Preparation and application of benzenesulfonic acid / benzenemethanesulfonic acid modified silica gel mixed mode chromatographic filler

Abstract

The invention discloses a preparation method of a sulfonic acid / benzene methanesulfonic acid modified silica gel mixed mode chromatographic filler, which comprises the following steps: reacting silica gel with (3-mercaptopropyl) triethoxy silane, then respectively reacting with 4-vinyl sodium benzenesulfonate and (4-vinyl phenyl) sodium methanesulfonate through a click reaction to obtain the sulfonic acid / benzene methanesulfonic acid modified silica gel mixed mode chromatographic filler. The mercaptopropyl bridged biphenyl sulfonic acid modified silica gel chromatographic packing material Sil-SSa and the mercaptopropyl bridged biphenyl sulfonic acid modified silica gel chromatographic packing material Sil-SMSa are prepared. Due to the introduction of methylene, the Sil-SMSa shows higher separation rate and higher heavy rare earth selectivity. The two fillers have hydrophilic and ion exchange effects, can efficiently separate base nucleoside, flavone, alkaloid and rare earth element mixtures, and are suitable for multi-mode chromatographic analysis of complex samples.

Description

Technical Field

[0001] The present invention relates to a preparation method of a silica gel mixed-mode chromatographic packing modified with benzenesulfonic acid / benzenesulfonic acid, and also relates to the application of the chromatographic packing in the separation of base nucleosides, alkaloids, flavonoids, and rare earth elements, belonging to the technical fields of the preparation of chromatographic packing and chromatographic separation. Background Art

[0002] In the field of chromatographic separation, traditional single-mode chromatographic packings usually rely only on a single retention mechanism and cannot simultaneously separate polar, non-polar, and ionic compounds on a single chromatographic column. For example, the reversed-phase chromatographic stationary phase C18 only provides effective retention for hydrophobic analytes, while strongly polar or ionized analytes are always weakly retained; the hydrophilic chromatographic stationary phase has good separation effects on strongly polar compounds and weakly ionized analytes, but has weak retention for charged substances (such as rare earth ions); the ion-exchange chromatographic stationary phase is suitable for charged analytes, but is prone to non-specific adsorption of hydrophobic compounds. As an alternative or supplement to single-mode chromatography, mixed-mode chromatography (MMC) can make it easier to separate and analyze complex analytes on a single chromatographic column through at least two retention mechanisms, avoiding the waste of chromatographic materials and frequent column changes, greatly reducing the analysis cost, and improving the working efficiency. Therefore, mixed-mode chromatography is an important tool for the analysis of complex samples.

[0003] Sulfonic acid group-modified silica gel (such as SCX packing) is commonly used for cation exchange, but the overly strong acidic group may cause over-retention of the target substance or peak tailing. The present invention has carried out research on the application of a silica gel mixed chromatographic packing modified with sodium 4-vinylbenzenesulfonate / (4-vinyl)phenylmethanesulfonate. By respectively carrying out click reactions of sodium 4-vinylbenzenesulfonate and (4-vinyl)phenylmethanesulfonate with mercaptopropyl silica gel and modifying them on the silica gel surface, new chromatographic packings Sil-SSa and Sil-SMSa are obtained. It is found that both of these two stationary phases have certain separation effects on base nucleosides, flavonoids, alkaloids, and rare earth elements. By introducing methylene groups, the separation performance of benzenesulfonic acid-modified silica gel can be regulated. The benzenesulfonic acid-modified silica gel chromatographic packing has a faster separation rate for base nucleosides, flavonoids, and alkaloids than the benzenesulfonic acid-modified silica gel chromatographic packing. In the ion-exchange mode, it also has a faster separation rate for rare earth elements, and has a higher resolution and better peak shape for heavy rare earths. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of a silica gel mixed-mode chromatographic packing modified with benzenesulfonic acid / benzenesulfonic acid bridged by a mercaptopropylsilane reagent; Another object of the present invention is to provide the application of benzenesulfonic acid / benzenecarboxylic acid modified silica gel mixed-mode chromatographic packing materials in the separation of base nucleosides, alkaloids, flavonoids and rare earth elements. Under the hydrophilic mode, good separation effects of base nucleosides, alkaloids and flavonoids can be achieved. Under the ion exchange mode, rare earth element mixtures can be effectively separated. The introduction of methylene can regulate the chromatographic separation performance of benzenesulfonic acid modified silica gel.

[0005] I. Preparation of chromatographic packing materials The preparation method of the benzenesulfonic acid / benzenecarboxylic acid modified silica gel chromatographic packing materials of the present invention includes the following steps: (1) Preparation of Sil-SH: Silica gel is dispersed in toluene, (3-mercaptopropyl)triethoxysilane is added, and the mixture is stirred and reacted at 100-110 °C for 24-48 h under nitrogen protection, washed and dried to obtain mercaptopropyl silica gel Sil-SH; the mass ratio of (3-mercaptopropyl)triethoxysilane to silica gel is 0.5:1-1:1.

[0006] (2) Preparation of chromatographic packing materials: Sodium 4-vinylbenzenesulfonate or sodium (4-vinyl)phenylmethanesulfonate and an initiator are dissolved in a methanol / water mixed solvent, then Sil-SH is added and dispersed evenly, and the mixture is stirred at 90-105 °C for 24-48 h under nitrogen protection, washed, and dried in vacuo to obtain benzenesulfonic acid modified silica gel chromatographic material Sil-SSa or benzenecarboxylic acid modified silica gel chromatographic material Sil-SMSa.

[0007] The mass ratio of sodium 4-vinylbenzenesulfonate or sodium (4-vinyl)phenylmethanesulfonate to Sil-SH is 0.2:1-0.5:1. The initiator is 2,2'-azobis(2-methylpropionamidine) dihydrochloride, and the mass ratio of sodium 4-vinylbenzenesulfonate or sodium (4-vinyl)phenylmethanesulfonate to the initiator is 1:0.01-1:0.1. The methanol / water mixed solvent has a volume ratio of methanol to water of 1:1.

[0008] II. Structure of chromatographic packing materials Figure 1 The infrared spectra of the chromatographic packing materials Sil-SSa and Sil-SMSa prepared in the present invention are shown, where (a), (b) and (c) are Sil-SH, Sil-SMSa and Sil-SSa respectively, which confirm the successful preparation of Sil-SSa and Sil-SMSa.

[0009] To investigate the bonding amount of the separation materials obtained by this method, a Vario EL elemental analyzer produced by Elementar Analysensysteme GmbH was used to quantitatively analyze the elements C and H in Sil-SMSa and Sil-SSa silica gels. Table 1 shows the elemental analysis results of Sil-SH, Sil-SSa, and Sil-SMSa. It can be seen from the elemental analysis results that Sil-SSa has a higher bonding amount than Sil-SMSa, which may be attributed to the steric hindrance of the methylene group.

[0010] III. Chromatographic Separation Performance of Chromatographic Packing Sil-TPE To investigate the chromatographic separation performance of the Sil-SSa stationary phase and the Sil-SMSa stationary phase, they were packed into a 5 cm * 2.1 cm chromatographic column, and the mixed chromatographic performance was investigated using base nucleosides, alkaloids, flavonoids, and rare earth element mixtures as analytes.

[0011] Figure 2 Shows the separation effects of a 16-rare earth element mixture (1. Scandium, 2. Lutetium, 3. Ytterbium, 4. Thulium, 5. Erbium, 6. Holmium, 7. Dysprosium and Yttrium, 8. Terbium, 9. Gadolinium, 10. Europium, 11. Samarium, 12. Neodymium, 13. Praseodymium, 14. Cerium, 15. Lanthanum) on the Sil-SSa and Sil-SMSa stationary phases respectively.

[0012] Figure 3 Shows the separation effects of 7 base nucleosides (1. Thymine, 2. Uracil, 3. Adenosine, 4. Adenine, 5. Inosine, 6. Cytosine, 7. Guanosine) on the Sil-SSa and Sil-SMSa stationary phases respectively.

[0013] Figure 4 Shows the separation effects of 4 flavonoids (1. Arbutin, 2. Cordycepin, 3. Naringin, 4. Wogonoside) on the Sil-SSa and Sil-SMSa stationary phases respectively, and the separation effects of 2 alkaloids (1. Berberine hydrochloride, 2. Fibrauretin) on the Sil-SSa and Sil-SMSa stationary phases respectively.

[0014] From the chromatographic separation effects of Sil-SSa and Sil-SMSa on the 16 rare earth element mixture, it can be seen that both Sil-SSa and Sil-SMSa have good separation potential for the rare earth element mixture, showing good ion exchange ability. Compared with Sil-SSa, Sil-SMSa has a faster separation rate, which may be due to the lower surface bonding amount of benzenesulfonic acid than that of phenylsulfonic acid, and has a higher separation degree for heavy rare earths, which may be due to the introduction of methylene resulting in a decrease in the acidity of sulfonic acid groups and a change in the spatial configuration; from the separation effects on 7 base nucleosides, 4 flavonoids and 2 alkaloids, it can be seen that both Sil-SSa and Sil-SMSa have a certain hydrophilic separation ability. The hydrophilic retention ability of Sil-SMSa is weaker than that of Sil-SSa, but Sil-SMSa has a faster separation ability, which may be due to the lower surface bonding degree of Sil-SMSa and the introduction of methylene reducing the hydrophilicity of sulfonic acid groups.

[0015] In summary, in the present invention, 3-mercaptopropyltriethoxysilane is used as a bridging agent, and 4-vinylbenzenesulfonic acid and (4-vinyl)phenylmethanesulfonic acid are respectively modified on the silica gel surface through a click reaction to synthesize the benzenesulfonic acid modified silica gel chromatographic packing Sil-SSa and the phenylmethanesulfonic acid modified silica gel chromatographic packing Sil-SMSa. Due to the sulfonic acid functional group and methylene, the Sil-SSa and Sil-SMSa chromatographic packings exhibit two separation mechanisms of hydrophilicity and ion exchange. In the hydrophilic mode, base nucleosides, flavonoids and alkaloids can be effectively separated; in the ion exchange mode, the rare earth element mixture can be better separated. Due to the introduction of methylene, Sil-SMSa shows a faster separation rate and higher heavy rare earth selectivity. Description of the Drawings

[0016] Figure 1 are the infrared spectra of the chromatographic packings Sil-SMSa and Sil-SSa prepared in the present invention.

[0017] Figure 2 are the separation results of the chromatographic packings Sil-SMSa and Sil-SSa prepared in the present invention on the 16 rare earth element mixture.

[0018] Figure 3 are the separation results of the chromatographic packings Sil-SMSa and Sil-SSa prepared in the present invention on 7 base nucleosides.

[0019] Figure 4 are the separation results of the chromatographic packings Sil-SMSa and Sil-SSa prepared in the present invention on 4 flavonoids and 2 alkaloids.

[0020] Figure 5This is the repeatability test of the chromatographic packing materials Sil-SMSa and Sil-SSa prepared by the present invention for 7 base nucleosides. Detailed implementation manners

[0021] The preparation of the benzenesulfonic acid / benzenemethanesulfonic acid modified silica gel chromatographic packing materials Sil-SSa and Sil-SMSa of the present invention will be further described below through specific examples.

[0022] Example 1 Preparation of chromatographic packing material Sil-SSa (1) Preparation of Sil-SH: Silica gel (2 g, 1.8 μm) was dispersed in toluene, and an excessive amount of (3-mercaptopropyl)triethoxysilane (2 mL) was added dropwise. Under nitrogen protection, the mixture was stirred at 100 - 105 °C for 48 h, and then washed successively with ethanol, ethanol / water (1:1, v / v) and methanol, and dried to obtain mercaptopropyl silica gel Sil-SH; (2) Preparation of Sil-SSa stationary phase: Sodium p-vinylbenzenesulfonate (1.6 mmol, 0.36 g) and initiator 2,2'-azobis(2-methylpropionamidine) dihydrochloride (0.01 g, 3%) were dissolved in a mixed solvent of methanol / water (1:1, v / v), and then mercaptopropyl silica gel (1 g) was added and dispersed evenly. Under nitrogen protection, the mixture was mechanically stirred at 95 °C for 48 h; after the reaction, it was washed with water and anhydrous methanol, and vacuum dried to obtain the chromatographic material Sil-SSa of sodium p-vinylbenzenesulfonate modified silica gel.

[0023] Example 2 Preparation of chromatographic packing material Sil-SMSa (1) Preparation of Sil-SH: The same as in Example 1; (2) Synthesis of sodium (4-vinyl)phenylmethanesulfonate: Na2SO3 (18 mmol, 2.27 g) and 2,6-di-tert-butyl-p-cresol (0.4 mmol, 0.08 g) were dissolved in 15 mL of water. Under nitrogen protection, an acetone solution of 15 mL of 1-chloromethyl-4-vinylbenzene (12 mmol, 2.04 g) was added, and the mixture was refluxed at 67 °C for 4 h, filtered, washed with ethanol, recrystallized with water / isopropanol (3 / 1, v / v), and then washed with ethanol and dried to obtain sodium (4-vinyl)phenylmethanesulfonate; the preparation of sodium (4-vinyl)phenylmethanesulfonate was prepared with reference to the patent (PCT / EP2022 / 065198); (3) Synthesis of Sil-SMSa stationary phase: Dissolve sodium (4-vinyl)phenylmethanesulfonate (1.6 mmol, 0.42 g) and initiator 2,2'-azobis(2-methylpropionamidine) dihydrochloride (0.01 g, 3%) in a mixed solvent of methanol / water (1:1, v / v), then add mercaptopropyl silica gel and disperse evenly. Under nitrogen protection, stir mechanically at 95 °C for 48 h; after the reaction is completed, wash with water and anhydrous methanol, and dry under vacuum to obtain the chromatographic material Sil-SMSa of silica gel modified with sodium (4-vinyl)phenylmethanesulfonate; The synthetic routes of chromatographic packings Sil-SSa and Sil-SMSa are as follows: .

Claims

1. A preparation method of a mixed-mode chromatographic packing material modified with benzenesulfonic acid / benzenecarboxylic acid, comprising the following steps: (1) Preparation of Sil-SH: Silica gel is dispersed in toluene, and (3-mercaptopropyl)triethoxysilane is added. Under nitrogen protection, the mixture is stirred and reacted at 100-110 °C for 24-48 h, washed and dried to obtain mercaptopropyl silica gel Sil-SH; (2) Preparation of the chromatographic packing material: Sodium 4-vinylbenzenesulfonate or sodium (4-vinyl)phenylmethanesulfonate and an initiator are dissolved in a methanol / water mixed solvent, and then Sil-SH is added and dispersed evenly. Under nitrogen protection, the mixture is stirred at 90-105 °C for 24-48 h, washed, and dried under vacuum to obtain a benzenesulfonic acid-modified silica gel chromatographic material Sil-SSa or a benzenecarboxylic acid-modified silica gel chromatographic material Sil-SMSa.

2. The preparation method of the benzenesulfonic acid / benzoic acid modified silica gel mixed-mode chromatographic packing material according to claim 1, characterized in that: In step (1), the mass ratio of (3-mercaptopropyl)triethoxysilane to silica gel is 0.5:1-1:

1.

3. The preparation method of the benzenesulfonic acid / benzoic acid modified silica gel mixed-mode chromatographic packing material according to claim 1, characterized in that: In step (2), the mass ratio of sodium 4-vinylbenzenesulfonate or sodium (4-vinyl)phenylmethanesulfonate to Sil-SH is 0.2:1-0.5:

1.

4. The preparation method of the benzenesulfonic acid / benzoic acid modified silica gel mixed-mode chromatographic packing material according to claim 1, wherein: In step (2), the initiator is azodiisobutylamidine hydrochloride, and the mass ratio of sodium 4-vinylbenzenesulfonate or sodium (4-vinyl)phenylmethanesulfonate to the initiator is 1:0.01-1:0.

1.

5. The preparation method of the benzenesulfonic acid / benzoic acid modified silica gel mixed-mode chromatographic packing material according to claim 1, characterized in that: In step (2), for the methanol / water mixed solvent, the volume ratio of methanol to water is 1:

1.

6. Application of the chromatographic packing material prepared by the method according to claim 1 in the separation of base nucleosides, alkaloids, flavonoids, and rare earth elements.

7. The application according to claim 6, wherein: Separation of base nucleosides, flavonoids, and alkaloids in the hydrophilic mode; separation of rare earth element mixtures in the ion exchange mode.

8. The application according to claim 6 or 7, characterized in that: The base nucleosides include thymine, uracil, adenosine, adenine, inosine, cytosine, guanosine; the alkaloids include berberine hydrochloride, palmatine; the flavonoids include arbutin, cordycepin, naringin, wogonoside; the rare earth elements include scandium, lutetium, ytterbium, thulium, erbium, holmium, dysprosium, yttrium, terbium, gadolinium, europium, samarium, neodymium, praseodymium, cerium, lanthanum.

Images