a nitric acid passivation. Passivated parts and a silicon wafer are then introduced to the vapor deposition chamber and vacuum is established. The first step is a 15 minute, 200 Watt, 200 cc/min oxygen plasma cleaning step. Next is the first vapor deposition cycle. Each vapor deposition cycle contains a silane vapor deposition, followed by the introduction of water vapor for silane hydrolysis. The silane vapor is delivered at a pressure of 2.0 Torr for 5 seconds, and then the water vapor is delivered at a pressure of 50 Torr for 5 seconds. Following delivery, the silane and water is left to react with the substrate for 15 minutes. This cycle is repeated to produce the desired number of layers and coating thickness. An additional processing cycle can be implemented to functionalize the coating with yet another silane. Moreover, a post coating annealing step can be used to further cross-link and increase the hydrophobicity of the coating. Typically, the annealing cycle involves subjecting the coating to 200° C. for 3 hours under vacuum.
A silicon wafer is used as a coupon to measure the thickness and contact angle of the coating. To measure the thickness, a Gaertner Scientific Corporation stokes ellipsometer model LSE is used. By analyzing the change in polarization of light, and comparing to a model, the film thickness can be established. To measure the contact angle, a Rame-Hart goniometer model 190 is used. After dropping a controlled amount of water onto a perfectly level silicon wafer, optical techniques are used to measure the contact angle.
Prior to coating, all metal components are passivated according to a nitric acid passivation. Passivated parts and a silicon wafer are then introduced to the vapor deposition chamber and vacuum is established. The first step is a 15 minute, 200 Watt, 200 cc/min oxygen plasma cleaning step. Next is the first vapor deposition cycle. Each vapor deposition cycle contains a silane vapor deposition, followed by the introduction of water vapor for silane hydrolysis. The silane vapor is delivered at a pressure of 2.0 Torr for 5 seconds, and then the water vapor is delivered at a pressure of 50 Torr for 5 seconds. Following delivery, the silane and water is left to react with the substrate for 15 minutes. This cycle is repeated to produce the desired number of layers and coating thickness. In this example, the bis(trichlorosilyl)ethane silane is used to build up an adhesion or primer layer of approximately 800 Å. After C2 deposition, the 3-(glycidoxypropyl)trimethoxysilane is delivered anhydrously to a pressure of 0.4 Torr in the vapor deposition chamber. This silane vapor is left to react with the C2 coated substrate for one hour. This process results in an epoxide terminated coating, with a contact angle of 50°. After deposition, the next step is to hydrolyze the epoxide groups. This is performed either in the liquid phase or the vapor phase, with 0.1M acetic acid. After epoxide hydrolysis, the contact angle is <20°. Contact angle measurements are taken on a silicon wafer using a Rame-Hart goniometer model 190.
It is relatively easy to measure the contact angle on the flat silicon wafers using a goniometer. However, not all our substrates have such smooth and flat surfaces. Frits can be considered a chromatography column's most important substrate, since the fluidic surface area to mass ratio is higher in the frit than in any other column hardware component. In order to measure the solid-liquid wetting properties of frit porosity, and confirm the presence of a coating, we can use the bubble point test. The bubble point test is used to determine the largest pore diameter of a frit structure, and the bubble point pressure is related to this diameter with the following equation:
P=(2 γcos Θ)/r
This equation is from ASTM E128 and is derived from the equilibrium condition of capillary rise.
Using the bubble point test to calculate a contact angle requires two steps. This first is to test the frit in IPA, and assume a 0 contact angle, since IPA has excellent wetting characteristics. This will yield a maximum pore diameter. The next step is to repeat the experiment with water as the test liquid, and the known pore radius. This will yield the contact angle with water, relative to the assumed 0 degree contact angle of IPA.
ASTM G48 Method A is used to rank the relative pitting corrosion performance of various grades of stainless steel. It consists of placing a part in ˜6% ferric chloride solution for 72 hours, and checking the mass loss of your component. The test can be run at room temperature, or at slightly elevated temperatures to increase the corrosion rate. The ferric chloride solution is similar to the environment inside a pit during “non-accelerated” pitting corrosion; an acidic, oxidizing, chloride containing environment. When an entire part of interest is submerged in the ferric chloride solution, pitting corrosion is greatly accelerated, with normal test times only being 72 hours.
A recombinant alpha-galactosidase was diluted to 2mg/mL. A 7.5 uL aliquot of the protein solution was then added to a 1mL reaction tube containing 15.3 μL of water and 6 μL of buffered 5% RapiGest SF solution-commercially available from Waters Corporation (Milford, Mass.) (50 mM HEPES-NaOH, pH 7.9). The mixture was placed in a heat block at 90° C. for 3 minutes. Thereafter, the reaction tube was allowed to cool at room temperature for 3 minutes. To the reaction tube, 1.2 μL of PNGase F was then added and incubated at 50° C. for 5 minutes. After incubation, the reaction was again allowed to cool at room temperature for 3 minutes. To a vial containing 9 mg of RapiFluor-MS reagent, 131 uL of anhydrous DMF was added and vortexed to create a labeling solution. A 12 uL volume of this labeling solution was next added to the reaction tube. This labeling reaction was allowed to proceed for 5 minutes to produce the final sample.
A fully porous amide HILIC stationary phase (1.7 um, 130 Å) was used in a 2.1×50 mm column dimension to chromatograph the samples at a flow rate of 0.4 mL/min and temperature of 60° C. The gradient flow conditions initiated with 75.0% organic solvent (Acetonitrile) and 25.0% aqueous eluent (50mM ammonium formate, pH 4.4) followed by a 11.66 min linear gradient to 54.0% organic/46% aqueous eluent. The column was then cycled through an aqueous regeneration step at 100% aqueous mobile phase at a flow rate of 0.2 mL/min for one minute. After the aqueous regeneration, the column was equilibrated at initial conditions for 4 minutes. Species eluting during the above separations were detected serially via fluorescence (Ex 265/Em 425, 2 Hz) followed by online ESI-MS. Mass spectra were acquired with a Xevo G2-XS QToF mass spectrometer operating with a capillary voltage of 2.2 kV, source temperature of 120° C., desolvation temperature of 500° C., and sample cone voltage of 50 V. Mass spectra were acquired at a rate of 2 Hz with a resolution of approximately 40,000 over a range of 700-2000 m/z.
A vial of phosphopeptide test standard (Waters Corporation, Milford, Mass.) was reconstituted with 50 uL of 0.1% formic acid. A fully porous CSH C18 stationary phase material (1.7 um, 130 Å) was used in 2.1×50 mm column dimensions to chromatograph the samples at a flow rate of 0.2 mL/min at a temperature of 60° C. The gradient flow conditions initiated with 0.7% organic mobile phase (0.075% formic acid in acetonitrile) and 99.3% aqueous mobile phase (0.1% formic acid) followed by a 30 min linear gradient to 50% organic/50% aqueous. Species eluting during the above separations were detected serially via UV (220 nm) followed by online ESI-MS. Mass spectra were acquired with a Xevo G2-XS QToF mass spectrometer operating with a capillary voltage of 1.5 kV, source temperature of 100° C., desolvation temperature of 350° C., and sample cone voltage of 50 V. Mass spectra were acquired at a rate of 2 Hz with a resolution of approximately 40,000 over a range of 500-6500 m/z.
RPLC/MS analyses of two nucleotides (adenosine monophosphate and adenosine triphosphate) and two sugar phosphates (glucose-6-phosphate and fructose-6-phosphate) were performed by reversed phase separations with an organosilica C18 stationary phase according to the methods parameters noted below.
RapiFluor-MS labeled N-glycans were prepared from bovine fetuin (Sigma F3004) according to a previously published protocol (Lauber, M. A.; Yu, Y. Q.; Brousmiche, D. W.; Hua, Z.; Koza, S. M.; Magnelli, P.; Guthrie, E.; Taron, C. H.; Fountain, K. J., Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection. Anal Chem 2015, 87 (10), 5401-9). Analyses of these released glycans were performed using a Waters ACQUITY UPLC H-Class Bio LC system and a separation method based on a previously described charged surface reversed phase chromatographic material described in International Application No. PCT/US2017/028856, entitled “CHARGED SURFACE REVERSED PHASE CHROMATOGRAPHIC MATERIALS METHOD FOR ANALYSIS OF GLYCANS MODIFIED WITH AMPHIPATHIC, STRONGLY BASED MOIETIES” (and incorporated by reference). Specifically, RapiFluor-MS labeled glycans (e.g., glycans labeled with the labeling reagent discussed in PCT/US2017/028856 were separated according to a mixed mode separation using a fully porous (130 Å) 1.7 μm diethylaminopropyl high purity chromatographic material (DEAP HPCM) in a 2.1×100 mm column configuration. Details of the method are described below.
Gradient Table:
Formulated NIST mAb Reference Material 8671 (an IgG1κ) was added to 100 units of IdeS and incubated for 30 minutes at 37° C. The resulting IdeS-digested mAb was then denatured and reduced by the addition of 1M TCEP and solid GuHCl. The final buffer composition for the denaturation/reduction step was approximately 6 M GuHCl, 80 mM TCEP, and 10 mM phosphate (pH 7.1). IdeS-digested NIST RM 8671 (1.5 mg/mL) was incubated in this buffer at 37° C. for 1 hour, prior to being stored at 4° C. Reversed phase (RP) separations of the reduced, IdeS-fragmented mAb were performed to demonstrate the effects of employing different vapor deposition coated column hardware pieces, namely the column tube and the frits that enclose the stationary phase into its packing.
A C4 bonded superficially porous stationary phase (2 μm, Rho 0.63, 290A) was used in a 2.1×50 mm column dimension to chromatograph the samples at a flow rate of 0.2 mL/min and temperature of 80° C. across a linear gradient consisting of a 20 min linear gradient from 15 to 55% organic mobile phase (aqueous mobile phase: 0.1% (v/v) formic acid in water; organic mobile phase: 0.1% (v/v) formic acid in acetonitrile). Species eluting during the above separations were detected serially via fluorescence (Ex 280/Em 320, 10 Hz) followed by online ESI-MS. Mass spectra were acquired with a Synapt G2-S mass spectrometer operating with a capillary voltage of 3.0 kV, source temperature of 150° C., desolvation temperature of 350° C., and sample cone voltage of 45 V. Mass spectra were acquired at a rate of 2 Hz with a resolution of approximately 20,000 over a range of 500-4000 m/z.
NIST mAb Reference Material 8671 (an IgG1κ) was separated using columns constructed from a 3 μm non-porous cation exchange stationary phase packed into either uncoated or vapor deposition coated hardware. Separations were performed with an ACQUITY UPLC H-Class Bio instrument according to the experimental conditions outlined below.
Testing has shown that flow paths modified with the vapor deposition coatings of this invention are also helpful in improving oligonucleotide separations. Example 11 provides evidence of such as observed in the form of improved recoveries and more accurate profiling of a sample's composition, particularly with respect to the first chromatograms obtained with a column.
In this work, a mixture of 15, 20, 25, 30, 35 and 40-mer deoxythymidine was separated using columns constructed from a 1.7 μm organosilica 130 Å C18 bonded stationary phase packed into either uncoated or vapor deposition coated hardware. Separations were performed with an ACQUITY UPLC H-Class Bio instrument according to the experimental conditions outlined below.
It should also be pointed out that the benefits of this invention are not limited to only biomolecules or phosphorylated/phospho group containing analytes. In fact, numerous types of so-called “small molecules” can be seen to have their separations improved through the adoption of vapor deposition coated flow paths and column hardware. One notable class of small molecules corresponds to compounds having a carboxylic acid moiety. By their nature, these are ubiquitous compounds and some, like citric acid and malic acid, are important metabolites of living organisms, given that they are constituents of the Kreb's cycle.
Herein, we have investigated the effects of separating citric acid and malic acid with untreated versus vapor deposition coated columns. Citric acid and malic acid were analyzed by LC-MS with columns constructed from a 1.8 μm silica 100 Å C18 bonded stationary phase packed into either uncoated or C2C3 vapor deposition coated hardware. Separations were performed with an ACQUITY UPLC I-Class PLUS instrument, and eluting analytes were detected with a Xevo TQ-S triple quadrupole mass spectrometer according to the experimental conditions outlined below.
Mobile Phase A: 0.1% formic acid, water
Glyphosate is non-selective broad spectrum herbicide which is widely used across the globe as a crop desiccant. Maximum residue limits (MRLs) are enforced globally on various commodities of interest because of the potential health consequences posed to consumers. Glyphosate and its metabolite aminomethylphosphonic acid (AMPA) require unique approaches for sample preparation and chromatography separation. Various methods can be employed for quantitation, whether they are based on reversed phase, porous graphitizes carbon, ion chromatography, hydrophilic interaction chromatography (HILIC) or mixed mode retention mechanisms. No matter the separation mode, assays for glyphosate and other related herbicide compounds can prove to be problematic. First, polar pesticides are difficult to retain on reversed phase columns without derivatization. Second, glyphosate interacts with active metal surfaces. As a result, it is notoriously observed in the form of a broad peak or one with pronounced tailing.
Herein, we have investigated the separation of glyphosate with untreated versus vapor deposition coated mixed mode HILIC columns. Glyphosate was analyzed by LC-MS with 1.7 μm diethylamine bonded organosilica 130 Å columns constructed from either uncoated or C2C10 vapor deposition coated stainless steel hardware. Separations were performed with an ACQUITY UPLC H-Class Bio coupled with a Xevo TQ-XS triple quadrupole mass spectrometer according to the experimental conditions outlined below.
Column Temperature: 50° C.
Immunoglobulin G (IgG) is the most common antibody found within blood circulation. Diagnostic testing of patient's blood samples analyzes the concentration of IgG to detect the possibility of certain diseases, infection, or cancer possibility. As a result, losses of IgG to labware and sample preparation devices can impact or have a negative effect on the results.
One piece of labware that is very frequently used to manipulate samples is the disposable pipette tip. While commonly used to aspirate, measure, transfer and dispense liquids, a pipette tip can also be used as a housing for solid phase extraction media or affinity resin. Most pipette tips are made of polypropylene, as this material exhibits chemical resistance to common acids, bases and organic solvents. However, the hydrophobicity of polypropylene is known to cause high levels of adsorptive losses when used with biological analytes, like proteins and peptides. Similarly, frits used to secure solid phase extraction media or affinity resin within a housing is also commonly made from polypropylene or polyethylene (another known material that causes adsorptive losses).
Herein, we have investigated the adsorptive losses with untreated versus vapor deposition coated fritted disposable pipette tips. To address adsorptive losses, the wetted surfaces of fritted pipette tips were modified with a C2-GPTMS-OH coating (See Example 2 above). The coating was applied using vapor deposition through the interior of fritted pipette. That is, the wetted surfaces of the pipette tips were coated with C2-GPTMS-OH with the frits installed within the devices; the vapors infiltrating into the pipette and coating walls and the frit along the fluidic path. Tests were performed on the C2-GPTMS-OH coated tips (in two sizes 200 μL and 1000 μL), and compared to plasma treated devices, and untreated devices.
The adsorptive losses resulting in the C2-GPTMS-OH coated tips, the plasma-treated (oxygen plasma with water vapor), and untreated tips were measured by passing 20 μg of rabbit IgG (rIgG, Equitech-Bio, SLR56) through each tip for 5 aspiration/dispensing cycles with a multi-channel pipette. The flow-through fraction was collected to determine the amount of IgG recovered from each tip. The concentration of rig in each fraction was measured using a fluorometer (Gemini XPS, Molecular Devices). The amount of igG lost during the sample preparation was calculated by comparing the amount of rIgG loaded versus the amount of IgG recovered in the flow-through.
In this comparative test, 200 μL of 0.1 mg/mL IgG solution was applied to the 200 μL injection-molded tips (in coated, plasma-treated, and untreated formats) and 1 mL of 0.02 mg/mL of IgG solution was applied to the 1000 μL tips. The rIgG solutions were first transferred to a 96-well plastic plate (commercially available from Waters Corporation, Milford, Mass.) using either 200 μL or 1000 μL untreated polypropylene pipette tips, then loaded to the test tips. Six replicates were tested for the coated and plasma treated tips and four replicates were tested for the untreated tips.
There are a number of alternative methods and uses for the present technology. While the above methods have generally been discussed with respect to chromatography, the use of a column, or labware, such as beakers, pipettes, and extraction devices, other types of fluid components having an internal flow path may benefit from the present technology. For example, it is generally thought that capillary electrophoresis, such as capillary zone electrophoresis, exhibits relatively poor reproducibility. Much of the reproducibility issues can be reasoned to originate from irreproducible surface chemistry on the inner diameter of the tubular capillaries that are used to perform the separation. A vapor deposition coating on capillaries intended for a CE separation may therefore circumvent reproducibility issues as it can yield an inordinately thick, rugged coating. The inventions described herein may consequently be applicable to improving CE separations of both small and large biomolecules.
Moreover, while many of the examples of the described aspects employ comparatively hydrophobic coatings, with water contact angles ranging from 50° to 115°, it is reasonable to suggest that some separations could be enhanced through the application of hydrophilic or other types of coatings, including but not restricted to diol, ethers, esters, amide/ureido type, and polyethylene oxide/glycol bondings and, in some embodiments, coatings having contact angles between 0.1 to about 60°.
Other analytes, not yet explicitly described, may also benefit from vapor deposition coated chromatographic flow paths, for instance phosphorothioated oligonucleotides. Nucleic acids inherently contain repeating phosphodiester bonds as part of their backbone. In some case, the phosphodiester backbone is replaced in part with a phosphorothioate backbone, which can impart in itself unique challenges for a separation. Similarly, intact and proteolytically digested antibody conjugates may benefit from methods entailing the use of vapor deposition chromatographic flow paths. Lastly, biomolecules containing histidine residues are likely to benefit from this invention as, like phosphorylated and carboxylate containing residues, they have a propensity for binding to metal.
This application is a continuation-in-part of U.S. application Ser. No. 16/133,089, filed on Sep. 17, 2018 and entitled “Use of Vapor Deposition Coated Flow Paths for Improved Chromatography of Metal Interacting Analytes”, which claims priority to and benefit of U.S. provisional application No. 62/559,895 filed Sep. 18, 2017, also entitled “Use of Vapor Deposition Coated Flow Paths for Improved Chromatography of Biomolecules.” The contents of each application are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62559895 | Sep 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16133089 | Sep 2018 | US |
| Child | 16746108 | US |
