Capillary electrochromatographic column for separating chiral tyrosine and preparation method thereof

Abstract

This invention discloses a capillary electrochromatographic column for the separation of chiral tyrosine and its preparation method, belonging to the field of chemical synthesis and analytical technology. It includes a capillary and a composite coating fixed to the inner wall of the capillary. The composite coating comprises chiral metal-organic frameworks (CMOFs) and chiral molecularly imprinted polymers (CMIPs) modified on the surface of the CMOFs. The CMOFs are formed by coordination reaction between MIL-101 (Cr) and L-tyrosine as a chiral ligand. The composite coating (CMIP@CMOF) is a multi-level composite structure formed by in-situ polymerization of the CMOFs as a support in a system containing a crosslinking agent, functional monomer, initiator, and L-tyrosine as a template molecule. The separation system constructed by this coated column exhibits excellent separation performance, with a resolution (Rs) of 5.83 and a selectivity factor (α) of 1.33 for DL-tyrosine enantiomers.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis and analysis technology, specifically relating to a capillary electrochromatographic column for the separation of chiral tyrosine and its preparation method. Background Technology

[0002] Chirality is a core characteristic in the life sciences, playing a decisive role in the function, metabolic processes, and physiological effects of bioactive substances, and widely influencing key areas such as drug development, disease diagnosis, and pathological mechanism research. L-tyrosine and D-tyrosine, as enantiomers, have completely identical chemical compositions and elemental ratios, but their mirror-image spatial configurations lead to drastically different physiological activities and pathological effects. L-tyrosine, as a core precursor in the synthesis of catecholamine neurotransmitters (including dopamine, adrenaline, and noradrenaline), directly affects the normal functioning of the nervous system due to its metabolic homeostasis. In Parkinson's disease patients, the metabolism of L-tyrosine is significantly disrupted, resulting in a 30% to 50% reduction in its concentration in cerebrospinal fluid compared to healthy individuals. This metabolic abnormality directly leads to dopamine deficiency, thereby impairing the signal transduction function of the nervous system, accelerating the degenerative process of nerve cells, and exacerbating the patient's pathological symptoms. Conversely, D-tyrosine is abnormally highly expressed in the tumor microenvironment, especially in breast cancer tissue, where its content can be 2-3 times that of normal tissue. D-tyrosine specifically activates the EGFR signaling pathway, regulates the expression of genes related to tumor cell proliferation, migration, and invasion, disrupts the normal cell growth regulation mechanism, and ultimately promotes abnormal proliferation and malignant progression of tumor cells.

[0003] Achieving efficient, rapid, and stable separation of DL-tyrosine is a crucial prerequisite for in-depth exploration of its related pathological mechanisms, clarifying its role in disease development, developing targeted therapies, and conducting clinical testing. It holds significant importance for life science research and the development of the pharmaceutical field. Capillary electrochromatography (CEC), as a novel and highly efficient chiral separation technique, cleverly combines the high separation efficiency and high analytical speed of capillary electrophoresis with the high selectivity and high separation precision of liquid chromatography. It has demonstrated excellent application potential and broad development prospects in the field of chiral compound separation and has become an important technical means in chiral separation research.

[0004] However, this technology still has significant limitations in the application of DL-tyrosine separation. The core problem lies in the inherent defects of traditional chiral stationary phases: for example, although single chiral metal-organic frameworks (CMOFs) have advantages such as structural stability and large specific surface area, their specific recognition ability for tyrosine enantiomers is insufficient, making it difficult to achieve efficient and accurate separation of the two, and the separation effect cannot meet the needs of practical applications; while single chiral molecularly imprinted polymers (CMIPs), although they have the characteristics of strong specific recognition ability, low preparation cost and simple preparation process, have poor stability of their porous structure. In buffer systems with different pH values ​​and different ionic strengths, they are prone to obvious expansion and contraction. This structural change will cause deformation of the recognition cavity, which will seriously affect the reproducibility and stability of the separation results, limiting their large-scale application in actual separation and detection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a capillary electrochromatographic column for the separation of chiral tyrosine and its preparation method, thereby solving the problems in the prior art.

[0006] The objective of this invention can be achieved through the following technical solutions: A capillary electrochromatographic column for the separation of chiral tyrosine comprises: a capillary and a composite coating fixed to the inner wall of the capillary; characterized in that the composite coating comprises CMOFs and chiral molecularly imprinted polymers (CMIPs) modified on the surface of the CMOFs. The CMOFs are formed by a coordination reaction between MIL-101 (Cr) and L-tyrosine, which acts as a chiral ligand. The composite coating (CMIP@CMOF) is a multi-level composite structure formed by in-situ polymerization of the CMOFs as a support carrier in a system containing crosslinking agents, functional monomers, initiators and L-tyrosine as template molecules.

[0007] Furthermore, the capillary is a fused silica capillary.

[0008] Furthermore, the functional monomer is (S)-3-phenyl-2-(undeca-10-enamide)propionic acid, the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is azobisisobutyronitrile.

[0009] The above-mentioned method for preparing a capillary electrochromatographic column for the separation of chiral tyrosine includes the following steps: S1, the capillary tube is rinsed sequentially with alkaline solution, deionized water and acid solution, deionized water and methanol, and then dried; S2, synthesize MIL-101(Cr) and activate it under vacuum heating. Then, remove the residual air under vacuum to obtain pure MIL-101(Cr). Mix the obtained pure MIL-101(Cr) with L-tyrosine dissolved in solvent and carry out a static reaction at an isothermal temperature. After separation and drying, CMOFs are obtained. S3. Prepare a CMOF dispersion and mix it evenly with functional monomers, crosslinking agents, initiators, and L-tyrosine as template molecules to obtain a polymerization mixture. Apply external pressure to draw the polymerization mixture into a washed and dried capillary for in-situ polymerization. After the reaction is completed, remove the template molecules to obtain the capillary electrochromatographic column.

[0010] Furthermore, the process for synthesizing MIL-101(Cr) includes: Cr(NO3)3·9H2O and 1,4-phthalic acid were dissolved in deionized water and subjected to a static reaction at a constant temperature. The reaction product was then washed, purified, centrifuged, and dried to obtain the final product.

[0011] Furthermore, the temperature for vacuum heating activation of MIL-101(Cr) was 120°C, and the activation time was 24 hours.

[0012] Furthermore, the isothermal static reaction is carried out at a temperature of 70°C for 3 days, and anhydrous ethanol is used as the solvent.

[0013] Furthermore, the applied external pressure is 750 mbar; the in-situ polymerization reaction is carried out under the condition of reacting in a constant temperature water bath at 70°C for 40 minutes.

[0014] The above-mentioned capillary electrochromatographic column for the separation of chiral tyrosine is applied to the enantiomeric separation of DL-tyrosine.

[0015] The applications include: During the capillary electrochromatographic separation process, the buffer solutions used include 10 mM Na2HPO4 and 10 mM NaH2PO4, and both the DL-tyrosine sample solution and the buffer solutions are filtered through a 0.45 µm organic filter membrane before use.

[0016] The beneficial effects of this invention are: 1. This invention employs in-situ polymerization to modify CMIPs onto the surface of CMOFs, constructing a multi-level composite structure. Due to the rigid framework structure of CMOFs, they provide strong physical stability and support for CMIPs. This effectively avoids the expansion and contraction phenomena that easily occur in buffer systems with different pH values ​​and ionic strengths for single CMIPs materials, preventing deformation of the recognition cavity, thereby significantly improving the reproducibility and stability of the separation process. 2. The core carrier of this invention is a MIL-101(Cr) metal-organic framework with a large specific surface area. This structural feature effectively increases the loading of CMIPs on the carrier surface. The abundant loading ensures that a sufficient number of specific recognition sites participate in the recognition of enantiomers during chromatographic separation, thereby effectively improving the separation efficiency of tyrosine enantiomers.

[0017] 3. This method generates a CMIPs polymer network in situ on the inner wall of the capillary and the surface of CMOFs. These CMIPs not only possess molecular recognition capabilities but also significantly enhance the adhesion stability of the composite stationary phase material on the inner wall of the capillary. This effectively prevents the stationary phase from detaching during chromatographic washing, further improving the stability and lifespan of the actual separation and detection operations.

[0018] 4. In the synthesis of CMOFs, this invention utilizes the strong coordination between the Cr center and L-tyrosine to precisely construct chiral recognition sites. Simultaneously, after activation and cooling in MIL-101, residual air is innovatively removed by slowly introducing N2 gas. This specific process eliminates interference from air impurities, effectively enhancing the coordination ability of the framework, thereby significantly improving the initial recognition specificity of the underlying skeleton for tyrosine enantiomers. 5. This invention integrates the structural advantages of CMOFs (high specific surface area, strong coordination, and rigid support) with the molecular-specific recognition characteristics of CMIPs, forming a synergistic system with complementary advantages. Experimental data show that, compared with using CMIPs alone (resolution 0.53) or CMOFs alone (resolution 0.61) as stationary phases, the CMIP@CMOF coated capillary electrochromatographic column prepared in this invention achieves baseline separation of DL-tyrosine racemic compounds, with a significant increase in resolution to 5.83, demonstrating extremely excellent chiral resolution performance. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the preparation process of L-Tyr@CMIP@MIL-101-L-Tyr(Cr)@capillary; Figure 2 This is a scanning electron microscope image of MIL-101-L-Tyr(Cr)@capillary; Figure 3This is a scanning electron microscope image of L-Tyr@CMIP@MIL-101-L-Tyr(Cr)@capillary; Figure 4 This is a scanning electron microscope image of MIL-101-L-Tyr(Cr) (CMOFs); Figure 5 This is a scanning electron microscope image of L-Tyr@CMIP@MIL-101-L-Tyr(Cr); Figure 6 The Fourier transform infrared absorption spectra of MIL-101-L-Tyr(Cr) and L-Tyr@CMIP@MIL-101-L-Tyr(Cr) are shown. Figure 7 The results are the CEC chiral resolutions of D-tyrosine, DL-tyrosine, and L-tyrosine by L-Tyr@CMIP@MIL-101-L-Tyr(Cr)@capillary. Figure 8 This is a comparison of the chiral resolution performance of DL-tyrosine on capillary coated columns with MIL-101-L-Tyr(Cr) and CMIP as stationary phases alone, and L-Tyr@CMIP@MIL-101-L-Tyr(Cr)@capillary. Figure 9 The results show the CEC chiral separation of DL-tyrosine by L-Tyr@CMIP@MIL-101-L-Tyr(Cr)@capillary at different CMOF concentrations. Figure 10 The results show the CEC chiral resolution of DL-tyrosine by L-Tyr@CMIP@MIL-101-L-Tyr(Cr) @capillary at different acetonitrile percentages. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] This invention, through in-depth research and repeated experiments, designs and synthesizes a novel L-Tyr@CMIP@MIL-101-L-Tyr(Cr) nanomaterial, aiming to achieve synergistic, efficient chiral recognition and precise separation of DL-tyrosine. This novel nanomaterial uses MIL-101(Cr) as the core carrier, fully utilizing its advantages of structural stability, large specific surface area, and strong coordination ability. By leveraging the strong coordination between Cr³⁺ and tyrosine, stable and efficient chiral recognition sites are precisely constructed on its surface, enhancing the recognition specificity for tyrosine enantiomers. Subsequently, in-situ polymerization is used to uniformly modify the surface of CMOFs with CMIPs, forming a structurally stable and complementary multi-level composite structure, efficiently leveraging the advantages of both materials. Among them, the high specific surface area of ​​CMOFs can effectively increase the loading capacity of CMIPs, ensuring that there are enough recognition sites to participate in enantiomer recognition. Its rigid framework structure can provide stable support for CMIPs, effectively preventing CMIPs from deforming in the buffer system. At the same time, CMIPs can significantly enhance the adhesion stability of the stationary phase on the inner wall of the capillary, prevent the stationary phase from falling off, and further improve the stability of the separation process. By effectively integrating the structural advantages of CMOFs with the molecular recognition characteristics of CMIPs, the separation efficiency, resolution effect and separation reproducibility of tyrosine enantiomers are significantly improved, meeting the needs of practical research and application.

[0023] like Figure 1 As shown, a method for preparing a capillary electrochromatographic column for the separation of chiral tyrosine includes the following steps: S1, Capillary Pretreatment First, rinse the capillary with sodium hydroxide solution for 1 hour, then rinse with deionized water for 5 minutes, followed by rinsing with hydrochloric acid solution for 30 minutes. Next, rinse with deionized water until neutral, rinse with methanol for 30 minutes, and then dry with nitrogen (N2). After treatment, place the capillary column in a forced-air drying oven at 100°C for 1 hour. Once drying is complete, remove and set aside for later use. In S1, the capillary is an unmodified fused silica capillary with an inner diameter of 75 μm, a total length of 40 cm, and an effective length of 8.5 cm; the concentrations of the sodium hydroxide solution and the hydrochloric acid solution are both 1 mol·L⁻¹. -1 .

[0024] Synthesis of S2,MIL-101-L-Tyr(Cr)(CMOFs) Cr(NO3)3·9H2O and 1,4-phthalic acid were added to deionized water, sonicated until completely dissolved and homogenized to ensure thorough mixing of the reactants. The mixture was then transferred to a polytetrafluoroethylene-lined autoclave and heated at a constant temperature. After the reaction was complete, it was allowed to cool naturally to room temperature to obtain a green solid product. The product was collected by high-speed centrifugation and washed with N,N-dimethylformamide (DMF) with stirring to remove unreacted 1,4-phthalic acid. The product was then dissolved in chloroform at room temperature and stirred overnight for further purification, followed by high-speed centrifugation again. The resulting green product was dried overnight in a vacuum drying oven to remove residual solvent. The dried product, MIL-101(Cr), was further activated by heating in a vacuum drying oven to enhance its coordination ability. After cooling to room temperature, N2 gas was slowly introduced under vacuum to remove residual air, yielding pure MIL-101(Cr). L-tyrosine was dissolved in a solvent and stirred until completely dissolved. Activated purified MIL-101(Cr) was added to the ligand solution, and the reaction was carried out at a constant temperature to ensure complete coordination between L-tyrosine and MIL-101(Cr). After the reaction was complete, the green product was recovered by high-speed centrifugation. Uncoordinated L-tyrosine was removed by washing, and the product was finally dried under vacuum to obtain CMOFs, which were then sealed and stored for later use.

[0025] In S2: the activation temperature of MIL-101(Cr) is 120℃, the activation time is 24h, and N2 gas needs to be introduced under vacuum after activation; the coordination reaction temperature of L-tyrosine with MIL-101(Cr) is 70℃, the reaction time is 3 days, and the coordination reaction solvent is anhydrous ethanol; the final drying conditions of CMOFs are vacuum drying at 80℃ for 8h, and they need to be washed with anhydrous ethanol and acetone in sequence before drying.

[0026] Preparation of S3, L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coated columns CMOFs were added to deionized water and sonicated to obtain a uniform dispersion, preventing CMOF aggregation. A coated column was prepared by in-situ polymerization using ethylene glycol dimethacrylate (EDMA) as a crosslinking agent, (S)-3-phenyl-2-(undec-10-enamide)propionic acid as a functional monomer, L-tyrosine as a template molecule, and azobisisobutyronitrile (AIBN) as an initiator. The specific steps are as follows: First, L-tyrosine and (S)-3-phenyl-2-(undec-10-enamide)propionic acid were added to a mixture of anhydrous ethanol and methanol and sonicated until completely dissolved, ensuring sufficient interaction between the template molecule and the functional monomer. Then, EDMA, AIBN, and the CMOF dispersion were added to the solution, and sonicated again to form a uniform polymerization mixture. External pressure was applied, and the polymerization mixture was slowly drawn into a pretreated silica capillary column, ensuring the mixture filled the entire capillary lumen. The capillary column was then placed in a constant-temperature water bath to allow the polymerization reaction to proceed fully, forming a stable coating. The capillary column was then rinsed with eluent to completely remove the template molecules, yielding an L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coated column for later use.

[0027] In S3: the in-situ polymerization reaction temperature is 70℃, and the reaction time is 40 min; the eluent for the template molecules is a mixture of methanol and acetic acid with a volume ratio of 90:10, the elution time is 2 h, and the elution flow rate is 0.5 mL / min; the ultrasonic treatment power is 200 W, and each ultrasonic treatment lasts for 5 min; the aspiration pressure of the polymerization mixture is 750 mbar. All solutions (including test samples) are filtered through a 0.45 μm organic filter membrane before use.

[0028] The application of L-Tyr@CMIP@MIL-101-L-Tyr (Cr) coated columns is described below: CEC resolution of tyrosine enantiomers: The L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coated column prepared by S3 above was used as the CEC separation column. The running buffer used with it was prepared by dissolving disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4) in deionized water. After preparation, ultrasonic degassing was required to remove air bubbles and avoid affecting the separation effect. L-tyrosine, DL-tyrosine, and D-tyrosine were prepared by dissolving them in a water-methanol (1:1, volume ratio) mixture at room temperature. During the separation process, the column temperature was strictly controlled at 25℃, the detection wavelength was fixed at 220nm, and the injection conditions were 30 mbar for 3 s. The separation voltage, acetonitrile volume fraction, CMOF concentration, and buffer pH were adjusted. All experimental solutions (including the test samples) were filtered through a 0.45 μm organic phase filter membrane before use.

[0029] The pH of the buffer solution is adjusted to 7.0; the buffer solution is subjected to ultrasonic degassing for 15 minutes after preparation; the sample solution is prepared at room temperature; the separation voltage is set to 20 kV; the organic modifier (acetonitrile volume fraction) is 60%~90%; the CMOFs concentration is 1~4 mg / mL; the sample solution (L-tyrosine, DL-tyrosine, D-tyrosine) concentration is 2 mg / mL.

[0030] The technical solution of the present invention will be described in detail below through the following embodiments, wherein the sources of the relevant raw materials are as follows: Fused silica capillary tube: purchased from Hebei Yongnian Ruifeng Chromatography Instruments Co., Ltd.; specifications: inner diameter 75μm, total length 40cm, effective length 8.5cm; L-Tyrosine: Purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number (L818844). DL-Tyrosine: Purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number (T6193). D-Tyrosine: Purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number (D818619).

[0031] Example 1 In this embodiment, the preparation of the L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coated column is described, starting with 1 mol·L⁻¹ - 1 Rinse the capillary tube with NaOH solution for 1 hour, then rinse with water for 5 minutes. (1 mol·L⁻¹) -1 The capillary was rinsed with HCl solution for 30 min, then rinsed with deionized water until neutral, rinsed with methanol for 30 min, and then dried with N2. The treated capillary column was placed in a forced-air drying oven at 100℃ for 1 h. After drying, it was taken out for use. The capillary used was an unmodified fused silica capillary with an inner diameter of 75 μm, a total length of 40 cm, and an effective length of 8.5 cm. 5 mmol of chromium nitrate nonahydrate (Cr(NO3)3・9H2O) and 5 mmol of terephthalic acid were added to 25 mL of water and sonicated for 30 minutes to ensure complete dissolution and homogeneity. The mixture was then transferred to a reaction vessel and hydrothermally reacted at 220 °C for 8 h, yielding a green product. The product was collected by centrifugation, washed three times with DMF (50 mL each time, stirred for 20 minutes each time), dissolved in chloroform at room temperature and stirred overnight, and then separated again by centrifugation. The resulting green product was dried overnight in a vacuum oven at 80 °C. The dried product was further heated in a vacuum oven at 120 °C for 24 h to activate 1.5 mmol of MIL-101. After cooling the activated product to room temperature, nitrogen gas was slowly introduced under vacuum to obtain MIL-101. Subsequently, 3.5 mmol of L-tyrosine was dissolved in ethanol, and 1.5 mmol of activated MIL-101 was added to this ligand solution. The reaction mixture was statically reacted at 70 °C for three days. After the reaction was completed, the green product was recovered by centrifugation, washed three times with ethanol and acetone respectively, and finally dried under vacuum at 80 °C for 8 h to obtain CMOFs, which were then sealed and stored for later use.

[0032] Subsequently, (2 mg) CMOFs were added to 1 mL of deionized water and sonicated for 5 min to obtain a uniform dispersion. Using EDMA as a crosslinking agent, (S)-3-phenyl-2-(undec-10-enamido)propionic acid as a functional monomer, L-tyrosine as a template molecule, and AIBN as an initiator, a coated column was prepared by in-situ polymerization. The specific steps are as follows: First, 2 mg L-tyrosine and 309 mg (S)-3-phenyl-2-(undec-10-enamido)propionic acid were dissolved in a mixture of 4.5 mL ethanol and 3 mL methanol and sonicated for 5 min until completely dissolved. Then, 339 μL of EDMA, 2 mg of AIBN, and 0.5 mL of CMOFs dispersion were added to the solution, and sonicated again for 5 min. In a CEC apparatus, the polymerization mixture was drawn into a pretreated quartz capillary column (effective length: 8.5 cm, total length: 40 cm) under an external pressure of 750 mbar. The capillary column was placed in a 70 °C constant temperature water bath for 40 min, and then the entire capillary column was rinsed with a methanol-acetic acid mixture (volume ratio 90:10) for 2 h to remove template molecules, and finally L-Tyr@CMIP@ MIL-101-L-Tyr(Cr) coated column was prepared for use.

[0033] Example 2 This embodiment describes how to perform CEC detection and splitting under different test conditions; Test condition 1: After accurately weighing the racemic DL-tyrosine, it was dissolved in a mixture of water and methanol (1:1, V / V) and stirred at room temperature until completely dissolved to prepare a homogeneous sample solution (2 mg / mL). All solutions (including the test sample) were filtered through a 0.45 μm organic filter membrane before use. The L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coated column prepared in Example 1 was used as a CEC column for CEC separation of DL-tyrosine enantiomers. The separation temperature was 25℃, and the column was run for 10 min to obtain a stable baseline. The capillary electrochromatographic detection performance conditions were as follows: the buffer composition was 10 mM Na2HPO4 and 10 mM NaH2PO4 (pH 7.0), and the buffer was degassed by sonication for 15 min after preparation. The separation voltage was 20 kV, the CMOF concentration was 2 mg / mL, the acetonitrile volume fraction was 80%, the injection pressure was 30 mbar, and the injection time was 3 s. All capillary electrochromatographic data were collected and analyzed at 220 nm.

[0034] Test condition 2: The only difference between test condition 2 and test condition 1 is that the concentration of CMOFs is 1 mg / mL; Test condition 3: The only difference between test condition 3 and test condition 1 is that the concentration of CMOFs is 3 mg / mL; Test condition 4: The only difference between test condition 4 and test condition 1 is that the concentration of CMOFs is 4 mg / mL. Test condition 5: The only difference between test condition 5 and test condition 1 is that the acetonitrile volume fraction is 60%. Test condition 6: The only difference between test condition 6 and test condition 1 is that the acetonitrile volume fraction is 70%. Test condition 7: The only difference between test condition 7 and test condition 1 is that the acetonitrile volume fraction is 90%. Test results are as follows Figure 9 and Figure 10 As shown, it can be seen that the L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coated column prepared in Example 1 under different test conditions (CMOFs 1~4 mg / mL, acetonitrile volume fraction 60%~90%): The effect of CMOF concentration (1.0–4.0 mg / mL) on the reaction was investigated under fixed conditions of buffer pH 7.0, acetonitrile volume fraction 80%, and separation voltage 20 kV. Figure 9 The results are as follows: When the concentration is 2.0 mg / mL, the resolution Rs reaches its maximum value of 5.83, and the selectivity factor α is 1.33, achieving baseline separation. At this point, the L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coated column achieves optimal chiral recognition ability for DL-tyrosine. When the concentration is too low (1.0 mg / mL), the chiral site density is insufficient, causing the resolution to drop sharply to 1.42, with significant peak overlap. When the concentration is too high (3.0–4.0 mg / mL), the CMOF nanoparticles aggregate, encapsulating the effective recognition sites, and the resolution drops to 3.42 and 1.82, respectively, with obvious peak tailing.

[0035] When the volume fraction of acetonitrile is 80% ( Figure 10 The hydrophobicity of the mobile phase is highly compatible with the porous structure of CMOFs and the microenvironment of the CMIPs cavity. The hydrophobic benzene ring group of the DL-tyrosine enantiomer interacts fully with the CMOF ligands, while the CMIPs cavity forms a specific recognition with the amino group of DL-tyrosine, achieving a balance between retention time and separation efficiency, with a resolution of 5.83. When the acetonitrile volume fraction increases to 90%, the mobile phase becomes too hydrophobic, causing the enantiomers to elute too quickly, and the resolution drops to 2.36. When the volume fraction decreases to 60%–70%, the hydrophilicity of the mobile phase increases, weakening the interaction between the enantiomers and the stationary phase, resulting in a resolution of 2.67–3.74, and a significantly prolonged separation time.

[0036] In summary, the optimal conditions for DL-tyrosine separation using an L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coated column in the CEC system were determined as follows: CMOF concentration of 2.0 mg / mL, acetonitrile volume fraction of 80%, buffer pH of 7.0, separation voltage of 20 kV, injection pressure of 30 mbar, and injection time of 3 s. The influence of each parameter on Rs is completely consistent with the experimental results, providing a clear reference for the separation of chiral compounds.

[0037] In addition, in this embodiment, the L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coated column prepared in Example 1 was used to perform CEC chiral resolution experiments on D-tyrosine, DL-tyrosine, and L-tyrosine. The experimental procedure included: First, the running buffer was prepared by dissolving 10 mM Na2HPO4 and 10 mM NaH2PO4 in deionized water. After preparation, ultrasonic degassing was performed to remove air bubbles and avoid affecting the separation effect. L-tyrosine, DL-tyrosine, and D-tyrosine were prepared by dissolving them in a water-methanol (1:1, volume ratio) mixture at room temperature. During the separation process, the column temperature was strictly controlled at 25℃, the detection wavelength was fixed at 220 nm, and the injection conditions were 30 mbar for 3 s. The separation voltage, acetonitrile content, and buffer pH were also set. All experimental solutions (including the test samples) were filtered through a 0.45 μm organic phase filter membrane before use.

[0038] The pH of the buffer solution is adjusted to 7.0; the buffer solution is subjected to ultrasonic degassing for 15 minutes after preparation; the sample solution is prepared at room temperature; the separation voltage is set to 20 kV; the acetonitrile volume fraction is 80%; the CMOF concentration is 2 mg / mL; and the concentrations of the sample solution (L-tyrosine, DL-tyrosine, and D-tyrosine) are all 2 mg / mL.

[0039] Experimental results are as follows Figure 7 As shown, D-tyrosine (green line) peaks at approximately 2.2 min, while L-tyrosine (blue line) peaks at approximately 2.8 min, indicating a significant difference in retention times. When resolving the racemic DL-tyrosine (red line), baseline separation was achieved, with the two characteristic peaks of D-tyrosine and L-tyrosine clearly distinguishable. The resolution (Rs) reached 5.83, and the selectivity factor (α) was 1.33, demonstrating that the L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coated column exhibits excellent chiral resolution capability for tyrosine enantiomers.

[0040] Example 3 This embodiment describes the preparation process of MIL-101-L-Tyr(Cr)@capillary (CMOFs@capillary, a capillary coated column with CMOFs as the stationary phase), specifically including: Preparation of MIL-101-L-Tyr(Cr)@capillary: 2.0 mg of CMOFs were added to 1 mL of deionized water and sonicated for 5 min to form a uniform dispersion; then the dispersion was pumped into a pretreated capillary column to obtain a coated column with CMOFs as the stationary phase.

[0041] Example 4 This embodiment describes the preparation process of a capillary coated column (L-Tyr@CMIPs@capillary) with CMIP as the stationary phase alone, specifically including: Preparation of L-Tyr@CMIPs@capillary: The preparation steps of L-Tyr@CMIP@MIL-101-L-Tyr(Cr)@capillary were followed, except that CMOFs were not added to the reaction system. The dispersion was then pumped into a pretreated capillary column to obtain a coated column with CMIPs as the stationary phase.

[0042] Example 5 In this embodiment, comparative experiments were conducted on the coated columns prepared in Examples 1, 3 and 4. 1) Scanning electron microscope image The scanning electron microscope image of MIL-101-L-Tyr(Cr)@capillary prepared in Example 3 is shown below. Figure 2 As shown, the scanning electron microscope image of the L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coated column prepared in Example 1 is as follows. Figure 3 As shown; a comparison reveals: From the low-loading MIL-101-L-Tyr(Cr) coating column (Figure 2), to the high-loading L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coating column (Figure 2) Figure 3 This indicates the preliminary synthesis of L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coated columns.

[0043] Furthermore, in Example 1, during the preparation of the L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coated column, the scanning electron microscope image of MIL-101-L-Tyr(Cr) is as follows: Figure 4 As shown, the scanning electron microscope image of the nanomaterial (L-Tyr@CMIP@MIL-101-L-Tyr(Cr)) inside the coated column is as follows. Figure 5 As shown in the figure, it can be seen that the nanoparticles have evolved from a conventionally arranged MOF structure (Figure 4) into a more dense and complex morphology. Figure 5 This further validates the preliminary synthesis of L-Tyr@CMIP@MIL-101-L-Tyr(Cr) coated columns.

[0044] 2) Fourier transform infrared absorption spectrum Fourier transform infrared absorption spectra of MIL-101-L-Tyr(Cr) and L-Tyr@CMIP@MIL-101-L-Tyr(Cr) were measured. The testing process included: First, wipe the mortar and tablet mold with ethanol, and dry the mold under a lamp. Take pure white potassium bromide (KBr) powder, grind it into a powder-like consistency, dry it, and press a KBr tablet as a background. After pressing the tablet to 90% full, tighten the top and bottom screws until the pointer is >1 and hold the pressure for 3 minutes. Release the bottom screw and then tighten the top screw again to remove the KBr tablet. Turn on the instrument and execute "Spectral Scan" → "Initialize". Place the KBr tablet in the sample chamber and complete the background acquisition with the default 16 scans. Then, change to a sample mixed with KBr tablet and perform sample scanning. After the scan is completed, select "View" → "Process" in the software to perform atmospheric correction to eliminate the influence of gases such as CO2 on the infrared spectrum. Set the baseline grid area to "3 points", the range to 4000→2000, 400→∞, and then select "Smooth" → "Smooth Points" → "Uncertain" and set the number of points to 13 to complete the baseline correction and smoothing process and obtain the results.

[0045] Test results are as follows Figure 6 As shown, comparing the Fourier transform infrared (FT-IR) spectra of CMOFs and L-Tyr@CMIP@MIL-101-L-Tyr(Cr), it can be seen that L-Tyr@CMIP@MIL-101-L-Tyr(Cr) exhibits several key differences: new characteristic peaks appear, including those at 3200–3300 cm⁻¹. -1 The amide N–H stretching vibration peak at 1650 cm⁻¹, the amide I band (C=O stretching vibration) at 1650 cm⁻¹, and the peak at 1540 cm⁻¹. -1 The amide II band (N–H bending vibration) at 1600–1450 cm⁻¹. These peaks all originate from the amide group in the functional monomer (S)-3-phenyl-2-(undecad-10-enamido)propionic acid, strongly demonstrating that the functional monomer has been successfully introduced into the synthetic system. -1 Within this range, the intensity of the benzene ring skeletal vibration peaks is significantly enhanced, especially in the range of 1000–900 cm⁻¹. -1 The appearance of bending vibration peaks of olefin double bonds (C=C) indicates that the benzene ring on the functional monomer and the olefin chain participate in the polymerization reaction. Notably, the characteristic peaks of the carboxyl groups in CMOFs (1650–1350 cm⁻¹) are also observed. -1 The intensity of L-Tyr@CMIP@MIL-101-L-Tyr (Cr) decreases and shifts to lower wavenumbers. This is most likely because the L-tyrosine template molecule is bonded to the functional monomer through hydrogen bonds or coordination, or the crosslinking agent EDMA is linked to the backbone through ester / amide bonds, thereby changing the chemical environment of the functional groups. This also reflects that intermolecular interactions play a key role in the formation of new structures.

[0046] In summary, infrared spectroscopy provides multifaceted evidence: the appearance of new characteristic peaks in amide bonds, benzene rings, and olefin groups, as well as shifts and intensity changes in the original carboxyl peak. From the perspectives of organic functional group modification and changes in the inorganic metal center structure, these spectral differences clearly indicate that L-Tyr@CMIP@MIL-101-L-Tyr (Cr) has been successfully synthesized via the expected reaction pathway.

[0047] 3) Comparison Experiment of Chiral Separation Performance Three types of coated columns prepared in Examples 1, 3, and 4 were used to perform chiral resolution experiments on DL-tyrosine, and their performance was compared. The comparative experiments included: First, a running buffer was prepared by dissolving 10 mM Na₂HPO₄ and 10 mM NaH₂PO₄ in deionized water. After preparation, ultrasonic degassing was performed to remove air bubbles and avoid affecting the separation effect. DL-tyrosine was prepared by dissolving it separately in a water-methanol (1:1, volume ratio) mixture at room temperature. During separation, the column temperature was strictly controlled at 25°C, the detection wavelength was fixed at 220 nm, and the injection conditions were 30 mbar for 3 s. The separation voltage, acetonitrile volume fraction, CMOF concentration, and buffer pH were adjusted accordingly. All experimental solutions (including the test samples) were filtered through a 0.45 μm organic phase filter membrane before use. The three coated columns prepared in Examples 1, 3, and 4 were used as CEC separation columns.

[0048] The pH of the buffer solution is adjusted to 7.0; the buffer solution is subjected to ultrasonic degassing for 15 minutes after preparation; the sample solution is prepared at room temperature; the separation voltage is set to 20 kV; the organic modifier (acetonitrile volume fraction) is 80%; the CMOFs concentration is 2 mg / mL; the sample solution (DL-tyrosine) concentration is 2 mg / mL.

[0049] Comparative experimental results as follows Figure 8As shown, the novel capillary coated column achieved baseline separation of tyrosine enantiomers. Under optimal conditions (buffer pH 7.0, separation voltage 20 kV, acetonitrile volume fraction 80%, CMOF concentration 2 mg / mL, column temperature 25℃, detection wavelength 220 nm, injection pressure 30 mbar, injection time 3 s), the resolution Rs reached 5.83, and the selectivity factor α was 1.33. In contrast, although the CMOF-coated column alone possessed chiral channels, its separation efficiency was weak (Rs=0.61, α=1.01). The CMIP-coated column alone could only separate a portion of the enantiomers (Rs=0.53, α=1.01), with an efficiency far lower than that of the L-Tyr@CMIP@MIL-101-L-Tyr (Cr) coated column. This indicates that CMOFs and CMIPs played an important synergistic role. L-Tyr@CMIP@MIL-101-L-Tyr (Cr) coated columns outperform single-component columns (CMIPs coated columns, CMOFs coated columns) in the separation of tyrosine enantiomers.

[0050] CMOFs provide a highly ordered porous framework and abundant chiral recognition sites, offering a stable microenvironment for molecular interactions; CMIPs, on the other hand, form specific template-bound cavities through molecular imprinting technology, achieving precise chiral recognition. The synthesized novel nanomaterials utilize the high specific surface area of ​​CMOFs to overcome the low loading problem of single CMIPs; simultaneously, the rigid MIL-101 framework within the CMOFs effectively suppresses the pore collapse phenomenon commonly seen in flexible CMIPs. Furthermore, the viscosity of CMIPs further enhances the adhesion stability of the stationary phase on the capillary inner wall. Most notably, the synergistic effect between CMOFs and CMIPs significantly enhances the chiral selectivity of this novel nanomaterial. The structural complementarity of CMOFs and CMIPs greatly improves the superior performance of the L-Tyr@CMIP@MIL-101-L-Tyr (Cr) coated column.

[0051] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0052] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A capillary electrochromatographic column for the separation of chiral tyrosine, comprising: A capillary and a composite coating fixed to the inner wall of the capillary; characterized in that the composite coating comprises CMOFs and chiral molecularly imprinted polymers (CMIPs) modified on the surface of the CMOFs; The CMOFs are formed by a coordination reaction between MIL-101 (Cr) and L-tyrosine, which acts as a chiral ligand. The composite coating is a multi-level composite structure formed by in-situ polymerization of the CMOFs as a support carrier in a system containing a crosslinking agent, functional monomer, initiator and L-tyrosine as a template molecule.

2. The capillary electrochromatographic column for the separation of chiral tyrosine according to claim 1, characterized in that, The capillary is a fused silica capillary.

3. A capillary electrochromatographic column for the separation of chiral tyrosine according to claim 1, characterized in that, The functional monomer is (S)-3-phenyl-2-(undecad-10-enamide)propionic acid, the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is azobisisobutyronitrile.

4. A method for preparing a capillary electrochromatographic column for the separation of chiral tyrosine according to any one of claims 1-3, characterized in that, Includes the following steps: S1, the capillary tube is rinsed sequentially with alkaline solution, deionized water and acid solution, deionized water and methanol, and then dried; S2, synthesize MIL-101(Cr) and activate it under vacuum heating. Then, remove the residual air under vacuum to obtain pure MIL-101(Cr). Mix the obtained pure MIL-101(Cr) with L-tyrosine dissolved in solvent and carry out a static reaction at an isothermal temperature. After separation and drying, CMOFs are obtained. S3. Prepare a CMOF dispersion and mix it evenly with functional monomers, crosslinking agents, initiators, and L-tyrosine as template molecules to obtain a polymerization mixture. Apply external pressure to draw the polymerization mixture into a washed and dried capillary for in-situ polymerization. After the reaction is completed, remove the template molecules to obtain the capillary electrochromatographic column.

5. The method for preparing a capillary electrochromatographic column for the separation of chiral tyrosine according to claim 4, characterized in that, The process of synthesizing MIL-101 (Cr) includes: Cr(NO3)3·9H2O and 1,4-phthalic acid were dissolved in deionized water and subjected to a static reaction at a constant temperature. The reaction product was then washed, purified, centrifuged, and dried to obtain the final product.

6. The method for preparing a capillary electrochromatographic column for the separation of chiral tyrosine according to claim 4, characterized in that, The temperature for vacuum heating activation of MIL-101(Cr) was 120℃, and the activation time was 24h.

7. The method for preparing a capillary electrochromatographic column for the separation of chiral tyrosine according to claim 4, characterized in that, The isothermal static reaction was carried out at a temperature of 70°C for 3 days, and anhydrous ethanol was used as the solvent.

8. The method for preparing a capillary electrochromatographic column for the separation of chiral tyrosine according to claim 4, characterized in that, The applied external pressure is 750 mbar; the in-situ polymerization reaction is carried out in a constant temperature water bath at 70°C for 40 min.

9. The use of the capillary electrochromatographic column for chiral tyrosine separation according to any one of claims 1-3 in the enantiomeric separation of DL-tyrosine.

10. The application according to claim 9, characterized in that, The applications include: During the capillary electrochromatographic separation process, the buffer solutions used include 10 mM Na2HPO4 and 10 mM NaH2PO4, and both the DL-tyrosine sample solution and the buffer solutions are filtered through a 0.45 µm organic filter membrane before use.

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