CN115245816A - Amino nicotinic acid mixed-mode chromatographic stationary phase and preparation method and application thereof - Google Patents
Amino nicotinic acid mixed-mode chromatographic stationary phase and preparation method and application thereof Download PDFInfo
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- KPIVDNYJNOPGBE-UHFFFAOYSA-N 2-aminonicotinic acid Chemical compound NC1=NC=CC=C1C(O)=O KPIVDNYJNOPGBE-UHFFFAOYSA-N 0.000 title claims abstract description 71
- 230000005526 G1 to G0 transition Effects 0.000 title claims abstract description 61
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 94
- 239000000741 silica gel Substances 0.000 claims description 70
- 229910002027 silica gel Inorganic materials 0.000 claims description 70
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 36
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 36
- 238000005406 washing Methods 0.000 claims description 35
- 238000005859 coupling reaction Methods 0.000 claims description 31
- 239000004593 Epoxy Substances 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- 239000012265 solid product Substances 0.000 claims description 20
- 238000007142 ring opening reaction Methods 0.000 claims description 19
- 239000000463 material Substances 0.000 claims description 18
- 238000000967 suction filtration Methods 0.000 claims description 18
- 230000008878 coupling Effects 0.000 claims description 17
- 238000010168 coupling process Methods 0.000 claims description 17
- 238000012434 mixed-mode chromatography Methods 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 17
- 239000002253 acid Substances 0.000 claims description 16
- 238000001291 vacuum drying Methods 0.000 claims description 16
- 239000007864 aqueous solution Substances 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- GYZLOYUZLJXAJU-UHFFFAOYSA-N diglycidyl ether Chemical compound C1OC1COCC1CO1 GYZLOYUZLJXAJU-UHFFFAOYSA-N 0.000 claims description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- 239000006087 Silane Coupling Agent Substances 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- -1 amino nicotinic acid compound Chemical class 0.000 claims description 10
- 238000004458 analytical method Methods 0.000 claims description 9
- 239000003054 catalyst Substances 0.000 claims description 9
- BYIORJAACCWFPU-UHFFFAOYSA-N 5-aminonicotinic acid Chemical compound NC1=CN=CC(C(O)=O)=C1 BYIORJAACCWFPU-UHFFFAOYSA-N 0.000 claims description 8
- ZCIFWRHIEBXBOY-UHFFFAOYSA-N 6-aminonicotinic acid Chemical compound NC1=CC=C(C(O)=O)C=N1 ZCIFWRHIEBXBOY-UHFFFAOYSA-N 0.000 claims description 8
- JXUKBNICSRJFAP-UHFFFAOYSA-N triethoxy-[3-(oxiran-2-ylmethoxy)propyl]silane Chemical compound CCO[Si](OCC)(OCC)CCCOCC1CO1 JXUKBNICSRJFAP-UHFFFAOYSA-N 0.000 claims description 8
- QMKZZQPPJRWDED-UHFFFAOYSA-N 2-aminopyridine-4-carboxylic acid Chemical compound NC1=CC(C(O)=O)=CC=N1 QMKZZQPPJRWDED-UHFFFAOYSA-N 0.000 claims description 7
- 239000003960 organic solvent Substances 0.000 claims description 4
- 238000005554 pickling Methods 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 3
- 239000003929 acidic solution Substances 0.000 claims description 2
- 230000002378 acidificating effect Effects 0.000 claims description 2
- 238000004811 liquid chromatography Methods 0.000 claims description 2
- 239000012046 mixed solvent Substances 0.000 claims description 2
- QQQSFSZALRVCSZ-UHFFFAOYSA-N triethoxysilane Chemical compound CCO[SiH](OCC)OCC QQQSFSZALRVCSZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000003828 vacuum filtration Methods 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 32
- 230000009471 action Effects 0.000 abstract description 6
- 239000001257 hydrogen Substances 0.000 abstract description 5
- 229910052739 hydrogen Inorganic materials 0.000 abstract description 5
- 238000013375 chromatographic separation Methods 0.000 abstract description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 239000000945 filler Substances 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- RQFQJYYMBWVMQG-IXDPLRRUSA-N chitotriose Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)[C@@H](CO)O1 RQFQJYYMBWVMQG-IXDPLRRUSA-N 0.000 description 30
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 27
- 238000000926 separation method Methods 0.000 description 26
- 230000014759 maintenance of location Effects 0.000 description 21
- 229920001661 Chitosan Polymers 0.000 description 11
- 238000005303 weighing Methods 0.000 description 10
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- 238000012360 testing method Methods 0.000 description 9
- QLTSDROPCWIKKY-PMCTYKHCSA-N beta-D-glucosaminyl-(1->4)-beta-D-glucosamine Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O)[C@@H](CO)O1 QLTSDROPCWIKKY-PMCTYKHCSA-N 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 235000000346 sugar Nutrition 0.000 description 8
- 238000010521 absorption reaction Methods 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 7
- 229910021641 deionized water Inorganic materials 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000012491 analyte Substances 0.000 description 6
- 230000007935 neutral effect Effects 0.000 description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 150000008040 ionic compounds Chemical class 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
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- 238000002156 mixing Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- HBAVVSYNWHLVDB-UDAFUQIYSA-N (2r,3r,4s,5r)-2-amino-4-{[(2s,3r,4r,5s,6r)-3-amino-5-{[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-4-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-3,5,6-trihydroxyhexanal Chemical compound O[C@@H]1[C@@H](N)[C@H](O[C@@H]([C@H](O)[C@H](C=O)N)[C@H](O)CO)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O)[C@@H](CO)O1 HBAVVSYNWHLVDB-UDAFUQIYSA-N 0.000 description 2
- 229910008051 Si-OH Inorganic materials 0.000 description 2
- 229910006358 Si—OH Inorganic materials 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000002013 hydrophilic interaction chromatography Methods 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- 238000004190 ion pair chromatography Methods 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
- 229910002808 Si–O–Si Inorganic materials 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000009878 intermolecular interaction Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
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- 230000007246 mechanism Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 238000005220 pharmaceutical analysis Methods 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention provides an amino nicotinic acid mixed-mode chromatographic stationary phase as well as a preparation method and application thereof. The chromatographic stationary phase with the amino nicotinic acid mixed mode, which is prepared by the invention, has the characteristics of zwitterion, contains various active groups and can provide various acting forces such as pi-pi, hydrogen bond, ionic action and the like for chromatographic separation. The preparation method of the amino nicotinic acid mixed-mode chromatographic stationary phase provided by the invention has the advantages of mild reaction conditions, simple synthesis steps, complete and uniform prepared filler, good stability and the like.
Description
Technical Field
The invention belongs to the technical field of liquid chromatogram stationary phase materials, and particularly relates to an amino nicotinic acid mixed mode chromatogram stationary phase and a preparation method and application thereof.
Background
In the modern chromatographic analysis field, high performance liquid chromatography is a conventional analysis method which is most widely applied, and is widely applied to the fields of food, agricultural products, aquatic products, pharmaceutical chemicals and the like. With the increase of sample types and the increasing complexity of sample matrixes, the requirements on chromatographic separation conditions are higher and higher, and the selection of a chromatographic stationary phase plays a key role in the separation selectivity and the separation efficiency of a sample.
At present, a commercially available chromatographic column is mainly in a single separation mode, has strong separation specificity, has respective defects under specific conditions, has poor universality and is easy to generate false positive when encountering a complex matrix sample. The C18 column is less retained for strongly polar and charged compounds; the positive phase column has strong retention on compounds with strong polarity and charges, but the mobile phase (normal hexane, ethyl acetate and the like) has poor solubility on the compounds; ion exchange chromatography can achieve separation of ionic compounds, but has poor separation of ionic compounds with the same charge.
Mixed mode chromatography is the preferred method for simultaneous analysis of neutral and ionic compounds by displacement ion pair chromatography. Unlike conventional single mode chromatography, mixed mode chromatography uses a multifunctional cluster chromatography stationary phase, the retention mechanism of which involves intermolecular interactions of two or more different types, and thus has a stronger separation ability. The most commonly used mixed mode chromatography is reverse phase/ion exchange chromatography, using a stationary phase with hydrophobic and dissociable functional groups on the surface, thus enabling the retention and simultaneous separation of neutral and ionic compounds by the synergistic effect of van der waals and electrostatic forces. Because the mobile phase does not need to add a surfactant, the problems existing in ion pair chromatography do not exist, and the gradient elution analysis can be used and can also be combined with mass spectrometry, so that the method has a wide application prospect in pharmaceutical analysis. However, the mixed-mode chromatography stationary phase has few varieties and single structure so far, and cannot adapt to the increasing complex components in the analysis field.
Therefore, how to provide a mixed-mode chromatographic column stationary phase to realize simultaneous separation of multiple components becomes a problem to be solved at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an amino nicotinic acid mixed mode chromatographic stationary phase and a preparation method and application thereof. The amino nicotinic acid mixed-mode chromatographic stationary phase provided by the invention has zwitterion characteristics, contains various active groups, can provide various acting forces such as pi-pi, hydrogen bonds, ionic action and the like for chromatographic separation, and can realize simultaneous separation of multiple components.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides an amino nicotinic acid mixed mode chromatography stationary phase, which has a structure represented by formula I below:
wherein R is selected from any one of the structures shown as the following formulas II-V:
In a second aspect, the present invention provides a method for preparing an amino nicotinic acid mixed-mode chromatography stationary phase according to the first aspect, comprising the following steps:
(1) Adding silica gel into an acid solution for acid washing to obtain activated silica gel;
(2) Carrying out coupling reaction on the activated silica gel obtained in the step (1) and an epoxy silane coupling agent to obtain epoxy coupling silica gel;
(3) And (3) carrying out ring-opening reaction on the epoxy coupling silica gel obtained in the step (2) and an amino nicotinic acid compound to obtain the amino nicotinic acid mixed mode chromatographic stationary phase.
In the present invention, in step (1), the silica gel comprises spherical silica gel having a diameter of 3 to 10 μm (which may be, for example, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, etc.).
Preferably, in step (1), the acidic solvent comprises 5-30% (e.g. 5%, 10%, 15%, 20%, 25%, 30% etc.) of aqueous hydrochloric acid by volume.
Preferably, in the step (1), the mass ratio of the silica gel to the acidic solution is 1 (5-10), wherein "5-10" can be 5, 6, 7, 8, 9, 10, etc.
In the present invention, in step (1), the temperature of the acid washing is 90 to 120 ℃ (for example, 90 ℃, 95 ℃, 100 ℃,105 ℃,110 ℃,115 ℃,120 ℃ and the like may be used), and the time of the acid washing is 5 to 18h (for example, 5h, 7h, 9h, 11h, 13h, 15h, 17h, 18h and the like may be used).
Preferably, in step (1), after the acid washing, a post-treatment is further required, wherein the post-treatment comprises the following steps: washing the material obtained after acid washing with water to obtain a washed material; and carrying out reduced pressure suction filtration on the material after washing to obtain a solid product, and carrying out vacuum drying on the solid product to obtain the activated silica gel.
Preferably, the pH of the washed material is 6.5-7.5 (e.g., can be 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, etc.).
Preferably, the vacuum drying temperature is 100-150 deg.C (such as 100 deg.C, 110 deg.C, 120 deg.C, 130 deg.C, 140 deg.C, 150 deg.C, etc.), and the time is 1.5-6h (such as 1.5h, 2.5h, 3.5h, 4.5h, 5.5h, 6h, etc.).
In the present invention, in the step (2), the epoxy silane coupling agent includes 3- (glycidyloxypropyl) triethoxysilane, and the 3- (glycidyloxypropyl) triethoxysilane has a structure represented by the following formula VI:
preferably, in step (2), the coupling reaction is carried out in a solvent comprising anhydrous toluene.
Preferably, the mass ratio of the activated silica gel to the epoxy silane coupling agent is 1 (1-4), wherein "1-4" can be 1, 2, 3, 4, etc.
Preferably, the mass ratio of the activated silica gel to the solvent is 1 (5-15), wherein the 5-15 can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and the like.
In the present invention, in the step (2), the temperature of the coupling reaction is 110 to 140 ℃ (for example, 110 ℃,115 ℃,120 ℃,125 ℃,130 ℃, 135 ℃, 140 ℃ and the like) and the time is 12 to 36h (for example, 12h, 15h, 20h, 25h, 30h, 35h, 36h and the like).
Preferably, in step (2), the coupling reaction is further followed by a post-treatment, wherein the post-treatment comprises the following steps: washing the material obtained after the coupling reaction with an organic solvent, and then carrying out suction filtration to obtain a solid product; and (4) carrying out vacuum drying on the solid product to obtain the epoxy coupling silica gel.
Preferably, the organic solvent includes a mixed solvent of absolute ethanol and toluene.
Preferably, the vacuum drying temperature is 100-150 deg.C (such as 100 deg.C, 110 deg.C, 120 deg.C, 130 deg.C, 140 deg.C, 150 deg.C, etc.), and the time is 1.5-6h (such as 1.5h, 2.5h, 3.5h, 4.5h, 5.5h, 6h, etc.).
In the present invention, in step (3), the amino nicotinic acid compound includes any one of 2-amino nicotinic acid, 2-amino isonicotinic acid, 5-amino nicotinic acid and 6-amino nicotinic acid.
Preferably, in the step (3), the mass ratio of the epoxy coupling silica gel to the amino nicotinic acid compound is (1.5-3): 1, wherein '1.5-3' can be 1.5, 2, 2.5, 3 and the like.
Preferably, in step (3), the ring-opening reaction is carried out in the presence of a catalyst comprising a volume fraction of 0.5-1.5% (e.g., may be 0.5%, 0.7%, 0.9%, 1.1%, 1.3%, 1.5%, etc.) of aqueous acetic acid.
Preferably, the mass ratio of the epoxy coupling silica gel to the catalyst is (3-10): 1, wherein "3-10" can be 3, 4, 5, 6, 7, 8, 9, 10, etc.
Preferably, in step (3), the ring-opening reaction is carried out in a solvent, the solvent comprising water.
Preferably, the mass ratio of the epoxy coupling silica gel to the solvent is 1 (3-10), wherein the '3-10' can be 3, 4, 5, 6, 7, 8, 9, 10 and the like.
In the present invention, in the step (3), the ring-opening reaction is carried out at a temperature of 50 to 90 ℃ (for example, 50 ℃, 60 ℃, 70 ℃,80 ℃, 90 ℃ or the like) for a period of 36 to 60 hours (for example, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours or the like).
Preferably, in step (3), after the ring-opening reaction, a post-treatment is required, and the post-treatment comprises the following steps: washing the material obtained after the ring-opening reaction to obtain a washed material; and carrying out vacuum filtration on the washed materials to obtain a solid product, and carrying out vacuum drying on the solid product to obtain the amino nicotinic acid mixed mode chromatographic stationary phase.
Preferably, the vacuum drying temperature is 100-150 deg.C (such as 100 deg.C, 110 deg.C, 120 deg.C, 130 deg.C, 140 deg.C, 150 deg.C, etc.), and the time is 1.5-6h (such as 1.5h, 2.5h, 3.5h, 4.5h, 5.5h, 6h, etc.).
As a preferable technical scheme of the invention, the preparation method of the amino nicotinic acid mixed mode chromatographic stationary phase comprises the following steps:
(1) Adding silica gel into an acid solution for acid washing to obtain activated silica gel;
wherein the pickling temperature is 90-120 ℃, and the pickling time is 5-18h;
(2) Carrying out coupling reaction on the activated silica gel obtained in the step (1) and an epoxy silane coupling agent to obtain epoxy coupling silica gel; the reaction formula is shown as follows:
wherein the epoxy silane coupling agent comprises 3- (glycidyl ether oxypropyl) triethoxysilane, and the coupling reaction is carried out at 110-140 ℃ for 12-36h;
(3) Performing ring-opening reaction on the epoxy coupling silica gel obtained in the step (2) and an amino nicotinic acid compound to obtain the amino nicotinic acid mixed mode chromatographic stationary phase; the reaction formula is shown as follows:
wherein the amino nicotinic acid compound comprises any one of 2-amino nicotinic acid, 2-amino isonicotinic acid, 5-amino nicotinic acid or 6-amino nicotinic acid; the ring-opening reaction is carried out in the presence of a catalyst, and the catalyst comprises an acetic acid aqueous solution with the volume fraction of 0.5-1.5%; the temperature of the ring-opening reaction is 50-90 ℃, and the time is 36-60h.
In the present invention, 2-aminonicotinic acid, 2-aminoisonicotinic acid, 5-aminonicotinic acid, 6-aminonicotinic acid have the following structures:
in the invention, the amino nicotinic acid mixed mode chromatography stationary phase is selected from any one of the following structures:
in the invention, firstly, activating silica gel to obtain activated silica gel; and bonding the activated silica gel and a silane coupling agent to prepare epoxy coupling silica gel, and finally grafting amino nicotinic acid onto the surface of the silica gel by taking amino nicotinic acid substances as functional ligands through amino-epoxy group ring-opening reaction to further obtain an amino nicotinic acid mixed mode chromatographic stationary phase, wherein the amino nicotinic acid substances comprise any one of 2-amino nicotinic acid, 5-amino nicotinic acid or 6-amino nicotinic acid.
In a third aspect, the present invention provides a use of the amino nicotinic acid mixed-mode chromatography stationary phase of the first aspect in liquid chromatography.
Compared with the prior art, the invention has the following beneficial effects:
the functional ligand selected by the invention is low in price and easy to obtain, and the prepared amino nicotinic acid mixed mode chromatographic stationary phase has zwitterion characteristics, contains various active groups and can provide various acting forces such as pi-pi, hydrogen bonds, ionic action and the like for chromatographic separation. The preparation method of the amino nicotinic acid mixed-mode chromatographic stationary phase provided by the invention has the advantages of mild reaction conditions, simple synthesis steps, complete and uniform prepared filler, good stability and the like.
Drawings
FIG. 1 is an infrared spectrum of 2-aminonicotinic acid grafted stationary phase;
wherein, (a) is 2-amino nicotinic acid grafted silica gel, (b) is epoxy coupled silica gel, and (c) is naked silica gel.
FIG. 2 is a graph of water content in the mobile phase as a function of retention time.
FIG. 3 is a graph of pH as a function of retention time in the mobile phase.
FIG. 4 is a diagram showing the separation of chitosan oligosaccharide standard in a 2-aminonicotinic acid grafted silica gel chromatographic column;
wherein 1 is chitobiose, 2 is chitobiose, 3 is chitotrisaccharide, 4 is chitotetrasaccharide, and 5 is chitopentasaccharide.
FIG. 5 is a diagram showing the separation of chitosan oligosaccharide standard in an amino hydrophilic chromatographic column;
wherein 1 is chitobiose, 2 is chitobiose, 3 is chitotriose, 4 is chitotetrasaccharide, and 5 is chitopentasaccharide.
FIG. 6 is a diagram showing the separation of a crude product of chitosan oligosaccharide in a 2-aminonicotinic acid-grafted silica gel chromatographic column;
wherein 1 is chitobiose, 2 is chitobiose, 3 is chitotrisaccharide, 4 is chitotetrasaccharide, and 5 is chitopentasaccharide.
FIG. 7 is a diagram showing the separation of a crude product of chitosan oligosaccharide in an amino hydrophilic chromatographic column;
wherein 1 is chitobiose, 2 is chitobiose, 3 is chitotriose, 4 is chitotetrasaccharide, and 5 is chitopentasaccharide.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The reaction temperatures described in the examples below are the temperatures of the reaction medium, and the manufacturer of the 5 μm amino hydrophilic column in the examples below is Shodex under the trade name F763001.
Example 1
The present embodiment provides an amino nicotinic acid mixed-mode chromatography stationary phase, where the amino nicotinic acid mixed-mode chromatography stationary phase has a structure as shown in the following:
the preparation method of the amino nicotinic acid mixed mode chromatographic stationary phase comprises the following steps:
(1) Weighing 15g of 10 μm silica gel into a 250mL single-neck flat-bottom flask, adding 100mL of 10% by volume hydrochloric acid solution, and refluxing at 110 ℃ for 6h. And after the reflux is finished, carrying out suction filtration, washing the mixture to be neutral by using a large amount of water, obtaining a solid product through suction filtration, and drying the solid product in a vacuum drying oven at 110 ℃ for 2 hours to obtain the activated silica gel.
(2) 10g of activated silica gel, 100mL of anhydrous toluene and 20mL of 3- (glycidyloxypropyl) triethoxysilane were placed in a 250mL single-neck flat-bottom flask, and subjected to reflux reaction at 125 ℃ for 24 hours, followed by suction filtration after the reaction was completed. Washing the product with ethanol/toluene solution, standing overnight, and drying at 120 deg.C for 2 hr to obtain 3- (glycidyl ether oxypropyl) triethoxy silica gel; the reaction formula is shown as follows:
(3) Weighing 2g of 3- (glycidyl ether oxypropyl) triethoxy silica gel, placing the 2-amino nicotinic acid triethoxy silica gel into a 50mL round-bottom flask, adding 10mL of aqueous solution containing 1g of 2-amino nicotinic acid solid into the flask, dropwise adding 0.5mL of acetic acid (1% volume fraction of acetic acid aqueous solution), keeping a closed reaction, carrying out magnetic stirring reaction at 65 ℃ for 48 hours, carrying out suction filtration after the reaction is finished, washing with a large amount of deionized water, and placing the mixture into an oven to be dried at 110 ℃ for 2 hours to obtain a 2-amino nicotinic acid grafted stationary phase, wherein the reaction formula is as follows:
example 2
The present embodiment provides an amino nicotinic acid mixed-mode chromatography stationary phase, where the amino nicotinic acid mixed-mode chromatography stationary phase has a structure as shown in the following:
the preparation method of the amino nicotinic acid mixed mode chromatographic stationary phase comprises the following steps:
(1) Weighing 14g 10 μm silica gel into a 250mL single-neck flat-bottom flask, adding 90mL 10% volume fraction hydrochloric acid solution, and refluxing at 105 ℃ for 6.5h. And after the reflux is finished, carrying out suction filtration, washing the product to be neutral by using a large amount of water, obtaining a solid product through suction filtration, and drying the solid product in a vacuum drying oven at the temperature of 120 ℃ for 1.8h to obtain the activated silica gel.
(2) 10g of activated silica gel, 100mL of anhydrous toluene and 22mL of 3- (glycidyloxypropyl) triethoxysilane were placed in a 250mL single-neck flat-bottomed flask, and the reaction was refluxed at 120 ℃ for 26 hours and then filtered with suction after the reaction was completed. Washing the product with ethanol/toluene solution, standing overnight, and drying at 125 deg.C for 2.1h to obtain 3- (glycidyl ether oxypropyl) triethoxy silica gel; the reaction formula is shown as follows:
(3) Weighing 2g of 3- (glycidyl ether oxypropyl) triethoxy silica gel, placing the 3- (glycidyl ether oxypropyl) triethoxy silica gel in a 50mL round-bottom flask, adding 10mL of aqueous solution containing 1.1g of 5-aminonicotinic acid solid, dropwise adding 0.55mL of acetic acid (aqueous solution of acetic acid with the volume fraction of 1%), keeping a closed reaction, magnetically stirring at 70 ℃ for reaction for 46 hours, performing suction filtration after the reaction is finished, washing with a large amount of deionized water, placing in an oven, drying at 115 ℃ for 1.8 hours, and obtaining the 5-aminonicotinic acid grafted stationary phase reaction formula shown as follows:
example 3
The present embodiment provides an amino nicotinic acid mixed-mode chromatography stationary phase, which has a structure shown in the following:
the preparation method of the amino nicotinic acid mixed mode chromatographic stationary phase comprises the following steps:
(1) 1lg 10 μm silica gel was weighed into a 250mL single-neck flat-bottomed flask, 105mL of 10% by volume hydrochloric acid solution was added, and the mixture was refluxed at 115 ℃ for 6 hours. And after the reflux is finished, carrying out suction filtration, washing the product to be neutral by using a large amount of water, obtaining a solid product through suction filtration, and drying the solid product in a vacuum drying oven at 115 ℃ for 2.1h to obtain the activated silica gel.
(2) 10g of activated silica gel, 100mL of anhydrous toluene and 23mL of 3- (glycidyloxypropyl) triethoxysilane were placed in a 250mL single-neck flat-bottomed flask, and the mixture was refluxed at 130 ℃ for 22 hours, and then filtered with suction after the reaction was completed. Washing the product with ethanol/toluene solution, standing overnight, and drying at 115 deg.C for 2.3h to obtain 3- (glycidyl ether oxypropyl) triethoxy silica gel; the reaction formula is shown as follows:
(3) Weighing 2g of 3- (glycidyl ether oxypropyl) triethoxy silica gel, placing the 2g of 3- (glycidyl ether oxypropyl) triethoxy silica gel into a 50mL round-bottom flask, adding 10mL of aqueous solution containing 1g of 6-aminonicotinic acid solid into the flask, dropwise adding 0.45mL of acetic acid (1% volume fraction of acetic acid aqueous solution), keeping a closed reaction, carrying out magnetic stirring reaction at 68 ℃ for 50 hours, carrying out suction filtration after the reaction is finished, washing with a large amount of deionized water, and placing the washed solution into an oven to be dried at 113 ℃ for 1.9 hours to obtain a 6-aminonicotinic acid grafted stationary phase reaction formula shown as follows:
example 4
The present embodiment provides an amino nicotinic acid mixed-mode chromatography stationary phase, which has a structure shown in the following:
the preparation method of the amino nicotinic acid mixed mode chromatographic stationary phase comprises the following steps:
(1) Weighing 15g 10 μm silica gel into a 250mL single-neck flat-bottom flask, adding 108mL 10% volume fraction hydrochloric acid solution, and refluxing at 110 ℃ for 6h. And after the reflux is finished, carrying out suction filtration, washing the mixture to be neutral by using a large amount of water, obtaining a solid product through suction filtration, and drying the solid product in a vacuum drying oven at 110 ℃ for 2.1h to obtain the activated silica gel.
(2) 10g of activated silica gel, 100mL of anhydrous toluene and 20mL of 3- (glycidyloxypropyl) triethoxysilane were placed in a 250mL single-neck flat-bottomed flask, and subjected to reflux reaction at 128 ℃ for 23 hours, followed by suction filtration after the reaction was completed. Washing the product with ethanol/toluene solution, standing overnight, and drying at 125 deg.C for 2.1h to obtain 3- (glycidyl ether oxypropyl) triethoxy silica gel; the reaction formula is shown as follows:
(3) Weighing 2g of 3- (glycidyl ether oxypropyl) triethoxy silica gel, placing the 2g of 3- (glycidyl ether oxypropyl) triethoxy silica gel into a 50mL round bottom flask, adding 10mL of aqueous solution containing 1.1g of 2-aminoisonicotinic acid solid into the flask, dropwise adding 0.5mL of acetic acid (1% volume fraction of acetic acid aqueous solution), keeping a closed reaction, carrying out magnetic stirring reaction at 70 ℃ for 49 hours, carrying out suction filtration after the reaction is finished, washing with a large amount of deionized water, and placing the mixture into an oven to be dried for 2 hours at 115 ℃ to obtain a 2-aminoisonicotinic acid grafted stationary phase reaction formula shown as follows:
test example 1
Structural verification
Testing a sample: example 1 provides the 2-amino nicotinic acid graft stationary phase.
The test method comprises the following steps: and (4) performing characterization by adopting infrared spectroscopy.
And (3) testing results: the infrared spectrum of the 2-amino nicotinic acid graft stationary phase is shown in figure 1; wherein, (a) is 2-amino nicotinic acid grafted silica gel, (b) is epoxy coupled silica gel, and (c) is naked silica gel.
Wherein, in the infrared curve of the bare silica gel of (c), 800cm -1 And 1100cm -1 The nearby absorption peak should be antisymmetric telescopic vibration absorption peak of Si-O-Si, 3500-3000cm -1 The nearby strong broad peak is the characteristic absorption peak of Si-OH, 1640cm -1 The nearby weak single peak is an H-O-H bending vibration peak of the silica gel without water after being dried at high temperature; comparing the infrared curve of epoxy-coupled silica gel in (b) to find that the infrared curve is 3500-3000cm -1 The intensity of the nearby absorption peak is obviously reduced, probably caused by consuming a large amount of Si-OH, 3000-2800cm -1 A new absorption peak appears nearby, probably caused by C-H carried by ethyl in the silane coupling agent, so that the successful bonding of the 3- (glycidyl ether oxygen propyl) triethoxysilane to the naked silica gel is proved.
Comparing the curves (a) and (b), 3500-3000cm in 2-aminonicotinic acid grafted silica gel -1 The absorption peak is obviously enhanced, and probably caused by the stretching vibration of groups such as-OH, NH and the like brought by 2-amino nicotinic acid；1640cm -1 The water peak nearby is 1700-1650cm -1 The absorption peak is covered and is 1700-1650cm -1 Has a relatively strong single peak, and is caused by C = O increase in carboxyl or amide group due to 2-amino nicotinic acid, 1550-1480cm -1 The appearance of a double peak in the vicinity is caused by the C = C bond on the pyridine ring in 2-aminonicotinic acid, thus demonstrating that 2-aminonicotinic acid is successfully immobilized on the silica gel surface.
Application example 1
The oligochitosan oligosaccharide (chitooligosaccharide 1-5 saccharide) was separated using 2-aminonicotinic acid grafted silica gel as the stationary phase as provided in example 1.
And filling the stationary phase in a 4.6X 150mm stainless steel analysis chromatographic column, connecting an Agilent 1260 type high performance liquid chromatograph, detecting by a differential detector, washing for 4 hours by taking a pure acetonitrile solution as a mobile phase, preparing to obtain a 2-amino nicotinic acid grafted silica gel chromatographic column, and testing the retention behavior of the prepared chromatographic column in a hydrophilic/ion exchange mode by taking the oligo-chitosan oligosaccharide as an analysis sample.
(1) Testing the Effect of changes in Water content of the Mobile phase on Retention
The oligochitosan standard (chitooligosaccharide 2-5 sugar) is used as the analyte. The method specifically comprises the following steps: weighing shell 2-5 sugar 50mg each, mixing in 10mL test tube, adding 5mL deionized water, and ultrasonic dissolving at 35 deg.C for 5min. Filtering with 0.22 μm water system filter membrane after completely dissolving to obtain chitosan oligosaccharide standard mixed solution with concentration of 50 mg/mL.
A2-aminonicotinic acid grafted silica gel chromatographic column is adopted, and the mobile phase is two phases, namely acetonitrile and an aqueous solution of ammonia water. Mobile phase conditions were set to acetonitrile/water (65%/35%), acetonitrile/water (70%/30%), acetonitrile/water (75%/25%), acetonitrile/water (80%/20%), respectively; the pH value of the mobile phase is 10.24, the flow rate of the mobile phase is 1.0mL/min, the column temperature is 35 ℃, and the detection wavelength is 254nm.
The change of the water content in the mobile phase along with the retention time is shown in figure 2, and the graph shows that the retention time of the chitosan oligosaccharide analyte is increased along with the decrease of the water content in the mobile phase, which shows that the retention of the chitosan oligosaccharide on a chromatographic column is influenced by the hydrophilic action, so that the amino nicotinic acid grafted stationary phase can be shown to have the typical hydrophilic action chromatographic retention behavior.
Reason analysis: this pattern of change is consistent with the retention characteristics of hydrophilic interaction chromatography. The retention of hydrophilic interaction chromatography is mainly achieved by establishing a "rich layer" on the surface of the stationary phase, into which the analyte can enter from the mobile phase. The main factor influencing the retention effect is hydrogen bond effect, and according to the structural analysis of the amino nicotinic acid stationary phase, amide and hydroxyl groups in the stationary phase have stronger capability of forming hydrogen bonds, so that the retention of an analyte on the surface of the stationary phase can be enhanced. For chitosan oligosaccharide, effective separation of chitosan 2-5 sugar can be realized according to different distribution acting forces of chitosan oligosaccharide with different polymerization degrees between a 'water-rich layer' and a flowing phase.
(2) Testing the Effect of pH change of the mobile phase on the Retention
Taking a chitosan oligosaccharide standard (chitosan oligosaccharide 2-5 sugar) as an analyte, specifically weighing 50mg of each chitosan oligosaccharide 2-5 sugar, mixing in a 10mL test tube, adding 5mL deionized water, and ultrasonically dissolving at 35 ℃ for 5min. Filtering with 0.22 μm water system filter membrane after completely dissolving to obtain chitosan oligosaccharide standard mixed solution with concentration of 50 mg/mL.
A2-aminonicotinic acid grafted silica gel chromatographic column is adopted, and the mobile phase is two phases, namely acetonitrile and an aqueous solution of ammonia water. Adjusting the pH of the mobile phase to 8.85, 9.42, 9.65, 9.85, 10.08, 10.24, 10.58 and 10.74 respectively, wherein the flow rate of the mobile phase is 1.0mL/min, the column temperature is 35 ℃, and the detection wavelength is 254nm.
The change of the pH value in the mobile phase along with the retention time is shown in FIG. 3, and the graph shows that the retention time of the chitosan oligosaccharide analyte is shortened along with the increase of the pH value in the mobile phase, which indicates that the retention of the chitosan oligosaccharide on the chromatographic column is influenced by the ion action, so that the amino nicotinic acid grafted stationary phase can be proved to have the typical retention behavior of the ion chromatography.
Reason analysis: on the one hand, it is likely that the pH rise enhances the interaction between the mobile phase and the stationary phase, resulting in a decrease in the adsorption of chitosan oligosaccharide by the stationary phase; on the other hand, the pH is increased to enable free amino groups in the chitosan oligosaccharide to be bound to H + The quantity is reduced, the positive charge on the surface is reduced, the binding capacity of the carboxyl group with negative charge on the surface of the stationary phase and the amino group with positive charge on the chitosan oligosaccharide is weakened, and the pH value is increasedThe retention time of the chitosan oligosaccharide on the chromatographic column is shortened.
(3) Comparison of the 10 μm 2-aminonicotinic acid-grafted silica gel column provided in application example 1 with a commercially available 5 μm aminohydrophilic column (Chitosan oligosaccharide Standard)
The preparation method of the chitosan oligosaccharide standard comprises the following steps: weighing shell 1-5 sugar 50mg each, mixing in 10mL test tube, adding 5mL deionized water, and ultrasonic dissolving at 35 deg.C for 5min. After the chitosan oligosaccharide is completely dissolved, filtering the solution by using a 0.22 mu m water system filter membrane to obtain a chitosan oligosaccharide standard mixed solution with the concentration of 50 mg/mL.
Chitosan oligosaccharide standard (chitosan oligosaccharide 1-5 sugar) was separated using a 10 μm 2-aminonicotinic acid grafted silica gel column and a commercially available 5 μm amino hydrophilic column simultaneously under the chromatographic conditions of acetonitrile/aqueous ammonia solution (70/30), mobile phase pH 10.24, mobile phase flow rate 1.0mL/min, column temperature 35 ℃, detection wavelength 254nm.
The separation results are shown in fig. 4 and 5. The 10 mu m 2-aminonicotinic acid grafted silica gel chromatographic column provided by the invention shows separation capacity similar to that of a commercially available 5 mu m amino hydrophilic column under the condition of greatly reduced preparation cost. In the 10 μm 2-aminonicotinic acid grafted silica gel chromatographic column separation chromatogram in fig. 4, the separation degrees of adjacent chromatographic peaks are respectively: the separation degree of the chitosan 2 and the chitosan 3 is 3.30, the separation degree of the chitosan 3 and the chitosan 4 is 2.72, and the separation degree of the chitosan 4 and the chitosan 5 is 2.62, which are both more than 1.5, namely, the novel amino nicotinic acid chromatographic column can realize the complete separation of the chitosan oligosaccharide mixture.
(4) Comparison of the 10 μm 2-aminonicotinic acid-grafted silica gel column provided in application example 1 with a commercially available 5 μm aminohydrophilic column (crude Chitosan oligosaccharide)
The crude product of chitosan oligosaccharide with unknown detailed components is separated by using a 10 mu m 2-aminonicotinic acid grafted silica gel chromatographic column and a commercially available 5 mu m amino hydrophilic column under the chromatographic conditions of acetonitrile/ammonia water solution (70/30), pH 10.24 of a mobile phase, flow rate of the mobile phase of 1.0mL/min, column temperature of 35 ℃ and detection wavelength of 254nm.
The separation results are shown in FIGS. 6 and 7, in which 10 μm 2-aminonicotinic acid grafted silica gel has fewer peaks in actual separation and better separation ability for basic sugars.
The applicant states that the present invention is illustrated by the above examples of the process of the present invention, but the present invention is not limited to the above process steps, i.e. it is not meant that the present invention must rely on the above process steps to be carried out. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (10)
1. An amino nicotinic acid mixed mode chromatographic stationary phase, which is characterized in that the amino nicotinic acid mixed mode chromatographic stationary phase has a structure shown as the following formula I:
wherein R is selected from any one of structures shown as formulas II-V below:
2. A method of preparing an amino nicotinic acid mixed mode chromatography stationary phase according to claim 1, comprising the steps of:
(1) Adding silica gel into an acid solution for acid washing to obtain activated silica gel;
(2) Carrying out coupling reaction on the activated silica gel obtained in the step (1) and an epoxy silane coupling agent to obtain epoxy coupling silica gel;
(3) And (3) carrying out ring-opening reaction on the epoxy coupling silica gel obtained in the step (2) and an amino nicotinic acid compound to obtain the amino nicotinic acid mixed mode chromatographic stationary phase.
3. The method according to claim 2, wherein in the step (1), the silica gel comprises spherical silica gel having a diameter of 3 to 10 μm;
preferably, in the step (1), the acidic solvent comprises 5-30% by volume of hydrochloric acid aqueous solution;
preferably, in the step (1), the mass ratio of the silica gel to the acidic solution is 1 (5-10).
4. The method according to claim 2 or 3, wherein in the step (1), the temperature of the acid washing is 90-120 ℃, and the time of the acid washing is 5-18h;
preferably, in step (1), after the acid washing, a post-treatment is further required, wherein the post-treatment comprises the following steps: washing the material obtained after acid washing to obtain a washed material; carrying out reduced pressure suction filtration on the material after washing to obtain a solid product, and carrying out vacuum drying on the solid product to obtain activated silica gel;
preferably, the pH of the material after water washing is 6.5-7.5;
preferably, the temperature of the vacuum drying is 100-150 ℃ and the time is 1.5-6h.
5. The method according to any one of claims 2 to 4, wherein in the step (2), the epoxy silane coupling agent comprises 3- (glycidyloxypropyl) triethoxysilane, and the 3- (glycidyloxypropyl) triethoxysilane has a structure represented by the following formula VI:
preferably, in step (2), the coupling reaction is carried out in a solvent comprising anhydrous toluene;
preferably, the mass ratio of the activated silica gel to the epoxy silane coupling agent is 1 (1-4);
preferably, the mass ratio of the activated silica gel to the solvent is 1 (5-15).
6. The process according to any one of claims 2 to 5, wherein in the step (2), the temperature of the coupling reaction is 110 to 140 ℃ and the time is 12 to 36 hours;
preferably, in step (2), the coupling reaction is further followed by a post-treatment, wherein the post-treatment comprises the following steps: washing the material obtained after the coupling reaction with an organic solvent, and then carrying out suction filtration to obtain a solid product; vacuum drying the solid product to obtain epoxy coupling silica gel;
preferably, the organic solvent comprises a mixed solvent of absolute ethyl alcohol and toluene;
preferably, the temperature of the vacuum drying is 100-150 ℃ and the time is 1.5-6h.
7. The method according to any one of claims 2 to 6, wherein in the step (3), the aminonicotinic acid compound comprises any one of 2-aminonicotinic acid, 2-aminoisonicotinic acid, 5-aminonicotinic acid and 6-aminonicotinic acid;
preferably, in the step (3), the mass ratio of the epoxy coupling silica gel to the amino nicotinic acid compound is (1.5-3): 1;
preferably, in the step (3), the ring-opening reaction is carried out in the presence of a catalyst, and the catalyst comprises an aqueous solution of acetic acid with a volume fraction of 0.5-1.5%;
preferably, the mass ratio of the epoxy coupling silica gel to the catalyst is (3-10): 1;
preferably, in step (3), the ring-opening reaction is carried out in a solvent comprising water;
preferably, the mass ratio of the epoxy coupling silica gel to the solvent is 1 (3-10).
8. The process according to any one of claims 2 to 6, wherein in the step (3), the ring-opening reaction is carried out at a temperature of 50 to 90 ℃ for 36 to 60 hours;
preferably, in step (3), after the ring-opening reaction, a post-treatment is required, and the post-treatment comprises the following steps: washing the material obtained after the ring-opening reaction to obtain a washed material; carrying out vacuum filtration on the material after water washing to obtain a solid product, and carrying out vacuum drying on the solid product to obtain the amino nicotinic acid mixed mode chromatographic stationary phase;
preferably, the temperature of the vacuum drying is 100-150 ℃, and the time is 1.5-6h.
9. The preparation method of any one of claims 2 to 8, wherein the preparation method of the amino nicotinic acid mixed mode chromatography stationary phase comprises the following steps:
(1) Adding silica gel into an acid solution for acid washing to obtain activated silica gel;
wherein the pickling temperature is 90-120 ℃, and the pickling time is 5-18h;
(2) Carrying out coupling reaction on the activated silica gel obtained in the step (1) and an epoxy silane coupling agent to obtain epoxy coupling silica gel;
wherein the epoxy silane coupling agent comprises 3- (glycidyl ether oxypropyl) triethoxysilane, and the coupling reaction is carried out at 110-140 ℃ for 12-36h;
(3) Performing ring-opening reaction on the epoxy coupling silica gel obtained in the step (2) and an amino nicotinic acid compound to obtain the amino nicotinic acid mixed mode chromatographic stationary phase;
wherein the amino nicotinic acid compound comprises any one of 2-amino nicotinic acid, 2-amino isonicotinic acid, 5-amino nicotinic acid or 6-amino nicotinic acid; the ring-opening reaction is carried out in the presence of a catalyst, and the catalyst comprises an acetic acid aqueous solution with the volume fraction of 0.5-1.5%; the temperature of the ring-opening reaction is 50-90 ℃, and the time is 36-60h.
10. Use of the amino nicotinic acid mixed-mode chromatography stationary phase according to claim 1 in liquid chromatography analysis.
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| WO2012020080A2 (en) * | 2010-08-12 | 2012-02-16 | Graffinity Pharmaceuticals Gmbh | LIGANDS FOR ANTIBODY AND Fc-FUSION PROTEIN PURIFICATION BY AFFINITY CHROMATOGRAPHY II |
| CN104324707A (en) * | 2014-09-16 | 2015-02-04 | 华东理工大学 | Amino diacid hydrophilic chromatographic stationary phase and preparation method thereof |
| CN108114707A (en) * | 2016-11-29 | 2018-06-05 | 中国科学院大连化学物理研究所 | A kind of silica matrix hydrophobic amino acid bonded stationary phase and its preparation and application |
| CN108906008A (en) * | 2018-07-19 | 2018-11-30 | 西南医科大学 | One kind multiple toothization amino polarity inline type mixed mode efficient liquid phase chromatographic stuffing and its preparation method and application |
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| WO2012020080A2 (en) * | 2010-08-12 | 2012-02-16 | Graffinity Pharmaceuticals Gmbh | LIGANDS FOR ANTIBODY AND Fc-FUSION PROTEIN PURIFICATION BY AFFINITY CHROMATOGRAPHY II |
| CN104324707A (en) * | 2014-09-16 | 2015-02-04 | 华东理工大学 | Amino diacid hydrophilic chromatographic stationary phase and preparation method thereof |
| CN108114707A (en) * | 2016-11-29 | 2018-06-05 | 中国科学院大连化学物理研究所 | A kind of silica matrix hydrophobic amino acid bonded stationary phase and its preparation and application |
| CN108906008A (en) * | 2018-07-19 | 2018-11-30 | 西南医科大学 | One kind multiple toothization amino polarity inline type mixed mode efficient liquid phase chromatographic stuffing and its preparation method and application |
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