Preparation method and application of a bio-inert chromatographic column

Abstract

The application belongs to the technical field of liquid chromatography column, and relates to a preparation method and application of a biologically inert chromatography column. A transition layer is formed by pre-modification through alkoxy silane liquid phase reaction, and then a silicon carbide coating is generated in situ by gas phase deposition and pyrolysis combined with hyperbranched polycarbosilane. The coating is very excellent in biologic inertia, extremely strong in chemical stability, free from ion elution, low in surface energy, weak in polarity, small in non-specific adsorption, and can construct a metal-transition layer-polycarbosilane ceramic layer gradient structure, optimize stress distribution and interface performance, and further enhance the shielding and protection effect on the metal base material.

Description

Technical Field

[0001] This invention belongs to the field of liquid chromatography column technology, and relates to a method for preparing and applying a biological inert chromatography column. Background Technology

[0002] Currently, biopharmaceuticals mainly include macromolecular drugs, enzymes, peptides, proteins, nucleic acids, vaccines, small molecule ligands, and other biological agents with specific structures and functions. Biopharmaceutical molecules typically contain electron donors (N, O, S, P), with P electron donors being particularly common in biopharmaceuticals. Because these drugs can form directional, saturable, and selective bindings with metal surfaces, more stringent requirements are placed on the application of chromatographic columns in biopharmaceuticals.

[0003] Polymer materials such as polyetheretherketone (PEEK) chromatographic columns can avoid the influence of metal ions, but the materials are expensive and have low pressure resistance. The accompanying polymer filter plates do not yet meet the requirements for high precision and high throughput, failing to provide suitable robust components for commercial chromatographic columns. Chinese patent CN111107931B proposes using alkylsilane compositions to coat the flow path of instruments and chromatographic devices to improve liquid chromatography separation. However, small-molecule alkylsilanes have high internal stress during film formation, and the thicker film layer is prone to cracking, curling, peeling, pinholes, voids, inclusions, etc., resulting in poor interfacial adhesion. Strict pretreatment is required to ensure that delamination at the interface occurs when the substrate surface energy is low. Chinese patent CN117248184A uses micro-arc ion plating technology, utilizing carbon targets and elemental metal targets to form a diamond-like coating. This type of coating still contains metal, and the coating exhibits a clustering phenomenon with obvious gaps between the clusters, making it difficult to control its uniform distribution on the surface of the chromatographic column substrate. Summary of the Invention

[0004] To address the need for low-specificity separation and purification in biopharmaceutical applications, this invention provides a novel method for preparing bio-inert chromatographic columns. A transition layer is formed through a liquid-phase reaction of alkoxysilanes, followed by in-situ generation of a silicon carbide coating using hyperbranched polycarbosilane via vapor deposition and pyrolysis. This coating exhibits excellent bioinertness, strong chemical stability, no ion dissolution, low surface energy, weak polarity, and minimal non-specific adsorption. It can construct a gradient structure of metal-transition layer-polycarbosilane (PCS) ceramic layer, optimizing stress distribution and interfacial properties, further enhancing the shielding and protection effect on the metal substrate.

[0005] Firstly, the preparation method of a biological inert chromatographic column includes the following steps: Step (1) is the process of cleaning the chromatographic column; Step (2) is a process of liquid-phase surface silanization of the cleaned chromatographic column; wherein, alkoxysilane is used to pre-modify the chromatographic column by liquid-phase reaction to form a transition layer; Step (3) is the process of washing the chromatographic column again after silanization of the liquid phase surface; Step (4) involves removing volatile substances from the washed chromatographic column; Step (5) is the process of depositing a hyperbranched polycarbosilane coating on the surface of the chromatographic column after the removal of volatile substances; Step (6) involves pyrolyzing the column after depositing a hyperbranched polysilane coating to form a silicon carbide film in situ.

[0006] In an optional embodiment, in step (2), the chemical composition of the alkoxysilane is (R1). n1 (R2) n2 Si(OR3) 4-n1-n2 R1 is selected from methyl, butyl, isobutyl, aminopropyl, glycidoxypropyl, methacryloyloxypropyl, and propylcarbamate; R2 is selected from methyl, butyl, isobutyl, aminopropyl, glycidoxypropyl, methacryloyloxypropyl, and propylcarbamate; R1 and R2 may be the same or different; R3 is selected from methyl and ethyl; preferably, n1 and n2 are independently selected from natural numbers from 0 to 2; more preferably, 0 < n1 + n2 ≤ 3.

[0007] In an optional embodiment, in step (2), the alkoxysilane is selected from one or more of γ-(2,3-epoxypropoxy)propyltrimethoxysilane, 3-(methacryloyloxy)propyltrimethoxysilane, 3-aminopropyltrimethoxysilane, n-butyldimethylmethoxysilane, tert-butyldimethylmethoxysilane, γ-glycidoxypropyltrimethoxysilane, and γ-methacryloyloxypropyltrimethoxysilane.

[0008] In an optional embodiment, in step (2), the cleaned chromatographic column is sealed and connected to the silane storage container of the delivery system. The silane storage container contains a silane system in which alkoxysilane, alcohol solvent and acid are mixed according to a stoichiometric relationship. The silane system is delivered to the chromatographic column. After the silane system flows through the chromatographic column, it is returned to the silane tank.

[0009] In an optional embodiment, the alcohol solvent is ethanol; the acid solution is 0.01-0.5 mol / L hydrochloric acid; preferably, the weight ratio of alkoxysilane: alcohol solvent: acid solution is 5-50:45-85:0.5-5.

[0010] In an optional implementation, in step (3), the delivery liquid of the delivery system is switched to a small molecule alcohol to perform flow rinsing on the chromatographic column.

[0011] In an optional embodiment, in step (4), the chromatographic column is removed from the delivery system, placed at room temperature to air dry, placed in the vapor deposition apparatus, sealed, and then replaced with an inert atmosphere to raise the temperature to 200-600°C according to a preset heating program.

[0012] In an optional embodiment, in step (5), the hyperbranched polycarbosilane is selected from one or more of allyl hyperbranched polycarbosilane, fully hydrogenated hyperbranched polycarbosilane, vinyl-terminated hydrogenated hyperbranched polycarbosilane, and alkyl hyperbranched polycarbosilane.

[0013] The hyperbranched polycarbosilane in step (5) and the alkoxysilane backbone in step (2) should have similar bonding ratios. In other words, the hyperbranched polycarbosilane in step (5) and the alkoxysilane backbone in step (2) should follow the principle of similar compatibility or be able to undergo chemical reactions.

[0014] In an optional embodiment, in step (5), the deposition temperature is 600-1000℃, the deposition pressure is 100-1000Pa, and the deposition time is 0.5-5h. The deposition may be chemical vapor deposition.

[0015] In an optional embodiment, in step (6), the pyrolysis temperature is 1000-1300℃ and the pyrolysis time is 1-4 h; preferably, the pyrolysis is carried out under an inert atmosphere; more preferably, the inert atmosphere flow rate is 50-200 sccm.

[0016] Secondly, the present invention provides the application of the bio-inert chromatographic column obtained by any of the above preparation methods in the separation of bioactive analytes.

[0017] Compared with the prior art, the present invention has the following advantages: 1. Hyperbranched polycarbosilanes are used to form silicon carbide coatings in situ via vapor deposition and pyrolysis. Hyperbranched polycarbosilanes have low viscosity, are easily volatile, easily transportable, easy to form films, low stress, high density, good step coverage, and high ceramic conversion rate. The silicon carbide coating formed after ceramic conversion has excellent bioinertness, strong chemical stability, no ion dissolution, low surface energy, weak polarity, and low non-specific adsorption. In addition, the silicon carbide coating has high hardness and wear resistance, making it more suitable for high-pressure packing shear friction of chromatographic columns.

[0018] 2. The core of alkoxy-based liquid-phase pre-modification lies in constructing molecular bridges with alkoxy groups, significantly enhancing interfacial bonding and optimizing the uniformity and density of hyperbranched polycarbosilane deposition. This addresses the shortcomings of simple hyperbranched polycarbosilane deposition, such as easy detachment, weak interfaces, and numerous film defects. Alkoxy groups act as a transition layer, connecting to a metal at one end and complementing the hyperbranched polycarbosilane at the other, reducing internal stress and minimizing cracking during pyrolysis after deposition. This lowers the barrier to entry for hyperbranched polycarbosilane deposition and enhances compatibility. Furthermore, alkoxy pre-modification can be completed under mild conditions ranging from room temperature to 150°C. After pre-modification, the deposition temperature of hyperbranched polycarbosilane can be appropriately reduced while maintaining film quality, reducing energy consumption and equipment requirements. Simultaneously, by selecting different alkoxy groups (such as methyl, amino, and epoxy groups), interfacial polarity, reactivity, and crosslinking density can be controlled to match the side group structures of different hyperbranched polycarbosilanes.

[0019] 3. Construct a gradient structure of metal-transition layer-hyperbranched polycarbosilane ceramic layer to further optimize stress distribution and interface properties; interfacial covalent bonding promotes cross-linking of hyperbranched polycarbosilane, resulting in more complete ceramicization during pyrolysis; improves the density, hardness, and high-temperature resistance of silicon carbide coating, reduces interface defects, prevents gas from penetrating along the interface at high temperatures, and enhances the shielding and protection effect on the metal substrate. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the bio-inert chromatographic column of the present invention.

[0021] Figure 2 These are morphological comparison images of Example 1 and Comparative Example 1. Among them, A (optical micrograph), B (optical micrograph), and C (electron scanning micrograph) are morphological images of Example 1; a (optical micrograph), b (optical micrograph), and c (electron scanning micrograph) are morphological images of Comparative Example 1.

[0022] Figure 3A This is the result of nucleotide analysis of the untreated column tube in Example 1.

[0023] Figure 3B This is the nucleotide analysis result of the vapor-deposited column in Example 1.

[0024] Figure 3C The results are from the nucleotide analysis of the liquid phase reaction + vapor phase deposition column in Example 1.

[0025] Figure 4A This is the result of the AMP / ADP / ATP analysis of the untreated column in Example 2.

[0026] Figure 4B This is a comparison of the AMP / ADP / ATP analysis results of the vapor-deposited column in Example 2.

[0027] Figure 4C The results are the AMP / ADP / ATP analysis results from the liquid-phase reaction + vapor-phase deposition column of Example 2.

[0028] Figure 5 This is a flowchart of the preparation process for a biological inert chromatographic column.

[0029] Figure 6 This is a diagram illustrating the effect of the shielding layer cracking due to the mismatch between the transition layer and the deposition layer in Example 5. Detailed Implementation

[0030] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the content of the present invention are within the scope of protection of the present invention.

[0031] The following combination Figure 1 and Figure 5 This invention describes the preparation and usage process of the bio-inert chromatographic column. The process includes column cleaning, column surface silanization in liquid chromatography, furnace inertization, hyperbranched polycarbosilane vapor deposition, silicon carbide ceramicization, packing, and biological sample separation and purification.

[0032] The procedure for cleaning the chromatographic column can be performed using any cleaning method commonly used in the art. For example, the column can be ultrasonically cleaned with an alkaline cleaning solution to remove oil, metal debris, or other impurities; then, it can be ultrasonically cleaned multiple times with deionized water to remove residual cleaning solution; subsequently, the column can be ultrasonically washed with an organic solvent (including but not limited to ethanol, acetone, etc.) to remove residual deionized water from the column surface.

[0033] A process for liquid-phase surface silanization of a chromatographic column. An alkoxysilane is used for pre-modification via a liquid-phase reaction to form a transition layer. In an optional embodiment, the chemical composition of the alkoxysilane is (R1). n1 (R2) n2 Si(OR3) 4-n1-n2 In a liquid-phase system, alkoxysilanes containing groups R1 and R2 react with active hydroxyl groups on a metal surface to form a silane-modified layer containing active groups. R1 is selected from methyl, butyl, isobutyl, aminopropyl, glycidoxypropyl, methacryloyloxypropyl, propylcarbamate, etc. R2 is selected from methyl, butyl, isobutyl, aminopropyl, glycidoxypropyl, methacryloyloxypropyl, propylcarbamate, etc. R1 and R2 can be the same or different. R3 is selected from methyl, ethyl, etc. Further, n1 and n2 are independently natural numbers from 0 to 2. More preferably, 0 < n1 + n2 ≤ 3.

[0034] In an optional embodiment, the cleaned chromatographic column (assembly) is sealed and connected to a delivery system. For example, it is connected to a high-pressure plunger pump or diaphragm pump of the delivery system. The silane tank of the delivery system contains a silane system in which alkoxysilane, alcohol solvent, and acid are mixed in stoichiometric proportions. As an example, but not limited to, the alcohol solvent can be ethanol, and the acid can be 0.01-0.5 mol / L hydrochloric acid. The weight ratio of alkoxysilane:alcohol solvent:acid can be 5-50:45-85:0.5-5. As an example, but not limited to, the weight ratio of alkoxysilane:alcohol solvent:acid is 10-50:45-89:1-5. The silane system is heated to 50-150°C, and the silane system is delivered to the chromatographic column using a high-pressure plunger pump / diaphragm pump. After flowing through the column, the silane system is returned to the silane tank. The flow rate of the silane system is maintained at 0.5-50 mL / min. The cycle time of the silane system is controlled between 3 and 24 hours.

[0035] The procedure involves rewashing the chromatographic column after pre-modification with alkoxysilane liquid chromatography. The delivery system is switched to a small molecule alcohol, and the column is flow-flushed. The flow rate of the small molecule alcohol can be 0.5-50 mL / min. The washing time can be 5-30 min.

[0036] The process involves removing volatiles from the washed column. The column is removed from the delivery system, allowed to air dry at room temperature, and placed in a vapor deposition apparatus (e.g., a vapor deposition furnace). After sealing the apparatus, an inert atmosphere is introduced. Optionally, after sealing the furnace, a vacuum of 5 Pa or less is applied, followed by the introduction of high-purity inert gas (including but not limited to argon and nitrogen) until the furnace pressure is positive. Vacuuming is then continued, and this process is repeated three or more times. The inert gas flow rate is controlled at 50-200 sccm. The temperature is then increased to 200-600°C according to a preset temperature program. For example, the temperature is increased to 200-600°C at a rate of 1-5°C / min, and then no further holding is performed after reaching the preset temperature.

[0037] The process of depositing a hyperbranched polycarbosilane (HBPCS) coating on the surface of a chromatographic column. Hyperbranched polycarbosilanes include, but are not limited to, one or more mixtures of allyl-substituted hydrogenated hyperbranched polycarbosilanes (AHPCS, Allied Signal / Starfire, also known as allyl hyperbranched polycarbosilane), fully hydrogenated hyperbranched polycarbosilanes (H-HPCS), vinyl-terminated hydrogenated hyperbranched polycarbosilanes (Vi-HPCS), and alkyl hyperbranched polycarbosilanes (Me-HPCS). As an example, alkyl hyperbranched polycarbosilanes (Me-HPCS) can be methyl hyperbranched polycarbosilanes. The selection of hyperbranched polycarbosilanes requires the application of alkoxysilanes according to the liquid phase to ensure reaction with or high compatibility with group R1. For example, in step (2), the alkoxysilane is selected as n-butyldimethylmethoxysilane, and in step (5), the hyperbranched polycarbosilane is selected as an alkyl (methyl) hyperbranched polycarbosilane. Two silanes should follow the principle of similarity and compatibility, or form a stabilizing force through a specific chemical reaction.

[0038] In an optional embodiment, the hyperbranched polycarbosilane is vaporized using a bubbling method. Vaporization conditions (pre-vaporization chamber setup conditions) are, for example, a temperature of 40-80°C, a pressure of 100-1000 Pa, and a high-purity inert gas (including but not limited to argon, nitrogen, etc.) as the carrier gas at a flow rate of 50-200 sccm. Deposition conditions may be, for example, a deposition temperature of 600-1000°C, a deposition pressure of 100-1000 Pa, and a deposition time of 0.5-5 h. During deposition, in addition to the aforementioned high-purity inert carrier gas, reducing / diluting hydrogen gas is also introduced through a branch pipe. The high-purity inert carrier gas serves to suppress oxidation / carbon enrichment. The reducing / diluting gas serves to reduce free carbon and improve purity. As an example, but not limited to, the volume ratio of reducing / diluting gas to high-purity inert carrier gas is 5%-30%:70%-95%. The total flow rate of hydrogen and argon can be 100-500 sccm.

[0039] The process of forming a silicon carbide film by pyrolysis. In an optional embodiment, the pyrolysis temperature is 1000-1300℃, and the pyrolysis time is 1-4 h. In another optional embodiment, the temperature is increased to 1000-1300℃ at a rate of 1-3℃ / min, held at that temperature for 1-4 h, and then cooled to room temperature in the furnace. This heating rate can prevent cracking or porosity. Pyrolysis is carried out under an inert gas atmosphere. The inert gas flow rate can be 50-200 sccm. Inert gases include, but are not limited to, argon and nitrogen.

[0040] This invention employs alkoxysilanes to pre-modify and form a transition layer via liquid-phase reaction, while simultaneously combining hyperbranched polycarbosilanes to generate a silicon carbide coating in situ through vapor deposition and pyrolysis. The core of the alkoxy liquid-phase pre-modification lies in using alkoxy groups to construct molecular bridges, significantly enhancing interfacial bonding and optimizing the uniformity and density of the hyperbranched polycarbosilane deposition. This addresses the shortcomings of simple hyperbranched polycarbosilane deposition, such as easy detachment, weak interfaces, and numerous film defects. The alkoxy layer, as a transition layer, matches the difference in thermal expansion coefficients between the metal and the hyperbranched polycarbosilane, reducing internal stress and minimizing cracking during pyrolysis after deposition. The bio-inert chromatographic column obtained by the preparation method described in this invention can be used to separate bioactive analytes (e.g., polysaccharides, peptides, oligonucleotides, etc.).

[0041] The following is an example illustrating the preparation method of a bioactive chromatographic column.

[0042] The required 316L chromatographic column was ultrasonically cleaned with an alkaline cleaning solution to remove oil, metal debris, or other impurities. Then, it was ultrasonically cleaned multiple times with deionized water to remove any remaining cleaning solution. Finally, the column was ultrasonically washed with a solvent (ethanol, acetone, etc.) to remove any remaining deionized water from the main column surface.

[0043] Seal the cleaned column assembly and connect it to the plunger pump of the delivery system. Mix alkoxysilane, alcohol molecules, and acidic solution according to stoichiometric ratios, heat to 50-150°C, place in a storage tank, and use a high-pressure plunger pump to deliver the silane system to the column. After circulating through the column, the silane system returns to the silane storage tank. The circulation time of the silane system is controlled within 3-24 hours.

[0044] Switch the delivery fluid of the plunger pump delivery system to ethanol at a flow rate of 20 mL / min and a washing time of 10 min.

[0045] Inertize the chemical vapor deposition (CVD) furnace. Remove the column from the plunger pump delivery system, allow it to air dry in a clean environment at room temperature, and place it in the CVD furnace. After sealing the furnace, evacuate it to a vacuum level of 5 Pa or below. Purge the furnace with high-purity inert gas (argon / nitrogen) until the gauge pressure inside the furnace is positive. Continue evacuating, repeating the above steps at least three times. Control the flow rate of the high-purity inert gas to 50-200 sccm. Proceed to 200-600℃ according to the preset temperature program.

[0046] The raw materials for hyperbranched polycarbosilanes include, but are not limited to, one or more of allyl hyperbranched polycarbosilanes, fully hydrogenated hyperbranched polycarbosilanes, vinyl-terminated hydrogenated hyperbranched polycarbosilanes, ethynyl-terminated hydrogenated hyperbranched polycarbosilanes, and alkyl hyperbranched polycarbosilanes. The vaporization method is bubbling. Vaporization chamber setup conditions: temperature 40-80℃, pressure 100-1000 Pa, high-purity inert gas carrier gas flow rate 50-200 sccm. Deposition conditions: deposition temperature 600-1000℃, system pressure 100-1000 Pa, deposition time 0.5-5 h. Atmosphere and ratio during deposition: inert atmosphere (Ar / N2) is the main carrier gas, and reducing / dilution (H2) is the auxiliary carrier gas, accounting for 5%-30%, with a total flow rate of 100-500 sccm.

[0047] Pyrolysis forms a silicon carbide film. The heating rate is 1-3℃ / min. The temperature is held at 1000-1300℃ for 1-4 h. The furnace is then cooled to room temperature. The entire pyrolysis process is carried out under inert gas (Ar / N2) protection. The inert gas flow rate is 50-200 sccm.

[0048] Column packing and characterization. The prepared chromatographic column was used as a container, packed under high pressure and homogenized, and the detection was performed according to the corresponding column detection conditions.

[0049] In summary, this invention relates to the formation of highly dense, bio-inert chromatographic columns through synergistic liquid-phase and gas-phase reactions, and its application in improving chromatographic methods. The liquid-phase reaction forms a transition layer, while gas-phase deposition forms a highly dense, bio-inert coating. By selecting different alkoxy groups (such as methyl, amino, and epoxy groups), the interfacial polarity, reactivity, and crosslinking density are controlled to match the side group structures of different hyperbranched polycarbosilanes. A gradient structure of metal-transition layer-hyperbranched polycarbosilane ceramic layer is constructed to further optimize stress distribution and interfacial properties; interfacial covalent bonding promotes crosslinking of hyperbranched polycarbosilanes, resulting in more complete ceramization during pyrolysis.

[0050] The present invention will be further described in detail below with reference to specific embodiments. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the range based on the description herein, and are not intended to limit themselves to the specific values ​​in the examples below. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Chemical vapor deposition (CVD) furnaces are used in all embodiments and comparative examples.

[0051] Example 1 The preparation method of a bioinert chromatographic column includes the following steps: Step (1) Cleaning the chromatographic column. Twenty sets of chromatographic columns (4.6 mm inner diameter, 250 mm length, each set including: 1 column tube, 316L material; 2 sieve plates, 316L material, 2 μm filtration accuracy; 2 column heads, 316L material, threads matching the column tube) were ultrasonically cleaned three times with alkaline cleaning solution, vertically suspended from the bottom, to remove oil, metal debris, or other impurities. Then, the columns were ultrasonically cleaned three times with deionized water to remove residual cleaning solution. Finally, the columns were ultrasonically washed three times with ethanol to remove residual deionized water from the main tube surface.

[0052] Step (2) Alkoxysilane pre-modification in liquid chromatography. Twenty sets of cleaned chromatographic columns were sealed and connected to the plunger pump of the delivery system. The weight ratio of γ-(2,3-epoxypropoxy)propyltrimethoxysilane / ethanol / hydrochloric acid (0.1 mol / L) in the silane tank of the delivery system was 20:78:2. The mixture was heated to 70°C, and the silane system was delivered to the chromatographic column using a high-pressure plunger pump at a flow rate of 20 mL / min. After the silane system passed through the column, it was returned to the silane tank, with the circulation time controlled at 4 hours.

[0053] Step (3) Wash the column. Switch the delivery fluid of the plunger pump system to ethanol at a flow rate of 20 mL / min and a washing time of 10 min.

[0054] Step (4) Removal of volatiles from the chromatographic column. Remove the column from the plunger pump delivery system, allow it to air dry at room temperature, place it in a vapor deposition furnace, seal it, and evacuate it to a vacuum level of 5 Pa. Then, purge with high-purity argon until the furnace pressure reaches 0.01 MPa. Continue evacuating, repeating the above steps three times. The flow rate of high-purity argon is 200 sccm. Increase the temperature to 300℃ at a rate of 1℃ / min according to the preset temperature program.

[0055] Step (5) Deposition of hyperbranched polycarbosilane. Methyl hyperbranched polycarbosilane (HMP) was used as the deposition material. Liquid hyperbranched polycarbosilane was vaporized using a bubbling method at a temperature of 80°C, a pressure of 100 Pa, and high-purity argon as the carrier gas at a flow rate of 100 sccm. The deposition conditions were a deposition temperature of 800°C, a deposition pressure of 100 Pa, and a deposition time of 1 h. During the deposition process, in addition to the aforementioned high-purity argon carrier gas, reducing / diluting hydrogen gas was also introduced through a branch pipe. The volume ratio of hydrogen to argon was 5%:95%. The total flow rate of hydrogen and argon was 110 sccm.

[0056] Step (6) Pyrolysis to form a silicon carbide film. The temperature was increased to 1100℃ at a rate of 1℃ / min, then held at 1100℃ for 3 h, and subsequently cooled to room temperature in the furnace. Pyrolysis was carried out under argon protection. The argon flow rate was 200 sccm.

[0057] Column packing and characterization. The prepared chromatographic column was used as a container and packed using a high-pressure homogenizer. After filling, the homogenizer container was sealed and tightened. Methanol was used as the displacement solvent for pressurization and pressure holding. The packing material in the homogenate was retained at the filter plate at the column tail, while the homogenate was expelled from the column. After the holding time was reached, the pressure was released, gradually reducing it to atmospheric pressure. The packed column was removed and a column head with a sieve plate (filter) was installed. Detection was performed according to the corresponding chromatographic column detection conditions.

[0058] Chromatographic conditions for the detection of bioactive nucleotide samples. Column: MicroPulite XP RP18 Plus 5μm 250*4.6mm; UV detector: 260nm; flow rate: 1.0 mL / min; column temperature: 30℃; mobile phase: 50mM TEAA (triethylamine-acetic acid) (pH 7.0)-acetonitrile.

[0059] The results showed that the column tailing decreased sequentially from the untreated group, the vapor deposition group, to the liquid-phase reaction transition layer + vapor deposition group. The presence of the inert column transition layer in this invention provides a synergistic shielding effect, resulting in less specific adsorption of biological samples. (See results below.) Figure 3A , Figure 3B and Figure 3C .

[0060] Comparative Example 1 It is basically the same as Example 1, except that steps (2) and (3) are omitted.

[0061] Figure 2 These are morphological images of Example 1 and Comparative Example 1. A (optical micrograph), B (optical micrograph), and C (electron scanning micrograph) are morphological images of Example 1; a (optical micrograph), b (optical micrograph), and c (electron scanning micrograph) are morphological images of Comparative Example 1. The results show that the bio-inert chromatographic column of Example 1 has a smooth surface, without cracks or warping; while the chromatographic column of Comparative Example 1 exhibits a sheet-like structure, with relatively poor shielding effect. This demonstrates that the transition layer can further improve the shielding effect.

[0062] Example 2

[0063] The preparation method of a bioinert chromatographic column includes the following steps: Step (1) Cleaning the chromatographic column. Twenty sets of chromatographic columns (4.6 mm inner diameter, 250 mm length, each set including: 1 column tube, material 316; 2 sieve plates, material 316L, filtration accuracy 2 μm; 2 column heads, material 316L, threads matching the column tube) were ultrasonically cleaned three times with alkaline cleaning solution, vertically suspended from the bottom, to remove oil, metal debris, or other impurities. Then, the columns were ultrasonically cleaned three times with deionized water to remove residual cleaning solution. Finally, the columns were ultrasonically washed three times with ethanol to remove residual deionized water from the main tube surface.

[0064] Step (2) Alkoxysilane pre-modification in liquid chromatography. Twenty sets of cleaned chromatographic columns were sealed and connected to the plunger pump of the delivery system. The weight ratio of 3-(methacrylamido)propyltrimethoxysilane / ethanol / hydrochloric acid (0.1 mol / L) in the silane tank of the delivery system was 20:78:2. The system was heated to 70°C, and the silane system was delivered to the chromatographic column using a high-pressure plunger pump at a flow rate of 20 mL / min. After the silane system passed through the column, it was returned to the silane tank, with the circulation time controlled at 4 hours.

[0065] Step (3) Wash the column. Switch the delivery fluid of the plunger pump system to ethanol at a flow rate of 20 mL / min and a washing time of 10 min.

[0066] Step (4) Removal of volatiles from the chromatographic column. Remove the column from the plunger pump delivery system, allow it to air dry at room temperature, place it in a vapor deposition furnace, seal it, and evacuate it to a vacuum level of 5 Pa. Then, purge with high-purity argon until the furnace pressure reaches 0.01 MPa. Continue evacuating, repeating the above steps three times. The flow rate of high-purity argon is 200 sccm. Increase the temperature to 300℃ at a rate of 1℃ / min according to the preset temperature program.

[0067] Step (5) Deposition of hyperbranched polycarbosilane. Vinyl-terminated hydrogenated hyperbranched polycarbosilane (Vi-HPCS) was used as the deposition material. The vinyl-terminated hydrogenated hyperbranched polycarbosilane was vaporized using a bubbling method at a temperature of 40°C, a pressure of 100 Pa, and high-purity argon as the carrier gas at a flow rate of 100 sccm. The deposition conditions were a deposition temperature of 800°C, a deposition pressure of 100 Pa, and a deposition time of 1 h. During the deposition process, in addition to the aforementioned high-purity argon carrier gas, reducing / diluting hydrogen gas was also introduced through a branch pipe. The volume ratio of hydrogen to argon was 5%:95%. The total flow rate of hydrogen and argon was 110 sccm.

[0068] Step (6) Pyrolysis to form a silicon carbide film. The temperature was increased to 1100℃ at a rate of 1℃ / min, then held at 1100℃ for 3 h, and subsequently cooled to room temperature in the furnace. Pyrolysis was carried out under argon protection. The argon flow rate was 200 sccm.

[0069] Column packing and characterization. The prepared column is used as a container, filled with high-pressure homogenate. After filling, the homogenate container is sealed and tightened. Methanol is used as the displacement solvent for pressurization and pressure maintenance. The packing material in the homogenate is retained at the filter plate at the column tail, while the homogenate is expelled from the column. After the pressure maintenance time is reached, the pressure is released gradually to atmospheric pressure. The packed column is removed and a column head with a sieve plate (filter) is installed. Detection is performed according to the corresponding chromatographic column detection conditions.

[0070] Chromatographic conditions for the detection of bioactive AMP / ADP / ATP samples. Column: MicroPulite XP RP18Plus 5μm 250*4.6mm; UV detector: 260 nm; flow rate: 1.0 mL / min; column temperature: 30℃; mobile phase: 50mM TTEAA (triethylamine-acetic acid) (pH 7.0) - acetonitrile.

[0071] The results showed that the column tailing decreased sequentially from the untreated group, the vapor deposition group, to the liquid-phase reaction transition layer + vapor deposition group. The presence of the inert column transition layer in this invention provides a synergistic shielding effect, resulting in less specific adsorption of biological samples. (See results below.) Figure 4A , Figure 4B and Figure 4C .

[0072] Example 3

[0073] The preparation method of a bioinert chromatographic column includes the following steps: Step (1) Cleaning the chromatographic column. Twenty sets of chromatographic columns (2.1 mm inner diameter, 250 mm length, each set including: 1 column tube, 316L material; 2 sieve plates, 316L material, 0.2 μm filtration accuracy; 2 column heads, 316L material, threads matching the column tube) were ultrasonically cleaned three times with alkaline cleaning solution, vertically suspended from the bottom, to remove oil, metal debris, or other impurities. Then, the columns were ultrasonically cleaned three times with deionized water to remove residual cleaning solution. Finally, the columns were ultrasonically washed three times with ethanol to remove residual deionized water from the main tube surface.

[0074] Step (2) Alkoxysilane pre-modification in liquid chromatography. Twenty sets of cleaned chromatographic columns were sealed and connected to the plunger pump of the delivery system. The γ-(2,3-epoxypropoxy)propyltrimethoxysilane / ethanol / hydrochloric acid (0.1 mol / L) ratio in the silane tank of the delivery system was 20:78:2. The mixture was heated to 70°C, and the silane system was delivered to the chromatographic column using a high-pressure plunger pump at a flow rate of 20 mL / min. After the silane system passed through the column, it was returned to the silane tank, with the circulation time controlled at 4 hours.

[0075] Step (3) Wash the column. Switch the delivery fluid of the plunger pump system to ethanol at a flow rate of 5 mL / min and a washing time of 10 min.

[0076] Step (4) Removal of volatiles from the chromatographic column. Remove the column from the plunger pump delivery system, allow it to air dry at room temperature, place it in a vapor deposition furnace, seal it, and evacuate it to a vacuum level of 5 Pa. Then, purge with high-purity argon until the furnace pressure reaches 0.01 MPa. Continue evacuating, repeating the above steps three times. The flow rate of high-purity argon is 200 sccm. Increase the temperature to 300℃ at a rate of 1℃ / min according to the preset temperature program.

[0077] Step (5) Deposition of hyperbranched polycarbosilane. Methyl hyperbranched polycarbosilane (HMP) was used as the deposition material. Liquid hyperbranched polycarbosilane was vaporized using a bubbling method at a temperature of 80°C, a pressure of 100 Pa, and high-purity argon as the carrier gas at a flow rate of 100 sccm. The deposition conditions were a deposition temperature of 800°C, a deposition pressure of 100 Pa, and a deposition time of 1 h. During the deposition process, in addition to the aforementioned high-purity argon carrier gas, reducing / diluting hydrogen gas was also introduced through a branch pipe. The volume ratio of hydrogen to argon was 5%:95%. The total flow rate of hydrogen and argon was 110 sccm.

[0078] Step (6) Pyrolysis to form a silicon carbide film. The temperature was increased to 1100℃ at a heating rate of 1℃ / min, and then held at 1100℃ for 3 h, followed by furnace cooling to room temperature. Pyrolysis was carried out under inert gas protection. The inert gas flow rate was 200 sccm.

[0079] Column packing and characterization. The prepared column was used as a container, filled with high-pressure homogenate. The prepared homogenate was immediately poured into the homogenate container, filled, and then the container was sealed and tightened. Methanol was used as the displacement solvent for pressurization and pressure holding. The packing material in the homogenate was retained on the filter plate at the column tail, while the homogenate was expelled from the column. After the holding time was reached, the pressure was released, gradually reducing it to atmospheric pressure. The packed column was then removed and fitted with a column head equipped with a sieve plate (filter). Detection was performed according to the corresponding chromatographic column detection conditions. Detection of bioactive nucleotide samples showed that the inert column packing resulted in less tailing, higher column efficiency, and no dead adsorption.

[0080] Example 4

[0081] The preparation method of a bioinert chromatographic column includes the following steps: Step (1) Cleaning the chromatographic column. Take 20 sets of chromatographic columns (4.6 mm inner diameter, 250 mm length, each set including: 1 column tube, 316L material; 2 sieve plates, 316L material, 2 μm filtration accuracy; 2 column heads, 316L material, threads matching the column tube), and use a hanger to vertically and clean the column tubes three times with alkaline cleaning solution to remove oil, metal debris, or other impurities. Then, use deionized water to ultrasonically clean the column three times to remove residual cleaning solution. Finally, use ethanol as a solvent to ultrasonically wash the column three times to remove residual deionized water from the main tube surface.

[0082] Step (2) Alkoxysilane pre-modification in liquid chromatography. Twenty sets of cleaned chromatographic columns were sealed and connected to the plunger pump of the delivery system. The weight ratio of 3-(methacryloyloxy)propyltrimethoxysilane / ethanol / hydrochloric acid (0.1 mol / L) in the silane tank of the delivery system was 20:78:2. The mixture was heated to 70°C, and the silane system was delivered to the chromatographic column using a high-pressure plunger pump at a flow rate of 20 mL / min. After the silane system passed through the column, it was returned to the silane tank, with the circulation time controlled at 4 hours.

[0083] Step (3) Wash the column. Switch the delivery fluid of the plunger pump system to ethanol at a flow rate of 20 mL / min and a washing time of 10 min.

[0084] Step (4) Removal of volatiles from the chromatographic column. Remove the column from the plunger pump delivery system, allow it to air dry at room temperature, place it in a vapor deposition furnace, seal it, and evacuate it to a vacuum level of 5 Pa. Then, purge with high-purity argon until the furnace pressure reaches 0.01 MPa. Continue evacuating, repeating the above steps three times. The flow rate of high-purity argon is 200 sccm. Increase the temperature to 300℃ at a rate of 1℃ / min according to the preset temperature program.

[0085] Step (5) Deposition of hyperbranched polycarbosilane. Allyl hyperbranched polycarbosilane (AHPCS) was used as the deposition material. The acetylene-terminated hydrogenated hyperbranched polycarbosilane was vaporized using a bubbling method at a temperature of 80°C, a pressure of 100 Pa, and high-purity argon as the carrier gas at a flow rate of 100 sccm. The deposition conditions were a deposition temperature of 800°C, a deposition pressure of 100 Pa, and a deposition time of 1 h. During the deposition process, in addition to the aforementioned high-purity argon carrier gas, reducing / diluting hydrogen gas was also introduced through a branch pipe. The volume ratio of hydrogen to argon was 5%:95%. The total flow rate of hydrogen and argon was 110 sccm.

[0086] Step (6) Pyrolysis to form a silicon carbide film. The temperature was increased to 1100℃ at a heating rate of 1℃ / min, and then held at 1100℃ for 3 h, followed by furnace cooling to room temperature. Pyrolysis was carried out under inert gas protection. The inert gas flow rate was 200 sccm.

[0087] Column packing and characterization. The prepared column was used as a container, filled with high-pressure homogenate, and the container was sealed tightly. Methanol was used as the displacement solvent for pressurization and pressure maintenance. The packing material in the homogenate was retained at the filter plate at the column tail, while the homogenate was expelled from the column. After the holding time was reached, the pressure was released, gradually reducing it to atmospheric pressure. The packed column was removed and a column head with a sieve plate (filter) was installed. Detection was performed according to the corresponding column detection conditions. Detection of bioactive nucleotide samples showed that the inert column packing resulted in less tailing, higher column efficiency, and no dead adsorption.

[0088] Example 5

[0089] The preparation method of a bioinert chromatographic column includes the following steps: Step (1) Cleaning the chromatographic column. Twenty sets of chromatographic columns (4.6 mm inner diameter, 250 mm length, each set including: 1 column tube, material 316; 2 sieve plates, material 316L, filtration accuracy 2 μm; 2 column heads, material 316L, threads matching the column tube) were ultrasonically cleaned three times with alkaline cleaning solution, vertically suspended from the bottom, to remove oil, metal debris, or other impurities. Then, the columns were ultrasonically cleaned three times with deionized water to remove residual cleaning solution. Finally, the columns were ultrasonically washed three times with ethanol to remove residual deionized water from the main tube surface.

[0090] Step (2) Alkoxysilane pre-modification in liquid chromatography. Twenty sets of cleaned chromatographic columns were sealed and connected to the plunger pump of the delivery system. The weight ratio of 3-aminopropyltrimethoxysilane / ethanol / hydrochloric acid (0.1 mol / L) in the silane tank of the delivery system was 20:78:2. The mixture was heated to 70°C, and the silane system was delivered to the chromatographic column using a high-pressure plunger pump at a flow rate of 20 mL / min. After the silane system passed through the column, it was returned to the silane tank, with the circulation time controlled at 4 hours.

[0091] Step (3) Wash the column. Switch the delivery fluid of the plunger pump system to ethanol at a flow rate of 20 mL / min and a washing time of 10 min.

[0092] Step (4) Removal of volatiles from the chromatographic column. Remove the column from the plunger pump delivery system, allow it to air dry at room temperature, place it in a vapor deposition furnace, seal it, and evacuate it to a vacuum level of 5 Pa. Then, purge with high-purity argon until the furnace pressure reaches 0.01 MPa. Continue evacuating, repeating the above steps three times. The flow rate of high-purity argon is 200 sccm. Increase the temperature to 300℃ at a rate of 1℃ / min according to the preset temperature program.

[0093] Step (5) Deposition of hyperbranched polycarbosilane. Vinyl-terminated hydrogenated hyperbranched polycarbosilane (Vi-HPCS) was used as the deposition material. The vinyl-terminated hydrogenated hyperbranched polycarbosilane was vaporized using a bubbling method at a temperature of 40°C, a pressure of 100 Pa, and high-purity argon as the carrier gas at a flow rate of 100 sccm. The deposition conditions were a deposition temperature of 800°C, a deposition pressure of 100 Pa, and a deposition time of 1 h. During the deposition process, in addition to the aforementioned high-purity argon carrier gas, reducing / diluting hydrogen gas was also introduced through a branch pipe. The volume ratio of hydrogen to argon was 5%:95%. The total flow rate of hydrogen and argon was 110 sccm.

[0094] Step (6) Pyrolysis to form a silicon carbide film. The temperature was increased to 1100℃ at a rate of 1℃ / min, then held at 1100℃ for 3 h, and subsequently cooled to room temperature in the furnace. Pyrolysis was carried out under argon protection. The argon flow rate was 200 sccm.

[0095] Column packing and characterization. The prepared column was used as a container, filled with high-pressure homogenate, and the container was sealed tightly after filling. Methanol was used as the displacement solvent for pressurization and pressure holding. The packing material in the homogenate was retained at the filter plate at the column tail, while the homogenate was expelled from the column. After the holding time was reached, the pressure was released and gradually reduced to atmospheric pressure. The packed column was removed and a column head with a sieve plate (filter) was installed. Detection was performed according to the corresponding chromatographic column detection conditions. The shielding layer on the column surface was cracked, resulting in poor shielding effectiveness and some specific adsorption of biological samples still occurred. Results are shown in […]. Figure 6 .

Claims

1. A method for preparing a biological inert chromatographic column, characterized in that, Includes the following steps: Step (1) is the process of cleaning the chromatographic column; Step (2) is a process of liquid-phase surface silanization of the cleaned chromatographic column; wherein, alkoxysilane is used to pre-modify the chromatographic column by liquid-phase reaction to form a transition layer; Step (3) is the process of washing the chromatographic column again after silanization of the liquid phase surface; Step (4) involves removing volatile substances from the chromatographic column after it has been washed again; Step (5) is the process of depositing a hyperbranched polycarbosilane coating on the surface of the chromatographic column after the removal of volatile substances; Step (6) involves pyrolyzing the column after depositing a hyperbranched polysilane coating to form a silicon carbide film in situ.

2. The preparation method according to claim 1, characterized in that, In step (2), the chemical composition of the alkoxysilane is (R1). n1 (R2) n2 Si(OR3) 4-n1-n2 R1 is selected from methyl, butyl, isobutyl, aminopropyl, glycidoxypropyl, methacryloyloxypropyl, and propylcarbamate; R2 is selected from methyl, butyl, isobutyl, aminopropyl, glycidoxypropyl, methacryloyloxypropyl, and propylcarbamate; R1 and R2 may be the same or different; R3 is selected from methyl or ethyl; preferably, n1 and n2 are independently selected from natural numbers from 0 to 2; more preferably, 0 < n1 + n2 ≤ 3.

3. The preparation method according to claim 1 or 2, characterized in that, In step (2), the cleaned chromatographic column is sealed and connected to the silane storage container of the delivery system. The silane storage container contains a silane system in which alkoxysilane, alcohol solvent and acid are mixed according to a stoichiometric relationship. The silane system is delivered to the chromatographic column. After the silane system flows through the chromatographic column, it is returned to the silane tank.

4. The preparation method according to claim 3, characterized in that, The alcohol solvent is ethanol; the acid solution is 0.01-0.5 mol / L hydrochloric acid; preferably, the weight ratio of alkoxysilane: alcohol solvent: acid solution is 5-50:45-85:0.5-5.

5. The preparation method according to any one of claims 1 to 4, characterized in that, In step (3), the delivery fluid of the delivery system is switched to small molecule alcohols to flush the chromatographic column.

6. The preparation method according to any one of claims 1 to 5, characterized in that, In step (4), the chromatographic column is removed from the delivery system, left to air dry at room temperature, placed in the vapor deposition apparatus, the vapor deposition apparatus is sealed and replaced with an inert atmosphere, and the temperature is raised to 200-600℃ according to the preset heating program.

7. The preparation method according to any one of claims 1 to 6, characterized in that, In step (5), the hyperbranched polycarbosilane is selected from one or more of allyl hyperbranched polycarbosilane, fully hydrogenated hyperbranched polycarbosilane, vinyl-terminated hydrogenated hyperbranched polycarbosilane, and alkyl hyperbranched polycarbosilane, or a mixture thereof.

8. The preparation method according to any one of claims 1 to 7, characterized in that, In step (5), the deposition temperature is 600-1000℃, the deposition pressure is 100-1000 Pa, and the deposition time is 0.5-5h.

9. The preparation method according to any one of claims 1 to 8, characterized in that, In step (6), the pyrolysis temperature is 1000-1300℃ and the pyrolysis time is 1-4 h; preferably, the pyrolysis is carried out under an inert atmosphere; more preferably, the inert atmosphere flow rate is 50-200 sccm.

10. The application of the bio-inert chromatographic column obtained by any one of claims 1 to 9 in the separation of bioactive analytes.

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