CN110013836B - Reversed phase/ion exchange mixed mode chromatographic stationary phase, preparation method and application - Google Patents

Reversed phase/ion exchange mixed mode chromatographic stationary phase, preparation method and application Download PDF

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CN110013836B
CN110013836B CN201910238170.6A CN201910238170A CN110013836B CN 110013836 B CN110013836 B CN 110013836B CN 201910238170 A CN201910238170 A CN 201910238170A CN 110013836 B CN110013836 B CN 110013836B
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octadecyl
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万谦宏
张双红
陈磊
张高乐
焦亚磊
丁超
李宏亮
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Tianjin University
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Abstract

The invention relates to a reversed phase/ion exchange mixed mode chromatographic stationary phase, a preparation method and application, wherein the reversed phase/ion exchange mixed mode chromatographic stationary phase is called as an octadecyl/sulfonic group mixed chromatographic stationary phase and consists of octadecyl and sulfonic group bonded on the surface of porous silica gel, and the preparation method comprises the following steps: bonding gamma- (2, 3-epoxypropoxy) propyl trimethoxy silane KH-560 on the surface of the porous silica gel particles by a chemical vapor deposition method, and respectively reacting with sodium bisulfite and octadecyl chloride by taking KH-560 as a connecting arm to prepare the octadecyl/sulfonic group mixed chromatographic stationary phase.

Description

Reversed phase/ion exchange mixed mode chromatographic stationary phase, preparation method and application
Technical Field
The invention relates to a liquid chromatogram stationary phase and a preparation method and application thereof.
Background
The drug analysis technology plays an important role in the quality control of the drug during the development, production, storage and circulation processes. In order to improve the treatment effect of the drugs on diseases, the combination of different single-component drugs becomes a widely applied strategy, but the combination causes the drug compliance to be reduced and the risk of patients to be increased. The fixed dose compound medicine developed in recent years is a single dosage form, such as a capsule or a pill, containing two or more active ingredients. The medicine can improve the treatment effect of the medicine by utilizing the synergistic effect of different medicine components and reduce the number of tablets taken by patients, so that the medicine is popular with doctors and patients, and the products comprise anti-AIDS medicines, anti-hypertension medicines and anti-cold medicines. Because the compound medicine contains a plurality of active ingredients, compared with a single-component medicine, the difficulty of quality control is greatly increased, and an applicable analysis method is urgently needed to be developed.
According to the records of the 'Chinese pharmacopoeia' 2015 edition and related documents, the most widely applied analysis technique in the quality control of single-component drugs, including identification, content determination and impurity analysis, is reverse phase liquid chromatography, for example, reverse phase chromatography using an octadecyl bonded silica gel stationary phase, but the reverse phase chromatographic column has weak retention on some polar or ionic compounds, and due to the electrostatic interaction between the dissociation of the residual silicon hydroxyl on the surface of the column and a basic drug, the chromatographic peak is trailing, the resolution of chromatographic separation is affected, and the simultaneous analysis of neutral and polar ionic compounds in compound drugs cannot be realized. To increase retention of ionic compounds, an ionic surfactant is typically added to the mobile phase of reverse phase chromatography to achieve simultaneous analysis of neutral and ionic compounds by forming an ion pair with the opposite ionic compound being analyzed. However, the ion pair chromatography has two fatal defects, so that the application of the ion pair chromatography in compound drug analysis is greatly limited. One is that the dynamic adsorption equilibrium of ions to reagents on the surface of a stationary phase is involved in the separation process, and the equilibrium time is long, so that gradient elution cannot be used in the actual drug separation process, and components with different polarities or retention capacities can be analyzed simultaneously. Secondly, because the ion pair reagent is difficult to volatilize, the ion source pollution is caused in the ionization process, so that the ion pair chromatography cannot be combined with the mass spectrum, and the application of the liquid chromatography-mass spectrum combination technology to the structure identification of different components is limited.
Mixed mode chromatography is the preferred method for simultaneous analysis of neutral and ionic compounds by displacement ion pair chromatography. Unlike conventional single mode chromatography, mixed mode chromatography uses a multifunctional cluster chromatography stationary phase, the retention mechanism of which involves intermolecular interactions of two or more different types, and thus has a stronger separation ability. The most commonly used mixed mode chromatography is reverse phase/ion exchange chromatography, using a stationary phase whose surface contains hydrophobic and dissociable functional groups, thus enabling the retention and simultaneous separation of neutral and ionic compounds by the synergistic effect of van der waals and electrostatic forces. Because the surface active agent does not need to be added into the mobile phase, the problems in ion pair chromatography do not exist, the gradient elution analysis can be used, and the mass spectrum can be combined with the mass spectrum, so that the method has wide application prospect in the drug analysis. However, the mixed-mode chromatography stationary phase has few varieties and single structure so far, and cannot meet the increasing analysis requirements of complex components, especially compound medicines, in the field of pharmaceutical analysis.
Disclosure of Invention
The invention aims to provide a novel mixed-mode chromatographic stationary phase, and provides a simple and feasible preparation method and application thereof in the field of pharmaceutical analysis.
A kind of reversed phase/ion exchange mixed mode chromatogram fixed phase, called octadecyl/sulfonic group mixed chromatogram fixed phase, is composed of octadecyl and sulfonic group bonded on the surface of porous silica gel, and its chemical structural formula is:
Figure BDA0002008849550000021
the preparation method of the chromatographic stationary phase comprises the following steps:
(1) preparation of activated silica gel: adding a certain amount of silica gel particles into HCl solution to carry out reflux reaction at the temperature of 80-105 ℃, washing with water to be neutral, and drying in vacuum;
(2) preparation of epoxy silica gel: adding 1.92mmol-3.6mmol of gamma- (2, 3-epoxypropoxy) propyl trimethoxy silane into each gram of the acidified silica gel particles, sealing and reacting at 140-160 ℃, washing with absolute ethyl alcohol, filtering and collecting a product, and drying in vacuum to obtain the epoxy group functionalized porous silica gel microspheres;
(3) preparation of sulfonic silica gel: dispersing the epoxy group functionalized porous silica gel microspheres into ultrapure water with the mass number of 8-10 times, adding sodium bisulfite according to the amount of 1.92-3.6 mmol sodium bisulfite added into each gram of epoxy group functionalized porous silica gel microspheres, uniformly mixing, adjusting the pH value to 7-8 by using triethylamine, reacting at 85 ℃, washing with water and drying in vacuum to obtain sulfonic group bonded silica gel microspheres;
(4) preparation of octadecyl/sulfonic acid group silica gel: dispersing the sulfonic bonded silica gel microspheres in N, N-Dimethylformamide (DMF) with the mass number of 8-10 times, and dropwise adding octadecyl chloride according to the amount of 1.92-3.6 mmol of octadecyl chloride added to each gram of sulfonic bonded silica gel microspheres under the ice bath condition; and after the dropwise addition is finished, stirring and reacting at 40-65 ℃, filtering and collecting a product, washing with toluene and ethanol in sequence, and drying in vacuum to obtain the octadecyl/sulfonic group mixed mode chromatographic stationary phase.
The chromatographic stationary phase can be used for preparing a reversed phase/ion exchange mixed liquid chromatographic column.
The preparation of the chromatographic column adopts a high-pressure homogenization method, and the process conditions are as follows: according to n-hexanol: the mass ratio of carbon tetrachloride is 6: 4 preparing homogenate liquid, taking normal hexane as a displacement liquid, and loading the column at the pressure of 300 bar.
The reversed phase/ion exchange mixed liquid chromatographic column is applied to medicine separation.
The mixed-mode stationary phase provided by the invention takes the epoxy group as a connecting arm, combines the octadecyl long chain, and has higher flexibility and hydrophobicity compared with the phenyl group in the traditional benzenesulfonic acid bonding phase, and stronger synergistic effect with the separated alkaline compound, so that the separation effect is better. In addition, since hydrophilic groups such as ether bonds and hydroxyl groups are present in the linker arms, polar molecules in the mobile phase are easily bound, and therefore the problem of decreased retention of the analyte caused by collapse of the octadecyl chain at higher water content in the mobile phase can be prevented.
The invention adopts a gas phase chemical deposition method to carry out silanization reaction on porous silica gel, so that the porous silica gel is combined with an epoxy group with larger chemical activity, and then the octadecyl/sulfonic group mixed mode chromatographic stationary phase is prepared by utilizing the ring opening reaction of the epoxy group and the condensation reaction of hydroxyl and acyl chloride. Compared with the conventional liquid phase reaction method, the chemical vapor deposition method avoids using toxic and harmful organic solvents such as toluene and the like, and greatly reduces the harm to human bodies and the pollution to the environment. The epoxy functional silica gel is used as a mesophase to bridge reversed phase and ion exchange groups, so that the synthesis steps are simplified, and the proportion of the reversed phase and the ion exchange groups is adjustable. The preparation method has the advantages of cheap and easily obtained raw materials, simple and feasible process route and easy large-scale industrial production.
The stationary phase can be applied to simultaneous separation and determination of multiple components in a fixed-dose compound medicine. Ion-pair chromatography or multiple separations are often required in current pharmacopoeias to perform multi-component analysis. The stationary phase of the invention can be used for simultaneous separation of multiple components, has a separation effect superior to that of the traditional ion pair reversed phase chromatography, can be used for performing qualitative and quantitative one-time analysis on compound medicines on line by adopting gradient elution and being directly combined with mass spectrometry, and saves a large amount of analysis time and operation cost.
Drawings
FIG. 1 shows a process for preparing a mixed-mode chromatographic stationary phase.
FIG. 2 example 1 Infrared characterization of activated microspheres
FIG. 3 Infrared characterization of epoxy-based silica gel microspheres of example 2
FIG. 4 example 3 chemical reaction scheme for preparation of diol-based functional group
FIG. 5 example 3 Infrared characterization of diol-based silica gel microspheres
FIG. 6 example 3 Nuclear magnetic characterization of diol-based silica gel microspheres
FIG. 7 example 4 chemical reaction formula for preparation of octadecyl functionalized microsphere
FIG. 8 example 4 Infrared characterization of octadecyl functionalized silica gel microspheres
FIG. 9 example 4 Nuclear magnetic characterization of octadecyl functionalized silica gel microspheres
FIG. 10 example 5 chemical reaction scheme for preparation of sulfonic acid functional group
FIG. 11 example 5 Infrared characterization of sulfonic acid functionalized silica gel microspheres
FIG. 12 example 5 Nuclear magnetic characterization of sulfonic acid functionalized silica gel microspheres
FIG. 13 chemical reaction formula for preparing octadecyl sulfonic acid group functionalized microsphere in example 6
FIG. 14 Infrared characterization of octadecyl/sulfo silica gel microspheres of example 6
FIG. 15 Nuclear magnetic characterization of octadecyl/sulfonic acid based silica gel microspheres of example 6
TABLE 1 elemental analysis characterization of examples 2-6
FIG. 16 separation of alkylbenzene homologs on octadecyl, sulfonic acid, and octadecyl/sulfonic acid chromatography columns.
Figure 17 separation of dicyandiamide, melamine and metformin on octadecyl, sulfonic and octadecyl/sulfonic chromatography columns.
FIG. 18 separation of aniline homologs on octadecyl, sulfonic acid, and octadecyl/sulfonic acid chromatography columns.
FIG. 19 separation of common basic drugs on octadecyl/sulfonic acid chromatography column.
FIG. 20 separation of Compound Methanosine capsules on octadecyl/sulfonic acid chromatography columns.
FIG. 21 separation of compound dihydralazine tablets on octadecyl/sulfonic acid chromatography column.
FIG. 22 separation of the compound reserpine triamterene pteridine tablet on an octadecyl/sulfonic acid group chromatographic column, the detection wavelength is 268 nm.
FIG. 23 separation of the compound reserpine triamterene pteridine tablet on an octadecyl/sulfonic acid group chromatographic column, the detection wavelength is 310 nm.
FIG. 24 is an ultraviolet chromatogram of the separation of the FUFANGLIXUEPING tablet on an octadecyl/sulfonic acid group chromatographic column under the condition of gradient elution.
FIG. 25 is an ion-flow diagram and corresponding mass spectrum of the organic components of the FUFANGLIXUEPING tablet.
Detailed Description
The invention provides a reversed phase/ion exchange mixed mode chromatographic stationary phase with a novel structure, wherein a hydrophobic and soft alkyl hydrocarbon long chain is bonded at the periphery of an ionic functional group, and can react with an analyzed compound through a synergistic reversed phase and ion exchange retention mechanism, so that the chromatographic stationary phase has high chromatographic selectivity and is suitable for simultaneous analysis and content determination of multiple components in a compound medicament. The invention provides a simple and feasible preparation technology of the mixed-mode chromatographic stationary phase, adopts dry derivatization reaction to replace the traditional wet method, avoids using toxic and harmful organic solvents, and protects health and environment. The preparation technology has simple and controllable reaction route, cheap and easily obtained raw materials, and is suitable for industrial large-scale production. The invention further aims to provide the unique application of the mixed-mode chromatographic stationary phase in pharmaceutical analysis, which can realize gradient elution analysis and mass spectrum combination, and realize rapid content determination and structure identification of multiple components in compound medicines.
The mixed chromatographic stationary phase prepared by the invention consists of octadecyl and sulfonic group bonded on the surface of porous silica gel. Firstly, gamma- (2, 3-epoxypropoxy) propyl trimethoxy silane (KH-560) is bonded on the surface of porous silica gel particles by a chemical vapor deposition method, and respectively reacts with sodium bisulfite and octadecyl chloride by taking KH-560 as a connecting arm to prepare an octadecyl/sulfonic group mixed chromatographic stationary phase which is applied to compound medicine separation. The preparation process is shown in figure 1. The specific operation steps are as follows:
preparation of activated silica gel: adding a certain amount of silica gel particles into 10-15% HCl solution with the mass 8-10 times of the silica gel particles, stirring, carrying out reflux reaction at 105 ℃ for 8h, washing with water to be neutral, and carrying out vacuum drying at 80 ℃.
Preparation of epoxy silica gel: putting the acidified silica gel particles into an autoclave with a Teflon liner, adding 1.92-3.6 mmol of gamma- (2, 3-epoxypropoxy) propyl trimethoxy silane per gram, sealing the autoclave, and reacting at 150 ℃ for 8 h. Washing away unreacted silanization reagent with absolute ethyl alcohol, filtering and collecting the product, and drying in vacuum at 80 ℃ to obtain the epoxy group functionalized porous silica gel.
Preparation of sulfonic silica gel: dispersing the bonded epoxy silica gel microspheres into ultrapure water with the mass number of 8-10 times, adding 1.92-3.6 mmol of sodium bisulfite into each gram, ultrasonically mixing uniformly, adjusting the pH value to 7-8 by triethylamine, reacting for 8 hours at 85 ℃, washing by distilled water, and drying in vacuum at 120 ℃ to obtain the sulfonic group bonded silica gel microspheres.
Preparation of octadecyl/sulfonic acid group silica gel: dispersing the sulfonic functional silica gel microspheres in N, N-Dimethylformamide (DMF) with the mass number of 8-10 times, and slowly dropwise adding octadecyl chloride with the mass number of 1.92-3.6 mmol per gram under the ice bath condition. And after the dropwise addition is finished, stirring and reacting for 9h at 60 ℃, filtering and collecting a product, washing the product with toluene and ethanol in sequence, and drying the product in vacuum at 80 ℃ to obtain the octadecyl/sulfonic group mixed mode chromatographic stationary phase.
Preparing a high-pressure liquid chromatographic column: and filling the chromatographic stationary phase into a stainless steel column tube by adopting a conventional high-pressure homogenization method to obtain the reversed-phase/ion exchange mixed liquid chromatographic column. The specific conditions are n-hexanol: carbon tetrachloride 6: 4, loading homogenate liquid, using normal hexane as displacement liquid, and loading column pressure of 300 bar.
Reverse phase chromatography behavior of high pressure liquid chromatography column: benzene homologues (toluene-pentylbenzene) with different chain lengths are used as molecular probes, and the retention values of the benzene homologues on an octadecyl stationary phase, a sulfonic stationary phase and an octadecyl/sulfonic stationary phase are measured, and the retention strengths of reversed phase chromatography of the stationary phases are compared.
Chromatographic conditions are as follows: a stationary phase, (a) an octadecyl bonding phase, (b) a sulfonic acid group bonding phase, (c) an octadecyl/sulfonic acid group bonding phase; 15x 0.46 cm; mobile phase, 60% methanol-40% water; flow rate, 1 mL/min; detection wavelength, 254 nm.
Ion exchange chromatography behavior of high pressure liquid chromatography column: dicyandiamide, melamine and metformin are taken as molecular probes, retention values of dicyandiamide, melamine and metformin on an octadecyl phase, a sulfonic acid phase and an octadecyl/sulfonic acid phase are measured, and the ion exchange retention strength of a stationary phase is compared.
Chromatographic conditions are as follows: a stationary phase, (a) an octadecyl bonding phase, (b) a sulfonic acid group bonding phase, (c) an octadecyl/sulfonic acid group bonding phase; 15x 0.46 cm; mobile phase, 30% acetonitrile-70% ammonium dihydrogen phosphate solution (1.7%, pH 3); flow rate, 1 mL/min; detection wavelength, 220 nm.
Reversed phase/ion exchange mixed chromatography retention behavior of high pressure liquid chromatography column: alkylanilines of different chain lengths (methylaniline-pentylaniline) have hydrophobic groups alkylbenzene and dissociable groups amine, and therefore, they were selected as molecular probes, their retention values on octadecyl, sulfonic, and octadecyl/sulfonic phases were determined, and mixed mode chromatography retention strengths of the stationary phases were compared.
Chromatographic conditions are as follows: a stationary phase, (a) an octadecyl bonding phase, (b) a sulfonic acid group bonding phase, (c) an octadecyl/sulfonic acid group bonding phase; 15x 0.46 cm; mobile phase, 40% acetonitrile-60% potassium dihydrogen phosphate solution (30mM, pH 2.5); flow rate, 1 mL/min; detection wavelength, 254 nm.
Isolation of common basic drugs: common basic drugs of salbutamol sulfate, clonidine, procainamide, propranolol and amitriptyline are used as model compounds, retention values of the common basic drugs on an octadecyl/sulfonic acid group phase are measured, and the separation effect of the fixed relative basic drugs is shown.
Chromatographic conditions are as follows: a stationary phase, an octadecyl/sulfonic acid group bonding phase; 15x 0.46 cm; mobile phase, 30% acetonitrile-70% potassium dihydrogen phosphate solution (30mM, pH 2.5); flow rate, 1 mL/min; detection wavelength, 220 nm.
Separating the compound methoxyphenamine capsules: precisely weighing the content of the compound methoxamine capsule, grinding to obtain a sample with concentration about equal to 0.1mg/mL-1mg/mL of methoxamine hydrochloride, filtering with 0.22m filter membrane, taking the subsequent filtrate as the sample, precisely measuring 10-30l, and injecting into a liquid chromatograph. Chromatographic conditions are as follows: a carbo-octadeca/sulfonic acid mixed mode chromatographic column; using 50% potassium dihydrogen phosphate buffer salt (30mM, pH2.5) -50% acetonitrile as mobile phase; flow rate, 1 mL/min; the detection wavelength was 230 nm.
Separating the compound dihydralazine sulfate tablets: taking compound dihydralazine sulfate tablets, grinding, precisely weighing a proper amount to prepare a sample with the concentration about equivalent to 0.1mg/mL-1mg/mL of dihydralazine, filtering with a 0.22m filter membrane, taking a subsequent filtrate as the sample, precisely weighing 10-30l of the subsequent filtrate, and injecting into a liquid chromatograph. Chromatographic conditions are as follows: a carbooctadecanesulfonic acid group mixed mode chromatographic column; using 50% potassium dihydrogen phosphate buffer salt (30mM, pH2.5) -50% acetonitrile as mobile phase; flow rate, 1 mL/min; the detection wavelength was 220 nm.
Analysis of reserpine in the compound reserpine and triamterene pteridine tablets: the compound reserpine and triamterene pteridine tablet is ground, a proper amount of 0.1mg/mL-1mg/mL is precisely weighed, the mixture is filtered through a 0.22m filter membrane, a subsequent filtrate is taken as a sample, and 10-30l is precisely weighed and injected into a liquid chromatograph. Chromatographic conditions are as follows: a carbooctadecanesulfonic acid group mixed mode chromatographic column; using 50% potassium dihydrogen phosphate buffer salt (30mM, pH2.5) -50% acetonitrile as mobile phase; flow rate, 1 mL/min; the detection wavelength was 268 nm.
The hydrochlorothiazide, the dihydralazine sulfate and the triamterene in the compound reserpine triamterene tablet are ground, a proper amount of 0.1-1 mg/mL is precisely weighed, the mixture is filtered through a 0.22m filter membrane, the subsequent filtrate is taken as a sample, and 10-30l is precisely measured and injected into a liquid chromatograph. Chromatographic conditions are as follows: a carbooctadecanesulfonic acid group mixed mode chromatographic column; using 50% potassium dihydrogen phosphate buffer salt (30mM, pH2.5) -50% acetonitrile as mobile phase; flow rate, 1 mL/min; the detection wavelength was 310 nm.
Separating the compound reserpine tablets: the compound reserpine tablet is ground, a proper amount of sample which is equivalent to the concentration of the dihydralazine of 0.1mg/mL-1mg/mL is precisely weighed, the sample is filtered through a filter membrane of 0.22m, the subsequent filtrate is taken as the sample, and 10-30l is precisely measured and injected into a liquid chromatograph. Chromatographic conditions are as follows: a carbooctadecanesulfonic acid group mixed mode chromatographic column; 50mM ammonium formate (pH3) is used as a mobile phase A, acetonitrile is used as a mobile phase B, and the elution gradient is 0-3min 0% B, 3-7min 40% B,7-40min 40% B, and 40-45min 0% B; flow rate, 1 mL/min; the detection wavelength is 268nm and the mass spectrum is combined to detect in a positive ion mode.
In order to better understand the technique of the present invention, the present invention is further illustrated by the following examples, but the present invention is not limited to the following examples.
Example 1 activation of silica gel microspheres
Adding 10g of silica gel microspheres into a 250mL three-necked bottle, adding 100mL of 10% HCl solution, mechanically stirring, refluxing at 105 ℃ for 8h, washing with water to neutrality, vacuum drying at 120 ℃ overnight to obtain activated silica gel microspheres, wherein the infrared characterization spectrum of the acidified silica gel microspheres is shown in FIG. 2 and is at 3400cm-1Has a larger absorption peak of silicon hydroxyl.
Example 2 Dry preparation of epoxy-bonded silica gel microspheres
10g of the activated silica gel microspheres prepared in example 1 were placed in an autoclave, and 5.31mL of gamma- (2, 3-glycidoxy) propyltrimethoxysilane was added and reacted at 150 ℃ for 8 hours. Washing with absolute ethyl alcohol, and drying in vacuum at 80 ℃ to obtain the epoxy group functionalized silica gel microspheres. A spectrum of infrared characterization of epoxy-functionalized silica gel at 2800cm after bonding and epoxy is shown in FIG. 3-1And 2890cm-1C-H stretching vibration peak appears nearby, which indicates epoxy bond and silica gel surface, the element analysis is characterized as shown in Table 1, the carbon content is 8.05%, and the calculated surface bond sum is 4.50mol/m2. The method has the outstanding advantages that harmful organic solvents such as toluene and the like are not used, and the method is favorable for protecting health and environment.
EXAMPLE 3 preparation of diol-bonded silica gel microspheres
10g of epoxy-functionalized microspheres obtained as in example 2 were dispersed in 100mL of 0.5% H2SO4Heating to react for 8h, washing to neutrality with water, vacuum drying at 80 deg.C to obtain diol-based bonded silica gel microsphere with reaction equation shown in FIG. 4, and infrared characterization of diol-based stationary phase shown in FIG. 5 at 3400cm-1Has a distinct O-H peak at 2800cm-1And 2890cm-1The C-H stretching vibration peak appears nearby, the nuclear magnetic characterization map has obvious resonance peak as shown in figure 6, the element analysis characterization data is shown in table 1, the carbon content is 5.52 percent, the surface bond and the density are 2.95mol/m2It shows that the epoxy chain has a certain shedding under the acidic condition.
Example 4 preparation of octadecyl functionalized microspheres
4g of the di-product prepared as in example 3 were takenDispersing the alcohol-based microspheres in 25mL of N, N-Dimethylformamide (DMF), and slowly dropwise adding 6mL of octadecyl chloride under the ice bath condition; after the dropwise addition, the mixture was heated to raise the temperature and reacted at 60 ℃ for 9 hours. Washing the octadecyl functionalized silica gel microspheres with DMF and ethanol in sequence, and drying at 80 ℃ to obtain the octadecyl functionalized silica gel microspheres, wherein the reaction chemical equation is shown in figure 7, the infrared characterization of the octadecyl functionalized microspheres is shown in figure 8, the octadecyl functionalized silica gel microspheres have obvious C-H stretching vibration peaks, the nuclear magnetic characterization map is shown in figure 9, the nuclear magnetic peaks of carbon with octadecyl chains are obvious, and the results of element analysis are shown in table 1, wherein the bond and the density of the octadecyl are 1.08mol/m2。
Example 5 preparation of sulfonic acid functionalized microspheres
Dispersing 10g of the epoxy-bonded silica gel microspheres prepared according to example 2 into 100mL of ultrapure water, sequentially adding 5g of sodium bisulfite, ultrasonically mixing, adjusting the pH value to 7-8 with triethylamine, heating, reacting at 85 ℃ for 8H, washing with distilled water, and vacuum drying at 120 ℃ for 8H to obtain the sulfonic-bonded silica gel microspheres, wherein the reaction chemical equation is shown in FIG. 10, the infrared characterization map is shown in FIG. 11, the microspheres have obvious C-H stretching vibration peaks and are 650cm in length-1The characteristic peak of sulfonic acid group is shown in figure 12, the nuclear magnetic resonance spectrum shows that two shoulder peaks are separated when the peak is about 40ppm, the sulfonic acid group is caused, and the element analysis and characterization are shown in table 1.
Example 6 preparation of octadecyl/sulfonic acid functionalized microspheres (octadecyl: sulfonic acid group ═ 1: 2)
4g of the sulfonic acid group microsphere prepared in example 5 was dispersed in 25mL of N, N-Dimethylformamide (DMF), and 6mL of octadecyl chloride was slowly added dropwise under ice bath conditions; after the dropwise addition, the mixture was heated to raise the temperature and reacted at 60 ℃ for 9 hours. Washing with DMF and ethanol in sequence, and drying at 80 ℃ to obtain the octadecyl/sulfonic group silica gel microspheres. The reaction equation is shown in figure 13, the infrared characterization map has C-H stretching vibration peak and sulfonic characteristic absorption peak as shown in figure 14, the nuclear magnetic characterization map has shown in figure 15, the nuclear magnetic resonance peak of octadecyl chain appears obviously around 60ppm, and the peak is split into two peaks around 50ppm, which indicates the influence of sulfonic acid group, the element analysis characterization result is shown in table 1, the carbon loading is 10.93%,the sulfur loading was 0.81% and the calculated bond sum amount for carbo-octadecane was 0.67mol/m2The sum of sulfonic acid groups being 1.13mol/m2。
TABLE 1 elemental analysis characterization
Figure BDA0002008849550000081
Example 7 Wet preparation of epoxy-bonded silica gel microspheres
10g of the activated porous silica gel prepared in example 1 was taken and placed in a 100mL three-necked flask, and a condenser tube and a drying tube were installed. 100mL of anhydrous toluene and 5.31mL of gamma- (2, 3-glycidoxy) propyltrimethoxysilane were added. Mechanically stirred and heated to 110 ℃ under the protection of nitrogen for reaction for 9 h. After cooling, the mixture was filtered and washed with toluene and methanol in this order. Drying at 60 deg.C under vacuum for 12 h. And obtaining the epoxy group functionalized silica gel. The disadvantage of this process is the consumption of toluene, a toxic solvent, in large quantities compared to the dry process of example 2.
Example 8 chromatographic evaluation: reversed phase retention behavior
Benzene homologues with different chain lengths are taken as molecular probes, retention values of the benzene homologues on a mesophase (octadecyl stationary phase and sulfonic stationary phase) and a mixed chromatographic stationary phase (octadecyl/sulfonic stationary phase) prepared by the method are measured, and reversed-phase chromatographic retention strength of the stationary phases is compared.
Sample preparation: 50L of toluene, ethylbenzene, propylbenzene, butylbenzene and pentylbenzene are respectively taken to be placed in a 10mL volumetric flask, methanol-water (60: 40) is added to be diluted to scale marks, and the mixture is filtered through a filter membrane of 0.22m to prepare an alkylbenzene homologous sample.
Chromatographic conditions are as follows: a stationary phase, (a) an octadecyl bonding phase, (b) a sulfonic acid group bonding phase, (c) an octadecyl/sulfonic acid group bonding phase; 15x 0.46 cm; mobile phase, 60% methanol-40% water; flow rate, 1 mL/min; detection wavelength, 254 nm. Solute: (1) toluene; (2) ethylbenzene; (3) propyl benzene; (4) butylbenzene; (5) and (3) pentylbenzene.
As shown in fig. 16, the hydrophobic retention strength of the stationary phase increased with the sequence of sulfonate phase < octadecyl/sulfonate phase < octadecyl phase, reflecting the decisive contribution of the strongly hydrophobic octadecyl to the non-polar interactions.
Example 9 chromatographic evaluation: ion exchange behavior
Dicyandiamide, melamine and metformin are taken as molecular probes, retention values of dicyandiamide, melamine and metformin on an octadecyl phase, a sulfonic acid phase and an octadecyl/sulfonic acid phase are measured, and the ion exchange retention strength of a stationary phase is compared.
Sample preparation: 10mg of dicyandiamide, 10mg of melamine and 10mg of metformin are respectively put into a 10mL volumetric flask, and a mixed solution of acetonitrile-pH 3 and 1.7% ammonium dihydrogen phosphate (30:70) is added to the scale mark to prepare a sample of 1 mg/mL.
Chromatographic conditions are as follows: a stationary phase, (a) an octadecyl bonding phase, (b) a sulfonic acid group bonding phase, (c) an octadecyl/sulfonic acid group bonding phase; 15x 0.46 cm; mobile phase, 30% acetonitrile-70% ammonium dihydrogen phosphate solution (1.7%, pH 3); flow rate, 1 mL/min; detection wavelength, 220 nm. Solute: (1) dicyandiamide; (2) melamine; (3) metformin is used as a binder.
As shown in fig. 17, the retention values for both melamine and metformin increased with the sequence of octadecyl phase < sulfonic phase < octadecyl/sulfonic phase >, reflecting the decisive contribution of the strong cation exchange group sulfonic groups to the electrostatic interaction.
Example 10 chromatographic evaluation: reversed phase/ion exchange mixed chromatography retention behavior
Alkyl anilines of different chain lengths have the hydrophobic group alkylbenzene and the dissociable group amine and therefore they were selected as molecular probes and their retention on the octadecyl, sulfonate and octadecyl/sulfonate phases was determined and the mixed mode chromatographic retention of the stationary phase was compared.
Sample preparation: 50mg of 4-methylaniline, 50mg of 4-ethylaniline, 4-propylaniline, 4-butylaniline and 50L of 4-pentylaniline are respectively taken out of a 10mL volumetric flask, diluted to a scale mark by adding acetonitrile-water (40: 60), and filtered by a filter membrane of 0.22m to prepare an alkylaniline homologous sample.
Chromatographic conditions are as follows: a stationary phase, (a) an octadecyl bonding phase, (b) a sulfonic acid group bonding phase, (c) an octadecyl/sulfonic acid group bonding phase; 15x 0.46 cm; mobile phase, 40% acetonitrile-60% potassium dihydrogen phosphate solution (30mM, pH 2.5); flow rate, 1 mL/min; detection wavelength, 254 nm. Solute: (1) 4-methylaniline; (2) 4-ethylaniline; (3) 4-propylaniline; (4) 4-butylaniline; (5) 4-pentylaniline.
As shown in fig. 18, under the same conditions, the alkylbenzene retained only weakly on the monomodal stationary phase, while the retention on the mixed-mode stationary phase increased greatly, and the order of appearance of the peaks was proportional to the length of the carbon chain on the alkylbenzene, reflecting the decisive contribution of the reversed phase/strong cation exchange mixed retention mechanism to the chromatographic retention value.
Example 11 chromatographic application: separation of common basic drugs
The chromatographic evaluation result shows that the octadecyl/sulfonic mixed stationary phase has excellent retention and separation capacity on the aniline compounds, and the application potential in pharmaceutical analysis is shown. Common basic drugs of salbutamol sulfate, clonidine, procainamide, propranolol and amitriptyline are used as model compounds, retention values of the common basic drugs on an octadecyl/sulfonic acid group phase are measured, and the separation effect of the fixed relative basic drugs is shown.
Sample preparation: respectively taking 10mg of salbutamol, clonidine, procainamide, propranolol and amitriptyline, putting the salbutamol, the clonidine, the procainamide, the propranolol and the amitriptyline into a 10mL volumetric flask, adding a mixed solution of acetonitrile-pH 3 and 30mM potassium dihydrogen phosphate (30:70) to a scale mark, and passing through 0.22
m filters, to make up 1mg/mL of sample.
Chromatographic conditions are as follows: a stationary phase, an eighteen/sulfonic acid group bonding phase; 15x 0.46 cm; mobile phase, 30% acetonitrile-70% potassium dihydrogen phosphate solution (30mM, pH 2.5); flow rate, 1 mL/min; detection wavelength, 220 nm. Solute: (1) salbutamol; (2) (ii) clonidine; (3) procainamide; (4) propranolol and (5) amitriptyline.
As shown in FIG. 19, the five drugs have stronger retention on the mixed-mode stationary phase, the peak patterns are relatively symmetrical, and the peak tailing phenomenon which often occurs on the conventional reversed-phase chromatographic column is not found. Under isocratic elution conditions, baseline separation was obtained for all compounds within 55 min.
Example 12 chromatographic application: chromatographic separation of compound methoxyphenamine capsules
The compound methoxyphenamine capsule contains four effective components. The unique advantages of the mixed chromatographic fixation of the invention relative to the multi-component separation in the compound medicine are shown by the separation of the main components of the compound metominone capsule.
Sample preparation: taking 20 grains of the product, precisely weighing, grinding, precisely weighing an appropriate amount (about equivalent to 25mg of methoxamine hydrochloride) in a 50ml volumetric flask, adding 20ml of acetonitrile, performing ultrasonic treatment for 5 minutes, adding 10ml of water, performing ultrasonic treatment for 5 minutes, adding acetonitrile, diluting to a scale, shaking uniformly, filtering through a 0.22m filter membrane, taking a subsequent filtrate as a sample, precisely weighing 20l, and injecting into a liquid chromatograph.
Chromatographic conditions are as follows: octadecyl/sulfonic acid bonded phase chromatography column, 15x 0.46 cm; mobile phase, 50% acetonitrile-50% potassium dihydrogen phosphate solution (30mM, pH 2.5); flow rate, 1 mL/min; detection wavelength, 254 nm. Solute: (1) aminophylline; (2) methoxyphenamine; (3) narcotine; (4) chlorpheniramine.
As shown in FIG. 20, the column efficiency of the four components in the compound methoxyphenamine capsules on the mixed-mode chromatographic column is more than 15000/m in terms of theoretical plate number, and the tailing factors are less than 1.4. The separation degree of all the components is more than 1.5. Can completely meet the requirements of identification and content determination of the compound methoxyphenamine.
Example 13 chromatographic application: chromatographic separation of compound dihydralazine sulfate tablets
The compound dihydralazine sulfate tablet contains three effective components. The unique advantages of the mixed chromatographic fixation of the invention relative to multi-component separation in compound medicines are further shown by the separation of the main components of the compound dihydralazine sulfate.
Sample preparation: taking 20 tablets of the product, grinding, precisely weighing a proper amount (about equivalent to 25mg of dihydralazine) and placing the tablets in a 50ml volumetric flask, adding 20ml of methanol for ultrasonic treatment for 5 minutes, adding 10ml of water for ultrasonic treatment for 5 minutes, adding methanol for diluting to a scale, shaking up, filtering through a 0.22m filter membrane, taking a subsequent filtrate as a sample, precisely weighing 20l, and injecting into a liquid chromatograph.
Chromatographic conditions are as follows: octadecyl/sulfonic mixed mode chromatography column, 15x 0.46 cm; mobile phase, 50% acetonitrile-50% potassium dihydrogen phosphate solution (30mM, pH 2.5); flow rate, 1 mL/min; detection wavelength, 220 nm. Solute: (1) hydrochlorothiazide, (2) clonidine, and (3) dihydralazine.
As shown in FIG. 21, the separation degree of 3 components in the compound dihydralazine sulfate on the mixed-mode chromatographic column is more than 1.5, the column efficiency of clonidine and dihydralazine is more than 20000/m, and the tailing factor is below 1.05.
Example 13 chromatographic application: chromatographic separation of compound reserpine and methotrexate tablet
The compound reserpine and triamterene pteridine tablet contains four effective components. The unique advantages of the mixed chromatographic fixation of the invention compared with the multi-component separation in the compound medicine are further shown by the separation of the main components of the compound reserpine and methotrexate.
Sample preparation: taking 20 tablets of the product, grinding, precisely weighing a proper amount (about equivalent to 25mg of dihydralazine) and placing the weighed tablets in a 50mL volumetric flask, adding 20mL of methanol for ultrasonic treatment for 5min, adding 10mL of water for ultrasonic treatment for 5min, adding methanol for diluting to a scale, shaking up, filtering through a 0.22m filter membrane, taking a subsequent filtrate as a sample, precisely weighing 20l, and injecting into a liquid chromatograph.
Chromatographic conditions are as follows: octadecyl/sulfonic mixed mode chromatography column, 15x 0.46 cm; mobile phase, 50% methanol-50% potassium dihydrogen phosphate solution (30mM, pH 2.5); flow rate, 1 mL/min; detection wavelength, 268 or 310 nm. Solute: (1) hydrochlorothiazide; (2) (ii) dihydralazine; (3) methotrexate and (4) reserpine.
As shown in FIG. 22, on the mixed-mode chromatographic column, at a detection wavelength of 268nm, the separation degrees of the four components in the compound reserpine and methotrexate are all more than 1.5, the column efficiencies of the dihydralazine, the methotrexate and reserpine are all more than 15000/m, and the tailing factors are all below 1.3.
FIG. 23 is a separation chromatogram of the compound reserpine and triamterene pteridine tablet at a detection wavelength of 310 nm. Under this condition, reserpine cannot be detected. The separation degrees of other three components in the compound reserpine and methotrexate tablet are all larger than 1.5, the column effects of the dihydralazine and the methotrexate are both larger than 15000/m, and the tailing factors are all about 1.0.
Example 14 chromatographic application: chromatographic separation and mass spectrum identification of effective components in compound reserpine tablets
The compound reserpine tablet is a compound preparation and contains reserpine, hydrochlorothiazide, vitamin B6, calcium DL-pantothenate, magnesium trisilicate, potassium chloride, vitamin B1, dihydralazine sulfate and promethazine hydrochloride. The main components in the compound reserpine tablet are separated and identified by utilizing the technology of combining gradient elution mode chromatography and mass spectrum, and the unique advantages of the mixed-mode stationary phase in compound medicine separation and analysis are further shown.
Sample preparation: taking 10 tablets of the product, adding 10mL of methanol into a 25mL volumetric flask, performing ultrasonic treatment for 5min, adding 10mL of water, performing ultrasonic treatment for 5min, adding methanol to dilute to a scale, shaking up, filtering with a 0.22m filter membrane, taking the subsequent filtrate as a sample, precisely measuring 20l, and injecting into a liquid chromatograph.
Chromatographic conditions are as follows: octadecyl/sulfonic mixed mode chromatography column, 15x 0.46 cm; mobile phase, 50mM ammonium formate (pH3) as mobile phase A, acetonitrile as mobile phase B, and elution gradient of 0-3min 0% B, 3-7min 40% B,7-40min 40% B,40-45min 0% B; flow rate, 1 ml/min; the wavelength of detection, 268nm,
mass spectrum detection: positive ion mode, scan range 100m/z-1000m/z.
FIG. 24 is an ultraviolet chromatogram of FUFANGLIXUEPING tablet. Under the condition of gradient elution, seven chromatographic peaks can be obviously separated.
The chromatographic peaks in FIG. 24 were identified using mass spectrometry. FIG. 25 is an ion flow diagram and its mass spectrum of the corresponding chromatographic peak. By spectrogram analysis, the components corresponding to the peaks in the ultraviolet chromatogram can be determined: (1) hydrochlorothiazide, (2) vitamin B6, (3) dihydralazine, (4) reserpine, (6) vitamin B1, and (7) promethazine, which encapsulates all organic components except pantothenic acid. Pantothenic acid has no absorption at the detection wavelength of 268 nm. Chromatographic peak 5 is an impurity peak, possibly from pharmaceutical excipients.

Claims (1)

1. A method for preparing chromatographic stationary phase of chromatographic stationary phase in reversed phase/ion exchange mixed mode, the stationary phase is named as octadecyl/sulfonic group mixed chromatographic stationary phase, and is composed of octadecyl and sulfonic group bonded on the surface of porous silica gel, and the chemical structural formula is:
Figure FDA0003255365200000011
the method comprises the following steps:
(1) preparation of activated silica gel: adding silica gel particles into HCl solution to carry out reflux reaction at 80-105 ℃, washing with water to be neutral, and drying in vacuum;
(2) preparation of epoxy silica gel: adding 1.92mmol-3.6mmol of gamma- (2, 3-epoxypropoxy) propyl trimethoxy silane into each gram of acidified silica gel particles, sealing and reacting at 140-160 ℃, washing with absolute ethyl alcohol, filtering and collecting a product, and drying in vacuum to obtain epoxy group functionalized porous silica gel particles;
(3) preparation of sulfonic silica gel: dispersing the epoxy group functionalized porous silica gel particles into ultrapure water with the mass number of 8-10 times, adding sodium bisulfite according to the amount of 1.92mmol-3.6mmol sodium bisulfite added into each gram of epoxy group functionalized porous silica gel particles, uniformly mixing, adjusting the pH value to 7-8 by using triethylamine, reacting at 85 ℃, washing with water and drying in vacuum to obtain sulfonic group bonded silica gel particles;
(4) preparation of octadecyl/sulfonic acid group silica gel: dispersing the sulfonic bonded silica gel particles in N, N-Dimethylformamide (DMF) with the mass number of 8-10 times, and dropwise adding octadecyl chloride according to the amount of 1.92mmol-3.6mmol of octadecyl chloride added to each gram of sulfonic bonded silica gel particles under the ice bath condition; and after the dropwise addition is finished, stirring and reacting at 40-65 ℃, filtering and collecting a product, washing with toluene and ethanol in sequence, and drying in vacuum to obtain the octadecyl/sulfonic group mixed mode chromatographic stationary phase.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6074555A (en) * 1991-09-13 2000-06-13 Merck Patent Gesellschaft Mit Beschrankter Haftung Modified chromatographic support materials
CN1421693A (en) * 2002-12-20 2003-06-04 中国科学院成都有机化学研究所 Alkyl silica gel bonded chromatographic fixed phase and its prepn process
CN101234339A (en) * 2007-11-14 2008-08-06 天津大学 Silica matrix chemically bonded phase packing
CN105833849A (en) * 2016-02-02 2016-08-10 大连依利特分析仪器有限公司 Anti-phase-strong cation exchange mixing mechanism chromatographic stationary phase preparation method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6074555A (en) * 1991-09-13 2000-06-13 Merck Patent Gesellschaft Mit Beschrankter Haftung Modified chromatographic support materials
CN1421693A (en) * 2002-12-20 2003-06-04 中国科学院成都有机化学研究所 Alkyl silica gel bonded chromatographic fixed phase and its prepn process
CN101234339A (en) * 2007-11-14 2008-08-06 天津大学 Silica matrix chemically bonded phase packing
CN105833849A (en) * 2016-02-02 2016-08-10 大连依利特分析仪器有限公司 Anti-phase-strong cation exchange mixing mechanism chromatographic stationary phase preparation method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Capillary electrochromatography with novel stationary phases. I. Preparation and characterization of octadecyl-sulfonated silica;Minquan Zhang等;《Electrophoresis》;19981231;第19卷;2068-2072 *
Minquan Zhang等.Capillary electrochromatography with novel stationary phases. I. Preparation and characterization of octadecyl-sulfonated silica.《Electrophoresis》.1998,第19卷 *

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