OPO column chromatography purified stationary phase, preparation and purification method and OPO product

Abstract

The application belongs to the field of separation and purification, and relates to a fixed phase for OPO column chromatography separation and purification, a preparation method and a separation and purification method and an OPO product. The structural formula of the fixed phase is: The fixed phase is used for column chromatography separation and purification of OPO, and can effectively separate OPO and OOP. Through the pi-complexation of silver ions and double bonds and the pi-pi stacking ability of the triazine structure, the fixed phase can distinguish the hydrophobicity and steric effect of the two isomers, so that high-efficiency separation is realized.

Description

Technical Field

[0001] This invention belongs to the field of separation and purification, specifically relating to the stationary phase for OPO column chromatography separation and purification, its preparation and separation and purification methods, and OPO products. Background Technology

[0002] 1,3-Dioleoyl-2-palmitoylglycerol (OPO), a key component of breast milk fat, accounts for approximately 16%-29% of its total composition. It is a triglyceride with a unique USU-type structure (where U represents unsaturated fatty acid and S represents saturated fatty acid). OPO plays a crucial role in infant nutrient absorption, promoting the absorption of fatty acids and minerals, softening stools, reducing infant crying and colic frequency, aiding in the colonization of beneficial gut microbiota, promoting metabolism, enhancing immunity, and reducing the incidence of related diseases. Therefore, OPO is artificially synthesized and widely used as a food fortifier in infant formula.

[0003] Currently, there are no published standards for OPO testing in food, and the OPO content in infant formula milk powder is often falsely labeled. Studies have shown that the OPO content in infant formula milk powder is only 28.4%-59.7% of the value stated on the nutrition label. Most OPO standard materials on the market are currently produced abroad, lack accurate quantitative values, and are expensive. This leads to a lack of assurance regarding the reliability, comparability, and traceability of domestic OPO measurement results. Therefore, there is a need to develop a simple, low-cost, and efficient preparation method for OPO standard materials.

[0004] Currently, crude OPO products are available on the market. It is hoped that OPO standard substances can be obtained through separation and purification. Molecular distillation, low-temperature crystallization, solvent crystallization, column chromatography, and supercritical extraction are common purification methods for glycerides. Since the physicochemical properties of OPO and its positional isomer OOP are similar, current purification methods are difficult to effectively separate OPO and OOP, and it is difficult to obtain OPO standard substances.

[0005] It should be noted that this part of the present invention only provides background technology related to the present invention, and does not necessarily constitute prior art or known technology. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing OPO crude product separation and purification methods, which are difficult to effectively separate OPO and OOP and difficult to use for obtaining OPO standard substances. This invention provides a stationary phase for OPO column chromatography separation and purification, its preparation and separation and purification method, and OPO products. This stationary phase can effectively separate OPO and OOP when used for column chromatography separation and purification, and the organic purity of the purified OPO can reach 98.7%.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a stationary phase for OPO column chromatography separation and purification, the structural formula of which is:

[0008]

[0009] It is understood that the stationary phase of this invention is silver (I)-dimercaptotriazine functionalized silica, and its core innovation lies in its unique molecular structure: two thiol groups are introduced at the meta position of the triazine ring, and through this rigid planar structure, they coordinate with silver ions, ultimately being immobilized on a silica support. This design, based on multi-dimensional considerations of separation mechanism, coordination chemistry, and material stability, solves the shortcomings of traditional silver ion chromatography in separating triglyceride positional isomers.

[0010] The key advantage of designing dithiol groups at the meta-position of triazine, compared to monothiol or flexible double-chain thiol groups, lies in forming a stable coordination mode with enhanced chelation effect. In existing technologies, monothiol groups and silver ions (Ag... + Ag typically forms linear coordination (Ag-SR), a relatively weak bonding mechanism. Under prolonged scouring with polar eluents (such as mixtures containing ethyl acetate) and photothermal conditions, Ag... + It readily dissociates or migrates from sulfur atoms, leading to a rapid decrease in column efficiency. Even when two independent mercaptopropyl chains are grafted onto silica via silanization, their spatial conformation remains highly flexible, with an inconsistent spacing between the two mercapto groups, making it difficult for them to coordinate with the same Ag. + Forming an ideal chelate ring, Ag + The coordination environment of the ligand is unstable, and the flexibility of the long chain may lead to uneven steric hindrance, affecting the reproducibility of the interaction with the target molecule. This invention uses the triazine ring as a rigid planar platform, fixing its two thiol groups at specific meta positions, thus forming an ideal bidentate ligand. When interacting with Ag... + During coordination, these two thiol groups can spatially coordinate with Ag. + This forms a stable, low-ring-strain five-membered chelate ring. This chelation greatly enhances the Ag... + The strength of coordination with the thiol group significantly reduced Ag + The leaching risk during operation is reduced. The stability of silver ions is thus greatly improved, allowing the stationary phase to withstand longer elution times and more purification cycles.

[0011] This invention chooses a triazine ring, particularly a mestriazine ring, as the framework of this bidentate ligand because of the unique electronic and steric effects of the triazine ring. The mestriazine ring is a typical electron-deficient aromatic system; its strong electron-withdrawing effect significantly reduces the electron cloud density of the sulfur atoms attached to it. This has two effects: first, it enhances the proton acidity of the thiol group (-SH), making it easier to deprotonate during the reaction, thus facilitating its reaction with Ag. + First, it forms stronger coordination bonds; second, the electron-deficient triazine ring interacts with Ag. + There are certain charge interactions between them, and this additional secondary force further strengthens the overall coordination structure, preventing Ag from... + The sliding or rotation of the rings. In contrast, other aromatic systems, such as benzene rings, exhibit different electronic effects and cannot provide significant synergistic stabilizing effects.

[0012] More importantly, the triazine ring in this invention, besides serving as an inert framework to stabilize silver ions, also acts as a rigid plane with strong electron deficiency, actively synergistically working with silver ions to act on the target analyte, thus achieving the identification and separation of subtle structural differences between OPO and OOP. This synergistic mechanism is mainly manifested on two levels:

[0013] Firstly, there is the synergy between electronic effects and polar recognition. The strong electron-withdrawing inductive effect of the triazine ring not only makes it easier for the thiol group to form a stable coordination bond with silver ions, but its own strong dipole moment plane can also interact with the ester group (-COO-) in the triglyceride molecular backbone through dipole-dipole and n-π interactions. For the OOP molecule (UUS structure), the two ester groups at the sn-1 and sn-2 positions are spatially adjacent, forming a localized, highly polar, and concentrated target site. However, this highly concentrated polarity may result in an uneven polarity distribution throughout the molecule, leading to a less than ideal spatial match between the dipole interaction and the electron-deficient triazine ring plane, thus weakening the overall affinity. Therefore, the OOP molecule mainly relies on Ag. + The double bond π-complexation results in a relatively weak overall binding force with the stationary phase, leading to a shorter retention time on the chromatographic column and subsequent elution. In contrast, the ester groups of the OPO molecule (USU structure) are more spatially dispersed and have a more uniform polarity distribution. Its molecular configuration is more conducive to generating comprehensive and stable dipole-dipole and n-π interactions with the triazine ring plane, enhancing the overall binding force with the stationary phase. Therefore, it has a longer retention time and is eluted later.

[0014] Secondly, the rigid plane filters spatial conformations. The fatty acid chains of triglycerides are not entirely flexible; their conformation is constrained by double bonds and ester groups. In the OOP molecule, two adjacent oleoyl groups may lead to a more compact and rigid conformation, which may have poor spatial matching with the triazine ring plane, hindering effective face-to-face π-π stacking and thus reducing retention. In contrast, the saturated palmitoyl chain at the sn-2 position in the OPO molecule provides better conformational flexibility, allowing it to adjust its orientation to better interact complementaryly with the triazine ring plane, resulting in stronger retention.

[0015] Finally, from the perspective of carrier linkage, the triazine ring structure also offers convenience. The remaining third position on the triazine ring (e.g., another substitution site relative to the two thiol groups) can be easily linked to the silica surface via coupling agents such as aminosilanes. This linkage method is mature and reliable, and the rigidity of the triazine ring helps to form a well-defined, spatially oriented functional layer on the silica surface, avoiding the functional group burial or uneven distribution problems often caused by long flexible chains, thus ensuring the active site (Ag) + Accessibility and uniformity of ).

[0016] In summary, the stationary phase of this invention uses a rigid, electron-deficient triazine ring as a platform. On one hand, it forms a high-strength, migration-resistant chelate structure with silver ions through its fixed-spacing dithiol groups; on the other hand, it utilizes its own planar structure to generate secondary interactions with the target molecule, synergizing with the primary force of silver ions, to achieve efficient and highly selective separation of OPO and OOP, a pair of difficult-to-separate positional isomers. In this separation system, OOP, due to its high polarity concentration and poor spatial conformational matching, has a weaker overall interaction with the stationary phase and is therefore eluted first; while OPO, due to its uniform polarity distribution and good conformational adaptability, interacts more strongly with the stationary phase and is eluted later. If only a single thiol group is used, the stability of silver ions is insufficient; if a flexible double-chain thiol group is used, the coordination environment is uncontrollable and lacks planar recognition function; if the triazine ring is replaced with other ring systems (such as a benzene ring), it cannot provide the same level of electronic effect and synergistic stabilizing effect.

[0017] In a second aspect, the present invention provides a method for preparing a stationary phase for OPO column chromatography separation and purification, the preparation method comprising:

[0018] Dithiotriazine-functionalized silica was obtained by reacting the dimeriotriazine-functionalized silica with silver nitrate solution and drying it to obtain the stationary phase.

[0019] Preferably, the preparation method specifically includes adding dimercaptotriazine-functionalized silica to a silver nitrate solution and reacting it at 20-30°C and 100-300 rpm in the dark for 15-40 hours. After the reaction is completed, the solution is filtered, washed alternately with methanol and deionized water, and dried under vacuum at 50-70°C for 15-30 hours to obtain the stationary phase.

[0020] Understandably, these preparation conditions are set to ensure sufficient reaction between silver ions and the dimercaptotriazine functional groups, while avoiding side reactions. The reaction temperature is controlled within the range of 20–30°C because excessively high temperatures may lead to silver ion reduction or functional group degradation, while excessively low temperatures result in slow reaction rates and incomplete loading. Light-protected operation prevents silver ions from being reduced to silver particles under light, affecting the stability of the stationary phase and separation performance. A rotation speed of 100–300 rpm ensures uniform mixing of the reaction system, avoiding agglomeration or uneven loading due to excessively high local concentrations. A reaction time of 15–40 h provides a sufficient kinetic window for silver ions to form stable coordination bonds with mercapto groups; too short a time may result in incomplete reaction, while too long a time increases energy consumption without significant benefits. The washing step uses alternating methanol and deionized water to remove unreacted silver nitrate and byproducts such as nitric acid, preventing residues from interfering with the separation process. The drying process was carried out under vacuum at 50–70 °C for 15–30 h to ensure complete removal of moisture and solvent from the stationary phase while avoiding damage to the silver-thiol bonds due to high temperatures. Excessive drying temperature or time may lead to functional group decomposition or silver ion aggregation; conversely, residual solvent may affect the equilibrium and elution of column chromatography. This preparation method, by optimizing these parameters, achieves efficient and reproducible production of the stationary phase, laying the foundation for large-scale application.

[0021] Preferably, the silver nitrate solution is obtained by dissolving silver nitrate in an aqueous methanol solution, and the volume ratio of methanol to water is (0.8~1.2):1.

[0022] Understandably, the choice of the methanol-to-water volume ratio is based on the solubility of silver nitrate and the polarity requirements of the reaction medium. A methanol-to-water ratio of (0.8–1.2:1) creates a suitable polar environment, effectively dissolving silver nitrate without excessively affecting the stability of the dimercaptotriazine functional group. Methanol, as an organic solvent, facilitates the dispersion and penetration of silver ions, while water promotes ion exchange and coordination reactions. If the methanol ratio is too high, it may lead to insufficient aqueous phase, inadequate dissolution of silver nitrate, and decreased reaction efficiency; if the water ratio is too high, the solubilizing effect of methanol is weakened, potentially causing silver ion precipitation or functional group hydrolysis. This ratio range ensures the homogeneity of the reaction system and the reaction rate, thereby optimizing the silver ion loading and the performance of the stationary phase.

[0023] Preferably, the mass ratio of silver nitrate to dimercaptotriazine-functionalized silica is (23~30):80.

[0024] Understandably, this mass ratio setting is intended to balance silver ion loading and functional group utilization. A mass ratio of (23~30):80 ensures a slight excess of silver ions, thereby covering all potential coordination sites of the dimercaptotriazine functional group and achieving maximum loading. If the mass ratio is too low, insufficient silver ions may result in some functional groups remaining unreacted, reducing the separation capacity of the stationary phase; if the mass ratio is too high, excess silver ions may exist in a physically adsorbed form, easily migrating during elution, causing column contamination and reduced separation efficiency. Within this range, the stationary phase exhibits optimal stability and separation performance.

[0025] Preferably, the dimercaptotriazine-functionalized silica has a particle size of 40-63 μm and a pore size of 40-80 Å.

[0026] Understandably, the selection of particle size and pore size for dimercaptotriazine-functionalized silica is a comprehensive optimization result based on the mass transfer efficiency and separation resolution requirements of column chromatography. The particle size referred to here is the diameter distribution range of the silica support particles, which directly affects the hydrodynamic performance and pressure drop of the packed column; while the pore size refers to the average diameter of the pores inside the silica particles, which determines whether target molecules can smoothly enter the pores and contact the active sites of the stationary phase, i.e., the mass transfer efficiency.

[0027] Specifically, controlling the particle size within the range of 40–63 μm is based on the balance considerations of preparative column chromatography. This range is considered a medium particle size, providing a sufficiently high specific surface area to ensure adequate loading capacity while maintaining a reasonable column inlet pressure, allowing for stable operation under conventional pressurization or gravity-driven conditions. If the particle size is too small, although it can significantly increase the theoretical plate number, it will lead to a sharp increase in column pressure, requiring a high-pressure system, which not only increases equipment costs and operational complexity but also easily leads to column bed compaction and channeling, making it completely unsuitable for conventional preparative purification. Conversely, if the particle size is too large, the specific surface area will be significantly reduced, the stationary phase loading will decrease, and the mass transfer path between particles will become longer, resulting in peak broadening and severely degraded separation resolution.

[0028] Setting the pore size to 40–80 Å is to match the hydrodynamic dimensions of triglyceride molecules. OPO and OOP, as relatively large organic molecules, require sufficiently large pores to diffuse smoothly into the interior. A pore size range of 40–80 Å ensures that these molecules can freely enter and exit the pores, contacting the numerous bonded silver (I)-dimercaptotriazine active sites within the pores, thus achieving efficient adsorption and desorption. If the pore size is too small, steric hindrance will repel most target molecules outside the pores, allowing only a limited number of sites on the outer surface of the particles to be utilized, greatly reducing the effective loading of the stationary phase and separation efficiency. If the pore size is too large, although molecule entry and exit are unimpeded, the specific surface area per unit volume will decrease sharply, similarly reducing the total number of active sites and potentially weakening the close-range interaction between the stationary phase and molecules, ultimately leading to poor separation and peak tailing.

[0029] This invention achieves control over particle size and pore size by selecting commercially available silica support raw materials with specific specifications. Specifically, before step S102, "acid washing with silica in an acidic solution," at the beginning of the synthesis route, spherical or amorphous silica with a particle size of 40–63 μm and a pore size of 40–80 Å is selected as the starting material. All subsequent functionalization reactions (silanization, triazine ring grafting, mercapto introduction, etc.) are chemical modifications based on this pre-defined physical framework. These reaction conditions (such as temperature, solvent, and reaction time) are optimized to maximize the transformation of surface functional groups while maintaining the inherent particle size and pore size distribution of the silica support itself. Through this strategy of combining standardized raw material selection with standardized synthesis processes, this invention ensures high batch-to-batch consistency and reproducibility of the prepared dimercaptotriazine-functionalized silica, and even the final stationary phase product, in terms of core physical parameters, guaranteeing the reliability and scalability of the separation and purification results.

[0030] It is understood that the present invention does not impose specific restrictions on the method of obtaining dimercaptotriazine-functionalized silica. The product can be obtained directly through conventional commercial channels or prepared using conventional reagents, methods and equipment in this technical field.

[0031] To better obtain the dithiotriazine-functionalized silica required by this invention, this invention provides a method for preparing dithiotriazine-functionalized silica, the method comprising:

[0032] S102. Add silicon dioxide to an acidic solution and wash it to obtain activated silicon dioxide;

[0033] S104. React the activated silica obtained in step S102 with the silane coupling agent to obtain silane-coupled silica.

[0034] S106. Connect the epoxy-coupled silica obtained in step S104 to a triazine functional ligand to obtain triazine-functionalized silica.

[0035] S108. The triazine-functionalized silica obtained in step S106 is introduced into a mercapto group to obtain dimercaptotriazine-functionalized silica.

[0036] It is understood that dimercaptotriazine-functionalized silica is the foundation for constructing the stationary phase described in this invention. The preparation method parameters provided by this invention have been designed and optimized to ensure that the final product possesses a stable chemical structure, uniform physical properties, and excellent chromatographic separation performance. Through multi-step functionalization reactions, the method precisely constructs a bidentate coordination structure with specific spatial configuration and electronic characteristics on a silica support, ensuring stable loading and high selective separation performance of subsequent silver ions.

[0037] Preferably, step S102 includes immersing silica in an acidic solution, acid washing at 90~100℃ for 8~12h, washing with hot deionized water (60~80℃) until neutral after acid washing, and vacuum drying at 100~150℃ for 1.5~6h to obtain activated silica; wherein, the acidic solution includes a nitric acid aqueous solution with a volume fraction of 2~10%, and the mass ratio of silica to acidic solution is 1:(5~10).

[0038] Understandably, the acid pickling activation step is a prerequisite for all subsequent functionalization reactions. Acid pickling with a 2-10% (v / v) nitric acid aqueous solution at 90-100°C for 8-12 hours aims to deeply clean the silica surface and maximize its silanol (-Si-OH) density. Within this temperature and time range, the oxidizing properties of nitric acid aid in surface cleaning and its volatility minimizes the risk of residue. If the pickling temperature is too low or the time too short, activation will be insufficient, resulting in inadequate surface silanol density, directly affecting the grafting density and uniformity of the subsequent silanization reaction. If the pickling temperature is too high or the acid concentration is too high, it may over-etch the silica framework, damaging its pore structure and mechanical strength. Controlling the mass ratio of silica to acid solution to 1:(5-10) ensures sufficient solid-liquid contact, allowing each silica particle to be effectively wetted and treated by the acid. Washing with water to neutrality after acid pickling thoroughly removes residual acid and soluble impurities, preventing interference with subsequent reactions or adverse effects on the stationary phase performance. Subsequently, vacuum drying at 100-150°C for 1.5-6 hours aims to completely remove physically adsorbed water, forming an activated, readily reacting silica surface to prepare for the next silanization reaction. Ultrasonic treatment can also be used in the initial pickling and washing stages to help disperse particles, remove internal impurities, and improve activation uniformity.

[0039] Preferably, step S104 includes refluxing activated silica and silane coupling agent in an organic solvent at 90-120°C for 12-36 h, washing with a mixed solvent of anhydrous ethanol and toluene after the reaction, and vacuum drying at 100-150°C for 1.5-6 h to obtain silane-coupled silica; wherein the silane coupling agent includes γ-aminopropyltriethoxysilane, the organic solvent includes anhydrous toluene, the mass ratio of activated silica to silane coupling agent is 1:(1-4), and the mass ratio of activated silica to organic solvent is 1:(5-15).

[0040] Understandably, the purpose of the silanization step is to establish a stable, covalent bond between the inorganic silica support and the organic functional layer. γ-aminopropyltriethoxysilane (APTES) is chosen as the coupling agent because its ethoxysilane group at one end can undergo a hydrolytic condensation reaction with the silanol groups on the activated silica surface, forming a strong Si-O-Si covalent bond; while the primary amino group (-NH2) at the other end provides a highly reactive site for subsequent linkage with the triazine ring. Preferably, γ-aminopropyltriethoxysilane can be a silane coupling agent of type KH550. The reflux reaction is carried out in anhydrous toluene to prevent water molecules from causing premature self-condensation of APTES, forming multilayers or oligomers, thereby ensuring the formation of a dense, ordered, and stable monolayer on the silica surface. The reaction temperature was controlled at 90–120°C, and the reaction time was set at 12–36 hours to provide sufficient energy and time to drive the hydrolysis and condensation reactions of APTES to near completion, achieving high grafting density and uniform coverage. The mass ratio of activated silica to APTES was controlled within the range of 1:(1–4) to provide an appropriate amount of coupling agent to ensure surface reaction saturation, while avoiding excessive APTES that could lead to physical adsorption or the formation of multilayer structures, affecting the accuracy of subsequent reactions. The mass ratio of activated silica to organic solvent was 1:(5–15) to provide sufficient reaction medium, ensuring good dispersion and mass transfer efficiency of the reactants. After the reaction, washing with a mixed solvent of anhydrous ethanol and toluene was used to effectively remove physically adsorbed, unreacted APTES and its byproducts. Finally, vacuum drying at 100–150°C was performed to thoroughly remove residual solvent and further stabilize the bond between the silane layer and the silica surface.

[0041] Preferably, step S106 includes dispersing silane-coupled silica and an acid-binding agent (such as triethylamine) in anhydrous toluene, and slowly adding an anhydrous toluene solution of cyanuric chloride under an inert atmosphere (such as nitrogen or argon). The reaction is stirred at room temperature (20-30°C) for 6-12 hours. After the reaction is complete, the silica is thoroughly washed successively with anhydrous toluene and anhydrous acetone to remove unreacted cyanuric chloride, acid-binding agent salt, and byproducts. Finally, it is vacuum dried at 40-60°C for 3-6 hours to obtain triazine-functionalized silica. The molar ratio of the acid-binding agent to the amino groups on the surface of the silane-coupled silica can be (1.0-1.5):1; the molar ratio of cyanuric chloride to the surface amino groups can be (0.8-1.2):1.

[0042] Understandably, the purpose of this step is to precisely introduce the rigid triazine ring planar structure with key functions onto the support surface. Cyanurium chloride was chosen as the triazine ring precursor because of the differentiated reactivity of its three chlorine atoms. Under strict anhydrous and inert atmosphere (such as nitrogen) protection and in the presence of an acid-binding agent (such as triethylamine), the reaction is carried out at room temperature (20–30 °C), achieving highly selective single-site nucleophilic substitution. Specifically, the primary amino group on the silane-coupled silica surface attacks the most reactive chlorine atom on the cyanurium chloride ring. The acid-binding agent promptly neutralizes the HCl generated in the reaction, driving the reaction forward and preventing acid damage to the support bonds, thereby forming a stable CN bond. This covalently anchors the triazine ring to the support in a single-site connection manner. This connection method retains the other two chlorine atoms in the triazine ring, creating the structural prerequisite for the subsequent introduction of a dithiol group at a specific position (meta). The reaction temperature and time settings (room temperature, 20–30 °C, reaction time 6–12 h) are based on optimization of reaction efficiency and selectivity. In the presence of an acid-binding agent, room temperature provides sufficient kinetics for nucleophilic substitution, avoiding side reactions (such as substitution of multiple chlorine atoms or self-polymerization of cyanuric chloride) that might be exacerbated by heating (e.g., 60-90°C). This mild condition, combined with sufficient reaction time, ensures the completeness and specificity of the grafting reaction. Controlling the molar ratio of acid-binding agent to surface amino groups at (1.0-1.5):1 provides sufficient basicity to neutralize the byproduct HCl while avoiding excess. Controlling the molar ratio of cyanuric chloride to surface amino groups at (0.8-1.2):1 aims to provide near-stoichiometric reactants, ensuring complete grafting while minimizing the purification burden and side reaction risks associated with excess reagents. Using anhydrous toluene as a solvent and maintaining an inert atmosphere are crucial measures to suppress the hydrolysis of cyanuric chloride. The presence of water causes chlorine atoms to hydrolyze into hydroxyl groups, which not only consumes reactants but also destroys the structural integrity of the triazine ring; therefore, an anhydrous environment is essential for the success of this step. Following the reaction, the product was thoroughly washed sequentially with anhydrous toluene and then with anhydrous acetone to completely remove unreacted cyanuric chloride, the generated triethylamine hydrochloride, and other organic byproducts. Washing with anhydrous organic solvents avoids introducing moisture during purification, thus protecting the grafted triazine ring intermediate still bearing active chlorine atoms. Subsequent vacuum drying at 40–60°C further enhances product stability while removing residual solvents.

[0043] Preferably, step S108 includes reacting triazine-functionalized silica with a thiolizing agent in an organic solvent at 60-90°C for 6-24 hours, washing alternately with water and acetone after the reaction, and vacuum drying at 100-150°C for 1.5-6 hours to obtain acetyl-protected dimercaptotriazine-functionalized silica; wherein the thiolizing agent includes potassium thioacetate, and the organic solvent includes N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO); the mass ratio of triazine-functionalized silica to thiolizing agent is 1:(1-5), and the mass ratio of triazine-functionalized silica to organic solvent is 1:(5-15).

[0044] Understandably, this step aims to introduce a thioacetyl group (-SAc), which serves as a thiol precursor, into the remaining specific chlorine atom position on the triazine ring. Potassium thioacetate was chosen as the thioacetylating agent because of its thioacetyl anion (CH3COS). - Potassium thioacetate (β-S-acetate) is an excellent nucleophile capable of efficiently replacing the remaining chlorine atoms on the triazine ring to form CS bonds in polar aprotic solvents (such as DMF or DMSO) at 60–90 °C. The key to using potassium thioacetate instead of directly using mercapto compounds (such as ethanethiol) lies in the protecting group strategy: the introduced thioacetyl group (-SAc) acts as a protecting group, preventing the free mercapto groups (-SH) from being oxidized to form disulfide bonds (-SS-) during synthesis, storage, and subsequent reactions, thus ensuring the integrity and reactivity of the two mercapto groups on each triazine ring. The reaction is carried out at 60–90 °C for 6–24 hours to ensure the nucleophilic substitution reaction proceeds fully and achieves a high conversion rate. Controlling the mass ratio of triazine-functionalized silica to potassium thioacetate to 1:(1–5) provides sufficient nucleophile to drive the reaction while also considering economic efficiency and the convenience of subsequent purification. Using high-boiling-point polar aprotic solvents such as DMF or DMSO helps dissolve potassium thioacetate and promotes mass transfer and reaction between the solid and liquid phases. After the reaction, alternating washing with water and acetone effectively removes unreacted reagents, byproduct salts (such as KCl), and solvent residues. Subsequent vacuum drying ensures stable storage of the intermediate and prepares it for the next step of hydrolysis to remove the protective layer.

[0045] Preferably, step S108 further includes hydrolyzing acetyl-protected dimercaptotriazine-functionalized silica in a hydrolysate at 20-40°C for 2-12 hours, washing alternately with water and acetone after hydrolysis, and vacuum drying at 100-150°C for 1.5-6 hours to obtain dimercaptotriazine-functionalized silica; wherein, the hydrolysate is a mixed solution of ammonia, methanol and water, or a mixed solution of sodium hydroxide, methanol and water; in the hydrolysate, the volume fraction of ammonia is 5%-15%, or the concentration of sodium hydroxide is 0.1-0.5 mol / L; the volume ratio of methanol to water is (7:3)-(9:1); the mass ratio of acetyl-protected dimercaptotriazine-functionalized silica to the hydrolysate is 1:(5-15).

[0046] Understandably, this hydrolysis step is crucial for removing the thioacetyl protecting group and exposing the active free thiol group (-SH), thereby obtaining the final dimercaptotriazine-functionalized silica precursor. Alkaline hydrolysis conditions (ammonia or sodium hydroxide) are used because, under a mild alkaline environment, the thioester bond (-SAc) can be selectively hydrolyzed and broken to generate free thiol groups and acetate ions without damaging the triazine ring structure, Si-C bonds, or Si-O-Si framework. Choosing a relatively mild alkali (ammonia or low-concentration NaOH) and reacting at a relatively low temperature (20–40°C) for 2–12 hours is to ensure the hydrolysis reaction proceeds smoothly while minimizing the potential degradation or damage to the silica support and its surface functional groups caused by strong alkali and high temperature. A methanol-water mixture was used as the hydrolysis medium, with a volume ratio controlled between 7:3 and 9:1. The main function of methanol was to promote the wetting and penetration of the hydrolysis reagent onto the hydrophobic functionalized silica surface, ensuring that the hydrolysis reaction could occur effectively within the particles. The mass ratio of acetyl-protected dimercaptotriazine functionalized silica to the hydrolysis solution was controlled at 1:(5-15) to ensure sufficient solid-liquid contact and provide a sufficiently homogeneous environment for the hydrolysis reaction. After hydrolysis, the silica was washed again alternately with water and acetone to thoroughly remove ammonium acetate (or sodium acetate), excess alkali, and solvent produced during hydrolysis. The final vacuum drying step removed all volatile components, yielding dry, stable, and free thiol-rich dimercaptotriazine functionalized silica, which provided an ideal precursor for the subsequent reaction with silver nitrate solution to prepare the final stationary phase.

[0047] Specifically, the reaction process of silver (I)-dimercaptotriazine functionalized silicon dioxide in this invention is as follows:

[0048]

[0049] Thirdly, the present invention provides a column chromatography separation and purification method for OPO, wherein the stationary phase prepared by the method of the first aspect or the preparation method of the second aspect is uniformly packed into a chromatography column, the chromatography column is pre-equilibrated with an eluent, crude OPO is dissolved in the eluent to obtain a sample to be loaded, the sample to be loaded is loaded onto the chromatography column, the substances adsorbed by the stationary phase are eluted with an eluent, and the fractions containing OPO are combined, evaporated and dried to obtain purified OPO.

[0050] Understandably, this separation and purification method fully utilizes the selective adsorption and elution kinetics of the stationary phase. Uniform stationary phase packing ensures mass transfer uniformity and separation efficiency within the column, avoiding peak broadening caused by channeling or bubbles. A pre-equilibration step uses eluent to bring the stationary phase to adsorption-desorption equilibrium, preparing it for sample loading. During sample loading, crude OPO is dissolved in the eluent, reducing the polarity difference between the sample solvent and the mobile phase and preventing band distortion. During elution, the hexane and ethyl acetate mixture of the eluent achieves gradient elution of OPO and OOP by adjusting their polarity: hexane, as a nonpolar component, promotes hydrophobic interactions, while ethyl acetate, as a polar component, regulates the elution intensity, allowing OPO and OOP to separate based on the difference in the number and position of their double bonds. After combining the fractions containing OPO, evaporation and drying remove the solvent, yielding a high-purity product. This method requires only one column chromatography step to achieve efficient separation, simplifying the process and reducing energy consumption and cost.

[0051] Preferably, the eluent is a mixture of n-hexane and ethyl acetate, with a volume ratio of n-hexane to ethyl acetate of (80:20) to (90:10), and the eluent flow rate is 2 to 6 ml / min; the inner diameter of the chromatography column is 10 to 20 mm, and the amount of stationary phase packed in the chromatography column is 13 to 18 g; the mass of crude OPO in the sample to be loaded is 300 to 700 mg, and the volume of the eluent is 0.5 to 2 ml.

[0052] Understandably, the selection of eluent ratio and flow rate is based on the polarity and adsorption characteristics of OPO and OOP. A volume ratio of n-hexane to ethyl acetate in the range of (80:20) to (90:10) provides moderate elution strength; the higher the ratio, the lower the eluent polarity, the longer the retention time of OPO, and the better the separation. A flow rate setting of 2–6 ml / min optimizes the mass transfer process: too low a flow rate improves separation but prolongs the experimental time; too high a flow rate may lead to peak overlap and decreased recovery. A column inner diameter of 10–20 mm and a packing volume of 13–18 g ensure a match between column efficiency and sample loading; too small an inner diameter or insufficient packing volume may limit the throughput, while too large an inner diameter reduces separation efficiency. A sample loading volume of 300–700 mg and an eluent volume of 0.5–2 ml are designed based on the adsorption capacity of the stationary phase; too low a loading volume wastes stationary phase, while too high a loading volume leads to overload and tailing. The synergistic effect of these parameters achieves an efficient and economical purification process.

[0053] Preferably, the crude OPO is crude OPO synthesized by lipase catalysis and has an OPO content of 40% to 60%.

[0054] Understandably, crude OPO synthesized using lipase catalysis is chosen because of its high reaction specificity, low byproduct production, ease of subsequent purification, and the environmentally friendly and low-cost lipase-catalyzed synthesis route. An OPO content within the range of 40%–60% ensures sufficient target molecules for separation from the crude product, while maintaining controllable impurity levels. If the content is below 40%, purification efficiency is low, and recovery rate decreases; if it is above 60%, elution conditions may need to be adjusted to prevent co-elution.

[0055] Fourthly, the present invention provides an OPO product obtained by column chromatography separation and purification using the OPO method described in the third aspect.

[0056] Understandably, this OPO product, prepared using the aforementioned optimized method, possesses high purity and accurate quantitative values, making it suitable as a standard substance. Its organic purity is high, and the residual impurities are primarily trace amounts of OOP, which do not affect its application. This product exhibits strong traceability and comparability, filling a gap in domestic OPO standard substances and providing a reliable reference for the quality control of infant formula milk powder.

[0057] The beneficial effects of this invention are as follows:

[0058] The stationary phase structure of this invention is silver(I)-dimercaptotriazine functionalized silica. Its core lies in the covalent fixation of silver ions onto the silica support, thereby solving the problems of silver ion migration and poor stability in existing silver-impregnated silica columns. In existing technologies, although silver ion chromatography columns can separate compounds containing varying numbers of double bonds, silver ions are easily migrated during elution because they are only loaded onto the silica surface through physical adsorption or weak interactions, leading to decreased separation efficiency and shortened column life. Furthermore, silver ions are sensitive to light and heat, and are easily reduced to silver particles under long-term use, affecting separation performance. This invention introduces a dimercaptotriazine functional group, utilizing the strong coordination bond between the thiol group and silver ions, combined with the rigid framework of the triazine structure, to achieve stable fixation of silver ions. This structure not only prevents silver ion loss but also enhances the stability of the stationary phase under thermal and light conditions, maintaining high separation efficiency during long-term use. Furthermore, the surface chemistry of the stationary phase is carefully designed, with the dimercaptotriazine functional group providing specific affinity sites that can selectively interact with the double bonds and ester groups in OPO and OOP molecules. As positional isomers, OPO and OOP differ primarily in the arrangement of their fatty acid chains: OPO has a USU structure (unsaturated-saturated-unsaturated), while OOP has a UUS structure (unsaturated-unsaturated-saturated). This subtle difference leads to their different polarities and spatial configurations. This stationary phase, through the π-complexation of silver ions with the double bonds and the π-π stacking ability of the triazine structure, can distinguish the hydrophobicity and steric effects of these two isomers, thus achieving efficient separation. Attached Figure Description

[0059] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0060] Figure 1 The liquid chromatogram of the raw material for the OPO purity standard substance prepared in this invention;

[0061] Figure 2 The image shows the 1H NMR spectrum of the purified OPO product from Example 7 of this invention. Detailed Implementation

[0062] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0063] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0064] Preparation Example

[0065] Preferably, the structural formula of the stationary phase used in the embodiment is:

[0066] .

[0067] The method for preparing the stationary phase includes the following steps:

[0068] S102. Immerse silica in an acidic solution and acid wash at 100°C for 10 hours. After acid washing, wash with hot deionized water at 70°C until neutral, and vacuum dry at 140°C for 4 hours to obtain activated silica. The acidic solution includes a 5% (v / v) aqueous solution of nitric acid. The mass ratio of silica to acidic solution is 1:8. The silica has a particle size of 55 μm and a pore size of 60 Å.

[0069] S104. Activated silica and silane coupling agent are refluxed in an organic solvent at 110°C for 24 h. After the reaction, the mixture is washed with a mixed solvent of anhydrous ethanol and toluene and dried under vacuum at 140°C for 4 h to obtain silane-coupled silica. The silane coupling agent includes γ-aminopropyltriethoxysilane, the organic solvent includes anhydrous toluene, the mass ratio of activated silica to silane coupling agent is 1:2, and the mass ratio of activated silica to organic solvent is 1:10.

[0070] S106. Silane-coupled silica and triethylamine are dispersed in anhydrous toluene. Under nitrogen protection, an anhydrous toluene solution of cyanuric chloride is slowly added. The reaction is stirred at room temperature (25°C) for 10 hours. After the reaction is complete, the silica is thoroughly washed successively with anhydrous toluene and anhydrous acetone to remove unreacted cyanuric chloride, acid-binding agent salt, and byproducts. Finally, it is vacuum dried at 50°C for 4 hours to obtain triazine-functionalized silica. The molar ratio of the acid-binding agent to the amino groups on the surface of the silane-coupled silica is 1:1; the molar ratio of cyanuric chloride to the surface amino groups is 1:1.

[0071] S108. Triazine-functionalized silica and a mercaptochemical reagent are reacted in an organic solvent at 70°C for 12 h. After the reaction, the silica is washed alternately with water and acetone and dried under vacuum at 140°C for 4 h to obtain acetyl-protected dimercaptotriazine-functionalized silica. The mercaptochemical reagent includes potassium thioacetate, and the organic solvent includes N,N-dimethylformamide. The mass ratio of triazine-functionalized silica to mercaptochemical reagent is 1:2.5, and the mass ratio of triazine-functionalized silica to organic solvent is 1:10.

[0072] Acetyl-protected dimercaptotriazine-functionalized silica was hydrolyzed in a hydrolysate at 30°C for 6 hours. After hydrolysis, the silica was washed alternately with water and acetone and then dried under vacuum at 140°C for 4 hours to obtain dimercaptotriazine-functionalized silica. The hydrolysate was a mixed solution of sodium hydroxide, methanol, and water. The concentration of sodium hydroxide in the hydrolysate was 0.25 mol / L. The volume ratio of methanol to water was 9:1. The mass ratio of acetyl-protected dimercaptotriazine-functionalized silica to the hydrolysate was 1:10.

[0073] The dimercaptotriazine-functionalized silica obtained by S108 has a particle size of 55 μm and a pore size of 60 Å.

[0074] S110. 16g of dimercaptotriazine-functionalized silica was added to a silver nitrate solution and reacted at 25°C and 200rpm in the dark for 24h. After the reaction, the mixture was filtered, washed alternately with methanol and deionized water, and dried under vacuum at 60°C for 24h to obtain the stationary phase. The silver nitrate solution was obtained by dissolving 4.6g of silver nitrate in 100mL of methanol-water solution, with a methanol-to-water volume ratio of 1:1.

[0075] The specific parameters for sample loading, eluent ratio, and elution flow rate for separation and purification in the following examples are shown in Table 1.

[0076] Table 1

[0077]

[0078] Example 1

[0079] A wet packing process was used. 16 g of the stationary phase prepared in the preparation example was uniformly packed into a 15 mm inner diameter sintered sand column. Brief ultrasonic-assisted pressure settling was performed to ensure no air bubbles formed. Finally, a 10 mm high layer of quartz sand was packed at the top of the column to obtain the chromatography column. Elution was performed in the column using an eluent with a hexane:ethyl acetate volume ratio of 80:20. The sample loading was 300 mg of crude OPO (OPO content 53%), dissolved in 1 mL of eluent. Before loading, the column was pre-equilibrated with 300 mL of eluent, and the elution flow rate was controlled at 5 mL / min. 40 mL of eluent was collected per tube.

[0080] Example 2

[0081] The crude OPO was purified using the method described in Example 1, except that an eluent with a volume ratio of n-hexane to ethyl acetate of 85:15 was used for elution.

[0082] Example 3

[0083] The crude OPO was purified using the method described in Example 1, except that an eluent with a volume ratio of n-hexane to ethyl acetate of 90:10 was used for elution.

[0084] Example 4

[0085] The crude OPO was purified using the method described in Example 3, except that the sample loading amount was 500 mg of crude OPO (OPO content 53%).

[0086] Example 5

[0087] The crude OPO was purified using the method described in Example 3, except that the sample loading amount was 700 mg of crude OPO (OPO content 53%).

[0088] Example 6

[0089] The crude OPO was purified using the method described in Example 4, except that the elution flow rate was controlled at 3 mL / min.

[0090] Example 7

[0091] The crude OPO was purified using the method described in Example 4, except that the elution flow rate was controlled at 4 mL / min.

[0092] Example 8

[0093] The crude OPO was purified using the method described in Example 7, except that the silica in S102 had a particle size of 40 μm and a pore size of 40 Å. The dimercaptotriazine-functionalized silica obtained in S108 had a particle size of 40 μm and a pore size of 40 Å.

[0094] Example 9

[0095] The crude OPO was purified using the method described in Example 7, except that the silica in S102 of the preparation example had a particle size of 63 μm and a pore size of 80 Å. The dimercaptotriazine-functionalized silica obtained in S108 had a particle size of 63 μm and a pore size of 80 Å.

[0096] Example 10

[0097] The crude OPO was purified using the method of Example 7, except that in the preparation example, the silver nitrate solution in S110 was obtained by dissolving 5.2 g of silver nitrate in 100 mL of methanol aqueous solution, and the volume ratio of methanol to water was 1:1.

[0098] Example 11

[0099] The crude OPO was purified using the method of Example 7, except that in the preparation example, the silver nitrate solution in S110 was obtained by dissolving 6.0 g of silver nitrate in 100 mL of methanol aqueous solution, and the volume ratio of methanol to water was 1:1.

[0100] Comparative Example 1

[0101] The crude OPO was purified using the method described in Example 7, except that the silica in S102 of the preparation example had a particle size of 5 μm and a pore size of 20 Å. The dimercaptotriazine-functionalized silica obtained in S108 had a particle size of 5 μm and a pore size of 20 Å.

[0102] Comparative Example 2

[0103] The crude OPO was purified using the method of Example 7, except that in the preparation example, the silver nitrate solution in S110 was obtained by dissolving 4.0 g of silver nitrate in 100 mL of methanol aqueous solution, and the volume ratio of methanol to water was 1:1.

[0104] Comparative Example 3

[0105] The crude OPO was purified using the method of Example 7, except that in the preparation example, the silver nitrate solution in S110 was obtained by dissolving 7.0 g of silver nitrate in 100 mL of methanol aqueous solution, and the volume ratio of methanol to water was 1:1.

[0106] Comparative Example 4

[0107] The crude OPO was purified using the method described in Example 7, except that the stationary phase used was silver(I)-mercaptopropyl-functionalized silica, which was prepared as follows:

[0108] 16 g of silica (particle size 55 μm, pore size 60 Å) was immersed in 128 mL of 5% (v / v) nitric acid aqueous solution and acid-washed at 100 °C for 10 h. After acid washing, it was repeatedly washed with hot deionized water at 70 °C until the filtrate was neutral, and then vacuum dried at 140 °C for 4 h to obtain activated silica.

[0109] The activated silica and 32 g of mercaptopropyltrimethoxysilane were added to 160 mL of anhydrous toluene and refluxed at 110 °C for 24 h. After the reaction was completed, the silica was thoroughly washed with a mixture of anhydrous ethanol and toluene, and finally dried under vacuum at 140 °C for 4 h to obtain mercaptopropyl-functionalized silica.

[0110] Add 4.6 g of silver nitrate to 100 mL of a methanol-water mixture (volume ratio 1:1), stir to dissolve, and then add 16 g of the previously prepared mercaptopropyl-functionalized silica. React at 25 °C and 200 rpm in the dark for 24 h to allow silver ions to bond with mercapto groups. After the reaction is complete, filter to collect the solid, and wash alternately with methanol and deionized water until no silver ions are detected in the washings. Finally, dry under vacuum at 60 °C for 24 h to obtain the stationary phase used in Comparative Example 4.

[0111] Test case

[0112] The eluent obtained in the above examples was analyzed by HPLC. The specific detection results are shown in Table 2. The specific detection method is as follows:

[0113] The purified OPO product at a concentration of 2 mg / mL was prepared and subjected to high-performance liquid chromatography (HPLC). The chromatographic column was a ChromSpher 5Lipids (4.6 × 250 mm, 5 μm), the mobile phase was n-hexane:acetonitrile = 99.5:0.5, the flow rate was 1 mL / min, the detection wavelength was 190 nm, and the injection volume was 5 μL. Figure 1 As shown, based on retention time, the peak at 17.797 min is OOP, and the peak at 18.990 min is OPO. OOP and OPO can be baseline separated, and the only impurity in the OPO product is a small amount of OOP.

[0114] The fractions containing OPO but not OOP as detected by the above method (the first 160~360mL fractions) were combined, rotary evaporated, freeze-dried, and then prepared into a 2mg / mL solution with n-hexane for instrumental testing.

[0115] Table 2

[0116]

[0117] The results in Table 2 comprehensively evaluate the impact of various process parameters and stationary phase structure on OPO purification. Regarding the eluent composition, the OPO purity significantly improved with increasing hexane to ethyl acetate ratio, indicating that a higher hexane ratio enhances separation selectivity. When the sample loading was 500 mg and the elution flow rate was 4 mL / min (Example 7), the OPO purity reached 98.7%, and the recovery rate was 89.6%, achieving a balance between high purity and high recovery. Further optimization of the stationary phase structure parameters showed that a support particle size of 55 μm and a pore size of 60 Å (Example 7) yielded optimal performance, while deviations from this range resulted in a slight decrease in purity and recovery. The silver ion loading was optimal at a mass ratio of 23:80. In particular, the separation effect of Comparative Example 1 was significantly worsened when a small particle size / small pore size carrier was used, while the purity and recovery rate of Comparative Example 4, which used a monomercaptopropyl stationary phase without a rigid triazine ring and a bidentate chelate structure, were much lower than those of the structure of the present invention. This fully demonstrates the structural advantages of the silver (I)-dimercaptotriazine functionalized silica of the present invention in terms of silver ion stability and isomer recognition.

[0118] To further illustrate the technical effects of the present invention, the OPO product of Example 7 after purification was characterized as follows:

[0119] Approximately 4 mg of sample was weighed into a brown glass sample vial, dissolved in 1 mL of deuterated chloroform by sonication, and then transferred to a 5 mm standard NMR tube. The test was performed on a 400 MHz superconducting NMR spectrometer with the following key parameters: spectral width 20 ppm (8000.256 Hz), excitation pulse 90°, zg pulse sequence, sampling time 4 s, relaxation delay 2 s, cumulative scans 128, time-domain data points 64 K, and probe temperature 299.0 K (25.85 °C). The instrument underwent automatic shimming and tuning before sampling, followed by manual fine shimming and tuning. After data acquisition, professional processing was performed using MestReNova 14.0 software: phase correction and baseline smoothing were first performed, followed by chemical shift correction using tetramethylsilane (TMS, δ=0 ppm) as an internal standard. The software automatically identified the solvent residue peak (CDCl3: δ=7.26 ppm) and marked the sample characteristic peaks. By integrating and calculating the area ratio of each characteristic peak, and combining the chemical shift value and coupling constant with the OPO standard spectrum, the sample's 1H NMR spectrum is as follows: Figure 2 As shown, the sample can be confirmed to be OPO.

[0120] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A stationary phase for OPO column chromatographic separation and purification, characterized in that, The stationary phase is prepared by the following method: Dithiotriazine-functionalized silica was obtained by reacting the dimeriotriazine-functionalized silica with silver nitrate solution and drying it to obtain the stationary phase. The structural formula of the dimercaptotriazine-functionalized silica is as follows: 。 2. The stationary phase of claim 1, wherein, The dimercaptotriazine-functionalized silica has a particle size of 40-63 μm and a pore size of 40-80 Å.

3. The stationary phase of claim 1, wherein, The preparation method specifically includes adding dimercaptotriazine-functionalized silica to a silver nitrate solution and reacting it at 20-30°C and 100-300 rpm in the dark for 15-40 hours. After the reaction is completed, the solution is filtered, washed alternately with methanol and deionized water, and dried under vacuum at 50-70°C for 15-30 hours to obtain the stationary phase.

4. The stationary phase of claim 3, wherein, Silver nitrate solution is obtained by dissolving silver nitrate in an aqueous methanol solution, with a volume ratio of methanol to water of (0.8~1.2):

1.

5. The stationary phase of claim 4, wherein, The mass ratio of silver nitrate to dimercaptotriazine-functionalized silica is (23~30):

80.

6. A method for the purification of OPO by column chromatography, characterized in that, The stationary phase according to any one of claims 1 to 5 is uniformly packed into the chromatography column, the chromatography column is pre-equilibrated with eluent, crude OPO is dissolved in eluent to obtain the sample to be loaded, the sample to be loaded is loaded onto the chromatography column, the substances adsorbed by the stationary phase are eluted with eluent, the fractions containing OPO are combined, evaporated and dried to obtain purified OPO.

7. The column chromatography separation and purification method according to claim 6, characterized in that, The eluent is a mixture of n-hexane and ethyl acetate, with a volume ratio of n-hexane to ethyl acetate of (80:20) to (90:10). The eluent flow rate is 2 to 6 ml / min. The inner diameter of the chromatography column is 10 to 20 mm, and the amount of stationary phase packed in the column is 13 to 18 g. The mass of crude OPO in the sample to be loaded is 300 to 700 mg, and the volume of the eluent is 0.5 to 2 ml.

8. The column chromatography separation and purification method according to claim 7, characterized in that, The crude OPO is a crude OPO synthesized by lipase catalysis and has an OPO content of 40% to 60%.

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