Ultra-efficient supercritical fluid chromatography stationary phase as well as preparation and application thereof

Abstract

The invention relates to an ultra-efficient supercritical fluid chromatography stationary phase as well as a preparation method and application thereof. The stationary phase is ordered mesoporous silica gel microspheres or ordered mesoporous inorganic / organic hybrid silica gel microspheres. The ultra-efficient supercritical fluid chromatography stationary phase is a material which is uniform in pore size distribution, ordered in pore structure and controllable in morphology and is obtained by a soft template method. The separation material prepared by the invention can obtain ultrahigh column efficiency in supercritical chromatography, meanwhile, due to the property of large specific surface area, the retention of weak polar compounds is enhanced, and the problems of weak retention and poor separation effect of different lipid-soluble samples in supercritical chromatography are solved. The preparation method is simple in process and mild in reaction condition, has good preparation reproducibility and stability, and is easy to popularize and apply.

Description

Technical Field

[0001] The present invention relates to a chromatographic stationary phase material and its preparation and application, and relates to the field of ultra-high performance supercritical fluid chromatography analysis materials. Technical Background

[0002] Supercritical fluid chromatography (SFC) is a chromatographic technique that uses a fluid above its critical temperature and critical pressure as the mobile phase for analysis. Analyzed from the perspective of the separation mode, SFC is very similar to high performance liquid chromatography (HPLC), and the mobile phase plays an important role. This is not only reflected in that the analyte will directly dissolve in the mobile phase, but also in that the mobile phase will compete with the analyte for the surface of the stationary phase, thus affecting the interaction between the analyte and the stationary phase. Currently, the stationary phase of SFC still uses the HPLC stationary phase, including particle size, pore size, and the morphology of the base sphere, etc. However, compared with the liquid mobile phase used in HPLC, the supercritical fluid has low viscosity, fast diffusion, and small surface tension, and the separation speed of the sample on SFC is faster. Therefore, developing a unique stationary phase type that is more compatible with SFC to promote the mass transfer process is one of the important research directions in the field of analytical chemistry.

[0003] Ordered mesoporous silica (OMS) has attracted extensive attention in the fields of catalysis, drug delivery, energy storage, and chromatography due to its unique porosity. The highly ordered pores can promote uniform and efficient mass transfer, thereby improving the efficiency and resolution of liquid chromatography columns. However, precisely controlling the morphology without losing the pore order is a huge challenge for chromatographic applications. MCM-41 is a classic OMS templated with cetyltrimethylammonium bromide surfactant. Even if monodisperse microspheres can be obtained while changing the pore size, the pore size is usually less than 5 nm. In HPLC, in order to ensure the mass transfer process of most samples, the pore size of the chromatographic material is generally 10 nm. Therefore, the application of materials such as MCM-41 in liquid chromatography is limited and only stays at separating small molecule benzene series compounds. However, when applied to SFC, when the mobile phase is a supercritical fluid with low viscosity and fast diffusion, the relatively small pore size of MCM-41 microspheres is no longer a limitation. At the same time, the uniform pore size distribution, ordered pore structure, controllable morphology, and large specific surface area can further promote the separation advantages of SFC. Although OMS has played an important role in various fields, there is little research in the field of chromatographic stationary phase materials, and there is no reported literature on using it as an SFC stationary phase. Therefore, OMS as an SFC stationary phase has important research significance and broad application prospects in the field of chromatographic separation and analysis. Summary of the Invention

[0004] The present invention discloses an ultra-high efficiency supercritical fluid chromatography stationary phase, a preparation method and an application thereof. The separation material prepared by the present invention can obtain ultra-high column efficiency in supercritical chromatography. At the same time, due to the property of large specific surface area, the retention of weakly polar compounds is enhanced, and the problems of weak retention and poor separation effect of different types of liposoluble samples in supercritical chromatography are solved. The preparation method has a simple process, mild reaction conditions, good preparation reproducibility and stability, and is easy to be popularized and applied.

[0005] Specifically, the technical solution of the ultra-high efficiency supercritical fluid chromatography stationary phase of the present invention is as follows:

[0006] An ultra-high efficiency supercritical fluid chromatography stationary phase, which is an ordered mesoporous silica microsphere or an ordered mesoporous inorganic / organic hybrid silica microsphere, and its structural schematic formula is:

[0007]

[0008] R is one of a hydroxyl group, a mercaptopropyl group, a vinyl group, a benzyl group, and a methyl group.

[0009] The microspheres have monodisperse particle size, and the particle diameter is 0.8 - 3 μm; the pore volume is 0.2 - 0.8 cm 3 / g.

[0010] The mesopore diameter is 1 - 5 nm.

[0011] The preparation method of the ultra-high efficiency supercritical fluid chromatography stationary phase includes the following steps: Measure methanol and water and mix them evenly, transfer them to a beaker for standby; transfer the beaker to an oil bath, heat it to 30 - 60 °C, and add dodecylamine to dissolve it fully; add tetraethyl orthosilicate to obtain a material with hydroxyl groups as functional groups, and then add different silane coupling agents to obtain 4 modified materials. After stirring and reacting for 5 - 15 min, stop stirring and let it stand for sedimentation at room temperature for 16 - 48 h; filter the mixture, take the solid and wash it twice with methanol and water respectively, and dry the obtained solid in a drying oven at 80 °C for 16 hours.

[0012] The silane coupling agents include: (3-mercaptopropyl)triethoxysilane, triethoxyvinylsilane, benzyltriethoxysilane, and methyltriethoxysilane.

[0013] In the preparation method, the volume ratio of methanol to water is 3:1 - 1:1; the molar ratio of tetraethyl orthosilicate to dodecylamine is 8:1 - 2:1; the molar ratio of tetraethyl orthosilicate to the silane coupling agent is 20:1 - 5:1; the dosage of tetraethyl orthosilicate is 0.01 - 0.06 mol (2.2 mL - 13.4 mL) contained in 100 mL of methanol.

[0014] The application of the described chromatographic stationary phase in the separation and detection of related lipophilic samples such as the ethanol extract of Salvia miltiorrhiza, the mixture of unsaturated fatty acids in fish oil, and vitamin E derivatives in soybean oil under the detection mode of supercritical fluid chromatography.

[0015] The present invention uses the soft template method to obtain materials with uniform pore size distribution, ordered pore structure, and controllable morphology. The separation material prepared by the present invention can achieve ultra-high column efficiency in supercritical chromatography. At the same time, due to the large specific surface area, the retention of weakly polar compounds is enhanced, solving the problems of weak retention and poor separation effect of different types of lipophilic samples in supercritical chromatography. The preparation method has a simple process, mild reaction conditions, good preparation reproducibility and stability, and is easy to promote and apply.

[0016] The present invention has the following advantages:

[0017] 1. The ordered mesoporous silica microspheres or ordered mesoporous inorganic / organic hybrid silica microspheres of the present invention have uniform particle size, pore size, and large specific surface area. The present invention first proposes to use them as SFC stationary phase materials. In the supercritical system, ultra-high column efficiency can be achieved, reaching 250,000 - 340,000 / m; and efficient separation of various lipophilic compounds can be realized.

[0018] 2. The preparation method of the supercritical chromatography stationary phase provided by the present invention has a simple process, mild reaction conditions, good preparation reproducibility and stability, is easy to promote and apply, and can realize industrialization. Description of the Drawings

[0019] Figure 1 It is the structural schematic formula of the stationary phase material, where R is one or more of hydroxyl, mercaptopropyl, vinyl, benzyl, and methyl;

[0020] Figure 2 It is the transmission electron microscope characterization diagram of the material in Example 1;

[0021] Figure 3 It is the transmission electron microscope characterization diagram of the material in Example 1;

[0022] Figure 4 It is the chromatogram of Example 6;

[0023] Figure 5 It is the chromatogram of Example 7;

[0024] Figure 6 It is the chromatogram of Example 8;

[0025] Figure 7 It is the chromatogram of Example 9.

[0026] Figure 8 It is the chromatogram of Example 10. Detailed Embodiments

[0027] The present invention will be further described below in conjunction with specific examples. The examples are only for illustrating the present invention and not for limiting the invention.

[0028] Example 1

[0029] Measure 540 mL of methanol and 225 mL of water, mix them evenly, and transfer to a 1 L beaker for standby; transfer the beaker to an oil bath, heat to 30 °C, add 16 mL of dodecylamine and dissolve it completely (n(tetraethyl orthosilicate):n(dodecylamine)=2.4:1); then add 40 mL of tetraethyl orthosilicate, stir and react for 8 minutes, stop stirring and let it stand and settle at room temperature for 24 hours; filter the mixture, take the solid and wash it twice with methanol and water respectively, and dry the obtained solid in an oven at 80 °C for 16 hours to obtain chromatographic stationary phase 1 (mesoporous silica microspheres), and its structural schematic formula is as follows. After characterization by transmission electron microscopy ( Figure 2 ) and scanning electron microscopy ( Figure 3 ), it shows that the microspheres have good monodispersity and uniform particle size distribution; the particle size distribution range measured by a laser particle size analyzer is 0.4 - 2.5 μm, and the average particle size is 1.03 μm; after nitrogen adsorption-desorption characterization, the pore size distribution range is 2.3 - 3.1 nm, the average pore size is 2.5 nm, and the pore volume is 0.56 cm 3 / g.

[0030]

[0031] Example 2

[0032] Measure 540 mL of methanol and 225 mL of water, mix them evenly, and transfer to a 1 L beaker for standby; transfer the beaker to an oil bath, heat to 30 °C, add 7 mL of dodecylamine and dissolve it completely (n(tetraethyl orthosilicate):n(dodecylamine)=5.4:1); then add 40 mL of tetraethyl orthosilicate and 4.4 mL of 3-mercaptopropyltriethoxysilane (n(tetraethyl orthosilicate):n(3-mercaptopropyltriethoxysilane)=10:1), stir and react for 8 minutes, stop stirring and let it stand and settle at room temperature for 24 hours; filter the mixture, take the solid and wash it twice with methanol and water respectively, and dry the obtained solid in an oven at 80 °C for 16 hours to obtain chromatographic stationary phase 2 (modified mesoporous inorganic / organic hybrid silica microspheres), and its structural schematic formula is as follows. After characterization by transmission electron microscopy and scanning electron microscopy, it shows that the microspheres have good monodispersity and uniform particle size distribution; the particle size distribution range measured by a laser particle size analyzer is 0.9 - 4.3 μm, and the average particle size is 2.5 μm; after nitrogen adsorption-desorption characterization, the pore size distribution range is 2.0 - 4.1 nm, the average pore size is 2.5 nm, and the pore volume is 0.53 cm 3 / g.

[0033]

[0034] Example 3

[0035] Measure 540 mL of methanol and mix it evenly with 225 mL of water, then transfer it to a 1 L beaker for standby; transfer the beaker to an oil bath, heat it to 30 °C, add 7 mL of dodecylamine and dissolve it fully (n(tetraethyl orthosilicate):n(dodecylamine) = 5.4:1); then add 40 mL of tetraethyl orthosilicate and 3.8 mL of triethoxyvinylsilane (n(tetraethyl orthosilicate):n(triethoxyvinylsilane) = 10:1), stir and react for 8 minutes, then stop stirring and let it stand and settle at room temperature for 24 hours; filter the mixture, take the solid and wash it twice with methanol and water respectively, and the obtained solid is dried in an oven at 80 °C for 16 hours to obtain chromatographic stationary phase 3 (modified mesoporous inorganic / organic hybrid silica microspheres), and its structural schematic formula is as follows. After characterization by transmission electron microscopy and scanning electron microscopy, it shows that the microspheres have good monodispersity and uniform particle size distribution; the particle size distribution range measured by a laser particle size analyzer is 0.7 - 4.1 μm, and the average particle size is 2.3 μm; after nitrogen adsorption and desorption characterization, the pore size distribution range is 2.0 - 4.3 nm, the average pore size is 2.7 nm, and the pore volume is 0.522 cm 3 / g.

[0036]

[0037] Example 4

[0038] Measure 540 mL of methanol and mix it evenly with 225 mL of water, then transfer it to a 1 L beaker for standby; transfer the beaker to an oil bath, heat it to 30 °C, add 7 mL of dodecylamine and dissolve it fully (n(tetraethyl orthosilicate):n(dodecylamine) = 5.4:1); then add 40 mL of tetraethyl orthosilicate and 4.7 mL of benzyltriethoxysilane (n(tetraethyl orthosilicate):n(benzyltriethoxysilane) = 10:1), stir and react for 8 minutes, then stop stirring and let it stand and settle at room temperature for 24 hours; filter the mixture, take the solid and wash it twice with methanol and water respectively, and the obtained solid is dried in an oven at 80 °C for 16 hours to obtain chromatographic stationary phase 4 (modified mesoporous inorganic / organic hybrid silica microspheres), and its structural schematic formula is as follows. After characterization by transmission electron microscopy and scanning electron microscopy, it shows that the microspheres have good monodispersity and uniform particle size distribution; the particle size distribution range measured by a laser particle size analyzer is 0.9 - 4.3 μm, and the average particle size is 2.3 μm; after nitrogen adsorption and desorption characterization, the pore size distribution range is 2.2 - 4.3 nm, the average pore size is 2.66 nm, and the pore volume is 0.53 cm 3 / g.

[0039]

[0040] Example 5

[0041] Measure 540 mL of methanol and mix it evenly with 225 mL of water, then transfer it to a 1 L beaker for standby; transfer the beaker to an oil bath, heat it to 30 °C, and add 7 mL of dodecylamine to dissolve it completely (n(tetraethyl orthosilicate):n(dodecylamine) = 5.4:1); then add 40 mL of tetraethyl orthosilicate and 3.6 mL of methyltriethoxysilane (n(tetraethyl orthosilicate):n(methyltriethoxysilane) = 10:1), stir and react for 8 minutes, stop stirring and let it stand and settle at room temperature for 24 hours; filter the mixture, wash the obtained solid with methanol and water twice respectively, and dry the obtained solid in an oven at 80 °C for 16 hours to obtain chromatographic stationary phase 5 (modified mesoporous inorganic / organic hybrid silica microspheres), and its structural schematic formula is as follows. After characterization by transmission electron microscopy and scanning electron microscopy, it shows that the microspheres have good monodispersity and uniform particle size distribution; the particle size distribution range measured by a laser particle size analyzer is 0.5 - 4.3 μm, and the average particle size is 2.3 μm; after nitrogen adsorption-desorption characterization, the pore size distribution range is 2.0 - 4.1 nm, the average pore size is 2.3 nm, and the pore volume is 0.63 cm 3 / g

[0042]

[0043] Example 6

[0044] Use the chromatographic stationary phase 1 obtained in Example 1 to pack a 4.6×100 mm (inner diameter × length) chromatographic column for column efficiency test. This stationary phase is tested with uracil, and according to the chromatographic data processing software (such as: Empower3 software), a super-high column efficiency of 340,000 / m is read. As Figure 4 shown, the chromatographic conditions are as follows:

[0045] Chromatographic column: 4.6 mm × 100 mm (inner diameter × length);

[0046] Sample: Uracil (dissolved in methanol, concentration is 1 mg / mL);

[0047] Mobile phase: A: CO 2 , B: 6% water / methanol (V / V);

[0048] Elution: A:B = 80:20 (V / V);

[0049] Supercritical back pressure BAR: 2100 psi;

[0050] Flow rate: 2 mL / min;

[0051] Column temperature: 40 °C;

[0052] Detection wavelength: PDA (200 - 400 nm) & UV (254 nm);

[0053] Example 7

[0054] The chromatographic stationary phase 2 obtained in Example 2 was used to pack a 4.6×100 mm (inner diameter × length) chromatographic column, which can be used for the separation of fat-soluble unsaturated fatty acids in fish oil. This stationary phase has good selectivity for the four main compounds in fish oil, and the elution order is as follows: 1. Ethyl oleate, 2. Ethyl linoleate, 3. Ethyl linolenate, 4. Ethyl arachidonate. As Figure 5 shown, the chromatographic conditions are as follows:

[0055] Chromatographic column: 4.6 mm × 100 mm (inner diameter × length);

[0056] Sample: A mixed standard of fat-soluble unsaturated fatty acids (ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate are dissolved in n-hexane together, and the concentration of each compound in their mixed solution is 1 mg / mL);

[0057] Mobile phase: A: CO 2 , B: Methanol;

[0058] Elution: A:B = 99:1 (V / V);

[0059] Supercritical back pressure BAR: 2000 psi;

[0060] Flow rate: 3 mL / min;

[0061] Column temperature: 35 °C;

[0062] Detection wavelength: PDA (200 - 400 nm) & UV (215 nm);

[0063] Example 8

[0064] The chromatographic stationary phase 3 obtained in Example 3 was used to pack a 4.6×100 mm (inner diameter × length) chromatographic column, which can be used for the separation of fat-soluble vitamin E derivatives in soybean oil. This stationary phase has good selectivity for the four main compounds among them, and the elution order is as follows: 1. α-Tocopherol, 2. β-Tocopherol, 3. γ-Tocopherol, 4. δ-Tocopherol. As Figure 6 shown, the selectivity is good. The chromatographic conditions are as follows:

[0065] Chromatographic column: 4.6 mm × 100 mm (inner diameter × length);

[0066] Sample: A mixed standard of fat-soluble vitamin E derivatives (α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol are dissolved in n-hexane together, and the concentration of each compound in their mixed solution is 1 mg / mL);

[0067] Mobile phase: A: CO 2 , B: Methanol;

[0068] Elution: 0 - 10 min, 1% B - 10% B (V / V, linear);

[0069] Supercritical back pressure BAR: 2000 psi;

[0070] Flow rate: 3 mL / min;

[0071] Column temperature: 35 °C;

[0072] Detection wavelength: PDA (200 - 400 nm) & UV (215 nm);

[0073] Example 9

[0074] Use the chromatographic stationary phase 1 obtained in Example 1 to fill a 4.6×100 mm (inner diameter × length) chromatographic column, which can be used for the separation of liposoluble phenanthraquinone compounds in the ethanol extract of Salvia miltiorrhiza Bunge. This stationary phase successfully separated 7 compounds among them, and the elution order is as follows: 1. Methyl tanshinonate, 2. Tanshinone new quinone A, 3. Tanshinone I, 4. Tanshinone IIA, 5. Cryptotanshinone, 6. Salvianol A, 7. Dihydrotanshinone I. As Figure 7 shown, the chromatographic conditions are:

[0075] Chromatographic column: 4.6 mm × 100 mm (inner diameter × length);

[0076] Sample: Mixed standard of liposoluble phenanthraquinone compounds (7 compounds are dissolved in methanol together, and the concentration of each of the 7 compounds in their mixed methanol solution is 1 mg / mL);

[0077] Mobile phase: A: CO 2 , B: Methanol;

[0078] Elution: 0 - 10 min, 5% B - 40% B (V / V, linear);

[0079] Supercritical back pressure BAR: 2100 psi;

[0080] Flow rate: 3 mL / min;

[0081] Column temperature: 35 °C;

[0082] Detection wavelength: PDA (200 - 400 nm) & UV (215 nm);

[0083] Example 10

[0084] Use commercial Fuji silica gel packing material (HS200860) to pack a 4.6×100 mm (inner diameter × length) chromatographic column. The particle size distribution range measured by a laser particle size analyzer is 1.2 - 6.2 μm, and the average particle size is 2.5 μm; the pore size distribution range characterized by nitrogen adsorption and desorption is 7.5 - 15 nm, the average pore size is 10 nm, and the pore volume is 0.88 cm 3 / g. Use this stationary phase to separate the sample in Example 9, and the complete separation effect cannot be achieved. As Figure 8 shown, the chromatographic conditions are as follows:

[0085] Chromatographic column: 4.6 mm × 100 mm (inner diameter × length);

[0086] Sample: Mixed standard of fat-soluble phenanthraquinone compounds (1. Methyl tanshinonate, 2. Tanshinone A, 3. Tanshinone I, 4. Tanshinone IIA, 5. Cryptotanshinone, 6. Tanshinol A, 7. Dihydrotanshinone I; The 7 compounds are dissolved in methanol together, and the concentrations of the 7 compounds in their mixed methanol solution are all 1 mg / mL respectively);

[0087] Mobile phase: A: CO 2 , B: Methanol;

[0088] Elution: 0 - 10 min, 5% B - 40% B (V / V, linear);

[0089] Supercritical back pressure BAR: 2100 psi;

[0090] Flow rate: 3 mL / min;

[0091] Column temperature: 35 °C;

[0092] Detection wavelength: PDA (200 - 400 nm) & UV (215 nm).

Claims

1. A method for preparing an ultra-high performance supercritical fluid chromatography stationary phase, characterized in that: The following steps are involved: Take methanol and water, mix them and heat them to 30-60°C, add dodecylamine to dissolve; add tetraethyl orthosilicate, without or with a silane coupling agent, stir to react for 5-15 minutes, stop stirring and let stand and settle at room temperature for 16-48 hours; separate the solid and liquid, wash and dry the solid to obtain a chromatographic stationary phase.

2. The preparation method according to claim 1, characterized in that: The solid-liquid separation method is filtration, and then the solid is washed with methanol and water in turn, and the obtained solid is dried at 60-90°C for 8-48h.

3. The preparation method according to claim 1, characterized in that: The stationary phase is mesoporous silica microspheres or modified mesoporous inorganic / organic hybrid silica microspheres, and the schematic structure thereof is as follows: R is one or more of hydroxyl, mercaptopropyl, vinyl, benzyl and methyl.

4. The preparation method according to claim 1, characterized in that: The silane coupling agent includes one or more of (3-mercaptopropyl)triethoxysilane, triethoxyvinylsilane, benzyltriethoxysilane and methyltriethoxysilane.

5. The preparation method according to claim 1 or 4, characterized in that: The volume ratio of methanol to water is 3:1 to 1:1; The molar ratio of tetraethyl orthosilicate to dodecylamine is 8:1 to 2:1; The molar ratio of tetraethyl orthosilicate to the silane coupling agent is 20:1 to 5:

1.

6. The preparation method according to claim 1, characterized in that: The amount of tetraethyl orthosilicate used is 0.01 to 0.06 mol (2.2 mL to 13.4 mL) in 100 mL of methanol.

7. The preparation method according to claims 1-6, characterized in that: The stationary phase is silica microspheres or modified silica microspheres, the microsphere particles are monodispersed, the particle size is 0.8 to 3 μm, and the pore volume is 0.2 to 0.8 cm 3 / g; the stationary phase is mesoporous silica microspheres or modified silica microspheres with a pore size of 1 to 5 nm.

8. A chromatographic stationary phase prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the chromatographic stationary phase according to claim 8 in the separation and detection of one or more related fat-soluble compounds among tanshinone, unsaturated fatty acids and vitamin E in the detection mode of supercritical fluid chromatography.

10. Use of the chromatographic stationary phase according to claim 9, wherein the chromatographic stationary phase can separate and detect related fat-soluble samples such as fat-soluble phenanthrenequinone compounds in Danshen medicinal material alcohol extract, unsaturated fatty acid mixtures in fish oil, or vitamin E derivatives in soybean oil under the detection mode of supercritical fluid chromatography.

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