Amphiphilic carboxylated covalent organic framework silica composite stationary phases based on chiral induction strategy and their application in chromatography.

Abstract

This invention belongs to the field of chromatographic separation materials technology, specifically relating to an amphiphilic carboxylated covalent organic framework composite stationary phase based on a chiral induction strategy and its application in chromatography. The stationary phase uses amino silica gel as a carrier and (R)-1-(1-naphthyl)ethylamine as a chiral inducer. The carboxylated functionalized covalent organic framework generated by the reaction of 2,4,6-tricarboxymethyl phloroglucinol with 4,4-diaminobiphenyl-2,2-dicarboxylic acid is in situ immobilized on the amino silica gel surface. The resulting stationary phase possesses both hydrophilic carboxyl groups and a hydrophobic aromatic skeleton, enabling the separation of hydrophobic and polar compounds in both reversed-phase and hydrophilic chromatographic modes, exhibiting excellent shape selectivity for isomers. This stationary phase features high column efficiency (up to 47,744 columns / meter), low column pressure, and good stability, making it suitable for the efficient separation of polar to nonpolar compounds in complex samples.

Description

Technical Field

[0001] This invention belongs to the field of chromatographic separation materials technology, specifically relating to an amphiphilic carboxylated covalent organic framework composite stationary phase based on a chiral induction strategy and its application in chromatography. Background Technology

[0002] Covalent organic frameworks (COFs) are a class of crystalline porous materials formed by organic structural units linked by strong covalent bonds. They possess advantages such as high specific surface area, regular channels, strong structural designability, and good stability, showing great potential in separation science. Using COFs as stationary phases in high-performance liquid chromatography (HPLC) has become a research frontier. However, existing COF stationary phases mostly achieve a single separation mode (such as reversed-phase or hydrophilic mode) by introducing specific functional groups through post-modification methods, making it difficult to simultaneously and efficiently separate hydrophilic and hydrophobic compounds, especially lacking the ability to recognize the shape of structural analogs (such as positional isomers).

[0003] Shape selectivity refers to the ability of a stationary phase to distinguish analyte molecules based on differences in their three-dimensional configurations, such as planarity and rigidity. This is crucial for the efficient separation of structure analogs. Efficient separation of structure analogs typically requires a stationary phase with highly ordered recognition sites and unique chemical environments. Chiral-induced synthesis strategies offer a new approach to constructing co-formed molecules (COFs) with specific topologies and surface microenvironments. By introducing chiral inducers, this strategy asymmetrically influences COF framework assembly, potentially producing localized structural distortions or unique pore properties that are difficult to achieve using traditional methods, thus enabling the realization of advanced shape selectivity. Summary of the Invention

[0004] To overcome the limitations of existing COF stationary phases, which suffer from limited separation modes and difficulty in effectively distinguishing structural analogs, this invention provides an amphiphilic carboxyl-functionalized covalent organic framework silica composite stationary phase synthesized using a chiral induction strategy. This stationary phase possesses both hydrophilic and hydrophobic properties and exhibits significant shape selectivity. It is structurally stable, has high column efficiency, and low column pressure, enabling the efficient separation of a wide range of compounds, from polar to nonpolar.

[0005] To achieve the above technical objectives, the present invention is implemented through the following technical solution:

[0006] This invention provides an amphiphilic carboxyl-functionalized covalent organic framework silica composite stationary phase based on a chiral induction strategy. The stationary phase uses amino silica gel as a carrier and (R)-1-(1-naphthyl)ethylamine as a chiral inducer to in situ immobilize the carboxyl-functionalized covalent organic framework on the surface of the amino silica gel, resulting in carboxyl-functionalized covalent organic framework-based silica microspheres, which are the amphiphilic carboxyl-functionalized covalent organic framework silica composite stationary phase and can be used as a high-performance liquid chromatography (HPLC) stationary phase. The carboxyl-functionalized covalent organic framework is generated from 2,4,6-triformylphloroglucinol (TFP) and 4,4-diaminobiphenyl-2,2-dicarboxylic acid (DPA). Among them, 2,4,6-tricarboxymethyl phloroglucinol with aldehyde group will be chemically bonded to the amino group on the surface of amino silicone, so as to realize the in-situ immobilization of carboxyl functionalized covalent organic framework on the surface of amino silicone.

[0007] The inherent carboxyl groups in the covalent organic framework structure of this invention improve its strong hydrophobicity, thereby imparting hydrophilic properties to enable the separation of both hydrophobic and polar compounds. Furthermore, the unique three-dimensional structure induced by chirality allows the covalent organic framework to engage in differentiated interactions with molecules of different shapes, thus exhibiting excellent shape selectivity for isomers.

[0008] In addition, this invention provides a method for preparing a mixed-mode chromatographic stationary phase of amphiphilic carboxyl-functionalized covalent organic framework, specifically including the following steps:

[0009] Step 1: Synthesis of SiO2-TFP microspheres: Using 1,4-dioxane as solvent and amino silica gel, 2,4,6-tricarboxymethyl phloroglucinol, and 1,3,5-trimethylbenzene as raw materials, SiO2-TFP microspheres were obtained by reaction under acetic acid catalysis. This step involved reacting at 80ºC for at least 24 hours.

[0010] The ratio of aminosilicone, 2,4,6-tricarboxymethyl phloroglucinol, 1,4-dioxane, 1,3,5-trimethylbenzene and acetic acid is 3.0 g : 1.0507 g : 50 mL : 50 mL : 6.87 mL.

[0011] Step 2: Synthesis of SiO2@COF-COOH microspheres: Using n-butanol and o-dichlorobenzene as solvents, and 2,4,6-tricarboxylic acid, 4,4-diaminobiphenyl-2,2-dicarboxylic acid, SiO2-TFP microspheres, and 2,4,6-trimethylaniline as raw materials, and R-1-(1-naphthyl)ethylamine as a chiral inducer, the reaction was carried out under acetic acid catalysis to obtain SiO2@COF-COOH microspheres, which serve as the amphiphilic carboxyl-functionalized covalent organic framework mixed-mode chromatographic stationary phase. 2,4,6-trimethylaniline acts as a hydrophobic substance to hinder the interlayer π-π stacking of the carboxyl-functionalized covalent organic framework.

[0012] The specific steps are as follows: 2,4,6-tricarboxymethyl phloroglucinol, 4,4-diaminobiphenyl-2,2-dicarboxylic acid, SiO2-TFP, n-butanol and o-dichlorobenzene are mixed, and then 2,4,6-trimethylaniline and R-1-(1-naphthyl)ethylamine are added; when heated to 40 ºC, acetic acid is added, and the mixture is reacted at 80 ºC for more than 48 h to obtain the final product.

[0013] The ratio of 2,4,6-tricarboxymethyl phloroglucinol, 4,4-diaminobiphenyl-2,2-dicarboxylic acid, SiO2-TFP, n-butanol, o-dichlorobenzene, 2,4,6-trimethylaniline, R-1-(1-naphthyl)ethylamine, and acetic acid was: 945.63 mg : 1.837 g : 3.0 g : 50 mL : 50 mL : 675 μL : 675 μL : 13.5 mL.

[0014] This invention also provides a high-performance liquid chromatography (HPLC) column comprising a mixed-mode chromatographic stationary phase (i.e., SiO2@COF-COOH microspheres) based on a chiral induction strategy and a method for preparing the HPLC column, comprising the following steps:

[0015] SiO2@COF-COOH microspheres were added to an organic solvent and ultrasonically dispersed to form a suspension. Then, the suspension was packed into a stainless steel column at a pressure of 40–80 MPa for 5–30 min using the organic solvent as a displacement solvent. The pressure was then reduced to 10–30 MPa, and the column was packed for another 3–20 min to obtain a SiO2@COF-COOH chromatographic column.

[0016] In preparing the suspension, the ratio of SiO2@COF-COOH microspheres to organic solvent is 2.5 g : 40 mL, and the organic solvent is methanol or acetonitrile.

[0017] This invention also provides the application of the aforementioned amphiphilic carboxylated covalent organic framework composite stationary phase in high performance liquid chromatography, specifically as follows:

[0018] The amphiphilic carboxylated covalent organic framework composite stationary phase material SiO2@COF-COOH described in this invention has a unique three-dimensional structure endowed by chirality, which can generate differentiated interactions with molecules of different shapes. It also has hydrophilic carboxyl groups and hydrophobic aromatic skeletons. Therefore, it can exhibit excellent separation selectivity for hydrophobic and polar compounds by high performance liquid chromatography, and also exhibits excellent shape selectivity for isomers.

[0019] The amphiphilic carboxylated covalent organic framework composite stationary phase SiO2@COF-COOH described in this invention can generate multiple interactions with the analytes, including hydrophobic, π-π, and hydrogen bonding, enabling the separation of hydrophobic compounds in reversed-phase chromatography. In a further embodiment, the hydrophobic compounds include monosubstituted benzenes and polycyclic aromatic hydrocarbons (PAHs). Monosubstituted benzenes include, but are not limited to, toluene, ethylbenzene, n-propylbenzene, n-butylbenzene, and n-pentylbenzene; PAHs include, but are not limited to, benzene, diphenylmethane, o-terphenyl, m-terphenyl, p-terphenyl, cis-stilbene, and fluorene. During separation in reversed-phase chromatography, the amphiphilic carboxylated covalent organic framework composite stationary phase SiO2@COF-COOH of this invention also exhibits excellent shape selectivity for isomers.

[0020] The amphiphilic carboxylated covalent organic framework composite stationary phase material SiO2@COF-COOH described in this invention possesses hydrophilic properties, enabling the separation of polar compounds in a hydrophilic mode. In a further embodiment, the polar compounds include nucleosides, bases, sulfonamide antibiotics, and analgesic and anti-inflammatory drugs, wherein nucleosides include, but are not limited to, 2'-deoxyuridine and thymidine, and bases include, but are not limited to, thymine, cytosine, and adenine; sulfonamide antibiotics include, but are not limited to, sulfonamides, sulfabenzoyl, sulfaguanidine, and sulfamethoxypyridazine; and analgesic and anti-inflammatory drugs include, but are not limited to, ketoibuprofen, acetaminophen, and carboprofen.

[0021] In summary, the stationary phase of this invention can separate polar compounds, such as nucleosides, bases, sulfonamides, and analgesics and anti-inflammatory drugs, in hydrophilic mode, and hydrophobic compounds such as monosubstituted benzenes and polycyclic aromatic hydrocarbons in reversed-phase mode. It also exhibits excellent shape selectivity on a range of structural analogs (terphenyl isomers and polycyclic aromatic hydrocarbons with significant differences in planarity). This is due to the unique three-dimensional structure generated by chirality, which allows it to interact differentially with molecules of different shapes.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The SiO2@COF-COOH material synthesized by the chiral induction strategy of the present invention can not only introduce multiple action sites by utilizing the hydrophilic properties of the carboxyl group, thus achieving efficient separation of various compounds from polar to nonpolar; in addition, due to the unique three-dimensional structure introduced by the chiral inducing agent, it also has excellent shape selectivity for the target analyte when separating isomers and other structural analogs.

[0024] (2) The stationary phase provided by the present invention can generate multiple interactions with the analyte, such as hydrophobic, π-π, hydrophilic, and hydrogen bond, and is expected to be widely used in the field of separation and analysis of complex systems.

[0025] (3) When the SiO2@COF-COOH microspheres provided by this invention are applied to high performance liquid chromatography columns, the column efficiency is as high as 47,744 pieces / meter; at the same time, they have excellent solvent resistance, good stability and repeatability when applied to mobile phases with high content of organic solvents, and the relative standard deviation of retention time is less than 0.93% after 10 consecutive injections.

[0026] (4) The SiO2@COF-COOH stationary phase provided by the present invention can achieve complete separation of nonpolar and weakly polar compounds (hydrophobic compounds) in reversed-phase chromatography separation mode, and can also achieve effective separation of polar compounds in hydrophilic interaction mode; the selectivity factor of SiO2@COF-COOH for o-terphenyl and triphenylene is as high as 21.04, which shows ultra-high shape selectivity compared with commercial C18 stationary phase (selectivity factor is 1.44). Attached Figure Description

[0027] Figure 1 This is a flowchart of the preparation process for a SiO2@COF-COOH chromatographic column.

[0028] Figure 2 Scanning electron microscope images of amino silica gel (a) and SiO2@COF-COOH (b) microspheres.

[0029] Figure 3 The isotherms (a) and pore size distribution diagram (b) of nitrogen adsorption-desorption on SiO2@COF-COOH microspheres are shown.

[0030] Figure 4 The chromatograms show the separation of ortho-terphenyl, meta-terphenyl, para-terphenyl, and triphenylene.

[0031] Figure 5 The SiO2@COF-COOH column was used in reverse phase mode for the chromatography of polycyclic aromatic hydrocarbons (benzene, diphenylmethane, cis-stilbene, o-terphenyl, fluorene, m-terphenyl and p-terphenyl).

[0032] Figure 6The graph shows the relationship between the capacity factor k of monosubstituted benzene on a SiO2@COF-COOH column and the acetonitrile content in the mobile phase (a), the chromatographic separation of monosubstituted benzene in reversed-phase mode (b), the relationship between the column pressure of the SiO2@COF-COOH column and the flow rate of the mobile phase (c), and the chromatogram of 10 consecutive injections of monosubstituted benzene on the SiO2@COF-COOH column (d).

[0033] Figure 7 The chromatographic separation of nucleosides / bases by the SiO2@COF-COOH column in hydrophilic mode is shown in (a), and the relationship between the nucleoside / base capacity factor k and the water content in the mobile phase is shown in (b).

[0034] Figure 8 This is a chromatographic diagram of the separation of sulfonamide antibiotics by a SiO2@COF-COOH column in hydrophilic mode.

[0035] Figure 9 This is a chromatographic diagram of analgesic and anti-inflammatory drugs separated by a SiO2@COF-COOH column in hydrophilic mode.

[0036] Figure 10 The chromatograms show the separation of fluorene, m-terphenyl, and p-terphenyl standard solutions (500 μg / mL), wastewater effluent from a wastewater treatment plant, and spiked wastewater effluent from a wastewater treatment plant (500 μg / mL) on a SiO2@COF-COOH column. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0038] Example 1

[0039] 1. Preparation of SiO2@COF-COOH chromatographic column

[0040] like Figure 1 As shown, the preparation of the SiO2@COF-COOH chromatographic column includes the following steps:

[0041] Step 1: 3 g of aminosilicone (5.2 μm, purchased from Suzhou Nanomicro Technology Co., Ltd.), 1.0507 g of 2,4,6-tricarboxymethyl phloroglucinol, 50 mL of 1,4-dioxane, and 50 mL of 1,3,5-trimethylbenzene were mixed and sonicated for 5 min to obtain a uniformly dispersed solution. Then, 6.87 mL of acetic acid was slowly added dropwise to the above mixed solution, and the reaction solution was stirred at 80 ºC for 24 h. After the reaction was completed, the solution was cooled to room temperature and washed three times with anhydrous ethanol. Finally, the solution was vacuum dried for 24 h to obtain SiO2-TFP microspheres.

[0042] Step 2: 945.63 mg of 2,4,6-tricarboxymethyl phloroglucinol, 1.837 g of 4,4-diaminobiphenyl-2,2-dicarboxylic acid, 3.0 g of SiO2-TFP, 50 mL of n-butanol, and 50 mL of o-dichlorobenzene were mixed and ultrasonically dispersed. Then, at room temperature, 675 μL of 2,4,6-trimethylaniline and 675 μL of R-1-(1-naphthyl)ethylamine were added sequentially to the above mixture. When the reaction temperature reached 40 ºC, 13.5 mL of acetic acid was added to the mixture. The solution was then stirred at 80 ºC for 48 h. After the reaction was complete, the mixture was cooled to room temperature and washed three times each with N,N dimethylformamide, acetone, and anhydrous ethanol. Finally, the mixture was vacuum dried for 24 h to obtain SiO2@COF-COOH microspheres.

[0043] Step 3: Weigh 2.5 g of SiO2@COF-COOH microspheres and add them to 40 mL of methanol. Disperse the suspension using ultrasonication. Quickly pour the suspension into a homogenizing vessel. Using methanol as the displacement solvent, pack the suspension onto a column at 60 MPa for 20 min. Then reduce the pressure to 20 MPa and continue packing for 5 min to obtain a SiO2@COF-COOH chromatographic column (150 mm × 4.6 mm). In Step 3, replacing methanol with acetonitrile will also successfully achieve column packing and use.

[0044] 2. Morphological characterization of SiO2@COF-COOH

[0045] The microstructure of the prepared SiO2@COF-COOH microspheres was observed using scanning electron microscopy. Figure 2 The average particle size of the amino silica gel is approximately 5.2 μm. Figure 2 b shows that the average particle size of the SiO2@COF-COOH microspheres is about 5.6 μm, and a large number of protruding COF-COOH (carboxyl functionalized covalent organic framework) particles can be observed on its rough surface.

[0046] 3. Pore size characterization of SiO2@COF-COOH

[0047] The pore size distribution of the prepared SiO2@COF-COOH microspheres was characterized by nitrogen adsorption-desorption. Figure 3 As can be seen from this, the nitrogen adsorption capacity of SiO2@COF-COOH increases sharply in the low-pressure region, and a significant hysteresis loop exists in the medium-pressure region. Figure 3 As can be seen from the pore size distribution diagram in b, new mesopores appeared in SiO2@COF-COOH at 2.04 nm and 2.40 nm.

[0048] 4. Evaluation of the shape selectivity of SiO2@COF-COOH chromatographic column

[0049] The prepared SiO2@COF-COOH column was used as a high-performance liquid chromatography column, and its shape-selective separation capability for structural analogs was evaluated in reversed-phase chromatography mode.

[0050] o-terphenyl, m-terphenyl, p-terphenyl, and triphenylene were selected as model analytes. The chromatographic separation conditions were: acetonitrile / dichloromethane = 90 / 10, flow rate: 1.2 mL / min, and detection wavelength: 254 nm. Figure 4 As shown, chromatographic peaks 1-4 represent o-terphenyl, m-terphenyl, p-terphenyl, and triphenylene, respectively. These four analytes were well separated on the prepared SiO2@COF-COOH column. Triphenylene, being a rigid planar molecule, exhibits strong π-π packing or spatial matching with the ordered channels or planar regions of COF-COOH, resulting in strong retention. In contrast, the o-terphenyl molecule can be twisted and deformed, leading to a weaker interaction with COF-COOH. Therefore, the selectivity factor between the two can be used to evaluate the shape recognition ability of the stationary phase.

[0051] The selectivity factor for o-terphenyl and triphenylene on the SiO2@COF-COOH column was 21.04, significantly higher than that of the commercially available Eclipse XDB-C18 column (Agilent, USA (selectivity factor 1.44)), demonstrating extremely high planar selectivity. Furthermore, the SiO2@COF-COOH column exhibited a selectivity factor of 1.45 for nonlinear m-terphenyl and linear p-terphenyl, higher than that of the Eclipse XDB-C18 column (Agilent, USA (selectivity factor 1.03)), further indicating that the SiO2@COF-COOH column possesses high linear selectivity.

[0052] 5. Separation of polycyclic aromatic hydrocarbons using SiO2@COF-COOH chromatographic column

[0053] The prepared SiO2@COF-COOH column was used as a high-performance liquid chromatography column, and its separation performance for polycyclic aromatic hydrocarbons benzene, diphenylmethane, cis-stilbene, o-terphenyl, fluorene, m-terphenyl and p-terphenyl was tested in reverse phase mode.

[0054] The chromatographic separation conditions for benzene, diphenylmethane, cis-stilbene, o-terphenyl, fluorene, m-terphenyl, and p-terphenyl were: acetonitrile / water = 70 / 30, flow rate: 1.0 mL / min, and detection wavelength: 254 nm. Figure 5 As shown, chromatographic peaks 1-7 represent benzene, diphenylmethane, cis-stilbene, o-terphenyl, fluorene, m-terphenyl, and p-terphenyl, respectively. Diphenylmethane and m-terphenyl were co-eluted on an Eclipse XDB-C18 column (Agilent, USA), but were efficiently separated on a SiO2@COF-COOH column. This is attributed to the hydrophobic and π-π interactions between the SiO2@COF-COOH stationary phase and the polycyclic aromatic hydrocarbons.

[0055] 6. Separation of monosubstituted benzenes using SiO2@COF-COOH chromatographic column

[0056] The prepared SiO2@COF-COOH column was used as a high-performance liquid chromatography (HPLC) column to test its separation performance, mechanical stability, and repeatability for five monosubstituted benzenes: toluene, ethylbenzene, n-propylbenzene, n-butylbenzene, and n-pentylbenzene. The chromatographic separation conditions were: mobile phase: acetonitrile / water; flow rate: 1.0 mL / min; detection wavelength: 254 nm.

[0057] like Figure 6 As shown in Figure a, within the acetonitrile volume fraction range of 55%-90% in the mobile phase, the capacity factor k of the five monosubstituted benzenes decreases with increasing acetonitrile content in the mobile phase, indicating that the stationary phase conforms to the reversed-phase chromatography mode; Figure 6 As shown in b, when the acetonitrile volume fraction in the mobile phase was 60%, the chromatographic peaks 1–5 were toluene, ethylbenzene, n-propylbenzene, n-butylbenzene, and n-pentylbenzene, respectively. These five monosubstituted benzenes were well separated on the SiO2@COF-COOH column, achieving high column efficiency (41819–47744 blocks / meter) and good peak shape. Furthermore, the relationship between column pressure and mobile phase flow rate was investigated within the acetonitrile volume fraction range of 55%–90% in the SiO2@COF-COOH column, as shown in Figure b. Figure 6 c shows a good linear relationship between column pressure and flow rate, indicating that the SiO2@COF-COOH column has excellent mechanical stability. Furthermore, with acetonitrile in the mobile phase at 60% volume, after 10 consecutive injections within one day, the relative standard deviations of the retention times of the five monosubstituted benzenes did not exceed 0.93%. Figure 6As shown in d, this indicates that SiO2@COF-COOH has good mechanical stability and repeatability.

[0058] 7. Separation of nucleosides / bases using SiO2@COF-COOH chromatographic columns

[0059] The prepared SiO2@COF-COOH column was used as a high-performance liquid chromatography (HPLC) column, and its separation performance for nucleosides / bases (2'-deoxyuridine, thymine, thymidine, cytosine, and adenine) was tested in hydrophilic mode. The chromatographic separation conditions were: mobile phase: acetonitrile / ammonium acetate solution (20 mmol / L), flow rate: 1.0 mL / min, detection wavelength: 254 nm.

[0060] like Figure 7 As shown in Figure a, when the volume fraction of ammonium acetate aqueous solution in the mobile phase is 40%, chromatographic peaks 1–5 represent 2'-deoxyuridine, thymine, thymidine, cytosine, and adenine, respectively. The SiO2@COF-COOH column achieved separation of these five nucleosides / bases within 10 min. However, under the same chromatographic conditions, the chromatographic peaks on the commercially available XBridge Amide column (Waters, USA) showed severe overlap. Furthermore, as shown in Figure a… Figure 7 As shown in b, when the volume fraction of ammonium acetate aqueous solution in the mobile phase is in the range of 10% to 45%, the retention time of the five nucleosides / bases on the SiO2@COF-COOH column decreases with the increase of the volume fraction of ammonium acetate aqueous solution, indicating that the SiO2@COF-COOH column exhibits typical characteristics of hydrophilic retention.

[0061] 8. Separation of sulfonamide antibiotics using SiO2@COF-COOH chromatographic column

[0062] The prepared SiO2@COF-COOH column was used as a high-performance liquid chromatography (HPLC) column, and its separation performance for sulfonamide antibiotics sulfabenzoyl, sulfonamide, sulfamethoxypyridazine, and sulfaguanidine was tested in hydrophilic mode. The chromatographic separation conditions were: acetonitrile / ammonium acetate solution (20 mmol / L) = 90 / 10, flow rate: 1.0 mL / min, and detection wavelength: 270 nm.

[0063] like Figure 8As shown, peaks 1-4 represent sulfabenzoyl, sulfonamide, sulfamethoxypyridazine, and sulfaguanidine, respectively. These four sulfonamide antibiotics were efficiently separated within 5 min on a SiO2@COF-COOH column, while sulfonamide and sulfamethoxypyridazine were co-eluted on an XBridge Amide (Waters, USA) column. The retention order of sulfabenzoyl (log P, 1.19), sulfonamide (log P, -0.72), and sulfaguanidine (log P, -1.22) was consistent with their polarity, indicating that hydrophilic interaction dominated their retention. Although sulfamethoxypyridazine (log P, 0.32) had a lower polarity than sulfonamide (log P, -0.72), its retention time on the SiO2@COF-COOH column was longer than that of sulfonamide. Structurally, sulfamethoxypyridazine has an additional pyridazine ring compared to sulfonamides. Therefore, in addition to hydrophilic interactions, sulfamethoxypyridazine can also form hydrophobic, π-π and hydrogen bond interactions with SiO2@COF-COOH.

[0064] 9. Separation of analgesic and anti-inflammatory drugs using SiO2@COF-COOH chromatographic column

[0065] The prepared SiO2@COF-COOH column was used as a high-performance liquid chromatography (HPLC) column, and its separation performance for the analgesic and anti-inflammatory drugs ketoibuprofen, acetaminophen, and carboprofen was tested in hydrophilic mode. The chromatographic separation conditions were: acetonitrile / ammonium acetate solution (20 mmol / L) = 90 / 10, flow rate: 1.0 mL / min, and detection wavelength: 270 nm.

[0066] like Figure 9 As shown, chromatographic peaks 1-3 are ketoibuprofen, acetaminophen, and carboprofen, respectively. The three analgesic and anti-inflammatory drugs were well separated on the SiO2@COF-COOH column. However, under the same conditions, ketoibuprofen and carboprofen were co-eluted on the Xbridge Amide column (Waters, USA).

[0067] 10. Testing of actual samples using SiO2@COF-COOH chromatographic column

[0068] Using the prepared SiO2@COF-COOH column as a high-performance liquid chromatography (HPLC) column, wastewater effluent from a wastewater treatment plant was selected as the test sample, and polycyclic aromatic hydrocarbons (fluorene, m-terphenyl, and p-terphenyl) were used as analytes. The application potential of this column in the separation and analysis of actual samples was tested in reversed-phase mode. The chromatographic separation conditions for fluorene, m-terphenyl, and p-terphenyl were: acetonitrile / water = 70 / 30, flow rate: 1.0 mL / min, and detection wavelength: 254 nm. The chromatograms of fluorene, m-terphenyl, and p-terphenyl standard solutions (500 μg / mL), wastewater effluent from a wastewater treatment plant, and spiked wastewater effluent from a wastewater treatment plant (500 μg / mL) on the SiO2@COF-COOH column are shown below. Figure 10 .

[0069] The results showed that polycyclic aromatic hydrocarbons (fluorene, m-terphenyl, and p-terphenyl) were not detected in the effluent water samples. The method for determining fluorene, m-terphenyl, and p-terphenyl in the effluent water samples was validated using high-performance liquid chromatography-ultraviolet detection (HPLC-UV) (Table 1). All analytes exhibited good linearity (r0) within the linear range of 0.05–25 mg / L. 2 The limits of detection for fluorene, m-terphenyl, and p-terphenyl were all 0.02 mg / L. The accuracy of the method was investigated by spiked wastewater effluent samples from wastewater treatment plants at three concentration levels (0.15, 5, and 20 mg / L). The recoveries ranged from 93.20% to 117.05%, and the intra-day relative standard deviations ranged from 0.27% to 2.79%. These results demonstrate that the separation and analysis method based on the SiO2@COF-COOH column can be successfully applied to the analysis of wastewater effluent samples from wastewater treatment plants.

[0070] Table 1. Analysis results of wastewater effluent from the wastewater treatment plant

[0071]

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An amphiphilic carboxyl-functionalized covalent organic framework silica composite stationary phase based on a chiral induction strategy, characterized in that, The stationary phase uses amino silica gel as a support and (R)-1-(1-naphthyl)ethylamine as a chiral inducer to in situ immobilize a carboxyl-functionalized covalent organic framework on the surface of amino silica gel, resulting in an amphiphilic carboxyl-functionalized covalent organic framework silica gel composite stationary phase; the carboxyl-functionalized covalent organic framework is generated by reacting 2,4,6-tricarboxyloyl-resorcinol with 4,4-diaminobiphenyl-2,2-dicarboxylic acid as raw materials.

2. The method for preparing the amphiphilic carboxyl-functionalized covalent organic framework mixed-mode chromatographic stationary phase based on a chiral induction strategy as described in claim 1, characterized in that, Includes the following steps: Step 1: Using 1,4-dioxane as solvent, and amino silica gel, 2,4,6-tricarboxymethyl phloroglucinol, and 1,3,5-trimethylbenzene as raw materials, SiO2-TFP microspheres were obtained by reacting under acetic acid catalysis. Step 2: Using n-butanol and o-dichlorobenzene as solvents, and 2,4,6-tricarboxyloyl-resorcinol, 4,4-diaminobiphenyl-2,2-dicarboxylic acid, SiO2-TFP microspheres, and 2,4,6-trimethylaniline as raw materials, and R-1-(1-naphthyl)ethylamine as a chiral inducer, the reaction is carried out under acetic acid catalysis to obtain SiO2@COF-COOH microspheres, which are the amphiphilic carboxyl-functionalized covalent organic framework mixed-mode chromatographic stationary phase.

3. The preparation method according to claim 2, characterized in that, Step 1 involves reacting at 80°C for at least 24 hours.

4. The preparation method according to claim 2, characterized in that, Step 2 includes: mixing 2,4,6-tricarboxyloyl-resorcinol, 4,4-diaminobiphenyl-2,2-dicarboxylic acid, SiO2-TFP microspheres, n-butanol and o-dichlorobenzene, then adding 2,4,6-trimethylaniline and R-1-(1-naphthyl)ethylamine, heating to 40 ºC, adding acetic acid, and then reacting at 80 ºC for more than 48 hours.

5. A high-performance liquid chromatography column, characterized in that, Includes the amphiphilic carboxyl-functionalized covalent organic framework mixed-mode chromatographic stationary phase based on the chiral induction strategy described in claim 1.

6. A high-performance liquid chromatography column according to claim 5, characterized in that, The method for preparing the high-performance liquid chromatography column includes: An amphiphilic carboxyl-functionalized covalent organic framework mixed-mode chromatographic stationary phase was added to an organic solvent and ultrasonically dispersed to form a suspension. Then, using the organic solvent as a displacement solvent, the suspension was packed into a stainless steel column at a pressure of 40–80 MPa for 5–30 min. The pressure was then reduced to 10–30 MPa, and the column was packed for another 3–20 min to obtain a SiO2@COF-COOH chromatographic column.

7. A high-performance liquid chromatography column according to claim 6, characterized in that, The organic solvent is methanol or acetonitrile.

8. The application of the amphiphilic carboxyl-functionalized covalent organic framework mixed-mode chromatographic stationary phase based on chiral induction strategy as described in claim 1 in high performance liquid chromatography.

9. The application according to claim 8, characterized in that, The separation of hydrophobic compounds is performed in reversed-phase chromatography mode. Preferably, the hydrophobic compounds include monosubstituted benzenes and polycyclic aromatic hydrocarbons. Preferably, the monosubstituted benzenes include toluene, ethylbenzene, n-propylbenzene, n-butylbenzene, and n-pentylbenzene. Preferably, the polycyclic aromatic hydrocarbons include benzene, diphenylmethane, o-terphenyl, m-terphenyl, p-terphenyl, cis-stilbene, and fluorene.

10. The application according to claim 8, characterized in that, The polar compounds are separated in hydrophilic chromatography mode; preferably, the polar compounds include nucleosides, bases, sulfonamide antibiotics, and analgesic and anti-inflammatory drugs; preferably, the nucleosides include 2'-deoxyuridine and thymidine; preferably, the bases include thymine, cytosine, and adenine; preferably, the sulfonamide antibiotics include sulfonamides, sulfabenzoyl, sulfaguanidine, and sulfamethoxypyridazine; preferably, the analgesic and anti-inflammatory drugs include ketoibuprofen, acetaminophen, and carboprofen.

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