Dehydroabietic acid cellulose ester (DCE) bonded silica gel stationary phase and preparation method thereof

Abstract

The invention discloses a preparation method of a dehydroabietic acid cellulose ester (DCE) bonded silica gel stationary phase, which comprises the following steps: firstly, carboxylating the surface of a SiO2 microsphere with a core-shell structure, then carrying out acylating chlorination, and then preparing amphiphilic DCE coated SiO2 solid particles with DCE in a chemical bonding manner. The DCE-SiO2 solid particle has the pore diameter of 5-30nm and the specific surface area of 50-400m < 2 > / g, has the characteristics of intact particle shape, uniform particle size, high mechanical strength, high pressure resistance and the like, and can be used as a liquid chromatographic column filler. The preparation method is simple and convenient, the column pressure is low, the repeatability is good, the chemically modified DCE has a phenanthrene ring skeleton and has good selective separation performance on saponin drugs containing ring structures, and the application prospect in the pharmaceutical industry is wide.

Description

Technical Field

[0001] The present invention relates to the field of high performance liquid chromatography, and particularly to a dehydroabietic acid cellulose ester (DCE) bonded silica stationary phase. Background Art

[0002] Cellulose is one of the most representative renewable resources of natural polysaccharides. Due to the unique spatial structure of cellulose, a large number of hydroxyl groups are arranged periodically, so cellulose has good chemical modification. Cellulose derivatization effectively improves the solubility of cellulose materials and endows new properties. Dehydroabietic acid is a natural compound extracted from pine plants. Its specific tricyclic phenanthrene skeleton has a high similarity with natural medicine saponins, and dehydroabietic acid has the advantages of easy degradation and natural renewability, making it have great advantages as a separation material. Therefore, in this study, based on the previous work of researchers, dehydroabietic acid cellulose ester and silane coupling agent were chemically bonded to obtain dehydroabietic acid cellulose ester bonded SiO 2 stationary phase.

[0003] Establishing the technical integration of high performance liquid chromatography (HPLC) for the separation and purification of natural products and accelerating the development speed of drug lead compounds and innovative drugs are the future development trends of HPLC separation and purification research. The chromatographic stationary phase is the core component of HPLC, and its retention mechanism, separation performance, separation selectivity and resolution will all affect the analysis effect of HPLC. Developing new stationary phases with high separation efficiency and high selectivity to meet various application requirements has always been the focus of researchers. Summary of the Invention

[0004] In order to solve the problem that the existing SiO 2 stationary phase has an unsatisfactory separation effect for saponin drugs, after amino-functionalization with a silanizing reagent, carboxyl-functionalization with succinic anhydride, and finally acylation with oxalyl chloride, dehydroabietic acid cellulose ester was bonded to the surface of the acylated porous SiO 2 microspheres to obtain a dehydroabietic acid cellulose ester chromatographic stationary phase (DCE@SiO 2 stationary phase). The amount of the silanizing reagent was adjusted to control the effective degree of the fixation of dehydroabietic acid cellulose ester; the hydrophilicity and hydrophobicity of the dehydroabietic acid cellulose ester chromatographic stationary phase were controlled by adjusting the degree of substitution of dehydroabietic acid cellulose ester. Modern analytical testing techniques were used to characterize the hydrophilicity and hydrophobicity, bonding amount, specific surface area, pore structure, surface and internal microtopography, and surface chemical structure of the stationary phase.

[0005] To achieve the above object, the specific solutions provided by the present invention are as follows:

[0006] A dehydroabietic acid cellulose ester (DCE) bonded silica stationary phase, comprising the following steps:

[0007] (1) A DCE-bonded SiO 2 stationary phase, after SiO 2 is aminated and modified with a silane coupling agent, it is carboxylated with succinic anhydride; finally, it is modified with oxalyl chloride by acylation and then reacts with DCE to form DCE@SiO 2 solid particles; the pore size of the DCE@SiO 2 solid particles is 5 - 30 nm, and the specific surface area is 50 - 400 m 2 / g;

[0008] (2) Further, the mass ratio of DCE to the SiO 2 particles modified with a silane coupling agent is 1:10 - 20;

[0009] (3) Further, the silane coupling agent is one of (3-aminopropyl)triethoxysilane (APTMS) silanes;

[0010] (4) Further, the catalyst is one of triethylamine, pyridine, and 4-methylpyridine.

[0011] The present invention has the following advantageous aspects:

[0012] 1. The DCE@SiO 2 stationary phase prepared by the present invention has a phenanthrene ring skeleton, and its structure is similar to the structure of the drug to be separated. By electrostatic shielding, the influence of the silanol groups on the surface of the SiO 2 core-shell structure on the separation performance is eliminated, and at the same time, the spatial selectivity for the separated substances is enhanced.

[0013] 2. The preparation method of the DCE@SiO 2 stationary phase is simple, the column pressure is low, the repeatability is good, and after chemical modification, DCE has a phenanthrene ring skeleton and has good selective separation for saponin drugs containing cyclic structures, and has a wide application prospect in the pharmaceutical industry. Description of the Drawings Figure 1 is the infrared spectrum of the DCE@SiO 2 stationary phase; Figure 2 is the thermogravimetric analysis chart of SiO 2 ; Figure 3 is the adsorption-desorption curve of the DCE@SiO 2 stationary phase under N 2 protection; Figure 4 is the pore size distribution chart of the DCE@SiO 2 stationary phase; Figure 5 is the scanning electron microscope image of the DCE@SiO 2 stationary phase; Figure 6 For DCE@SiO 2 X-ray diffraction pattern of the stationary phase Figure 7 is the chromatographic column flow performance diagram; Figure 8 is the chromatographic column repeatability and stability diagram. Specific implementation mode

[0014] The following further illustrates the technical process of the present invention in combination with specific implementation examples:

[0015] Preparation Example 1

[0016] An amphiphilic SiO 2 Preparation method of the stationary phase, specifically including the following steps:

[0017] (1) (8.00 g) SiO 2 Particles were activated with hydrochloric acid, washed with deionized water until neutral, dried, and dispersed in toluene. After being treated with the silane coupling agent (3-aminopropyl)triethoxysilane (APTMS) and heated to 80 °C for reflux reaction for 12 h, after the reaction ended, it was washed with methanol and acetone to obtain amino-modified SiO 2 microspheres, named SiO 2 -NH 2 .

[0018] (2) (5.00 g) SiO 2 -NH 2 , anhydrous toluene, triethylamine, and succinic anhydride were added to a container and heated to 50 °C for reflux reaction for 6 h. After the reaction ended, it was washed with methanol and acetone to obtain carboxylated SiO 2 microspheres, named SiO 2 -NH-COOH.

[0019] (3) SiO 2 -NH-COOH, anhydrous toluene, and oxalyl chloride were added, and the mixture was refluxed for 1 - 5 h. After the reaction ended, precipitation was obtained by centrifugation, and the precipitate was washed three times with ether to obtain acylated SiO 2 microspheres, named the final product SiO 2 -NH-COCl.

[0020] (4) Dehydroabietic acid cellulose ester was dissolved in a 10 wt% DMAc / LiCl transparent solution, then SiO 2 -NH-COCl and pyridine were added, and the mixture was heated to 90 °C for reflux reaction for 12 h. After the reaction ended, precipitation was obtained by centrifugation and washed three times with ether and ethanol to obtain the precipitate, which was the DCE@SiO 2 solid particles.

[0021] Figure 1 is the infrared spectrum obtained in Example 1, where the stretching vibration peak of Si-OH can be observed at 3425 cm -1 ; the stretching vibration peak of Si-O-Si is at 1050 cm -1 ; the stretching vibration peak of SiO -1 is the asymmetric stretching vibration peak of silanol O-H on the surface of the microspheres, and this characteristic peak significantly decreases after amination, carboxylation, and acyl chlorination; in the DCE@SiO 2 stationary phase sample, the stretching vibration peaks of methyl and methylene are at 2960 cm 2 and 2850 cm -1 , and the stretching vibration peak of the carbonyl group in the newly formed ester group is shown at 1726 cm -1 . -1

[0022] Figure 2 is the thermogravimetric analysis chart obtained in Example 1. It can be seen from the figure that the weight loss of SiO 2 microspheres is about 3% within 100 °C because there is a small amount of moisture on the surface of the silica gel, and the increase in temperature causes the evaporation of the moisture. The mass loss intensifies at 100 - 200 °C, corresponding to the decomposition of MCC; the mass loss is more severe at 200 - 335 °C, corresponding to the decomposition of DA; the mass loss at 300 - 500 °C is caused by the thermal decomposition of the bonded DCE.

[0023] Figure 3 is the adsorption - desorption curve of the stationary phase obtained in Example 1 under N 2 protection, which is a type IV curve. This indicates that the stationary phase is a mesoporous material. Compared with the SiO 2 microspheres, the specific surface area of the stationary phase shows a decreasing trend, which is due to the successful bonding of APTMS and dehydroabietic acid cellulose ester on the surface of the microspheres during the chemical bonding process.

[0024] Figure 4 is the pore size distribution chart obtained in Example 1. Its pore size distribution is relatively concentrated, indicating that the pore size distribution of the microspheres is narrow. The specific surface areas of the SiO 2 microspheres and the stationary phase are 271.34 m 2 / g and 263.52 m 2 / g respectively; the average pore volumes are 0.74 cm -1 / g and 0.61 cm -1 / g respectively; the average pore diameters are 9.36 nm and 8.12 nm;. These data indicate that the successful bonding of dehydroabietic acid cellulose ester to the SiO 2 microspheres causes this.

[0025] Preparation Example 2

[0026] ​(1)Disperse (10.00 g) SiO 2 particles after activation with hydrochloric acid, washing with deionized water until neutral, and drying, in toluene. React with the silane coupling agent (3-aminopropyl)triethoxysilane (APTMS) by heating to 80 °C and refluxing for 12 h. After the reaction, wash with methanol and acetone to obtain amino-modified SiO 2 microspheres, named SiO 2 -NH 2 .

[0027] Steps (2), (3), and (4) are exactly the same as those in Example 1.

[0028] Figure 5 Figure 15 is the SEM image of Example 2. It can be seen from the figure that the DCE@SiO2 stationary phase prepared by the present invention has a complete spherical shape, uniform particle size, and rich surface pores.

[0029] Preparation Example 3

[0030] Steps (1), (2), and (3) are exactly the same as those in Example 1.

[0031] (4) Dissolve cellulose dehydroabietate (0.40 g) in a 10 wt% DMAc / LiCl transparent solution, then add SiO 2 -NH-COCl (1.00 g) and pyridine, and then heat to 90 °C and reflux for 12 h. After the reaction, centrifuge and wash three times with ether and ethanol to obtain a precipitate, which is the DCE@SiO 2 solid particles.

[0032] Figure 6 Figure 16 is the XRD pattern of Example 3. It can be seen from the figure that when the chemically bonded stationary phases are at different molar ratios, the mesoporous structure of SiO 2 is not changed.

[0033] Application Example of the Prepared DCE@SiO 2 Stationary Phase

[0034] 1. Packing of the Chromatographic Column

[0035] The DCE@SiO 2 stationary phase packing prepared by the present invention is used as the displacement liquid with methanol, and the column is packed at a pressure of 3000 - 6000 psi for 50 min. The chromatographic column is connected to the EClassical3100 system and washed with methanol at a flow rate of 0.1 mL / min to remove impurities until a stable baseline is obtained.

[0036] 2. Evaluation of the Flowability of the Chromatographic Column

[0037] The rosin-based bonded silica stationary phase prepared in Preparation Example 2 was connected to a liquid chromatograph according to the methods of steps (1) to (4) above to evaluate its flow performance. The results are as Figure 7 shown.

[0038] Figure 7 is the chromatographic column flow performance diagram of DCE@SiO obtained in Preparation Example 2. It can be seen from the figure that the background pressure of the chromatographic column was tested in the flow rate range of 0.1 - 2.0 mL / min. As the flow rate increased, the background pressure also increased, and there was a good linear relationship between the two. The linear correlation coefficient R 2 > 0.99, indicating that the chromatographic column has good flow performance and meets the analysis requirements of high performance liquid chromatography. The mobile phase of the chromatographic column has a larger slope for methanol because the viscosity of methanol is greater than that of acetonitrile. 2

[0039] 3. Evaluation of the repeatability and stability of the chromatographic column

[0040] The rosin-based bonded silica stationary phase prepared in Preparation Example 3 was connected to a liquid chromatograph according to the methods of steps (1) to (4) above to evaluate its repeatability and stability. The results are as Figure 8 shown.

[0041] Figure 8 is the chromatographic column repeatability and stability diagram of DCE@SiO obtained in Preparation Example 3. It can be seen from 2 that when uracil, caffeine, phenol, and toluene were used as probes and continuously injected 45 times, the chromatographic peaks basically overlapped. This shows that the chromatographic column has good repeatability and stability. Figure 8

[0042] For the DCE@SiO 2 stationary phases prepared in the above Examples 1 - 3, by changing the substitution degree of DCE to regulate the hydrophilicity and hydrophobicity of the DCE@SiO 2 stationary phase, and at the same time adjusting the different molar ratios of DCE to adjust the bonding amount of the DCE@SiO 2 stationary phase, the amphiphilicity of the stationary phase can be adjusted and controlled.

Claims

1. A dehydroabietic cellulose ester (DCE) bonded silica gel stationary phase, characterized in that: After SiO2 particles are modified with a silane coupling agent, they react with DCE through a chemical bonding reaction to form a DCE@SiO2 stationary phase material; the particle size distribution of the rosin-based bonded silica stationary phase is 2-10 μm, the average pore size is 3-50 nm, and the specific surface area is 50-500 m 2 / g; the DCE@SiO2 solid particles have a pore size of 5-30nm and a specific surface area of ​​50-400m 2 / g. The mass ratio of DCE to SiO2 particles modified by silane coupling agent is 1:10-20; A method for preparing an amphiphilic SiO2 stationary phase, characterized by the following specific steps: (1) SiO2 particles were activated with hydrochloric acid, washed with deionized water until neutral, dried, and dispersed in toluene. The particles were reacted with silane coupling agent (3-aminopropyl) triethoxysilane (APTMS) under reflux for 1-12 h. After the reaction, the particles were washed with solvent A to obtain amino-modified SiO2 microspheres, named SiO2-NH2. (2) SiO2-NH2, anhydrous toluene, triethylamine, and succinic anhydride are added to a container and refluxed for 1-6 hours. After the reaction is completed, the carboxylated SiO2 microspheres are washed with solvent B to obtain SiO2-NH-COOH. (3) Add oxalyl chloride to SiO2-NH-COOH and anhydrous toluene, and reflux for 1-5 hours. After the reaction is completed, centrifuge to obtain a precipitate, and wash the precipitate with solvent C to obtain chlorinated SiO2 microspheres, which are named the final product SiO2-NH-COCl. (4) Dissolve dehydroabietic cellulose ester in a mixed solvent D, then add SiO2-NH-COCl and pyridine and reflux for 1-5 hours. After the reaction is completed, centrifuge and wash with solvent E to obtain a precipitate, which is DCE@SiO2 solid particles.

2. The method for preparing an amphiphilic SiO2 stationary phase according to claim 1, characterized in that The pore size of SiO2 stationary phase is 5-30nm, and the specific surface area is 50-400m 2 / g.

3. The method for preparing an amphiphilic SiO2 stationary phase according to claim 1, characterized in that The pore size of SiO2 stationary phase is 8.12nm and the specific surface area is 263.52m 2 / g.

4. The method for preparing an amphiphilic SiO2 stationary phase according to claim 1, characterized in that (1) SiO2 particles were activated with hydrochloric acid, washed with deionized water until neutral, dried, and dispersed in toluene. The particles were reacted with silane coupling agent (3-aminopropyl) triethoxysilane (APTMS) under reflux for 1-12 h. After the reaction, the particles were washed with solvent A to obtain amino-modified SiO2 microspheres, named SiO2-NH2. (2) SiO2-NH2, anhydrous toluene, triethylamine, and succinic anhydride are added to a container and refluxed for 1-6 hours. After the reaction is completed, the carboxylated SiO2 microspheres are washed with solvent B to obtain SiO2-NH-COOH. (3) Add oxalyl chloride to SiO2-NH-COOH and anhydrous toluene, and reflux for 1-5 hours. After the reaction is completed, centrifuge to obtain a precipitate, and wash the precipitate with solvent C to obtain chlorinated SiO2 microspheres, which are named the final product SiO2-NH-COCI. (4) Dissolve dehydroabietic cellulose ester in a mixed solvent D, then add SiO2-NH-COCl and pyridine and reflux for 1-5 hours. After the reaction is completed, centrifuge and wash with solvent E to obtain a precipitate, which is DCE@SiO2 solid particles.

5. A dehydroabietic cellulose ester (DCE) bonded silica gel stationary phase according to claim 4, characterized in that The mass ratio of activated SiO2 particles: anhydrous toluene: silane coupling agent in step (2) is 1:1-40:1-10.

6. The method for preparing the dehydroabietic cellulose ester (DCE) bonded silica gel stationary phase according to claim 4, characterized in that The organic solvent A in step (1) is one or two of methanol, acetone, and toluene; the organic solvent B in step (2) is one or two of methanol, acetone, ethanol, or toluene; the organic solvent C in step (3) is one or two of methanol, acetone, ethanol, or diethyl ether; the organic solvent D in step (4) is one or two of N,N-dimethylacetamide and lithium chloride; the organic solvent E is one or two of methanol, ethanol, diethyl ether, water, or N,N-dimethylacetamide.

7. The method for preparing the dehydroabietic cellulose ester (DCE) bonded silica gel stationary phase according to claim 4, characterized in that The gas is nitrogen.

Images