A method for separating EHB enantiomers based on a synergic chiral recognition chromatographic system

Abstract

The application discloses a method for separating EHB enantiomers based on a synergistic chiral recognition chromatographic system, and belongs to the technical field of chiral compound separation. The method comprises the following steps: dissolving L-proline and a copper salt in a mixed solution of an organic solvent and water, adding alkali to adjust the pH to 7.0-7.8 to prepare an L-proline-copper (II) chiral complex mobile phase; flowing the mobile phase through a chromatographic column filled with a polysaccharide derivative chiral stationary phase to establish a synergistic chiral recognition environment; introducing a (R,S)-EHB sample for elution and separation, collecting (R)-EHB and (S)-EHB fractions respectively, and removing copper ions and recovering L-proline through a cation exchange resin. The application utilizes the chiral synergistic effect of the stationary phase and the mobile phase, significantly enhances the enantiomer separation effect, realizes optical purity ee value of greater than or equal to 99.0% and recovery rate of greater than or equal to 95%, and can recover L-proline, and has the advantages of high efficiency, environmental protection and low cost.

Description

Technical Field

[0001] This invention relates to the field of chiral compound separation technology, and specifically to a method for separating EHB enantiomers based on a synergistic chiral recognition chromatographic system. Background Technology

[0002] Ethyl 3-hydroxybutyrate (EHB) is an important chiral building block. Its chiral hydroxyl (-OH) and ester (-COOEt) groups serve as active sites in various chemical reactions, thus playing an irreplaceable role in the synthesis of β-lactam antibiotics, lipid-lowering drugs, high-value fragrances, and biodegradable materials such as polyhydroxyalkanoates. Among these, (R)-EHB exhibits superior bioactivity and economic value due to its higher spatial matching with enzymes and receptors in vivo.

[0003] However, (R)-EHB and (S)-EHB differ only in the spatial configuration of their chiral centers. Their physicochemical properties, such as boiling point, solubility, and density, are very similar. Conventional separation methods, such as distillation and crystallization, cannot effectively separate them. Therefore, developing efficient chiral separation methods has become a key industry requirement.

[0004] Currently, methods for separating EHB enantiomers mainly include enzymatic resolution, chemical resolution, and chromatography. However, all of these methods have significant drawbacks: enzymatic methods require harsh conditions, have long cycles, and exhibit strong substrate specificity; chemical resolution involves cumbersome steps, low yields, and the use of large amounts of organic solvents can easily cause environmental pollution; chromatography, especially high-performance liquid chromatography (HPLC), is an effective means of separating enantiomers, but existing technologies mostly employ expensive chiral stationary phases, such as columns based on cyclodextrin, cellulose, or polysaccharide derivatives. While these chiral columns are effective, they suffer from high costs, limited lifespans, and sometimes difficulty in balancing separation efficiency and selectivity. This is particularly problematic for molecules with small molecular weights and weak chiral polarity, such as EHB, and for large-scale preparations, where high solvent consumption and operating costs are significant.

[0005] Therefore, developing a separation method for ethyl (R,S)-3-hydroxybutyrate that is highly efficient, low-cost, easy to scale up, and environmentally friendly is of great industrial significance. Summary of the Invention

[0006] The purpose of this invention is to provide a method for separating EHB enantiomers based on a synergistic chiral recognition chromatography system. By constructing a synergistic separation system consisting of a chiral stationary phase and a chiral complexed mobile phase, the synergistic effect of the two is utilized to amplify the enantiomer retention differences. At the same time, combined with online mobile phase recovery technology, efficient separation and low-cost operation of (R,S)-EHB can be achieved.

[0007] To achieve the above objectives, the present invention provides a method for separating EHB enantiomers based on a synergistic chiral recognition chromatographic system, comprising the following steps: S1. L-proline and copper salt were dissolved in a mixed solution of organic solvent and water, and the pH was adjusted with alkali to prepare an L-proline-copper(II) chiral complex mobile phase. S2. The chiral complex mobile phase obtained in step S1 is passed through a chromatographic column packed with a chiral stationary phase of polysaccharide derivatives. The stationary phase and the mobile phase reach adsorption equilibrium, thereby establishing a dynamic synergistic chiral recognition chromatographic separation environment. S3. Introduce the (R,S)-EHB sample solution into the chromatographic separation environment established in step S2 for elution separation. S4. Collect the elution fractions of (R)-EHB and (S)-EHB based on the online detection signal.

[0008] Preferably, in step S1, the molar ratio of L-proline to copper salt is (1.8~2.2):1; the copper salt is one of copper sulfate, copper acetate or copper chloride.

[0009] Preferably, in step S1, the volume percentage of the organic solvent in the mixed solution of organic solvent and water is 85% to 92%, and the organic solvent is one of acetonitrile, methanol or ethanol.

[0010] Preferably, in step S1, alkali is added to adjust the pH to 7.0-7.8 to obtain an L-proline-copper(II) chiral complex mobile phase.

[0011] Preferably, in step S2, the chiral stationary phase of the polysaccharide derivative is amylose-tris(3,5-dimethylphenylcarbamate) or cellulose-tris(3,5-dimethylphenylcarbamate).

[0012] Preferably, in step S3, the column temperature is 20-30℃ during the elution process.

[0013] Preferably, in step S4, the optical purity ee value of the collected (R)-EHB and (S)-EHB elution fractions is ≥99.0%, and the recovery rate is ≥95%.

[0014] Preferably, the method further includes step S5: subjecting the fraction collected in step S4 to ion exchange treatment via a cation exchange resin recovery column to remove copper ions and recover L-proline.

[0015] Preferably, the cation exchange resin is a strongly acidic styrene-based cation exchange resin.

[0016] This invention constructs a dynamic, synergistic chiral recognition chromatographic system. Instead of simply using a single chiral stationary phase or mobile phase, it organically combines a polysaccharide derivative chiral stationary phase with an L-proline-copper(II) chiral complex mobile phase. This system simultaneously provides two chiral recognition environments. The stationary phase interacts with EHB molecules through its own chiral cavity via hydrogen bonds, dipole-dipole, π-π interactions, and steric hindrance, representing a "static" chiral selection effect. In the mobile phase, Cu... 2+ It forms a chiral complex with L-proline, and then coordinates with the chiral hydroxyl group and oxygen atom on the ester group of the EHB enantiomer molecule to form a diastereomer ternary complex. This is a "dynamic" chiral selection. The stereoselectivity provided by the chiral cavities of the stationary phase, combined with the dynamic coordination between the complex and the enantiomer in the mobile phase, works synergistically and in the same direction. Both tend to "prefer" or "reject" the same enantiomer. This superposition of forces significantly amplifies the difference in retention behavior between the two enantiomers on the chromatographic column (i.e., the retention time difference ΔtR increases), achieving a multiplier effect in separation efficiency and solving the problem of difficult separation of small chiral compounds due to their simple structure. The entire system is in dynamic equilibrium, with the EHB enantiomer molecule distributing between the stationary and mobile phases, while simultaneously undergoing reversible coordination and dissociation with the chiral complex in the mobile phase. By controlling key parameters such as the molar ratio of L-proline to copper salt, the proportion of organic solvent, and the pH of the mobile phase, the strength and balance of the two chiral recognition mechanisms can be adjusted, thereby finding the optimal separation conditions and achieving efficient and rapid separation.

[0017] After separation, this invention connects a strongly acidic cation exchange resin column, rich in L-proline and Cu, in series after the chromatographic column. 2+ When the effluent flows through this column, Cu 2+ With H on the resin + Or Na + An exchange occurs, and the substance is adsorbed onto the resin, thus achieving removal. L-proline, being a neutral molecule, is not retained by the resin and flows out with the solution, thus being recovered. This step transforms the traditional consumption-waste process into a green and economical process of recycling and reuse, significantly reducing the cost of the core chiral additive (L-proline) and the consumption of copper salts, as well as reducing the discharge of heavy metal wastewater.

[0018] Therefore, the present invention provides a method for separating EHB enantiomers based on a synergistic chiral recognition chromatographic system, which has the following beneficial effects: (1) Based on the principle of cooperative chirality recognition, this invention can efficiently achieve complete baseline separation of (R,S)-EHB, obtain (R)-type and (S)-type products with an optical purity ee value of not less than 99.0%, and the enantiomeric recovery rate can reach more than 95%. It can meet the high purity requirements of key chiral building blocks in fields such as chiral drug synthesis.

[0019] (2) The chromatographic separation conditions of this invention are mild and the process is simple, and the operating cost is controlled through multiple pathways. The synergistic effect reduces the single dependence on the top chiral stationary phase, which helps to extend the column life; the ion exchange treatment realizes the recovery of the chiral ligand L-proline, reducing reagent consumption and waste liquid treatment costs. At the same time, this process reduces the use of organic solvents and the discharge of waste liquid containing heavy metals, which is in line with the principles of green chemistry and has both economic and environmental benefits.

[0020] The technical solution of the present invention will be further described in detail below through embodiments. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the invention should be considered equivalent substitutions and are included within the scope of protection of the invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims and are all within the scope of protection of the invention.

[0022] In this document, the term "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The term "embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment, nor does it specifically limit its independence or connection with other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0023] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.

[0024] Unless otherwise specified, the reagents, instruments, and equipment used in this invention are all commonly used by those skilled in the art, and the testing standards all use national or international standards commonly used in the field, without further explanation.

[0025] Example 1 This embodiment provides a method for separating EHB enantiomers based on a synergistic chiral recognition chromatography system. The experimental materials and instruments used include: Experimental materials: The chiral stationary phase consisted of amylose-tris(3,5-dimethylphenylcarbamate) (packed on a 4.6 mm × 250 mm column, particle size 5 μm), L-proline (purity ≥ 99%), copper sulfate (CuSO4·5H2O, analytical grade), acetonitrile (chromatographic grade), water, and (R,S)-EHB standard (ee value 50%, purity ≥ 98%).

[0026] Instruments: High performance liquid chromatograph (with UV detector), pH meter, ultrasonic cleaner, cation exchange resin column (packed with strong acid styrene-based cation exchange resin 001×7, column size 10mm×200mm).

[0027] The separation method specifically includes the following steps: S1. Preparation of L-proline-copper(II) chiral complex mobile phase: Mix 880 mL of acetonitrile with 120 mL of water to prepare an acetonitrile-water mixed solution; weigh 0.112 g of copper sulfate (0.5 mmol) and 0.115 g of L-proline (1.0 mmol) and add them to the above mixed solution, sonicate until completely dissolved, and adjust the pH to 7.4 with 0.1 mol / L NaOH solution to obtain the L-proline-copper(II) chiral complex mobile phase.

[0028] S2. Establish a synergistic chiral recognition chromatographic environment: Pass the above-mentioned chiral complex mobile phase into a chromatographic column packed with chiral polysaccharide derivatives at a flow rate of 1.0 mL / min. Set the column temperature to 25℃ and the UV detector wavelength to 210 nm. Continue eluting for 30 min until the chromatographic baseline is stable (the stationary phase and the mobile phase reach adsorption equilibrium).

[0029] S3. Elution and separation of (R,S)-EHB: Weigh 10 mg of (R,S)-EHB standard, dissolve it in acetonitrile and dilute to 10 mL to prepare a 1.0 mg / mL sample solution; inject 10 μL of the sample solution into the chromatograph for elution and separation, and record the chromatogram.

[0030] S4. Collect and analyze the elution fractions: Based on the UV detection signal, collect the elution fractions of (R)-EHB (retention time tR1=8.5min) and (S)-EHB (retention time tR2=10.8min) respectively; the optical purity of the fractions is detected by chiral-HPLC and the recovery rate is calculated by gravimetric method.

[0031] S5. Mobile phase recovery: The collected fraction (containing L-proline and Cu) is recovered.2+ The effluent was passed through a cation exchange resin column at a flow rate of 0.5 mL / min and collected. Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to detect the Cu content in the effluent. 2+ The concentration of L-proline was determined by high performance liquid chromatography.

[0032] The separation and recovery results of this embodiment are shown in Table 1 below.

[0033] Table 1. Separation and recovery results of Example 1

[0034] Example 2 This embodiment provides a method for separating EHB enantiomers based on a synergistic chiral recognition chromatography system. The experimental materials and instruments used include: Materials: The chiral stationary phase consisted of cellulose-tris(3,5-dimethylphenylcarbamate) (packed on a 4.6 mm × 250 mm column with a particle size of 5 μm), L-proline (purity ≥ 99%), copper acetate (analytical grade), methanol (chromatographic grade), water, and (R,S)-EHB standard (ee value 50%, purity ≥ 98%). Instruments: Same as in Example 1.

[0035] The separation method specifically includes the following steps: S1. Preparation of L-proline-copper(II) chiral complex mobile phase: Mix 880 mL of methanol with 120 mL of water to prepare a methanol-water mixed solution; weigh 0.100 g of copper acetate (0.5 mmol) and 0.104 g of L-proline (0.9 mmol) and add them to the above mixed solution, sonicate until completely dissolved, and adjust the pH to 7.0 with 0.1 mol / L NaOH solution to obtain the L-proline-copper(II) chiral complex mobile phase.

[0036] S2. Establish a synergistic chiral recognition chromatographic environment: Pass the chiral complex mobile phase into the chromatographic column packed with the chiral stationary phase of the polysaccharide derivative at a flow rate of 1.0 mL / min. Set the column temperature to 20℃ and the UV detector wavelength to 210 nm. Continue eluting for 30 min until the chromatographic baseline is stable (the stationary phase and the mobile phase reach adsorption equilibrium).

[0037] S3, Elution and separation (R,S)-EHB: Same as Example 1.

[0038] S4. Collect and analyze the elution fractions: Based on the UV detection signal, collect the elution fractions of (R)-EHB (retention time tR1=9.2min) and (S)-EHB (retention time tR2=11.3min) respectively; the optical purity of the fractions is detected by chiral-HPLC and the recovery rate is calculated by gravimetric method.

[0039] S5. Mobile phase recovery: Same as in Example 1.

[0040] The separation and recovery results of this embodiment are shown in Table 2 below.

[0041] Table 2. Separation and recovery results of Example 2

[0042] Example 3 This embodiment provides a method for separating EHB enantiomers based on a synergistic chiral recognition chromatography system. The experimental materials and instruments used include: Experimental materials: chiral fixation as in Example 1, L-proline (purity ≥99%), copper chloride (analytical grade), ethanol (chromatographic grade), water, (R,S)-EHB standard (ee value 50%, purity ≥98%).

[0043] Instruments: Same as in Example 1.

[0044] The separation method specifically includes the following steps: S1. Preparation of L-proline-copper(II) chiral complex mobile phase: Mix 850 mL of ethanol with 150 mL of water to prepare an ethanol-water mixed solution; weigh 0.085 g of copper chloride (0.5 mmol) and 0.126 g of L-proline (1.1 mmol) and add them to the above mixed solution, sonicate until completely dissolved, and adjust the pH to 7.8 with 0.1 mol / L NaOH solution to obtain the L-proline-copper(II) chiral complex mobile phase.

[0045] S2. Establish a synergistic chiral recognition chromatographic environment: Pass the chiral complex mobile phase into the chromatographic column packed with the chiral stationary phase of the polysaccharide derivative at a flow rate of 1.0 mL / min. Set the column temperature to 30℃ and the UV detector wavelength to 210 nm. Continue eluting for 30 min until the chromatographic baseline is stable (the stationary phase and the mobile phase reach adsorption equilibrium).

[0046] S3, Elution and separation (R,S)-EHB: Same as Example 1.

[0047] S4. Collect and analyze the elution fractions: Based on the UV detection signal, collect the elution fractions of (R)-EHB (retention time tR1=11.5min) and (S)-EHB (retention time tR2=14.5min) respectively; the optical purity of the fractions is detected by chiral-HPLC and the recovery rate is calculated by gravimetric method.

[0048] S5. Mobile phase recovery: Same as in Example 1.

[0049] The separation and recovery results of this embodiment are shown in Table 3 below.

[0050] Table 3. Separation and recovery results of Example 3

[0051] Comparative Example 1 This comparative example illustrates the decrease in separation efficiency when a chiral complexing mobile phase is not used. The experimental materials and instruments used included: the same chiral fixation as in Example 1, acetonitrile (chromatographic grade), water, and (R,S)-EHB standard; the instruments were the same as in Example 1 (excluding step S5).

[0052] The separation method specifically includes the following steps: S1. Preparation of mobile phase: Mix 880 mL of acetonitrile with 120 mL of water to prepare an acetonitrile-water mixed solution. Do not add L-proline or copper salt. Use this solution directly as the mobile phase.

[0053] S2. Establish the chromatographic environment: Same as in Example 1.

[0054] S3, Elution and separation (R,S)-EHB: Same as Example 1.

[0055] S4. Collect and analyze the elution fractions: Based on the UV detection signal, attempt to collect the elution fractions with retention times tR1 = 7.2 min and tR2 = 7.8 min; use chiral-HPLC to detect the optical purity of the fractions and calculate the recovery rate by weighing.

[0056] The separation results of this comparative example are shown in Table 4.

[0057] Table 4 Separation results of Comparative Example 1

[0058] Comparative Example 2 This comparative example is used to illustrate the negative impact of an improper molar ratio of L-proline to copper salt on the separation effect. The experimental materials and instruments used are the same as in Example 1.

[0059] The separation method specifically includes the following steps: S1. Preparation of L-proline-copper(II) chiral complex mobile phase: Mix 880 mL of acetonitrile with 120 mL of water; weigh 0.112 g of copper sulfate (0.5 mmol) and 0.058 g of L-proline (0.5 mmol), add them to the above mixed solution, sonicate to dissolve, adjust the pH to 7.4, and obtain the chiral complex mobile phase with a molar ratio of L-proline to copper salt of 1.0:1. Filter and set aside for later use.

[0060] S2. Establish a synergistic chiral recognition chromatographic environment: Same as Example 1.

[0061] S3, Elution and separation (R,S)-EHB: Same as Example 1.

[0062] S4. Collect and detect the elution fractions: Based on the UV detection signal, collect the elution fractions of (R)-EHB (retention time tR1=8.9min) and (S)-EHB (retention time tR2=9.8min) respectively, and detect them.

[0063] S5. Mobile phase recovery: Same as in Example 1.

[0064] The separation results of this comparative example are shown in Table 5.

[0065] Table 5 Separation results of Comparative Example 2

[0066] As shown in Tables 1-5, firstly, comparing the data from Examples 1-3 using the synergistic system with Comparative Example 1 using only the chiral stationary phase and without the chiral complexing mobile phase, it is evident that the synergistic effect of the chiral stationary phase and the chiral complexing mobile phase is the core key to achieving effective separation of EHB enantiomers. Examples 1-3 achieved a resolution (Rs) of 2.3-2.6 (complete baseline separation), optical purity (ee%) ≥99.0%, and recovery rate ≥95.9%; while Comparative Example 1 had an Rs of only 0.8 (peak overlap, ineffective separation), an ee value of 83.9%-85.2% (insufficient purity), and a recovery rate of 76.8%-78.5% (high material loss). This verifies that the static chiral cavity effect of the chiral stationary phase and the dynamic coordination effect of the L-proline-copper(II) mobile phase can be superimposed to amplify the enantiomer retention differences, solving the problem of difficult separation of small molecule chiral compounds. Secondly, the differences between Examples 1-3 and Comparative Example 2 indicate that the molar ratio of L-proline to copper salt is a key parameter affecting the separation effect. Examples 1-3 achieve Rs of 2.3-2.6, ee ≥ 99.0%, and recovery rate ≥ 95.9%; Comparative Example 2 has an Rs of only 1.2 (incomplete baseline separation), ee of 89.5%-90.1% (not meeting high purity standards), and a recovery rate of 80.7%-82.3% (poor economic efficiency). This is because an excessively low molar ratio leads to the release of free Cu. 2+Excessive amounts disrupt the dynamic chiral recognition balance, while an optimal ratio ensures that Cu... 2+ A stable chiral complex is fully formed, ensuring separation selectivity. Thirdly, the recovery data from Examples 1-3 and Comparative Example 2 are consistent, indicating that the recovery effect of the strongly acidic styrene-based cation exchange resin is not affected by the initial separation parameters. On the one hand, it can efficiently adsorb Cu. 2+ This method avoids the discharge of heavy metal waste liquid; on the other hand, it enables the recycling of L-proline, reducing the cost of chiral additives and transforming the traditional consumption-waste model into a recovery-recycling model, which aligns with green chemistry principles. Fourth, Examples 1-3 used different chiral stationary phases, organic solvents, and column temperatures, but all maintained excellent separation performance (ee≥99%, recovery rate≥95.9%, Rs≥2.3). This indicates that the method is adaptable to different experimental conditions and has strong applicability.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for separating enantiomers of ethyl 3-hydroxybutyrate based on a synergistic chiral recognition chromatographic system, characterized in that, Includes the following steps: S1. L-proline and copper salt are dissolved in a mixed solution of organic solvent and water, and the pH is adjusted to 7.0-7.8 with alkali to obtain an L-proline-copper(II) chiral complex mobile phase; The molar ratio of L-proline to copper salt is (1.8~2.2):1; in the mixed solution of organic solvent and water, the volume percentage of organic solvent is 85%~92%, and the organic solvent is one of acetonitrile, methanol or ethanol; S2. The chiral complex mobile phase obtained in step S1 is passed through a chromatographic column packed with a chiral stationary phase of polysaccharide derivatives. The stationary phase and the mobile phase reach adsorption equilibrium, thereby establishing a dynamic synergistic chiral recognition chromatographic separation environment. S3. The sample solution of (R,S)-3-hydroxybutyrate ethyl ester is introduced into the chromatographic separation environment established in step S2 for elution separation. S4. Collect the elution fractions of (R)-3-hydroxybutyrate ethyl ester and (S)-3-hydroxybutyrate ethyl ester based on the online detection signal; In step S1, the copper salt is one of copper sulfate, copper acetate, or copper chloride; In step S2, the chiral stationary phase of the polysaccharide derivative is either amylose-tris(3,5-dimethylphenylcarbamate) or cellulose-tris(3,5-dimethylphenylcarbamate).

2. The method for separating ethyl 3-hydroxybutyrate enantiomers based on a synergistic chiral recognition chromatographic system according to claim 1, characterized in that: In step S3, the column temperature is 20-30℃ during the elution process.

3. The method for separating ethyl 3-hydroxybutyrate enantiomers based on a synergistic chiral recognition chromatographic system according to claim 1, characterized in that: In step S4, the optical purity ee value of the collected (R)-3-hydroxybutyrate ethyl ester and (S)-3-hydroxybutyrate ethyl ester elution fractions is ≥99.0%, and the recovery rate is ≥95%.

4. The method for separating ethyl 3-hydroxybutyrate enantiomers based on a synergistic chiral recognition chromatographic system according to claim 1, characterized in that, The method further includes step S5: passing the fraction collected in step S4 through a cation exchange resin recovery column for ion exchange treatment to remove copper ions and recover L-proline.

5. The method for separating ethyl 3-hydroxybutyrate enantiomers based on a synergistic chiral recognition chromatographic system according to claim 4, characterized in that: The cation exchange resin is a strongly acidic styrene-based cation exchange resin.

Images