Ionic liquid bridged hybrid silicon oxide as water-rich chromatographic stationary phase and preparation method and application thereof
阅读说明：本技术 作为富水色谱固定相的离子液体桥联杂化氧化硅及其制备方法与应用 (Ionic liquid bridged hybrid silicon oxide as water-rich chromatographic stationary phase and preparation method and application thereof ) 是由 陈桐 宋广三 肖震 李炳祥 徐亮 徐虹 沈伟健 杨敏 吴艳玲 于 2020-07-21 设计创作，主要内容包括：本发明公开了一种作为富水色谱固定相的离子液体桥联杂化氧化硅及其制备方法与应用。本发明提供了一种新的环境友好的亲水固定相PMO-ILs-Au NPs。该新型固定相具有HILIC和PALC的特征,而且与HILIC相同,PALC可以对极性化合物产生保留。该固定相可以分离有机酸类、生物胺类等极性化合物,分离度大、选择性好。本发明还建立了水产品中8种生物胺化合物的PALC-MS/MS检测方法,操作简便、准确、快速、分离效果好,能实现8种生物胺类化合物的同步分析,可以满足食品中常用生物胺类化合物的检测需求。(The invention discloses ionic liquid bridged hybrid silicon oxide serving as a water-rich chromatographic stationary phase, and a preparation method and application thereof. The invention provides a novel environment-friendly hydrophilic stationary phase PMO-ILs-Au NPs. The novel stationary phase has the characteristics of HILIC and PALC, and like HILIC, PALC can retain polar compounds. The stationary phase can separate polar compounds such as organic acids, biogenic amines and the like, and has high separation degree and good selectivity. The invention also establishes a PALC-MS/MS detection method of 8 biological amine compounds in aquatic products, has simple, convenient, accurate and rapid operation and good separation effect, can realize synchronous analysis of 8 biological amine compounds, and can meet the detection requirement of common biological amine compounds in food.)
1. A method of preparing a water-rich chromatographic stationary phase comprising:
1) suspending Au NPs-COOH in a solvent, and carrying out carboxyl activation reaction on the surface of the Au NPs with EDC.HCl and NHS;
edc. hcl represents 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride;
the NHS represents N-hydroxysuccinimide;
the Au NPs-COOH is a gold nanoparticle with functionalized carboxyl;
2) dispersing amino-functionalized PMO-ILs hydrophilic microspheres in a dispersing agent, carrying out esterification reaction with the solution system obtained in the step 1), and obtaining the water-rich chromatographic stationary phase after the reaction is finished.
2. The method of claim 1, wherein: in the step 1), the solvent is at least one selected from DMF, ethanol and DMSO;
the dosage ratio of the Au NPs-COOH to the solvent is 10-20 mg: 10 mL;
the dosage ratio of the solvent to the EDC.HCl is 10-30 mL: 90 mg;
the mass ratio of EDC, HCl and NHS is 1-3: 1;
in the step of carboxyl activation reaction, the temperature is room temperature; the time is 2-5 h;
in the step 2), the dispersing agent is at least one selected from anhydrous DMF, ethanol and DMSO;
the dosage ratio of the amino functionalized PMO-ILs hydrophilic microspheres to the dispersing agent is 1-4 g: 20 mL;
In the step of esterification, the temperature is room temperature; the time is 12-48 h.
3. A water-rich chromatographic stationary phase prepared by the method of any one of claims 1-2.
4. A method of making PMO-ILs hydrophilic microspheres comprising:
1) preparing BTMSPICl:
dissolving imidazole sodium and (3-chloropropyl) trimethoxy silane for substitution reaction, cooling to room temperature after the reaction is finished, performing rotary evaporation, and performing reflux reaction on the obtained reaction mixture and newly added (3-chloropropyl) trimethoxy silane in a solvent to obtain the compound;
2) preparing PMO-ILs hydrophilic microspheres:
a. c is to be18Dissolving TACL in a solvent, and adding a catalyst to be uniform to obtain a solution a;
b. dissolving BTSE and BTMSPICl in ethanol to obtain a solution b;
c. mixing and stirring the solution b and the solution a, heating and standing for carrying out a co-condensation polymerization reaction, cooling after the reaction is finished, filtering, collecting filter residues, washing and drying, adding the obtained solid into a concentrated hydrochloric acid/ethanol solution, extracting a template agent by refluxing, filtering and collecting the filter residues to obtain the PMO-ILs hydrophilic microspheres;
3) pore expansion of PMO-ILs hydrophilic microspheres:
stirring the PMO-ILs hydrophilic microspheres obtained in the step 2) and a pore-expanding agent in ultrapure water, and standing to obtain the pore-expanded PMO-ILs hydrophilic microspheres.
5. The method of claim 4, wherein: in the step 1), the feeding molar ratio of the imidazole sodium to the (3-chloropropyl) trimethoxysilane is 1: 1-3; specifically 1: 1; the substitution reaction is carried out in a solvent and under an inert atmosphere; the inert atmosphere is specifically nitrogen atmosphere;
the solvent is at least one of anhydrous tetrahydrofuran and toluene;
in the step of substitution reaction, the temperature is 60-110 ℃; in particular 65 ℃; the time is 24-72 h; specifically 24 h;
in the step of reflux reaction, the time is 12-48 h;
in the step 2) a, the solvent is selected from a mixed solution consisting of absolute ethyl alcohol and ultrapure water or a mixed solution consisting of DMF and ultrapure water;
the catalyst is selected from NaOH |, KOH and NH3·H2At least one of O;
in the steps 2) a and b, the feeding molar ratio of BTSE to BTMSPICl is (0.7-0.9): (0.1-0.3);
the NaOH and the C18The mass ratio of TACL is 200 mg: 225-600 mg;
total amount of ethanol used and said C18The dosage ratio of TACL to ultrapure water is 20 mL: (225- > 600 mg): (30-50 mL);
in the step 2) c, the temperature for mixing and stirring the solution b and the solution a is room temperature; the time is 30-90 min;
the temperature of the copolycondensation reaction is 80-100 ℃; the time is 15-25 h;
The dosage ratio of the solid to the concentrated hydrochloric acid/ethanol solution is 1 g: 140-160 mL;
in the step of collecting and washing filter residues, the solvent is selected from at least one of methanol, ultrapure water and ethanol;
the drying is vacuum drying; the drying temperature is 60 ℃;
in the step of refluxing and extracting the template agent, the refluxing time is 6-24 h;
in the step 3), the pore-expanding agent is DMDA;
in the stirring step, the temperature is room temperature; the time is 30-90 min;
in the standing step, the temperature is 100-150 ℃; the time is 36-96 h;
the mass ratio of the PMO-ILs hydrophilic microspheres to the pore-expanding agent is 1: 1-3.
6. Hydrophilic PMO-ILs microspheres obtainable by the process according to claim 4 or 5.
7. Use of the water-rich chromatographic stationary phase of claim 3 as a stationary phase in a chromatographic column for separating polar substances;
use of the stationary phase for water-rich chromatography according to claim 3 as a stationary phase of a chromatography column for detecting the content of polar substances.
8. Use according to claim 7, characterized in that: the polar substance is selected from at least one of organic acid and biological amine;
the organic acid is at least one selected from succinic acid, malic acid, tartaric acid, lactic acid, citric acid and fumaric acid;
The biogenic amines are specifically selected from at least one of tryptamine, beta-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine.
9. Use according to claim 8, characterized in that: when the polar substance is the organic acid, the chromatographic conditions are as follows:
mobile phase A: 0.01mol/L disodium hydrogenphosphate-phosphate buffer (pH 2.0);
mobile phase B: ACN;
isocratic elution: a: and B is 98: 2 (v/v);
flow rate: 1.0 mL/min;
detection wavelength: 210 nm;
when the polar substance is the biogenic amine, the chromatographic mass spectrum conditions for separation are as follows:
stationary phase in the column used: the water-rich chromatographic stationary phase of claim 3;
the specification of the chromatographic column: 150mm × 4.6mm, i.d.5 μm;
flow rate: 0.35 mL/min;
column temperature: room temperature;
sample introduction amount: 10 uL;
mobile phase: a is acetonitrile, B is 10mM ammonium acetate aqueous solution;
the gradient elution conditions were as follows:
in the gradient elution condition, the acetonitrile (%) represents a volume percentage of acetonitrile in the mobile phase;
the mass spectrometry conditions used were as follows:
ionization mode: electrospray ionization positive ion mode;
the scanning mode is as follows: monitoring multiple reactions;
resolution ratio: unit mass resolution;
Capillary voltage: 4000V;
capillary temperature: 350 ℃;
collision gas: nitrogen gas.
10. Use according to claim 8 or 9, characterized in that: in the step of detecting the content of the biogenic amines, linear equations corresponding to the standard curves are as follows in sequence:
in the linear equation, Y is the peak area of the quantitative ions;
and X is the concentration of the biogenic amines and has the unit of mu g/kg.
Technical Field
The invention belongs to the field of food detection, and relates to ionic liquid bridged hybrid silicon oxide serving as a water-rich chromatographic stationary phase, and a preparation method and application thereof.
Background
Food additives are widely used in food production and are an important component of the modern food industry. According to the national regulation, the food additives allowed to be used in food are more than 2000, and some illegal manufacturers lose integrity and add some illegal components in the processed food to increase the color, taste or counterfeit main components of the food. The inundation of illegal additives brings great harm to the food safety of China. In recent years, the influence caused by frequent exposure of food safety problems, such as dicyandiamide and melamine events in dairy products, leather milk events, malachite green events in aquatic products, clenbuterol in feeds, and events such as adding sodium copper chlorophyllin serving as olive oil in soybean oil, is continuously upgraded, and the public pays much attention to the result of food safety detection. In the current food production situation of China at the present stage, the requirements on the capability and the quick response of a detection mechanism are higher and higher.
Whether it is a food additive or an illegal additive, most of them are small molecular compounds, and they are classified into three types of strong polarity, medium polarity and weak polarity according to their chemical properties. Of these three classes, most of the less and moderately polar compounds can be separated by reverse-phase chromatography (RPLC). RPLC, however, does not retain or retains very little polar food additives and illegal additives. Although normal phase chromatography can be used for analysis, normal phase chromatography generally uses a nonpolar solvent as a mobile phase, and polar analytes are difficult to dissolve in the nonpolar mobile phase. Therefore, the main means for separating polar food additives and illegal additives is hydrophilic interaction chromatography (HILIC). On hydrophilic chromatographic columns, the non-polar mobile phase system used in normal phase chromatography is replaced by a water and organic solvent system, which solves the problem of insolubility of polar analytes in normal phase chromatography systems. Relevant documents prove that the polar compound has strong retention on a hydrophilic chromatographic column and better separation effect.
However, both normal phase, reverse phase and hydrophilic chromatography often require the use of large amounts of organic solvents. In HILIC in particular, the mobile phase generally uses a high proportion of Acetonitrile (ACN), about 70-95%. ACN is a harmful solvent, and its use in large amounts can negatively affect the environment and human beings. To solve this problem, water-rich liquid chromatography (PALC) has been developed. The method is a green chromatographic separation technology, not only inherits the advantages of HILIC, but also can replace HILIC to efficiently separate polar compounds; in the PALC mode, the mobile phase consists of water with high proportion (water content is more than 90 percent in general), the use of harmful solvent is reduced, the realization of green liquid chromatogram is facilitated, the environment is protected, the sustainable development concept is met, the detection cost can be greatly reduced, and the participation in market competition is facilitated. At present, few literature reports on PALC at home and abroad exist, and the research on the application of PALC in the separation and detection of polar additives and illegal additives in food is less. The types of the stationary phases are very limited, and in the face of a complex separation system, effective separation of complex samples is realized, and more novel PALC stationary phases with good stability and higher separation efficiency must be developed.
In PALC mode, a high proportion of water is used as the mobile phase, which is a great challenge to the service life of the separation material and needs to solve several problems: (1) the column efficiency of a general stationary phase, such as C18, is seriously reduced after a water-rich mobile phase is used for a long time, particularly when silicon hydroxyl groups are exposed. (2) The usable pH range is narrow; when the pH is more than 8, the silica gel is easy to dissolve; at pH <2, the bound silica gel stationary phase will be lost, changing the retention characteristics and peak shape of the analyte, while the selectivity of the column is reduced. (3) When alkaline substances are analyzed, the silicon hydroxyl groups exposed on the surface of the silica gel have irreversible adsorption on the alkaline substances, particularly N-containing compounds, and biological macromolecules, particularly polypeptides, proteins and the like, are easily denatured to generate nonspecific adsorption, so that a chromatographic peak is seriously trailing, and the recovery rate is reduced. Therefore, when a water-rich mobile phase is used, good stability of the stationary phase is required.
Disclosure of Invention
The invention aims to provide ionic liquid bridged hybrid silicon oxide serving as a water-rich chromatographic stationary phase, and a preparation method and application thereof.
The method for preparing the water-rich chromatographic stationary phase comprises the following steps:
1) Suspending Au NPs-COOH in a solvent, and carrying out carboxyl activation reaction on the surface of the Au NPs with EDC.HCl and NHS;
edc. hcl represents 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride;
the NHS represents N-hydroxysuccinimide;
the Au NPs-COOH is a gold nanoparticle with functionalized carboxyl;
2) dispersing amino-functionalized PMO-ILs hydrophilic microspheres in a dispersing agent, and carrying out esterification reaction with the solution system obtained in the step 1) to obtain the water-rich chromatographic stationary phase after the reaction is finished;
in step 1) of the above method, the solvent is at least one selected from DMF, ethanol and DMSO;
the dosage ratio of the Au NPs-COOH to the solvent is 10-20 mg: 10 mL; in particular 15 mg: 10 mL;
the dosage ratio of the solvent to the EDC.HCl is 10-30 mL: 90 mg; specifically 10 mL: 90 mg;
the mass ratio of EDC, HCl and NHS is 1-3: 1; specifically 1.8: 1;
in the step of carboxyl activation reaction, the temperature is room temperature; the time is 2-5 h; in particular for 2 h;
the Au NPs-COOH, namely the carboxyl functionalized gold nanoparticles can be subjected to carboxyl functionalization according to various conventional methods;
it can be functionalized by carboxyl groups, for example, as follows: 20mg of Au NPs were dispersed in 100mL of ultrapure water . Then 340. mu.L thioglycolic acid was added to the aqueous Au NPs solution, N2The reaction was stirred at room temperature for 24h under ambient. Filling the reaction solution into a dialysis bag with the cut-off quantity of 3KDa, dialyzing in deionized water for 48 hours, and removing residual thioglycollic acid to obtain Au NPs-COOH;
the gold nanoparticles used can be prepared according to various conventional methods, for example, according to the following reduction method: 100mL of 0.01% chloroauric acid (HAuCl) was taken4) Heating the aqueous solution to boiling, quickly adding 1mL of 1% sodium citrate solution into the aqueous solution, stirring at constant temperature for 30min, dialyzing, centrifuging and drying to obtain the Au NPs.
In the step 2), the amino functionalization method in the amino functionalized PMO-ILs hydrophilic microspheres is a conventional method, for example, the amino functionalization can be performed according to the following method:
2.0g of the pore-expanded hydrophilic microspheres of PMO-ILs were dispersed in 30mL of anhydrous toluene, and 2.0mL of 3-aminopropyltrimethoxysilane ((3-Aminopropyl) trimethoxysilane, APTMS) was added thereto and stirred to mix well, followed by stirring under reflux for 20 hours under nitrogen. After the reaction is finished, filtering the mixture, washing the solid material by dichloromethane, acetone and methanol respectively, and drying the obtained solid in vacuum at the temperature of 60 ℃ overnight to obtain the product.
The dispersing agent is at least one of anhydrous DMF, ethanol and DMSO;
the dosage ratio of the amino functionalized PMO-ILs hydrophilic microspheres to the dispersing agent is 1-4 g: 20 mL; specifically, 2.0 g: 20 mL;
in the step of esterification, the temperature is room temperature; the time is 12-48 h; specifically 24 h;
the method further comprises the following steps: after the reaction in the step 2) is finished, filtering the reaction system, collecting filter residues, washing with ethanol and drying.
The water-rich chromatographic stationary phase prepared by the method also belongs to the protection scope of the invention.
The invention also claims a PMO-ILs hydrophilic microsphere used in the preparation of the water-rich chromatographic stationary phase and a preparation method thereof, wherein; the preparation method of the PMO-ILs hydrophilic microspheres comprises the following steps:
1) preparation of BTMSPICl (i.e.1, 3-bis (trimethoxysilylpropyl) imidazolium chloride [1,3-bis (trimethylsilylpropyl) imidazolium chloride, BTMSPICl ]):
dissolving imidazole sodium and (3-chloropropyl) trimethoxy silane for substitution reaction, cooling to room temperature after the reaction is finished, performing rotary evaporation, and performing reflux reaction on the obtained reaction mixture and newly added (3-chloropropyl) trimethoxy silane in a solvent to obtain the compound;
the BTMSPICl represents an ionic liquid silicon source;
2) Preparing PMO-ILs hydrophilic microspheres:
a. c is to be18Dissolving TACL in a solvent, and adding a catalyst to be uniform to obtain a solution a;
b. dissolving BTSE and BTMSPICl in ethanol to obtain a solution b;
c. mixing and stirring the solution b and the solution a, heating and standing for carrying out a co-condensation polymerization reaction, cooling after the reaction is finished, filtering, collecting filter residues, washing and drying, adding the obtained solid into a concentrated hydrochloric acid/ethanol solution, extracting a template agent by refluxing, filtering and collecting the filter residues to obtain the PMO-ILs hydrophilic microspheres;
3) pore expansion of PMO-ILs hydrophilic microspheres:
stirring the PMO-ILs hydrophilic microspheres obtained in the step 2) and a pore-expanding agent in ultrapure water, and standing to obtain the pore-expanded PMO-ILs hydrophilic microspheres.
In step 1) of the above method, the molar ratio of the input materials of the imidazole sodium and the (3-chloropropyl) trimethoxysilane is 1: 1-3; specifically 1: 1; the substitution reaction is carried out in a solvent and under an inert atmosphere; the inert atmosphere is specifically nitrogen atmosphere;
the solvent is at least one of anhydrous tetrahydrofuran and toluene;
in the step of substitution reaction, the temperature is 60-110 ℃; in particular 65 ℃; the time is 24-72 h; specifically 24 h;
in the step of reflux reaction, the time is 12-48 h;
In the step 2) a, the solvent is selected from a mixed solution consisting of absolute ethyl alcohol and ultrapure water or a mixed solution consisting of DMF and ultrapure water;
the catalyst is selected from NaOH |, KOH and NH3·H2At least one of O;
in the steps 2) a and b, the feeding molar ratio of BTSE to BTMSPICl is (0.7-0.9): (0.1-0.3); specifically, 0.7: 0.3;
the NaOH and the C18The mass ratio of TACL is 200 mg: 225-600 mg; in particular 200 mg: 300 mg;
total amount of ethanol used and said C18The dosage ratio of TACL to ultrapure water is 20 mL: (225- > 600 mg): (30-50 mL); specifically 20 mL: 300 mg: 30-50 mL;
in the step 2) c, the temperature for mixing and stirring the solution b and the solution a is room temperature; the time is 30-90 min;
the temperature of the copolycondensation reaction is 80-100 ℃; in particular to 90 ℃; the time is 15-25 h; in particular 20 h;
the dosage ratio of the solid to the concentrated hydrochloric acid/ethanol solution is 1 g: 140-160 mL; specifically, the content is 1 g: 150 mL; in the concentrated hydrochloric acid/ethanol solution, the volume ratio of the concentrated hydrochloric acid to the ethanol solution is specifically 5: 95;
in the step of collecting and washing filter residues, the solvent is selected from at least one of methanol, ultrapure water and ethanol; specifically, methanol, ultrapure water and ethanol are used for washing in sequence;
The drying is vacuum drying; the drying temperature is 60 ℃;
in the step of refluxing and extracting the template agent, the refluxing time is 6-24 h;
the method further comprises the following steps: after the step 2) c and before the step 3), washing the filter residue obtained in the step c with ethanol, ultrapure water and methanol in sequence, and drying;
in the step 3), the pore-expanding agent is DMDA;
in the stirring step, the temperature is room temperature; the time is 30-90 min;
in the standing step, the temperature is 100-150 ℃; in particular to 120 ℃; the time is 36-96 h; in particular 72 h;
the mass ratio of the PMO-ILs hydrophilic microspheres to the pore-expanding agent is 1: 1-3; specifically, 1: 1.25;
the method further comprises the following steps: and after the step 3) of standing, washing with ultrapure water and methanol in sequence, and drying.
The PMO-ILs hydrophilic microspheres prepared by the method also belong to the protection scope of the invention.
Specifically, the specific surface area of the PMO-ILs hydrophilic microspheres is 300-700m2(ii)/g; specifically, it may be 417m2/g；
The aperture is 6.0-10.0 nm; specifically 8.7 nm;
the pore volume is 0.55-1.20cm3(ii)/g; specifically, it may be 0.93cm3/g。
In addition, the invention also claims the application of the water-rich chromatographic stationary phase as the stationary phase of the chromatographic column in separating polar substances and the application of the water-rich chromatographic stationary phase as the stationary phase of the chromatographic column in detecting the content of the polar substances.
Specifically, the polar substance is selected from at least one of organic acids and biological amines;
the organic acid is at least one selected from succinic acid, malic acid, tartaric acid, lactic acid, citric acid and fumaric acid;
the biogenic amines are specifically selected from at least one of tryptamine, beta-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine.
More specifically, when the polar substance is the organic acid, the chromatographic conditions used are as follows:
mobile phase A: 0.01mol/L disodium hydrogenphosphate-phosphate buffer (pH 2.0);
mobile phase B: ACN;
isocratic elution: a: and B is 98: 2 (v/v);
flow rate: 1.0 mL/min;
detection wavelength: 210 nm;
when the polar substance is the biogenic amine, the chromatographic mass spectrum conditions for separation are as follows:
stationary phase in the column used: the water-rich chromatographic stationary phase provided by the invention;
the specification of the chromatographic column: 150mm × 4.6mm, i.d.5 μm;
flow rate: 0.35 mL/min;
column temperature: room temperature;
sample introduction amount: 10 mu L of the solution;
mobile phase: a is acetonitrile, B is 10mM ammonium acetate aqueous solution;
the gradient elution conditions were as follows:
in the gradient elution condition, the acetonitrile (%) represents a volume percentage of acetonitrile in the mobile phase;
The mass spectrometry conditions used were as follows:
ionization mode: electrospray ionization (ESI) positive ion mode;
the scanning mode is as follows: multiple Reaction Monitoring (MRM);
resolution ratio: unit mass resolution;
capillary voltage: 4000V;
capillary temperature: 350 ℃;
collision gas: nitrogen gas;
the mass spectrum parameters of the biogenic amines are shown in table 6;
specifically, in the step of detecting the content of the biogenic amines, linear equations corresponding to the standard curves are as follows in sequence:
histamine Y-19665.7X-12765.1
Cadaverine Y ═ 13265.1X +11967.2
Putrescine Y-104434.0X +8778.1
Spermine Y-144735.3X-1197.2
Spermidine Y-19726.7X-4728.0
2-phenylethylamine Y ═ 12767.1X-15613.2
Tyramine Y ═ 10315.5X +17200.2
Tryptamine Y-4120.8X-1767.5
In the linear equation, Y is the peak area of the quantitative ions;
and X is the concentration of the biogenic amines and has the unit of mu g/kg.
In addition, when the polar substance is the biogenic amine, the separating and detecting step further comprises: before the separation step, the sample to be detected is pretreated as follows:
accurately weighing 2g (accurate to 0.01g) of the minced or homogenized sample to be detected into a 50mL polypropylene centrifuge tube, adding 20mL of acetonitrile-methanol-2: 8(v: v) extract, performing vortex for 3min, adding 5g of anhydrous sodium sulfate, continuing to perform vortex mixing, performing ultrasonic extraction for 10min, shaking at least for 2 times in the ultrasonic process, performing centrifugal 5min at 5000r/min, and taking 10mL of supernatant into a 15mL centrifuge tube for purification;
Purifying: adding 100mg PSA adsorbent and 100mg C18 adsorbent into 10mL supernatant, vortexing for 3min, centrifuging at 5000r/min for 5min, blowing nitrogen, redissolving with 1mL methanol, filtering with 0.22 μm organic filter membrane, and analyzing with liquid chromatography-tandem mass spectrometer.
In the separation or detection method, the sample to be detected can be food; more particularly, the aquatic product; more specifically, the compound may be a food or a marine product containing or suspected of containing the organic acids or the biogenic amine compounds.
The invention provides a new environment-friendly hydrophilic stationary phase. The stationary phase takes Ionic Liquid (ILs) bridged silane and 1,2-bis (triethoxysilyl) ethane [1,2-bis (triethoxysilyl) ethane, BTSE ] as a diatomic source, and prepares novel PMO hydrophilic microspheres suitable for being used as liquid chromatography packing through co-condensation, so that ILs can be completely incorporated into a mesoporous framework of PMO. The invention synthesizes a
The PMO-ILs-Au NPs filler is prepared as a water-rich stationary phase, and is characterized by utilizing infrared spectroscopy, elemental analysis, a transmission electron microscope, a scanning electron microscope and the like, and the chromatographic behavior of the PMO-ILs-Au NPs filler is researched. The novel stationary phase has the characteristics of HILIC and PALC, and the PALC can retain polar compounds and greatly improve acid and alkali resistance. The stationary phase can separate polar compounds such as organic acids, biogenic amines and the like, and has high separation degree and good selectivity. The immobilization is less retention of relatively non-polar and less polar compounds than the common C18 column, and more retention of more polar compounds. PALC, a green chromatographic mode, is expected to be an alternative to HILIC and a complementary mode to RPLC. The invention also establishes a PALC-MS/MS detection method of 8 biological amine compounds in aquatic products, has simple, convenient, accurate and rapid operation and good separation effect, can realize synchronous analysis of 8 biological amine compounds, and can meet the detection requirement of common biological amine compounds in food.
Drawings
FIG. 1 is an infrared spectrum of stationary phase synthesis at various stages. (A) PMO-ILs without template removal; (B) removing PMO-ILs of the template; (C) reaming the PMO-ILs; (D) PMO-ILs-NH 2(ii) a (E) PMO-ILs-Au NPs hydrophilic stationary phase;
FIG. 2 is a scanning electron micrograph of PMO-ILs prepared under different conditions; in the figure, A-F correspond to samples of numbers 1-6 in Table 1, namely PMO-ILs-A to PMO-ILs-F in sequence;
FIG. 3 is a transmission electron micrograph of PMO-ILs (A and B) and PMO-ILs-Au NPs (C);
FIG. 4 is a comparison of column pressure/flow rate performance of a PMO-ILs-Au NPs column with a commercial C18 column;
FIG. 5 is a chromatogram of 5 batches of PMO-ILs-Au NPs on hydrophilic stationary phase (A-E) for sodium benzoate and potassium sorbate. Chromatographic separation conditions: 10mM aqueous ammonium acetate solution: ACN 95:5(v/v), flow rate: 1.0 mL/min. Detection wavelength: 230 nm. (1. sodium benzoate; 2. potassium sorbate);
FIG. 6 is a chromatogram of 6 organic acids. A is PMO-ILs-Au NPs column separation chromatogram, B is C18 column separation chromatogram; wherein, 1, tartaric acid; 2. malic acid; 3. lactic acid; 4. citric acid; 5. succinic acid; 6. fumaric acid;
FIG. 7 is a total ion flow diagram of HPLC-MS/MS separation of 8 kinds of biogenic amines. A is a PMO-ILs-Au NPs chromatographic column separation total ion flow diagram, and B is a C18 column separation total ion flow diagram. (1, putrescine; 2, cadaverine; 3, histamine; 4, tyramine; 5, 2-phenylethylamine; 6, tryptamine; 7, spermidine; 8, spermine).
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.