CN103357390A - Multi-layer structure bonded silica gel liquid chromatography packing and synthesis method thereof - Google Patents
Multi-layer structure bonded silica gel liquid chromatography packing and synthesis method thereof Download PDFInfo
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- CN103357390A CN103357390A CN2012104469739A CN201210446973A CN103357390A CN 103357390 A CN103357390 A CN 103357390A CN 2012104469739 A CN2012104469739 A CN 2012104469739A CN 201210446973 A CN201210446973 A CN 201210446973A CN 103357390 A CN103357390 A CN 103357390A
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Abstract
The invention discloses a type of novel multi-layer structure bonded silica gel liquid chromatography packing and a synthesis method thereof. The method comprises the following steps of: performing surface alkylation treatment on silica gel matrix particles by utilizing an alkylsilane reagent; reacting silica gel matrix alkylation packing and polar fluorophenyl silane; finally, further reacting the prepared dried silica gel packing with a silane blocking agent to prepare a type of novel high performance liquid chromatography stationary phases with high inertness and excellent chromatographic separation performance. Because a novel combination of polar fluorophenyl and alkyl functional groups serves as a silica gel surface bonded phase, the selectivity, separation degree and reproducibility higher than those in the traditional C18 chromatographic column are obtained. The bonded phase has hydrophobic interaction, dipole-dipole interaction and pi-pi interaction and forms a hydrogen bond and other multi-action mechanisms, and the prepared chromatography packing can be effectively used for separation analysis of acidic, neutral and alkaline compounds under proper chromatographic conditions.
Description
Technical field
The present invention relates to a kind of efficient liquid phase chromatographic stuffing and preparation method thereof, be particularly related to the alkyl and the polarity difluorophenyl that adopt the preparation of multilayer bonding techniques and make up bonded silica gel as efficient liquid phase chromatographic stuffing and preparation method thereof, belong to high performance liquid chromatography packing material preparing technical field.This chromatograph packing material can be used for positive, anti-phase and three kinds of clastotypes of hydrophilic Interaction Chromatography, is mainly used in the compartment analysis of multicomponent organic compound in organic synthesis, food, environment and the pharmaceutical industry.
Background technology
Liquid chromatogram is a kind of widely used analysis chromatographic technique, and its basic principle is to come component in the separating mixture by mobile phase and fixing separating effect mutually, more specifically says the lewis' acid of separate dissolved in solvent.Chromatographic column is used for filling fixedly phase, and mobile phase carries the mixture fixedly phase of flowing through.In the sample with fixing have mutually than the component of strong affinity fixing mutually in the time of staying longer, the component weak with fixing phase affinity then flows out chromatographic column quickly, so just reached the effect of separation.
It is the research contents that the chromatogram worker pays close attention to all the time that high performance liquid chromatography separates detection compound of different nature simultaneously, and the chromatographic stationary phases material that preparation has the multiple mechanism of action is the key that realizes this separation.People once attempted ining all sorts of ways to realize having hydrophobic effect, and ion-exchange is analyzed in the time of the acidity of the multiple action mechanism such as p-p effect and hydrogen bond action, neutrality, alkalescence and ionic compound.As chromatographic column of different nature is carried out parallel connection, the post switching method of series connection or pre-stop etc. realizes the separation of heterogeneity compound; Be filled in to make in the same root chromatogram column when mixed bed is fixing to carry out acidic, neutral and basic compounds mutually after two kinds of fillers of different performance are mixed and separate; Another kind method is to coat the material with suitable multi-functional base at fixing phase surface, separates when realizing the heterogeneity compound.All these methods all can not fundamentally solve the problem that the heterogeneity compound separates simultaneously.
At Pharmaceutical Analysis and purifying, metabolite analysis, in the analysis of food, environmental analysis and pesticide residue, the chromatography of polar compound is a difficult problem in the analytical test field always.For easier Ionized organic acid or organic base, generally be in mobile phase, to add ion-pairing agent, to reach the purpose of separation.But there are many shortcomings that self can't overcome in this method, and for example system is complicated, the method poor reproducibility, and equilibration time is long etc.Along with the development trend of current LC-MS, the ion pair method is difficult to the online use at LC-MS.Thereby people begin to research and develop the reverse-phase chromatographic column that can be complementary with 100% aqueous solution and the forward chromatographic column that is complementary mutually with high water flow, so that the scope of application of positive and reverse-phase chromatographic column is expanded, thereby can be to hydrophily under the condition of not using ion-pairing agent, strong polar compound carries out liquid-phase chromatographic analysis.Yet this two classes chromatographic column of using at present all has stability and poor reproducibility, the shortcomings such as separating mechanism complexity.
Along with developing rapidly of the research fields such as proteomics, metabolism group, the modernization of Chinese medicine, environmental protection; strong polarity and hydrophilic small-molecule substance become rapidly the important research object of analytical chemistry and biochemical field, obtain effective separation but this class material is often difficult in liquid chromatogram.Traditional reverse-phase chromatography to the reservation of these materials and separating power a little less than, because the semipolar functional group of this class sample molecule is easy to form the dipole moment effect with mobile phase, namely by " solvation ", and with the nonpolar fixing this effect that lacks mutually, cause sample to rest in the mobile phase and can't with fixing mutually effect, therefore usually flow out and can't obtain separating in the dead time.For the polar molecule with one or more ionogenic functional groups, no doubt can adopt ion-exchange chromatography or ion pair chromatogram to replace reverse-phase chromatography to carry out compartment analysis, but these two kinds of clastotypes because of with the compatible relatively poor limitation that causes its application in the complex sample analysis of mass detector.The normal-phase chromatography technology can reach in the fixedly phase of strong polarity and distribution between the stronger eluant, eluent of polarity by sample separates purpose, but in application process, normal-phase chromatography has some shortcomings that are difficult to avoid, easily protract or trail such as the sample peak shape, retention time is drifted about with sample size, and minor amount of water exists appreciable impact reservation etc. in the mobile phase.The more important thing is that for the strong sample of most of hydrophilies, the mobile phase of normal-phase chromatography can't provide enough solubility, these problems all cause its limitation in actual applications.Hydrophilic Interaction Chromatography (HILIC) can be used as the important supplement of positive, reversed-phase liquid chromatography as a kind of liquid chromatogram pattern of separating polar compound, solves the separation problem of polar compound, thereby becomes one of focus of in recent years chromatographic field research.Hydrophilic Interaction Chromatography has and the similar retention behavior of normal-phase chromatography, can provide suitable reservation for the testing sample of strong polarity; It adopts water-water-soluble organic phase as mobile phase, can significantly improve the solubility of sample in mobile phase again.In addition, it is embodying and reverse-phase chromatography quadrature while optionally, have the post effect and the symmetrical peak shape that compare favourably with reverse-phase chromatography, has good compatibility with multiple detector especially mass detector again, therefore be a kind of liquid chromatogram substitute technology that is very suitable for strong polarity and hydrophily sample qualitative and quantitative analysis, and obtain paying attention to more and more widely and using.In the HILIC separation system, generally adopt the acetonitrile-water system as mobile phase, wherein the ratio of water is that 5-40% is to guarantee its significant hydrophilic interaction.Such mobile phase composition is particularly advantageous in the sensitivity that improves electro-spray ionization mass spectrum (ESI-MS) so that HILIC can be compatible with multiple detector.In the HILIC separation system, generally use at present the normal-phase chromatography fillers such as silica gel, amino, glycol-based, cyano group, be suitable for the special-purpose chromatograph packing material of HILIC separation seldom, thereby the chromatograph packing material finishing of development of new functional group, that have good reproduction and stability has great importance.
Summary of the invention
It is wide to the purpose of this invention is to provide a class range of application, the post effect is high, and column capacity is large, and separative efficiency is high, separating degree is good, can be used for the novel multi-layer structure bonded silica gel performance liquid chromatographic column filler that positive, anti-phase and three kinds of clastotypes of hydrophilic Interaction Chromatography such as are applicable at degree or the gradient analysis.
Another object of the present invention provides the preparation method of above-mentioned multilayer structure bonded silica gel performance liquid chromatographic column filler, to overcome the deficiencies in the prior art.
The present invention seeks to realize like this:
The present invention is on silica matrix, and the method for employing multistep bonding obtains the silica matrix Bonded Phase of a class sandwich construction, and such material is well behaved efficient liquid phase chromatographic stuffing.
The preparation method of multilayer structure bonded silica gel performance liquid chromatographic column filler may further comprise the steps:
(1) utilizes alkyl silane reagent that the silica matrix particle is carried out surperficial alkylation processing and obtain silica matrix alkylation filler;
(2) silica matrix alkylation filler and polarity difluorophenyl silane reaction obtain silica filler;
(3) with the silica filler of drying further with Silante terminated reagent reacting, obtain the silicone fluid phase chromatography stuffing.
Described silica matrix particle is spherical porous silica gel, and metals content impurity is less than 30 ppm, and the particle size range of silica matrix particle is 1-60 mm; Pore diameter range is 50-1000, and the specific area scope is 50-500 m
2/ g.
Described alkyl silane reagent is the C of single or two or trifunctional
1-C
30Organosilan, its chemical structure of general formula is
R wherein
1=C
1-C
30Replace or substituted alkyl not;
R
2=C
1– C
20Replace or substituted alkyl not cycloalkyl, Heterocyclylalkyl, phenyl, substituted-phenyl;
α=?0,?1,?2;
X=halogen, alkoxyl, acyloxy or amido.
Described polarity difluorophenyl silane is obtained by 2,3,4,5,6-phenyl-pentafluoride formyl chloride and amino silane reaction, and polarity difluorophenyl silane chemical structure of general formula is:
R wherein
1=C
6F
5-, C
6F
5CONH-, C
6F
5CONHC
6H
4-or C
6F
5CONHC
6H
4O-;
α=?0~30；β=?0,?1,?2;
R
2=C
1– C
20Replace or substituted alkyl not cycloalkyl, Heterocyclylalkyl, phenyl, substituted-phenyl;
X=halogen, alkoxyl, acyloxy or amido.
Described Silante terminated reagent is single, in two, three, four or five silane one or more, described single silane is trim,ethylchlorosilane, N, N-dimethyl trimethyl silicane amine, trimethyl silicon based imidazole, methyl trichlorosilane, dimethyldichlorosilane, dimethoxy dimethylsilane, trimethyl silanol or N-trimethyl silicane yl acetamide; Described disilane is HMDS or 1,3-dimethoxy tetramethyl disiloxane; Described three silane are hexamethyl cyclotrisiloxanes; Described tetrasilane is octamethylcy-clotetrasiloxane; Described five silane are decamethylcyclopentaandoxane.
The silicone fluid phase chromatography stuffing for preparing according to above-mentioned silicone fluid phase chromatography stuffing preparation method can be used for positive, anti-phasely separates with hydrophilic Interaction Chromatography.
The present invention is take silica gel particle as matrix, utilize corresponding alkyl silane reagent, adopting the liquid phase reactor method that silica gel particle is carried out surperficial alkylation processes, obtain the alkyl silica gel filler, further obtain multilayer structure bonded silica filler with polarity difluorophenyl silane reaction, again through being hydrolyzed, further making the novel efficient liquid phase chromatographic stuffing of a class with Silante terminated reagent reacting.Key of the present invention is to have united novel polarity difluorophenyl and alkyl functional group as the Silica Surface Bonded Phase, thereby obtains comparing traditional C
18Selective and the separating degree that chromatographic column is better.Be characterized in that alkyl functional group has hydrophobic effect, polarity difluorophenyl functional group has the dipole-dipole effect, the multiple mechanisms of action such as π-π effect and hydrogen bond action can be separated detection of acidic, neutrality and alkali compounds effectively simultaneously, particularly polar compound there is very strong separating power, can with contain oxygen, nitrogen, phosphorus, the organic compound of sulphur forms hydrogen bond, thereby has good application potential.
Chromatographic column of the present invention can be used for positive, anti-phasely separates with hydrophilic Interaction Chromatography, and degree or the gradient analysis such as is applicable to, and namely the component ratio of mobile phase can keep constant or change according to certain rules in whole separation process.Can contain 0-100% water or 0-100% organic solvent in the mobile phase.When moisture, other composition Ying Yushui dissolves each other.Organic solvent commonly used has (but being not limited to) methyl alcohol, acetonitrile, isopropyl alcohol, ethanol, oxolane etc.Can add the acid of 0-100 mmol/L solubility in the mobile phase, alkali or other buffer salt.Mobile phase pH scope between pH 2-8 to guarantee certain Column stability.The temperature range of using can be at 5-60
0C is preferably in 20-40
0C.When LC-MS is online, uses high organic enough increasing ionization process of being on good terms that flow, thereby improve detection sensitivity.
The Bonded Phase that deactivation processing (end-blocking) is crossed has higher inertia, it is compared than the Bonded Phase silica filler of not deactivation processing mixture uracil-pyridine-phenol chromatographic isolation analysis, the peak shape of pyridine and retention time be improved significantly, its interaction between alkaline matter and chromatographic stationary phases residual silicon hydroxyl of effectively having weakened is described.Under suitable chromatographic condition, the fixedly phase that deactivation was processed can be used for the compartment analysis of acidic, neutral and basic compounds effectively.
Chromatograph packing material of the present invention and traditional C
18Chromatograph packing material is compared and is had the following advantages:
(1) alkyl and polarity difluorophenyl combination bonded silica gel is the novel liquid chromatography stuffing of a class, has good potentiality to be exploited.New material has U-shaped chromatographic isolation characteristic, and being expected to provides strong instrument for the compartment analysis of present insoluble complicated polarity and hydrophily sample; Because alkyl and polarity difluorophenyl combination bonded silica gel are the fillers that a class has the multiple mechanism of action, thereby can separate simultaneously detection to the mixture of opposed polarity compound;
(2) fixing have positive, anti-phase function of separating with hydrophilic Interaction Chromatography when identical.Both can use pure water mobile phase separating polar, alkalescence and hydrophily organic compound, reduce the pollution of organic solvent; Can use again pure organic mobile phase, strengthen the detection sensitivity of LC-MS;
(3) the filler synthesis step is simple, easily operation, good reproducibility;
(4) chromatographic performance of filler is superior, and the post effect is high, and column capacity is large, and is selectively good, and separating degree is high.Embody and the reverse-phase chromatography quadrature optionally simultaneously, have the post effect that compares favourably with reverse-phase chromatography and the peak shape of symmetry, can be used as the important supplement of positive and reverse-phase chromatography.
Description of drawings
Fig. 1 be among the embodiment 6 acetonitrile concentration on the impact of pindolol retention factors;
Fig. 2 is the chromatogram that traditional C 18 posts and chromatographic column of the present invention are separated pyridine-phenol among the embodiment 7; 1 represents pyridine, and 2 represent phenol;
Fig. 3 is the chromatogram that chromatographic column of the present invention is separated uracil-butyl p-hydroxybenzoate-Propranolol-dipropyl phthalate-naphthalene-acenaphthene-amitriptyline mixture among the embodiment 8; 1 represents uracil, and 2 represent butyl p-hydroxybenzoate, and 3 represent Propranolol, and 4 represent dipropyl phthalate, and 5 represent naphthalene, and 6 represent acenaphthene, and 7 represent amitriptyline;
Fig. 4 is the chromatogram that chromatographic column of the present invention is separated cytimidine-thiocarbamide-adenine-uridine mixture among the embodiment 9; 1 represents cytimidine, and 2 represent thiocarbamide, and 3 represent adenine, and 4 represent uridine.
The specific embodiment
In order to understand better technology of the present invention, give further instruction by example.
Specifically, the synthetic concrete steps of the multilayer structure bonded silica filler of the present invention are as follows:
The silica gel of activation is added in the reactor, pass into dry inert gas, add dimethylbenzene and pyridine, mechanical agitation is even, then adds alkyl silane reagent, adds hot reflux, stop reaction, vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash, drying.Alkyl linked silica gel is put into reactor, pass into dry inert gas, add dimethylbenzene and pyridine, mechanical agitation is even, then adds polarity difluorophenyl silane reagent, adds hot reflux, stop reaction, vacuum filtration is used toluene, carrene successively, acetone, methanol-water (1:1, v/v), methanol wash.Then material uses 0.1% trifluoracetic acid methanol aqueous solution (5:1) at room temperature to be hydrolyzed, and vacuum filtration is used acetone successively, methanol-water (1:1, v/v), methanol wash, drying.Bonded silica gel is placed in the reactor, passes into dry inert gas, adds dimethylbenzene, mechanical agitation is even, then adds a certain amount of terminated silane reagent, adds hot reflux, stop reaction, vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash, drying.In silica filler of the present invention synthetic, related reactive material optimal proportion is, silica gel, and silane, the mol ratio between pyridine and the solvent is 1:1.5:1.5:5, the return stirring reaction time is 24-48 hour.
Embodiment 1
(1) take by weighing aminopropyl trimethoxysilane 18 grams, put into there-necked flask, condenser pipe and dropping funel are installed, pass into dry argon gas, add 100 milliliters of dry toluene, triethylamine 13 grams drip 2,3,4,5,6-phenyl-pentafluoride formyl chloride, 23 grams, mechanical agitation 16 hours.Roughage distillating method purifying.
(2) take by weighing 10 gram spherical silica gel (5 μ m, 100,450 m
2/ g, metals content impurity is less than 30 ppm), 120
0C vacuum drying 24 hours is inserted after the cooling in the reactor, passes into dry argon gas, condenser pipe and drying tube are installed, are added 50 milliliters of dimethylbenzene, 4 milliliters of pyridines, mechanical agitation is even, then adds n-octadecane base dimethylchlorosilane 18 grams, is heated to the also isothermal reaction 24 hours that refluxes, stop reaction, vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
(3) take by weighing the dry n-octadecane base key of 10 grams and close silica gel, put into reactor, pass into dry argon gas, condenser pipe and drying tube are installed, are added 50 milliliters of dimethylbenzene, 4 milliliters of pyridines, mechanical agitation is even, then adds the synthetic silane of step (1) 20 grams, is heated to the also isothermal reaction 48 hours that refluxes, stop reaction, vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash.
(4) step (3) bonded silica gel (10 gram) is put into reactor, add 50 milliliter of 0.1% trifluoracetic acid methanol aqueous solution (5:1, v/v), at room temperature reacted 24 hours, stop reaction, vacuum filtration is used acetone successively, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
(5) get step (4) bonded silica gel (10 gram) and put into reactor, pass into dry argon gas, condenser pipe and drying tube are installed, add 50 milliliters of dimethylbenzene, 10 milliliters of HMDSs, (
N, N-dimethylamino) trimethyl silane is 8 milliliters, and mechanical agitation is even, is heated to the also isothermal reaction 48 hours that refluxes, and stops reaction, and vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
Embodiment 2
(1) takes by weighing 10 gram spherical silica gel (5 μ m, 100,450 m
2/ g, metals content impurity is less than 30 ppm), 120
0C vacuum drying 24 hours is inserted after the cooling in the reactor, passes into dry argon gas, condenser pipe and drying tube are installed, are added 50 milliliters of dimethylbenzene, 4 milliliters of pyridines, mechanical agitation is even, then adds n-octadecane base dimethylchlorosilane 18 grams, is heated to the also isothermal reaction 24 hours that refluxes, stop reaction, vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
(2) take by weighing the dry n-octadecane base key of 10 grams and close silica gel, put into reactor, pass into dry argon gas, condenser pipe and drying tube are installed, are added 50 milliliters of dimethylbenzene, 4 milliliters of pyridines, mechanical agitation is even, then adds aminopropyl trimethoxysilane 3 grams, is heated to the also isothermal reaction 24 hours that refluxes, stop reaction, vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
(3) get step (2) bonded silica gel (10 gram) and put into reactor, pass into dry argon gas, condenser pipe and drying tube are installed, add 50 milliliters of toluene, triethylamine 4 grams drip 2,3,4,5,6-phenyl-pentafluoride formyl chloride, 8 grams, mechanical agitation 16 hours.Stop reaction, vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash.
(4) step (3) bonded silica gel is put into reactor, add 50 milliliter of 0.1% trifluoracetic acid methanol aqueous solution (5:1, v/v), at room temperature reacted 24 hours, stop reaction, vacuum filtration is used acetone successively, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
(5) get step (4) bonded silica gel (10 gram) and put into reactor, pass into dry argon gas, condenser pipe and drying tube are installed, add 50 milliliters of dimethylbenzene, two (the chloro dimethyl is silica-based) ethane 5 grams of 1,2-,
N-trimethyl silicon based imidazole 7 grams, mechanical agitation is even, is heated to the also isothermal reaction 48 hours that refluxes, and stops reaction, and vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
Embodiment 3
(1) take by weighing [3-(
p-anilino-) propyl group] trimethoxy silane 26 grams, put into there-necked flask, condenser pipe and dropping funel are installed, pass into dry argon gas, add 100 milliliters of dry toluene, triethylamine 13 grams drip 2,3,4,5,6-phenyl-pentafluoride formyl chloride, 23 grams, mechanical agitation 16 hours.Roughage distillating method purifying.
(2) take by weighing 10 gram spherical silica gel (5 μ m, 100,450 m
2/ g, metals content impurity is less than 30 ppm), 120
0C vacuum drying 24 hours is inserted after the cooling in the reactor, passes into dry argon gas, condenser pipe and drying tube are installed, are added 50 milliliters of dimethylbenzene, 4 milliliters of pyridines, mechanical agitation is even, then adds n-octadecane ylmethyl dichlorosilane 20 grams, is heated to the also isothermal reaction 24 hours that refluxes, stop reaction, vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
(3) take by weighing the dry n-octadecane base key of 10 grams and close silica gel, put into reactor, pass into dry argon gas, condenser pipe and drying tube are installed, are added 50 milliliters of dimethylbenzene, 4 milliliters of pyridines, mechanical agitation is even, then adds the synthetic silane of step (1) 7 grams, is heated to the also isothermal reaction 48 hours that refluxes, stop reaction, vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash.
(4) step (3) bonded silica gel (10 gram) is put into reactor, add 50 milliliter of 0.1% trifluoracetic acid methanol aqueous solution (5:1, v/v), at room temperature reacted 24 hours, stop reaction, vacuum filtration is used acetone successively, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
(5) get step (4) bonded silica gel (10 gram) and put into reactor, pass into dry argon gas, condenser pipe and drying tube are installed, add 50 milliliters of dimethylbenzene, two (the chloro dimethyl is silica-based) ethane 5 grams of 1,2-, 10 milliliters of HMDSs, mechanical agitation is even, is heated to the also isothermal reaction 48 hours that refluxes, stop reaction, vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
Embodiment 4
(1) take by weighing [8-(
p-anilino-) octyl group] dimethylchlorosilane 30 grams, put into there-necked flask, condenser pipe and dropping funel are installed, pass into dry argon gas, add 100 milliliters of dry toluene, triethylamine 13 grams drip 2,3,4,5,6-phenyl-pentafluoride formyl chloride, 23 grams, mechanical agitation 16 hours.Roughage distillating method purifying.
(2) take by weighing 10 gram spherical silica gel (5 μ m, 100,450 m
2/ g, metals content impurity is less than 30 ppm), 120
0C vacuum drying 24 hours is inserted after the cooling in the reactor, passes into dry argon gas, condenser pipe and drying tube are installed, are added 50 milliliters of dimethylbenzene, 4 milliliters of pyridines, mechanical agitation is even, then adds positive eight alkyl dimethyl chlorosilanes, 11 grams, is heated to the also isothermal reaction 24 hours that refluxes, stop reaction, vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
(3) take by weighing positive eight dry alkyl linked silica gel of 10 grams, put into reactor, pass into dry argon gas, condenser pipe and drying tube are installed, are added 50 milliliters of dimethylbenzene, 4 milliliters of pyridines, mechanical agitation is even, then adds the synthetic silane of step (1) 27 grams, is heated to the also isothermal reaction 48 hours that refluxes, stop reaction, vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
(4) get step (3) bonded silica gel (10 gram) and put into reactor, pass into dry argon gas, condenser pipe and drying tube are installed, add 50 milliliters of dimethylbenzene, two (the chloro dimethyl is silica-based) ethane 5 grams of 1,2-,
N, O12 milliliters of-two (trimethyl silicon based) acetamides, mechanical agitation is even, is heated to the also isothermal reaction 48 hours that refluxes, and stops reaction, and vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
Embodiment 5
(1) takes by weighing 10 gram spherical silica gel (5 μ m, 100,450 m
2/ g, metals content impurity is less than 30 ppm), 120
0C vacuum drying 24 hours is inserted after the cooling in the reactor, passes into dry argon gas, condenser pipe and drying tube are installed, are added 50 milliliters of dimethylbenzene, 4 milliliters of pyridines, mechanical agitation is even, then adds dodecyl dimethylchlorosilane 14 grams, is heated to the also isothermal reaction 24 hours that refluxes, stop reaction, vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
(2) take by weighing the dry dodecyl bonded silica gel of 10 grams, put into reactor, pass into dry argon gas, condenser pipe and drying tube are installed, add 50 milliliters of dimethylbenzene, 4 milliliters of pyridines, mechanical agitation is even, then adds (2,3,4,5,6-, five fluoro propyl group) dimethylchlorosilane 11 grams, be heated to the also isothermal reaction 48 hours that refluxes, stop reaction, vacuum filtration is used toluene successively, carrene, acetone, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
(3) get step (2) bonded silica gel (10 gram) and put into reactor, pass into dry argon gas, condenser pipe and drying tube are installed, add 50 milliliters of dimethylbenzene, 10 milliliters of hexamethyl cyclotrisiloxanes, 10 milliliters of HMDSs, mechanical agitation is even, is heated to the also isothermal reaction 48 hours that refluxes, and stops reaction, vacuum filtration is used toluene, carrene successively, acetone, methanol-water (1:1, v/v), methanol wash, 80
0Dry 24 hours of C.
Embodiment 6
Get bonded silica gel filler 0.5 gram of embodiment 1 gained, the homogenate method is filled in the stainless steel column of 50 x, 2.0 mm ID.Use pindolol as specimen, take acetonitrile: 20 mM ammonium acetates (pH 5.6) are as mobile phase, 30
0C detects at Agilent 1200 liquid chromatographs chromatographic column under 0.2 milliliter of flow velocity of per minute, and detecting wavelength is 220 nm.Fig. 1 has shown the impact of acetonitrile concentration on the pindolol retention factors.As can be seen from Figure 1, when the organic phase concentration in the mobile phase increases (0-30%), the retention factors of solute descends, and shows the character of reverse-phase chromatography; And along with the concentration of organic phase in the mobile phase continues to increase (30-80%), it is constant that the retention factors of solute keeps substantially; When the concentration of organic phase in the mobile phase continues to increase (80-100%), the retention factors of solute increases fast, shows the character of normal-phase chromatography.The efficient liquid phase chromatographic stuffing of the present invention's preparation has anti-phase function of separating with normal-phase chromatography simultaneously, thereby can separate simultaneously the mixture that detects the opposed polarity compound.For traditional anti-phase C
18Chromatographic column is always the retention time of solute reduces with the increase of organic phase concentration in the mobile phase.
Embodiment 7
Get bonded silica gel filler 2 grams of embodiment 1 gained, the homogenate method is filled in the stainless steel column of 150 x, 4.6 mm ID.The gained chromatographic column is for separating of blend sample.Use 1 pyridine, 2 phenol are as specimen, take 50% acetonitrile and 50% water as mobile phase, 30
0C detects at Agilent 1200 liquid chromatographs chromatographic column under one milliliter of flow velocity of per minute, and detecting wavelength is 254 nm.The gained spectrogram is seen accompanying drawing 2.Its middle and upper part is traditional C
18Chromatographic column test result, bottom are chromatographic column test result of the present invention.The resulting peak shape of chromatographic column of the present invention obviously is better than traditional C
18The accordingly result of chromatographic column gained.
Embodiment 8
With 150 x, the 4.6 mm ID chromatographic columns of embodiment 7 preparations, separated 1 uracil, 2 butyl p-hydroxybenzoates, 3 Propranolols, 4 dipropyl phthalates, 5 naphthalenes, 6 acenaphthenes, 7 amitriptyline mixtures, Fig. 3 are its chromatogram.Chromatographic condition is as follows: mobile phase, methyl alcohol: 20 mmol/L phosphate buffers (pH 7.0)=80:20 (v/v); Flow velocity, 1 mL/min; Column temperature, 30
0C; Detect wavelength, UV254 nm.
Embodiment 9
With 150 x, the 4.6 mm ID chromatographic columns of embodiment 7 preparations, separated 1 cytimidine, 2 thiocarbamides, 3 adenines, 4 uridine mixtures, Fig. 4 are its chromatogram.Chromatographic condition is as follows: mobile phase, the 10 mmol/L ammonium formate aqueous solution (pH 3.0); Flow velocity, 1 mL/min; Column temperature, 30
0C; Detect wavelength, UV254 nm.
Claims (7)
1. the preparation method of a silicone fluid phase chromatography stuffing is characterized in that may further comprise the steps:
(1) utilizes alkyl silane reagent that the silica matrix particle is carried out surperficial alkylation processing and obtain silica matrix alkylation filler;
(2) silica matrix alkylation filler and polarity difluorophenyl silane reaction obtain silica filler;
(3) silica filler with drying further obtains the silicone fluid phase chromatography stuffing with Silante terminated reagent reacting.
2. the preparation method of silicone fluid phase chromatography stuffing according to claim 1, it is characterized in that: described silica matrix particle is spherical porous silica gel, metals content impurity is less than 30 ppm, the particle size range of silica matrix particle is 1-60 mm, pore diameter range is 50-1000, and the specific area scope is 50-500 m
2/ g.
3. the preparation method of silicone fluid phase chromatography stuffing according to claim 1, it is characterized in that: the chemical structure of general formula of described alkyl silane reagent is
R wherein
1=C
1-C
30Replace or substituted alkyl not;
R
2=C
1– C
20Replace or substituted alkyl not cycloalkyl, Heterocyclylalkyl, phenyl, substituted-phenyl;
α=?0,?1,?2;
X=halogen, alkoxyl, acyloxy or amido.
4. the preparation method of silicone fluid phase chromatography stuffing according to claim 1, it is characterized in that: the chemical structure of general formula of polarity difluorophenyl silane is:
R wherein
1=C
6F
5-, C
6F
5CONH-, C
6F
5CONHC
6H
4-or C
6F
5CONHC
6H
4O-;
α=?0~30；β=?0,?1,?2;
R
2=C
1– C
20Replace or substituted alkyl not cycloalkyl, Heterocyclylalkyl, phenyl, substituted-phenyl;
X=halogen, alkoxyl, acyloxy or amido.
5. the preparation method of silicone fluid phase chromatography stuffing according to claim 1 is characterized in that: described Silante terminated reagent is single, two, three, in four or five silane one or more, described single silane is trim,ethylchlorosilane, N, N-dimethyl trimethyl silicane amine, trimethyl silicon based imidazole, methyl trichlorosilane, dimethyldichlorosilane, the dimethoxy dimethylsilane, trimethyl silanol or N-trimethyl silicane yl acetamide; Described disilane is HMDS or 1,3-dimethoxy tetramethyl disiloxane; Described three silane are hexamethyl cyclotrisiloxanes; Described tetrasilane is octamethylcy-clotetrasiloxane; Described five silane are decamethylcyclopentaandoxane.
6. the silicone fluid phase chromatography stuffing for preparing to 5 one of any described silicone fluid phase chromatography stuffing preparation methods according to claim 1.
7. the described silicone fluid phase chromatography stuffing of claim 6 is in positive, anti-phase application with during hydrophilic Interaction Chromatography is separated.
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| CN111632582B (en) * | 2019-03-01 | 2023-02-28 | 月旭科技（上海）股份有限公司 | Silica gel reverse phase chromatographic packing and preparation method and application thereof |
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