CN110013836A - Reverse phase/ion exchange mixed mode chromatographic stationary phases, preparation method and application - Google Patents
Reverse phase/ion exchange mixed mode chromatographic stationary phases, preparation method and application Download PDFInfo
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- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
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Abstract
The present invention relates to a kind of reverse phase/ion exchange mixed mode chromatographic stationary phases, preparation method and application, such reverse phase/ion exchange mixed mode chromatographic stationary phases, referred to as octadecyl/sulfonic group mixed chromatogram stationary phase, it is made of the octadecyl and sulfonic group that are bonded in Bio-sil surface, the preparation method comprises the following steps: passing through chemical vapour deposition technique for γ-(2, the third oxygen of 3- epoxy) propyl trimethoxy silicane KH-560 is bonded to Bio-sil microparticle surfaces, it is reacted respectively with sodium hydrogensulfite and octadecyl chloride using KH-560 as linking arm, prepare octadecyl/sulfonic group mixed chromatogram stationary phase.
Description
Technical field
The present invention relates to Stationary Phase for HPLC and the preparation method and application thereof.
Background technique
Quality control of the Pharmaceutical Analysis technology in the development & production and the storage process of circulation of drug plays an important role.For
Drug is improved to the therapeutic effect of disease, the drug combination of different single component drugs becomes widely applied strategy, still
Drug combination causes drug compliance to reduce, and increases the risk of patient.Fixed dosage compound medicine developed in recent years is
Single formulation containing two or more effective component, such as capsule or pill.It can be made using the collaboration of different pharmaceutical ingredient
With, the therapeutic effect of drug is improved, and can be reduced the tablet quantity that patient takes and therefore receive the welcome of doctor and patient,
Product covers AIDS drugs, antihypertensive, Coritab.It is and single due to containing a variety of active ingredients in compound medicine
Component drug is compared, and the difficulty of quality control is significantly increased, and there is an urgent need to develop applicable analysis methods.
It is recorded according to " Chinese Pharmacopoeia " 2015 editions and pertinent literature, in one-component Control of drug quality, including identifies, contains
Measuring fixed and impurity analysis, the analytical technology being most widely used is reversed-phase liquid chromatography, such as uses octadecyl bonded silica
The reverse-phase chromatography of glue stationary phase, but this kind of reverse-phase chromatographic column is weaker for the reservation of some polarity or ionic compound, and
And cause chromatographic peak to trail since with alkaline drug electrostatic interaction occurs for its silicone hydroxyl dissociation remained on surface, influence color
Isolated resolution ratio is composed, analysis while cannot achieve neutral and polar ion type compound in compound medicine.In order to increase from
Usually ionic surfactant is added in reverse-phase chromatography mobile phase in the reservation of subtype compound, by with it is analyzed anti-
Number ionic compound forms ion pair and realizes neutral with analysis while ionic compound.But Ion-pair chromalography technology
Because it is fatal to have the shortcomings that two, so that it is in compound medicine analysis using being extremely limited.One is separating
For ion-pairing agent involved in process in the Dynamic Adsorption balance of fixed phase surface, equilibration time is longer, causes in actual drug
Gradient elution cannot be used in separation process, and the component of opposed polarity or reserve capability is carried out while being analyzed.Secondly as from
Son is difficult to volatilize to reagent, and pollution of ion source is caused in ionization process, prevent Ion-pair chromalography from mass spectrometry, limit
The application that liquid chromatography-mass spectrometry carries out Structural Identification to heterogeneity is made.
Mixed mode chromatography is substitution ion to chromatography, the preferred side for carrying out while analyzing to neutral and ionic compound
Method.Different from traditional single-mode chromatography, mixed mode chromatography uses function groups chromatographic stationary phases, and retention mechanism is related to
Two or more different types of intermolecular interaction, therefore there is stronger separating capacity.The most commonly used mixing
Mode chromatographic is reverse phase/ion-exchange chromatography, and used fixed phase surface contains hydrophobic and can dissociate functional group, therefore energy
Enough by the synergistic effect of your power and electrostatic force of Van der Waals, neutral and ionic compound is realized and retains and separates simultaneously.By
Addition surfactant is not needed in mobile phase, therefore the problem of there is no in the presence of Ion-pair chromalography, can not only be made
With gradient elution analysis, can also and mass spectrometry wide application prospect is therefore presented in Pharmaceutical Analysis.But it arrives
So far, the kind of mixed mode chromatographic stationary phases is few, and structure is single, does not adapt to growing in Pharmaceutical Analysis field
Complicated ingredient, especially compound medicine, analysis demand.
Summary of the invention
The purpose of the present invention is to provide a kind of new mixed mode chromatographic stationary phases, and provide its simple and feasible preparation
Method and its application in Pharmaceutical Analysis field.
A kind of reverse phase/ion exchange mixed mode chromatographic stationary phases, referred to as octadecyl/sulfonic group mixed chromatogram are fixed
Phase is made of, chemical structural formula the octadecyl and sulfonic group that are bonded in Bio-sil surface are as follows:
。
The preparation method of the chromatographic stationary phases, comprises the following steps that
(1) preparation of activated silica gel: the reflux that 80 DEG C -105 DEG C of progress in HCl solution is added in a certain amount of aerosil particles is anti-
It answers, is washed with water to neutrality, vacuum drying；
(2) preparation of epoxy group silica gel: by the aerosil particles of above-mentioned acidification, every gram is added 1.92mmol-3.6mmol's
γ-(2,3- the third oxygen of epoxy) propyl trimethoxy silicane seals reaction at 140 DEG C -160 DEG C, is washed with dehydrated alcohol, filters
It collects product and is dried in vacuo, obtain Epoxy functionalized Bio-sil microballoon；
(3) preparation of sulfonic group silica gel: it is 8-10 that above-mentioned Epoxy functionalized Bio-sil microballoon, which is distributed to mass number,
In ultrapure water again, 1.92mmol-3.6mmol sodium hydrogensulfite is added according to every gram of Epoxy functionalized Bio-sil microballoon
Sodium hydrogensulfite is added in amount, mixes, and using triethylamine tune pH value to 7-8,85 DEG C of reactions are washed with water and are dried in vacuo, obtain
Sulfonic group bonded silica gel microballoon；
(4) octadecyl/sulfonic group silica gel preparation: it is 8- that above-mentioned sulfonic group bonded silica gel microballoon, which is dispersed in mass number,
In 10 times of n,N-Dimethylformamide (DMF), under condition of ice bath, it is added according to every gram of sulfonic group bonded silica gel microballoon
Octadecyl chloride is added dropwise in the amount of 1.92mmol-3.6mmol octadecyl chloride；After completion of dropwise addition, 40-65 DEG C is stirred to react,
Product is collected by filtration, after successively with toluene and ethanol washing and be dried in vacuo, obtain octadecyl/sulfonic group mixed mode chromatography
Stationary phase.
Reverse phase/ion exchange blended liquid phase chromatographic column can be made using the chromatographic stationary phases.
The preparation of the chromatographic column uses high-pressure homogenization, process conditions are as follows: according to n-hexyl alcohol: the quality of carbon tetrachloride
Proportion 6:4 prepares homogenate dress, and using n-hexane as displacement fluid, dress column pressure is 300bar.
The reverse phase/ion exchange blended liquid phase chromatographic column is applied to medical separation.
Mixed mode stationary phase of the invention is using epoxy group as linking arm, in conjunction with octadecyl long-chain, relative to traditional benzene
The flexibility of phenyl in sulfonic acid Bonded Phase is bigger, and hydrophobicity is higher, with by the synergistic effect between isolated alkali compounds more
By force, thus separating effect is more preferable.In addition, because being easy to combine mobile phase there are hydrophilic radicals such as ehter bond and hydroxyls in linking arm
In polar molecule, therefore when can prevent octadecyl chain water content is higher in mobile phase the lodging of octadecyl chain and cause
The problem of reservation decline of analyte.
The present invention carries out Silanization reaction to Bio-sil using chemical vapor deposition method, makes it combine with larger chemistry
Octadecane is prepared then using the condensation reaction of the ring-opening reaction of epoxy group and hydroxyl and acyl chlorides in active epoxy group
Base/sulfonic group mixed mode chromatographic stationary phases.Compared with conventional liquid phase reaction method, chemical vapour deposition technique is avoided using such as toluene
Etc. poisonous and harmful organic solvent, the harm to human body and the pollution to environment are significantly reduced.Utilize Epoxy functionalized silicon
Glue bridges reverse phase and ion-exchange group, simplifies synthesis step, and the ratio of reverse phase and ion-exchange group as interphase
Example is adjustable.The preparation method invented has raw material cheap and easy to get, and process route is simple and feasible, is easy to the industrialization for the scale of amplifying
Production.
Stationary phase of the invention can be applied to multi-component in fixed dosage compound medicine while separation and measurement.And mesh
Ion-pair chromalography is generally required in preceding pharmacopeia or separating for several times just can be carried out multicomponent analysis.Not using stationary phase of the invention
It is only able to carry out multicomponent while separating, and isolated effect is better than conventional ion to reverse-phase chromatography, and can be using ladder
Degree elution directly and mass spectrometry carries out qualitative, quantitative to compound medicine online and disposably analyzes, save a large amount of analysis time
And operating cost.
Detailed description of the invention
The preparation flow of Fig. 1 mixed mode chromatographic stationary phases.
The IR Characterization of the activation microballoon of Fig. 2 embodiment 1
The IR Characterization of 2 epoxy group silica gel microball of Fig. 3 embodiment
The chemical equation of the preparation of Fig. 4 embodiment 3 glycol-based functional group
The IR Characterization of 3 glycol-based silica gel microball of Fig. 5 embodiment
The nuclear-magnetism of 3 glycol-based silica gel microball of Fig. 6 embodiment characterizes
Fig. 7 embodiment 4 prepares the chemical equation of octadecyl functionalized microsphere
The IR Characterization of 4 octadecyl functional silica gel microballoon of Fig. 8 embodiment
The nuclear-magnetism of 4 octadecyl functional silica gel microballoon of Fig. 9 embodiment characterizes
The chemical equation of the preparation sulfonate functional groups of Figure 10 embodiment 5
The IR Characterization of 5 sulfonic functional silica gel microball of Figure 11 embodiment
The nuclear-magnetism of 5 sulfonic functional silica gel microball of Figure 12 embodiment characterizes
Figure 13 embodiment 6 prepares the chemical equation of octadecyl sulfonic acid base functionalized microsphere
6 octadecyls of Figure 14 embodiment/sulfonic group silica gel microball IR Characterization
6 octadecyls of Figure 15 embodiment/sulfonic group silica gel microball nuclear-magnetism characterization
The elemental analysis of 1 embodiment 2-6 of table
Separation of Figure 16 alkyl benzene homologue in octadecyl, sulfonic group and octadecyl/sulfonic group chromatographic column.
Figure 17 dicyandiamide, melamine and melbine are in octadecyl, sulfonic group and octadecyl/sulfonic group chromatographic column
On separation.
Separation of Figure 18 aniline homologue in octadecyl, sulfonic group and octadecyl/sulfonic group chromatographic column.
Separation of Figure 19 common alkali drug in octadecyl/sulfonic group chromatographic column.
Separation of Figure 20 compound methoxyphenamine capsules in octadecyl/sulfonic group chromatographic column.
Separation of Figure 21 compound Dihydralazine piece in octadecyl/sulfonic group chromatographic column.
Figure 22 compound compound hypotensive is talked endlessly separation of the pyridine piece in octadecyl/sulfonic group chromatographic column, and Detection wavelength is
268nm。
Figure 23 compound compound hypotensive is talked endlessly separation of the pyridine piece in octadecyl/sulfonic group chromatographic column, and Detection wavelength is
310nm。
Figure 24 FUFANG LIXUEPING PIAN under condition of gradient elution, separation in octadecyl/sulfonic group chromatographic column it is ultraviolet
Chromatogram.
The ion flow graph of organic component and corresponding mass spectrogram in Figure 25 FUFANG LIXUEPING PIAN.
Specific embodiment
The present invention provides a kind of reverse phase/ion exchange mixed mode chromatographic stationary phases of structure novel, in ionic function
The periphery of group is bonded hydrophobic and soft alkyl hydrocarbon long-chain, can pass through the reverse phase and ion exchange retention mechanism and quilt of collaboration
Analysis of compounds is had an effect, to have high chromatography selectivity, multicomponent suitable for compound medicine while analysis and is contained
It is fixed to measure.The present invention provides the technology of preparing of its simple and feasible mixed mode chromatographic stationary phases, using dry method derivative reaction
Traditional wet process is substituted, is avoided using poisonous and harmful organic solvent, protection health and environment.The technology of preparing reaction route is simple
Controllably, raw material is cheap and easy to get, is suitable for industrial amplification production.It is solid that it is also another object of the present invention to provide mixed mode chromatographies
The fixed mutually unique application in Pharmaceutical Analysis, not only may be implemented gradient elution analysis and can and mass spectrometry, realize compound
Multi-component quick assay and Structural Identification in drug.
Mixed chromatogram stationary phase prepared by the present invention is made of the octadecyl and sulfonic group for being bonded in Bio-sil surface.
γ-(the third oxygen of 2,3- epoxy) propyl trimethoxy silicane (KH-560) is bonded to by porous silicon by chemical vapour deposition technique first
Glue microparticle surfaces are reacted with sodium hydrogensulfite and octadecyl chloride respectively using KH-560 as linking arm, prepare octadecyl/
Sulfonic group mixed chromatogram stationary phase, and be applied to compound medicine and separate.Its preparation flow is as shown in Figure 1.Concrete operation step is such as
Under:
The preparation of activated silica gel: a certain amount of aerosil particles are added to the HCl solution of 8-10 times of quality of 10%-15%, are stirred
It mixes, 105 DEG C of back flow reaction 8h are washed with water to neutrality, 80 DEG C of vacuum drying.
The preparation of epoxy group silica gel: the aerosil particles of above-mentioned acidification are put into the autoclave with Teflon liner, often
Gram be added 1.92mmol-3.6mmol γ-(2,3- the third oxygen of epoxy) propyl trimethoxy silicane, sealing autoclave, at 150 DEG C
Lower reaction 8h.Unreacted silicon Alkylators are washed away with dehydrated alcohol, product is collected by filtration, is dried in vacuo at 80 DEG C, obtains epoxy group
Functionalization Bio-sil.
The preparation of sulfonic group silica gel: it is 8-10 times ultrapure that the silica gel microball of above-mentioned bonding epoxy group, which is distributed to mass number,
In water, every gram plus 1.92mmol-3.6mmol sodium hydrogensulfite, ultrasound are mixed, triethylamine tune pH value to 7-8,85 DEG C of reaction 8h,
Water washing is distilled, 120 DEG C of vacuum drying obtain sulfonic group bonded silica gel microballoon.
The preparation of octadecyl/sulfonic group silica gel: it is 8- that above-mentioned sulfonic functional silica gel microball, which is dispersed in mass number,
In 10 times of n,N-Dimethylformamide (DMF), under condition of ice bath, every gram of 1.92mmol-3.6mmol octadecane is slowly added dropwise
Base acyl chlorides.After completion of dropwise addition, 60 DEG C are stirred to react 9h, and product is collected by filtration, after successively use toluene and ethanol washing, 80 DEG C of vacuum
It is dry, obtain octadecyl/sulfonic group mixed mode chromatographic stationary phases.
The preparation of high pressure liquid chromatography column: chromatographic stationary phases are filled by stainless steel tube using conventional high-pressure homogenate method
In, obtain reverse phase/ion exchange blended liquid phase chromatographic column.Actual conditions are n-hexyl alcohol: carbon tetrachloride 6:4 be homogenate dress, just oneself
Alkane is displacement fluid, and dress column pressure is 300bar.
The reverse-phase chromatography behavior of high pressure liquid chromatography column: being molecule spy with the benzene homologue (- penta benzene of toluene) of different chain length
Needle measures their retentions in octadecyl stationary phase, sulfonic group stationary phase and octadecyl/sulfonic group stationary phase, than
Compared with the reverse-phase chromatography strength retention of stationary phase.
Chromatographic condition: stationary phase, (a) Octadecylsilane bonded phase, (b) sulfonic group Bonded Phase, (c) octadecyl/sulfonic group
Bonded Phase；15x 0.46cm；Mobile phase, -40% water of 60% methanol；Flow velocity, 1mL/min；Detection wavelength, 254nm.
The ion-exchange chromatography behavior of high pressure liquid chromatography column: with dicyandiamide, melamine and melbine are molecule spy
Needle measures their retentions in octadecane base phase, sulfonic acid base phase and octadecyl/sulfonic acid base phase, compare stationary phase from
The intensity that son exchange retains.
Chromatographic condition: stationary phase, (a) Octadecylsilane bonded phase, (b) sulfonic group Bonded Phase, (c) octadecyl/sulfonic group
Bonded Phase；15x 0.46cm；Mobile phase, -70% biphosphate amine aqueous solution (1.7%, pH 3) of 30% acetonitrile；Flow velocity, 1mL/
min；Detection wavelength, 220nm.
The reverse phase of high pressure liquid chromatography column/ion exchange mixed chromatogram retention behavior: the alkyl benzene amine (methyl of different chain length
Aniline-amyl aniline) there is hydrophobic grouping alkylbenzene and dissociable group amine, therefore, select them as molecular probe, measurement
Their retentions in octadecane base phase, sulfonic acid base phase and octadecyl/sulfonic acid base phase, compare the mixed mode color of stationary phase
Compose strength retention.
Chromatographic condition: stationary phase, (a) Octadecylsilane bonded phase, (b) sulfonic group Bonded Phase, (c) octadecyl/sulfonic group
Bonded Phase；15x 0.46cm；Mobile phase, -60% potassium dihydrogen phosphate of 40% acetonitrile (30mM, pH 2.5)；Flow velocity, 1mL/
min；Detection wavelength, 254nm.
The separation of common alkali drug: with common alkali drug salbutamol sulfate, clonidine, procainamide, general naphthalene
Luo Er, amitriptyline are model compound, measure their retentions in octadecyl/sulfonic acid base phase, show stationary phase pair
The separating effect of alkaline drug.
Chromatographic condition: stationary phase, octadecyl/sulfonic group Bonded Phase；15x 0.46cm；Mobile phase, 30% acetonitrile -70%
Potassium dihydrogen phosphate (30mM, pH 2.5)；Flow velocity, 1mL/min；Detection wavelength, 220nm.
The separation of compound methoxyphenamine capsules: taking the content of compound methoxyphenamine capsules, accurately weighed, finely ground, prepares
At the sample for being approximately equivalent to methoxyphenamine hydrochloride concentration 0.1mg/mL-1mg/mL, 0.22m filter membrane is crossed, takes subsequent filtrate as sample,
Precision measures 10-30l and injects liquid chromatograph.Chromatographic condition: carbon 18/sulfonic group mixed mode chromatographic column；With 50% phosphoric acid
- 50% acetonitrile of potassium dihydrogen buffer salt (30mM, pH 2.5) is mobile phase；Flow velocity, 1mL/min；Detection wavelength is 230nm.
The separation of dihydralazine sulfate,clonidine and hydrochlorothiazine piece: taking dihydralazine sulfate,clonidine and hydrochlorothiazine piece, finely ground, and precision weighs in right amount, prepares
At the sample for being approximately equivalent to Dihydralazine concentration 0.1mg/mL-1mg/mL, 0.22m filter membrane is crossed, takes subsequent filtrate as sample, it is accurate
It measures 10-30l and injects liquid chromatograph.Chromatographic condition: 18 sulfonic group mixed mode chromatographic column of carbon；With 50% potassium dihydrogen phosphate
- 50% acetonitrile of buffer salt (30mM, pH 2.5) is mobile phase；Flow velocity, 1mL/min；Detection wavelength is 220nm.
Compound compound hypotensive is talked endlessly the analysis of reserpine in pyridine piece: compound compound hypotensive is talked endlessly pyridine piece, and finely ground, precision weighs
Appropriate 0.1mg/mL-1mg/mL crosses 0.22m filter membrane, takes subsequent filtrate as sample, and precision measures 10-30l and injects liquid chromatogram
Instrument.Chromatographic condition: 18 sulfonic group mixed mode chromatographic column of carbon；With 50% potassium dihydrogen phosphate buffer salt (30mM, pH2.5)-
50% acetonitrile is mobile phase；Flow velocity, 1mL/min；Detection wavelength is 268nm.
Compound compound hypotensive is talked endlessly Hydrochioro, dihydralazine sulfate and dyrenium in pyridine piece, and finely ground, precision weighs suitable
0.1mg/mL-1mg/mL is measured, 0.22m filter membrane is crossed, takes subsequent filtrate as sample, precision measures 10-30l and injects liquid chromatograph.
Chromatographic condition: 18 sulfonic group mixed mode chromatographic column of carbon；With -50% second of 50% potassium dihydrogen phosphate buffer salt (30mM, pH2.5)
Nitrile is mobile phase；Flow velocity, 1mL/min；Detection wavelength is 310nm.
The separation of FUFANG LIXUEPING PIAN: FUFANG LIXUEPING PIAN, finely ground, precision weighs in right amount, is equivalent to Dihydralazine concentration
The sample of 0.1mg/mL-1mg/mL crosses 0.22m filter membrane, takes subsequent filtrate as sample, and precision measures 10-30l and injects liquid chromatogram
Instrument.Chromatographic condition: 18 sulfonic group mixed mode chromatographic column of carbon；50mM ammonium formate (pH 3) is mobile phase A, and acetonitrile is mobile phase
B, gradient are 0-3min 0%B, 3-7min 40%B, 7-40min 40%B, 40-45min 0%B；Flow velocity, 1mL/
min；Detection wavelength is 268nm and mass spectrometry positive ion mode detects.
Technology for a better understanding of the present invention further illustrates the present invention below with embodiment, but the present invention is not
It is limited to following embodiment.
The activation of 1 silica gel microball of embodiment
10g silica gel microball is taken to be added in 250mL there-necked flask, the HCl solution of addition 100mL 10%, mechanical stirring, 105
DEG C back flow reaction 8h, is washed with water to neutrality, and 120 DEG C are dried in vacuum overnight, and obtains activated silica gel microballoon, the silica gel microball after acidification
IR Characterization map is as shown in Fig. 2, in 3400cm-1There is biggish silicone hydroxyl absorption peak.
The dry process of 2 epoxy group bonded silica gel microballoon of embodiment
It takes 10g to be put into autoclave by activated silica gel microballoon prepared by embodiment 1,5.31mL γ-(2,3- epoxies is added
Third oxygen) propyl trimethoxy silicane, 150 DEG C of reaction 8h.It is washed with dehydrated alcohol, 80 DEG C of vacuum drying obtain Epoxy functionalized
Silica gel microball.The characterization map of this infrared method of Epoxy functionalized silica gel as shown in figure 3, after key and epoxy group
2800cm-1And 2890cm-1Nearby there is the stretching vibration peak of C-H, show epoxy group key and arrives Silica Surface, elemental analysis table
As shown in table 1, carbon content 8.05%, calculated surface key and amount are 4.50mol/m to sign2.This method has the prominent advantages that
Without using harmful organic solvents such as toluene, be conducive to protection health and environment.
The preparation of 3 glycol-based bonded silica gel microballoon of embodiment
It takes 10g by Epoxy functionalized microballoon obtained in embodiment 2, is dispersed in the 0.5%H of 100mL2SO4, heating is instead
8h is answered, is washed with water to neutrality, 80 DEG C of vacuum drying obtain glycol-based bonded silica gel microballoon, reaction equation is as shown in figure 4, two
Alcohol radical stationary phase IR Characterization is as shown in figure 5, in 3400cm-1There is the obvious peak O-H, and in 2800cm-1And 2890cm-1Near
There is the stretching vibration peak of C-H, nuclear-magnetism characterization map has apparent formant as shown in Figure 6, and elemental analysis data are such as
Shown in table 1, carbon content 5.52%, surface key and density 2.95mol/m2, show that reacting epoxy base chain has in acid condition
Certain falls off.
The preparation of 4 octadecyl functionalized microsphere of embodiment
4g is taken to be dispersed in 25mL n,N-Dimethylformamide (DMF) by glycol-based microballoon prepared in embodiment 3,
Under condition of ice bath, 6mL octadecyl chloride is slowly added dropwise；After being added dropwise to complete, heat temperature raising, in 60 DEG C of reaction 9h.Successively use
DMF and ethanol washing, 80 DEG C of dryings obtain octadecyl functional silica gel microballoon, and reactive chemistry equation is as shown in fig. 7, ten
For the IR Characterization of eight alkyl functional microballoons as shown in figure 8, there is apparent C-H stretching vibration peak, nuclear-magnetism characterizes map such as Fig. 9 institute
It is shown with the nuclear-magnetism peak of the carbon of apparent octadecyl chain, the key and density of elemental analysis the results are shown in Table 1 octadecyl are
1.08mol/m2。
The preparation of 5 sulfonic functional microballoon of embodiment
It takes 10g to be distributed in 100mL ultrapure water by epoxy group bonded silica gel microballoon prepared by embodiment 2, sequentially adds
5g sodium hydrogensulfite, ultrasound mix, triethylamine tune pH value to 7-8, heat temperature raising, in 85 DEG C of reaction 8h, distillation water washing, and 120
DEG C vacuum drying 8h, obtain sulfonic group bonded silica gel microballoon, reactive chemistry equation is as shown in Figure 10, and IR Characterization map is such as
Shown in Figure 11, there is apparent C-H stretching vibration peak, in 650cm-1There are sulfonic characteristic peak, nuclear magnetic resonance map such as Figure 12 in place
It is shown, there is apparent peak in 40ppm or so and divide out two acromions, is due to sulfonic group, elemental analysis such as 1 institute of table
Show.
6 octadecyls of embodiment/sulfonic functional microballoon preparation (octadecyl: sulfonic group=1:2)
4g is taken to be dispersed in 25mL n,N-Dimethylformamide (DMF) by sulfonic group microballoon prepared by embodiment 5,
Under condition of ice bath, 6mL octadecyl chloride is slowly added dropwise；After being added dropwise to complete, heat temperature raising, in 60 DEG C of reaction 9h.Successively use DMF
And ethanol washing, 80 DEG C of dryings obtain octadecyl/sulfonic group silica gel microball.Its reaction equation is as shown in figure 13, infrared table
There is the stretching vibration peak and sulfonic group characteristic absorption peak of C-H in sign map, nuclear-magnetism characterization map is as shown in figure 15 as shown in figure 14,
There is the nuclear magnetic resonance peak of apparent octadecyl chain in 60ppm or so, and occurs peak in 50ppm or so and split to be divided into two
Peak shows there is sulfonic influence, and the results are shown in Table 1 for elemental analysis, carbon carrying capacity 10.93%, and sulphur carrying capacity 0.81% is counted
It calculates the key for obtaining carbon 18 and amount is 0.67mol/m2, sulfonic group key and amount are 1.13mol/m2。
1 elemental analysis of table
It is prepared by the wet process of 7 epoxy group bonded silica gel microballoon of embodiment
It takes 10g by activation Bio-sil prepared by embodiment 1, is placed in the there-necked flask of 100mL, condenser pipe is installed and does
Dry pipe.Dry toluene 100mL, γ-(the third oxygen of 2,3- epoxy) propyl trimethoxy silicane 5.31mL is added.It is mechanical under nitrogen protection
It is stirred and heated to 110 DEG C of reaction 9h.Cooled and filtered, and successively washed with toluene, methanol.12h is dried in vacuo at 60 DEG C.
Obtain Epoxy functionalized silica gel.Compared with the dry method of embodiment 2, the shortcomings that this method is to need to consume a large amount of toxic solvent first
Benzene.
8 chromatographic evaluation of embodiment: reverse phase retention behavior
Using the benzene homologue of different chain length as molecular probe, their interphases (18 prepared in the present invention are measured
Alkyl stationary phase, sulfonic group stationary phase) and mixed chromatogram stationary phase (octadecyl/sulfonic group stationary phase) on retention, than
Compared with the reverse-phase chromatography strength retention of stationary phase.
Sample preparation: toluene is taken respectively, ethylbenzene, propyl benzene, butylbenzene and penta benzene 50L add methanol-water in 10mL volumetric flask
(60:40) is diluted to graduation mark, crosses the filter membrane of 0.22m, is configured to alkyl benzene homologue sample.
Chromatographic condition: stationary phase, (a) Octadecylsilane bonded phase, (b) sulfonic group Bonded Phase, (c) octadecyl/sulfonic group
Bonded Phase；15x 0.46cm；Mobile phase, -40% water of 60% methanol；Flow velocity, 1mL/min；Detection wavelength, 254nm.Solute: (1)
Toluene；(2) ethylbenzene；(3) propyl benzene；(4) butylbenzene；(5) penta benzene.
As shown in figure 16, the hydrophobic strength retention of stationary phase is with sulfonic acid base phase < octadecyl/sulfonic acid base phase < octadecyl
The sequence of phase increases, and reflects decisive contribution of the strong hydrophobic octadecyl to apolar interaction.
9 chromatographic evaluation of embodiment: ion exchange behavior
With dicyandiamide, melamine and melbine are molecular probe, measure they octadecane base phase, sulfonic acid base phase and
Retention in octadecyl/sulfonic acid base phase compares the intensity that the ion exchange of stationary phase retains.
Sample preparation: dicyandiamide, melamine and melbine 10mg are taken respectively, is placed in the volumetric flask of 10mL, adds second
Nitrile-pH 3, the mixed solution of 1.7% ammonium dihydrogen phosphate (30:70) are configured to the sample of 1mg/mL to graduation mark.
Chromatographic condition: stationary phase, (a) Octadecylsilane bonded phase, (b) sulfonic group Bonded Phase, (c) octadecyl/sulfonic group
Bonded Phase；15x 0.46cm；Mobile phase, -70% ammonium dihydrogen phosphate of 30% acetonitrile (1.7%, pH 3)；Flow velocity, 1mL/
min；Detection wavelength, 220nm.Solute: (1) dicyandiamide；(2) melamine；(3) melbine.
As shown in figure 17, the retention of melamine and melbine is all with octadecane base phase < sulfonic acid base phase < octadecane
Base/sulfonic acid base phase > sequence increase, reflect decisive tribute of the strong cation exchange group group's sulfonic group to electrostatic interaction
It offers.
10 chromatographic evaluation of embodiment: reverse phase/ion exchange mixed chromatogram retention behavior
The alkyl benzene amine of different chain length have hydrophobic grouping alkylbenzene and dissociable group amine, therefore, select they as
Molecular probe measures their retentions in octadecane base phase, sulfonic acid base phase and octadecyl/sulfonic acid base phase, compares fixation
The mixed mode chromatography strength retention of phase.
Sample preparation: 4- methylaniline 50mg, 4- ethyl aniline, 4- propyl aniline, 4- butylaniline and 4- penta are taken respectively
Base aniline 50L crosses the filter membrane of 0.22m, is configured to alkyl in 10mL volumetric flask, adding acetonitrile-water (40:60) to be diluted to graduation mark
Aniline homologue sample.
Chromatographic condition: stationary phase, (a) Octadecylsilane bonded phase, (b) sulfonic group Bonded Phase, (c) octadecyl/sulfonic group
Bonded Phase；15x 0.46cm；Mobile phase, -60% potassium dihydrogen phosphate of 40% acetonitrile (30mM, pH 2.5)；Flow velocity, 1mL/
min；Detection wavelength, 254nm.Solute: (1) 4- methylaniline；(2) 4- ethyl aniline；(3) 4- propyl aniline；(4) 4- butyl benzene
Amine；(5) 4- amyl aniline.
As shown in figure 18, under the same conditions, alkylbenzene only has faint reservation in single mode stationary phase, and in hybrid guided mode
Reservation in formula stationary phase greatly increases, and appearance order is directly proportional to the carbon chain lengths on alkylbenzene, reflects reverse phase/Qiang Yang
Decisive contribution of the ion exchange mixed-mode retention mechanism to chromatographic retention.
11 chromatographic applications of embodiment: common alkali medical separation
Chromatographic evaluation the result shows that, octadecyl/sulfonic group, which mixes stationary phase, has excellent guarantor to amino benzenes compounds
It stays and separating capacity, presents the application potential in Pharmaceutical Analysis.With common alkali drug salbutamol sulfate, clonidine,
Procainamide, Propranolol, amitriptyline are model compound, measure their reservations in octadecyl/sulfonic acid base phase
Value shows stationary phase to the separating effect of alkaline drug.
Sample preparation: salbutamol, clonidine, procainamide, Propranolol and amitriptyline 10mg are taken respectively, is placed in
In the volumetric flask of 10mL, add acetonitrile-pH 3, the mixed solution of 30mM potassium dihydrogen phosphate (30:70) crosses 0.22 to graduation mark
The filter membrane of m is configured to the sample of 1mg/mL.
Chromatographic condition: stationary phase, 18/sulfonic group Bonded Phase；15x 0.46cm；Mobile phase, -70% phosphoric acid of 30% acetonitrile
Dihydro potassium solution (30mM, pH 2.5)；Flow velocity, 1mL/min；Detection wavelength, 220nm.Solute: (1) salbutamol；(2) laughable
It is fixed；(3) procainamide；(4) Propranolol and (5) amitriptyline.
As shown in figure 19, five drugs have stronger reservation in mixed mode stationary phase, and peak type is more symmetrical, does not have
It is found the peak trailing phenomenon often occurred on conventional reverse-phase chromatographic column.Under isocratic condition, whole compounds are in 55min
Inside obtain baseline separation.
12 chromatographic applications of embodiment: the chromatographic isolation of compound methoxyphenamine capsules
Compound methoxyphenamine capsules are containing there are four effective components.Pass through point of the main component to compound methoxyphenamine capsules
From showing mixed chromatogram stationary phase of the invention to the unique advantage of multicomponent separation in compound medicine.
Sample preparation: taking this product 20 contents, accurately weighed, finely ground, and precision, which weighs, (is approximately equivalent to hydrochloric acid first in right amount
Oxygen that bright 25mg) it sets in 50ml volumetric flask, add acetonitrile 20ml ultrasound 5 minutes, add water 10ml ultrasound 5 minutes, adds dilution in acetonitrile extremely
Scale shakes up, and crosses 0.22m filter membrane, takes subsequent filtrate as sample, and precision measures 20l and injects liquid chromatograph.
Chromatographic condition: octadecyl/sulfonic group bonded-phase chromatography column, 15x 0.46cm；Mobile phase, 50% acetonitrile -50%
Potassium dihydrogen phosphate (30mM, pH 2.5)；Flow velocity, 1mL/min；Detection wavelength, 254nm.Solute: (1) aminophylline；(2) first
That is bright for oxygen；(3) coscopin；(4) chlorphenamine.
As shown in figure 20, the column of four components in mixed mode chromatographic column in compound methoxyphenamine capsules is imitated with theory
Number of plates meter is all larger than 15000/m, and tailing factor is respectively less than 1.4.Separating degree between component is all larger than 1.5.It is able to satisfy completely multiple
The identification of square methoxyphenamine and the requirement of assay.
13 chromatographic applications of embodiment: the chromatographic isolation of dihydralazine sulfate,clonidine and hydrochlorothiazine piece
Dihydralazine sulfate,clonidine and hydrochlorothiazine piece is containing there are three effective components.Pass through the main component to dihydralazine sulfate,clonidine and hydrochlorothiazine
Separation, further shows mixed chromatogram stationary phase of the invention to the unique advantage of multicomponent separation in compound medicine.
Sample preparation: taking 20 tablets of tablets of this product, finely ground, and precision weighs appropriate (about suitable Dihydralazine 25mg) and sets 50ml appearance
In measuring bottle, add methanol 20ml ultrasound 5 minutes, add water 10ml ultrasound 5min, adds methanol dilution to scale, shake up, cross 0.22m filter
Film takes subsequent filtrate as sample, and precision measures 20l and injects liquid chromatograph.
Chromatographic condition: octadecyl/sulfonic group mixed mode chromatographic column, 15x 0.46cm；Mobile phase, 50% acetonitrile-
50% potassium dihydrogen phosphate (30mM, pH 2.5)；Flow velocity, 1mL/min；Detection wavelength, 220nm.Solute: (1) Hydrochioro,
(2) clonidine, (3) Dihydralazine.
As shown in figure 21, the separating degree of 3 components in mixed mode chromatographic column in dihydralazine sulfate,clonidine and hydrochlorothiazine is both greater than
1.5, the column effect of clonidine and Dihydralazine is greater than 20000/m, and tailing factor is below 1.05.
13 chromatographic applications of embodiment: compound compound hypotensive is talked endlessly the chromatographic isolation of pyridine piece
Compound compound hypotensive talks endlessly pyridine piece containing there are four effective components.By talking endlessly main group of pyridine to compound compound hypotensive
The separation divided, further shows mixed chromatogram stationary phase of the invention to the unique advantage of multicomponent separation in compound medicine.
Sample preparation: taking 20 tablets of tablets of this product, finely ground, and precision weighs appropriate (about suitable Dihydralazine 25mg) and sets 50mL appearance
In measuring bottle, add methanol 20mL ultrasound 5min, add water 10mL ultrasound 5min, methanol dilution is added to shake up to scale, crosses 0.22m filter membrane,
Take subsequent filtrate as sample, precision measures 20l and injects liquid chromatograph.
Chromatographic condition: octadecyl/sulfonic group mixed mode chromatographic column, 15x 0.46cm；Mobile phase, 50% methanol-
50% potassium dihydrogen phosphate (30mM, pH 2.5)；Flow velocity, 1mL/min；Detection wavelength, 268 or 310nm.Solute: (1) hydrogen chlorine
Thiazine；(2) Dihydralazine；(3) dyrenium and (4) reserpine.
As shown in figure 22, in mixed mode chromatographic column, when Detection wavelength is 268nm, compound compound hypotensive is talked endlessly in pyridine
Four component separating degrees be both greater than 1.5, Dihydralazine, dyrenium and its column of reserpine effect are both greater than 15000/m, hangover because
Son is below 1.3.
Figure 23 is that compound compound hypotensive is talked endlessly separation chromatogram of the pyridine piece when Detection wavelength is 310nm.With this condition,
Reserpine cannot detect.Compound compound hypotensive other three component separating degrees in pyridine piece of talking endlessly are both greater than 1.5, Dihydralazine and
Dyrenium column effect is both greater than 15000/m, and tailing factor is 1.0 or so.
14 chromatographic applications of embodiment: the chromatographic isolation and Mass Spectrometric Identification of effective component in FUFANG LIXUEPING PIAN
FUFANG LIXUEPING PIAN is compound preparation, contains reserpine, Hydrochioro, vitamin B6, Calciipantothenas Racemicus, three silicic acid
Magnesium, potassium chloride, vitamin B1, dihydralazine sulfate and promethazine hydrochloride.Utilize gradient elution mode chromatography and mass spectrometry skill
Art is separated and is identified to the main component in FUFANG LIXUEPING PIAN, is further showed mixed mode stationary phase of the invention and is existed
Unique advantage in compound medicine separation analysis.
Sample preparation: taking this product 10, in 25mL volumetric flask, adds methanol 10mL ultrasound 5min, adds water 10mL ultrasound 5min,
Add methanol dilution to scale, shakes up, cross 0.22m filter membrane, take subsequent filtrate as sample, precision measures 20l and injects liquid chromatograph.
Chromatographic condition: octadecyl/sulfonic group mixed mode chromatographic column, 15x 0.46cm；Mobile phase, 50mM ammonium formate
It (pH3) is mobile phase A, acetonitrile is Mobile phase B, and gradient is 0-3min 0%B, 3-7min 40%B, 7-40min 40%
B, 40-45min 0%B；Flow velocity, 1ml/min；Detection wavelength, 268nm,
Mass Spectrometer Method: positive ion mode, scanning range 100m/z-1000m/z.
Figure 24 is the ultraviolet chromatogram of FUFANG LIXUEPING PIAN.Under condition of gradient elution, seven colors can be clearly separated out
Spectral peak.
The chromatographic peak in Figure 24 is identified using mass spectrometric hyphenated technique.Figure 25 be corresponding chromatographic peak ion flow graph and
Its mass spectrogram.By spectrum analysis, component corresponding to peak in ultraviolet chromatogram: (1) Hydrochioro, the life of (2) dimension can be determined
Plain B6, (3) Dihydralazine, (4) reserpine, (6) vitamin B1, (7) fenazil, enumerate in addition to pantothenic acid it is all it is organic at
Point.Pantothenic acid is in Detection wavelength 268nm without absorption.Chromatographic peak 5 is impurity peaks, may be from excipient substance.
Claims (8)
1. a kind of reverse phase/ion exchange mixed mode chromatographic stationary phases, referred to as octadecyl/sulfonic group mixed chromatogram are fixed
Phase is made of, chemical structural formula the octadecyl and sulfonic group that are bonded in Bio-sil surface are as follows:
。
2. chromatographic stationary phases according to claim 1, which is characterized in that preparation method are as follows: pass through chemical vapor deposition
γ-(2,3- the third oxygen of epoxy) propyl trimethoxy silicane KH-560 is bonded to Bio-sil microparticle surfaces by method, is with KH-560
Linking arm is reacted with sodium hydrogensulfite and octadecyl chloride respectively, prepares octadecyl/sulfonic group mixed chromatogram stationary phase.
3. the preparation method of chromatographic stationary phases described in claim 1, comprises the following steps that
(1) a certain amount of aerosil particles: being added the back flow reaction of 80 DEG C -105 DEG C of progress in HCl solution by the preparation of activated silica gel,
It is washed with water to neutrality, vacuum drying；
(2) preparation of epoxy group silica gel: by the aerosil particles of above-mentioned acidification, the γ-of every gram of addition 1.92mmol-3.6mmol (2,
The third oxygen of 3- epoxy) propyl trimethoxy silicane, reaction is sealed at 140 DEG C -160 DEG C, is washed with dehydrated alcohol, production is collected by filtration
Product are simultaneously dried in vacuo, and obtain Epoxy functionalized Bio-sil microballoon；
(3) preparation of sulfonic group silica gel: it is 8-10 times that above-mentioned Epoxy functionalized Bio-sil microballoon, which is distributed to mass number,
In ultrapure water, add according to the amount that 1.92mmol-3.6mmol sodium hydrogensulfite is added in every gram of Epoxy functionalized Bio-sil microballoon
Enter sodium hydrogensulfite, mix, using triethylamine tune pH value to 7-8,85 DEG C of reactions are washed with water and are dried in vacuo, obtain sulfonic acid
Base bonded silica gel microballoon；
(4) octadecyl/sulfonic group silica gel preparation: it is 8-10 times that above-mentioned sulfonic group bonded silica gel microballoon, which is dispersed in mass number,
N,N-Dimethylformamide (DMF) in, under condition of ice bath, be added according to every gram of sulfonic group bonded silica gel microballoon
Octadecyl chloride is added dropwise in the amount of 1.92mmol-3.6mmol octadecyl chloride；After completion of dropwise addition, 40-65 DEG C is stirred to react,
Product is collected by filtration, after successively with toluene and ethanol washing and be dried in vacuo, obtain octadecyl/sulfonic group mixed mode chromatography
Stationary phase.
4. utilizing reverse phase/ion exchange blended liquid phase chromatographic column made from chromatographic stationary phases described in claim 1.
5. chromatographic column according to claim 4, which is characterized in that the preparation of the chromatographic column uses high-pressure homogenization,
Process conditions are as follows: according to n-hexyl alcohol: the quality proportioning 6:4 of carbon tetrachloride prepares homogenate dress, using n-hexane as displacement fluid, fills column
Pressure is 300bar.
6. reverse phase described in claim 1/ion exchange blended liquid phase chromatographic column is applied to medical separation.
7. application according to claim 1, which is characterized in that the drug is salbutamol sulfate, clonidine, Pu Lu
Cacaine amine, Propranolol or amitriptyline.
8. application according to claim 6, which is characterized in that the drug is compound medicine, is Compound Methoxyphenamine
Capsule, dihydralazine sulfate,clonidine and hydrochlorothiazine piece, compound compound hypotensive are talked endlessly reserpine in pyridine piece, and compound compound hypotensive is talked endlessly hydrogen in pyridine piece
Chlorothiazide, dihydralazine sulfate or dyrenium.
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