A method for preparing a cyclodextrin chromatography stationary phase

Abstract

The present application belongs to a novel chromatographic stationary phase, and particularly relates to a preparation method of a cyclodextrin chromatographic stationary phase. The present application adopts tetrafluoro-p-phenylenedinitrile as a representative spacer, is easy to be crosslinked with cyclodextrin to generate a crosslinked cyclodextrin compound with a high specific surface area, and solves the problem of low cyclodextrin bonding amount of the traditional cyclodextrin chromatographic stationary phase. The chromatographic stationary phase can provide pi-pi interaction, hydrogen bond interaction and steric hindrance effect for the separation and analysis of structural analogs, and the inclusion effect of the cyclodextrin hydrophobic cavity is mutually coordinated and cooperated, the selectivity for the structural analogs is improved, and a unique separation effect is generated. As shown in the experimental example 1 of the present application, the peak orders of the compounds with similar structures on the present chromatographic column and the C18 column are different, and the separation selectivity is different. For the compounds which are difficult to separate, a new selectable chromatographic stationary phase is provided, and the separation efficiency of the C18 chromatographic stationary phase is improved.

Description

Technical Field

[0001] This invention relates to novel chromatographic stationary phases, and specifically to a method for preparing a cyclodextrin chromatographic stationary phase, namely, using tetrafluoroterephthalonitrile and other substances as spacers to prepare a cyclodextrin chromatographic stationary phase with the ability to separate structural analogs. Background Technology

[0002] The separation and purification of compounds are the cornerstones of modern chemistry in laboratory research and industrial applications. Biomass, coal, and other hydrocarbon resources produce large amounts of tar during thermal conversion. This tar contains a significant amount of high-value-added substances, phenols. Therefore, the efficient and economical extraction and separation of phenolic compounds is of great practical importance for the utilization of tar.

[0003] Cyclodextrins are cyclic oligomers composed of multiple D-pyranose glucose units (commonly 6-8 units). The hydrophobic cavities of cyclodextrins can accommodate guest molecules of a certain size. The primary hydroxyl group at the 6-position exhibits the highest reactivity and basicity, readily undergoing nucleophilic reactions with isocyanates, acyl halides, and epoxides. Its chiral recognition also stems from its cavity stereostructure. However, the current bottleneck in the preparation of cyclodextrin chromatographic stationary phases lies in the low bonding capacity, slow recognition speed, and limited recognition ability of cyclodextrins, leading to poor separation results.

[0004] Tetrafluoroterephthalonitrile (TFTPN) is a rigid, multi-substituted aromatic compound. It readily bonds to the surface of silica spheres and readily crosslinks with cyclodextrin to form crosslinked cyclodextrin complexes with high specific surface area, solving the problem of low cyclodextrin bonding amounts commonly found in traditional cyclodextrin chromatographic stationary phases. Secondly, this cyclodextrin chromatographic stationary phase with TFTPN as the spacer arm possesses a high-porosity structure, enabling rapid identification. Thirdly, this chromatographic stationary phase provides π-π interactions, hydrogen bonding, and steric exclusion for the separation and analysis of structural analogs, which synergistically enhance the inclusion effect of the hydrophobic cavities of cyclodextrin, improving the selectivity for structural analogs. Summary of the Invention

[0005] The present invention aims to provide a novel method for preparing cyclodextrin-based chromatographic stationary phases. The stationary phase prepared by this method has a high cyclodextrin bonding amount and specific recognition ability, enabling rapid separation and analysis of structural analogs.

[0006] The cyclodextrin chromatographic stationary phase was obtained according to the preparation method of the present invention:

[0007] A cyclodextrin chromatographic stationary phase can be prepared by a one-step or two-step method, and the reaction process is as follows:

[0008] (a) The silicon spheres were uniformly dispersed in a mixed solution of anhydrous ethanol, water, and ammonia, and then a silanizing reagent was added. The mixture was stirred at room temperature. After the reaction was completed, the mixture was filtered, washed with water and ethanol respectively, and then dried under vacuum at 40-70℃ to obtain the silanized silicon spheres.

[0009] (b1) One-step preparation: Silanized silica spheres, polysubstituted aromatic compounds, and cyclodextrin are uniformly dispersed in an organic solution, transferred to a reaction apparatus, and heated and stirred under nitrogen for 3-10 min. After the reaction is complete, the mixture is filtered and washed with tetrahydrofuran, N,N-dimethylformamide, and dichloromethane, respectively. Finally, it is dried under vacuum at 40-70℃ to obtain the product.

[0010] (b2) Two-step preparation: Silanized silica spheres and polysubstituted aromatic compounds were uniformly dispersed in an organic solvent, heated and stirred. After the reaction was complete, the mixture was filtered, washed sequentially with acetonitrile and ethanol, and finally dried under vacuum at 40-70°C to obtain the product of the first step reaction. Then, the product of the first step reaction and cyclodextrin were uniformly dispersed in an organic solvent, transferred to a reaction apparatus, and nitrogen gas was introduced for 3-10 min, followed by heating and stirring. After the reaction was complete, the mixture was filtered, washed with tetrahydrofuran, N,N-dimethylformamide, and dichloromethane, respectively. Finally, it was dried under vacuum at 40-70°C to obtain the final product.

[0011] The method is characterized in that the silanizing agent in step (a) is 3-aminopropyltriethoxysilane, (3-mercaptopropyl)triethoxysilane, 3-chloropropyltriethoxysilane, propyltriethoxyisocyanate, 3-(2-aminoethylamino)propyltriethoxysilane, triethoxy(3-epoxypropyloxypropyl)silane, etc.

[0012] The method is characterized in that the cyclodextrin in steps (b1) and (b2) can be: α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or hydroxypropyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, dialdehyde-β-cyclodextrin, polyamine-β-cyclodextrin, mono-(6-deoxy-6-p-toluenesulfonyl)-β-cyclodextrin, sulfobutyl ether-β-CD, methylated-β-CD and other cyclodextrin derivatives.

[0013] The method is characterized in that the representative polysubstituted aromatic compounds in steps (b1) and (b2) should simultaneously possess groups capable of reacting with cyclodextrin (such as -F, -COOH, -Cl, -OH, -COOH) and groups capable of reacting with silanized modified silicon spheres (such as -F, -Cl, -OH). Representative substances include tetrafluoroterephthalonitrile, tetrachloroterephthalonitrile, tetrachloroisophthalonitrile, 4-bromo-3,5-dihydroxybenzoic acid, m-trichlorophenol, 4-fluoro-2-nitrophenol, etc., with the following chemical structures:

[0014]

[0015]

[0016] The method is characterized in that the organic solvent in steps (b1) and (b2) can be acetonitrile, N,N-dimethylformamide, tetrahydrofuran, or a mixture thereof.

[0017] The method is characterized in that the reaction temperature in step (b1) can be 40℃-100℃.

[0018] The method is characterized in that the reaction temperature of the two-step process in step (b2) can be 40℃-100℃.

[0019] The method is characterized in that, in step (b1) of the one-step process, the mass ratio of SiO2@APTES to tetrafluoroterephthalonitrile is 0.1:0.2-0.1:4, and the mass ratio of tetrafluoroterephthalonitrile to cyclodextrin is 0.5:3-0.5:5. The reaction time is 5-24 h.

[0020] The method is characterized in that, in the two-step process of step (b2) above, the mass ratio of SiO2@APTES to tetrafluoroterephthalonitrile is 0.1-0.2:0.1-4. The reaction time is 5-36 h. The mass ratio of the first step product to cyclodextrin is 0.2:0.4-0.3:5, and the reaction time is 12-36 h.

[0021] The two methods involved in this invention each have their advantages and disadvantages: the one-step method has the advantage of fewer steps and simpler preparation. The two-step method has the advantage of producing fewer byproducts in the resulting cyclodextrin stationary phase.

[0022] The role of silica spheres: Nanoscale silica spheres are common stationary phase supports. 3-10µm microparticle silica and various chemically bonded stationary phases based on them are currently the dominant chemical types of packing materials in high-performance liquid chromatography (HPLC). This is due to silica's advantages such as good mechanical strength, easily controllable pore structure and surface area, good chemical stability, and specificity for surface chemical reactions. However, its selectivity is limited; for example, it cannot effectively separate certain structurally similar compounds. Therefore, further modification is necessary to meet practical requirements.

[0023] Traditional cyclodextrin chromatography stationary phases consist of silica spheres and cyclodextrin, but this traditional structure generally suffers from low cyclodextrin bonding. Therefore, we developed a novel stationary phase by introducing a polysubstituted aromatic compound, such as tetrafluoroterephthalonitrile, as a spacer arm. This not only increased the cyclodextrin bonding but also improved the rapid recognition performance of the stationary phase. However, tetrafluoroterephthalonitrile cannot directly react with the hydroxyl groups on the silica spheres. Therefore, we modified the silica spheres with amino groups to facilitate the introduction of tetrafluoroterephthalonitrile, ultimately forming a stationary phase with the following structure: silica spheres + NH2 + tetrafluoroterephthalonitrile + cyclodextrin.

[0024] Advantages and effects of the present invention:

[0025] 1. This invention uses tetrafluoroterephthalonitrile as a representative spacer arm, which readily crosslinks with cyclodextrin to form a crosslinked cyclodextrin complex with a high specific surface area, thus solving the problem of low cyclodextrin bonding amount commonly found in traditional cyclodextrin chromatographic stationary phases.

[0026] 2. Cyclodextrin chromatographic stationary phases with tetrafluoroterephthalonitrile as spacers have a high pore structure, resulting in faster recognition speeds compared to traditional cyclodextrin chromatographic stationary phases.

[0027] 3. This chromatographic stationary phase provides π-π interactions, hydrogen bonding, and steric exclusion for the separation and analysis of structural analogs. These interactions synergize with the inclusion effect of the hydrophobic cavity of cyclodextrin, improving selectivity for structural analogs and producing unique separation effects. As shown in Experimental Example 1 of this invention, compounds with similar structures exhibit different peak sequences on this column and the C18 column, demonstrating different separation selectivity. For compounds that are difficult to separate, this provides a new and alternative chromatographic stationary phase, improving the separation efficiency of the C18 stationary phase. Attached Figure Description

[0028] Figure 1 The diagram shows two synthetic routes of the present invention. A is a one-step preparation method, and B is a two-step preparation method.

[0029] Figure 2 The column efficiency of the chromatographic column obtained in Example 1 was evaluated using naphthalene.

[0030] Figure 3 To obtain the separation results of phenolic compounds with similar structures by the chromatographic column in Example 1;

[0031] Figure 4 Results of separating phenolic compounds with similar structures using a commercially available ordinary C18 column (a) and the chromatographic column (b) of Example 1. Detailed Implementation

[0032] The following embodiments are examples of the present invention and should not be regarded as limiting the present invention.

[0033] Example 1: One-step preparation

[0034] 0.1 g of 5 μm silicon spheres were uniformly dispersed in 40 mL of anhydrous ethanol, 8 mL of water, and 0.2 mL of ammonia water. Then, 0.2 mL of APTES (silane coupling agent) was added, and the reaction was carried out at room temperature for 6 h. After the reaction was completed, the mixture was filtered, washed with water and ethanol respectively, and dried under vacuum at 60 °C to obtain the product SiO2@APTES.

[0035] 0.1 g of SiO2@APTES, 0.2 g of tetrafluoroterephthalonitrile, and 0.2 g of α-cyclodextrin were uniformly dispersed in a mixed solution of 50 mL of tetrahydrofuran and 6 mL of N,N-dimethylformamide. The mixture was transferred to a serum bottle, and nitrogen gas was introduced for 5 min. The mixture was then heated and stirred at 80 °C for 24 h. After the reaction was completed, the mixture was filtered and washed with tetrahydrofuran, N,N-dimethylformamide, and dichloromethane, respectively. The mixture was then sieved and finally dried under vacuum at 60 °C to obtain the product.

[0036] Example 2 is the same as Example 1 except that the tetrafluoroterephthalonitrile in Example 1 is replaced with tetrachloroisophthalonitrile.

[0037] Example 3

[0038] Simply replace α-cyclodextrin with β-cyclodextrin in Preparation Example 1, and the rest of the operation is the same as in Example 1.

[0039] Example 4

[0040] Simply replace tetrafluoroterephthalonitrile with tetrachloroisophthalonitrile in Preparation Example 1, and replace α-cyclodextrin with β-cyclodextrin, and the rest of the operation is the same as in Example 1.

[0041] Example 5

[0042] Simply replace α-cyclodextrin with γ-cyclodextrin in Example 1, and the rest of the operation is the same as in Example 1.

[0043] Example 6

[0044] Simply replace tetrafluoroterephthalonitrile with tetrachloroisophthalonitrile in Example 1, and replace α-cyclodextrin with γ-cyclodextrin, and the rest of the operation is the same as in Example 1.

[0045] Example 7 Two-step preparation

[0046] 0.1 g of 5 μm silicon spheres were uniformly dispersed in 40 mL of anhydrous ethanol, 8 mL of water, and 0.2 mL of ammonia solution. Then, 0.2 mL of APTES was added, and the reaction was carried out at room temperature for 6 h. After the reaction was completed, the mixture was filtered, washed with water and ethanol respectively, and dried under vacuum at 60 °C to obtain the product SiO2@APTES.

[0047] 0.1 g of SiO2@APTES and 0.2 g of tetrafluoroterephthalonitrile were uniformly dispersed in 30 mL of acetonitrile solution and reacted at 80 °C for 10 h. After the reaction was completed, the mixture was filtered, washed with acetonitrile and ethanol, and finally dried under vacuum at 60 °C for 10 h to obtain product 1.

[0048] 0.1 g of Product 1 and 0.2 g of α-cyclodextrin obtained in the above steps were uniformly dispersed in a mixed solution of 50 mL tetrahydrofuran and 6 mL N,N-dimethylformamide. The mixture was transferred to a serum bottle, nitrogen gas was introduced for 5 min, and the mixture was heated and stirred at 85 °C for 24 h. After the reaction was completed, the mixture was filtered and washed with tetrahydrofuran, N,N-dimethylformamide, and dichloromethane, respectively. Finally, it was dried under vacuum at 60 °C to obtain the product.

[0049] Example 8

[0050] Simply replace tetrafluoroterephthalonitrile in Example 7 with tetrachloroisophthalonitrile, and the rest of the operation is the same as in Example 7.

[0051] Example 9

[0052] Simply replace α-cyclodextrin with β-cyclodextrin in Example 7, and the rest of the operation is the same as in Example 7.

[0053] Example 10

[0054] Simply replace tetrafluoroterephthalonitrile with tetrachloroisophthalonitrile in Example 7, and replace α-cyclodextrin with β-cyclodextrin, and the rest of the operation is the same as in Example 7.

[0055] Example 11

[0056] Simply replace α-cyclodextrin with γ-cyclodextrin in Example 7, and the rest of the operation is the same as in Example 7.

[0057] Example 12

[0058] Simply replace tetrafluoroterephthalonitrile with tetrachloroisophthalonitrile in Example 7, and replace α-cyclodextrin with γ-cyclodextrin, and the rest of the operation is the same as in Example 7.

[0059] Experimental Example 1

[0060] A chromatographic column was prepared using the packing material obtained in Example 1. The packing method was conventional, and the column efficiency was evaluated using naphthalene. The results are as follows: Figure 2 As shown: Acetonitrile was used as the mobile phase, the injection volume was 5 μL, the UV detection wavelength was 254 nm, the column temperature was 30 ℃, the flow rate was 1.0 mL / min, and the chromatogram is shown below. Figure 2 As shown, the theoretical plate number of naphthalene was measured to be 26266 / m, and the asymmetry factor was 0.943.

[0061] This chromatographic column exhibits a certain ability to separate structurally similar phenolic compounds. Taking phenol, 1-naphthol, and 2-naphthol as examples, the separation effect is as follows: Figure 3 As shown, when the mobile phase ratio is 50 / 50 (v / v), the β-cyclodextrin stationary phase not only exhibits good separation from phenol, 1-naphthol and 2-naphthol structural analogs, but also has a short retention time, achieving complete peak elution and baseline separation within 15 min.

[0062] Meanwhile, a comparative experiment was conducted between the chromatographic column and a commercially available C18 column for three types of phenols. The results are as follows: Figure 4 As shown in the figure, 'a' represents the separation of this product, with the peaks eluting in the order of phenol, 2-naphthol, and 1-naphthol; 'b' represents the separation of the C18 column, with the peaks eluting in the order of phenol, 1-naphthol, and 2-naphthol. The results indicate that the C18 column and the C18 column have comparable column efficiency and resolution. Particularly noteworthy is the difference in the peak order of the three substances on the two columns, indicating that the two stationary phases have different separation selectivity.

Claims

1. A method for preparing a cyclodextrin chromatographic stationary phase in one or two steps, characterized in that, a. Disperse the silicon spheres in a mixed solution of anhydrous ethanol, water, and ammonia, then add a silanizing reagent to react and obtain silanized modified silicon spheres; b1. One-step method: Silanized silicon spheres, polysubstituted aromatic compounds, and cyclodextrin are uniformly dispersed in an organic solvent, nitrogen gas is introduced, and the reaction is carried out to obtain the final product. or b2. Two-step method: Silanized modified silicon spheres and polysubstituted aromatic compounds are dispersed in an organic solvent to obtain the product of the first step reaction; the product of the first step reaction and cyclodextrin are dispersed in an organic solvent, nitrogen gas is introduced, and the reaction is carried out to obtain the final product; In step a, the silanizing agent is 3-aminopropyltriethoxysilane, (3-mercaptopropyl)triethoxysilane, 3-chloropropyltriethoxysilane, propyltriethoxysilane isocyanate, 3-(2-aminoethylamino)propyltriethoxysilane, or triethoxy(3-epoxypropyloxypropyl)silane. The cyclodextrin in step b1 or b2 is: α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, dialdehyde-β-cyclodextrin, polyamine-β-cyclodextrin, mono-(6-deoxy-6-p-toluenesulfonyl)-β-cyclodextrin, sulfobutyl ether-β-CD, or methylated-β-CD; The polysubstituted aromatic compound in step b1 or b2 is: tetrafluoroterephthalonitrile, tetrachloroterephthalonitrile, tetrachloroisophthalonitrile, 4-bromo-3,5-dihydroxybenzoic acid, m-trichlorophenol, or 4-fluoro-2-nitrophenol.

2. The method according to claim 1, characterized in that, The organic solvent in step b1 or b2 is acetonitrile, N,N-dimethylformamide, tetrahydrofuran, or a mixture of two or more thereof. The reaction temperature in step b1 above is 40℃-100℃ for the one-step process; The reaction temperature for the two-step process in b2 is 40℃-100℃.

3. The method according to claim 1, characterized in that, In step b1, the mass ratio of silanized modified silicon spheres to tetrafluoroterephthalonitrile is 0.1:0.2-0.1:4, and the mass ratio of tetrafluoroterephthalonitrile to cyclodextrin is 0.5:3-0.5:5; the reaction time is 5-24 h.

4. The method according to claim 1, characterized in that, In step b2, the mass ratio of silanized modified silicon spheres to tetrafluoroterephthalonitrile is 0.1-0.2:0.1-4, and the reaction time is 5-36 h; the mass ratio of the product from the first step reaction to cyclodextrin is 0.2:0.4-0.3:5, and the reaction time is 12-36 h.

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