Preparation method of capillary chromatographic column capable of being used for chiral separation and based on metal organic framework material

Abstract

The invention discloses a preparation method of a capillary chromatographic column based on a metal organic framework material for chiral separation, which comprises the following steps: slowly dropwise adding a cupric acetate monohydrate aqueous solution into a gelatinous ligand product aqueous solution for reaction to obtain a metal organic framework material solution; and introducing a metal organic framework material solution into the obtained ligand pre-anchored capillary column, closing after introducing for 0.5-2 hours, standing and reacting in the column, and washing with ultrapure water to obtain the metal organic framework material-based capillary chromatographic column capable of being used for chiral separation. The prepared capillary chromatographic column has good thermal stability and chemical stability, the structure is still complete under the conditions of high temperature, meta-acid-base and long-time storage, and the capillary chromatographic column still has good stability after running for 100 times.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical synthesis and analysis, and in particular relates to a method for preparing a capillary chromatographic column based on a metal organic framework material and capable of being used for chiral separation. Background Art

[0002] Chirality is one of the most prominent features of nature. The pharmacology and toxicology of enantiomers often have great differences in properties, so the separation of chiral substances is crucial in various fields such as medicine, chemical industry, and agriculture. It is very important and meaningful to develop simple and effective strategies for the separation of chiral compounds. Capillary electrochromatography is a new type of efficient and rapid separation technology. It has been developed by combining the advantages of high efficiency of capillary electrophoresis and high selectivity of high performance liquid chromatography. It has the advantages of small sample amount, simple operation, high separation efficiency, and multiple substitution modes. It is widely used in the separation of chiral substances. The core of capillary electrochromatography technology is the capillary electrochromatographic column. Therefore, the preparation of electrochromatographic columns with high column efficiency and good separation effect is the focus of research.

[0003] Many compounds have been successfully developed for chiral fixation in capillary columns, including cyclodextrin and its derivatives, proteins, sugars, crown ethers, antibiotics and surfactants. The sufficient specific surface area of ​​metal organic framework materials can well compensate for the defects of small sample loading of capillary columns, and they have the characteristics of complex structure and high porosity. At the same time, the porous materials of metal organic framework materials can effectively improve the sample loading capacity of capillary columns, making capillary electrophoresis chromatography gradually emerge in the field of chiral separation. The chiral separation range of metal organic framework materials is wide, including drugs, small biological molecules and other chiral intermediates used for synthetic raw materials. Metal organic framework materials have the characteristics of ultra-low density, adjustable porosity, good solvent stability, controllable topological structure and high specific surface area, and have been widely used as chiral stationary phases in capillary columns. Due to the above advantages, the introduction of metal organic framework materials in capillary electrochromatography is an effective way to increase the surface area of ​​the stationary phase and the action sites of small molecules, showing broad application prospects in the field of chiral compound separation.

[0004] The patent application with publication number CN114504843A discloses a method for preparing a capillary open-tube chromatographic column based on metal organic framework materials, comprising the following steps: dissolving an organic ligand and an inorganic metal ion reagent in a solvent, uniformly mixing by ultrasonication to form a uniform and clear solution, injecting into a carboxyl-modified capillary column, and then sealing both ends of the capillary, performing a heat treatment bonding reaction, and after the reaction is completed, taking out the capillary, cutting off the sealed parts at both ends, and washing with methanol to obtain the capillary open-tube chromatographic column based on metal organic framework materials. The present invention adopts an in-situ polymerization method to prepare a capillary open-tube chromatographic column based on metal organic framework materials. Since the metal organic framework material is directly bonded to the inner wall of the capillary, it has the advantages of high mechanical strength, not easy to break, and not easy to retain bubbles. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a capillary chromatographic column based on a metal organic framework material that can be used for chiral separation. The prepared capillary column can be used to separate chiral compounds, solving technical problems such as the difficulty in separating chiral amino acids and the temporary inability to separate some chiral drugs, and low separation efficiency.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect of the present invention, there is provided 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 3-aminopropyltriethoxysilane solution with a concentration of 40 to 60% (preferably 50%) is introduced into the pretreated capillary column, and after 10 to 30 minutes (preferably 15 minutes), both ends of the capillary column are sealed with rubber, and the reaction is heated at a temperature of 50 to 60° C. (preferably 55° C.) for 1 to 12 hours (preferably 12 hours), and the rubber sealing parts at both ends are cut off after taking out, and the column is rinsed with methanol to complete the amino modification, thereby obtaining a capillary column modified with a functional group;

[0009] Dissolve the 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) in a sodium dihydrogen phosphate buffer solution with a concentration of 50-150 mM (preferably 100 mM), and activate by ultrasound for 10-30 min (preferably 15 min) to obtain a mixed solution, and then flush the mixed solution into a functional group-modified capillary column by nitrogen blowing to react, and after each time the mixed solution is passed into the capillary for a period of time (preferably 1 h), turn off the nitrogen purge and let it stand (preferably 15 min), repeat at least three times, and then rinse with ultrapure water to obtain a ligand pre-anchored capillary column;

[0010] Slowly drop a monohydrated copper acetate aqueous solution into a gel-like ligand product aqueous solution, wherein the molar ratio of the monohydrated copper acetate to the gel-like ligand product is 1:1 to 3 (preferably 1:2), and react for 10 to 60 minutes (preferably 30 minutes) 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, and the column is closed after passing for 0.5 to 2 hours (preferably 1 hour). The column is allowed to react for 10 to 60 minutes (preferably 30 minutes), and rinsed with ultrapure water for 10 to 60 minutes (preferably 30 minutes) to obtain the capillary chromatographic column based on the metal organic framework material that can be used for chiral separation.

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

[0013] Using nitrogen purging, a sodium hydroxide aqueous solution with a concentration of 0.5 to 1.5 mol / L (preferably 1 mol / L), a hydrochloric acid aqueous solution with a concentration of 0.5 to 1.5 mol / L (preferably 1 mol / L), and methanol are sequentially introduced into a capillary column with a length of 1.5 m to 3 m for rinsing for 50 to 80 min (preferably 60 min), 10 to 30 min (preferably 20 min), and 20 to 40 min (preferably 30 min); the volume ratio of the sodium hydroxide aqueous solution, the hydrochloric acid aqueous solution, and the methanol is 1:1:1, and ultrapure water is used to rinse for 10 to 30 min (preferably 15 min) between each two solvents before rinsing the next reagent, and when the volume ratio of the ultrapure water used to the sodium hydroxide aqueous solution is 1:5 to 20 (preferably 1:10), after the methanol rinsing is completed, the residual solvent in the capillary column is blown dry with nitrogen, and dried at a temperature of 95 to 110° C. (preferably 100° C.) for 0.5 to 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-dimethylhydrazineamide and the ligand in ethanol at a molar ratio of 1 to 8:1 (preferably 4.4:1, 2.2:1), heat and stir under reflux for 1 to 48 hours (preferably 48 hours), remove the solvent, and obtain the gel ligand product.

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

[0017] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0018] The present invention adopts a pre-anchored ligand column modification strategy to prepare a capillary chromatographic column based on a metal organic framework material. Different from the traditional metal organic framework capillary chromatographic column, the capillary chromatographic column prepared by the present invention has mild synthesis conditions and uses cheap and easily available amino acids as ligand raw materials, which is more environmentally friendly and economical, helps to reduce production costs, and reduces the difficulty of industrial amplification. In addition, the chromatographic column has good chiral separation ability, is more stable, and has good repeatability.

[0019] The capillary chromatographic column prepared by the present invention has good thermal stability and chemical stability, and its structure remains intact under high temperature, partial acid and alkaline conditions and long-term storage conditions, and it still has good stability after running 100 times.

[0020] In the present 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 the present invention has good compatibility with biological samples, is particularly suitable for separation and analysis of biological small molecule samples, and has broad application prospects in the research of chiral compound separation and metabolomics.

[0022] The present invention achieves high-efficiency and high-resolution separation of chiral amino acid enantiomers and chiral drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the overall morphology of the capillary chromatographic column prepared in Example 1 observed under a scanning electron microscope at a scale of 3 microns.

[0024] Figure 2 Schematic diagram of the separation spectrum of chiral compounds by the capillary chromatographic 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 observed under a scanning electron microscope.

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

[0027] Figure 5 Schematic diagram of nitrogen adsorption-desorption curve and pore size analysis of the capillary chromatographic column prepared in Example 1.

[0028] Figure 6 Schematic diagram of nitrogen adsorption-desorption curve of unmodified empty column and pore size analysis.

[0029] Figure 7 Schematic diagram of the electroosmotic flow results of three different capillary columns at different pH values.

[0030] Figure 8 Schematic diagram of thermogravimetric analysis of the metal organic framework material prepared in Example 1.

[0031] Fig. 9 Schematic diagram of the acid-base stability of the metal organic framework material prepared in Example 1.

[0032] Fig.10 Schematic diagram of the storage stability of the metal organic framework material prepared in Example 1.

[0033] Fig.11 A schematic diagram showing the results of verifying the stability of the capillary column prepared in Example 1 using chiral proline as an example.

[0034] Fig.12 Schematic diagram of the overall morphology of the metal organic framework material prepared in Example 3 observed under a scanning electron microscope at scales of 1, 3, 5, and 10 microns. DETAILED DESCRIPTION

[0035] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit 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 that can be used for chiral separation comprises the following steps:

[0038] The first step is capillary pretreatment

[0039] Using nitrogen purge, pass 1.5 mL of 1 mol / L sodium hydroxide aqueous solution, 1.5 mL of 1 mol / L hydrochloric acid aqueous solution, and 1.5 mL of methanol into a 2 m long capillary column in turn and rinse for 60 min, 20 min, and 30 min. Between each two solvents, rinse with 15 mL of ultrapure water for 15 min before flushing the next reagent. After the methanol flushing is complete, blow dry the residual solvent in the capillary column with nitrogen and place it in an oven at 100 ° C to dry for 1 hour.

[0040] Step 2: Functional group modification

[0041] 15 mL of 50% 3-aminopropyltriethoxysilane aqueous solution was introduced into the pretreated capillary column. After 15 minutes, both ends of the capillary column were sealed with rubber and placed in a water bath at 55°C for heating reaction for 12 hours. After taking out, the rubber sealing parts at both ends were cut off, and the unreacted 3-aminopropyltriethoxysilane in the column was flushed out with 30 mL of methanol to complete the amino modification and obtain a capillary column modified with functional groups.

[0042] Step 3: Preparation of ligands

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

[0044] Step 4: Ligand pre-anchoring

[0045] The gel-like ligand product (0.83mmol, 207mg) obtained in the third step, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (1mmol, 191.7mg) and N-hydroxysuccinimide ester (0.2mmol, 23mg) were dissolved in 5mL of 100mM sodium dihydrogen phosphate buffer solution, and ultrasonically activated for 15min to obtain a mixed solution, and then the mixed solution was flushed into the functional group-modified capillary column obtained in the second step by nitrogen blowing for reaction. The nitrogen purge was closed for 15min every time the mixed solution was passed into the capillary for 1h, so that the ligand reacted fully with the amino group on the inner wall of the capillary. After repeating three times, it was rinsed with ultrapure water for 30min to clean the residual mixed solution and obtain a capillary column pre-anchored with the ligand.

[0046] Step 5: Preparation of chromatographic columns

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

[0048] The above metal organic framework material solution is passed into the ligand pre-anchored capillary column obtained in the fourth step. After 1 hour of passing, the gas valve is closed, and the column is allowed to react for 30 minutes, and then rinsed with ultrapure water for 30 minutes to obtain the capillary chromatographic column based on metal organic framework materials that can be used for chiral separation, which is recorded as NLMOF chiral column.

[0049] The prepared NLMOF chiral column was characterized by scanning electron microscopy. Figure 1 As shown, Figure 1 This is a schematic diagram of the overall morphology of the capillary chromatographic column prepared in Example 1 observed under a scanning electron microscope at a scale of 3 microns. 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) has been successfully modified to the inner surface of the capillary.

[0050] The prepared NLMOF chiral column was installed on a capillary electrophoresis instrument (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 is methanol / 0.1M HCl aqueous solution, with a volume ratio of 3:7. The buffer solution is 20mM sodium dihydrogen phosphate aqueous solution, pH 7; the electrophoresis separation voltage is 15kV. Separation conditions: methanol / 0.1M HCl aqueous solution (pH=7) = 30 / 70, injection time: 5s, separation temperature: room temperature.

[0052] Donepezil separation conditions: the solvent for dissolving donepezil is 50% methanol aqueous solution; the buffer solution is 15mM sodium hydrogen phosphate aqueous solution, pH 10; the electrophoresis separation voltage is 9kV. Separation conditions: methanol / water = 50 / 50, injection time: 5s, separation temperature: room temperature.

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

[0054] Figure 3 The overall morphology of the metal organic framework material prepared in Example 1 observed under a scanning electron microscope is shown in the figure. It can be seen from the figure that the NLMOF crystal has the best shape, which is a regular hexagonal prism, proving that the NLMOF material was successfully synthesized.

[0055] Figure 4 Schematic diagram of the results detected by Fourier transform infrared spectrometer of the metal organic framework material prepared in Example 1. It was observed that the C=N characteristic peak of NL ligand and NLMOF appeared at 1690 cm -1-1590cm -1 The NL ligand after L-lysine modification of the triazole ring showed a strong C=N bond characteristic peak at 1639cm-1 and a strong C=N bond characteristic peak at 1360cm-1. -1 -1020cm -1 Multiple CN bond characteristic peaks that were not previously found appeared at the site, indicating that the triazole ring was successfully modified onto L-lysine. The C=N bond of NLMOF synthesized by the coordination of NL ligand and divalent copper ion showed a blue shift (1639cm -1 →1626cm -1 ), so it is judged that NL is successfully coordinated with metal ions, that is, the NLMOF material is successfully synthesized.

[0056] Table 1 shows the separation of chiral amino acids and chiral drugs by the empty tube column, ligand column (ligand pre-anchored capillary column obtained in the fourth step) and NLMOF chiral column prepared in Example 1, wherein t1 and t2 are the retention times of two monomers after the chiral compound is separated, and Rs is the separation degree between monomers after the chiral compound is separated. As can be seen from Table 1, the unmodified empty tube column and the ligand column modified with ligand have no separation phenomenon for dansyl amino acid and chiral drug, while the NLMOF chiral column can achieve enantiomer separation, indicating that the NLMOF modified capillary column has good separation efficiency and separation ability for chiral compounds. In Table 1, the Rs value of the NLMOF chiral column for separating compounds is greater than 1.5, achieving baseline separation, while the Rs value of the empty tube column and the ligand column is 0.

[0057] Table 1 Separation data of chiral compounds

[0058]

[0059] Table 2 shows the repeatability data of NLMOF chiral column:

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

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

[0062]

[0063] Testing method for nitrogen adsorption-desorption curve: 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, and the adsorption amount at the partial pressure point is obtained by the gas state equation; the adsorption equilibrium pressure is increased by gradually adding adsorbate gas to obtain the adsorption isotherm; the adsorption equilibrium pressure is reduced by gradually withdrawing the adsorbate gas to obtain the desorption isotherm.

[0064] Figure 5 Schematic diagram of nitrogen adsorption-desorption curve and pore size analysis of the capillary chromatographic column prepared in Example 1. Figure 6 The figure shows the nitrogen adsorption-desorption curve of the unmodified empty column and the schematic diagram of its pore size analysis. Figure 5 and Figure 6 It can be seen that the specific surface area of ​​the capillary chromatographic column prepared in Example 1 of the present invention is 4.61672 m 2 / g, which is much larger than the specific surface area of ​​the unmodified empty column (0.44048m 2 The above data prove that the modification of NLMOF significantly increases the pore adsorption volume of the empty tube column.

[0065] Figure 8 This is a schematic diagram of the thermogravimetric analysis of the metal organic framework material prepared in Example 1. As can be seen from the figure, NLMOF only shows a large mass loss when it is close to 200°C, indicating that the formed structure of NLMOF is more stable. In actual application, CEC is basically operated at room temperature, and the material can show good thermal stability under conventional application environments.

[0066] Fig. 9 Schematic diagram of the acid-base stability of the metal organic framework material prepared in Example 1. The NLMOF metal organic framework material was placed in a sodium dihydrogen phosphate buffer solution with a pH of 4 or pH of 9 for one week, and then washed with ultrapure water and dried. The peak position and intensity of the infrared characteristic peak measured did not change significantly compared with the initial peak, indicating that the material has the potential to be suitable for long-term separation operations in a wide range of pH buffer environments.

[0067] Fig.10 This is a schematic diagram of the storage stability of the metal organic framework material prepared in Example 1. The infrared characteristic peaks of NLMOF did not change significantly when placed at room temperature for up to six months, indicating that the material has good storage stability.

[0068] Fig.11The schematic diagram of the results of the capillary column stability verification using chiral proline as an example for preparing the NLMOF chiral column in Example 1. The results show that the NLMOF chiral column can stably run 100 times for the separation of chiral proline and racemic donepezil, and the separation effect is stable. The peak times are slightly different but within the acceptable fluctuation range, and the peak shape is still relatively sharp, so the NLMOF chiral column also shows good operation stability.

[0069] Example 2

[0070] A method for preparing a capillary chromatographic column based on a metal organic framework material that can be used for chiral separation comprises the following steps:

[0071] The first step is capillary pretreatment

[0072] Using nitrogen purge, pass 1.5 mL of 1 mol / L sodium hydroxide aqueous solution, 1.5 mL of 1 mol / L hydrochloric acid aqueous solution, and 1.5 mL of methanol into a 2 m long capillary column in turn and rinse for 60 min, 20 min, and 30 min. Between each two solvents, rinse with 15 mL of ultrapure water for 15 min before flushing the next reagent. After the methanol flushing is complete, blow dry the residual solvent in the capillary column with nitrogen and place it in an oven at 100 ° C to dry for 1 hour.

[0073] Step 2: Functional group modification

[0074] 15 mL of 50% 3-aminopropyltriethoxysilane aqueous solution was introduced into the pretreated capillary column. After 15 minutes, both ends of the capillary column were sealed with rubber and placed in a water bath at 55°C for heating reaction for 12 hours. After taking out, the rubber sealing parts at both ends were cut off, and the unreacted 3-aminopropyltriethoxysilane in the column was flushed out with 30 mL of methanol to complete the amino modification and obtain a capillary column modified with functional groups.

[0075] Step 3: Preparation of ligands

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

[0077] Step 4: Ligand pre-anchoring

[0078] The gel-like ligand product (0.83mmol, 207mg) obtained in the third step, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (1mmol, 191.7mg) and N-hydroxysuccinimide ester (0.2mmol, 23mg) were dissolved in 5mL of 100mM sodium dihydrogen phosphate buffer solution, and ultrasonically activated for 15min to obtain a mixed solution, and then the mixed solution was flushed into the functional group-modified capillary column obtained in the second step by nitrogen blowing for reaction. The nitrogen purge was closed for 15min every time the mixed solution was passed into the capillary for 1h, so that the ligand reacted fully with the amino group on the inner wall of the capillary. After repeating three times, it was rinsed with ultrapure water for 30min to clean the residual mixed solution and obtain a capillary column pre-anchored with the ligand.

[0079] Step 5: Preparation of chromatographic columns

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

[0081] The above metal organic framework material solution is passed into the ligand pre-anchored capillary column obtained in the fourth step. After 1 hour of passing, the gas valve is closed, the column is allowed to react for 30 minutes, and then rinsed with ultrapure water for 30 minutes to obtain the capillary chromatographic column based on metal organic framework materials that can be used for chiral separation.

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

[0083] Figure 7 The figure is a schematic diagram of the electroosmotic flow results of three different capillary columns at different pH values. The three different capillary columns are an empty column, a ligand column (a capillary column pre-anchored with ligands obtained in the fourth step) and a capillary chromatographic column prepared in an embodiment of the present invention. The electroosmotic flow of the three capillary columns at different pH values ​​is different, and the change trend is consistent. This further proves that the NL ligand and NLMOF are successfully modified and coated on the inner wall of the capillary column, and the NLMOF chiral column is successfully prepared.

[0084] Compared with the existing public documents, the capillary prepared by the present 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 that can be used for chiral separation comprises the following steps:

[0087] The first step is capillary pretreatment

[0088] Using nitrogen purge, pass 1.5 mL of 1 mol / L sodium hydroxide aqueous solution, 1.5 mL of 1 mol / L hydrochloric acid aqueous solution, and 1.5 mL of methanol into a 2 m long capillary column in turn and rinse for 60 min, 20 min, and 30 min. Between each two solvents, rinse with 15 mL of ultrapure water for 15 min before flushing the next reagent. After the methanol flushing is complete, blow dry the residual solvent in the capillary column with nitrogen and place it in an oven at 100 ° C to dry for 1 hour.

[0089] Step 2: Functional group modification

[0090] 15 mL of 50% 3-aminopropyltriethoxysilane aqueous solution was introduced into the pretreated capillary column. After 15 minutes, both ends of the capillary column were sealed with rubber and placed in a water bath at 55°C for heating reaction for 12 hours. After taking out, the rubber sealing parts at both ends were cut off, and the unreacted 3-aminopropyltriethoxysilane in the column was flushed out with 30 mL of methanol to complete the amino modification and obtain a capillary column modified with functional groups.

[0091] Step 3: Preparation of ligands

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

[0093] Step 4: Ligand pre-anchoring

[0094] The gel-like ligand product (0.83mmol, 207mg) obtained in the third step, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (1mmol, 191.7mg) and N-hydroxysuccinimide ester (0.2mmol, 23mg) were dissolved in 5mL of 100mM sodium dihydrogen phosphate buffer solution, and ultrasonically activated for 15min to obtain a mixed solution, and then the mixed solution was flushed into the functional group-modified capillary column obtained in the second step by nitrogen blowing for reaction. The nitrogen purge was closed for 15min every time the mixed solution was passed into the capillary for 1h, so that the ligand reacted fully with the amino group on the inner wall of the capillary. After repeating three times, it was rinsed with ultrapure water for 30min to clean the residual mixed solution and obtain a capillary column pre-anchored with the ligand.

[0095] Step 5: Preparation of chromatographic columns

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

[0097] An appropriate amount of metal organic framework material solution is passed into the ligand pre-anchored capillary column obtained in the fourth step. After 1 hour of passing, the gas valve is closed, the column is allowed to react for 30 minutes, and then 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 Fig.12 As shown, Fig.12 This is a schematic diagram of the overall morphology of the metal organic framework material prepared in Example 3 observed under a scanning electron microscope at 1, 3, 5, and 10 micrometer scales. Fig.12 It can be clearly seen that the appearance morphology of the metal-organic framework material is spherical with a diameter of about 1μm to 3μm, indicating that the metal-organic framework material was successfully synthesized.

[0099] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution 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: The following steps are involved: A 40-60% 3-aminopropyltriethoxysilane solution is introduced into the pretreated capillary column, and after 10-30 minutes, both ends of the capillary column are sealed with rubber, and the column is heated at 50-60° C. for reaction for 1-12 hours, and the rubber sealing parts at both ends are cut off after the column is taken out, and the column is rinsed with methanol to complete the amino modification, thereby obtaining a capillary column modified with functional groups; The gel-like ligand product, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide ester in a molar ratio of 0.1 to 1:1:0.05 to 0.5 are dissolved in a sodium dihydrogen phosphate buffer solution with a concentration of 50 to 150 mM, and ultrasonically activated for 10 to 30 minutes to obtain a mixed solution, and then the mixed solution is flushed into a functional group-modified capillary column by nitrogen blowing to react, and the nitrogen purge is closed for a period of time after the mixed solution is passed into the capillary and allowed to stand, and after repeating at least three times, it is rinsed with ultrapure water to obtain a ligand pre-anchored capillary column; Slowly drop a monohydrated copper acetate aqueous solution into a gel-like ligand product aqueous solution, wherein the molar ratio of the monohydrated copper acetate to the gel-like ligand product is 1:1-3, and react for 10-60 minutes to obtain a metal organic framework material solution; The metal organic framework material solution is passed into the obtained ligand pre-anchored capillary column, and the column is closed after 0.5 to 2 hours of passing. The column is allowed to react for 10 to 60 minutes and rinsed with ultrapure water for 10 to 60 minutes to obtain the capillary chromatographic column based on the metal organic framework material 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 of the pretreated capillary column is as follows: Using nitrogen purging, pass a 0.5-1.5 mol / L sodium hydroxide aqueous solution, a 0.5-1.5 mol / L hydrochloric acid aqueous solution and methanol into a 1.5 m to 3 m long capillary column in turn and rinse for 50-80 min, 10-30 min and 20-40 min; the volume ratio of the sodium hydroxide aqueous solution, the hydrochloric acid aqueous solution and the methanol is 1:1:1, and between each two solvents, rinse with ultrapure water for 10-30 min and then rinse the next reagent. When the volume ratio of the ultrapure water to the sodium hydroxide aqueous solution is 1:5-20 and the methanol is rinsed, use nitrogen to dry the residual solvent in the capillary column, and dry it at a temperature of 95-110°C for 0.5-2h.

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-dimethylmethylhydrazineamide and the ligand in a molar ratio of 1 to 8:1 are dissolved in ethanol, heated, stirred and refluxed for 1 to 48 hours, and the solvent is removed to obtain the gel 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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