CN109174048B - Silica gel chromatographic packing and preparation method thereof - Google Patents
Silica gel chromatographic packing and preparation method thereof Download PDFInfo
- Publication number
- CN109174048B CN109174048B CN201811339007.0A CN201811339007A CN109174048B CN 109174048 B CN109174048 B CN 109174048B CN 201811339007 A CN201811339007 A CN 201811339007A CN 109174048 B CN109174048 B CN 109174048B
- Authority
- CN
- China
- Prior art keywords
- silica gel
- coupling agent
- silane coupling
- packing
- chromatographic packing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- 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
- B01J20/30—Processes for preparing, regenerating, or reactivating
Abstract
The invention provides a silica gel chromatographic packing, which comprises: the silica gel comprises silica gel and an organosilane coupling agent bonded on the surface of the silica gel, wherein the organosilane coupling agent comprises a stereo silane coupling agent and other silane coupling agents. According to the silica gel chromatographic packing provided by the invention, the steric structural group with the steric hindrance effect is introduced, so that the hydrophobicity of the silica gel chromatographic packing is kept, the polarity of the surface of the packing is improved, and the polarity range of the analyzable compound is greatly improved; in addition, certain hydrophobicity is considered while the polarity is improved, so that the complex components can be well retained and separated in the silica gel chromatographic packing. The preparation method of the silica gel chromatographic packing has the advantages of simple method, good stability, good reproducibility and the like, and is suitable for large-scale production.
Description
Technical Field
The invention relates to a liquid chromatography packing material, in particular to a silica gel chromatography packing and a preparation method thereof.
Background
High Performance Liquid Chromatography (HPLC) is one of the fastest-developing separation technologies in the analytical chemistry field since the seventies of the twentieth century, and has become an indispensable means in the fields of biochemistry, medicine, pharmaceutical clinic, chemical industry, food, sanitation, environmental protection detection and the like through development and improvement for decades. The chromatographic column is of course central in the overall HPLC system. In the past 40 years, the development of HPLC is mainly attributed to two aspects of chromatographic instruments and chromatographic column packing technology, and research and development of various chromatographic packing with different properties are the basis for the wide application of HPLC methods. Currently, Reverse Phase Liquid Chromatography (RPLC) is the most widely used mode of chromatography in current separation analysis and separation preparation, relying on hydrophobic interactions between a hydrophobic stationary phase and a solute to achieve high efficiency separation of weakly polar and moderately polar compounds. However, RPLC has poor or no retention of strongly polar compounds (such as oligosaccharides, glycosides, and strongly polar oligopeptides), and thus strongly polar compounds cannot be separated well in reverse phase chromatography mode.
Hydrophilic interaction chromatography (HILIC), the concept of which was first proposed by Alpert in 1990, was used as a liquid chromatography model for the separation of polar compounds. The main feature of HILIC is the use of a polar stationary phase similar to normal phase chromatography and a mobile phase of aqueous organic solvent (usually acetonitrile) where water is a strong eluting solvent. Similar to normal phase chromatography, the retention time of a compound in the HILIC mode increases with increasing polarity of the compound. The HILIC mode chromatographic separation material is mainly prepared by bonding molecules containing strong polar groups such as amide groups, hydroxyl groups, zwitter ions and the like to silica gel.
Zwitterionic hydrophilic silica gel chromatography packing prepared by merck corporation
HILIC) has a better separation effect on strongly polar or hydrophilic ionic compounds (US20100300971), the filler being in siliconThe gel or polymer surface is bonded to a zwitterionic bonding phase resembling a sulfobetaine structure, and in this separation mode, the filler surface forms a water-rich liquid layer, and the analyte partitions between the mobile phase and the aqueous layer, and is retained and separated. However, the electrostatic interaction between the compound in the filler and the stationary phase is too strong, the compound is not easy to elute, high-concentration buffer salt is required to be adopted for adjustment, mass spectrum signals can be inhibited, the sensitivity is reduced, and the like; chinese patent (CN106669637A) also discloses a preparation method of a hydrophilic polymer filler, the chromatographic filler can be used for retaining and separating polar compounds such as saccharides, polypeptides, nucleosides and the like, but the preparation process steps of the chromatographic filler are complicated, and the filler needs to be prepared by using a complex reversible addition-fragmentation chain transfer polymerization (RAFT), thereby bringing great difficulty to the large-scale production of the filler. In addition, although according to the idea proposed by Alpert, the retention of the compound in the HILIC mode mainly depends on the distribution mechanism of the solute in the "water-rich layer" adsorbed on the surface of the stationary phase and the mobile phase, due to the existence of the direct interaction between the solute and the stationary phase, the retention properties of different stationary phases show larger difference, and meanwhile, the stability and reproducibility of the HILIC chromatographic mode are also worse than those of the traditional reversed-phase chromatographic mode, so that a series of problems such as unstable retention time, unsatisfactory peak shape, unstable peak area and the like occur in chromatographic separation.
In conclusion, a need exists for developing a novel silica gel chromatographic packing to solve the limitations of reversed phase chromatographic mode on retention of strong polar compounds and the problems of insufficient stability, unsatisfactory reproducibility and the like of HILIC chromatographic packing in the prior art.
It is noted that the information disclosed in the foregoing background section is only for enhancement of background understanding of the invention and therefore it may contain information that does not constitute prior art that is already known to a person of ordinary skill in the art.
Disclosure of Invention
The invention aims to provide a silica gel chromatographic packing and a preparation method thereof, and aims to solve the problems of poor retention and separation selectivity of a traditional reversed-phase chromatographic packing on polar compounds and a series of problems of unsatisfactory target peak type, unstable retention time, unstable peak area, high baseline noise and the like in an HILIC chromatographic mode.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a silica gel chromatographic packing, which comprises: silica gel and an organosilane coupling agent bonded on the surface of the silica gel, wherein the organosilane coupling agent comprises a stereo silane coupling agent and other silane coupling agents, and the general chemical structure of the stereo silane coupling agent is as follows:
wherein R is1Is hydrogen or alkyl with 1-2 carbon atoms; r2、R3、R4Independently selected from methyl, chloro and alkoxy, and R2、R3、R4At least one of which is chlorine or alkoxy.
According to one embodiment of the present invention, the other silane coupling agent has a chemical formula of: xn(Si)(CH3)3- nR5Wherein X is a hydrolyzable group, n is 1. ltoreq. n.ltoreq.3; r5Selected from monohaloalkyl groups containing 1 to 5 carbon atoms.
According to one embodiment of the invention, the further silane coupling agent is selected from chloromethyldimethylchlorosilane, bromomethyldimethylchlorosilane, chloroethyltrichlorosilane, chloroethylmethyldichlorosilane, bromoethyltrichlorosilane, 3-chloropropyldimethylchlorosilane, 3-chloropropyldimethylmethoxysilane, 3-chloropropyldimethylethoxysilane, 3-chloropropyltrichlorosilane, 3-chloropropylmethyldimethoxysilane, 3-chloropropylmethylethylmethoxysilane, 3-bromopropyltrichlorosilane, 3-bromopropyltrimethoxysilane, 4-chlorobutyldimethylchlorosilane, 4-bromobutyldimethylchlorosilane, 4-bromobutyldichlorosilane, 4-bromobutyldimethoxysilane, 5-bromopentyltrichlorosilane and 5-bromopentyltrimethoxysilane.
According to one embodiment of the invention, the stereo silane coupling agent is selected from the group consisting of bicyclo [2.2.1] hex-ethyltrichlorosilane, bicyclo [2.2.1] hex-ethyldimethylchlorosilane, 5- (bicycloheptyl) trichlorosilane, 5- (bicycloheptyl) methyldichlorosilane, 5- (bicycloheptyl) triethoxysilane, 5- (bicycloheptyl) methyldiethoxysilane, 5- (bicycloheptyl) dimethylethoxysilane, 2- (bicyclo [2.2.1] hept-5-en-2-ylethyl) trichlorosilane, 2- (bicyclo [2.2.1] hept-5-en-2-ylethyl) methyldichlorosilane, 2- (bicyclo [2.2.1] hept-5-en-2-ylethyl) dimethylchlorosilane and 2- (bicyclo [2.2.1] hept-5-en-2-ylethyl) trimethoxysilane One or more of alkanes.
The invention provides a method for preparing the silica gel chromatographic packing, which comprises the following steps:
activating and pretreating silica gel to obtain activated silica gel with silicon hydroxyl on the surface;
placing the activated silica gel in water and stirring to obtain hydrated silica gel;
and (3) placing the hydrated silica gel, the stereo silane coupling agent and other silane coupling agents into an organic solvent for mixing, and carrying out stirring reflux reaction to obtain the silica gel chromatographic packing.
According to one embodiment of the invention, the molar ratio of the silicon hydroxyl groups of the surface of the activated silica gel to the organosilane coupling agent is 1: (0.3-1.2), wherein the molar ratio of the other silane coupling agent to the stereo silane coupling agent is 1: (2-8).
According to one embodiment of the present invention, the ratio of the mass of the silica gel to the volume of the organic solvent is 1: (4-10) g/ml, wherein the organic solvent is a nonpolar solvent.
According to one embodiment of the invention, the stirring speed of the activated silica gel in water is 150-300 r/min, and the mass ratio of the activated silica gel to the water is 1: (0.005-0.1).
According to one embodiment of the invention, the silica gel has a particle size of 3 μm to 10 μm and a pore size of
Specific surface area of 180m2/g～400m2/g。
According to one embodiment of the invention, the stirring reflux reaction is carried out at 80-150 ℃ for 24-48 hours.
The invention has the beneficial effects that:
the silica gel chromatographic packing provided by the invention selects a stereo-silane coupling agent with a stereo structure and other silane coupling agents as bonding reaction reagents, and forms a stereo-structure group and a hydrophilic group with certain polarity on a silica gel substrate through a reaction mode of combining monofunctional group bonding and trifunctional group bonding. The silica gel chromatographic packing keeps hydrophobicity by introducing a three-dimensional structure group with steric hindrance effect, and simultaneously improves the polarity of the surface of the packing, so that the polarity range of an analyzable compound is greatly improved; in addition, certain hydrophobicity is considered while the polarity is improved, so that the complex components can be well retained and separated in the silica gel chromatographic packing. The preparation method of the silica gel chromatographic packing has the advantages of simple method, good stability, good reproducibility and the like, and is suitable for large-scale production.
Drawings
FIGS. 1a and 1b are schematic structural diagrams of a silica gel chromatographic packing according to an embodiment of the present invention;
FIG. 2 is a comparison chromatogram of the separation analysis of the retention effect of ribavirin by the silica gel chromatographic packing prepared in example 1 and the common silica gel chromatographic packing;
FIG. 3 is a comparison chromatogram of the retention effect of febuxostat and related substances in the separation analysis of the silica gel chromatographic packing prepared in example 2 and the common silica gel chromatographic packing;
FIG. 4 is a comparison chromatogram of the separation and analysis of 19 phenolic retention effects of silica gel chromatographic packing prepared in example 3 and common silica gel chromatographic packing.
Detailed Description
Exemplary embodiments that embody features and advantages of the present disclosure are described in detail below in the specification. It is to be understood that the disclosure is capable of various modifications in various embodiments without departing from the scope of the disclosure, and that the description and drawings are to be regarded as illustrative in nature, and not as restrictive.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to yield one or more new ranges of values, which ranges of values should be considered as specifically disclosed herein.
The invention provides a silica gel chromatographic packing, which comprises: silica gel and an organosilane coupling agent bonded on the surface of the silica gel, wherein the organosilane coupling agent comprises a stereo silane coupling agent and other silane coupling agents, and the general chemical structure of the stereo silane coupling agent is as follows:
wherein R is1Is hydrogen or alkyl with 1-2 carbon atoms; r2、R3、R4Independently selected from methyl, chloro and alkoxy, and R2、R3、R4At least one of which is chlorine or alkoxy.
Specifically, the "stereosilane coupling agent" has both a stereostructure (bridged cyclic hydrocarbon or norbornene structure) and a trifunctional group (R) as shown in the general formula2、R3、R4) The "other silane coupling agent" means other silane coupling agent than the stereo silane coupling agent.
The invention adopts a reaction mode of combining monofunctional group bonding and trifunctional group bonding by selecting a stereo silane coupling agent and other silane coupling agents as bonding reaction reagents to form chromatographic packing with a stereo structure group and a hydrophilic group with certain polarity on a silica gel substrate. The filler introduces a three-dimensional structure group with steric hindrance effect, so that the filler keeps hydrophobicity and simultaneously improves the polarity of the surface of the filler, breaks through the defects that the traditional chromatographic filler mainly based on the reversed-phase octadecyl group filler analyzes polar compounds in a reversed-phase chromatographic retention mode, and greatly improves the polarity range of the analyzable compounds; and meanwhile, the silica gel chromatographic packing material also overcomes the problems of unstable retention time, unsatisfactory peak pattern and the like and poor stability in a pure HILIC chromatographic retention mode.
In addition, the chemical modification carried out on the surface of the silica gel substrate does not need to carry out end-capping treatment like the preparation of the traditional reversed-phase chromatographic packing except that the stereo silane coupling agent and other silane coupling agents are mixed and bonded for modification.
In some embodiments, the other silane coupling agent has the general chemical formula: xn(Si)(CH3)3-nR5Wherein X is a hydrolyzable group, n is 1. ltoreq. n.ltoreq.3; monofunctional group R5Selected from monohaloalkyl groups containing 1 to 5 carbon atoms. As shown in fig. 1a and fig. 1b, which are respectively schematic structural diagrams of a silica gel chromatographic packing according to an embodiment of the present invention, wherein black microspheres represent a silica gel substrate, the fig. 1a and fig. 1b are only schematic diagrams of bonding of a stereo silane coupling agent and other silane coupling agents on the silica gel substrate, and are not used to limit the number and position relationship of bonding grafts. The chromatographic packing has a three-dimensional structure group and a halogenated alkane group simultaneously in structure, and can form various acting forces such as hydrophobic action, polar dipole action, hydrogen bond action and the like with a target object in a separation process, so that the chromatographic packing has more excellent separation selectivity compared with the traditional reversed-phase chromatographic packing.
In some embodiments, the other silane coupling agents include, but are not limited to, chloromethyldimethylchlorosilane, bromomethyldimethylchlorosilane, chloroethyltrichlorosilane, chloroethylmethyldichlorosilane, bromoethyltrichlorosilane, 3-chloropropyldimethylchlorosilane, 3-chloropropyldimethylmethoxysilane, 3-chloropropyldimethylethoxysilane, 3-chloropropyltrichlorosilane, 3-chloropropylmethyldimethoxysilane, 3-chloropropylmethylethylmethoxysilane, 3-bromopropyltrichlorosilane, 3-bromopropyltrimethoxysilane, 4-chlorobutyldimethylchlorosilane, 4-bromobutyldimethylchlorosilane, 4-bromobutyldichlorosilane, 4-bromobutyldimethoxysilane, 5-bromopentyltrichlorosilane and 5-bromopentyltrimethoxysilane.
In some embodiments, the stereo silane coupling agent includes, but is not limited to, bicyclo [2.2.1] hex-ethyltrichlorosilane, bicyclo [2.2.1] hex-ethyldimethylchlorosilane, 5- (bicycloheptyl) trichlorosilane, 5- (bicycloheptyl) methyldichlorosilane, 5- (bicycloheptyl) triethoxysilane, 5- (bicycloheptyl) methyldiethoxysilane, 5- (bicycloheptyl) dimethylethoxysilane, 2- (bicyclo [2.2.1] hept-5-en-2-ylethyl) trichlorosilane, 2- (bicyclo [2.2.1] hept-5-en-2-ylethyl) methyldichlorosilane, 2- (bicyclo [2.2.1] hept-5-en-2-ylethyl) dimethylchlorosilane, and 2- (bicyclo [2.2.1] hept-5-en-2-ylethyl) trimethoxysilane One or more of (a).
Through the mode of bonding and combining the monofunctional silane coupling agent and the trifunctional silane coupling agent, the problems of poor reproducibility, unsatisfactory stability and reproducibility, difficulty in large-scale production expansion and the like of chromatographic packing caused by pure trifunctional bonded silica gel can be greatly improved.
The invention also provides a preparation method of the silica gel chromatographic packing, which comprises the following steps:
s1: activating and pretreating silica gel to obtain activated silica gel with silicon hydroxyl on the surface;
s2: carrying out hydration treatment, namely placing the activated silica gel in water and stirring to obtain hydrated silica gel; the water is preferably ultrapure water.
S3: and (3) placing the hydrated silica gel, the stereo silane coupling agent and other silane coupling agents into an organic solvent for mixing, and carrying out stirring reflux reaction to obtain the silica gel chromatographic packing.
Specifically, the silica gel is preferably a fully porous spherical silica gel, and more preferably a silica gel for ultra-high purity fully porous spherical chromatography. Silicon as a starting material for activationThe particle size of the glue is preferably 3-10 μm, and the pore diameter is preferably
The specific surface area is preferably 180-400 m2/g。
In some embodiments, in step S1, the process of activating the silica gel pretreatment includes: adding a hydrofluoric acid aqueous solution with the mass concentration of 0.04-0.08% into the silica gel, and performing reflux reaction to obtain the activated silica gel; the ratio of the mass of the silica gel to the volume of the hydrofluoric acid aqueous solution is preferably 1: (8-10) g/ml. Preferably, the silica gel and the hydrofluoric acid aqueous solution react for 22-24 hours under the stirring, heating and refluxing mode, then the filtration is carried out under the reduced pressure suction filtration mode, the silica gel and the hydrofluoric acid aqueous solution are separated, the silica gel is cleaned by ultrapure water (secondary distilled water) to remove the residual hydrofluoric acid on the surface until the aqueous solution used for cleaning is neutral, and the activated silica gel is prepared by vacuum drying at 90-110 ℃ to constant weight.
In some embodiments, the molar ratio of silicon hydroxyl groups on the surface of the activated silica gel to the organosilane coupling agent is 1: (0.3-1.2), wherein the molar ratio of the other silane coupling agent to the stereo silane coupling agent is 1: (2-8). By controlling the other silane coupling agents and the stereo silane coupling agents which are bonded on the surface of the silica gel within the range, the filler can be ensured to have better hydrophilicity and certain hydrophobicity and polarity.
In some embodiments, the ratio of the mass of the silica gel to the volume of the organic solvent is 1: (4-10) g/ml, wherein the organic solvent is a nonpolar solvent. The non-polar solvent includes, but is not limited to, toluene, xylene, n-hexane, or n-heptane.
In some embodiments, in step S2, the activated silica gel is placed in water and stirred at a stirring rate of 150 rpm to 300 rpm, and the mass ratio of the activated silica gel to the water is 1: (0.005-0.1). The traditional process for preparing the hydrated silica gel is generally prepared by a method of introducing nitrogen with the relative humidity of 20-80%, and the method has the problems of complicated operation steps, labor waste, time waste and the like. The hydrated silica gel of the invention is prepared by directly adding ultrapure water into silica gel reaction raw materials and simply stirring, so that the activated hydrated silica gel can be obtained, the operation method is simpler and more convenient, and the hydration effect is better.
In some embodiments, in step S3, the stirring and refluxing are performed at a temperature of 80 ℃ to 150 ℃ for about 24 to 48 hours.
In some embodiments, the preparation method of the silica gel chromatographic packing further comprises filtering a product after the reflux reaction is completed after the reflux reaction is performed, and sequentially washing the product with toluene, tetrahydrofuran/water, methanol and acetonitrile to prepare the silica gel chromatographic packing. Specifically, the solution in the vessel after the completion of the reflux reaction was cooled to a temperature below the reflux temperature of the organic solvent, filtered under reduced pressure, and then washed with toluene, tetrahydrofuran/water, methanol, and acetonitrile in this order. Preferably, the obtained solid product is dried in vacuum at the temperature of 90-200 ℃ for more than 12 hours, and then the filler is prepared. Further preferably, the volume ratio of tetrahydrofuran/water may be 8: 2. the temperature of the toluene, tetrahydrofuran/water, methanol and acetonitrile is 40-80 ℃.
The silica gel chromatography packing of the present invention is further illustrated below with reference to specific examples:
example 1
(1) To a 2L glass reaction vessel, 100g of silica gel (5 μm, available from DAISO Co., Japan) was added, 1000mL of distilled water was added, and the mixture was stirred for 30 minutes, filtered, and the procedure was repeated twice. Then adding 1000mL of hydrofluoric acid aqueous solution with the mass ratio of 0.08%, stirring for 24 hours, cleaning with distilled water to be neutral, finally adding 1000mL of acetone for washing, filtering, and drying at 110 ℃ for 12 hours to obtain activated silica gel;
(2) 100g (5 μm,
specific surface area: 300m2(g) in a 2L glass reaction vessel; adding 5g of ultrapure water into the reaction vessel, and stirring at 170 revolutions per minute for more than 30 minutes to obtain hydrated silica gel;
(3) adding the hydrated silica gel prepared in the step (2) into a glass material reaction container, adding 800mL of dry anhydrous toluene solvent, uniformly stirring at 170 r/min, adding a mixture containing two silane reagents of 44.08g of bicyclo [2.2.1] hexane-ethyl trichlorosilane and 6.87g of chloromethyl dimethylchlorosilane, heating to 110 ℃ by using an electric heating sleeve, and carrying out stirring reflux reaction for 24 hours;
(4) after the reaction is finished, the electric heating sleeve is turned off, the glass material reaction vessel is cooled to about 60 ℃ when the temperature of the solvent is reduced, a decompression filter funnel is used for filtering, then toluene, tetrahydrofuran/water solution (v/v ═ 8/2), methanol and acetonitrile are sequentially used for washing, and the product is dried in vacuum at 110 ℃ for 12 hours to obtain the silica gel chromatographic packing.
Example 2
(1) To a 2L glass reaction vessel, 100g of silica gel (5 μm, available from DAISO Co., Japan) was added, 1000mL of distilled water was added, and the mixture was stirred for 30 minutes, filtered, and the procedure was repeated twice. Then adding 900mL of hydrofluoric acid aqueous solution with the mass ratio of 0.06%, stirring for 24 hours, cleaning with distilled water to be neutral, finally adding 1000mL of acetone for washing, filtering, and drying at 110 ℃ for 12 hours to obtain activated silica gel;
(2) 100g (5 μm,
specific surface area: 300m2Per gram) in a 2L reaction vessel; adding 8g of ultrapure water into a glass reaction vessel, and stirring at 250 revolutions per minute for 30 minutes to obtain hydrated silica gel;
(3) adding the hydrated silica gel prepared in the step (2) into a glass material reaction container, adding 800mL of dry anhydrous toluene solvent, uniformly stirring at 170 r/min, adding a mixture containing two silane reagents of 44.08g of bicyclo [2.2.1] hexane-ethyl trichlorosilane and 8.52g of chloroethyl methyl dichlorosilane, heating to 110 ℃ by using an electric heating sleeve, and carrying out stirring reflux reaction for 24 hours;
(4) after the reaction is finished, turning off the electric heating sleeve, cooling the solvent in the glass material reaction vessel to 60 ℃, filtering by using a decompression filter funnel, and washing twice by using toluene respectively; washing with tetrahydrofuran/water solution (v/v-8/2), methanol and acetonitrile, and vacuum drying at 110 deg.C for 12 hr to obtain silica gel chromatographic packing.
Example 3
(1) To a 2L glass reaction vessel, 100g of silica gel (5 μm, available from DAISO Co., Japan) was added, 1000mL of distilled water was added, and the mixture was stirred for 30 minutes, filtered, and the procedure was repeated twice. Then adding 800mL of hydrofluoric acid aqueous solution with the mass ratio of 0.04%, stirring for 24 hours, cleaning with distilled water to be neutral, finally adding 1000mL of acetone for washing, filtering, and drying at 110 ℃ for 12 hours to obtain activated silica gel;
(2) 100g (5 μm,
specific surface area: 300m2(g) in a 2L glass reaction vessel; adding 8g of ultrapure water into the reaction vessel, and stirring at 250 revolutions per minute for 30 minutes to obtain hydrated silica gel;
(3) adding the hydrated silica gel prepared in the step (2) into a glass reaction vessel, adding 800mL of dry anhydrous toluene solvent, uniformly stirring at 170 r/min, adding a mixture containing two silane reagents of 43.70g of 5- (bicycloheptyl) trichlorosilane and 8.21g of 3-chloropropyldimethylchlorosilane, heating to 110 ℃ by using an electric heating sleeve, and stirring and refluxing for 24 hours:
(4) after the reaction is finished, turning off the electric heating sleeve, cooling the solvent in the glass material reaction vessel to 60 ℃, filtering by using a decompression filter funnel, and washing twice by using toluene respectively; washing with tetrahydrofuran/water solution (v/v-8/2), methanol and acetonitrile, and vacuum drying at 110 deg.C for 12 hr to obtain silica gel chromatographic packing.
Example 4
(1) To a 2L glass reaction vessel, 100g of silica gel (5 μm, available from DAISO Co., Japan) was added, 1000mL of distilled water was added, and the mixture was stirred for 30 minutes, filtered, and the procedure was repeated twice. Then adding 800mL of hydrofluoric acid aqueous solution with the mass ratio of 0.08%, stirring for 24 hours, then cleaning with distilled water to be neutral, finally adding 1000mL of acetone for washing, filtering, and drying at 110 ℃ for 12 hours to obtain activated silica gel;
(2) 100g (5 μm,
specific surface area: 300m2(g) in a 2L glass reaction vessel; adding 10g of ultrapure water into the reaction vessel, and stirring at 300 revolutions per minute for more than 30 minutes to obtain hydrated silica gel;
(3) adding the hydrated silica gel prepared in the step (2) into a glass material reaction container, adding 800mL of dry anhydrous toluene solvent, stirring uniformly, adding 49.08g of 2- (bicyclo [2.2.1] hept-5-en-2-yl ethyl) dimethylchlorosilane and 12.31g of 3-bromopropyltrichlorosilane, mixing, adding into a reactor, heating to 110 ℃ by using an electric heating sleeve, and stirring and refluxing for 24 hours;
(4) after the reaction is finished, turning off the electric heating sleeve, cooling the solvent in the glass material reaction vessel to about 60 ℃, filtering by using a decompression filter funnel, and washing twice by using toluene respectively; washing tetrahydrofuran/water solution, methanol and acetonitrile, and vacuum drying the product at 110 deg.c for 12 hr to obtain silica gel chromatographic stuffing.
Example 5
(1) To a 2L glass reaction vessel, 100g of silica gel (5 μm, available from DAISO Co., Japan) was added, 1000mL of distilled water was added, and the mixture was stirred for 30 minutes, filtered, and the procedure was repeated twice. Then adding 800mL of hydrofluoric acid aqueous solution with the mass ratio of 0.06%, stirring for 24 hours, cleaning with distilled water to be neutral, finally adding 1000mL of acetone for washing, filtering, and drying at 110 ℃ for 12 hours to obtain activated silica gel;
(2) 100g (5 μm,
specific surface area: 300m2(g) in a 2L glass reaction vessel; adding 10g of ultrapure water into the reaction vessel, and stirring at 300 revolutions per minute for more than 30 minutes to obtain hydrated silica gel;
(3) adding the hydrated silica gel prepared in the step (2) into a reaction vessel, adding 800mL of dry anhydrous toluene solvent, uniformly stirring at 170 revolutions per minute, adding a mixture containing two silane reagents of 49.08g of 2- (bicyclo [2.2.1] hept-5-en-2-yl ethyl) methyldichlorosilane and 13.02g of 5-bromopentyltrimethoxysilane, heating to 110 ℃ by using an electric heating sleeve, and carrying out stirring reflux reaction for 24 hours;
(4) after the reaction is finished, turning off the electric heating sleeve, cooling the solvent in the glass material reaction vessel to 60 ℃, filtering by using a decompression filter funnel, and washing twice by using toluene respectively; washing tetrahydrofuran/water solution, methanol and acetonitrile, and vacuum drying the product at 110 deg.c for 12 hr to obtain silica gel chromatographic stuffing.
Test example 1
The silica gel chromatography packing prepared in example 1 was packed into a column having a length of 250mm and an inner diameter of 4.6 mm. The column packing conditions were as follows: the column loading pressure is 3000-7000psi, and the homogenizing agent: chloroform and tetrahydrofuran in the material-liquid ratio of 1 to 10, and finally replacing with methanol for 30 min. The methanol-water solution is used as a separated mobile phase, the flow rate of the mobile phase is 1.0mL/min, the detection temperature is 35 ℃, the ultraviolet detection wavelength is 207nm, the separated object is a strong polar compound ribavirin, and the LogP value of the ribavirin is predicted to be-2.26.
FIG. 2 is a comparison chromatogram of the separation analysis of the silica gel chromatographic packing prepared in example 1 and a common silica gel chromatographic packing for the retention effect of ribavirin. The abscissa is retention time in minutes (min), and the ordinate is response value of detection signal in μ V (microvolts). Wherein the common silica gel chromatographic packing is traditional reversed-phase octadecyl bonded silica gel chromatographic packing (obtained by directly carrying out bonding reaction on octadecyl trichlorosilane and silicon hydroxyl of silica gel).
As shown in fig. 2, 2a represents the sample chromatographic effect of the silica gel chromatographic packing prepared in example 1, and 2a represents the sample chromatographic effect of the general silica gel chromatographic packing. The chromatographic column packed by the silica gel chromatographic packing of the embodiment 1 is used for separating ribavirin, the retention time of the ribavirin is 7.3 minutes, the column efficiency is 12576 theoretical plate number, and the tailing factor is 0.98, which shows that the chromatographic packing has excellent retention capacity, higher column efficiency and good peak type (as shown in 2 a) on the ribavirin and can separate in a shorter time; whereas under the exact same mobile phase test conditions, it is difficult for a conventional C18 column to effectively retain ribavirin, which elutes from the column in a relatively short period of time (as shown in FIG. 2 b).
Test example 2
The silica gel column packing obtained in example 2 was packed to have a column length of 250mm and a column inner diameter of 4.6 mm. The column packing conditions were as follows: column pressure 3000 + 7000psi, homogenate: chloroform and isopropanol with a feed-liquid ratio of 1:10, and finally replacing with methanol for 30 min. Acetonitrile-10 mM potassium dihydrogen phosphate (pH 2.5) buffer solution is used as a separated mobile phase, the flow rate of the mobile phase is 1.0mL/min, the detection temperature is 25 ℃, the ultraviolet detection wavelength is 220nm, and the separated objects are febuxostat and related substances thereof.
FIG. 3 is a comparison chromatogram of the separation and analysis of the retention effects of febuxostat and related substances by using the silica gel chromatographic packing prepared in example 2 and a common silica gel chromatographic packing. The abscissa is retention time in min, and the ordinate is the response value of the detection signal in μ V (microvolts). Wherein the common silica gel chromatographic packing is traditional reversed-phase octadecyl bonded silica gel chromatographic packing (obtained by directly carrying out bonding reaction on octadecyl trichlorosilane and silicon hydroxyl of silica gel).
As shown in fig. 3, 3a represents the sample chromatographic effect of the silica gel chromatographic packing prepared in example 2, and 3b represents the sample chromatographic effect of the general silica gel chromatographic packing. By adopting the chromatographic column packed by the silica gel chromatographic packing of the embodiment 2 of the invention, the febuxostat main peak and the related impurity B peak can obtain better separation effect, the separation degree is 1.96, the impurity B peak is before, and the febuxostat main peak is after (as shown in 3 a); and a chromatographic column filled with common silica gel chromatographic packing is adopted, the separation degree of the febuxostat main peak and the related impurity B peak is 1.57, the febuxostat main peak is in front of the main peak, and the impurity B peak is behind the main peak (shown as 3B). The above results fully demonstrate that the separation selectivity of the silica gel chromatography packing of the present invention is superior to that of the traditional reversed-phase octadecyl bonded silica gel chromatography packing.
Test example 3
The isolated basic compound obtained in example 3 was packed in a column with a silica gel packing, the column length was 250mm, and the column inner diameter was 4.6 mm. The column packing conditions were as follows: column pressure 3000 + 7000psi, homogenate: carbon tetrachloride and isopropanol in a feed-liquid ratio of 1:10, and finally replacing with methanol for 30 min. Acetonitrile-0.2% phosphoric acid water solution is used as a separated mobile phase, the flow rate of the mobile phase is 1.0mL/min, the detection temperature is 30 ℃, the ultraviolet detection wavelength is 280nm, the separated objects are 19 phenolic substances, and the substances from No. 1 peak to No. 19 peak are respectively: gallic acid, protocatechuic acid, gentisic acid, chlorogenic acid, vanillic acid, epicatechin, caffeic acid, syringic acid, benzoic acid, scopoletin, ferulic acid, rutin, hyperoside, isoquercitrin, astragalin, quercitrin, quinoa alcohol, quercetin and kaempferide.
FIG. 4 is a comparison chromatogram of the separation and analysis of 19 phenolic retention effects of silica gel chromatographic packing prepared in example 3 and common silica gel chromatographic packing. The abscissa is retention time in min, and the ordinate is the response value of the detection signal in μ V (microvolts). Wherein the common silica gel chromatographic packing is traditional reversed-phase octadecyl bonded silica gel chromatographic packing (obtained by directly carrying out bonding reaction on octadecyl trichlorosilane and silicon hydroxyl of silica gel).
As shown in fig. 4, 4a represents the sample chromatographic effect of the silica gel chromatographic packing prepared in example 3, and 4b represents the sample chromatographic effect of the general silica gel chromatographic packing. With the column packed with silica gel chromatography packing of example 3 of the present invention, 19 phenolic compounds were able to achieve substantially better separation, with only the separation degrees of peak 6 and peak 7 being slightly lower than 1.12 (as shown in 4 a). While the separation degree of the No. 3 peak and the No. 4 peak is 1.29 by adopting the chromatographic column filled with the common silica gel chromatographic packing, the separation degree of the No. 14 peak and the No. 15 peak is only 0.73 (as shown in 4 b), the separation degree is obviously not the same as the separation effect of the silica gel chromatographic packing prepared in the example 3, and the chromatographic column can not elute the No. 19 chromatographic peak. The above results further fully demonstrate that the separation selectivity of the silica gel chromatography packing of the present invention is superior to that of the traditional reversed-phase octadecyl bonded silica gel chromatography packing.
Therefore, the stability of the filler is effectively improved by introducing a three-dimensional structure group with a steric hindrance effect and a polar group with various acting forces such as a hydrophilic action, a dipole-dipole action, an electrostatic attraction action and the like, so that the polar range of the analyzable compound is greatly improved; and moreover, certain hydrophobicity is considered while the polarity is improved, so that the complex components from the strong polar compound to the hydrophobic compound can be well retained and separated while the silica gel chromatographic packing is bonded with the three-dimensional structure group.
Unless otherwise defined, all terms used herein have the meanings commonly understood by those skilled in the art. It should be noted by those skilled in the art that the described embodiments of the present invention are merely exemplary and that various other substitutions, alterations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the above-described embodiments, but is only limited by the claims.
Claims (9)
1. A silica gel chromatography packing comprising: silica gel and bonded in the organosilane coupling agent on the silica gel surface, the organosilane coupling agent includes stereo silane coupling agent and other silane coupling agent, wherein: the chemical structural general formula of the stereo silane coupling agent is as follows:
wherein R is1Is hydrogen or alkyl with 1-2 carbon atoms; r2、R3、R4Independently selected from methyl, chloro and alkoxy, and R2、R3、R4At least one of which is chlorine or alkoxy;
the chemical formula of the other silane coupling agent is as follows: xn(Si)(CH3)3-nR5Wherein X is a hydrolyzable group, n is 1. ltoreq. n.ltoreq.3; r5Selected from monohaloalkyl groups containing 1 to 5 carbon atoms.
2. The silica gel chromatography packing material of claim 1, the other silane coupling agent is selected from one or more of chloromethyldimethylchlorosilane, bromomethyldimethylchlorosilane, chloroethyltrichlorosilane, chloroethylmethyldichlorosilane, bromoethyltrichlorosilane, 3-chloropropyldimethylchlorosilane, 3-chloropropyldimethylmethoxysilane, 3-chloropropyldimethylethoxysilane, 3-chloropropyltrichlorosilane, 3-chloropropylmethyldimethoxysilane, 3-bromopropyltrichlorosilane, 3-bromopropyltrimethoxysilane, 4-chlorobutyldimethylchlorosilane, 4-bromobutyldimethylchlorosilane, 4-bromobutyltrimethoxysilane, 5-bromopentyltrichlorosilane and 5-bromopentyltrimethoxysilane.
3. The silica gel chromatographic filler according to claim 1, wherein the stereo silane coupling agent is selected from the group consisting of bicyclo [2.2.1] hex-ethyltrichlorosilane, bicyclo [2.2.1] hex-ethyldimethylchlorosilane, 5- (bicycloheptyl) trichlorosilane, 5- (bicycloheptyl) methyldichlorosilane, 5- (bicycloheptyl) triethoxysilane, 5- (bicycloheptyl) methyldiethoxysilane, 5- (bicycloheptyl) dimethylethoxysilane, 2- (bicyclo [2.2.1] hept-5-en-2-ylethyl) trichlorosilane, 2- (bicyclo [2.2.1] hept-5-en-2-ylethyl) methyldichlorosilane, 2- (bicyclo [2.2.1] hept-5-en-2-ylethyl) dimethylchlorosilane, and 2- (bicyclo [2.2.1] hept-5-en-2-yl) 2-ethylchlorosilane -methylethyl) trimethoxysilane.
4. A process for preparing a silica gel chromatography packing material as claimed in any one of claims 1 to 3, comprising the steps of:
activating and pretreating silica gel to obtain activated silica gel with silicon hydroxyl on the surface;
placing the activated silica gel in water and stirring to obtain hydrated silica gel;
and (3) placing the hydrated silica gel, the stereo silane coupling agent and other silane coupling agents into an organic solvent for mixing, and carrying out stirring reflux reaction to obtain the silica gel chromatographic packing.
5. The method according to claim 4, wherein the molar ratio of the silicon hydroxyl groups on the surface of the activated silica gel to the organosilane coupling agent is 1: (0.3-1.2), wherein the molar ratio of the other silane coupling agent to the stereo silane coupling agent is 1: (2-8).
6. The method according to claim 4, wherein the ratio of the mass of the silica gel to the volume of the organic solvent is 1: (4-10) g/ml, wherein the organic solvent is a nonpolar solvent.
7. The method as claimed in claim 4, wherein the stirring rate of the activated silica gel in water is 150-300 r/min, and the mass ratio of the activated silica gel to the water is 1: (0.005-0.1).
8. The method according to claim 4, wherein the silica gel has a particle size of 3 to 10 μm and a pore size of
The specific surface area is 180m2/g～400m2/g。
9. The method according to claim 4, wherein the stirring reflux reaction is carried out at 80 to 150 ℃ for 24 to 48 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811339007.0A CN109174048B (en) | 2018-11-12 | 2018-11-12 | Silica gel chromatographic packing and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811339007.0A CN109174048B (en) | 2018-11-12 | 2018-11-12 | Silica gel chromatographic packing and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109174048A CN109174048A (en) | 2019-01-11 |
| CN109174048B true CN109174048B (en) | 2021-12-28 |
Family
ID=64938836
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811339007.0A Active CN109174048B (en) | 2018-11-12 | 2018-11-12 | Silica gel chromatographic packing and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109174048B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113318714A (en) * | 2021-05-26 | 2021-08-31 | 浙江月旭材料科技有限公司 | Monodisperse silicon dioxide microsphere for chromatography and preparation method and application thereof |
| CN113786817B (en) * | 2021-08-31 | 2022-06-14 | 浙江月旭材料科技有限公司 | Modified silica gel material and preparation method and application thereof |
| CN114405489B (en) * | 2022-01-12 | 2022-08-09 | 浙江月旭材料科技有限公司 | Filler for trapping liquid chromatography impurities and preparation method and application thereof |
| CN115646473A (en) * | 2022-10-27 | 2023-01-31 | 青岛邦凯高新技术材料有限公司 | Electronic-grade silicon dioxide base material chromatographic filler, and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101218290A (en) * | 2005-05-26 | 2008-07-09 | 罗狄亚化学公司 | Use of a combination of two types of different coupling agents as coupling system (white filler-elastomer) in rubber compositions comprising an inorganic filler |
| CN105080492A (en) * | 2015-08-20 | 2015-11-25 | 浙江月旭材料科技有限公司 | Silica gel chromatographic packing for separation of alkaline compound and preparation method of silica gel chromatographic packing |
| CN108176387A (en) * | 2018-03-08 | 2018-06-19 | 浙江月旭材料科技有限公司 | A kind of silica gel chromatographic column filling material, preparation method and applications |
-
2018
- 2018-11-12 CN CN201811339007.0A patent/CN109174048B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101218290A (en) * | 2005-05-26 | 2008-07-09 | 罗狄亚化学公司 | Use of a combination of two types of different coupling agents as coupling system (white filler-elastomer) in rubber compositions comprising an inorganic filler |
| CN105080492A (en) * | 2015-08-20 | 2015-11-25 | 浙江月旭材料科技有限公司 | Silica gel chromatographic packing for separation of alkaline compound and preparation method of silica gel chromatographic packing |
| CN108176387A (en) * | 2018-03-08 | 2018-06-19 | 浙江月旭材料科技有限公司 | A kind of silica gel chromatographic column filling material, preparation method and applications |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109174048A (en) | 2019-01-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109174048B (en) | Silica gel chromatographic packing and preparation method thereof | |
| JP6401151B2 (en) | Chromatographic material | |
| AU2005215377B2 (en) | Polar-modified bonded phase materials for chromatographic separations | |
| JP6181014B2 (en) | Novel organosilane, substrate covalently bonded to the organosilane, and methods of synthesis and use | |
| JP7005515B2 (en) | Analytical method for glycans modified with amphipathic strong bases using charged surface reverse phase chromatography material | |
| US4847159A (en) | Substrates coated with organo-silanes that are sterically-protected | |
| JP6768790B2 (en) | High-purity chromatography material containing ion-paired phase for supercritical fluid chromatography | |
| JP6557306B2 (en) | Apparatus and method for performing size exclusion chromatography | |
| Calleri et al. | Evaluation of a monolithic epoxy silica support for penicillin G acylase immobilization | |
| US20140116932A1 (en) | Novel Liquid Chromatographic Media and Methods of Synthesizing the Same | |
| AU751568B2 (en) | Recovery of organic solutes from aqueous solutions | |
| Yasmin et al. | Synthesis and characterization of surface modified SBA-15 silica materials and their application in chromatography | |
| WO2011012019A1 (en) | Separation material based on silica gel having copolymerization reaction on surface and preparation method thereof | |
| JP2007523331A (en) | Porous hybrid monolithic material with organic groups removed from the surface | |
| US9925515B1 (en) | Alkyl functionalized porous silica liquid chromatographic stationary phases and solid phase extraction sorbents | |
| Chen et al. | Preparation and application of covalently bonded polysaccharide-modified stationary phase for per aqueous liquid chromatography | |
| CA2846685A1 (en) | Sorbent comprising on its surface a cationic or protonizable aliphatic residue for the purification of organic molecules | |
| Bocian et al. | Synthesis and characterization of phosphodiester stationary bonded phases for liquid chromatography | |
| Hsieh et al. | Single‐step approach to β‐cyclodextrin‐bonded silica as monolithic stationary phases for CEC | |
| EP1496356A1 (en) | Optical isomer separation packing for simulated moving bed chromatography | |
| JP2010052986A (en) | Surface-treated silica and method for producing the same | |
| CN111250069B (en) | Silica gel chromatographic packing and preparation method and application thereof | |
| JP4700171B2 (en) | Method for producing hydrophilic ODS filler for liquid chromatography | |
| JPH06211902A (en) | Substituted aromatic carbamate derivative of polysaccharide, and separating agent | |
| CN112755985B (en) | Liquid chromatographic separation medium and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20210601 Address after: 1001b, area a, building 1, 3000 Longdong Avenue, China (Shanghai) pilot Free Trade Zone, Pudong New Area, Shanghai 203201 Applicant after: WELCH MATERIALS (SHANGHAI) Inc. Applicant after: ZHEJIANG YUEXU MATERIAL TECHNOLOGY Co.,Ltd. Applicant after: Shanghai wellrich Testing Technology Co.,Ltd. Address before: 1001b, area a, building 1, 3000 Longdong Avenue, China (Shanghai) pilot Free Trade Zone, Pudong New Area, Shanghai 203201 Applicant before: WELCH MATERIALS (SHANGHAI) Inc. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |