A zwitterionic ion-exchange microsphere adsorbent material, its preparation method and application

Abstract

This invention provides a zwitterionic ion-exchange microsphere material, its preparation method, and its applications, relating to the field of adsorption material preparation technology. The zwitterionic ion-exchange microsphere adsorbent material provided by this invention simultaneously contains quaternary ammonium functional groups and carboxyl functional groups, enabling the simultaneous selective retention of acidic and basic analytes in samples. The appropriate combination of porogens, dispersants, and initiators results in a porous structure on the microsphere surface, increasing the surface area for ion exchange and improving the material's adsorption capacity. The surface charge of the microspheres and the benzene ring structure they contain also endow the zwitterionic ion-exchange microsphere adsorbent material with both hydrophilicity and lipophilicity, thus broadening its applicability. Furthermore, the preparation method of the zwitterionic ion-exchange microsphere material provided by this invention is simple, uses mild conditions, and is conducive to large-scale production.

Description

Technical Field

[0001] This invention relates to the field of adsorption material preparation technology, and in particular to a zwitterionic ion exchange microsphere adsorption material, its preparation method, and its application. Background Technology

[0002] Solid-phase extraction (SPE) is a widely used sample pretreatment technique for enriching and separating analytes from complex samples. It can enrich analytes and eliminate matrix interference, thus it is widely used in sample pretreatment for mass spectrometry. The core of SPE technology is the adsorbent packing material, which often possesses high specific surface area and hydrophilic / lipophilic properties or ion exchange characteristics. Most current adsorbents combine a polymer backbone with ion exchange groups to form hybrid adsorbents, namely reversed-phase and ion exchange adsorption.

[0003] In the field of biological detection, it is often necessary to simultaneously perform quantitative analysis of acidic and basic analytes. For example, serotonin and catecholamines are important neurotransmitters in the human body and are both monoamine compounds, belonging to basic analytes. However, the metabolites of serotonin and some metabolites of catecholamines are carboxyl-containing compounds, belonging to acidic analytes. Currently, when detecting serotonin and its metabolites, and catecholamines and their metabolites in urine, a two-step solid-phase extraction method is typically used for pretreatment. First, a packing material containing weakly cationic groups is used to adsorb and separate compounds containing amino groups, and then a packing material containing strongly anionic groups is used to adsorb and separate compounds containing carboxyl groups. This is because most commercially available solid-phase extraction packing materials can only retain compounds containing one type of ionic group, and can only adsorb and separate acidic or basic analytes, not both simultaneously. Furthermore, insufficient retention has been observed when extracting highly polar compounds.

[0004] Currently, no suitable solid-phase extraction (SPE) adsorption materials have been reported that can simultaneously adsorb and separate acidic and basic analytes. Therefore, it is essential to develop a SPE adsorption material capable of simultaneously adsorbing and separating acidic and basic analytes. Summary of the Invention

[0005] The purpose of this invention is to provide a zwitterionic ion-exchange microsphere adsorption material, its preparation method, and its application, so as to solve the technical problem that existing solid-phase extraction adsorption materials can only adsorb compounds with a single property.

[0006] To solve the above-mentioned technical problems, the present invention provides a zwitterionic ion exchange microsphere adsorption material containing quaternary ammonium functional groups and carboxyl functional groups;

[0007] The structural formula of the microsphere adsorption material is as follows:

[0008] .

[0009] Furthermore, the density of the quaternary ammonium functional group is 0.5-2 mmol / g;

[0010] The density of the carboxyl functional group is 1-5 mmol / g.

[0011] The oxygen atom in the carboxyl functional group has a strong electron absorption capacity and readily undergoes electron transfer with cations or positively charged groups in basic compounds to form a stable adsorption structure. The successful introduction of carboxyl functional groups into microsphere adsorption materials enhances the selective adsorption of compounds containing basic functional groups in the analytes.

[0012] Quaternary ammonium functional groups contain nitrogen atoms and possess a lone pair of electrons. They are fully ionized in solution and carry a positive charge, allowing for the selective retention of acidic substances. This application creatively introduces both carboxyl and quaternary ammonium functional groups into microsphere adsorbent materials, expanding the application range of these materials and achieving simultaneous adsorption of both acidic and basic substances.

[0013] Furthermore, the particle size of the microsphere adsorbent material is 10 μm - 300 μm;

[0014] The microsphere adsorption material has a pore size of 1 nm - 50 nm.

[0015] The microspheres have a porous structure on their surface, which increases the specific surface area of ​​the material and ensures sufficient contact between the analyte and the microsphere adsorption material.

[0016] The present invention also provides a method for preparing the above-mentioned microsphere adsorbent material, the specific steps of which are as follows:

[0017] Step 1: Add the dispersant to distilled water and stir thoroughly to obtain a dispersant aqueous solution with a concentration of 0.1%-20%; the concentration of the dispersant aqueous solution is preferably 3%-10%.

[0018] The dispersant is hydroxypropyl methylcellulose and / or polyvinyl alcohol.

[0019] Dispersants have excellent dispersing ability. Appropriate dispersants can help monomers form stable and uniform droplets and control the collision of particles after polymerization, keeping them in a stable state.

[0020] Step 2: Weigh 50-90 parts of the polymer monomer mixture, 1-90 parts of the pore-forming agent, and 0.01-1 parts of the initiator according to the mass ratio, and stir them thoroughly to obtain 100 parts of the oil phase mixture.

[0021] The polymer monomer mixture includes a first monomer and a second monomer;

[0022] The first monomer is selected from o-divinylbenzene, m-divinylbenzene, and p-divinylbenzene;

[0023] The second monomer is selected from p-chloromethylstyrene and 4-chlorostyrene.

[0024] The polymer monomer contains a benzene ring, which is lipophilic and helps to improve the adsorption effect on hydrophobic substances.

[0025] Step 3: The acrylate monomers are thoroughly mixed with the oil phase mixture and the dispersant aqueous solution, and the mixture is refluxed at 50-80°C for 4-20 hours. After the reaction is completed, the mixture is cooled to room temperature, washed with water and ethanol, and dried at 60°C to obtain the first reaction product.

[0026] The acrylate monomers are selected from methyl acrylate, methyl methacrylate, and ethyl methacrylate;

[0027] The acrylate monomer is added at a mass of 1%-20% of the polymer monomer mixture; preferably 8%-15%.

[0028] The mass ratio of the oil phase mixture to the dispersant aqueous solution is (30-60):100.

[0029] Step 4: Place the first reaction product in a toluene solution and allow it to swell for 0.5-1.5 hours to obtain a swollen mixture;

[0030] A tertiary amine monomer is added to the swollen mixture, and the polymerization reaction is carried out by continuous and thorough stirring at 80-90°C for 4-20 hours to obtain the second reaction product.

[0031] The tertiary amine monomers are selected from N,N-dimethylbutylamine, trimethylamine, and triethylamine;

[0032] The amount of the tertiary amine monomer added is 1-10 times the mass of the second monomer; preferably 6-8 times.

[0033] Step 5: Add 100 parts of alkaline methanol solution to the second reaction product, stir at low speed at 65-75℃, and carry out ester hydrolysis reaction for 20-30h; after the reaction is completed, cool to room temperature, wash with ethanol, sodium bicarbonate solution and water in sequence, and dry to obtain the zwitterionic ion exchange microsphere adsorbent material.

[0034] After grafting quaternary ammonium functional groups, the ester groups grafted onto the microspheres in step 3 undergo ester hydrolysis in an alkaline environment to obtain carboxyl functional groups, ultimately yielding weak cation-strong anion zwitterion microspheres that simultaneously possess both carboxyl and quaternary ammonium functional groups.

[0035] The microsphere adsorption material is a weak cation-strong anion exchange microsphere. The surface of the microsphere is attached with charges and has hydrophilic properties, which is beneficial to improving the adsorption effect on hydrophilic substances.

[0036] Further, the dispersant in step 1 is hydroxypropyl methylcellulose and / or polyvinyl alcohol.

[0037] Furthermore, in step 2, the mass ratio of the first monomer to the second monomer is 1:(1-3).

[0038] Preferably, the pore-forming agent in step 2 is one or more of dichloromethane, n-dodecane, toluene, o-dichlorobenzene, and liquid paraffin;

[0039] The initiator is azobisisobutyronitrile and / or benzoyl peroxide.

[0040] Preferably, in step 2, the mass ratio of the first monomer: the second monomer: the porogen: the initiator is (17-30): (34-60): (9-50): (0.01-1).

[0041] Further, the specific steps for thorough mixing in step 3 are as follows: adding the acrylate monomer and the oil phase mixture together into the dispersant aqueous solution and stirring until homogeneous;

[0042] Alternatively, the oil phase mixture can be added to the dispersant aqueous solution first, and then acrylate monomers can be added at 50-80°C and mixed evenly.

[0043] Furthermore, the stirring rate described in steps 1 and 2 is 200-500 r / min;

[0044] An appropriate stirring rate can promote the dispersion of the oil phase into small droplets during the phase mixing process.

[0045] The low-speed stirring rate mentioned in step 5 is 80-120 r / min.

[0046] Preferably, the alkaline methanol solution in step 5 is selected from KOH-methanol solution and NaOH-methanol solution, and more preferably KOH-methanol solution;

[0047] The concentration of the alkaline methanol solution is 3%wt-5%wt.

[0048] On the other hand, the present invention also provides a solid-phase extraction device for simultaneously separating acidic and basic analytes, using the above-mentioned zwitterionic ion-exchange microsphere adsorbent material or the zwitterionic ion-exchange microsphere adsorbent material prepared by the above method as a packing material.

[0049] By adopting the above technical solution, the present invention has the following beneficial effects:

[0050] This invention provides a zwitterionic ion-exchange microsphere material that creatively incorporates both carboxyl and quaternary ammonium functional groups into the microsphere adsorption material, enabling the simultaneous selective retention of both acidic and basic analytes in the sample. Through the appropriate use of porogens, dispersants, and initiators, the microsphere material surface acquires a porous structure, increasing the surface area for ion exchange and enhancing the material's adsorption capacity. The surface charge of the microspheres and the benzene ring structure inherent in the microspheres themselves endow the zwitterionic ion-exchange microsphere adsorption material with both hydrophilicity and lipophilicity, thus broadening its applicability. Furthermore, the preparation method of the zwitterionic ion-exchange microsphere material provided by this invention is simple, operates under mild conditions, and is conducive to large-scale production. Attached Figure Description

[0051] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0052] Figure 1 A scanning electron microscope image of the zwitterionic ion exchange microsphere adsorption material provided by the present invention;

[0053] Figure 2 The particle size distribution diagram of the zwitterionic ion exchange microsphere adsorption material provided by the present invention;

[0054] Figure 3 The nitrogen adsorption-desorption isotherm curve of the microsphere adsorption material;

[0055] Figure 4 (a) is a scanning electron microscope image of zwitterionic exchange microspheres prepared using hydroxypropyl methylcellulose as a dispersant;

[0056] Figure 4 (b) is a scanning electron microscope image of zwitterionic exchange microspheres prepared using 3% polyvinyl alcohol as a dispersant;

[0057] Figure 4 (c) is a scanning electron microscope image of zwitterionic ion-exchange microspheres prepared using 5% polyvinyl alcohol as a dispersant;

[0058] Figure 4 (d) is a scanning electron microscope image of zwitterionic ion-exchange microspheres prepared using 10% polyvinyl alcohol as a dispersant;

[0059] Figure 5(a) is a scanning electron microscope image of zwitterionic exchange microspheres prepared using benzoyl peroxide as an initiator;

[0060] Figure 5 (b) is a scanning electron microscope image of zwitterionic exchange microspheres prepared using azobisisobutyronitrile as an initiator;

[0061] Figure 6 (a) is a scanning electron microscope image of zwitterionic ion exchange microspheres obtained at a stirring rate of 250 r / min;

[0062] Figure 6 (b) is a scanning electron microscope image of the zwitterionic ion exchange microspheres obtained at a stirring rate of 300 r / min;

[0063] Figure 6 (c) is a scanning electron microscope image of the zwitterionic ion exchange microspheres obtained at a stirring rate of 350 r / min;

[0064] Figure 6 (d) is a scanning electron microscope image of the zwitterionic ion exchange microspheres obtained at a stirring rate of 400 r / min;

[0065] Figure 7 This is a graph showing the trend of reaction yield versus reaction time in Example 9;

[0066] Figure 8 Scanning electron microscope image of zwitterionic ion-exchange microspheres obtained by the preparation method provided in Example 10;

[0067] Figure 9 This is a schematic diagram illustrating the selective adsorption principle of the zwitterionic ion-exchange microsphere adsorption material provided by the present invention. Detailed Implementation

[0068] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0069] The present invention will be further explained below with reference to specific embodiments.

[0070] The preparation method of the zwitterionic ion exchange microsphere adsorption material provided by the present invention is as follows, and the specific steps are as follows:

[0071] Step 1: Add the dispersant to distilled water and stir at a rate of 200-500 r / min to obtain a dispersant aqueous solution with a concentration of 0.1%-20%;

[0072] The dispersant is hydroxypropyl methylcellulose and / or polyvinyl alcohol;

[0073] Step 2: Weigh 50-90 parts of polymer monomer mixture, 1-90 parts of pore-forming agent, and 0.01-1 parts of initiator, and stir at a rate of 200-500 r / min to obtain an oil phase mixture;

[0074] The polymer monomer mixture includes a first monomer and a second monomer; the molar ratio of the first monomer to the second monomer is 1:(1-3).

[0075] The first monomer is selected from o-divinylbenzene, m-divinylbenzene, and p-divinylbenzene;

[0076] The second monomer is selected from p-chloromethylstyrene and 4-chlorostyrene;

[0077] The pore-forming agent is one or more of dichloromethane, n-dodecane, toluene, o-dichlorobenzene, and liquid paraffin;

[0078] The initiator is azobisisobutyronitrile (AIBN) and / or benzoyl peroxide;

[0079] Step 3: Mix the acrylate monomers with the oil phase mixture and the dispersant aqueous solution thoroughly, and reflux the mixture at 50-80℃ for 4-20 hours. After the reaction is completed, cool to room temperature, wash with water and ethanol successively, and dry at 60℃ for 10-12 hours to obtain the first reaction product.

[0080] The acrylate monomers are selected from methyl acrylate, methyl methacrylate, and ethyl methacrylate;

[0081] The amount of acrylate monomer added is 1%-20% of the mass of the polymer monomer mixture;

[0082] Step 4: Place the first reactant in a toluene solution and allow it to swell for 0.5-1.5 hours to obtain a swollen mixture;

[0083] A tertiary amine monomer is added to the swollen mixture, and the mixture is stirred thoroughly at 80-90°C for 4-20 hours to obtain the second reactant.

[0084] The tertiary amine monomers are selected from N,N-dimethylbutylamine, trimethylamine, and triethylamine;

[0085] The amount of the tertiary amine monomer added is 1-10 times the mass of the second monomer.

[0086] Step 5: Add a 3%wt-5%wt alkaline methanol solution to the second reaction product, stir at 80-120 r / min at 65-75℃, and carry out ester hydrolysis reaction for 20-30 h; after the reaction is completed, cool to room temperature, wash with ethanol, sodium bicarbonate solution and water in sequence, and dry at 60℃ for 10-12 h to obtain the zwitterionic ion exchange microsphere adsorption material;

[0087] All components are listed in parts by weight.

[0088] Methods for observing the morphology of microsphere materials:

[0089] The surface of the microsphere adsorbent material was sputter-coated with gold; then baked with an infrared lamp for 20 minutes, and then placed in a sample chamber and evacuated; the morphology and surface state of the microsphere material under a scanning electron microscope are as follows: Figure 1 As shown.

[0090] Methods for determining the particle size of microsphere materials:

[0091] An appropriate amount of microspheres were ultrasonically dispersed in a mixture of water and ethanol.

[0092] Then, take an appropriate amount of the mixed liquid and further determine the particle size distribution range using an MS3000 micron particle size analyzer, such as... Figure 2 As shown.

[0093] Methods for determining the pore size of microsphere materials:

[0094] The microsphere material is heated and degassed under vacuum to remove the substances adsorbed on its surface.

[0095] Then it was weighed, and finally placed in liquid nitrogen. Using a V-Sorb280OTP fully automated specific surface area and pore size analyzer, the high pressure and absorption of the test sample were calculated according to the specified pressure points, and the results were obtained as follows: Figure 3 The adsorption isotherm curves are shown. Specific surface area, pore size, and pore size distribution range are calculated using the adsorption isotherms, and the average pore size of the microsphere material is calculated using the Barrett-Joyner-Halenda method.

[0096] Methods for determining the density of carboxyl and quaternary ammonium functional groups:

[0097] (1) Standardization of sodium hydroxide solution: First, take a certain amount of potassium hydrogen phthalate in a beaker and dry it at 105-110℃ for 2-3 hours for later use.

[0098] Blank: Take 50 ml of deionized water and titrate with sodium hydroxide solution. Perform three parallel determinations and take the average value as V blank. The endpoint pH of the titration is 9.0-9.1.

[0099] Standardization: Weigh 0.5000g of potassium hydrogen phthalate, dissolve it in 50 ml of deionized water, and titrate with the sodium hydroxide solution to be tested. The titration endpoint is pH 9.0-9.1. Perform three parallel determinations and take the average value V1. Calculate the standard concentration of the sodium hydroxide solution using the following formula:

[0100] .

[0101] (2) Standardization of hydrochloric acid solution: Accurately transfer 25 ml of the hydrochloric acid solution to be tested into an Erlenmeyer flask using a pipette, add 25 ml of deionized water, and titrate with a pre-standardized sodium hydroxide solution. The titration endpoint is pH 7.0. Perform three parallel determinations and take the average value V2. Calculate the standardized concentration of the hydrochloric acid solution according to the following formula:

[0102] .

[0103] (3) Determination of carboxyl content in samples:

[0104] Titration experiment: Weigh 0.5000 g / 1.0000 g of the microsphere adsorbent material provided in this invention into an Erlenmeyer flask, add 100 ml of 0.1 mol / L sodium hydroxide solution, shake well, seal, and place in a 40°C water bath for 2 h. Remove and cool to room temperature. Transfer 25 ml of the supernatant to 25 ml of deionized water using a pipette, and titrate with standardized hydrochloric acid solution. The endpoint is indicated by an indicator, and the pH at the equivalence point is 7. Perform three parallel titrations and take the average value V3.

[0105] Blank experiment: Weigh 0.5000 g / 1.0000 g of the microsphere adsorbent material precursor provided by this invention into an Erlenmeyer flask, add 100 ml of 0.1 mol / L sodium hydroxide solution, shake well, seal, and place in a 40°C water bath for 2 h. Remove and cool to room temperature. Accurately pipette 25 ml of 0.1 mol / L sodium hydroxide solution and add 25 ml of deionized water. Titrate with standardized hydrochloric acid solution, using an indicator to indicate the endpoint. The pH at the equivalence point is 7. Perform three parallel titrations and take the average value V4. Calculate the carboxyl content W (carboxyl groups) of the microspheres according to the following formula based on the volume of hydrochloric acid consumed:

[0106] .

[0107] (4) Determination of quaternary ammonium group content in samples:

[0108] Titration experiment: Weigh 0.5000 g / 1.0000 g of the microsphere adsorbent material provided in this invention into an Erlenmeyer flask, add 100 ml of 0.1 mol / L hydrochloric acid solution, shake well, seal, and place in a 40°C water bath for 2 h. Remove and cool to room temperature. Transfer 25 ml of the supernatant to an Erlenmeyer flask using a pipette, add 25 ml of deionized water, and titrate with standardized sodium hydroxide solution. The endpoint is indicated by an indicator, and the pH at the equivalence point is 7. Perform three parallel titrations and take the average value V5.

[0109] Blank experiment: Weigh 0.5000 g / 1.0000 g of the microsphere adsorbent material precursor provided by this invention into an Erlenmeyer flask, add 100 ml of 0.1 mol / L hydrochloric acid solution, shake well, seal, and place in a 40°C water bath for 2 h. Remove and cool to room temperature. Accurately transfer 25 ml of 0.1 mol / L hydrochloric acid solution using a pipette, add 25 ml of deionized water, and titrate with standardized sodium hydroxide solution. The endpoint is indicated by an indicator, and the pH at the equivalence point is 7. Perform three parallel titrations and take the average value V6. Calculate the quaternary ammonium group content W (quaternary ammonium group) of the microspheres according to the following formula based on the volume of sodium hydroxide consumed.

[0110] .

[0111] Example 1

[0112] According to the method provided by the present invention, a zwitterionic ion exchange microsphere adsorption material is prepared.

[0113] Step 1: Add 6g of polyvinyl alcohol to 120ml of distilled water and stir thoroughly at a rate of 350r / min to obtain an aqueous dispersant solution for later use.

[0114] Step 2: Weigh 6.7g of o-divinylbenzene, 20.1g of p-chloromethylstyrene, 20ml of toluene, and 0.1848g of azobisisobutyronitrile, and stir at 350r / min for 30min to obtain an oil phase mixture for later use.

[0115] Step 3: The oil phase mixture obtained in Step 2 is thoroughly mixed with the dispersant aqueous solution obtained in Step 1, and stirred at a rate of 350 r / min for 30 min; 2.5 ml of methyl methacrylate is added at 70 °C, and the mixture is refluxed for 12 h; after the reaction is completed, the mixture is cooled to room temperature, washed with water and ethanol successively, and dried at 60 °C for 12 h to obtain the first reactant.

[0116] Step 4: Take 2g of the first reactant obtained in Step 3 and place it in 20ml of toluene solution to swell for 1h to obtain a swollen mixture; add 12.5ml of N,N-dimethylbutylamine and stir at 350r / min at 85℃ for 18h. After the reaction is completed, the second reactant is obtained.

[0117] Step 5: Cool the second reactant obtained in Step 4 to 70℃; add 100 ml of 4% KOH-methanol solution under low-speed stirring at 100 r / min, and react for 24 h; after the reaction is completed, cool to room temperature, wash the product with ethanol, sodium bicarbonate solution and water respectively, and vacuum dry at 60℃ for 12 h to obtain zwitterionic exchange microsphere adsorption material.

[0118] The most probable pore size of the zwitterionic ion exchange microsphere adsorbent material obtained in this embodiment is 2.3525 nm, the particle size is 30 μm-60 μm, the span value is 0.757, the carboxyl group density is 4.8972 mmol / g, and the quaternary ammonium group density is 1.3564 mmol / g.

[0119] Example 2

[0120] This embodiment verifies the effect of the type and amount of porogen added on the pore size of zwitterionic exchange microspheres.

[0121] The preparation conditions in this embodiment are basically the same as those in Example 1. The difference is that different types and amounts of porogens were used in step 2 to obtain 6 groups of zwitterionic exchange microspheres. The specific conditions for using each group of porogens are shown in Table 1.

[0122] Table 1 shows the conditions for the six different porogen formulations and addition ratios in Example 2.

[0123]

[0124] The pore size of the six groups of zwitterionic ion exchange microsphere adsorbent materials obtained in this embodiment was determined using a V-Sorb280OTP fully automated surface area and pore size analyzer. The results are shown in Table 2.

[0125] Table 2. Pore size measurement results of the six groups of microsphere materials obtained in this embodiment.

[0126]

[0127] As shown in Table 2, when the porogen contains n-dodecane or liquid paraffin, the pore size of the resulting zwitterionic ion exchange microspheres is significantly larger. After appropriately adding toluene, dichloromethane, and o-dichlorobenzene, the pore size of the resulting zwitterionic ion exchange microspheres decreases to varying degrees. As mentioned above, when only toluene and dichloromethane are used as porogens in the preparation of the samples in Examples 2-6, the pore size of the resulting zwitterionic ion exchange microspheres can be reduced to below 10 nm. For microsphere adsorption materials, the smaller the pore size, the larger the specific surface area of ​​the material, the more grafted carboxyl and quaternary ammonium functional groups, and the greater the adsorption capacity of the microsphere material. In practical use, different types of porogens can be selected to adjust the pore size of the resulting zwitterionic ion exchange microspheres as needed. However, considering that excessively large pore sizes may adsorb impurities such as large molecular proteins, causing a decrease in the specificity of the zwitterionic ion exchange microspheres, the preferred pore size of the zwitterionic ion exchange microspheres provided by this invention is 1 nm - 50 nm.

[0128] Example 3

[0129] like Figure 4 (a)- Figure 4 As shown in (d), this embodiment verifies the effect of the type and proportion of dispersant added on the particle size of zwitterionic exchange microspheres.

[0130] The preparation process in this embodiment is basically the same as that in Example 1, except that in step 1, aqueous solutions of 5% hydroxypropyl methylcellulose, 3% polyvinyl alcohol, 5% polyvinyl alcohol, and 10% polyvinyl alcohol by mass concentration were prepared as dispersants to prepare four groups of zwitterionic exchange microspheres. The particle size distribution range of the four groups of microsphere samples was determined using an MS3000 micron particle size analyzer, as shown in Table 3.

[0131] Table 3. Results of particle size range determination for the four groups of zwitterionic ion exchange microspheres obtained in this embodiment.

[0132]

[0133] like Figure 4 As shown in (a), the microspheres obtained using hydroxypropyl methylcellulose as a dispersant exhibit uneven particle size distribution, complex morphology, and large fragments under a scanning electron microscope. Figure 4 (b)- Figure 4 (d) and the data in Table 3 show that the microspheres obtained using polyvinyl alcohol as a dispersant have a uniform particle size distribution and no fragmentation. The particle size is significantly correlated with the concentration of polyvinyl alcohol added. In the preparation method of zwitterionic exchange microspheres provided by the present invention, the dispersant is preferably polyvinyl alcohol with a concentration of 0.1%-20%, more preferably 3-10%.

[0134] Example 4

[0135] like Figure 5 (a)- Figure 5 As shown in (b), this embodiment verifies the effect of adding different types of initiators on the structure of the prepared zwitterionic ion exchange microspheres.

[0136] The preparation process in this embodiment is basically the same as that in Example 1. The difference is that in step 2, benzoyl peroxide is used as an initiator to prepare zwitterionic exchange microspheres.

[0137] It can be clearly seen under an electron microscope, such as Figure 5 As shown in (a), the zwitterionic exchange microspheres prepared in Example 4 using benzoyl peroxide as an initiator have uneven particle size distribution, rough surfaces, and fragmented depressions; while... Figure 5 As shown in (b), the zwitterionic microspheres prepared in Example 1 using azobisisobutyronitrile as an initiator have a uniform particle size distribution between 30 μm and 60 μm and a smooth surface without depressions. This may be because the polymerization reaction temperature is 70°C, and at 70°C, the decomposition rate of azobisisobutyronitrile is significantly faster than that of benzoyl peroxide, resulting in more spherical microspheres. Therefore, in the method for preparing zwitterionic microspheres provided by this invention, azobisisobutyronitrile is preferably used as the initiator.

[0138] Example 5

[0139] like Figure 6 (a)- Figure 6 As shown in (d), this embodiment verifies the effect of stirring rate on the particle size of zwitterionic ion exchange microspheres.

[0140] The preparation process in this embodiment is basically the same as in Example 1, except that the stirring rates during the thorough stirring process in steps 1 and 2 are 250 r / min, 300 r / min, and 400 r / min, respectively, to prepare three sets of zwitterionic ion exchange microspheres. Figure 6 As shown in (a), when stirred at a low speed of 250 r / min, the weak circulation and insufficient shear force of the system result in uneven particle size distribution of the obtained microspheres; Figure 6 As shown in (b), when the stirring speed is 300 r / min, the large droplets break apart under high-speed shear force, resulting in more regular microspheres. However, due to insufficient shear force, some microspheres still exhibit adhesion. When the stirring speed reaches 350 r / min, as shown in (b), Figure 6 As shown in (c), the microspheres obtained in Example 1 have a particle size between 30 μm and 60 μm, and are regularly shaped and uniformly distributed; however, as the stirring speed is further increased to 400 r / min, as... Figure 6As shown in (d), excessive shear force leads to smaller particle size, uneven morphology, and fragmentation of the resulting microspheres. Therefore, in the preparation method of zwitterionic ion exchange microspheres provided by this invention, the stirring rate in steps 1 and 2 is 200-500 r / min, preferably 350 r / min.

[0141] Example 6

[0142] This embodiment verifies the effect of different acrylate monomers and their addition amounts on the functional group density in the obtained zwitterionic exchange microspheres.

[0143] The preparation process in this embodiment is basically the same as that in Example 1, except that the types and amounts of acrylate monomers added in step 3 are shown in Table 4:

[0144] Table 4 Comparison of sample preparation conditions in this embodiment

[0145]

[0146] The functional group densities of the six groups of zwitterionic ion exchange microspheres obtained in this embodiment are shown in Table 5 after measurement.

[0147] Table 5 shows the results of functional group density determination for the six groups of microspheres obtained in this embodiment.

[0148]

[0149] As shown in Table 5, the carboxyl density of the resulting zwitterionic ion-exchange microspheres continuously increases with the increase of acrylate monomer addition. This is because when the monomer addition is small, there are sufficient vinyl active sites in the microspheres, resulting in an increase in the carboxyl density of the microspheres with the increase of acrylate monomer addition. When the addition reaches 8% of the total mass of the polymer monomer mixture, the grafting active sites in the microspheres tend to saturate, and the number of grafted ester bonds also tends to stabilize, ultimately showing a gradual flattening of the measured carboxyl density growth trend, approaching saturation. On the other hand, due to the different molecular structures, under the same addition ratio, the zwitterionic ion-exchange microspheres obtained in the example with added methyl methacrylate have a higher carboxyl density. Therefore, in the preparation method provided by the present invention, methyl methacrylate is preferred as the acrylate monomer, and the addition ratio is preferably 8%-15% of the total mass of the polymer monomer mixture.

[0150] Example 7

[0151] This embodiment verifies the effect of the type and proportion of tertiary amine monomers added on the quaternary ammonium group density of the obtained zwitterionic exchange microspheres.

[0152] The preparation conditions provided in this embodiment are basically the same as those in Example 1, except that in step 4, three different tertiary amine monomers are added, and the amounts added are shown in Table 6:

[0153] Table 6 shows the specific conditions for adding tertiary amine monomers in this embodiment.

[0154]

[0155] The functional group densities of the six groups of zwitterionic ion-exchange microspheres prepared in this embodiment are shown in Table 7 after measurement.

[0156] Table 7 shows the measurement results of the functional group density of microspheres obtained in each group in this embodiment.

[0157]

[0158] Based on the measurement data in Table 7, it can be seen that as the amount of tertiary amine monomer added increases, the quaternary ammonium group density of the obtained zwitterionic exchange microspheres also gradually increases. When the amount of tertiary amine monomer added reaches 8 times the total mass of the second monomer added, the quaternary ammonium group density of the obtained microspheres no longer increases significantly, possibly because the active sites suitable for grafting quaternary ammonium functional groups in the microsphere material tend to be saturated at this point. Therefore, in the preparation method provided by this invention, the amount of tertiary amine monomer added is preferably 1-10 times the total mass of the second monomer, and from an economic point of view, it is preferably 6-8 times.

[0159] Example 8

[0160] This embodiment obtains different zwitterionic ion-exchange microsphere materials by adjusting the total mass of the polymer monomer mixture.

[0161] The preparation conditions in this embodiment are basically the same as those in Example 1, except that the addition mass of o-divinylbenzene and p-chloromethylstyrene in step 2 adopts the two schemes shown in Table 8:

[0162] Table 8. Addition scheme of o-divinylbenzene and p-chloromethylstyrene in this embodiment

[0163]

[0164] The two sets of zwitterionic ion-exchange microspheres were labeled as Example 8-1 and Example 8-2, respectively.

[0165] Example 9

[0166] like Figure 7 As shown, this embodiment verifies the effect of polymerization reaction time on reaction yield.

[0167] The preparation conditions in this embodiment are basically the same as in Example 1, except that the continuous stirring reaction time in step 4 is 4h, 8h, 12h, 16h, and 20h, respectively. Five groups of zwitterionic ion-exchange microspheres were obtained, each group was repeated three times, and the average yield of the three trials was calculated. The yield variation curve with reaction time was obtained, as shown below. Figure 7 As shown, before the reaction time reaches 12 hours, the reaction yield increases with increasing reaction time, reaching 84.6% at 12 hours. After the reaction time exceeds 12 hours, the reaction yield remains stable, and even slightly decreases due to prolonged high temperature. After 20 hours, the yield is 81.3%, with a relative standard deviation of 2.55%. Therefore, in the preparation method of zwitterionic ion exchange microspheres provided by this invention, the polymerization reaction time is preferably 12 hours.

[0168] Example 10

[0169] like Figure 8 As shown, this embodiment verifies the effect of the addition step of acrylate monomers on the structure of zwitterionic ion exchange microspheres.

[0170] The preparation process of this embodiment is basically the same as that of Example 1. The difference is that in step 3, 2.5 ml of methyl methacrylate and the oil phase mixture obtained in step 2 are added to the dispersant aqueous solution obtained in step 1 and mixed thoroughly. The mixture is stirred at a rate of 350 r / min and refluxed for 12-16 h. After the reaction is completed, the mixture is cooled to room temperature, washed with water and ethanol, and dried at 60 °C for 12 h to obtain the first reactant.

[0171] The zwitterionic ion-exchange microspheres obtained in this embodiment were observed under a scanning electron microscope, such as... Figure 8 As shown, the microspheres generated in the reaction exhibited a large aggregation, poor dispersibility, and inconsistent particle size, showing a significant difference in morphology compared to the microspheres obtained in Example 1. This phenomenon occurs because the addition of acrylate monomers to the oil phase mixture accelerates the polymerization reaction, leading to the rapid formation of a large number of microspheres that adhere together, resulting in a complex morphology of the final reaction product. Therefore, in the preparation method of zwitterionic ion exchange microspheres provided by this invention, the preferred step for adding acrylate monomers is to first thoroughly mix the oil phase mixture with the dispersant aqueous solution, and then add the acrylate monomers at 50-80°C and mix thoroughly.

[0172] Experimental Example 1

[0173] This experimental example verifies the recovery rates of polar and nonpolar analytes by the zwitterionic ion-exchange microspheres obtained in this invention.

[0174] Step S1: Weigh appropriate amounts of the nonpolar analyte docetaxel and the polar analyte ranitidine, dissolve them in methanol and prepare 1 g / L standard stock solutions respectively; then, take appropriate amounts of the two standard stock solutions and prepare a 10 mg / L mixed standard working solution.

[0175] Step S2: Select samples 6-1, 6-4, 7-1, 7-5, 8-1, and 8-2 from the zwitterionic ion exchange microsphere samples prepared in Examples 6-8 as packing materials for the solid-phase extraction process. Accurately weigh 20 mg of each of the above six groups of samples and place them in six 1 mL solid-phase extraction columns with sieve plates at both ends.

[0176] Step S3: First, activate the solid-phase extraction (SPE) material with 500 μL of methanol, then balance it with 500 μL of deionized water. Next, take 1 mL of the mixed standard solution from step S1 and pass it through the SPE column. Control the sample flow rate at 5 mL / min. After passing through the column, wash the SPE column with 1 mL of 50% acetonitrile aqueous solution, then elute the SPE column with the eluent. Finally, elute the SPE column a second time with 1 mL of deionized water to obtain 6 sets of eluents. Blow the eluents to near dryness using a nitrogen blower, then make up to 1 mL with methanol. Perform three parallel sets.

[0177] Step S4: Ultra-high performance liquid chromatography (UHPLC) is used for detection. Gradient elution is performed using 0.1% (v / v) formic acid aqueous solution and methanol as the mobile phase. The elution gradient is: 0-5 min 2% methanol; 5-10 min 60% methanol; 10-13 min 100% methanol; 13-15 min 100% methanol; 15-18 min 2% methanol. Quantification is performed using the external standard method.

[0178] (5) The recoveries of docetaxel and ranitidine were calculated based on peak area, as shown in Table 9:

[0179] Table 9. Results of recovery of docetaxel and ranitidine in each group of samples described in this embodiment.

[0180]

[0181] Because it contains both lipophilic benzene rings and hydrophilic charges attached to its surface, the zwitterionic ion-exchange microspheres provided by this invention, as packing materials, can efficiently extract and separate polar and nonpolar analytes simultaneously during solid-phase extraction. As shown in Table 9, Examples 6-1 and 7-1 showed relatively low recoveries of polar analytes. This is because these two groups of microspheres contain lower densities of carboxyl and quaternary ammonium functional groups, resulting in fewer surface charges and thus lower hydrophilicity compared to other groups. With increasing density of carboxyl and quaternary ammonium functional groups, the recoveries of polar analytes significantly improved; the zwitterionic ion-exchange microspheres prepared in Example 7-5 achieved a recoveries of 99.0% for polar analytes. Examples 8-1 and 8-2 showed significant improvements in the recovery of nonpolar analytes. The adsorption effect is excellent, especially the recovery rate of docetaxel by the zwitterionic ion exchange microspheres obtained in Examples 8-2, which reached 96.1%. This indicates that the greater the total amount of polymer monomer mixture in step 2 of the preparation method provided by the present invention, the more benzene rings are contained in the zwitterionic ion exchange microspheres generated in the final reaction, the stronger the lipophilicity, and the better the adsorption effect on non-polar analytes. On the other hand, the increase in the total amount of monomers also increases the grafting sites of carboxyl and quaternary ammonium functional groups, so the density of carboxyl and quaternary ammonium functional groups is also increased, and the recovery rate of polar analytes is also improved.

[0182] Experimental Example 2

[0183] This experimental example verifies that the zwitterionic ion-exchange microsphere material provided by this invention can achieve simultaneous solid-phase extraction separation of acidic and basic analytes.

[0184] Step A1: Take 200 μL of spiked sample containing 5-hydroxytryptamine and its metabolites, add it to 20 μL of internal standard working solution; then add 400 μL of pH buffer, vortex mix for 1 min to obtain the pre-treated solution;

[0185] Step A2: From the zwitterionic ion exchange microsphere samples prepared in Examples 6 and 7, samples 6-1, 6-4, 7-1, and 7-5, along with commercially available adsorption microspheres, were selected as packing materials for the solid-phase extraction process. 3 mg of each sample was added to a 96-well solid-phase extraction plate. 600 μL of methanol was added to the solid-phase extraction plate for activation, and the plate was pressed dry under a positive pressure device. Then, 600 μL of deionized water was added to balance the solid-phase extraction plate, and the plate was pressed dry under a positive pressure device.

[0186] Step A3: Take 600 μL of the pre-treated solution obtained in step A1 and add it to the solid phase extraction plate. Press dry with a positive pressure device to control the flow rate. Wash with 600 μL of deionized water and 600 μL of methanol solution in sequence, and press dry with a positive pressure device to control the flow rate.

[0187] Finally, add 200 μL of 2% formic acid-methanol solution for elution, collect the eluent into a 96-well plate, and press dry under a positive pressure device to control the flow rate; dry the collected eluent under nitrogen, and reconstitute it with 100 μL of deionized water;

[0188] Step A4: High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used. A Phenomenex Kinetex F5 column (2.6 μm, 3.0 × 100 mm) was used at 35 °C. The injection volume was 10 μL. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was methanol solution. Elution was performed according to the gradient parameters in Table 10.

[0189] Table 10 Gradient Parameters

[0190]

[0191] The recoveries of 5-hydroxytryptamine and its metabolites for each group of packing materials are shown in Table 11 below:

[0192] Table 11. Recovery rates of 5-hydroxytryptamine and its metabolites from the above five groups of microspheres.

[0193]

[0194] like Figure 9 The selective adsorption principle illustrated in this invention allows for the simultaneous recovery of both acidic and basic analytes from the zwitterionic ion-exchange microspheres. Due to the simultaneous presence of carboxyl and quaternary ammonium functional groups, these microspheres achieve high recovery rates for both. Table 11 shows that a higher carboxyl group density in the microspheres significantly improves the recovery rate of the basic analyte 5-hydroxytryptamine; conversely, a higher quaternary ammonium group density significantly improves the recovery rate of 5-hydroxyindoleacetic acid. Furthermore, Table 11 also demonstrates that commercially available adsorption microspheres can only separate and recover basic analytes, while the zwitterionic ion-exchange microspheres provided by this invention exhibit high recovery rates for both basic and acidic analytes.

[0195] In summary, by simultaneously introducing carboxyl and quaternary ammonium functional groups into the porous microsphere structure, the zwitterionic ion-exchange microsphere adsorbent material provided by this invention possesses the ability to efficiently and simultaneously recover acidic and basic analytes. Furthermore, due to its benzene ring structure and the presence of a large number of surface charges, the zwitterionic ion-exchange microspheres also exhibit the ability to efficiently and simultaneously recover polar and non-polar analytes, demonstrating high substance retention. The zwitterionic ion-exchange microsphere adsorbent material provided by this invention is particularly suitable for the simultaneous recovery and adsorption of acidic and basic analytes, effectively solving the technical challenge of existing technologies requiring two types of packing materials and two separate adsorption-desorption processes for recovery and determination.

[0196] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a zwitterionic ion-exchange microsphere adsorbent material, characterized in that, The preparation method of the microsphere adsorption material is as follows: Step 1: Add 6g of polyvinyl alcohol to 120ml of distilled water and stir thoroughly at a rate of 350r / min to obtain an aqueous dispersant solution for later use. Step 2: Weigh 6.7g of o-divinylbenzene, 20.1g of p-chloromethylstyrene, 20ml of toluene, and 0.1848g of azobisisobutyronitrile, and stir at 350r / min for 30min to obtain an oil phase mixture for later use. Step 3: The oil phase mixture obtained in Step 2 is thoroughly mixed with the dispersant aqueous solution obtained in Step 1, and stirred at a rate of 350 r / min for 30 min; 2.5 ml of methyl methacrylate is added at 70 °C, and the mixture is refluxed for 12 h; after the reaction is completed, the mixture is cooled to room temperature, washed with water and ethanol successively, and dried at 60 °C for 12 h to obtain the first reactant. Step 4: Take 2g of the first reactant obtained in Step 3 and place it in 20ml of toluene solution, and allow it to swell for 1h to obtain a swollen mixture; add 12.5ml of N,N-dimethylbutylamine, and stir at 350r / min at 85℃ for 18h. After the reaction is completed, the second reactant is obtained. Step 5: Cool the second reactant obtained in Step 4 to 70℃; add 100 ml of 4% KOH-methanol solution under low-speed stirring at 100 r / min, and react for 24 h; after the reaction is completed, cool to room temperature, wash the product with ethanol, sodium bicarbonate solution and water respectively, and vacuum dry at 60℃ for 12 h to obtain zwitterionic exchange microsphere adsorption material. The obtained zwitterionic ion exchange microsphere adsorbent material was determined to have a most probable pore size of 2.3525 nm, a particle size of 30 μm-60 μm, a span value of 0.757, a carboxyl group density of 4.8972 mmol / g, and a quaternary ammonium group density of 1.3564 mmol / g.

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