Patent Publication Number: US-2021164094-A1

Title: Use of vapor deposition coated flow paths for improved chromatography of metal interacting analytes

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation-in-part of U.S. utility application Ser. No. 16/133,089, entitled “Use of Vapor Deposition Coated Flow Paths for Improved Chromatography of Metal Interacting Analytes” filed on Sep. 17, 2018, which claims priority to and benefit of U.S. provisional application No. 62/559,895 filed Sep. 18, 2017 entitled “Use of Vapor Deposition Coated Flow Paths for Improved Chromatography of Biomolecules.” This application also claims the benefit of and priority to U.S. provisional application No. 63/092,789 filed Oct. 16, 2020 entitled “Device Including a Hydrophilic, Non-Ionic Coating for Size Exclusion Chromatography.” The contents of each of the foregoing applications are incorporated herein by reference in their entirety. 
    
    
     FIELD OF THE TECHNOLOGY 
     This technology relates to the use of vapor deposition coated frit(s) in a liquid flow path for improved chromatography. More specifically, this technology relates to liquid chromatographic devices for separating analytes in a sample having coated frit(s) within the fluidic flow path, methods of separating analytes in a sample (for example, glycans, peptides, pesticides, and citric acid cycle metabolites) using a fluidic system that includes a coated metallic frit within a liquid flow path, and methods of tailoring a liquid flow path for separation of a sample. 
     BACKGROUND OF THE TECHNOLOGY 
     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 (including frits, connectors, and other devices within the wetted path) 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. 
     In a chromatographic system, the sample fluidic pathway is often comprised of several components, such as needles, connecting tubing, unions, valves, and chromatographic columns (including, column tubing and porous frit elements). Each of these components contributes surface area with which injected sample can interact as it passes through the system and ultimately into a detector or mass spectrometer. These components are typically made of metal. 
     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. 
     Ongoing efforts to reduce chelation and secondary chromatographic interactions of analytes with metal chromatographic surfaces in an effort to facilitate chromatographic separation having higher resolutions are therefore needed. 
     SUMMARY OF THE TECHNOLOGY 
     One advantage of the coated frits of the present technology is that metal chromatographic flow paths (e.g., column tubes or channels formed from stainless steel, titanium, such as pure titanium or a titanium alloy) can be used while minimizing the interactions between analytes and the chromatographic device. Coating the frits disposed within chromatographic devices with certain organosilica (e.g., alkylsilyl) 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 flow through components can be manufactured out of stainless steel or other metallic or high pressure material. 
     Provided herein, therefore, are methods for isolating analytes comprising the use of vapor depositing one or more alkylsilyl derivatives to at least one component of a fluidic system (e.g., frit used within the liquid chromatographic column) to form a bioinert or low-bind coating, and eluting the analyte through the system. Unlike ambient, liquid phase silanization, coatings of the present technology 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, such as the complex shapes of a frit. This advantage allows for coatings to be applied to flow paths with narrow internal diameters, curved surfaces, and fine or mesh-like structures, 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 on just the frits disposed within the fluidic system, and eluting the sample through the system. 
     The disclosed methods can be applied to stainless steel, titanium (pure or alloy), 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 column including an uncoated tube (or channel) comprising stainless steel or titanium or both (e.g., stainless steel tube, a titanium tube, a titanium alloy channel, such as a MP35NLT channel, a titanium stabilized alloy such as a 316Ti stainless steel tube). A coated frit comprising a coating is disposed proximate to an inlet of the uncoated tube. The uncoated tube does not have the coating. The coating reduces binding of one or more sample components to the coated frit relative to the binding of the one or more sample components to the uncoated tube. 
     The above aspect can include one or more of the following features. The chromatographic column can include two or more coated frits. The at least one coated frit can include an underlying substrate of stainless or titanium. The coating of the coated frit substantially covers the underlying substrate such that the coating is in contact with sample components. The coating can have a contact angle of at least 150 with respect to the underlying substrate. In some embodiments, the coating has a contact angle less than or equal to 300 or less than or equal to 90 to the underlying substrate. 
     In some embodiments, the uncoated tube (or uncoated channel) has a length to diameter ratio of between 40 and 4. The coating can have a thickness of at least 100 Å. 
     In certain embodiments, the coating is an organosilica, and in some instances an alkylsilyl. In some embodiments, the alkylsilyl has the structure of Formula I: 
     
       
         
         
             
             
         
       
     
     Where at least one of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  is OR A . When R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  are not OR A , then R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  are each independently selected from (C 1 -C 6 )alkoxy, —NH(C 1 -C 6 )alkyl, —N((C 1 -C 6 )alkyl) 2 , OH, and halo. R A  represents a point of attachment to the interior surfaces of the fluidic system. X is (C 1 -C 20 )alkyl, —O[(CH 2 ) 2 O] 1-20 —, —(C 1 -C 10 )[NH(CO)NH(C 1 -C 10 )] 1-20 —, or —(C 1 -C 10 )[alkylphenyl(C 1 -C 10 )alkyl] 1-20 —. The device can include one or more of the following embodiments in any combination thereof. 
     In some embodiments, X is (C 2 -C 10 )alkyl. In other embodiments, X is ethyl. R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  can each be methoxy or chloro. In some embodiments, the coating (which is an alkylsilyl coating of Formula I) is formed using a vapor reagent of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane. 
     In some embodiments, the coating of the chromatographic column includes a primer layer and a top layer. In certain embodiments. The primer layer is an alkylsilyl coating of Formula I. In certain embodiments, the top layer is an alkylsilyl having a structure of Formula II: 
     
       
         
         
             
             
         
       
     
     R 7 , R 8 , and R 9  are each independently selected from —NH(C 1 -C 6 )alkyl, —N[(C 1 -C 6 )alkyl]2, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkenyl, OH, and halo. R 10  is selected from (C 1 -C 6 )alkyl, —OR B , [O(C 1 -C 3 )alkyl] 1-10 O(C 1 -C 6 )alkyl, —[O(C 1 -C 3 )alkyl] 1-10 OH, and phenyl, wherein said (C 1 -C 6 )alkyl is optionally substituted with one or more halo and wherein said phenyl is optionally substituted with one or more groups selected from (C 1 -C 3 )alkyl, hydroxyl, fluorine, chlorine, bromine, cyano, —C(O)NH 2 , and carboxyl. R B  is —(C 1 -C 3 )alkyloxirane, —(C 1 -C 3 )alkyl-3,4-epoxycyclohexyl, or —(C 1 -C 4 )alkylOH. The hashed bond to R 10  represents an optional additional covalent bond between R 10  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 some embodiments, y is an integer from 2 to 9. In some embodiments, is 9, R 10  is methyl, and R 7 , R 8 , and R 9  are each ethoxy or chloro. 
     In some embodiments, in which the top layer is an alkylsilyl of Formula II, a vapor reagent of (3-glycidyloxypropyl)trimethoxysilane, n-decyltrichlorosilane, trimethylchlorosilane, trimethyldimethyaminosilane, methoxy-polyethyleneoxy(1-10) propyl trichlorosilane, or methoxy-polyethyleneoxy(1-10) propyl trimethoxysilane can be used to form the top layer. In some embodiments, a vapor reagent of (3-glycidyloxypropyl)trimethoxysilane followed by hydrolysis is used to form the top layer. 
     In some embodiments which have a dual layer coating, the vapor reagent used to form the primer layer is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and the second or different vapor reagent used to form the top layer is (3-glycidyloxypropyl)trimethoxysilane. In some embodiments, the vapor reagent used to form the primer layer is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and the vapor reagent used to form the top layer is (3-glycidyloxypropyl)trimethoxysilane followed by hydrolysis. In some embodiments, vapor reagent used to form the primer layer is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and vapor reagent used to form the top layer is n-decyltrichlorosilane. The vapor reagent used to form the primer layer can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and the vapor reagent used to form the top layer can be trimethylchlorosilane or trimethyldimethyaminosilane. In some embodiments, the vapor reagent used to form the primer layer is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and vapor reagent used to form the top layer is methoxy-polyethyleneoxy(3)silane. 
     The top layer of the coating, in some embodiments, can be an alkylsilyl having the structure Formula III, 
     
       
         
         
             
             
         
       
     
     R 11 , R 12 , R 13 , R 14 , R 15 , and R 16  are each independently selected from (C 1 -C 6 )alkoxy, —NH(C 1 -C 6 )alkyl, —N((C 1 -C 6 )alkyl) 2 , OH, and halo. Z is (C 1 -C 20 )alkyl, —O[(CH 2 ) 2 O] 1-20 —, —(C 1 -C 10 )[NH(CO)NH(C 1 -C 10 )] 1-20 —, or —(C 1 -C 10 )[alkylphenyl(C 1 -C 10 )alkyl] 1-20 —. In some of these embodiments, the top layer is formed using a vapor reagent of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane. The primer layer can be formed of a vapor reagent of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane. 
     In some embodiments, the top layer is an organosilica, such as a polyethylene glycol (PEG). The PEG top layer is typically deposited using a liquid (wet) deposition as compared to the vapor/gas phase deposition of the primer layer (e.g., the alkylsilyl coating). The PEG top layer is deposited after the alkylsilyl coating coats the exposed surfaces of the underlying metal substrate (i.e., metal frit). As a result, the PEG layer is in contact with the fluid path during operation/use of the chromatographic device. That is the PEG layer is on the fluid exposed surfaces. In certain embodiments, the top layer has a structure of Formula IV: 
     
       
         
         
             
             
         
       
     
     wherein: 
     R 17 , R 18 , and R 19  are each independently selected from the group consisting of C 1 -C 6  alkyl, (C 1 -C 6 )alkenyl, —NHR 10 , —NR 10 R 11 , OH, OR 10 , and halo; 
     R 10  and R 11  are each independently selected from C 1 -C 6  alkyl; 
     P is —(C 1 -C 6 )alkyl-OR 21 , —[O(C 1 -C 3 )alkyl] 1-10 O(C 1 -C 6 )alkyl, —[O(C 1 -C 3 )alkyl] 1-10 OH or phenyl, wherein said (C 1 -C 6 )alkyl is substituted with one or more halo or one or more functional groups selected from the group consisting of OH, amino, (C 1 -C 6 )alkylamino, di(C 1 -C 6 )alkylamino, cyano, —C(O)NH 2 , sulfonic acid, and carboxyl, and wherein said phenyl is optionally substituted with one or more groups selected from the group consisting of (C 1 -C 3 )alkyl, OH, halo, cyano, —C(O)NH 2 , and carboxyl; R 21  is —(C 1 -C 3 )alkyloxirane, —(C 1 -C 3 )alkyl-3,4-epoxycyclohexyl, or —(C 1 -C 4 )alkylOH. In some embodiments an organosilane reagent used to deposit the top layer is 2-[methoxy(polyethyleneoxy) 6-9 propyl]tris(dimethylamino)silane—and in some instances, the vapor reagent used to form the primer layer is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic of a chromatographic flow system including a chromatography column and various other components, in accordance with an illustrative embodiment of the technology. A fluid is carried through the chromatographic flow system with a fluidic flow path extending from a fluid manager to a detector. 
         FIG. 2  depicts a non-limiting illustration of a modified metallic surface according to an embodiment in accordance with an embodiment of the present technology. 
         FIG. 3A  is a flow chart showing a method of tailoring wetted surfaces of a flow path, in accordance with an illustrative embodiment of the technology. 
         FIG. 3B  is a flow chart showing a method of tailoring a fluidic flow path for separation of a sample including a biomolecule, in accordance with an illustrative embodiment of the technology. 
         FIG. 4A  shows a fluorescence chromatogram obtained using uncoated stainless steel hardware, in accordance with an illustrative embodiment of the technology. 
         FIG. 4B  shows a fluorescence chromatogram obtained using hardware coated with exemplary vapor deposited alkylsilyl, in accordance with an illustrative embodiment of the technology. 
         FIG. 4C  shows a fluorescence chromatogram obtained using hardware coated with exemplary vapor deposited alkylsilyl, in accordance with an illustrative embodiment of the technology. 
         FIG. 5A  is a schematic of exemplified bioinert alkysilyl coated stainless steel sample flow path components, including column inlet tubing, in accordance with an illustrative embodiment of the technology. 
         FIG. 5B  is a schematic of exemplified bioinert alkysilyl coated stainless steel sample flow path components, including a sample needle, in accordance with an illustrative embodiment of the technology. 
         FIG. 6A  shows a fluorescence chromatogram obtained using an untreated flow path and untreated tube and frit combination in accordance with an embodiment of the technology. 
         FIG. 6B  shows a fluorescence chromatogram obtained using an untreated flow path and coated tube and frit combination, in accordance with an embodiment of the technology. 
         FIG. 6C  shows a fluorescence chromatogram obtained using a coated flow path and coated tube and frit combination, in accordance with an embodiment of the technology. 
         FIG. 7A  shows a UV chromatogram obtained using an untreated stainless steel tube/frit combination. 
         FIG. 7B  shows a UV chromatogram obtained using a C 2  vapor deposition coated tube/frit combination, in accordance with an embodiment of the technology. 
         FIG. 7C  shows a UV chromatogram obtained using a C 2 C 10  vapor deposition coated tube/frit combination, in accordance with an embodiment of the technology. 
         FIG. 8A  is a chromatogram showing the effects of employing vapor deposition coated column hardware for the reversed phase LC analyses of glucose-6-phosphate, in accordance with an illustrative embodiment of the technology. 
         FIG. 8B  is a chromatogram showing the effects of employing untreated column hardware for the reversed phase LC analyses of glucose-6-phosphate. 
         FIG. 9A  is a chromatogram showing the effects of employing vapor deposition coated column hardware for the reversed phase LC analyses of fructose-6-phosphate, in accordance with an illustrative embodiment of the technology. 
         FIG. 9B  is a chromatogram showing the effects of employing untreated column hardware for the reversed phase LC analyses of fructose-6-phosphate. 
         FIG. 10A  is a chromatogram showing the effects of employing vapor deposition coated column hardware for the reversed phase LC analyses of adenosine triphosphate, in accordance with an illustrative embodiment of the technology. 
         FIG. 10B  is a chromatogram showing the effects of employing untreated column hardware for the reversed phase LC analyses of adenosine triphosphate. 
         FIG. 11A  is a chromatogram showing the effects of employing vapor deposition coated column hardware for the reversed phase LC analyses of adenosine monophosphate, in accordance with an illustrative embodiment of the technology. 
         FIG. 11B  is a chromatogram showing the effects of employing untreated column hardware for the reversed phase LC analyses of adenosine monophosphate. 
         FIG. 12A  is a fluorescence chromatogram for fetuin N-glycans obtained with untreated stainless steel hardware. 
         FIG. 12B  is a fluorescence chromatogram for fetuin N-glycans obtained with vapor deposition coated hardware, in accordance with an illustrative embodiment of the technology. 
         FIG. 13A  is a graph showing fluorescence peak areas for disialyated glycans obtained with untreated stainless steel hardware compared to stainless steel hardware coated different types of vapor deposited coatings, in accordance with an illustrative embodiment of the technology. 
         FIG. 13B  is a graph showing fluorescence peak areas for trisialyated glycans obtained with untreated stainless steel hardware compared to stainless steel hardware coated different types of vapor deposited coatings, in accordance with an illustrative embodiment of the technology. 
         FIG. 13C  is a graph showing fluorescence peak areas for tetrasialyated glycans obtained with untreated stainless steel hardware compared to stainless steel hardware coated different types of vapor deposited coatings, in accordance with an illustrative embodiment of the technology. 
         FIG. 13D  is a graph showing fluorescence peak areas for pentasialyated glycans obtained with untreated stainless steel hardware compared to stainless steel hardware coated different types of vapor deposited coatings, in accordance with an illustrative embodiment of the technology. 
         FIG. 14A  is a reversed phase fluorescence chromatogram of reduced, IdeS-digested NIST Reference Material 8671 obtained with column hardware components treated with coatings in accordance with illustrative embodiments of the technology. 
         FIG. 14B  is a reversed phase fluorescence chromatogram of reduced, IdeS-digested NIST Reference Material 8671 obtained with column hardware components treated with coatings in accordance with illustrative embodiments of the technology. 
         FIG. 15A  is a reversed phase total ion chromatogram for columns constructed with stainless steel alternatives, namely polyether ether ketone (PEEK) and a low titanium, nickel cobalt alloy (MP35NLT) with various components coated, in accordance with an illustrative embodiment of the technology. 
         FIG. 15B  is a reversed phase total ion chromatogram for column components (i.e., frits) constructed with stainless steel, C 2  coatings and C 2 C 10  coatings, in accordance with an illustrative embodiment of the technology. 
         FIG. 16A  shows fluorescence chromatograms of reduced, IdeS-digested NIST Reference Material 8671 and the effect on baseline return when various components of the system are coated, in accordance with an illustrative embodiment of the technology. 
         FIG. 16B  shows reversed phase total ion chromatograms (TICs) reduced, IdeS-digested NIST Reference Material 8671 and the effect on baseline return when various components of the system are coated, in accordance with an illustrative embodiment of the technology. 
         FIG. 16C  is a schematic of the column tube and frits that were coated and used to obtain the chromatograms of  FIGS. 16A and 16B , in accordance with an illustrative embodiment of the technology. 
         FIG. 17A  shows fluorescence chromatograms of reduced, IdeS-digested NIST Reference Material 8671 and the effect on baseline return when various components of the system are coated, in accordance with an illustrative embodiment of the technology. 
         FIG. 17B  shows reversed phase total ion chromatograms (TICs) of reduced, IdeS-digested NIST Reference Material 8671 and the effect on baseline return when various components of the system are coated, in accordance with an illustrative embodiment of the technology. 
         FIG. 17C  is a schematic of the column tube and frits that were coated and used to obtain the chromatograms of  FIGS. 17A and 17B , in accordance with an illustrative embodiment of the technology 
         FIG. 18  is a bar graph showing bubble point pressure in each of water and IPA for a non-coated stainless steel frit and stainless steel frits coated in accordance with one or more illustrative embodiments of the technology. The bubble point in water is provided as the left bar, and the bubble point in IPA is provided as the right bar for each type of frit. 
         FIG. 19  is a bar graph showing a comparison of frit porosity contact angle with water for a non-coated stainless steel frit versus stainless steel frits coated in accordance with one or more embodiments of the technology. 
         FIG. 20  is a bar graph showing a comparison of mass loss test according to ASTM G48 Method A for a bare or uncoated stainless steel frit versus stainless steel frits coated in accordance with one or more embodiments of the technology. 
         FIG. 21A  is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection  1  using hardware A, and uncoated. 
         FIG. 21B  is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection  2  using hardware A, uncoated. 
         FIG. 21C  is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection  3  using hardware A, uncoated. 
         FIG. 21D  is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection  1  using hardware A, with a C 2  coating, in accordance with an illustrative embodiment of the technology. 
         FIG. 21E  is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection  2  using hardware A, with C 2  coating, in accordance with an illustrative embodiment of the technology. 
         FIG. 21F  is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection  3  using hardware A, with C 2  coating, in accordance with an illustrative embodiment of the technology. 
         FIG. 21G  is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection  1  using hardware B, and uncoated. 
         FIG. 21H  is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection  2  using hardware B, uncoated. 
         FIG. 21I  is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection  3  using hardware B, uncoated. 
         FIG. 21J  is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection  1  using hardware B, with a C 2 -GPTMS-OH coating, in accordance with an illustrative embodiment of the technology. 
         FIG. 21K  is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection  2  using hardware B, with a C 2 -GPTMS-OH coating, in accordance with an illustrative embodiment of the technology. 
         FIG. 21L  is a chromatogram of NIST reference material 8671, an IgG1κmAb, as obtained from injection  3  using hardware B, with a C 2 -GPTMS-OH coating, in accordance with an illustrative embodiment of the technology. 
         FIG. 22  presents a bar graph showing peak areas of NIST reference materials 8671 obtained from sequential cation exchange separations over three injections of the sample. This bar graph compares the peak areas for four different constructions in which the left most bar in each injection is an uncoated hardware A construction. The second from the left is a coated version of hardware A. The third bar from the left is an uncoated hardware B construction and the fourth or last bar per injection is a coated hardware B construction. 
         FIG. 23A  is a reversed-phase chromatogram of the first injection of 5 picomoles of deoxythymidine oligomers (15, 20, 25, 30, and 35-mer) obtained from a 2.1×50 mm 1.7 μm organosilica 130 Å C 18  column constructed with an untreated stainless steel (SS) tube and frits. 
         FIG. 23B  is a reversed-phase chromatogram of the second injection of 5 picomoles of deoxythymidine oligomers (15, 20, 25, 30, and 35-mer) obtained from a 2.1×50 mm 1.7 μm organosilica 130 Å C 18  column constructed with an untreated stainless steel (SS) tube and frits. 
         FIG. 23C  is a reversed-phase chromatogram of the third injection of 5 picomoles of deoxythymidine oligomers (15, 20, 25, 30, and 35-mer) obtained from a 2.1×50 mm 1.7 μm organosilica 130 Å C 18  column constructed with an untreated stainless steel (SS) tube and frits. 
         FIG. 23D  is a reversed-phase chromatogram of the first injection of 5 picomoles of deoxythymidine oligomers (15, 20, 25, 30, and 35-mer) obtained from a column constructed with a C 2 C 10  vapor deposition coated tube and frits, in accordance with an illustrative embodiment of the technology. 
         FIG. 23E  is a reversed-phase chromatogram of the second injection of 5 picomoles of deoxythymidine oligomers (15, 20, 25, 30, and 35-mer) obtained from a column constructed with a C 2 C 10  vapor deposition coated tube and frits, in accordance with an illustrative embodiment of the technology. 
         FIG. 23F  is a reversed-phase chromatogram of the third injection of 5 picomoles of deoxythymidine oligomers (15, 20, 25, 30, and 35-mer) obtained from a column constructed with a C 2 C 10  vapor deposition coated tube and frits, in accordance with an illustrative embodiment of the technology. 
         FIG. 24  is a graph showing the average UV peak areas of a 15-mer deoxythymidine analyte as observed during reversed phase chromatography and initial injections onto either a 2.1×50 mm 1.7 μm organosilica 130 Å C 18  column constructed with untreated stainless steel (SS) or C 2 C 10  vapor deposition coated components, in accordance with an illustrative embodiment of the technology. Analyses were performed in duplicate using two untreated columns and two C 2 C 10  vapor deposition coated columns. 
         FIG. 25A  is a reversed phase MRM (multiple reaction monitoring) chromatogram obtained for citric acid with the use of a 2.1×50 mm 1.8 μm silica 100 Å C 18  1.8 m column constructed with C 2 C 3  vapor deposition coated components, in accordance with an illustrative embodiment of the technology. 
         FIG. 25B  is a reversed phase MRM (multiple reaction monitoring) chromatogram obtained for citric acid with the use of a 2.1×50 mm 1.8 μm silica 100 Å C 18  1.8 m column constructed with untreated components. 
         FIG. 25C  is a reversed phase MRM (multiple reaction monitoring) chromatogram obtained for malic acid with the use of a 2.1×50 mm 1.8 μm silica 100 Å C 18  1.8 m column constructed with C 2 C 3  vapor deposition coated components, in accordance with an illustrative embodiment of the technology. 
         FIG. 25D  is a reversed phase MRM (multiple reaction monitoring) chromatogram obtained for malic acid with the use of a 2.1×50 mm 1.8 μm silica 100 Å C 18  1.8 m column constructed with untreated components. 
         FIG. 26A  is a mixed mode hydrophilic interaction chromatography (HILIC) MRM (multiple reaction monitoring) chromatogram of glyphosate showing MRM peak intensities obtained for glyphosate with the use of a 2.1×100 mm 1.7 μm diethylamine bonded organosilica 130 Å column constructed with C 2 C 10  vapor deposition coated components, in accordance with an illustrative embodiment of the technology. 
         FIG. 26B  is a mixed mode hydrophilic interaction chromatography (HILIC) MRM (multiple reaction monitoring) chromatogram of glyphosate showing MRM peak intensities obtained for glyphosate with the use of a 2.1×100 mm 1.7 μm diethylamine bonded organosilica 130 Å column with uncoated components. 
         FIG. 27A  is a graph showing the average peak areas of glyphosate as observed during mixed mode HILIC using either a 2.1×100 mm 1.7 μm diethylamine bonded organosilica 130 Å column constructed with either untreated or C 2 C 10  vapor deposition coated components in accordance with an illustrative embodiment of the technology. The analyses were performed with six replicate injections. 
         FIG. 27B  is a graph showing the average peak widths of glyphosate as observed during mixed mode HILIC using either a 2.1×100 mm 1.7 μm diethylamine bonded organosilica 130 Å column constructed with either untreated or C 2 C 10  vapor deposition coated components, in accordance with an illustrative embodiment of the technology. The analyses were performed with six replicate injections. 
         FIG. 28  is a graph showing average peak area for dexamethasone sodium phosphate versus uncoated total surface area for three different system configurations. Zero mm 2  of uncoated surface area corresponds to a fully coated system. 
         FIG. 29  is graph showing a calibration curve for dexamethasone sodium phosphate peak area versus the amount of dexamethasone injected on a fully coated system having a coated column. 
         FIG. 30  is graph showing ATP peak area versus the amount of ATP injected (i.e., per injection) for three different column assemblies. 
         FIG. 31  is a graph showing ATP peak area after the first injection versus coated surface area for three different system configurations. Zero mm 2  of coated surface area corresponds to an uncoated assembly. 
         FIG. 32  is a graph showing potential loss of dexamethasone sodium phosphate in uncoated flow paths for three different system configurations. 
     
    
    
     DETAILED DESCRIPTION 
     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 liquid flow path for isolation of an analyte, in particular a metal-interacting analyte; (3) method of isolating an analyte in a sample, in particular a metal-interacting analyte; and (4) kits comprising various 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, or at least a portion of the flow path, to address flow path interactions with an analyte. That is, the bioinert, low-bind coating minimizes surface reactions with the metal 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 a metal-interacting analyte, for example biomolecules, proteins, glycans, peptides, oligonucleotides, pesticides, bisphosphonic acids, 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) modify the fluidic path and decrease secondary interactions. In one embodiment, the alkylsilyl coating is vapor deposited on a portion (e.g., 40%, 45, 50%, 55%, 60% or more) of the exposed surface area defining or contributing to the flow path (e.g., column or tubing walls, entire surface area of frits, etc.). As such, the coatings 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. 
     Due to the generally high surface area nature of frits residing within the interior of a chromatographic column, their contribution to the available surface area in the sample fluidic pathway (fluid contacting surface area) can be substantial (e.g. frit surface area can make up 25% to almost 97% of the fluid contacting surface area of a system). Moreover, sample interaction with frit surfaces is inherently high since the sample is generally distributed over the entire cross-sectional area of the frit and then travels through a complex pathway of micro-channels through the thickness of the frit. By contrast, sample interaction with a column tube wall is limited, as the vast majority of sample travels through the packed bed of stationary phase, avoiding contact with the column walls. Moreover, the amount of available fluid contacting surface area attributable to the frits (i.e., a column interior can have one, two, or multiple frits residing therein) changes with column diameter. For example, for a 2.1 mm UHPLC column for use with a Waters Acquity UPLC system (150 mm column length), the amount of available surface area attributable to the system is calculated to be 20%, attributable to the column tubing is 40%, and attributable to the frits is 40%; for a 4.6 mm UHPLC column (also having 150 mm length) for use on a Waters ARC system, the amount of available surface area attributable to the system is approximately 15%, attributable to the column tubing 20%, and attributable to the frits is 65%; for a 7.8 mm UHPLC column (150 mm column length) for use on a Waters ARC system, the amount of available surface area attributable to the system is approximately 8%, attributable to the column tubing 12%, and attributable to the frits is 80%; and for a 30 mm preparatory column (150 mm length), the amount of available surface area attributable to both the system and column tubing combined is less than 5%, leaving over 95% attributable to the frits. As a result, as the column diameter increases, so does the amount of available fluid contacting surface area attributable to the frits (i.e., from 40% to 65% to 80%, to over 95% in the above example). 
     While frits can be made from polymeric materials to avoid metal interactions altogether, polymeric frits lack the strength to withstand very high operating pressures and are easily distorted or deformed. Polymeric frits also tend to be less consistent in their flow and permeability properties. Metal frits can be made thinner to reduce surface area, but this also comes at the expense of structural strength. Thin metal frits are susceptible to deformation under high pressure or mechanical force, especially when the column inner diameter is large. Furthermore, very thin frits are in general less capable of holding back fine stationary phase particles, allowing them to escape the column and foul downstream instrument components. 
     Devices 
       FIG. 1  is a representative schematic of a chromatographic flow system/device  100  that can be used to separate analytes in a sample. Chromatographic flow system  100  includes several components including a fluid manager system  105  (e.g., controls mobile phase flow through the system), tubing  110  (which could also be replaced or used together with microfabricated fluid conduits), fluid connectors  115  (e.g., fluidic caps), frits  120 , a chromatography column  125 , a sample injector  135  including a needle (not shown) to insert or inject the sample into the mobile phase, a vial, sinker, or sample reservoir  130  for holding the sample prior to injection, a detector  150  and a pressure regulator  140  for controlling pressure of the flow. Interior surfaces of the components of the chromatographic system/device form a fluidic flow path that has wetted surfaces. The fluidic flow path can have a length to diameter ratio of at least 20, at least 25, at least 30, at least 35 or at least 40. 
     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. 
     In some embodiments, only a portion of the wetted surfaces include the alkylsilyl coating. For example, not all wetted surfaces (i.e., surface area that will be in contact with liquids during use) needs to be coated to tailor a flow path and/or to improve chromatographic results. In some embodiments, 40% or more (e.g., 45%, 50%, 55%, 60%, 65%, 67%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 77%, 80%, 90% or more) of the surface area defining or contributing to the flow path is coated with an alkylsilyl coating. In certain embodiments, 40% or more of surface area defining or contributing to a metallic flow path (i.e., parts including stainless steel, titanium, titanium alloys, etc.) is coated with an alkylsilyl coating. In some embodiments, 60% or more of the surface area defining or contributing to a metallic flow path is coated with an alkylsilyl coating to provide improved chromatographic results. 
     In some embodiments, frits  120  contained within the chromatographic column  125  provide the greatest amount of wetted surface area to the flow path. Fluids in the system pass through the frits  120 , which have fluid facing and fluid flow through surfaces composed of mesh-like or fine features. As the fluid passes all around these features, the frits  120  contribute greatly to the surface area defining or contributing to the flow path. In particular, systems having two or more frits in short columns (e.g., &lt;150 mm), or columns with large diameters (e.g., &gt;2 mm) tend to have a majority portion (i.e., 51% or more) of the wetted surface area originating just from the frits. For example, for a 4.6 mm by 150 mm column including two frits, approximately 65% of the wetted surface area originates from the two frits. The remaining 35% or less is from the column/tubing walls and the remainder of the system. 
     In one embodiment, the at least a portion of the wetted surfaces of the fluidic flow path consist of the frits alone. 
     There are many applications where the highest sensitivity and best analyte recovery are desired. In general, these applications, may include complete or substantially complete coverage (e.g., 93% or more) of metallic surfaces with the alkylsilyl coating. For example, low level drug impurity analysis would likely require a fully coated sample fluidic path to ensure highest possible sensitivity and recovery of analytes. However, there are some embodiments where acceptable performance or improved performance is achieved by applying the vapor deposited coating to the frits alone (i.e., column tubing and connecting tubes and ports are uncoated). Once such embodiment is preparative chromatography, which often utilizes wide (&gt;10 mm) inner diameter columns, and which have very large and thick frits. Another possible embodiment is when the number of frits in the flow path is increased, such as when multiple columns are connected in series or when guard columns are utilized. 
     In an embodiment, the at least a portion of the wetted surfaces of the fluidic flow path consist of the frits (i.e., column frits  120 ) and some portion of the column walls and/or tubing upstream of the column. In an embodiment, the at least a portion of the wetted surfaces of the fluidic flow path consist of frits and the sample injector  135 . In certain embodiments, the at least a portion of the wetted surfaces of the fluidic flow path consists of frits, the sample injector  135 , and the sample reservoir  130 . Other components forming or contained within the flow path that are not specifically recited in the foregoing embodiments are not coated (i.e., less than 100% coverage of wetted surfaces, less than 80% coverage of wetted surfaces, less than 75% coverage of wetted surfaces, 65% covered). In some embodiments, just the frits are coated. An exemplary embodiment includes coating just the frits in a preparatory column or coating just the frits in a system that includes more than two frits. 
     At least a portion of the wetted surfaces of the fluidic flow path 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: 
     
       
         
         
             
             
         
       
     
     R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  are each independently selected from (C 1 -C 6 )alkoxy, —NH(C 1 -C 6 )alkyl, —N((C 1 -C 6 )alkyl) 2 , OH, OR A , and halo (i.e., a halogen, for example chloro). R A  represents a point of attachment to the interior surfaces of the fluidic system. At least one of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  is OR A . X is (C 1 -C 20 )alkyl, —O[(CH 2 ) 2 O] 1-20 —, —(C 1 -C 10 )[NH(CO)NH(C 1 -C 10 )] 1-20 —, or —(C 1 -C 10 )[alkylphenyl(C 1 -C 10 )alkyl] 1-20 —. 
     When used in the context of a chemical formula, a hyphen (“-”) indicates the point of attachment. For example, when X is —[(C 1 -C 10 )alkylphenyl(C 1 -C 10 )alkyl] 1-20 —, that means that X is connected to SiR 1 R 2 R 3  via the (C 1 -C 10 )alkyl and connected to SiR 4 R 5 R 6  via the other (C 1 -C 10 )alkyl. This applies to the remaining variables. 
     In one aspect, X in Formula I is (C 1 -C 15 )alkyl, (C 1 -C 12 )alkyl, or (C 1 -C 10 )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 R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  is (C 1 -C 6 )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 R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  is (C 1 -C 6 )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 R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  is (C 1 -C 6 )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 R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  is (C 1 -C 6 )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 R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  is (C 1 -C 6 )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 R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  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 R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  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 R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  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 R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  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 R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  is halo, e.g., chloro, wherein the values for X are described in Formula I or the preceding paragraphs above. 
     In another aspect, R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  are each methoxy or chloro. 
     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 90°. In some embodiments, the contact angle of the alkylsilyl coating of Formula I is between about 15 to about 105°. 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°, or 105°. 
     The thickness of the alkylsilyl coating can be at least about 100 Å. For example, the thickness can be between about 100 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 of the fluidic flow path are coated with multiple layers of the same or different alkylsilyls, where the thickness of the alkylsilyl coatings correlate with the number of layering steps performed (e.g., the number of deposited layers of alkylsilyl coating on wetted surface of the fluidic flow path of the chromatographic system/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 R 7 , R 8 , and R 9  are each independently selected from —NH(C 1 -C 6 )alkyl, —N[(C 1 -C 6 )alkyl]2, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkenyl, OH, and halo; R ° is selected from (C 1 -C 6 )alkyl, —OR B , —[O(C 1 -C 3 )alkyl] 1-10 O(C 1 -C 6 )alkyl, —[O(C 1 -C 3 )alkyl] 1-10 OH and phenyl. (C 1 -C 6 )alkyl is optionally substituted with one or more halo. The phenyl is optionally substituted with one or more groups selected from (C 1 -C 3 )alkyl, hydroxyl, fluorine, chlorine, bromine, cyano, —C(O)NH 2 , and carboxyl. R B  is —(C 1 -C 3 )alkyloxirane, —(C 1 -C 3 )alkyl-3,4-epoxycyclohexyl, or —(C 1 -C 4 )alkylOH. The hashed bond to R 10  represents an optional additional covalent bond between R 10  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 R 10  in Formula II is methyl and y is as described above for Formula II or the preceding paragraph. 
     In one aspect, R 7 , R 8 , and R 9  in Formula II are each the same, wherein R 10  and y are as described above. In one aspect, R 7 , R 8 , and R 9  are each halo (e.g., chloro) or (C 1 -C 6 )alkoxy such as methoxy, wherein R 10  and y are as described above. 
     In one aspect, y in Formula II is 9, R 10  is methyl, and R 7 , R 8 , and R 9  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)trichlorosilane, (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 90°. In some embodiments, the contact angle of the alkylsilyl coating of Formula I and II is between about 15 to about 105°. 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°, or 105°. 
     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 trichlorosilane 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 Formula I. 
     
       
         
         
             
             
         
       
     
     R 11 , R 12 , R 13 , R 14 , R 15 , and R 16  are each independently selected from (C 1 -C 6 )alkoxy, —NH(C 1 -C 5 )alkyl, —N((C 1 -C 6 )alkyl) 2 , OH, and halo (i.e., a halogen, for example, chloro). Z is (C 1 -C 20 )alkyl, —O[(CH 2 ) 2 O] 1-20 —, —(C 1 -C 10 )[NH(CO)NH(C 1 -C 10 )] 1-20 —, or —(C 1 -C 10 )[alkylphenyl(C 1 -C 10 )alkyl] 1-20 —. 
     In some aspects, Z in Formula III is (C 1 -C 10 )alkyl; and R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  are each methoxy or chloro. In other aspects, Z in Formula III is (C 2 -C 10 )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, C 2 C 2 ) 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 105°. 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 III is between about 15 to about 105°. 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°, or 105°. 
     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 II is applied directly to wetted surfaces of the fluidic flow path. Therefore, in some embodiments, one of R 7 , R 8 , and R 9  of Formula II can also include OR A , where R A  represents a point of attachment to the interior surfaces (e.g., wetted surfaces) of the fluidic system. In other embodiments, R 7 , R 8 , and R 9  of the alkylsilyl coating of Formula II does not include OR A , 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 system or sample vials connectable to the injection needle are coated. 
     In yet a further aspect, metallic flow path components, such as the frits, are coated with two layers. A primer layer, or layer in direct contact with the metal substrate (i.e., metallic frit) is an alkylsilyl coating that is vapor deposited, such as, for example, an alkylsilyl coating having Formula I. The second layer is deposited using wet chemistry—in that a liquid deposition is used to apply a layer of polyethylene glycol (PEG) silane on top of the alkylsilyl base coating. That is, the second layer is deposited using a solution based PEG silane. As a result, the PEG layer is deposited on the primer layer and is exposed to the fluidic path. 
     The PEG layer is in direct contact with the first coating (i.e., primer coating). In general, the first coating comprises a structure according to Formula I. An exemplary primary coating is a vapor deposited layer of a C2 (ethylene) bridged siloxane coating that is bound to surface hydroxyl groups of a metallic frit. In some embodiments, the second coating, is liquid deposited (instead of vapor deposited) and comprises a structure according to Formula IV 
     
       
         
         
             
             
         
       
     
     wherein: 
     R 17 , R 18 , and R 19  are each independently OH or OR B ; 
     R B  represents a point of attachment to the wetted surface of the fluidic flow path, or represents a point of attachment to the first coating, wherein in either instance, at least one of R 17 , R 18 , and R 19  is OR B ; 
     P is —(C 1 -C 6 )alkyl-OR 21 , —[O(C 1 -C 3 )alkyl] 1-10 O(C 1 -C 6 )alkyl, —[O(C 1 -C 3 )alkyl] 1-10 OH, or phenyl, wherein said (C 1 -C 6 )alkyl is substituted with one or more halo or one or more functional groups selected from the group consisting of OH, amino, (C 1 -C 6 )alkylamino, di(C 1 -C 6 )alkylamino, cyano, —C(O)NH 2 , sulfonic acid, quaternary ammonium, phosphate, and carboxyl, and wherein said phenyl is optionally substituted with one or more groups selected from the group consisting of (C 1 -C 3 )alkyl, OH, halo, cyano, —C(O)NH 2 , and carboxyl; and 
     R 21  is —(C 1 -C 3 )alkanediol, —(C 1 -C 3 )alkyl-3,4-cyclohexanediol, or —(C 2 -C 4 )alkylOH. 
     In some embodiments, R B  represents a point of attachment to the wetted surface of a metallic substrate. 
     In some embodiments, R B  represents a point of attachment to the first coating. In such embodiments, one of skill in the art will recognize that the attachment is to a hydroxyl group associated with the coating of Formula I, such as one or more of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  when said one or more of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  represents one or more hydroxyl groups. 
     A non-limiting, simplified, illustrative embodiment is provided in  FIG. 2 . Referring to  FIG. 2 , an alkylsilyl polyethyleneglycol coating is bound to free hydroxyl groups of a C 2  (ethylene) bridged siloxane coating according to an embodiment of the disclosure. 
     In some embodiments, P is —[O(C 1 -C 3 )alkyl] 1-10 O(C 1 -C 6 )alkyl or —[O(C 1 -C 3 )alkyl] 1-10 OH. In some embodiments, Y is —[O(C 1 -C 3 )alkyl] 1-10 O(C 1 -C 6 )alkyl. In some embodiments, Y is —[O(C 1 -C 3 )alkyl] 1-10 OH. 
     Exemplary coatings with their respective approximate thickness and contact angle are provided in Table 1. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                 Alternative 
                 Approximate 
                 Approximate 
               
               
                   
                   
                 Coating 
                 Thickness of 
                 Contact 
               
               
                 VPD# 
                 Vapor Deposited Material 
                 Abbreviation 
                 Product 
                 Angle 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 bis(trichlorosilyl)ethane or 
                 C 2 —GPTMS—OH 
                  500 Å 
                 15° 
               
               
                   
                 bis(trismethoxysilyl)ethane as a 
                   
                   
                   
               
               
                   
                 first layer followed by GPTMS 
                   
                   
                   
               
               
                   
                 followed by hydrolysis to form 
                   
                   
                   
               
               
                   
                 GPTMS—OH 
                   
                   
                   
               
               
                 2 
                 bis(trichlorosilyl)ethane or 
                 C 2   
                  500 Å 
                 35° 
               
               
                   
                 bis(trimethoxysilyl)ethane 
                   
                   
                   
               
               
                 3 
                 bis(trichlorosilyl)ethane or 
                 C 2 —C 2   
                 1600 Å 
                 35° 
               
               
                   
                 bis(trimethoxysilyl)ethane as a 
                   
                   
                   
               
               
                   
                 first layer followed by 
                   
                   
                   
               
               
                   
                 bis(trichlorosilyl)ethane or 
                   
                   
                   
               
               
                   
                 bis(trimethoxysilyl)ethane as a 
                   
                   
                   
               
               
                   
                 second layer. 
                   
                   
                   
               
               
                 4 
                 bis(trichlorosilyl)ethane or 
                 C 2 —GPTMS 
                  500 Å 
                 50° 
               
               
                   
                 bis(trimethoxysilyl)ethane as a 
                   
                   
                   
               
               
                   
                 first layer followed by GPTMS 
                   
                   
                   
               
               
                   
                 as a second layer 
                   
                   
                   
               
               
                 5 
                 Annealed 
                 Annealed C 2   
                  500 Å 
                 95° 
               
               
                   
                 bis(trichlorosilyl)ethane or 
                   
                   
                   
               
               
                   
                 bis(trimethoxysilyl)ethane 
                   
                   
                   
               
               
                 6 
                 Annealed 
                 Annealed 
                 1600 Å 
                 95° 
               
               
                   
                 bis(trichlorosilyl)ethane or 
                 C 2 —C 2   
                   
                   
               
               
                   
                 bis(trimethoxysilyl)ethane as a 
                   
                   
                   
               
               
                   
                 first layer followed by annealed 
                   
                   
                   
               
               
                   
                 bis(trichlorosilyl)ethane or 
                   
                   
                   
               
               
                   
                 bis(trimethoxysilyl)ethane as a 
                   
                   
                   
               
               
                   
                 second layer 
                   
                   
                   
               
               
                 7 
                 bis(trichlorosilyl)ethane or 
                 C 2 C 10   
                  500 Å 
                 105°  
               
               
                   
                 bis(trimethoxysilyl)ethane as a 
                   
                   
                   
               
               
                   
                 first layer followed by n- 
                   
                   
                   
               
               
                   
                 decyltrichlorosilane as a second 
                   
                   
                   
               
               
                   
                 layer 
                   
                   
                   
               
               
                 8 
                 Annealed 
                 Annealed 
                  500 Å 
                 105°  
               
               
                   
                 bis(trichlorosilyl)ethane or 
                 C 2 C 10   
                   
                   
               
               
                   
                 bis(trimethoxysilyl)ethane as a 
                   
                   
                   
               
               
                   
                 first layer followed by annealed 
                   
                   
                   
               
               
                   
                 n-decyltrichlorosilane as a 
                   
                   
                   
               
               
                   
                 second layer 
                   
                   
                   
               
               
                 9 
                 GPTMS 
                 GPTMS 
                 100 to 200 Å 
                 ~50°   
               
               
                 10 
                 GPTMS followed by  
                 GPTMS—OH 
                 100 to 200 Å 
                 ~20°   
               
               
                   
                 hydrolysis to form 
                   
                   
                   
               
               
                   
                 GPTMS—OH 
                   
                   
                   
               
               
                 11 
                 bis(trichlorosilyl)ethane or 
                 C 2 C 3   
                  500 Å 
                 40-90° 
               
               
                   
                 bis(trimethoxysilyl)ethane as a 
                   
                   
                   
               
               
                   
                 first layer followed by 
                   
                   
                   
               
               
                   
                 trimethylchlorosilane or 
                   
                   
                   
               
               
                   
                 trimethyldimethylaminosilane 
                   
                   
                   
               
               
                 12 
                 annealed 
                 Annealed 
                  500 Å 
                 95° 
               
               
                   
                 bis(trichlorosilyl)ethane or 
                 C 2 C 3   
                   
                   
               
               
                   
                 bis(trimethoxysilyl)ethane as a 
                   
                   
                   
               
               
                   
                 first layer followed by 
                   
                   
                   
               
               
                   
                 trimethylchlorosilane or 
                   
                   
                   
               
               
                   
                 trimethyldimethylaminosilane 
                   
                   
                   
               
               
                 13 
                 bis(trichlorosilyl)ethane or 
                 C 2 PEO 
                  500 Å 
                 15° 
               
               
                   
                 bis(trimethoxysilyl)ethane as a 
                   
                   
                   
               
               
                   
                 first layer followed by a 
                   
                   
                   
               
               
                   
                 methoxy-polyethyleneoxy(3) 
                   
                   
                   
               
               
                   
                 propyl trichlorosilane or 
                   
                   
                   
               
               
                   
                 methoxy-polyethyleneoxy(3) 
                   
                   
                   
               
               
                   
                 propyl trimethoxysilane 
                   
                   
                   
               
               
                   
               
            
           
         
       
     
     Referring to VPD #1 (C 2 -GPTMS-OH), the first coating layer, C 2  shown below, is a layer according to Formula I, described above. or 
     
       
         
         
             
             
         
       
     
     The second layer of VPD #1, GPTMS-OH, shown below, is a layer according to Formula II. 
     
       
         
         
             
             
         
       
     
     VPD #3 (C 2 -C 2 ) is an example of a coating of Formula I and then a coating for Formula III. 
     VPD #7 (C 2 C 10 ) 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 (C 2 ) is shown above. The structure of C 10  is shown below. 
     
       
         
         
             
             
         
       
     
     VPD #11 (C 2 C 3 ) 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 (C 2 ) is shown above. The structure of C 3  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 (C 2 ) is shown above. The structure of methoxy-polyethyleneoxy(3)propyl trichlorosilane (PEG) 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® (commercially available from SilcoTek Corporation, Bellefonte, Pa.). 
     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 C 2  and GPTMS (C 2 -GPTMS) followed by hydrolysis to form C 2 -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&gt;2&gt;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. 
     Methods of Tailoring a Fluidic Flow Path 
     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). 
     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. 
       FIG. 3B  is a flow chart illustrating method  200  for tailoring a fluidic flow path for separation of a sample including biomolecules, proteins, glycans, peptides, oligonucleotides, pesticides, bisphosphonic acids, anionic metabolites, and zwitterions like amino acids and neurotransmitters. 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. 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 ). 
       FIG. 3B  provides a flow chart illustrating a method ( 300 ) of tailoring a fluidic flow path for separation of a sample including a biomolecule or a metal interacting analyte. The method can be used to tailor a flow system for use in isolating, separating, and/or analyzing the biomolecule or metal interacting analyte. In step  305 , the analyte is assessed to determine its polarity. Understanding the polarity will allow an operator to select (by either look up table or make a determination) a desired coating chemistry and, optionally, contact angle as shown in step  310 . In some embodiments, in addition to assessing the polarity of the biomolecule or metal interacting analyte, the polarity of a stationary phase to be used to separate the biomolecule or metal interacting analyte (e.g., stationary phase to be included in at least a portion of the fluidic flow path) is also assessed. A chromatographic media can be selected based on the analyte in the sample. Understanding the polarity of both the analyte and the stationary phase is used in certain embodiments, by the operator to select the desired coating chemistry and contact angle in step  310 . The components to be tailored can then be positioned within a chemical infiltration system with environmental control (e.g., pressure, atmosphere, temperature, etc.) and precursor materials are infiltrated into the flow path of the component to deposit one or more coatings along the wetted surfaces to adjust the hydrophobicity as shown in step  315 . During any one of infiltration, deposition, and condition steps (e.g. annealing), coatings deposited from the infiltration system can be monitored and if necessary precursors and or depositing conditions can be adjusted if required allowing for fine tuning of coating properties. The alkylsilyl coating material selected in step  310  can be the alkylsilyl compounds of Formulas I, II and/or III. 
     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. 
     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)ethane). 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)ethane). 
     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 105°. 
     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 separation of 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 separation of 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. 
     Methods of Isolating an Analyte 
     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. 
     Methods of Improving Baseline Returns 
     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 110 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. 
     Kits 
     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. In addition to including the alkylsilyl coating of Formulas I, II, and/or III as a primer layer, the chromatographic component (i.e., frit) can include a second layer deposited on the top of the primer layer. The second layer can be a second alkylsilyl coating or the second layer can be a PEG coating deposited utilizing a liquid silane. 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. 
     Exemplary Separations 
     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&#39;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.  FIG. 4A-4C  show fluorescence chromatograms obtained with these types of column hardware. From these data, it was found that use of coated column hardware significantly improved the recovery of each phosphorylated N-glycan species. For example, there was a marked increase in the peak area of Man7-PP, a high mannose glycan with two mannose-6-phosphate residues, mannose-7-bisphosphate. Where Man7-PP could not be detected with stainless steel column hardware, it was easily detected with vapor deposition coated column hardware. This indicated that this species of N-glycan was interacting with the metallic surfaces of the column hardware in such a way that prevented it from reaching the detector. When using vapor deposition coated hardware, the peak area ratio of Man-7-PP to Man5 (a high mannose glycan without phosphorylation) was 0.24:1 ( FIG. 4A-4C ). 
     The increased recovery of phosphorylated glycans using coated column hardware and frits 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 ( FIGS. 5A and 5B ).  FIG. 6A-6C  show fluorescence chromatograms obtained using coated LC system components in conjunction with coated stainless steel column hardware. Phosphoglycan recovery improved even more with the use of coated column hardware and C 2 C 10  vapor deposition coated flow path components. Most notably, the peak area ratio of Man7-PP to Man5 increased to 0.8:1 from the ratio of 0.24:1 that was obtained by using coated column hardware alone. The observed relative abundance for Man7-PP with the coated system and coated column hardware is indicative of full recovery for the phosphorylated glycans, as can be determined by orthogonal assays to HILIC of RapiFluor-MS labeled released glycans. In sum, these results confirm that the loss of phosphorylated N-glycan species to sample flow path surfaces can be alleviated with the use of vapor deposition coatings. 
     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.  FIG. 7A-7C  show UV chromatograms obtained with these types of column hardware. In each case, the addition of the VPD #2, and the VPD #7 coatings increased the recovery of the singly phosphorylated peptides by at least 13% over the stainless steel alone ( FIG. 4A-4C ). The impact of coating the chromatographic flow path was much more pronounced with the doubly phosphorylated peptide. When using the stainless steel column hardware, there was no detectable recovery of the doubly phosphorylated peptide. However, when using either type of coated column hardware (VPD #2, and the VPD #7), this peptide became clearly visible in the obtained chromatograms. This result indicates, once again, that vapor deposition coatings can be used to minimize undesirable interactions with the metallic surfaces of chromatographic flow paths and in doing so allow for improved analyses of phosphorylated biomolecules. 
     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  FIGS. 8-11 . Interestingly, these data indicate that the use of the vapor deposition coated column hardware can yield a significant improvement in both the overall recovery and peak shape of these phosphate containing small biomolecules. Thus, it is foreseeable that this disclosure could also be used to improve the chromatography of non-biomolecules, such as small-molecule pharmaceuticals containing either phospho or phosphonate functional groups. 
     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  FIG. 12 , wherein the effect on peak shape and recovery of tetra- and penta-sialylated glycans is easily visualized. The observed chromatographic differences are likewise easily quantified. In particular, fluorescence peak areas for the most abundant di-, tri-, tetra- and penta-sialylated glycans showed there were indeed very distinct differences in recoveries ( FIG. 13 ). This testing was also used to demonstrate that other, chemically unique vapor deposition coating could be used with equally good effect. Much like the VPD #7 coated hardware, VPD #2 and SilcoTek Dursan® coated hardware showed equivalent capabilities in improving peak shape and recovery of the tetra- and penta-sialylated N-glycans. Interestingly though, it was not found to be necessary to use a coated flow through needle or column inlet in order to optimize peak shape and recovery. 
     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.  FIGS. 14A and 14B  show fluorescence chromatograms obtained with these column hardware materials. From these data, it was found that hardware coated with VPD #7, but not hardware coated with VPD #2, was uniquely able to improve the baseline quality of the model separation, particularly in providing quicker returns to baseline. This improvement to the chromatographic performance of the separation is underscored by the fact that the chromatogram produced with the VPD #7 coated column also shows higher peak intensities for some of the subunits. The nature of this baseline issue, as it exists with stainless steel hardware, can be reasoned to be a result of the protein analytes undergoing problematic secondary interactions and not homogenously eluting at one particular eluotropic strength. Interestingly, in this example, the VPD #7 MVD hardware did not appear to significantly improve half height peak capacity nor the carryover of the columns, which was universally found to be ˜0.9%. That is to say, for protein reversed phase chromatography, it would seem that vapor deposition coatings improve the quality of separation predominately through affecting baseline properties. 
     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.  FIG. 15A  presents total ion chromatograms (TICs) for some of these materials, including columns constructed with stainless steel alternatives, namely polyether ether ketone (PEEK) and a low titanium, nickel cobalt alloy (MP35NLT). Surprisingly, columns constructed of VPD #7 coated hardware were the only found to give uniquely quick returns to baseline. Stainless steel, PEEK, and VPD #2 coated hardware showed comparatively slower returns to baseline. In addition, control experiments showed that the improvement to baseline quality can be achieved through the use of a VPD #7 coated frit alone and that coated tubing is not required to achieve an effect. Further experimentation culminating in the chromatograms of  FIG. 15B  has made it possible to glean additional insights. One of which is that it does not matter if the frit has a 0.2 or 0.5 m porosity or if the VPD #7 coating has been thermally cured in the form of an annealing process (resulting in a VPD #8 coating). In contrast, neither a thicker VPD #3 coating (˜1800 Å thickness) nor a cured coating (VPD #5) with an increased contact angle of 90° (up from 35°) were able to produce the effect. Accordingly, VPD #7 coated frits are very unique in their ability being to affect the baseline of the example protein separation. While not limited to theory, it would seem reasonable to suggest that this effect derives from the hydrophobicity/contact angle of this coating. It could be that these coated frits closely mimic the surface chemistry of the reversed phase stationary phase. Consequently, a column with VPD #7 coated frits might exhibit adsorption sites (particularly those near the frit surface) that are more uniform in their chemical properties. Testing has shown that this effect on the protein reversed phase separation can be localized to the inlet frit of the column, lending credence to this hypothesis ( FIG. 16 ). Indeed, one hydrophobic VPD #7 vapor deposition coated frit at the column inlet is sufficient to produce uniquely quick returns to baseline for the example mAb subunit separations. Proteins undergo reversed phase chromatography via fairly discrete adsorption/desorption events. Consequently, upon loading, protein analytes will be most concentrated at and likewise spend a significant amount of time at the head of the column, where an interface exists between the inlet frit and the packed bed of the stationary phase. At this interface, a protein analyte would have an opportunity to establish undesired secondary interactions that would be cumulative to and energetically different than the desired hydrophobic interaction with the stationary phase. It is plausible that using a frit with surface properties similar to the stationary phase mitigates any chromatographic problems related to there being energetically and chemically diverse adsorption sites present at this packed bed interface. While not limited to theory, it may also be possible that a frit, such as the C 2 C 10  vapor deposition coated inlet frit (e.g., frit coated with VPD #7), imparts an entirely novel focusing effect to protein reversed phase separations that cannot be explained by the understanding and descriptions noted above. In addition, it is possible that a frit, such as the VPD #7 vapor deposition coated inlet frit, makes a unique contribution to how a stationary phase packs into a column. Use of a vapor deposition coated frit as the substrate for building a packed column bed may advantageously impact the properties of a stationary phase and resultant chromatography. 
     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 &gt;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 ( FIG. 17 ). Frits of alternative compositions are also relevant to this invention. In yet another embodiment, parylene, that is poly p-xylene polymer, coatings could be used treat column frits and to thereby improve the properties of a protein reversed phase separation. In addition, glass membranes could be used as the basis of a frit material. Onto the glass membrane substrate, silanes could be bonded to advantageously manipulate the hydrophobicity and contact angle of the material. These and other such membranes could also be used in conjunction with a backing material, like a porous polymer sheet, to lend physical rigidity to the apparatus. 
     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 deamidation 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.  FIGS. 21A-21L  presents chromatograms of NIST reference material 8671, an IgG1κmAb, as obtained from sequential cation exchange separations and repeat injections of sample. In this evaluation, columns derived from four different constructions were tested. These columns varied with respect to both hardware design and vapor deposition coating. From the observed results, it was most apparent that uncoated hardware showed a prominent conditioning effect, as manifest in there having been low peak areas on initial injections. While not limited to theory, it is believed that the metallic surfaces of the uncoated column hardware imposed adsorptive losses on these separations and thereby hindered recovery of the sample. In contrast, vapor deposition coated hardware, both C 2  or C 2 -GPTMS-OH chemistries, yielded comparatively high peak areas even on the very first runs of the columns ( FIG. 22 ). That is, coated hardware showed no evidence of requiring a passivation step, giving it the unique advantage of more quickly providing accurate chromatographic data. Here, it is clear that the noted vapor deposition coatings enhance the chromatographic properties of metallic hardware. Little can be seen in the way of distinguishing the chromatographic performance of the two tested vapor deposition coatings, namely the C 2  and C 2 -GPTMS-OH chemistries. However, the C 2 -GPTMS-OH coating has an inordinately low contact angle (as does C 2 PEO). It is foreseeable that certain types and classes of biomolecules will require a highly hydrophilic flow path. One such example could indeed be aqueous protein separations in which hydrophobic interactions could lead to poor recovery or peak tailing. As a whole, it is believed that vapor deposition coated hardware will show advantages for numerous forms of aqueous separations, including but not limited to ion exchange, size exclusion and hydrophobic interaction chromatography, and that the most ideal vapor deposition coating would be one that is very hydrophilic. Accordingly, in an embodiment of this invention, a vapor deposition coated column is used to improve the recovery of samples from aqueous chromatographic separations. In a more specific embodiment, a vapor deposition coating with a contact angle less than 200 is used to improve the recovery of biomolecules in ion exchange, size exclusion or hydrophobic interaction chromatography. 
     EXAMPLES 
     Example 1A 
     C 2  and C 2 C 10  Vapor Deposition Coatings 
     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 (if two layers of different materials is desired—this is optional). 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 Ramé-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. 
     Example 1B 
     C 2 -GPTMS-OH Vapor Deposition Coatings 
     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 C 2  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 C 2  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 &lt;20°. Contact angle measurements are taken on a silicon wafer using a Ramé-Hart goniometer model 190. 
     Example 2 
     C 2  Primer Coating (Vapor Deposition) Followed by PEG Liquid Phase Deposition 
     Metal components are subjected to vapor deposition of an organosilane reagent to form a primer layer. Prior to coating, all metal components are passivated according to a nitric acid passivation. Passivated parts and a silicon wafer are then introduced to a vapor deposition chamber and vacuum is established. The first step is a plasma cleaning step. Next is the first vapor deposition cycle. Each vapor deposition cycle comprises an organosilane vapor deposition, followed by the introduction of water vapor for organosilane hydrolysis. The organosilane vapor is delivered at a first pressure, and then the water vapor is delivered at a second pressure. Following delivery, the organosilane and water are left to react with the substrate. This cycle is repeated to produce the desired number of layers and coating thickness. In this example, bis(trichlorosilyl)ethanesilane is used to build up an adhesion or primer layer of approximately 800 Å. 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 an elevated temperature under vacuum. 
     The metal components with the vapor deposited C 2  are then treated with a solution of a polyethyleneglycol (PEG) organosilane reagent (liquid). In this case, C 2 -coated 4.6 mm, 0.2 m porous titanium frits were stirred with a dilute toluene solution of the organosilane reagent; for example 2-[methoxy(polyethyleneoxy) 6-9 propyl]tris(dimethylamino)silane; Gelest SIM6492.77). The reagent solution was allowed to react with the frits for approximately three days. The solution was discarded, and the frits washed (dipped) in toluene three times, and in isopropanol three times. The frits were vacuum dried at 70° C., then cooled. 
     Example 3 
     Alternative Contact Angle Measurement 
     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&#39;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 =(2γ cos θ)/ r  
 
     Where, 
     P=bubble point pressure, Pa (measured)
 
γ=surface tension of test liquid, N/m (known)
 
Θ=contact angle between test liquid and pore material (calculated)
 
r=largest pore radius, m (calculated)
 
     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.  FIG. 18  displays the different bubble point pressures recorded versus coating composition.  FIG. 19  displays the derived contact angles versus coating composition. These values correlate well with measurements taken with a goniometer on a flat silicon wafer. 
     Example 4 
     Corrosion Performance of Silane Coatings 
     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.  FIG. 20  displays the corrosion performance of a non-coated column tube, and various coatings on a column tube. The improvement ranges from ˜10× to −100×. 
     Example 5 
     HILIC-Fluorescence-MS of Phosphoglycans 
     A recombinant alpha-galactosidase was diluted to 2 mg/mL. A 7.5 uL aliquot of the protein solution was then added to a 1 mL 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 (50 mM 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.  FIGS. 4A-4C  present comparisons of this HILIC separation of RapiFluor-MS labeled released N-glycans from a recombinant alpha-galactosidase as performed with columns constructed of varying coatings and materials.  FIGS. 6A-6C  present comparisons of this HILIC separation as performed with columns constructed of varying coatings and materials in conjunction with a sample needle and column inlet tubing constructed of varying coatings. 
     Example 6 
     RPLC-UV-MS of Phosphopeptides 
     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.  FIGS. 7A-7C  present comparisons of this reversed phase separation of the phosphopeptide standard performed with columns constructed of varying coatings and materials. 
     Example 7 
     RPLC/MS of Small Biomolecules (Nucleotides and Sugar Phosphates) 
     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.  FIGS. 8-11  present comparisons of these reversed phase separations as performed with columns constructed of varying coatings and materials. 
     LC Conditions 
     
         
         Columns: BEH C 18  130 Å 1.7 μm 2.1×100 mm 
         Mobile Phase A: 0.25% Octylamine in H 2 O pH adjusted to 9 with acetic acid 
         Mobile Phase B: ACN 
         Column Temperature: 35° C. 
         Injection Volume: 10 μL (100 ng/mL sample concentrations) 
         Sample Diluent: Water 
         Detection: Tandem quadrupole mass spectrometer operating in ESI negative ionization mode and with MRM acquisition. 
       
    
     
       
         
           
               
            
               
                   
               
               
                 Gradient Table: 
               
            
           
           
               
               
               
               
               
            
               
                 Time(min) 
                 Flow Rate(mL/min) 
                 % A 
                 % B 
                 Curve 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Initial 
                 0.450 
                 95 
                 5 
                 Initial 
               
               
                 5 
                 0.450 
                 75 
                 25 
                 6 
               
               
                 10 
                 0.450 
                 75 
                 25 
                 6 
               
               
                 20 
                 0.450 
                 50 
                 50 
                 6 
               
               
                 21 
                 0.450 
                 5 
                 95 
                 6 
               
               
                 22 
                 0.450 
                 95 
                 5 
                 6 
               
               
                 30 
                 0.450 
                 95 
                 5 
                 6 
               
               
                   
               
            
           
         
       
     
     Example 8 
     LC-Fluorescence-MS of Highly Sialylated Glycans Using Charge Surface Reversed Phase Chromatography 
     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.  FIGS. 12 and 13  present comparisons of this mixed mode separation of sialylated glycans as performed with columns constructed of varying coatings and materials. 
     LC Conditions 
     
         
         Column: DEAP HPCM 130 Å 1.7 m 2.1×100 mm 
         Mobile Phase A: Water and 100 mM Formic Acid/100 mM Ammonium Formate in 60% 
         Mobile Phase B: 100 mM Formic Acid/100 mM Ammonium Formate in 60% ACN 
         Column Temperature: 60° C. 
         Injection Volume: 4 μL 
         Sample Concentration: 10 pmol/μL 
         Sample Diluent: Water 
         Fluorescence Detection: Ex 265 nm/Em 425 nm (10 Hz) 
       
    
     
       
         
           
               
            
               
                   
               
               
                 Gradient Table: 
               
            
           
           
               
               
               
               
               
            
               
                 Time(min) 
                 Flow Rate(mL/min) 
                 % A 
                 % B 
                 Curve 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Initial 
                 0.400 
                 100.0 
                 0.00 
                 Initial 
               
               
                 24.00 
                 0.400 
                 78.0 
                 22.0 
                 6 
               
               
                 24.20 
                 0.400 
                 0.0 
                 100.0 
                 6 
               
               
                 24.40 
                 0.400 
                 0.0 
                 100.0 
                 6 
               
               
                 24.60 
                 0.400 
                 100.0 
                 0.0 
                 6 
               
               
                 30.00 
                 0.400 
                 100.0 
                 0.0 
                 6 
               
               
                   
               
            
           
         
       
     
     Example 9 
     LC/MS of a Reduced, IdeS Digested Monoclonal Antibody (mAb) 
     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, 290 Å) 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.  FIGS. 14-17  present comparisons of this reversed phase C4 separation of reduced, IdeS-digested NIST Reference Material 8671 as performed with columns constructed of varying coatings and materials. 
     Example 10 
     Ion Exchange Chromatography 
     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.  FIGS. 21 and 22  present comparisons of these separations and their resulting data as obtained with columns constructed of varying coatings and materials. 
     LC Conditions 
     
         
         Columns: 3 μm non-porous cation exchange stationary phase in a 2.1×50 mm column dimension 
         Sample: NIST mAb Reference Material 8671 diluted to 2.5 mg/mL with 20 mM MES pH 6.0 buffer 
         Gradient: 20 mM MES pH 6.0, 10-200 mM NaCl in 7.5 min 
         Flow Rate: 0.2 mL/min 
         Column Temperature: 30° C. 
         Injection Volume: 1 μL (2.1 mm ID columns) 
         Detection: 280 nm 
         Hardware Design: Hardware A—Identical to ACQUITY UPLC BEH C18 column hardware
       Hardware B—A design with an alternative sealing mechanism and some alternative material compositions.   
     
       
    
     Example 11 
     Oligonucleotide Ion Pair RPLC 
     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&#39;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 C 18  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.  FIGS. 23A-F  and  24  present comparisons of these separations and their resulting data as obtained with columns constructed of varying coatings and materials. 
     LC Conditions 
     
         
         Columns: 1.7 μm organosilica 130 Å C 18  bonded stationary phase in a 2.1×50 mm column dimension 
         Sample: 15, 20, 25, 30, 35 and 40-mer deoxythymidine (0.5 pmol/μL) 
         Column Temperature: 60° C. 
         Flow Rate: 0.2 mL/min 
         Mobile Phase A: 400 mM HFIP, 15 mM TEA in water 
         Mobile Phase B: 400 mM HFIP, 15 mM TEA in methanol 
         Gradient: 18 to 28% B in 5 min 
         Injection volume: 10 μL 
         UV Detection: 260 nm 
       
    
     Example 12 
     RPLC of Citric and Malic Acid 
     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&#39;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 C 18  bonded stationary phase packed into either uncoated or C 2 C 3  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.  FIGS. 25A-D  presents a comparison of these separations and their resulting data. It can be observed from these results that use of the vapor deposition coated column hardware led to improvements in recovery and peak shape and thus sizable increases in MS intensity. This is noteworthy as it highlights the fact that the vapor deposition coating can be used to facilitate the development of a more sensitive and more accurate quantitation assay of these and other chemically similar compounds, including but not limited to isocitric acid, α-ketoglutaric acid, succinic acid, fumaric acid, lactic acid, aconitic acid, itaconic acid, oxaloacetic acid, pyruvic acid, pantothenic acid, biotin, and folic acid. It is reasonable to assume that even zwitterionic small molecules would benefit from this invention. This class of compounds includes but is not limited to amino acids and neurotransmitters. Likewise, it is envisioned that this invention will be advantageous to be used to separate and analyze compounds containing metal binding moieties, such as cobalamin and the various types of porphyrins. Lastly, these same compounds would exhibit improved separations whether analyzed by RPLC or other modes of chromatography, such as hydrophilic interaction chromatography (HILIC), ion exchange, or mixed mode LC separations (i.e. ion exchange/reversed phase or ion exchange/HILIC). 
     LC Conditions 
     
         
         Columns: 1.8 μm silica 100 Å C 18  bonded stationary phase in a 2.1×50 mm column dimension 
         Sample: Citric acid
       Malic acid   
     
         Column Temperature: 30° C. 
         Flow Rate: 0.5 mL/min 
         Mobile Phase A: 0.1% formic acid, water 
         Mobile Phase B: 0.1% formic acid, acetonitrile 
         Injection volume: 2 μL 
       
    
     
       
         
           
               
            
               
                   
               
               
                 Gradient Table: 
               
            
           
           
               
               
               
               
               
            
               
                 Time(min) 
                 Flow Rate(mL/min) 
                 % A 
                 % B 
                 Curve 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Initial 
                 0.500 
                 100.0 
                 0.00 
                 Initial 
               
               
                 0.25 
                 0.500 
                 100.0 
                 0.00 
                 6 
               
               
                 2.00 
                 0.500 
                 75.0 
                 25.0 
                 6 
               
               
                 2.50 
                 0.500 
                 5.0 
                 95.0 
                 6 
               
               
                 3.00 
                 0.500 
                 5.0 
                 5.0 
                 6 
               
               
                 3.10 
                 0.500 
                 100.0 
                 0.0 
                 6 
               
               
                   
               
            
           
         
       
     
     MS Conditions 
     
         
         MS1 Resolution: 1 Da 
         MS2 Resolution: 0.75 Da 
         Capillary Voltage: 1 kV 
         Source Offset: 50 
         Desolvation Temp.: 600° C. 
         Desolvation Gas Flow: 1000 L/hr 
         Cone Gas: 150 L/hr 
         Nebulizer: 7 bar 
         Source Temp.: 150° C. 
         MRM (Citric Acid): 191.2&gt;87.1 
         MRM (Malic Acid): 133.2&gt;115.2 
       
    
     Example 13 
     Mixed Mode Chromatography of Pesticides 
     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 C 2 C 10  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. 
       FIGS. 26A-B  and  27 A-B show a comparison of coated and uncoated column performance for glyphosate in a solvent standard. As seen in  FIG. 26B , glyphosate appears as a severely tailing, broad peak. In contrast, as seen in  FIG. 26B , glyphosate is separated with much improved peak shape on the vapor deposition coated column. It can be observed from these results that the use of the vapor deposition coated column hardware led to significant improvements in peak shape, reduced peak widths and thus sizable increases in MS intensity ( FIGS. 27A-B ). It is reasonable to assume that the vapor deposition coated column also yielded higher recovery. These results are noteworthy as they demonstrate a means to developing more sensitive and more accurate quantitation assays for glyphosate and other chemically similar compounds, including but not limited to pesticides such as Ethephon, 2-Hydroxyethyl phosphonic acid (HEPA), Glufosinate-Ammonium, N-Acetyl-glufosinate, 3-Methylphosphinicopropionic acid (MPPA), Aminomethylphosphonic acid (AMPA), N-Acetyl-glyphosate, N-Acetyl-AMPA, Fosetyl-aluminium, Phosphonic acid, Maleic hydrazide, Perchlorate, and Chlorate. 
     LC Conditions 
     
         
         Columns: 1.7 μm diethylamine bonded organosilica 130 Å stationary phase in a 2.1×100 mm column dimension 
         Sample: Glyphosate 
         Column Temperature: 50° C. 
         Flow Rate: 0.5 mL/min 
         Mobile Phase A: 0.9% formic acid, water 
         Mobile Phase B: 0.9% formic acid, acetonitrile 
         Injection volume: 10 μL 
       
    
     
       
         
           
               
            
               
                   
               
               
                 Gradient Table: 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Time (min) 
                 Flow Rate (mL/min) 
                 % A 
                 % B 
                 Curve 
               
               
                   
                   
               
               
                   
                 Initial 
                 0.500 
                 10 
                 90 
                 Initial 
               
               
                   
                 4.00 
                 0.500 
                 85 
                 15 
                 2 
               
               
                   
                 10.0 
                 0.500 
                 85 
                 15 
                 6 
               
               
                   
                 16.0 
                 0.500 
                 10 
                 90 
                 1 
               
               
                   
                 20.0 
                 0.500 
                 10 
                 90 
                 1 
               
               
                   
                   
               
            
           
         
       
     
     MS Conditions 
     
         
         MS1 Resolution: 1 Da 
         MS2 Resolution: 0.75 Da 
         Capillary Voltage: 2.4 kV 
         Ionization: ESI− 
         Desolvation Temp.: 600° C. 
         Desolvation Gas Flow: 1000 L/hr 
         Cone Gas: 150 L/hr 
         Nebulizer: 7 bar 
         Source Temp.: 150° C. 
         MRM 1: 168.0&gt;62.6 
         MRM 2: 168.0&gt;149.8 
       
    
     Example 14 
     Interaction of Dexamethasone Sodium Phosphate Across Multiple Column Configurations 
     Different chromatographic systems (e.g., a UHPLC system such as ACQUITY UPLC from Waters Technologies Corporation versus a HPLC system such as Arc also from Waters Technologies Corporation) have a different amount of wetted surface area. In addition, different column configurations (e.g., 2.1×50 mm vs. 4.6×150 mm) also provide a different amount of wetted surface area. This example presents a comparison of the amount of interaction of dexamethasone sodium phosphate versus an amount of uncoated metallic wetted surface area in a chromatographic system. The three chromatographic system configurations include: system 1: a stainless steel UHPLC system with a stainless steel column having uncoated frits (SS ACQUITY I-Class with SS column, from Waters Technologies Corporation); system 2: a stainless steel UHPLC system with a C 2  coated stainless steel column (SS ACQUITY I-Class with C2 column with coated frits, from Waters Technologies Corporation); and system 3: a C 2  coated UHPLC system with C 2  coated column and frits (ACQUITY PREMIER and PREMIER COLUMN from Waters Technologies Corporation). A 100 ng on column injections of dexamethasone sodium phosphate on each of system 1, system 2, and system 3 was performed for the comparison. 
     The wetted surface area of the system components is as follows: column surface area including the surface area of two frits is 1236 mm 2  and the surface area of the system not including the column and its internal components is 513 mm 2 . Thus, system 1 has an uncoated surface area of 1748 mm 2 ; system 2 has an uncoated amount of 513 mm 2 ; and system 3 is coated (e.g., approximately 0 to 10 mm 2 ). The peak area for the dexamethasone sodium phosphate separation after three injections on each of the three columns was as follows: system 1 had a peak area average of 3873.0; system 2 had a peak area average of 5152.3; whereas system 3 had a peak area average of 5793.3. The data was plotted and is presented in  FIG. 28  to show the relationship between peak area and surface area. If each of the systems were fully inert, the line would be horizontal or parallel with the x-axis (uncoated total surface area in mm 2 ). However, system 3 is the most inert, followed by system 2, and then last system 1. The difference between observed and expected area is the amount of analyte lost to the uncoated surface area. 
     This data was used to calculate the binding capacity of a system. Using the difference between system 3 (coated system and column) and system 2 (uncoated system and coated column), the binding capacity of the system was determined. From the data above, system 3 had a peak area of 5793.3, whereas system 2 had a peak of 5151.3. Both systems utilize coated columns. The difference between the two systems resides in that system 3 utilizes a coated tubing and components upstream of the column, whereas the upstream tubing and components of system 2 are uncoated. To calculate the binding capacity attributable to the system without a column (i.e., upstream tubing and components), the difference in peak areas for system 3 versus system 2 was calculated to be 642 (5793.3-5151.3). Using a calibration curve (shown in  FIG. 29 ), area was converted back to mass and the calculated binding capacity for system 2 (attributable to upstream of the column tubing and components) was 513 mm 2 . This is the mass loss due to metallic surface area, which can then be used to extrapolate binding capacity for any calculated metallic system surface area. 
     Example 15 
     Effect of Coating Frits Separately from Column Tubes 
     In Example 14, the binding capacity of an uncoated system was investigated. In this example, the effect of coating components within the column itself (e.g., frits  120 ) was evaluated. In this Example, three different column assemblies were tested. Assembly 1 was an uncoated 2.1 mm×150 mm column packed with 1.7 micrometer BEH C18, with uncoated stainless steel frits. Assembly 2 was an uncoated 2.1 mm×150 mm column packed with 1.7 micrometer BEH C18, with C2 coated stainless steel frits. And assembly 3 was a C2 coated 2.1 mm×150 mm column packed with 1.7 micrometer BEH C18, with C2 coated stainless steel frits. To form the coated frit, the uncoated stainless steel frit was coated via vapor deposition of C2. That is the only difference between the uncoated stainless steel frits and the coated stainless steel frits is the C2 coating. The two frits in each assembly have a total surface area of 1,376 mm 2  (673 mm 2  per frit). The total wetted surface area of a 2.1 mm×150 mm column is 519 mm 2 . As each column assembly is formed of the same parts (with the exception of the vapor deposited C2 coatings), the total wetted surface area is 1,865 mm 2 . As a result, the frits represent 72% of the calculated surface area, whereas the column tube represents 28%. The different column assemblies were evaluated using ATP as a sample for chromatographic separation. The following ATP test parameters were applied to evaluate each column assembly (assembly 1, assembly 2, and assembly 3) using an inert system including a PEEK injection needle, a C2 coated 10 microliter stainless steel loop, and a C2 coated MP35N (titanium alloy) APH. 
     LC Conditions 
     
         
         Columns: 1.7 μm BEH C18 stationary phase in a 2.1×150 mm with the following assemblies
       Assembly 1: uncoated stainless steel column, 2 uncoated stainless steel frits   Assembly 2: uncoated stainless steel column, 2 C2 coated stainless steel frits   Assembly 3: C2 coated stainless steel column, 2 C2 coated stainless steel frits   
     
         Sample: ATP 
         Column Temperature: 30° C. 
         Flow Rate: 025 mL/min 
         Mobile Phase: 10 mM ammonium acetate in 50/50 methanol/water 
         Sample Manager Temperature: 4° C. 
         Injection volume: 0.2 μL of 0.25 mg/mL ATP in water per injection (total 5 injections) 
       
    
       FIG. 30  plots peak area per injection number (i.e., first injection, second injection, etc.) for each of assembly 1 (circle), assembly 2 (square), and assembly 3 triangle). And Table 2 below provides the ATP peak area values and deviations for comparison purposes. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 ATP Peak Areas 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Injection # 
                 Assembly 1 
                 Assembly 2 
                 Assembly 3 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 1 
                 100600 
                 241858 
                 329660 
               
               
                   
                 2 
                 146717 
                 245130 
                 331658 
               
               
                   
                 3 
                 164607 
                 257430 
                 340328 
               
               
                   
                 4 
                 178101 
                 267658 
                 341251 
               
               
                   
                 5 
                 183585 
                 264845 
                 343807 
               
               
                   
                 Average 
                 154722 
                 255384.2 
                 337340.8 
               
               
                   
                 StDev 
                 33431.4 
                 11537.38 
                 6271.24 
               
               
                   
                 % RSD 
                 22% 
                 5% 
                 2% 
               
               
                   
                   
               
            
           
         
       
     
       FIG. 31  is a graph of peak area after the first injection versus the amount of coated surface area. For assembly 1 there is no coated surface area (i.e., 0 mm 2 ). For assembly 2, which has coated frits but an uncoated column tube, the amount of coated surface area is 1,346 mm 2  and for assembly 3, a fully coated assembly, the amount of coated surface area is 1,865 mm 2 . Comparing assemblies  1 ,  2 , and  3 , it was determined that the frits account for 62% of the ATP adsorption compared to an estimated 72% of the surface area. Thus, the column frits account for a majority of the metal interaction. Just coating the column frits with an alkylsilyl vapor deposited coating (e.g., vapor deposited C 2 ) can dramatically improve chromatographic performance over an untreated system. 
     Example 16 
     Effect of Coating Frits for Various Column Formats 
     In Example 14, the binding capacity of uncoated versus coated systems was investigated utilizing dexamethasone sodium phosphate. In this example, the potential loss of dexamethasone sodium phosphate to frits in three different chromatographic formats (i.e., three different sizes) is presented. 
     The relationship between available metallic surface area and analyte loss ( FIG. 28 ) was determined by plotting the peak area of a 100 ng injection of dexamethasone sodium phosphate versus the calculated uncoated metal surface area of the three different systems provided in Example 14. The slope of the linear curve fit represents the amount of analyte lost per additional square mm of exposed metal surface. 
     The direct relationship between analyte loss and available metal surface area can then be used to extrapolate the potential for analyte loss for other combinations of system and column configurations. This is shown in  FIG. 32  for two different LC systems (Acquity and ARC systems, both available from Waters Corporation, Milford, Mass.) and for three different column configurations (i.e., 2.1×150 mm; 4.6×150 mm, and 7.8×150 mm). Injecting anything less than the total potential analyte loss shown on the left axis of  FIG. 32  for each configuration would result in complete loss of analyte. In such a scenario, the frits of an uncoated 2.1×150 mm column would be responsible for 38% of dexamethasone sodium phosphate loss when tested on an uncoated Acquity system. The frits in a 4.6×150 mm column configuration would be responsible for 65% of the loss of dexamethasone sodium phosphate when tested on an uncoated Arc system. The frits from a 7.8×150 mm column configuration would be responsible for 79% of the loss of dexamethasone sodium phosphate when tested on an uncoated Arc system. In the cases of using the 4.6 or 7.8 mm ID columns, applying the C2 coating to at least the frits in these columns provide a significant benefit (prevent 65% or more of the analyte loss). 
     Alternatives: 
     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 or the use of a column. 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. 
     Other types of sample preparation devices, such as sample plates, vials, and extraction devices may also include a vapor deposited coating along wetted surfaces (i.e., internal pathways/surfaces in contact with the sample). 
     Moreover, while the examples of the described aspects employ comparatively hydrophobic coatings, with water contact angles ranging from 150 to 110°, it is reasonable to suggest that some separations could be enhanced through the application of hydrophilic coatings, including but not restricted to diol, amide/ureido type, and polyethylene oxide/glycol bondings. 
     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.