Preparation method and application of an amide type rosin-based bonded silica gel mixed mode stationary phase

Abstract

The application discloses a preparation method and application of an amide type rosin-based bonded silica gel mixed mode stationary phase. The method uses hydrogenated rosin or dehydroabietic acid as a starting material, and generates a corresponding acyl chloride compound through reaction with oxalyl chloride; subsequently, nucleophilic acyl substitution reaction occurs between the acyl chloride compound and amino groups on the surface of activated silica gel modified by an amino silane coupling agent, and then end capping treatment is performed to obtain the amide type rosin-based bonded silica gel mixed mode stationary phase. The preparation method is simple, and the obtained stationary phase has excellent sphericity, mechanical strength and reproducibility. The stationary phase has the advantages of both reversed phase (RPLC) and hydrophilic interaction (HILIC) chromatographic modes, can efficiently separate alkylbenzenes, polycyclic aromatic hydrocarbons and base nucleotides, and can realize good separation of saponins and flavone mixtures in panax notoginseng in the mixed mode.

Description

Technical Field

[0001] This invention belongs to the field of high-performance liquid chromatography (HPLC). Specifically, it relates to a method for preparing and applying an amide-type rosin-based bonded silica gel mixed-mode stationary phase. Background Technology

[0002] High-performance liquid chromatography (HPLC), as a core separation technique in modern analytical chemistry, has been widely applied in fields such as drug analysis, environmental monitoring, food safety, and natural product separation due to its advantages of high separation efficiency, wide applicability, and fast analysis speed. The stationary phase is the core material for achieving high-efficiency separation in HPLC; its structure and performance directly determine separation selectivity, column efficiency, and detection reproducibility. Silica gel possesses advantages such as high mechanical strength, large specific surface area, and controllable pore structure, making it the most widely used chromatographic matrix material. Currently, commercially available silica gel-based chemically bonded stationary phases mostly use petroleum-based carbon chains such as C8 and C18 as functional ligands. These traditional chromatographic columns rely solely on a single hydrophobic retention mechanism for component separation, resulting in poor selectivity for complex samples with significant polarity differences, strongly polar compounds, and basic compounds. Furthermore, residual silanol groups on the silica gel surface easily undergo secondary adsorption with basic analytes, readily causing peak tailing, reduced column efficiency, and poor analytical reproducibility, greatly limiting its application in complex sample systems.

[0003] Rosin is a green, natural, and renewable resource. Its main component is resin acid, possessing a rigid phenanthrene ring skeleton, good stereoselectivity, and natural hydrophobicity. Through chemical modification such as hydrogenation, disproportionation, polymerization, and esterification, a series of structurally diverse functionalized rosin-based derivatives can be prepared. Rosin's combination of green and renewable characteristics with structural advantages has shown excellent application potential in the field of novel chromatographic stationary phase modification. Currently, research reports on rosin-based silica gel stationary phases mainly focus on reversed-phase separation mechanisms, making it difficult to construct a mixed separation mode that combines reversed-phase and hydrophilic interactions, resulting in limited retention and separation resolution for polar compounds. Therefore, developing an amide-type rosin-based bonded silica gel reversed-phase / hydrophilic mixed-mode stationary phase can achieve synergistic complementarity of various intermolecular forces, effectively improving the separation and analytical performance of complex sample systems. This is of great significance for the development and application of novel green chromatographic stationary phases. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing and applying an amide-based rosin-bonded silica gel hybrid stationary phase. This invention chemically grafts polar functional groups of amide onto the silica gel surface in a synergistic manner with the rigid phenanthrene ring framework of rosin, constructing a hybrid separation mode that combines hydrophobic interactions, hydrogen bonding, and π-π interactions. Simultaneously, the amide groups effectively shield residual silanol groups on the silica gel, inhibiting the adsorption and tailing of basic substances and improving peak symmetry. This stationary phase has a simple preparation process, high bonding stability, and can achieve efficient separation of complex polar, weakly polar, and basic samples on a single chromatographic column. It overcomes the shortcomings of existing rosin-based stationary phases, which have limited functionality and separation modes, and has promising application prospects and widespread value.

[0005] This invention provides a method for using hydrogenated rosin or dehydroabsic acid as starting materials, reacting with oxalyl chloride to generate an acyl chloride compound, and then nucleophilically substituting this compound into activated silica gel modified with an aminosilane coupling agent to form a stable amide-type rosin-based bonded silica gel mixed stationary phase. Characterization and performance evaluation show that this invention combines the advantages of both reversed-phase chromatography (RPLC) and hydrophilic separation modes.

[0006] The aforementioned amide-type rosin-based bonded silica microspheres have the following structural formulas I or II:

[0007] This invention also provides a method for preparing an amide-type rosin-based bonded silica gel mixed stationary phase, the steps of which are as follows:

[0008] Step 1, Activating silica gel: Add bare silica gel to a round-bottom flask containing hydrochloric acid solution, disperse ultrasonically, mix well, and then heat and stir under nitrogen protection. After the reaction is complete, cool to room temperature, wash the product with deionized water to remove hydrochloric acid until the aqueous solution is neutral, and finally dry the product in a vacuum drying oven to obtain activated silica gel.

[0009] The second step, amino-modified silica gel: The activated silica gel prepared in the first step was placed in a flask containing toluene and ultrasonically dispersed until uniform. Then, a silane coupling agent was added, and the mixture was heated and stirred under nitrogen protection. After the reaction was completed, the mixture was cooled, filtered, and washed successively with methanol, ethanol, and acetone. Finally, the product was dried in a vacuum drying oven to obtain amino-modified silica gel.

[0010] Step 3: Hydrogenated rosin (or dehydroabsic acid) acyl chloride: Add hydrogenated rosin (or dehydroabsic acid) and toluene to a three-necked flask equipped with a spherical condenser, thermometer, and tail gas absorber in an ice-water bath. Stir magnetically until the hydrogenated rosin (or dehydroabsic acid) is completely dissolved. Then, add oxalyl chloride dropwise. After the addition is complete, heat and stir under reflux to remove the solvent toluene and any unreacted oxalyl chloride. Seal and store for later use.

[0011] Step 4, Amide-type Rosin-based Bonded Silica Gel: The amino-modified silica gel prepared in Step 2 was placed in a three-necked flask, and toluene and triethylamine were added. The mixture was then magnetically stirred. The hydrogenated rosin (or dehydroabietic acid) acyl chloride prepared in Step 3 was added to a constant-pressure dropping funnel, and the mixture was heated and stirred under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed successively with water and ethanol. Finally, the product was dried in a vacuum drying oven.

[0012] Step 5, End-capping: The dried product was placed in a round-bottom flask, and toluene, triethylamine, and end-capping agent were added. The mixture was heated and stirred under nitrogen protection. After the reaction was completed, it was cooled to room temperature, filtered, and washed successively with dichloromethane, methanol, and ethanol. The dried solid was wrapped in filter paper and extracted with ethanol using a Soxhlet extraction method. After extraction, it was filtered again, and the resulting solid was dried in a vacuum drying oven to obtain an amide-type rosin-based bonded silica gel mixed-mode stationary phase.

[0013] As a preferred embodiment of the present invention, the concentration of hydrochloric acid in the first step is 2-5 mol / L, the activation time is 8-12 h, the temperature is 70-100 °C, the vacuum drying time is 12-24 h, and the temperature is 60-80 °C.

[0014] As a preferred embodiment of the present invention, the amino reagent in the second step is selected from 3-aminopropyltrimethoxysilane or 3-aminopropyltriethoxysilane, the reaction time is 12-24 h, and the reaction temperature is 90-110 °C.

[0015] As a preferred embodiment of the present invention, the molar ratio of hydrogenated rosin (or dehydroabsic acid) to oxaloyl chloride in the third step is 1:(1.10-1.25), the reaction time is 8-12 h, the temperature is 80-110 °C, and the rotary evaporation temperature is 400-60 °C.

[0016] As a preferred embodiment of the present invention, in the fourth step, the mass ratio of amino-modified silica gel to hydrogenated rosin (or dehydroabietic acid) acyl chloride is 1:(1-3), the reaction time is 8-12 h, the temperature is 70-100 °C, the vacuum drying time of the bonded stationary phase is 12-24 h, and the temperature is 60-80 °C.

[0017] As a preferred embodiment of the present invention, the end-capping agent in the fifth step is trimethylchlorosilane. The mass ratio of the amide-type rosin-based bonded stationary phase to trimethylchlorosilane is 1:(0.2-0.4), the reaction time is 6-8 h, and the temperature is 80-100 °C; the vacuum drying time of the bonded stationary phase is 12-24 h, and the temperature is 60-0-80 °C.

[0018] As a preferred embodiment of the present invention, the Soxhlet extraction time in the fifth step is 12–24 h. The vacuum drying time of the bonded stationary phase is 12–24 h, and the temperature is 60–80 °C.

[0019] The present invention also provides a method for packing a chromatographic column with an amide-type rosin-based bonded silica gel mixed stationary phase.

[0020] As a preferred embodiment of the present invention, a wet packing method is adopted. The aforementioned amide-type rosin-based bonded silica gel is ultrasonically and uniformly dispersed in a solvent of methanol, chloroform, or isopropanol. The column is packed at a constant pressure of 4000–6500 psi for 20–60 min using a column packer. After packing is completed and the column pressure stabilizes, the chromatographic column is removed to obtain the target chromatographic column.

[0021] This invention also provides a method for separating alkylbenzenes, polycyclic aromatic hydrocarbons, basic nucleotides, total saponins of Panax notoginseng, and flavonoids using an amide-type rosin-based bonded silica gel mixed stationary phase column (Sil-HRAA).

[0022] Furthermore, the aforementioned alkylbenzenes are mixtures of benzene, toluene, ethylbenzene, propylbenzene, butylbenzene, and pentabenzene; the polycyclic aromatic hydrocarbons are naphthalene, fluorene, anthracene, pyrene, and triphenylene; the basic nucleotides are thymine, sulfadiazine, adenine, inosine, and guanine; the saponins in Panax notoginseng are ginsenosides Rb1, Rd, Rg1, Re, and notoginsenoside R1; and the flavonoids in Panax notoginseng are glycyrrhizin, quercetin, and kaempferol.

[0023] Compared with existing technologies, the present invention has the following beneficial effects:

[0024] 1. The chromatographic stationary phase prepared in this invention uses hydrogenated rosin or dehydroabsic acid as raw materials, and introduces amide groups to bond to the silica gel surface, thus broadening the application of rosin derivatives in chromatographic separation materials.

[0025] 2. The chromatographic stationary phase Sil-HRAA prepared in this invention possesses hydrogen bonding, π-π interactions, and rosin hydrophobic stereorecognition, giving it certain advantages in separating some specific complex and difficult-to-separate substances. In practical applications, it exhibits good separation performance for polycyclic aromatic hydrocarbon isomers and basic nucleotide compounds, and can also be applied to the separation and detection of saponins and flavonoids in Panax notoginseng.

[0026] 3. The chromatographic stationary phase prepared by this invention makes great use of green and environmentally friendly natural raw materials, which are inexpensive; at the same time, the preparation method is simple, has good reproducibility, and can be mass-produced. Attached Figure Description

[0027] Figure 1 This describes the synthetic route for the amide-type rosin-based bonded silica gel mixed stationary phase prepared in this invention.

[0028] Figure 2 The infrared spectrum of the mixed stationary phase of activated silica gel, amino-modified silica gel, and amide-type rosin-based bonded silica gel in this invention is shown.

[0029] Figure 3 This is a thermogravimetric diagram of the mixed stationary phase of activated silica gel, amino-modified silica gel, and amide-type rosin-based bonded silica gel in this invention.

[0030] Figure 4 This is a scanning electron microscope image of the mixed stationary phase of activated silica gel and amide-type rosin-based bonded silica gel prepared in this invention.

[0031] Figure 5 The pore size distribution (a) and nitrogen adsorption-desorption curve (b) of the amide-type rosin-based bonded silica gel mixed stationary phase prepared in this invention are shown.

[0032] Figure 6 The image shows the chromatographic separation of six alkylbenzenes on an amide-type rosin-bonded silica gel mixed stationary phase.

[0033] Figure 7 The effect of acetonitrile content on the retention time of alkylbenzene is shown in the separation effect diagram of amide-type rosin-bonded silica gel mixed stationary phase.

[0034] Figure 8 The image shows the chromatographic separation effect of a mixed stationary phase of five polycyclic aromatic hydrocarbon amide-type rosin-based bonded silica gels.

[0035] Figure 9 The image shows the chromatographic separation of five base nucleotides on an amide-type rosin-bonded silica gel mixed stationary phase and an amino column.

[0036] Figure 10 The effect of acetonitrile content on the retention time of base nucleotides is shown in the separation effect diagram of amide-type rosin-bonded silica gel mixed stationary phase.

[0037] Figure 11 The image shows the chromatographic separation effect of saponins and flavonoids in Panax notoginseng on an amide-type rosin-bonded silica gel mixed stationary phase.

[0038] Figure 12 The batch-to-batch reproducibility (a) and stability test (b) diagrams for amide-type rosin-based bonded silica gel mixed stationary phases are shown. Specific implementation methods

[0039] The technical solutions of this application are described in detail with reference to the embodiments of the present invention, so as to facilitate the intuitive understanding of the present invention by those skilled in the art. It should also be noted that the specific embodiments described herein are only for illustrating the present invention and are not intended to limit the present invention.

[0040] Example 1

[0041] An amide-type rosin-based bonded silica gel mixed stationary phase is prepared by the following steps:

[0042] (1) Weigh 5.0 g of 5 μm bare silica gel and add it to a 250 mL round-bottom flask containing 100 mL of 2.0 mol / mL hydrochloric acid solution. Disperse the mixture by sonication, mix well, and heat to 75 °C under nitrogen protection while stirring for 8 h. After the reaction is complete, cool to room temperature, wash the product with deionized water to remove hydrochloric acid until the aqueous solution is neutral, and finally place the product in a vacuum drying oven at 80 °C for 24 h to obtain activated silica gel (SiO2).

[0043] (2) Weigh 4.5 g of the activated silica gel (SiO2) prepared in step (1) and place it in a flask containing 50 mL of toluene. Disperse it evenly by ultrasonication, then add 4.5 mL of 3-aminopropyltrimethoxysilane. Heat the mixture to 110 °C under nitrogen protection and stir for 12 h. After the reaction is complete, cool to room temperature, filter, and wash with methanol, ethanol, and acetone successively. Finally, place the product in a vacuum drying oven and dry at 80 °C for 24 h to obtain amino-modified silica gel (Sil-NH2).

[0044] (3) Add 5.3 g of hydrogenated rosin and 15 mL of toluene to a 250 mL three-necked flask equipped with a spherical condenser, thermometer, and tail gas absorber in an ice-water bath. Stir magnetically until the hydrogenated rosin is completely dissolved. Then, add 1.79 mL of oxaloyl chloride dropwise at a rate of 2 drops per second. After the addition is complete, heat to 70 °C and reflux with stirring for 4 hours. After the reaction is complete, remove the solvent toluene and unreacted oxaloyl chloride by rotary evaporation under reduced pressure. Seal and store for later use.

[0045] (4) Weigh 4.0 g of the 5 μm amino-modified silica gel (Sil-NH2) prepared in step (2), add 80 mL of toluene and 0.8 mL of triethylamine, stir magnetically, and add 4.0 g of the hydrogenated rosinyl chloride prepared in step (3) to a constant pressure dropping funnel. Add the solution to the flask at a dropping rate of 1 drop per second, keeping the temperature below 10 °C. After the addition is complete, heat to 75 °C under nitrogen protection and stir for 8 h. After the reaction is complete, cool to room temperature, filter, and wash three times with water and ethanol. Finally, place the product in a vacuum drying oven and dry at 80 °C for 24 h.

[0046] (5) Weigh 3.5 g of the dried product into a 250 mL round-bottom flask, add 40 mL of toluene, 0.7 mL of triethylamine, and 1.33 mL of trimethylchlorosilane. Heat to 90 °C and stir for 7 h under nitrogen protection. After the reaction is complete, cool to room temperature, filter, and wash successively with dichloromethane, methanol, and ethanol. After drying, wrap with filter paper and extract with ethanol using a Soxhlet extractor for 24 h. Finally, filter the product and dry it in a vacuum drying oven at 80 °C for 24 h to obtain the amide-type rosin-based bonded silica gel mixed stationary phase Sil-HRAA.

[0047] Example 2

[0048] An amide-type rosin-based bonded silica gel mixed stationary phase is prepared by the following steps:

[0049] (1) Weigh 5.0 g of 5 μm bare silica gel and add it to a 250 mL round-bottom flask containing 100 mL of 3.0 mol / mL hydrochloric acid solution. Disperse the mixture by sonication, mix well, and heat to 75 °C under nitrogen protection while stirring for 8 h. After the reaction is complete, cool to room temperature, wash the product with deionized water to remove hydrochloric acid until the aqueous solution is neutral, and finally place the product in a vacuum drying oven at 80 °C for 24 h to obtain activated silica gel (SiO2).

[0050] (2) Weigh 4.0 g of the activated silica gel (SiO2) prepared in step (1) and place it in a flask containing 50 mL of toluene. Disperse it evenly by ultrasonication, then add 4.0 mL of 3-aminopropyltrimethoxysilane. Heat the mixture to 110 °C under nitrogen protection and stir for 12 h. After the reaction is complete, cool to room temperature, filter, and wash with methanol, ethanol, and acetone successively. Finally, place the product in a vacuum drying oven and dry at 80 °C for 24 h to obtain amino-modified silica gel (Sil-NH2).

[0051] (3) Add 4.0 g of hydrogenated rosin and 10 mL of toluene to a 250 mL three-necked flask equipped with a spherical condenser, thermometer, and tail gas absorber in an ice-water bath. Stir magnetically until the hydrogenated rosin is completely dissolved. Then, add 1.34 mL of oxaloyl chloride dropwise at a rate of 2 drops per second. After the addition is complete, heat to 80 °C and reflux with stirring for 4 hours. After the reaction is complete, remove the solvent toluene and unreacted oxaloyl chloride by rotary evaporation under reduced pressure. Seal and store for later use.

[0052] (4) Weigh 3.5 g of the 5 μm amino-modified silica gel (Sil-NH2) prepared in step (2), add 80 mL of toluene and 0.6 mL of triethylamine, stir magnetically, and add 3.5 g of the hydrogenated rosinyl chloride prepared in step (3) to a constant pressure dropping funnel. Add the mixture to the flask at a dropping rate of 1 drop per second, keeping the temperature below 10 °C. After the addition is complete, heat to 80 °C under nitrogen protection and stir for 8 h. After the reaction is complete, cool to room temperature, filter, and wash three times with water and ethanol. Finally, place the product in a vacuum drying oven and dry at 80 °C for 24 h.

[0053] (5) Weigh 3.0 g of the dried product into a 250 mL round-bottom flask, add 40 mL of toluene, 0.6 mL of triethylamine, and 1.2 mL of trimethylchlorosilane. Heat to 90 °C and stir for 7 h under nitrogen protection. After the reaction is complete, cool to room temperature, filter, and wash successively with dichloromethane, methanol, and ethanol. After drying, wrap with filter paper and extract with ethanol using a Soxhlet extractor for 24 h. Finally, filter the product and dry it in a vacuum drying oven at 80 °C for 24 h to obtain the amide-type rosin-based bonded silica gel mixed stationary phase Sil-HRAA.

[0054] Example 3

[0055] An amide-type rosin-based bonded silica gel mixed stationary phase is prepared by the following steps:

[0056] (1) Weigh 10.0 g of 5 μm bare silica gel and add it to a 500 mL round-bottom flask containing 100 mL of 2.0 mol / mL hydrochloric acid solution. Disperse the mixture by sonication, mix well, and heat to 75 °C under nitrogen protection while stirring for 8 h. After the reaction is complete, cool to room temperature, wash the product with deionized water to remove hydrochloric acid until the aqueous solution is neutral, and finally place the product in a vacuum drying oven at 80 °C for 24 h to obtain activated silica gel (SiO2).

[0057] (2) Weigh 9.0 g of the activated silica gel (SiO2) prepared in step (1) and place it in a flask containing 50 mL of toluene. Disperse it evenly by ultrasonication, then add 9.0 mL of 3-aminopropyltrimethoxysilane. Heat the mixture to 110 °C under nitrogen protection and stir for 12 h. After the reaction is complete, cool to room temperature, filter, and wash with methanol, ethanol, and acetone successively. Finally, place the product in a vacuum drying oven and dry at 80 °C for 24 h to obtain amino-modified silica gel (Sil-NH2).

[0058] (3) Add 30.3 g of hydrogenated rosin and 80 mL of toluene to a 250 mL three-necked flask equipped with a spherical condenser, thermometer, and tail gas absorber in an ice-water bath. Stir magnetically until the hydrogenated rosin is completely dissolved. Then, add 10.15 mL of oxaloyl chloride dropwise at a rate of 2 drops per second. After the addition is complete, heat to 70 °C and reflux with stirring for 4 hours. After the reaction is complete, remove the solvent toluene and unreacted oxaloyl chloride by rotary evaporation under reduced pressure. Seal and store for later use.

[0059] (4) Weigh 8.0 g of the 5 μm amino-modified silica gel (Sil-NH2) prepared in step (2), add 160 mL of toluene and 1.6 mL of triethylamine, stir magnetically, and add 8.0 g of the hydrogenated rosinyl chloride prepared in step (3) to a constant pressure dropping funnel. Add the solution to the flask at a dropping rate of 1 drop per second, keeping the temperature below 10 °C. After the addition is complete, heat to 75 °C under nitrogen protection and stir for 8 h. After the reaction is complete, cool to room temperature, filter, and wash three times with water and ethanol. Finally, place the product in a vacuum drying oven and dry at 80 °C for 24 h.

[0060] (5) Weigh 7.0 g of the dried product into a 500 mL round-bottom flask, add 80 mL of toluene, 1.4 mL of triethylamine, and 2.66 mL of trimethylchlorosilane. Heat to 90 °C and stir for 7 h under nitrogen protection. After the reaction is complete, cool to room temperature, filter, and wash successively with dichloromethane, methanol, and ethanol. After drying, wrap with filter paper and extract with ethanol using a Soxhlet extractor for 24 h. Finally, filter the product and dry it in a vacuum drying oven at 80 °C for 24 h to obtain the amide-type rosin-based bonded silica gel mixed stationary phase Sil-HRAA.

[0061] The results of characterizing an amide-type rosin-based bonded silica gel mixed stationary phase are as follows:

[0062] The SiO2, Sil-NH2, and Sil-HRAA synthesized in Example 1 were characterized by infrared spectroscopy, and the results are as follows: Figure 2 As shown in the figure, after the silica gel reacts with 3-aminopropyltrimethoxysilane, the bending vibration absorption peak of Si-OH at 960 cm⁻¹ in Sil-NH₂ shows obvious signs of disappearance, and the absorption peak at 1569 cm⁻¹ disappears. -1 The presence of a bending vibration peak of NH indicates that 3-aminopropyltrimethoxysilane was successfully grafted onto the silica gel surface. In Sil-HRAA, the peak is 1569 cm⁻¹. -1 The stretching vibration peaks of C=O and NH in the vicinity are enhanced, and the vibration peak of CN appears at 1464 cm⁻¹, and also at 2967 cm⁻¹. -1 2880cm -1 The stretching vibration peak at the point is significantly enhanced, further indicating that hydrogenated rosin acyl chloride has been successfully bonded to the silicone surface.

[0063] Thermogravimetric characterization was performed on the SiO2, Sil-NH2, and Sil-HRAA synthesized in Example 1. Thermogravimetric analysis is as follows: Figure 3 As shown, Sil-NH2 loses 7.04% of its weight compared to SiO2. This is due to the decomposition of the aminated modifying agent at high temperature, indicating that Sil-NH2 was successfully prepared. The Sil-HRAA stationary phase loses little weight before 300℃, indicating good thermal stability. After 300℃, it begins to decompose and lose weight rapidly, gradually stabilizing near 700℃. This indicates that the rosin monomer grafted onto Sil-NH2 loses weight due to high-temperature decomposition, resulting in a weight loss of approximately 16.51% compared to SiO2. In conclusion, Sil-HRAA was successfully prepared.

[0064] The SiO2 and Sil-HRAA synthesized in Example 2 were characterized by scanning electron microscopy, and the results are as follows: Figure 4As shown in the figure, the Sil-HRAA stationary phase prepared in this invention has uniform size and morphology, abundant pores, and high sphericity. Compared with activated silica gel, the modified silica gel did not show any morphological change, meeting the basic requirements for chromatographic packing material.

[0065] The pore size distribution of SiO2, Sil-NH2, and Sil-HRAA synthesized in Example 3 was analyzed. Figure 5 (a) and nitrogen adsorption-desorption isotherms Figure 5 (b) Characterization. For example... Figure 5 (a) The specific surface area, pore volume, and pore size of SiO2, Sil-NH2, and Sil-HRAA all decreased, indicating that although the rosin-based monomers were bonded to the surface and pores of silica gel, the final stationary phase still had a large specific surface area, pore volume, and pore size, which met the requirements of silica gel chromatography materials. Figure 5 (b) clearly shows that SiO2, Sil-NH2, and Sil-HRAA belong to type IV isotherms and are mesoporous materials, indicating that their structure was not destroyed during the synthesis process.

[0066] Elemental (EA) characterization was performed on the SiO2, Sil-NH2, and Sil-HRAA synthesized in Example 2. The elemental analysis of the SiO2, Sil-NH2, and Sil-HRAA stationary phases is shown in Table 1. Compared with silica gel, the nitrogen content of the aminosilicone increased by 2.30%, indicating that the amination reagent was successfully bonded to the silica gel. The carbon content of the Sil-HRAA stationary phase was 16.14%, which increased by 9.29% compared with aminosilicone, indicating that the Sil-HRAA stationary phase was successfully prepared.

[0067] Table 1 compares the elemental analysis of SiO2, Sil-NH2, and Sil-HRAA stationary phases.

[0068] Another object of the present invention is to provide a specific application of the above-mentioned amide-type hydrogenated rosin-bonded silica gel mixed mode stationary phase.

[0069] The Sil-HRAA stationary phase was packed into an empty stainless steel column (150 mm × 4.6 mm) using a homogenization method. 2.5 g of the Sil-HRAA stationary phase and 50 mL of methanol were added to a beaker and sonicated for 5 min to ensure uniform dispersion. Using a column packing apparatus, methanol was used as the displacement solvent, and the column was continuously pressurized at 65.00 MPa for 20 min. After the pressure dropped to 0 MPa, the column was allowed to stand for another 30 min to obtain the Sil-HRAA column. The prepared column was connected to an LC-16C high-performance liquid chromatograph and washed overnight with methanol at a flow rate of 0-1 mL / min.

[0070] 1. Flowability of the stationary phase in a mixed-mode system (Application Example 1)

[0071] Methanol and acetonitrile were used as mobile phases, respectively, to investigate the relationship between the flow rate (0.1–1.0 mL / min) and background pressure of the SiI-HRAA column. As shown in the figure, the flow rate of SiI-HRAA exhibited a good linear relationship with the background pressure, and its linear correlation coefficient (R0) was [value missing]. 2 If the value reaches 0.999 or higher, it indicates that the stationary phase has good flowability and meets the requirements for use as a high-performance liquid chromatography column.

[0072] 2. Study on the RPLC chromatographic performance of mixed-mode stationary phases (Application Example 2)

[0073] In RPLC chromatography mode, alkylbenzenes and polycyclic aromatic hydrocarbons were separated using a Sil-HRAA column.

[0074] The separation conditions for both were 65% acetonitrile (A) and 35% water (B) as the mobile phase, 1.0 mL / min as the flow rate, 30 °C as the temperature, and 254 nm as the detection wavelength.

[0075] The hydrophobic retention of the Sil-HRAA column was investigated using alkylbenzenes (benzene, toluene, ethylbenzene, propylbenzene, butadiene, and pentabenzene) as probes. The results are as follows: Figure 6 As shown, this indicates that alkylbenzenes have symmetrical and sharp peaks on this column.

[0076] In addition, the effect of acetonitrile content on the retention time of alkylbenzenes was investigated. Figure 7 As shown, the retention factors of all six alkylbenzenes decreased with increasing acetonitrile content in the mobile phase, consistent with the retention pattern of reversed-phase chromatography columns.

[0077] To further evaluate the separation performance of the Sil-HRAA column in RPLC mode, five polycyclic aromatic hydrocarbons (naphthalene, fluorene, anthracene, pyrene, and triphenylene) were also separated. The separation results are as follows: Figure 8 As shown, the peaks of the five compounds are sharp and symmetrical, enabling rapid separation and determination.

[0078] 3. Study on the HILIC chromatographic performance of mixed-mode stationary phase (Application Example 3)

[0079] In HILIC mode, six base nucleotides were separated using a Sil-HRAA column.

[0080] The separation conditions were: mobile phase of 88% acetonitrile (A) and 12% water (B), flow rate of 0.8 mL / min, temperature of 30 °C, and detection wavelength of 254 nm.

[0081] The hydrophilic retention of the Sil-HRAA column was investigated using five nucleotide bases (thymine, sulfadiazine, adenine, inosine, and guanine) as probes. The results are as follows: Figure 9 As shown, the peaks of the five highly polar nucleotide bases are symmetrical and sharp on this column, enabling rapid separation.

[0082] Furthermore, the effect of acetonitrile content on the retention time of base nucleotides was investigated. Figure 10 As shown, the retention factors of the five base nucleotides all increased with the increase of acetonitrile content, which is consistent with the HILIC retention mechanism, indicating that the Sil-HRAA column has hydrophilic interaction.

[0083] 4. Separation of saponins and flavonoids from Panax notoginseng using a mixed-mode stationary phase (Application Example 4)

[0084] Chromatographic conditions: The mobile phase was adjusted according to the Chinese Pharmacopoeia as follows: acetonitrile (A) 23% for 0-20 min, 45% for 20-22.5 min, 55% for 22.5-45 min, 77% for 0-20 min, 55% for 20-22.5 min, and 45% for 22.5-45 min; the flow rate was 0.5 mL / min; the temperature was 30℃; and the detection wavelength was 203 nm.

[0085] The crude extract of Panax notoginseng was separated on Sil-HRAA, and the separation effect is shown in the figure below. Figure 11 As shown, the resolution of notoginsenoside R1 to Rg1 was 6.164, the resolution of notoginsenoside Rg1 to Re was 2.180, the resolution of notoginsenoside Re to glycyrrhizin was 6.716, the resolution of glycyrrhizin to quercetin was 3.617, the resolution of notoginsenoside Rb1 to Rd was 7.450, and the resolution of notoginsenoside Rd to kaempferol was 12.028.

[0086] 5. Column reproducibility and stability of mixed-mode stationary phase (Application Example 5)

[0087] To evaluate the batch-to-batch reproducibility of the stationary phase, two batches of stationary phase were prepared using the same experimental method. A mixture of five polycyclic aromatic hydrocarbons (PAHs) was used as the test analyte, and the samples were injected consecutively 10 times under the same chromatographic conditions to obtain the corresponding chromatograms. Figure 12 (a) The RSDs of the retention times for the first batch were 0.06%, 0.07%, 0.08%, 0.12%, and 0.12%, respectively, and the RSDs for the retention times for the second batch were 0.05%, 0.06%, 0.05%, 0.06%, and 0.07%, respectively, indicating that the stationary phase has good batch-to-batch reproducibility. Using five nucleoside bases as analytes, injections were performed three times daily under the same conditions for seven consecutive days to obtain the corresponding chromatograms. Figure 12(b) The RSDs of the retention times were 0.29%, 1.89%, 0.63%, 1.06%, and 1.52%, respectively, indicating that the stationary phase has good stability.

[0088] In summary, the amide-type rosin-based bonded silica gel mixed stationary phase prepared by this invention exhibits uniform shape, high sphericity, good thermal stability, and a mixed hydrophilic and hydrophobic chromatographic separation mode, showing promising application prospects. Combined with green and natural forest chemical raw materials, it meets the current research and development needs of environmentally friendly products. Furthermore, this stationary phase boasts advantages such as good reproducibility, high stability, and non-toxicity, and can be prepared in a simple and convenient manner, possessing significant market potential.

Claims

1. An amide-type rosin-based bonded silica gel mixed-mode stationary phase, characterized in that: Starting with hydrogenated rosin or dehydroabsic acid, the mixture reacts with oxalyl chloride to generate the corresponding acyl chloride compound. Subsequently, the acyl chloride compound undergoes a nucleophilic acyl substitution reaction with the amino groups on the surface of activated silica gel modified with an aminosilane coupling agent. After end-capping treatment, an amide-type rosin-based bonded silica gel mixed-mode stationary phase is obtained. Its structural formula is shown in I or II.

2. The amide-type rosin-based bonded silica gel mixed-mode stationary phase as described in claim 1, characterized in that: The silica gel particles are 3-10 μm in size, with an average pore size ranging from 3-20 mm and a specific surface area of ​​200-700 m². 2 / g.

3. The method as described in claim 1, characterized in that: The method for preparing amide-type rosin-based bonded silica microspheres includes the following steps: Step 1, Activating silica gel: Add bare silica gel to a round-bottom flask containing hydrochloric acid solution, disperse ultrasonically, mix well, and then heat and stir under nitrogen protection. After the reaction is complete, cool to room temperature, wash the product with deionized water to remove hydrochloric acid until the aqueous solution is neutral, and finally dry the product in a vacuum drying oven to obtain activated silica gel. The second step, amino-modified silica gel: The activated silica gel prepared in the first step was placed in a flask containing toluene and ultrasonically dispersed until uniform. Then, a silane coupling agent was added, and the mixture was heated and stirred under nitrogen protection. After the reaction was completed, the mixture was cooled, filtered, and washed successively with methanol, ethanol, and acetone. Finally, the product was dried in a vacuum drying oven to obtain amino-modified silica gel. Step 3: Hydrogenated rosin (or dehydroabsic acid) acyl chloride: Add hydrogenated rosin (or dehydroabsic acid) and toluene to a three-necked flask equipped with a spherical condenser, thermometer, and tail gas absorber in an ice-water bath. Stir magnetically until the hydrogenated rosin (or dehydroabsic acid) is completely dissolved. Then, add oxalyl chloride dropwise. After the addition is complete, heat and stir under reflux to remove the solvent toluene and any unreacted oxalyl chloride. Seal and store for later use. Step 4, Amide-type Rosin-based Bonded Silica Gel: The amino-modified silica gel prepared in Step 2 was placed in a three-necked flask, and toluene and triethylamine were added. The mixture was then magnetically stirred. The hydrogenated rosin (or dehydroabietic acid) acyl chloride prepared in Step 3 was added to a constant-pressure dropping funnel, and the mixture was heated and stirred under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed successively with water and ethanol. Finally, the product was dried in a vacuum drying oven. Step 5, End-capping: The dried product was placed in a round-bottom flask, and toluene, triethylamine, and end-capping agent were added. The mixture was heated and stirred under nitrogen protection. After the reaction was completed, it was cooled to room temperature, filtered, and washed successively with dichloromethane, methanol, and ethanol. The dried solid was wrapped in filter paper and extracted with ethanol using a Soxhlet extraction method. After extraction, it was filtered again, and the resulting solid was dried in a vacuum drying oven to obtain an amide-type rosin-based bonded silica gel mixed-mode stationary phase.

4. The method as described in claim 3, characterized in that: The concentration of hydrochloric acid in the first step is 2-5 mol / L, the activation time is 8-12 h, the temperature is 70-100℃, the vacuum drying time is 12-24 h, and the temperature is 60-80℃.

5. The method as described in claim 3, characterized in that: The amino reagent used in the second step is 3-aminopropyltrimethoxysilane or 3-aminopropyltriethoxysilane, and the reaction time is 12-24 h and the reaction temperature is 90-110 °C.

6. The method as described in claim 3, characterized in that: In the third step, the molar ratio of hydrogenated rosin (or dehydroabsic acid) to oxaloyl chloride is 1:(1.10-1.25), the reaction time is 8-12 h, the temperature is 80-110 °C, and the rotary evaporation temperature is 40-60 °C.

7. The method as described in claim 3, characterized in that: In the fourth step, the mass ratio of amino-modified silica gel to hydrogenated rosin (or dehydroabietic acid) acyl chloride is 1:(1-3), the reaction time is 8-12 h, the temperature is 70-100 °C, the bonded stationary phase is vacuum dried for 12-24 h, and the temperature is 60-80 °C.

8. The method as described in claim 3, characterized in that: In step 5, the end-capping agent is trimethylchlorosilane. The mass ratio of the amide-type rosin-based bonded stationary phase to trimethylchlorosilane is 1:(0.2-0.4), the reaction time is 6-8 h, and the temperature is 80-100 °C; the bonded stationary phase is vacuum dried for 12-24 h at a temperature of 60-80 °C.

9. The method as described in claim 3, characterized in that: In step 5, the Soxhlet extraction time is 12–24 h. The bonded stationary phase is vacuum dried for 12–24 h at a temperature of 60–80 °C.

10. The amide-type rosin-based bonded silica gel mixed-mode stationary phase prepared by any of the methods of claims 3 to 9 is characterized in that, The stationary phase is used as a mixed-mode chromatographic packing material. In hydrophilic mode (HILIC), it can be used to separate mixtures of base nucleotides. In reversed-phase mode (RPLC), it can be used to separate mixtures of alkylbenzenes and polycyclic aromatic hydrocarbons. In mixed mode, it can be used to separate ginsenosides Rb1, Rd, Rg1, Re and notoginsenoside R1 from Panax notoginseng, as well as flavonoids such as glycyrrhizin, quercetin and kaempferol from Panax notoginseng.

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