A method for preparing a capillary chromatographic column based on metal-organic frameworks for chiral separation.

Abstract

The present invention discloses a method for preparing a capillary chromatographic column based on a metal-organic framework material for chiral separation, comprising the following steps: slowly adding a monohydrated copper acetate aqueous solution to a gel-like ligand product aqueous solution for reaction to obtain a metal-organic framework material solution; passing the metal-organic framework material solution through a capillary column pre-anchored with the obtained ligand for 0.5 to 2 hours, closing the column, allowing the column to stand for reaction, and rinsing with ultrapure water to obtain the capillary chromatographic column based on the metal-organic framework material for chiral separation. The capillary chromatographic column prepared by the present invention has good thermal and chemical stability, maintains structural integrity under high temperature, partial acid and alkaline conditions, and long-term storage conditions, and maintains good stability after 100 runs.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis and analysis technology, specifically, it relates to a method for preparing a capillary chromatographic column based on metal-organic framework materials that can be used for chiral separation. Background Technology

[0002] Chirality is one of the most prominent characteristics of nature. Enantiomers often exhibit significant differences in pharmacological and toxicological properties, making the separation of chiral substances crucial in various fields such as medicine, chemical industry, and agriculture. Developing simple and effective separation strategies for chiral compounds is therefore very important and meaningful. Capillary electrochromatography (CEC) is a novel, highly efficient, and rapid separation technique. It combines the advantages of high efficiency of capillary electrophoresis and high selectivity of high-performance liquid chromatography (HPLC), offering benefits such as small sample size, simple operation, high separation efficiency, and multiple alternative modes, leading to its widespread application in the separation of chiral substances. The core of capillary electrochromatography is the capillary column. Therefore, the preparation of electrochromatographic columns with high column efficiency and good separation performance is a key research focus.

[0003] Many compounds have been successfully developed for chiral stationary phases in capillary chromatography columns, including cyclodextrins and their derivatives, proteins, carbohydrates, crown ethers, antibiotics, and surfactants. Metal-organic frameworks (MOFs) offer ample specific surface area, effectively compensating for the limited sample loading capacity of capillary columns, and possess characteristics such as complex structures and high porosity. Simultaneously, the porous nature of MOFs effectively enhances the sample carrying capacity of capillary columns, allowing capillary electrophoresis chromatography to gradually emerge as a promising technology in chiral separation. MOFs have a wide range of chiral separation applications, including drugs, small biomolecules, and other chiral intermediates used in synthetic raw materials. MOFs possess characteristics such as ultra-low density, tunable porosity, good solvent stability, controllable topology, and high specific surface area, and have been widely used as chiral stationary phases in capillary columns. Due to these advantages, introducing MOFs into capillary electrochromatography is an effective way to increase the specific surface area of ​​the stationary phase and the interaction sites for small molecules, demonstrating broad application prospects in the separation of chiral compounds.

[0004] Patent application CN114504843A discloses a method for preparing a capillary open-tube chromatographic column based on a metal-organic framework (MOF) material. The method includes the following steps: dissolving an organic ligand and an inorganic metal ion reagent in a solvent, mixing them uniformly by ultrasonication to form a clear solution, injecting the solution into a carboxyl-modified capillary column, sealing both ends of the capillary, and performing a heat treatment bonding reaction. After the reaction is complete, the capillary is removed, the sealed ends are cut off, and the column is rinsed with methanol to obtain the aforementioned capillary open-tube chromatographic column based on the MOF material. This invention uses an in-situ polymerization method to prepare the capillary open-tube chromatographic column based on the MOF material. Because the MOF material is directly bonded to the inner wall of the capillary, it has advantages such as high mechanical strength, resistance to breakage, and minimal air bubble retention. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a capillary chromatographic column based on metal-organic framework materials that can be used for chiral separation. The prepared capillary column can be used to separate chiral compounds, solving the technical problems of difficult separation of chiral amino acids and the inability to separate some chiral drugs temporarily, as well as low separation efficiency.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for preparing a capillary chromatographic column based on a metal-organic framework material that can be used for chiral separation, comprising the following steps:

[0008] A 40-60% (preferably 50%) solution of 3-aminopropyltriethoxysilane was passed into a pretreated capillary column. After 10-30 min (preferably 15 min), the two ends of the capillary column were sealed with rubber. The column was heated at 50-60℃ (preferably 55℃) for 1-12 h (preferably 12 h). After removal, the rubber-sealed portions at both ends were cut off, and the column was rinsed with methanol to complete the amino modification and obtain a functionalized capillary column.

[0009] A gel-like ligand product, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide ester in a molar ratio of 0.1–1:1:0.05–0.5 (preferably 0.83:1:0.2) are dissolved in a disodium hydrogen phosphate buffer solution with a concentration of 50–150 mM (preferably 100 mM). The mixture is ultrasonically activated for 10–30 min (preferably 15 min) to obtain a mixture. The mixture is then purged into a functionalized capillary column by nitrogen blowing to carry out the reaction. The mixture is passed through the capillary for a period of time (preferably 1 h) and then the nitrogen purging is turned off and the column is allowed to stand (preferably 15 min). This process is repeated at least three times. The column is then rinsed with ultrapure water to obtain a capillary column with ligand pre-anchored.

[0010] A copper acetate monohydrate aqueous solution is slowly added dropwise to an aqueous solution of a gel-like ligand product. The molar ratio of copper acetate monohydrate to the gel-like ligand product is 1:1 to 3 (preferably 1:2). The reaction is carried out for 10 to 60 min (preferably 30 min) to obtain a metal-organic framework material solution.

[0011] The metal-organic framework material solution is passed into the obtained ligand-pre-anchored capillary column for 0.5 to 2 hours (preferably 1 hour) and then turned off. The column is allowed to stand for 10 to 60 minutes (preferably 30 minutes) and then rinsed with ultrapure water for 10 to 60 minutes (preferably 30 minutes) to obtain the metal-organic framework-based capillary chromatography column that can be used for chiral separation.

[0012] The pretreatment method for the pretreated capillary column is as follows:

[0013] Using nitrogen purging, sequentially pass a 0.5–1.5 mol / L (preferably 1 mol / L) sodium hydroxide aqueous solution, a 0.5–1.5 mol / L (preferably 1 mol / L) hydrochloric acid aqueous solution, and methanol into a 1.5 m to 3 m long capillary column for 50–80 min (preferably 60 min), 10–30 min (preferably 20 min), and 20–40 min (preferably 30 min) respectively. The volume ratio of sodium hydroxide aqueous solution, hydrochloric acid aqueous solution, and methanol is 1:1:1. Rinse with ultrapure water for 10–30 min (preferably 15 min) between each two solvents before rinsing with the next reagent. Continue rinsing until the volume ratio of ultrapure water to sodium hydroxide aqueous solution is 1:5–20 (preferably 1:10). After methanol rinsing, dry the remaining solvent in the capillary column with nitrogen and dry at 95–110 °C (preferably 100 °C) for 0.5–2 h (preferably 1 h).

[0014] The preparation method of the gel-like ligand product is as follows:

[0015] Dissolve (E)-N'-((E)-(dimethylamino)methylene))-N,N-dimethylmethylhydrazine amide and ligand in ethanol at a molar ratio of 1 to 8:1 (preferably 4.4:1 or 2.2:1), heat and stir under reflux for 1 to 48 hours (preferably 48 hours), remove the solvent, and obtain the gel-like ligand product.

[0016] The ligand is selected from L-lysine or L-histidine.

[0017] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0018] This invention employs a pre-anchored ligand in-column modification strategy to prepare a capillary column based on metal-organic frameworks. Unlike traditional metal-organic framework capillary columns, the capillary column prepared in this invention utilizes mild synthesis conditions and employs inexpensive and readily available amino acids as ligand raw materials, making it more environmentally friendly and economical, thus reducing production costs and simplifying industrial scale-up. Furthermore, this column exhibits good chiral separation capability, greater stability, and better reproducibility.

[0019] The capillary chromatographic column prepared by this invention has good thermal and chemical stability. Its structure remains intact under high temperature, acidic and alkaline conditions and long-term storage conditions, and it still has good stability after 100 runs.

[0020] In this invention, the stationary phase is directly used as the separation material, which greatly increases the column capacity and effectively improves the separation efficiency.

[0021] The capillary chromatographic column material prepared by this invention has excellent compatibility with biological samples, and is particularly suitable for the separation and analysis of small biological molecules. It has broad application prospects in the separation of chiral compounds and metabolomics research.

[0022] This invention achieves efficient and high-resolution separation of chiral amino acid enantiomers and chiral drugs. Attached Figure Description

[0023] Figure 1 A schematic diagram of the overall morphology of the capillary column prepared in Example 1 as observed under a scanning electron microscope at the 3-micrometer scale.

[0024] Figure 2 This is a schematic diagram of the separation spectrum of chiral compounds using the capillary chromatography column prepared in Example 1.

[0025] Figure 3 This is a schematic diagram of the overall morphology of the metal-organic framework material prepared in Example 1 as observed under a scanning electron microscope.

[0026] Figure 4 This is a schematic diagram of the Fourier transform infrared spectroscopy results detected by the metal-organic framework material prepared in Example 1.

[0027] Figure 5 The nitrogen adsorption-desorption curves and pore size analysis diagrams of the capillary column prepared in Example 1 are shown.

[0028] Figure 6 This is a schematic diagram of the nitrogen adsorption-desorption curves and pore size analysis of an unmodified empty tubular column.

[0029] Figure 7 This is a schematic diagram showing the electroosmotic flow results of three different types of capillary columns at different pH values.

[0030] Figure 8 This is a schematic diagram of the thermogravimetric analysis of the metal-organic framework material prepared in Example 1.

[0031] Figure 9 This is a schematic diagram illustrating the acid-base stability of the metal-organic framework material prepared in Example 1.

[0032] Figure 10 This is a schematic diagram illustrating the storage stability of the metal-organic framework material prepared in Example 1.

[0033] Figure 11 The diagram shows the results of verifying the stability of the capillary column prepared in Example 1 using chiral proline as an example.

[0034] Figure 12 This is a schematic diagram of the overall morphology of the metal-organic framework material prepared in Example 3 as observed by scanning electron microscopy at the 1, 3, 5, and 10 micrometer scales. Detailed Implementation

[0035] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] A method for preparing a capillary chromatographic column based on a metal-organic framework material for chiral separation includes the following steps:

[0038] The first step is the pretreatment of capillaries.

[0039] Purging with nitrogen, 1.5 mL of a 1 mol / L sodium hydroxide aqueous solution, 1.5 mL of a 1 mol / L hydrochloric acid aqueous solution, and 1.5 mL of methanol were sequentially passed into a 2 m long capillary column for 60 min, 20 min, and 30 min, respectively. Between each two solvents, the column was rinsed with 15 mL of ultrapure water for 15 min before moving on to the next reagent. After methanol rinsing, the remaining solvent in the capillary column was dried with nitrogen and then placed in an oven at 100 °C for 1 h.

[0040] The second step is functional group modification.

[0041] 15 mL of a 50% aqueous solution of 3-aminopropyltriethoxysilane was passed into the pretreated capillary column. After 15 min, both ends of the capillary column were sealed with rubber, and the column was heated in a water bath at 55 °C for 12 h. After removal, the rubber-sealed portions at both ends were cut off, and the unreacted 3-aminopropyltriethoxysilane in the column was flushed out with 30 mL of methanol, completing the amino modification and obtaining a functionalized capillary column.

[0042] The third step is the preparation of ligands.

[0043] (E)-N'-((E)-(dimethylamino)methylene))-N,N-dimethylmethylhydrazine amide (0.044 mol, 6.3 g) and L-lysine (0.01 mol, 1.46 g) were dissolved in 75 mL of EtOH and heated under reflux with stirring for 48 h. After the reaction was complete, the solvent in the mixture was removed by vacuum rotary evaporation at 45 °C. The collected orange gel-like solid was washed three times successively with ethanol and diethyl ether, and dried in air to obtain a gel-like ligand product.

[0044] Step 4: Ligand pre-anchoring

[0045] The gel-like ligand product obtained in step 3 (0.83 mmol, 207 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1 mmol, 191.7 mg), and N-hydroxysuccinimide ester (0.2 mmol, 23 mg) were dissolved in 5 mL of 100 mM disodium hydrogen phosphate buffer solution and activated by sonication for 15 min to obtain a mixture. This mixture was then purged into the functionalized capillary column obtained in step 2 by nitrogen blowing. Nitrogen was turned off and the mixture was purged for 15 min every 1 h to ensure sufficient reaction between the ligand and the amino group on the capillary wall. This process was repeated three times. After rinsing with ultrapure water for 30 min to remove residual mixture, the pre-anchored capillary column was obtained.

[0046] Step 5: Preparation of the chromatographic column

[0047] 10 mL of copper acetate monohydrate (2.4 mmol, 479.16 mg) aqueous solution was slowly added dropwise to 10 mL of the gel-like ligand product (4.8 mmol, 1200 mg) aqueous solution obtained in step 3, and the reaction was carried out for 30 min to obtain the metal-organic framework material solution.

[0048] The above metal-organic framework material solution was passed into the capillary column pre-anchored with ligands obtained in step four. After passing through the column for 1 hour, the gas valve was closed, and the column was allowed to stand for 30 minutes. Then, it was rinsed with ultrapure water for 30 minutes to obtain the capillary chromatographic column based on metal-organic framework material that can be used for chiral separation, denoted as NLMOF chiral column.

[0049] The prepared NLMOF chiral column was characterized by scanning electron microscopy, and the results are as follows: Figure 1 As shown, Figure 1 The diagram shows the overall morphology of the capillary column prepared in Example 1 as observed by scanning electron microscopy at the 3-micrometer scale. It can be seen that there are obvious spherical protrusions on the inner surface of the capillary column, indicating that the metal-organic framework material (NLMOF) was successfully modified onto the inner surface of the capillary.

[0050] The prepared NLMOF chiral column was installed on a capillary electrophoresis apparatus (CE, Beijing Huayang Limin Instrument CL1030, China) for capillary electrochromatographic separation to separate D / L-amino acids and donepezil.

[0051] Amino acid separation conditions: The solvent for dissolving amino acids was methanol / 0.1M HCl aqueous solution, with a volume ratio of 3:7. The buffer solution was 20mM disodium hydrogen phosphate aqueous solution with a pH of 7; the electrophoretic separation voltage was 15kV. Separation conditions: methanol / 0.1M HCl aqueous solution (pH=7) = 30 / 70, injection time: 5s, separation temperature: room temperature.

[0052] Donepezil separation conditions: Donepezil was dissolved in a 50% methanol-water solution; the buffer solution was a 15mM disodium hydrogen phosphate aqueous solution at pH 10; the electrophoretic separation voltage was 9kV. Separation conditions: methanol / water = 50 / 50, injection time: 5s, separation temperature: room temperature.

[0053] The obtained electrochromatogram is as follows Figure 2 As shown. Figure 2 This is a schematic diagram of the separation chromatogram of chiral compounds using the capillary column prepared in Example 1. The chromatogram shows that the NLMOF chiral column can separate 10 derivatized chiral amino acids and 1 chiral drug. In the figure, a represents dansyl-threonine, b represents dansyl-histidine, c represents dansyl-asparagine, d represents dansyl-alanine, e represents dansyl-valine, f represents dansyl-proline, g represents dansyl-tryptophan, h represents dansyl-phenylalanine, i represents dansyl-leucine, j represents dansyl-serine, and k represents donepezil.

[0054] Figure 3 This is a schematic diagram of the overall morphology of the metal-organic framework material prepared in Example 1 as observed under a scanning electron microscope. The figure shows that the NLMOF crystals have the best shape, exhibiting a regular hexagonal prism shape, proving the successful synthesis of the NLMOF material.

[0055] Figure 4 This is a schematic diagram of the Fourier transform infrared spectroscopy results detected by the metal-organic framework material prepared in Example 1. The characteristic C=N peaks of the NL ligand and NLMOF were observed to appear at 1690 cm⁻¹. -1-1590cm -1 The NL ligand modified with L-lysine and a triazole ring exhibits a strong C=N bond characteristic peak at 1639 cm⁻¹, and a peak at 1360 cm⁻¹. -1 -1020cm -1 The appearance of multiple previously unseen C=N bond characteristic peaks indicates that the triazole ring was successfully modified onto the L-lysine. A characteristic peak of the C=N bond in the synthesis of NLMOF via coordination of the NL ligand with divalent copper ions showed a blue shift (1639 cm⁻¹). -1 →1626cm -1 Therefore, it can be determined that NL has successfully coordinated with metal ions, that is, the NLMOF material has been successfully synthesized.

[0056] Table 1 shows the separation of chiral amino acids and chiral drugs using the empty column, ligand column (the ligand-pre-anchored capillary column obtained in step four), and NLMOF chiral column prepared in Example 1. Here, t1 and t2 are the retention times of the two monomers after separation of the chiral compound, respectively, and Rs is the degree of separation between the monomers after separation of the chiral compound. As can be seen from Table 1, the unmodified empty column and the ligand-modified ligand column showed no separation of dansyl amino acids and chiral drugs, while the NLMOF chiral column achieved enantiomeric separation. This indicates that the NLMOF-modified capillary column has good separation efficiency and ability for chiral compounds. In Table 1, the Rs value of the compounds separated by the NLMOF chiral column is greater than 1.5, achieving baseline separation, while the Rs value of the empty column and ligand column is 0.

[0057] Table 1 Resolution data of chiral compounds

[0058]

[0059] Table 2 shows the repeatability data for NLMOF chiral columns:

[0060] Repeatability testing procedure: Dansylproline and donepezil were used as samples to determine the repeatability of the chromatographic column. The separation conditions for dansylproline were: solvent: methanol / 0.1M HCl aqueous solution, volume ratio 3:7; disodium hydrogen phosphate buffer concentration: 20mM, pH 7; electrophoretic separation voltage: 15kV. The separation conditions for donepezil were: drug solvent: 50% methanol aqueous solution; disodium hydrogen phosphate buffer concentration: 15mM, pH 10; electrophoretic separation voltage: 9kV. The data in Table 2 show that the NLMOF chiral column prepared in this invention has good repeatability.

[0061] Table 2. Repeatability verification of dansylproline and donepezil separation using NLMOF chiral columns (n=3)

[0062]

[0063] Test method for nitrogen adsorption-desorption curves: Under low temperature (liquid nitrogen bath) conditions, adsorbate gas (N2) is introduced into the sample tube. The adsorption partial pressure is directly measured by controlling the equilibrium pressure in the sample tube. The adsorption amount at that partial pressure point is obtained by the gas law. The adsorption isotherm is obtained by gradually introducing adsorbate gas to increase the adsorption equilibrium pressure. The desorption isotherm is obtained by gradually withdrawing adsorbate gas to decrease the adsorption equilibrium pressure.

[0064] Figure 5 The nitrogen adsorption-desorption curves and pore size analysis diagrams of the capillary column prepared in Example 1 are shown. Figure 6 This is a schematic diagram showing the nitrogen adsorption-desorption curves and pore size analysis of an unmodified empty tubular column. From... Figure 5 and Figure 6 As can be seen from the data, the specific surface area of ​​the capillary chromatographic column prepared in Example 1 of this invention is 4.61672 m². 2 / g, which is much larger than the specific surface area of ​​0.44048m² of an unmodified empty column. 2 / g. The above data demonstrate that NLMOF modification significantly improves the pore adsorption volume of the empty column.

[0065] Figure 8 This is a schematic diagram of the thermogravimetric analysis (TGA) of the metal-organic framework (NLMOF) material prepared in Example 1. As shown in the figure, NLMOF only exhibits significant mass loss near 200°C, indicating that the NLMOF structure is more stable. In practical applications, CEC is generally operated at room temperature, demonstrating good thermal stability under typical application conditions.

[0066] Figure 9 This is a schematic diagram illustrating the acid-base stability of the metal-organic framework material prepared in Example 1. After being placed in sodium dihydrogen phosphate buffer solutions at pH 4 or 9 for one week, and then washed with ultrapure water and dried, the infrared characteristic peak positions and intensities of the NLMOF metal-organic framework material showed no significant changes compared to the initial values. This indicates that the material has the potential for long-term separation operations in a wide range of pH buffer environments.

[0067] Figure 10 This is a schematic diagram illustrating the storage stability of the metal-organic framework material prepared in Example 1. The infrared characteristic peaks of NLMOF showed no significant change after being stored at room temperature for up to six months, indicating that the material has good storage stability.

[0068] Figure 11This is a schematic diagram illustrating the results of verifying the stability of the capillary column using chiral proline as an example, prepared in Example 1. The results show that the NLMOF chiral column can stably separate chiral proline and racemic donepezil for 100 cycles, maintaining stable separation performance. While the peak times varied slightly, they remained within acceptable fluctuations, and the peak shapes were still relatively sharp. Therefore, the NLMOF chiral column also exhibited good operational stability.

[0069] Example 2

[0070] A method for preparing a capillary chromatographic column based on a metal-organic framework material for chiral separation includes the following steps:

[0071] The first step is the pretreatment of capillaries.

[0072] Purging with nitrogen, 1.5 mL of a 1 mol / L sodium hydroxide aqueous solution, 1.5 mL of a 1 mol / L hydrochloric acid aqueous solution, and 1.5 mL of methanol were sequentially passed into a 2 m long capillary column for 60 min, 20 min, and 30 min, respectively. Between each two solvents, the column was rinsed with 15 mL of ultrapure water for 15 min before moving on to the next reagent. After methanol rinsing, the remaining solvent in the capillary column was dried with nitrogen and then placed in an oven at 100 °C for 1 h.

[0073] The second step is functional group modification.

[0074] 15 mL of a 50% aqueous solution of 3-aminopropyltriethoxysilane was passed into the pretreated capillary column. After 15 min, both ends of the capillary column were sealed with rubber, and the column was heated in a water bath at 55 °C for 12 h. After removal, the rubber-sealed portions at both ends were cut off, and the unreacted 3-aminopropyltriethoxysilane in the column was flushed out with 30 mL of methanol, completing the amino modification and obtaining a functionalized capillary column.

[0075] The third step is the preparation of ligands.

[0076] (E)-N'-((E)-(dimethylamino)methylene))-N,N-dimethylmethylhydrazine amide (0.044 mol, 6.3 g) and L-lysine (0.01 mol, 1.46 g) were dissolved in 75 mL of EtOH and heated under reflux with stirring for 48 h. After the reaction was complete, the solvent in the mixture was removed by vacuum rotary evaporation at 45 °C. The collected orange gel-like solid was washed three times successively with ethanol and diethyl ether, and dried in air to obtain a gel-like ligand product.

[0077] Step 4: Ligand pre-anchoring

[0078] The gel-like ligand product obtained in step 3 (0.83 mmol, 207 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1 mmol, 191.7 mg), and N-hydroxysuccinimide ester (0.2 mmol, 23 mg) were dissolved in 5 mL of 100 mM disodium hydrogen phosphate buffer solution and activated by sonication for 15 min to obtain a mixture. This mixture was then purged into the functionalized capillary column obtained in step 2 by nitrogen blowing. Nitrogen was turned off and the mixture was purged for 15 min every 1 h to ensure sufficient reaction between the ligand and the amino group on the capillary wall. This process was repeated three times. After rinsing with ultrapure water for 30 min to remove residual mixture, the pre-anchored capillary column was obtained.

[0079] Step 5: Preparation of the chromatographic column

[0080] 10 mL of copper acetate monohydrate (4.8 mmol, 958.32 mg) aqueous solution was slowly added dropwise to 10 mL of the gel-like ligand product (9.6 mmol, 2400 mg) aqueous solution obtained in step 3, and the reaction was carried out for 30 min to obtain the metal-organic framework material solution.

[0081] The above metal-organic framework material solution was passed into the capillary column pre-anchored with ligands obtained in step four. After passing through the column for 1 hour, the gas valve was closed, and the column was allowed to stand for 30 minutes. Then, it was rinsed with ultrapure water for 30 minutes to obtain the capillary chromatographic column based on metal-organic framework material that can be used for chiral separation.

[0082] The electroosmotic flow determination method is as follows: using a 0.5 mg / ml thiourea aqueous solution as a neutral marker, the electroosmotic flow of the empty column, the ligand column and the chromatographic column prepared in the embodiments of the present invention are determined. The separation conditions are: methanol / 10 mM phosphate (pH=7)=1 / 9, injection time: 5 s, separation voltage: 17.5 kV, and separation temperature: room temperature.

[0083] Figure 7 This diagram illustrates the electroosmotic flow results of three different types of capillary columns at different pH values. The three types of capillary columns are an empty column, a ligand column (the capillary column pre-anchored with ligands obtained in step four), and the capillary chromatography column prepared in this embodiment of the invention. The electroosmotic flow of the three types of capillary columns differs at different pH values, but the trends are consistent. This further proves that the NL ligand and NLMOF were successfully modified and coated on the inner wall of the capillary column, and the NLMOF chiral column was successfully prepared.

[0084] Compared with existing literature, the capillary tube prepared by this invention has high separation degree and good separation effect for chiral compounds.

[0085] Example 3

[0086] A method for preparing a capillary chromatographic column based on a metal-organic framework material for chiral separation includes the following steps:

[0087] The first step is the pretreatment of capillaries.

[0088] Purging with nitrogen, 1.5 mL of a 1 mol / L sodium hydroxide aqueous solution, 1.5 mL of a 1 mol / L hydrochloric acid aqueous solution, and 1.5 mL of methanol were sequentially passed into a 2 m long capillary column for 60 min, 20 min, and 30 min, respectively. Between each two solvents, the column was rinsed with 15 mL of ultrapure water for 15 min before moving on to the next reagent. After methanol rinsing, the remaining solvent in the capillary column was dried with nitrogen and then placed in an oven at 100 °C for 1 h.

[0089] The second step is functional group modification.

[0090] 15 mL of a 50% aqueous solution of 3-aminopropyltriethoxysilane was passed into the pretreated capillary column. After 15 min, both ends of the capillary column were sealed with rubber, and the column was heated in a water bath at 55 °C for 12 h. After removal, the rubber-sealed portions at both ends were cut off, and the unreacted 3-aminopropyltriethoxysilane in the column was flushed out with 30 mL of methanol, completing the amino modification and obtaining a functionalized capillary column.

[0091] The third step is the preparation of ligands.

[0092] (E)-N'-((E)-(dimethylamino)methylene))-N,N-dimethylmethylhydrazine (0.044 mol, 6.3 g) and L-histidine (0.02 mol, 3.1 g) were dissolved in 150 mL of EtOH and heated under reflux with stirring for 48 h. After the reaction was complete, the solvent in the mixture was removed by vacuum rotary evaporation at 45 °C. The collected orange gel-like solid was washed three times with ethanol and diethyl ether, and dried in air to obtain a gel-like ligand product.

[0093] Step 4: Ligand pre-anchoring

[0094] The gel-like ligand product obtained in step 3 (0.83 mmol, 207 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1 mmol, 191.7 mg), and N-hydroxysuccinimide ester (0.2 mmol, 23 mg) were dissolved in 5 mL of 100 mM disodium hydrogen phosphate buffer solution and activated by sonication for 15 min to obtain a mixture. This mixture was then purged into the functionalized capillary column obtained in step 2 by nitrogen blowing. Nitrogen was turned off and the mixture was purged for 15 min every 1 h to ensure sufficient reaction between the ligand and the amino group on the capillary wall. This process was repeated three times. After rinsing with ultrapure water for 30 min to remove residual mixture, the pre-anchored capillary column was obtained.

[0095] Step 5: Preparation of the chromatographic column

[0096] 10 mL of copper acetate monohydrate (2.4 mmol, 479.16 mg) aqueous solution was slowly added dropwise to 10 mL of the gel-like ligand product (4.8 mmol, 1200 mg) aqueous solution obtained in step 3, and the reaction was carried out for 30 min to obtain the metal-organic framework material.

[0097] An appropriate amount of metal-organic framework material solution was introduced into the capillary column pre-anchored with ligands obtained in step four. After 1 hour of introduction, the gas valve was closed, and the column was allowed to stand for 30 minutes. Then, it was rinsed with ultrapure water for 30 minutes to obtain the capillary chromatographic column based on metal-organic framework material that can be used for chiral separation.

[0098] The results are as follows Figure 12 As shown, Figure 12 This is a schematic diagram showing the overall morphology of the metal-organic framework material prepared in Example 3 as observed by scanning electron microscopy at the 1, 3, 5, and 10 micrometer scales. From... Figure 12 The appearance and morphology of the metal-organic framework material can be clearly seen. The metal-organic framework material has a spherical shape with a diameter of about 1 μm to 3 μm, indicating that the metal-organic framework material has been successfully synthesized.

[0099] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing a capillary chromatographic column based on a metal-organic framework material for chiral separation, characterized in that, Includes the following steps: A 40-60% solution of 3-aminopropyltriethoxysilane was passed into a pretreated capillary column. After 10-30 minutes, the two ends of the capillary column were sealed with rubber. The column was heated at 50-60°C for 1-12 hours. After removal, the rubber-sealed portions at both ends were cut off, and the column was rinsed with methanol to complete the amino modification and obtain a functionalized capillary column. A gel-like ligand product, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide ester in a molar ratio of 0.1–1:1:0.05–0.5 were dissolved in a 50–150 mM disodium hydrogen phosphate buffer solution and ultrasonically activated for 10–30 min to obtain a mixture. The mixture was then purged into a functionalized capillary column by nitrogen blowing for reaction. The nitrogen purging was stopped after a period of time in the capillary, and the mixture was allowed to stand. This process was repeated at least three times. The column was then rinsed with ultrapure water to obtain a capillary column with ligand pre-anchored. A copper acetate monohydrate aqueous solution was slowly added dropwise to an aqueous solution of a gel-like ligand product. The molar ratio of copper acetate monohydrate to the gel-like ligand product was 1:1 to 3. The reaction was carried out for 10 to 60 minutes to obtain a metal-organic framework material solution. The metal-organic framework material solution was passed into the obtained ligand-pre-anchored capillary column for 0.5–2 h and then turned off. The column was allowed to stand for 10–60 min and then rinsed with ultrapure water for 10–60 min to obtain the metal-organic framework-based capillary chromatography column that can be used for chiral separation.

2. The method for preparing a capillary chromatographic column based on a metal-organic framework material for chiral separation according to claim 1, characterized in that, The pretreatment method for the pretreated capillary column is as follows: Purging with nitrogen gas, sequentially pass 0.5–1.5 mol / L sodium hydroxide aqueous solution, 0.5–1.5 mol / L hydrochloric acid aqueous solution, and methanol into a 1.5 m to 3 m long capillary column for 50–80 min, 10–30 min, and 20–40 min, respectively. The volume ratio of sodium hydroxide aqueous solution, hydrochloric acid aqueous solution, and methanol is 1:1:

1. Rinse with ultrapure water for 10–30 min between each two solvents before rinsing with the next reagent. When the volume ratio of ultrapure water to sodium hydroxide aqueous solution is 1:5–20, after methanol rinsing, blow nitrogen gas to dry the residual solvent in the capillary column and dry at a temperature of 95–110 °C for 0.5–2 h.

3. The method for preparing a capillary chromatographic column based on a metal-organic framework material for chiral separation according to claim 1, characterized in that, The preparation method of the gel-like ligand product is as follows: (E)-N'-((E)-(dimethylamino)methylene))-N,N-dimethylmethylhydrazine amide and ligand in a molar ratio of 1 to 8:1 were dissolved in ethanol, heated and stirred under reflux for 1 to 48 hours, and the solvent was removed to obtain the gel-like ligand product.

4. The method for preparing a capillary chromatographic column based on a metal-organic framework material for chiral separation according to claim 3, characterized in that, The ligand is selected from L-lysine or L-histidine.

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