Hydrophilic interaction microfluidic chromatography

Abstract

To provide a microfluidic chromatography column including a stationary phase substrate and a method for functionalizing the chromatography column.SOLUTION: The stationary phase substrate has a fluid channel defined by a channel wall, the channel having an inlet and an outlet. Pillars are positioned within the channels on the substrate. The stationary phase substrate of the chromatography column can comprise silicon oxide. At least a portion of the surface of the stationary phase substrate may be bound to a linker, wherein the linker is bound to the polymer.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present disclosure relates to the fields of liquid chromatography and microfluidics. [Background technology]

[0002] Microfluidic high pressure liquid chromatography High-performance liquid chromatography (HPLC) columns provide high-resolution separation and analysis at very small sample sizes. Microfluidic columns have been prepared using stationary phases containing hydrophobic organosilane chemistry useful for reversed-phase (RP) chromatography. While reversed-phase chromatography is useful for separating some analytes, it would be beneficial to have alternatives to increase the applicability of HPLC devices to other analytes. Summary of the Invention

[0003] To fulfill a long-standing need in the art, a microfluidic chromatography column is provided that includes a stationary phase substrate. The stationary phase substrate has a liquid channel defined by a channel wall, the channel having an inlet and an outlet. Pillars are positioned within the channel on the substrate. The stationary phase substrate of the chromatography column can include silicon oxide. At least a portion of the surface of the stationary phase substrate is attached to a linker, and the linker is attached to a polymer.

[0004] Also provided are methods for functionalizing a chromatography column, including depositing a linker and forming a polymer. The linker depositing comprises flowing a first mixture containing an acrylate silane, an acid catalyst, and a first solvent through the column to deposit a pre-linker on the stationary phase substrate of the chromatography column. The polymer formation comprises flowing a second mixture containing a monomer, a second solvent, and a radical initiator through the chromatography column under conditions sufficient to react the monomer with the pre-linker to form a polymer attached to the linker.

[0005] An advantage of this process is that the performance of columns produced by this method is improved compared to conventional non-functionalized columns in separating mixtures, especially mixtures containing polar molecules. Separation of mixtures, such as mixtures containing polar molecules, on these functionalized columns is improved compared to conventional reversed-phase columns or non-functionalized silica. [Brief explanation of the drawings]

[0006] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments and, together with the general description given above and the detailed description of the embodiments given below, serve to explain the principles of the present disclosure. [Figure 1] 1 is a chromatographic trace showing different retention times for uridine (3) and 2'-deoxyuridine (2) using toluene (1) as a void marker on a non-functionalized column compared to a functionalized column. [Figure 2] 1 is a chromatographic trace showing different retention times for uridine (3) and 5-methyluridine (2) using toluene (1) as a void marker on a non-functionalized column compared to a functionalized column. [Figure 3] Chromatography traces showing different retention times for sodium p-toluenesulfonate (2) and uracil (3) with toluene (1) as a void marker on a non-functionalized column compared to a functionalized column. [Figure 4] FIG. 1 is a chromatographic trace showing different retention times for trimethylphenylammonium chloride (TMPAC) (3) and uracil (2) with toluene (1) as a void marker on a non-functionalized column compared to a functionalized column. [Figure 5]Chromatography traces showing different retention times for theophylline (Tp) (2) and theobromine (Tb) (3) using toluene (1) as a void marker on a non-functionalized column compared to a functionalized column. [Figure 6] FIG. 1 is a schematic diagram of solvents and reagents used in an embodiment of the linker placement process. [Figure 7] FIG. 1 is a schematic diagram of solvents and reagents used in an embodiment of the polymer formation process. DETAILED DESCRIPTION OF THE INVENTION

[0007] The chromatography columns and methods for their manufacture taught herein provide neutral hydrophilic polymer stationary phases suitable for use in hydrophilic interaction chromatography (HILIC). Exemplary columns taught herein employ microfluidic channels, and the stationary phase includes microfabricated structural features, such as pillars, suitable for low flow rates (e.g., nanoflow) using very small sample volumes. Traditional HILIC columns use capillary flow (typical flow rates of 1-10 μL / min) and higher flow rates, making them unsuitable for nanoflow liquid chromatography applications (typical flow rates below 1000 nL / min). These columns are also typically fabricated by batch processing of the stationary phase (e.g., beads) followed by packing of the beads into a sheath or tubing. Such traditional fabrication techniques are unsuitable for microfabricated flow channels (e.g., created using lithographic processes) because the stationary phase is defined directly within the flow channel and not separately processed and packed. The chromatography columns and methods for their manufacture taught herein overcome these problems by utilizing a two-step in situ flow-through deposition process to functionalize microfabricated structural features in a controlled manner to prevent clogging or subsequent non-uniform flow characteristics.

[0008] Described herein are, among other things, microfluidic chromatography columns comprising a stationary phase substrate having a liquid channel defined by channel walls, the channel having an inlet and an outlet. Pillars may be present within the channel on the substrate. Examples of devices suitable for use as chromatography columns in embodiments taught herein are described, for example, in U.S. Patent Application Publication No. 2016 / 0001199 and International Patent Application Publication No. 2022 / 190056, each of which is incorporated herein by reference in its entirety. In some embodiments, the stationary phase substrate of the chromatography column comprises silicon oxide.

[0009] At least a portion of the surface of the stationary phase substrate can be attached to a linker, which can be attached to a polymer. Without being bound to any particular theory or embodiment, the polymer enables the microfluidic chromatography column to be used for hydrophilic interaction chromatography. However, although hydrophilic interaction chromatography is mentioned as an exemplary application, it should be understood that the disclosed microfluidic chromatography columns can be used for other forms of polymer-based chromatography.

[0010] As described above, microfabricated structural features, such as pillar structures, can be positioned within a liquid channel. In some embodiments, the microfabricated structural features can comprise a conductive or semiconductive material, such as silicon, and can be produced using microfabrication or nanofabrication techniques known to those skilled in the art, including, for example, using photobeam or electron beam lithography and deep reactive ion etching. Thus, in some examples, the microfabricated structural features are integral with the liquid channel, e.g., immobile or fixed relative to the liquid channel. In some embodiments, stationary phase elements can be added in a manufacturing process after formation of the liquid channel (e.g., using a packed-bed column). In some examples, the microfabricated structural features (e.g., pillars) can form the stationary phase of a chromatography column. In some embodiments, the pillars can be positioned in a regular pattern, although this is not a requirement, as the pillars can be arranged in an irregular or asymmetric pattern. For example, the pillars can be arranged in a first pattern along a first portion of the channel and a second pattern along a second portion of the channel.

[0011] In some embodiments, the silicon stationary phase substrate can be porous. The porosity can be created, for example, by electrochemically anodizing the substrate. After anodization, the substrate can be subjected to a heat treatment to obtain a silicon oxide surface layer on the entire substrate and its pillar structures, and to generate reactive silanol groups on at least a portion of the substrate and / or pillar structures, the heat treatment being carried out at a temperature, duration, and atmosphere such that any silicon oxide layer formed has a thickness of at least 10 nm, e.g., in the range of 10 nm to 20 nm or 10 nm to 30 nm. Non-limiting examples of creating stationary phase substrates containing microfabricated structural features (such as pillars) can be found in U.S. Pat. No. 11,491,458, International Patent Application Publication Nos. 2022 / 144786, and 2022 / 190056, which are incorporated by reference in their entireties.

[0012] The pillars can be of various shapes and can extend from the bottom to the top of the liquid channel. The pillars can have various cross-sectional shapes, for example, circular (for cylindrical pillars), square, rectangular, triangular, oval, American football-shaped, and chevron-shaped.

[0013] In some embodiments, the liquid channel includes wider sections (e.g., where the liquid is spread over a larger cross-sectional area to increase interactions between the liquid and features within the channel) and narrower sections (e.g., ducts for directing liquid from point to point within a chromatographic column, etc.) The liquid channel can have a width, for example, between 1 μm and 75 μm, e.g., between 1 μm and 5 μm, between 5 μm and 10 μm, between 10 μm and 15 μm, between 15 μm and 20 μm, between 20 μm and 25 μm, between 25 μm and 30 μm, between 30 μm and 35 μm, between 35 μm and 40 μm, between 40 μm and 45 μm, between 45 μm and 50 μm, between 50 μm and 55 μm, between 55 μm and 60 μm, between 60 μm and 65 μm, between 65 μm and 70 μm, or between 70 μm and 75 μm. In some embodiments, the liquid channels have a width in the range of 50 μm to 5 cm, such as 50 μm to 200 μm, 200 μm to 500 μm, 500 μm to 1 cm, or 1 cm to 5 cm.

[0014] The channels may have depths ranging from 1 μm to 750 μm, such as 1 μm to 5 μm, 5 μm to 10 μm, 10 μm to 15 μm, 15 μm to 20 μm, 20 μm to 50 μm, 50 μm to 75 μm, 75 μm to 100 μm, 100 μm to 150 μm, 150 μm to 200 μm, 200 μm to 250 μm, 250 μm to 300 μm, 300 μm to 500 μm, and 500 μm to 750 μm. In some embodiments, the channel depth may be less than half the thickness of the substrate to ensure that the substrate remains thick enough to provide mechanical strength and a solid foundation for microfabricated structures (e.g., pillars). In some embodiments, the wafer thickness is approximately 500 μm. The pillars may have typical diameters of 500 nm to 3 mm, e.g., 500 nm to 100 μm. The inter-pillar distance between adjacent pillar structures can be less than 10 micrometers, e.g., less than 5 micrometers. In some embodiments, the pillars can have sloping sidewalls, the slope of the sidewalls of the pillar structures being less than 2°, e.g., less than 1°, e.g., less than 0.5°. In some embodiments, the pillars are vertical.

[0015] A portion of the surface of the stationary phase substrate can be bonded to a linker, which in turn can be bonded to a polymer. The linker is formed upon reaction of the grafted prelinker with a monomer of the polymer. Examples of prelinkers are acrylate silanes, such as alkylacryloxypropyltrialkoxysilanes, or analogs containing terminal alkenes. In some embodiments, the prelinker comprises 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropyltrisdimethylaminosilane, allyltrimethoxysilane, 5-hexenyltrimethoxysilane, vinyltrimethoxysilane, or mixtures thereof. In some embodiments, the prelinker comprises 3-methacryloxypropyltrimethoxysilane.

[0016] Formula 1 below provides exemplary, non-limiting linkers and polymers.

[0017] [ka] wherein R1 and R4 are independently an alkyl group and R5 and R6 are a polymer. In some embodiments, R1 and R4 are independently a C alkyl group such as methyl, ethyl, propyl, or butyl. 1~4 In some embodiments, R is alkyl. In some embodiments, R is butyl. In some embodiments, R is methyl. In some embodiments, R and R are polyacrylamide. In some embodiments, the polyacrylamide is formed from monomers including acrylamide, N,N-dimethylacrylamide, or N,N-diethylacrylamide.

[0018] An exemplary embodiment of Equation 1 is as follows:

[0019] [ka]

[0020] A chromatography column can be functionalized by depositing a linker and forming a polymer. Linker deposition can include flowing a first mixture containing an acrylate silane, an acid catalyst, and a first solvent through the column to deposit a pre-linker on the stationary phase substrate of the chromatography column. The pre-linker is formed into a linker upon formation of the polymer. Figure 6 is a schematic diagram of an embodiment of the linker deposition process.

[0021] The polymer can be formed by flowing a second mixture comprising the monomer, a second solvent, and a radical initiator through a chromatography column under conditions sufficient to react the monomer with the pre-linker to form a linker-attached polymer.

[0022] In some embodiments, the solvent used in the linker placement reaction (first solvent) is a hydrophobic solvent capable of dissolving the acrylate silane. Examples of hydrophobic solvents include toluene, hexane, and xylene.

[0023] In the linker placement reaction, an organic acid catalyst can be used to catalyze the coupling of acrylate silane to the silica surface to form the pre-linker. The acid catalyst can include, for example, any one or more of acetic acid, formic acid, lactic acid, and citric acid.

[0024] In an example of a linker placement reaction, an acrylate silane is attached to the silica surface of the stationary phase substrate to form a pre-linker. Exemplary, non-limiting acrylate silanes or analogs with terminal alkenes include 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropyltrisdimethylaminosilane, allyltrimethoxysilane, 5-hexenyltrimethoxysilane, vinyltrimethoxysilane, or mixtures thereof.

[0025] In some embodiments, the temperature of the linker placement reaction is from about 50° C. to about 95° C., or from about 50° C. to about 150° C. Without being bound to any particular theory or embodiment, the temperature can affect the yield of linker placement, with higher temperatures resulting in higher yields. In some embodiments, the temperature of the linker placement reaction can be selected based on considerations of the boiling points of the reagents or solvents and / or based on the fluid pressure applied to the solution as it flows through the microfabricated chromatography column.

[0026] An exemplary pre-linker placement reaction is as follows:

[0027] [ka]

[0028] As shown, polymer formation creates a polymer attached to a linker. In some embodiments, the polymer provides hydrophilic interactions, and thus the microfluidic device can be used for hydrophilic interaction chromatography. In some embodiments, the monomer is acrylamide or N-vinylacetamide. Examples of monomers include acrylamide, N,N-dimethylacrylamide, or N,N-diethylacrylamide. In some embodiments, the monomer comprises acrylamide. In some embodiments, the monomer comprises N-vinylacetamide. The monomer may be at a concentration of 0.01 M to 1 M, such as 0.01 M to 0.05 M, 0.05 M to 0.1 M, 0.1 M to 0.2 M, 0.2 M to 0.3 M, 0.3 M to 0.4 M, 0.4 M to 0.5 M, 0.5 M to 0.6 M, 0.6 M to 0.7 M, 0.7 M to 0.8 M, 0.8 M to 0.9 M, and 0.9 M to 1.0 M. The concentration of the monomer may vary depending on the initiator concentration, temperature, flow rate, and other parameters. Figure 7 is a schematic diagram of an embodiment of the polymer formation process.

[0029] In some embodiments, the solvent used in the polymer-forming reaction (second solvent) is a hydrophilic solvent. An example of a hydrophilic solvent is a mixture of methanol and water.

[0030] The combination of solvent and catalyst affects the grafting reaction of a prelinker, such as 3-methacryloxypropyltrimethoxysilane (MPS), onto the silica surface. Using different solvents and catalysts can increase or decrease yield and density. In some embodiments, toluene was the solvent and acetic acid was the catalyst, which resulted in higher grafting yields compared to other solvent / catalyst systems, such as DMF / pyridine. It is hypothesized that the hydrophobic nature of toluene drives the hydrophilic alkoxy tails of the MPS organosilane toward the silica surface, where the acetic acid (also hydrophilic) present dissociates and catalyzes the reaction toward covalent grafting.

[0031] A radical initiator initiates the polymerization reaction. Examples of radical initiators include potassium persulfate, ammonium persulfate, tert-butyl hydroperoxide, and azobisisobutyronitrile (AIBN). In some embodiments, the radical initiator is selected from potassium persulfate or ammonium persulfate. These persulfate initiators can be controlled by their temperature sensitivity. At room temperature and neutral pH levels, there is no generation of primary free radicals, and therefore no monomer conversion occurs. This helps prevent undesired polymerization in the reagent vessels that feed the chromatography column. Monomer conversion can be limited to the column by simply heating the column. Without being bound by any particular theory or embodiment, the amount of radical initiator present in the second mixture affects the yield, rate, and size of the polymer produced. In some embodiments, the second mixture comprises about 2 mol % to about 10 mol % initiator relative to the monomer, e.g., 2-3 mol %, 3-4 mol %, 4-5 mol %, 5-6 mol %, 6-7 mol %, 7-8 mol %, 8-9 mol %, 9-10 mol %, 3-5 mol % initiator.

[0032] Experiments were performed using monomer:initiator (e.g., acrylamide:potassium persulfate) ratios to target polymerization with a high degree of initiation as well as termination. Ratios that are too high or too low can result in clogging of the microfluidic channels or insufficient polymer formation and poor chromatographic separation. Examples of monomer:initiator molar ratios are 0.01:1 to 0.1:1, such as 0.01:1 to 0.02:1, 0.02:1 to 0.03:1, 0.03:1 to 0.04:1, 0.04:1 to 0.05:1, 0.05:1 to 0.06:1, 0.06:1 to 0.07:1, 0.07:1 to 0.08:1, 0.08:1 to 0.09:1, and 0.09:1 to 0.1:1.

[0033] The first and second mixtures can be continuously flowed through the chromatography column with intermittent flushing steps for proper transition from the first solvent to the second solvent. Flowing through the column allows fresh reagents to react with sites on the stationary phase substrate and prelinker for each of the first and second mixtures. In some embodiments, the first mixture can be flowed at a flow rate of about 0.5 mm / s to about 10 mm / s. In some embodiments, the second mixture can be flowed at a flow rate of about 1 mm / s to about 7 mm / s. In some embodiments, a solvent or solvent mixture is flushed through the chromatography column before and after the first and second mixtures are flowed through the chromatography column. Examples of solvents that can be used to flush the chromatography column are acetonitrile and mixtures of acetonitrile with the solvent used in the reaction.

[0034] An example of a polymer-forming reaction is as follows:

[0035] [ka]

[0036] In this disclosure, the singular forms "a," "an," and "the" include the plural, and reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise. Thus, for example, reference to "a material" is a reference to at least one of such material and equivalents thereof known to those skilled in the art, and so forth.

[0037] The modifier "about" should be considered to disclose a range defined by the absolute values ​​of the two endpoints. For example, the phrase "about 2 to about 4" also discloses the range "2 to 4." When used to modify a single number, the term "about" can refer to plus or minus 10% of the recited number, including the recited number. For example, "about 10%" can indicate a range of 9% to 11%, and "about 1" means 0.9 to 1.1.

[0038] When a list is given, it is to be understood that each individual element of that list and every combination of that list is to be construed as a separate embodiment unless otherwise stated. For example, a list of embodiments given as "A, B, or C" shall be construed to include the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0039] In this regard, all ranges are inclusive and combinable, i.e., reference to values ​​stated in ranges includes every value within that range. For example, a range defined as 400-450 ppm includes 400 ppm and 450 ppm as independent embodiments. The ranges 400-450 ppm and 450-500 ppm can be combined to form the range 400-500 ppm.

[0040] It should also be understood that certain features of the invention that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. That is, unless clearly incompatible or excluded, each individual embodiment is considered combinable with any other embodiment, and such combinations are considered to be separate embodiments. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination. It should further be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for any use of such exclusive language, such as "solely," "only," etc., in connection with the recitation of claimed elements or the use of "negative" limitations. Finally, while an embodiment may be described as part of a series of steps or part of a more general structure, each step in question can also be considered a standalone embodiment in itself.

[0041] While this disclosure is illustrated by describing several embodiments, and exemplary embodiments are described in considerable detail, it is not the intention of the applicant to restrict or in any way limit the scope of the appended claims to such details. Additional advantages and modifications may be readily apparent to those skilled in the art. Furthermore, features from the individual listings may be combined, and features from the examples may be generalized throughout the disclosure.

[0042] Example

[0043] Evaluation Procedure The properties of HILIC stationary phases were tested using the so-called "Tanaka test probe" as described by Tanaka et al., Journal of Chromatography A, 1218 (2011) 5903-5919. These methods use the selectivity (α) of different sets of components to investigate the chemical properties of the stationary phase. Selectivity is expressed as the quotient of two retention factors (e.g., αOH = k'(U) / k'(2'-dU)). Standard experimental conditions were as follows: Equipment: Ultimate 3000, 4nL injection valve Mobile phase: acetonitrile - 90% by volume, water, 20 mM NH4OAc (aqueous solution pH: 4.7) - 10% by volume Gradient: Isocratic ·Flow rate: 250nL / min ·Temperature: 30℃ Detection: UV 260nm Void Marker: Toluene

[0044] Example 1 "Early" microfluidic chromatography columns (e.g., μPAC™ high-pressure liquid chromatography columns from Thermo Fisher Scientific, Inc. (Waltham, MA)) comprise a microfluidic column with a selected architecture, secured at the inlet and outlet with fused silica capillaries of selected dimensions (length, inner / outer diameter). The starting surface of the microfluidic chromatography column can be porous or non-porous, and it can be oxidized to various degrees as a result of prior processing parameters.

[0045] The initial microfluidic chromatography column was connected at one end via its capillary to a liquid displacement device, either single or in parallel with additional columns utilizing a flow-splitting manifold. This displacement device could be, but is not limited to, either a piston pump and / or a pressure-driven flow device. The connection points could be fittings of any desired type and material, as long as they provide a leak-tight fit to the device. All materials used should be chemically resistant to the chemicals used in the grafting protocol.

[0046] The microfluidic chromatography column is placed on or in a heating device, which may be, but is not limited to, a thermostatically controlled heating plate or a heating chamber. Individual microfluidic chromatography columns may be secured in dedicated casings of any desired design and material, and their capillaries may be sleeved with any material selected for robustness. Thermally conductive materials, such as pads, may be present in the microfluidic chromatography column container for optimal heat transfer between the container and the column.

[0047] The capillary serving as the outlet was positioned so that effluent could be collected during the grafting protocol, such as through connection to a collection vessel (vial, flask, etc.). This primarily served as a control action. In some cases, the outlet capillary was connected to a further capillary of a specific length and inner / outer diameter, which could act as a flow restrictor.

[0048] After one or more columns were properly connected to the apparatus, the columns were heated to the desired temperature and the first sequence of grafting / rinsing steps was initiated.

[0049] For the first step of the functionalization of the microfluidic chromatography column, i.e., the deposition of acrylate-type silanes onto the surface, the following grafting protocol was used.

[0050] (1) The column was first "wetted" by flowing pure acetonitrile ("ACN"). A liquid displacement device provided the flow of ACN at a selected flow rate and / or pressure, depending on the column architecture. Since this rinse step served purely as a wetting phase, there was no strict time window for the duration of this rinse step. It was typically carried out for at least 1 hour to also monitor the leak tightness of the device and its various connections.

[0051] (2) The column was then flushed with pure toluene ("TOL") to prepare it for the next grafting step. This so-called "solvent switch" involved appropriate priming procedures of the liquid displacement device and the fluid pathway toward the column. TOL was then flowed through the column at a selected flow rate and / or pressure, depending on the column architecture. The flushing time is typically determined by the design of the specific column architecture and the column volume. A typical TOL flushing sequence was performed for 1 to 24 hours.

[0052] (3) In the next step, the grafting solution was passed through the column at a flow rate and / or pressure selected to obtain the desired flow rate. A toluene-based solution containing the desired acrylate organosilane and organic acid catalyst in a selected molar ratio was prepared. A typical example includes a solution of 5% (v / v) 3-methacryloxypropyltrimethoxysilane and 1% (v / v) glacial acetic acid in toluene. The grafting step was carried out for a selected duration within a time frame of 2 hours to over 48 hours. Typically, a contact time of 24 to 48 hours was selected for maximum grafting yield (depending on the column architecture).

[0053] (4) After grafting, the column was again flushed with pure TOL to remove unreacted reagents from the column surface. The column was flushed at a selected flow rate and / or pressure depending on the column architecture. The flushing time is typically determined by the specific column architecture design and column volume. A typical TOL flushing step was performed for 1 to 24 hours.

[0054] (5) The final flushing step of this grafting sequence was performed with ACN, similar to the initial wetting step. The ACN displaced the hydrophobic TOL, which served as a preparation for the subsequent flushing step with a hydrophilic solvent. The column was flushed at a selected flow rate and / or pressure, depending on the column architecture. The flushing time was typically determined by the specific column architecture design and column volume. A typical ACN flushing step lasted from 1 to 24 hours.

[0055] The alkene-functionalized microfluidic chromatography column obtained in the first grafting step was subjected to a second and final grafting sequence in which the available alkene functional groups on the surface were utilized to in situ polymerize suitable amide monomers and immobilize them on the surface. Before starting this sequence, the temperature was adjusted to the desired value.

[0056] (1) The column was prepared for the subsequent grafting step by flushing it with a methanol / water mixture of selected composition. Depending on the column architecture, the solvent was passed through the column at a selected flow rate and / or pressure. The flushing time is typically determined by the specific column architecture design and column volume. A typical solvent flushing sequence lasted from 1 to 24 hours.

[0057] (2) For the subsequent grafting step, a solution containing the appropriate alkene-containing amide monomer and radical initiator dissolved in a selected molar ratio of methanol / water was prepared. A typical example involves a solution of 0.2 M acrylamide and 0.006 M potassium persulfate in 50 / 50 (v / v) methanol / water, resulting in a monomer:initiator molar ratio of 1:0.03. The solution was run through the column at a flow rate and / or pressure selected depending on the column architecture to achieve the desired linear velocity. The grafting step was carried out for a selected duration within a time frame ranging from 2 hours to over 48 hours. Typically, a contact time of 24 to 48 hours was selected for optimal grafting yield (depending on the column architecture and process parameters).

[0058] (3) The column was then flushed with a methanol / water mixture of selected composition to remove unreacted reagents from the column surface. The solvent was passed through the column at a selected flow rate and / or pressure depending on the column architecture. The flushing time was typically determined by the design of the specific column architecture and the column volume. A typical solvent flushing sequence lasted from 1 to 24 hours.

[0059] (4) A final flushing step was performed in pure ACN at a selected flow rate and / or pressure, depending on the column architecture. This step is optional because it helps prepare the column for chromatographic evaluation in HILIC mode, which primarily utilizes ACN / water mixtures as the mobile phase. The flushing time was typically determined by the specific architecture design and column volume. A typical solvent flushing sequence was performed for 1 to 24 hours.

[0060] Example 2 - Hydrophilic Ability The selectivity for uridine and 2'-deoxyuridine demonstrates the hydrophilic capabilities / nature of the column. The HILIC column of Example 1 was compared to a non-functionalized column as shown in Figure 1 using an evaluation procedure. Not only does the amide-functionalized column exhibit higher retention for these nucleosides compared to the bare silica column, but the selectivity between them also corresponds well to that typically observed for neutral amide phases.

[0061] Example 3 - Hydrophobic Capability The selectivity for uridine and 5-methyluridine demonstrates the hydrophobic capabilities / nature of the column. The HILIC column of Example 1 was compared to a non-functionalized column as shown in Figure 2 using an evaluation procedure. Again, the differences in retention between both columns are notable, as are the selectivities between the nucleoside probes.

[0062] Example 4 - Anion Selectivity The selectivity of sodium p-toluenesulfonate (SPTS) demonstrates the column's anion-selective ability / nature. The HILIC column from Example 1 was compared to a non-functionalized column, as shown in Figure 3, using the evaluation procedure. Positive retention times were observed on the functionalized column, whereas negative retention times were observed on the non-functionalized column. This can be explained by the grafting process, which functionalizes the silanol surface and protects the residual silanols with a polymeric amide phase. This largely negates the electrostatic repulsion with anionic probes such as SPTS. However, non-functionalized columns with accessible silanols exhibit this repulsive behavior, eluting anionic probes before the neutral void marker.

[0063] Example 5 - Cation Selectivity The selectivity for trimethylphenylammonium chloride (TMPAC) demonstrates the cation selectivity capability / nature of the column. The HILIC column of Example 1 was compared to a non-functionalized column as shown in Figure 4 using the evaluation procedure. Shorter retention times were observed for the functionalized column, while longer retention times were observed for the non-functionalized column. This is again due to ionic interaction behavior; the functionalized column exhibits less retention for cationic probes such as TMPAC, while the non-functionalized silanol surface exhibits a strong electrostatic attraction for such probes.

[0064] Example 6 - Acid-Base Selectivity The selectivity of theophylline (Tp) and theobromine (Tb) indicates the acid-base capabilities / properties of the column. The HILIC column of Example 1 was compared to a non-functionalized column as shown in Figure 5 using an evaluation procedure. A basic stationary phase gives an α(Tb / Tp) < 1, a neutral phase gives an α(Tb / Tp) = 1, and an acidic phase gives an α(Tb / Tp) > 1. Despite being a neutral phase, amide columns often behave as acidic columns, resulting in α(Tb / Tp) values ​​> 1 due to the acidic (and cation-exchange) nature of their carboxyl-like moieties. (Journal of Chromatography A, 1218 (2011) 5903-5919).

Claims

1. 1. A method for functionalizing a chromatography column, comprising: linker placement, the linker placement comprising flowing a first mixture comprising an acrylate silane, an acid catalyst, and a first solvent through the chromatography column to place a pre-linker on a stationary phase substrate of the chromatography column; forming a polymer comprising flowing a second mixture comprising a monomer, a second solvent, and a radical initiator through the chromatography column under conditions sufficient to react the monomer with the pre-linker to form a linker-attached polymer.

2. The method of claim 1 , wherein the stationary phase substrate comprises pillars comprising silicon oxide.

3. The method of claim 1 , wherein the first solvent is a hydrophobic solvent.

4. 10. The method of claim 1, wherein the acid catalyst comprises any one or more of acetic acid, formic acid, lactic acid, and citric acid.

5. 2. The method of claim 1, wherein the acrylate silane comprises 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropyltrisdimethylaminosilane, allyltrimethoxysilane, 5-hexenyltrimethoxysilane, vinyltrimethoxysilane, or mixtures thereof.

6. 10. The method of claim 1, wherein the linker placement is carried out at a temperature of about 50°C to about 150°C.

7. 10. The method of claim 1, wherein the second mixture has a molar ratio of initiator to monomer of from 0.01:1 to 0.1:

1.

8. The method of claim 1 , wherein the second solvent is a hydrophilic solvent.

9. The method of claim 1 , wherein the monomer comprises acrylamide or N-vinylacetamide.

10. The method of claim 1 wherein the monomer is acrylamide.

11. The method of claim 1 , wherein the radical initiator comprises potassium persulfate.

12. The method of claim 1 , wherein the second mixture is flowed at a flow rate of about 1 mm / sec to about 7 mm / sec.

13. The method of claim 1 , wherein the chromatography column is a microfluidic device.

14. 1. A chromatography column, comprising: a stationary phase substrate having a liquid channel defined by a channel wall, the liquid channel having an inlet and an outlet; pillars positioned within the liquid channels on the stationary phase substrate; A chromatography column, wherein at least a portion of the surface of the stationary phase substrate is attached to a linker, the linker being attached to a polymer.

15. 15. The chromatography column of claim 14, wherein the stationary phase substrate of the chromatography column comprises silicon oxide.

16. 15. The chromatography column of claim 14, wherein the linker is a reaction product of a pre-linker and a monomer of the polymer.

17. 17. The chromatography column of claim 16, wherein the prelinker is an acrylate silane.

18. 18. The chromatography column of claim 17, wherein the linker is formed from an alkylacryloxypropyltrialkoxysilane.

19. The linker and the polymer are represented by Formula 1: 【Chemistry 1】 In the formula, R １ and R ４ are independently an alkyl group, and R ５ and R ６ 15. The chromatography column of claim 14, wherein is a polymer.

20. R ５ and R ６ 20. The chromatography column of claim 19, wherein is polyacrylamide.

21. 21. The chromatography column of claim 20, wherein the polyacrylamide is formed from monomers including acrylamide, N,N-dimethylacrylamide, or N,N-diethylacrylamide.

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