A molecularly imprinted polymer-chitosan composite microsphere, its preparation method and application

Abstract

This invention provides a molecularly imprinted polymer-chitosan composite microsphere, its preparation method, and its application, relating to the field of solid-phase extraction separation. The preparation method of the molecularly imprinted polymer-chitosan composite microsphere uses tris(2-chloropropyl) phosphate as a template molecule, acrylamide as a functional monomer, ethylene glycol dimethacrylate as a crosslinking agent, azobisisobutyronitrile as an initiator, and anhydrous methanol as a solvent. After prepolymerization, the system undergoes a deoxygenation reaction to prepare tris(2-chloropropyl) phosphate-molecularly imprinted polymer powder, which then reacts with chitosan to form molecularly imprinted polymer-chitosan hydrogel beads, yielding the composite microspheres. Using these composite microspheres as solid-phase extraction packing material can improve the selective enrichment of target analytes and increase the recovery rate of large-volume solid-phase extraction methods. Furthermore, this invention solves the problem of low recovery rates in seawater using current large-volume solid-phase extraction methods and simultaneously lowers the detection limit of analytical methods, improving detection capabilities.

Description

Technical Field

[0001] This invention relates to the field of solid-phase extraction and separation, and particularly to a molecularly imprinted polymer-chitosan composite microsphere, its preparation method, and its application. Background Technology

[0002] Rapid societal development has brought about dramatic changes in human life, but environmental pollution has also increased significantly. With the implementation of various policies, brominated flame retardants, represented by PBDEs, are gradually being controlled and phased out, while organophosphate esters (OPEs) have emerged as a replacement for PBDEs. Due to the widespread use of OPEs, they are now prevalent in the environment, with tris(2-chloropropyl) phosphate being the most common and recalcitrant OPE component. The main methods for analyzing and detecting OPEs in environmental media include gas chromatography-mass spectrometry (GC-MS / MS) and liquid chromatography-mass spectrometry (LC-MS / MS). However, the low concentration and complex matrix of OPEs in the environment hinder effective analysis and detection. Solid-phase extraction (SPE) based on packing materials such as HLB is a mature and widely used sample pretreatment technique, commonly used for the enrichment, separation, and concentration of OPEs in water, bringing the OPE concentration in the sample to the instrument's detection and quantitation limits while significantly reducing matrix effects to improve analytical capabilities. In marine environments, especially seawater, OPE concentrations are generally at the ppt level, requiring large volumes (10-200L) of seawater for enrichment and separation to reach the lower limit of instrument analytical capability, which exceeds the applicability of traditional SPE columns. Large-volume SPE is a novel extraction technique for organic pollutants in seawater and can be used for seawater sample pretreatment experiments in the laboratory environments of research vessels and polar research stations.

[0003] However, the above methods still have problems. Currently, large-volume solid-phase extraction (SPE) technology lacks dedicated pretreatment and analytical methods for OPE components, resulting in low recovery rates and poor performance. Firstly, the packing material used in this technology is XAD resin. XAD resin lacks specific sites and structures, exhibiting poor selectivity for certain target analytes and unsatisfactory recovery rates in complex matrix samples—a significant drawback of this type of material. Molecularly imprinted polymers are a new type of organic material with structures similar to biological antibody-antigen systems. They can be synthesized for almost any target structure, exhibiting high target recognition capabilities. Powdered molecularly imprinted polymer materials are ideal for improving the selectivity of solid-phase extraction and are often used in traditional SPE. However, using molecularly imprinted polymer powder as packing material in large-volume SPE columns can lead to excessively high negative pressure, even clogging and packing material loss. Furthermore, dedicated and mature large-volume SPE methods and instrumental analysis methods for OPE components have not yet been established, resulting in poor recovery rates and detection limits for OPEs. Summary of the Invention

[0004] In view of this, the present invention proposes a molecularly imprinted polymer-chitosan composite microsphere, its preparation method and application.

[0005] To address the aforementioned problems, this invention increases the overall volume of the molecularly imprinted polymer (MIP) filler by combining MIPs with chitosan materials, thereby enhancing chitosan's selective enrichment capacity for target analytes. This yields an ideal large-volume solid-phase extraction filler, solving the current issues of poor selectivity and low recovery rates in this technology. Chitosan is highly flexible, allowing for the synthesis of chitosan-based materials with different morphologies and sizes to meet various needs. It can also be combined with other materials to form composite materials with diverse properties. The chitosan molecular chain possesses numerous amino and hydroxyl functional groups, exhibiting excellent adsorption performance. Using chitosan as a substrate to prepare bio-based aerogels retains the advantages of chitosan itself, such as abundant site sites, strong adsorption capacity, and diverse modification methods, while significantly improving the material's mechanical strength and stability. This allows for applications in the adsorption and removal of pollutants in environmental media, as well as solid-phase extraction. The incorporation of MIPs into chitosan enhances its selective enrichment capacity for target analytes, thereby increasing the recovery rate of large-volume solid-phase extraction methods. By using molecularly imprinted polymers and chitosan composite microspheres as column packing materials, a pretreatment method for large-volume solid-phase extraction was optimized, and an analytical technique based on high-resolution mass spectrometry coupled with liquid chromatography-quadrupole / orbital ion trap was established. This technique addresses the problem of low recovery rates in seawater in current large-volume solid-phase extraction methods, while simultaneously lowering the detection limit and improving detection capabilities.

[0006] The technical solution of this invention is implemented as follows:

[0007] A method for preparing molecularly imprinted polymer-chitosan composite microspheres involves using tris(2-chloropropyl) phosphate as a template molecule, acrylamide as a functional monomer, ethylene glycol dimethacrylate (EGDMA) as a crosslinking agent, azobisisobutyronitrile (AIBN) as an initiator, and anhydrous methanol as a solvent. After prepolymerization, the system undergoes a deoxygenation reaction to prepare tris(2-chloropropyl) phosphate-molecularly imprinted polymer powder. The molecularly imprinted polymer powder then reacts with chitosan to form molecularly imprinted polymer-chitosan hydrogel beads, yielding molecularly imprinted polymer-chitosan composite microspheres (MIP-CS microspheres).

[0008] Furthermore, the preparation method of the molecularly imprinted polymer-chitosan composite microspheres of the present invention specifically includes the following steps:

[0009] (1) Tris(2-chloropropyl) phosphate, acrylamide and anhydrous methanol are mixed and prepolymerized first; then ethylene glycol dimethacrylate and azobisisobutyronitrile are added and prepolymerized again.

[0010] (2) Deoxygenate the system and react at 55-65℃. After the reaction is completed, wash and Soxhlet extract with methanol-acetic acid solution to remove the template. After washing, vacuum drying and grinding, tri(2-chloropropyl) phosphate-molecularly imprinted polymer powder is obtained.

[0011] (3) Dissolve chitosan in acetic acid solution, mix it with the molecularly imprinted polymer powder, add it dropwise to sodium hydroxide solution to form hydrogel beads, continue stirring, let stand, wash with pure water until neutral, pre-freeze, freeze dry to obtain molecularly imprinted polymer-chitosan composite microspheres (i.e. MIP-CS microspheres).

[0012] Furthermore, the molar ratio of tris(2-chloropropyl) phosphate, acrylamide, and ethylene glycol dimethacrylate is 1:(2.7-3.3):(22-26).

[0013] The total molar amount of tri(2-chloropropyl) phosphate, acrylamide, and ethylene glycol dimethacrylate is in the ratio of the mass of azobisisobutyronitrile to 1 mmol: (4-6) mg.

[0014] Furthermore, the ratio of the total mass of the tri(2-chloropropyl) phosphate and acrylamide to the volume of anhydrous methanol is 1g:90-120mL.

[0015] Furthermore, the mass ratio of the molecularly imprinted polymer powder to chitosan is 1:(1.8-2.2).

[0016] Furthermore, in step (1), the prepolymerization time is 5-7 h, and the prepolymerization time is 2-4 h.

[0017] Furthermore, in step (2), the reaction time is 12-20 hours.

[0018] Furthermore, in step (2), the methanol-acetic acid solution is prepared by methanol and acetic acid in a volume ratio of (3.5-4.5:(0.9-1.1).

[0019] Furthermore, in step (2), the Soxhlet extraction time is 110-130 hours.

[0020] Furthermore, in step (2), the vacuum drying temperature is 55-65℃ and the drying time is 20-28h.

[0021] Furthermore, in step (3), the volume concentration of the acetic acid solution is 1%-3%, and the mass volume of the chitosan and the acetic acid solution is 2.0g:90-120ml.

[0022] Furthermore, in step (3), the molar concentration of the sodium hydroxide solution is 2-3 mol / L; the ratio of the total mass of the molecularly imprinted polymer powder and chitosan to the volume of the sodium hydroxide solution is 3.0 g: (350-450) ml.

[0023] Furthermore, in step (3), the mixing is a stirring mixture, and the stirring time is 20-28 hours;

[0024] Furthermore, in step (3), the stirring time is continued for 4-6 hours.

[0025] Furthermore, in step (3), the settling time is 0.8-1.2h.

[0026] Furthermore, in step (3), the pre-freezing temperature is -24~-18℃ and the pre-freezing time is 1.8-2.2h.

[0027] A molecularly imprinted polymer-chitosan composite microsphere is prepared by any one of the methods described in this invention.

[0028] The application of any of the molecularly imprinted polymer-chitosan composite microspheres described in this invention in solid-phase extraction.

[0029] Furthermore, the application of the molecularly imprinted polymer-chitosan composite microspheres described in any one of the inventions in the preparation of large-volume solid-phase extraction columns, wherein the large volume is 10-200L.

[0030] Furthermore, a method for preparing a large-volume solid-phase extraction column based on molecularly imprinted polymer-chitosan microsphere packing includes the following steps: The empty column and connector are sonicated with detergent water for 10-20 min, then rinsed sequentially with tap water, ultrapure water, and methanol 2-4 times each; calcined in a muffle furnace at 400-500℃ for 3-5 h to remove organic impurities; MIP-CS microspheres are added and packed while continuously immersed in ultrapure water to avoid drying the column and separation; the column is rinsed with ultrapure water 3-5 times and dried at 55-65℃ for 20-28 h. The column is placed in a stainless steel sleeve, sonicated with 40-50 mL of methanol for 8-15 min, the liquid is discarded, and this process is repeated 2-4 times. After drying, the column is sealed for later use.

[0031] Furthermore, a method for large-volume solid-phase extraction and high-performance liquid chromatography-high-resolution mass spectrometry analysis of tris(2-chloropropyl) phosphate in seawater includes the following steps:

[0032] (1) Large-volume solid-phase extraction: Seawater samples were spiked with 40-60 ng of tris(2-chloropropyl) phosphate-d 18Then, the sample was loaded at a flow rate of 400-800 mL / min; the sample was freeze-dried for 20-28 h to remove water; in-situ ultrasonic desorption: 30-50 mL of methanol was added to the large-volume solid-phase extraction column, and ultrasonic elution was performed for 10-15 min. The elution was repeated 2-4 times to obtain the eluent; the eluent was concentrated by rotary evaporation, purged with nitrogen to near dryness, and diluted to volume with methanol. The eluent was then filtered through an organic filter membrane to obtain the sample to be tested.

[0033] (2) Instruments: High-performance liquid chromatography-quadrupole / orbital ion trap mass spectrometry; Hypersil GOLD column, 100 mm × 2.1 mm, 1.9 μm; column temperature 24-26℃; injection volume 4-6 μL; flow rate 0.2-0.4 mL / min; mobile phase A: 4-6 mM ammonium acetate aqueous solution; mobile phase B: methanol; gradient elution.

[0034] Gradient elution program: 0-2 min, 90% v / v mobile phase A; 5 min, 10% v / v mobile phase A; 5.1-10 min, 1% v / v mobile phase A; 10.1-12 min, 90% v / v mobile phase A.

[0035] Mass spectrometry conditions: H-ESI source, positive ion 3.5 kV, negative ion 3.0 kV; sheath gas value 45, auxiliary gas value 10; ion transmission tube temperature 320 ℃, vaporization temperature 350 ℃; DDA full scan, resolution 120000, m / z 100-1000.

[0036] Compared with the prior art, the beneficial effects of the present invention are:

[0037] This invention uses tris(2-chloropropyl) phosphate as the target molecule, acrylamide as the functional monomer, and EGDMA as the crosslinking agent to synthesize a molecularly imprinted polymer powder, which is then mixed with chitosan. Through pH adjustment, chitosan is crosslinked to form hydrogel microspheres, locking the molecularly imprinted polymer within them. A freeze-drying process creates a porous structure in the chitosan, increasing its specific surface area and improving its adsorption capacity. The molecularly imprinted material enhances the selectivity of chitosan adsorption, and the tunability of chitosan allows for the expansion of the molecularly imprinted material's volume, making it suitable for large-volume solid-phase extraction columns. A large-volume solid-phase extraction column was fabricated using the molecularly imprinted polymer-chitosan composite microspheres prepared for tris(2-chloropropyl) phosphate as a packing material. This packing material exhibits selective enrichment and separation of tris(2-chloropropyl) phosphate. Subsequently, the extraction conditions, elution conditions, and concentration methods were optimized based on the characteristics of substances like tris(2-chloropropyl) phosphate to obtain a highly efficient method with optimal recovery. Then, a high-performance liquid chromatography-quadrupole / orbital ion trap mass spectrometry (HPLC-QIPMS) technique was used to establish an analytical method, further reducing the method's detection limit. By combining novel sample pretreatment and analytical techniques, a highly sensitive and selective sample analysis method was established. Attached Figure Description

[0038] Figure 1 This is a scanning electron microscope image of the molecularly imprinted polymer powder of the present invention.

[0039] Figure 2 This is the X-ray diffraction pattern of the molecularly imprinted polymer powder of the present invention.

[0040] Figure 3 This is a scanning electron microscope image of the molecularly imprinted polymer-chitosan composite microspheres of the present invention.

[0041] Figure 4 The images show the Raman scattering spectra of the molecularly imprinted polymer, chitosan, and molecularly imprinted polymer-chitosan composite microspheres of this invention.

[0042] Figure 5 The results show the optimization of the types of elution solvents.

[0043] Figure 6 , Figure 7 , Figure 8 3D response surface plot for optimizing large-volume solid-phase extraction conditions, where Figure 6 Extraction time-flow rate, Figure 7 Extraction number - flow rate Figure 8 Extraction number minus extraction time. Detailed Implementation

[0044] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods;

[0045] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0046] The Chinese meanings of some of the abbreviations in this invention are as follows:

[0047] PBDEs: Polybrominated diphenyl ethers;

[0048] OPEs: Organic phosphate esters;

[0049] EGDMA: Ethylene glycol dimethacrylate;

[0050] AIBN: Azobisisobutyronitrile;

[0051] CS: Chitosan.

[0052] The ultrasonic cell disruptor used in this invention has a maximum power of 650W. "Ultrasonic 90%" means that the ultrasonic power reaches 90% of the equipment's maximum ultrasonic power.

[0053] Example 1: Preparation of tris(2-chloropropyl) phosphate-molecularly imprinted polymer

[0054] Raw materials: Tris(2-chloropropyl) phosphate, acrylamide, EGDMA, AIBN, anhydrous methanol, glacial acetic acid.

[0055] Preparation method: Weigh 0.0656 g (approximately 0.2 mmol) of tris(2-chloropropyl) phosphate and 0.0426 g (approximately 0.6 mmol) of acrylamide and place them in a 100 mL three-necked flask; add 10 mL of anhydrous methanol, dissolve, and magnetically stir for 6 h for prepolymerization. Weigh 0.9504 g (approximately 4.8 mmol) of EGDMA and 140 mg of AIBN and add them to the flask, continuing prepolymerization for 3 h. Deoxygenate the system and react in an oil bath at 60 °C for 16 h. After the reaction is complete, wash three times with ethanol. Soxhlet extract the material with methanol-acetic acid (4:1, v / v) for 120 h to wash away the template. After Soxhlet extraction, wash three times each with ethanol and water. Vacuum dry at 60 °C for 24 h. After drying, grind into powder to obtain tris(2-chloropropyl) phosphate-molecularly imprinted polymer powder (MIP).

[0056] like Figure 1 As shown, Figure 1 It can be seen that the molecularly imprinted polymer powder is in the form of uniform spherical particles with a particle size of approximately 0.5 μm.

[0057] like Figure 2 As shown, Figure 2Clear peaks for O, N, C, and Cl elements can be seen in the X-ray diffraction pattern. (a) is the X-ray diffraction pattern of the molecularly imprinted polymer powder before the template is cleaned with acetic acid methanol solution. P and Cl peaks can be seen, indicating the presence of tris(2-chloropropyl) phosphate. (b) is the powder material after the template is cleaned. The P and Cl peaks have disappeared, indicating that the template has been successfully cleaned.

[0058] Example 2: Preparation of molecularly imprinted polymer-chitosan microspheres

[0059] Raw materials: Tris(2-chloropropyl) phosphate-molecularly imprinted polymer powder, chitosan, glacial acetic acid, NaOH, ultrapure water.

[0060] Weigh two 2.0 g portions of chitosan and add them to 100 ml of acetic acid solution (2%, v / v). Stir magnetically at room temperature until homogeneous. Weigh 1.0 g of MIP (Example 1) and add it to the solution, stirring for 24 h to ensure homogeneity. Prepare 400 ml of NaOH solution (2M). Using a syringe, draw the above mixture and add it dropwise to the stirred NaOH solution to form molecularly imprinted polymer-chitosan hydrogel beads. Continue stirring for 5 h, let stand for 1 h, and rinse the gel beads with ultrapure water until neutral. Pre-freeze at -20°C for 2 h and freeze-dry. Obtain molecularly imprinted polymer-chitosan microspheres (MIP-CS).

[0061] like Figure 3 As shown, Figure 3 (a) It can be seen that the microspheres are irregular spheres with a large number of wrinkles on the surface; (b) It can be seen that the surface of the microspheres is uneven and doped with molecularly imprinted polymer powder; (c) It shows that after freeze-drying, a large number of pores are distributed on the surface of the spheres; further magnification, (d) and (e) show that the pores have a cross-linked structure and are attached to or embedded in the MIPs spherical particles.

[0062] like Figure 4 As shown, Figure 4 As can be seen, the surface of the MIP-CS composite microspheres simultaneously exhibits Raman shift characteristic peaks of both the molecularly imprinted polymer and chitosan, indicating that the MIP and CS materials have been thoroughly mixed. In the Raman spectrum of chitosan, the peak at 941 cm⁻¹ is also observed. -1 The characteristic peak at 2890 cm⁻¹ corresponds to the vibration of the CN bond. -1 The characteristic peak at 811 cm⁻¹ corresponds to the vibration of the CH₂ group; in the Raman spectrum of MIP, the peak at 811 cm⁻¹ corresponds to the vibration of the CH₂ group. -1 The characteristic peak at 1118 cm⁻¹ corresponds to the vibration of the OC=O bond. -1 The characteristic peak at 1725 cm⁻¹ corresponds to the vibration of the C-C bond. -1 The characteristic peak at 1455 cm⁻¹ corresponds to the stretching vibration of the C=O group. -1The characteristic peak at 2958 cm⁻¹ corresponds to the shear vibration of the CH₃ group. -1 The characteristic peak at that location corresponds to the vibration of the CH2 group.

[0063] Example 3: Preparation of a large-volume solid-phase extraction column based on molecularly imprinted polymer-chitosan microspheres

[0064] Raw materials: ultrapure water, methanol (chromatographic grade), MIP-CS microbeads

[0065] The sampling column (63.6 cm) without filler was used. 3 The connector was ultrasonically cleaned with water containing detergent for 15 minutes, rinsed with tap water, rinsed three times with ultrapure water, and rinsed three times with methanol, then air-dried. It was then calcined in a muffle furnace at 450°C for 4 hours to remove organic impurities. Using a stainless steel spatula, the treated MIP-CS microspheres (Example 2) were loaded into the sampling column. During loading, ultrapure water was added simultaneously, ensuring the packing material in the column cavity was always just submerged in ultrapure water, allowing the packing material to settle evenly downwards without drying out or stratifying. After the packing material is packed, rinse it with ultrapure water from top to bottom 3-5 times. Then, place the sampling column in an electric heating drying oven and dry it at 60°C for 24 hours. After drying, place it in a stainless steel sleeve that has been ultrasonically cleaned with methanol, add 45 mL of methanol, and place it in an ultrasonic cell disruptor for ultrasonication (ultrasonic power ratio 90%, 10 min). During the ultrasonication process, methanol needs to be continuously added to the sleeve to keep the sampling column completely immersed in the liquid. After the ultrasonication is completed, discard the extract and repeat 3 times. After the sampling column is dried, reconnect the connector, seal and store it for later use.

[0066] Example 4: Establishment of a large-volume solid-phase extraction method and instrumental analysis method for tris(2-chloropropyl) phosphate in seawater based on molecularly imprinted polymer-chitosan microspheres

[0067] 1. Large-volume solid-phase extraction method

[0068] 1) Spiking: Add 50 ng of tris(2-chloropropyl) phosphate-d to the seawater sample. 18 (Deuterated form of the target object);

[0069] 2) Sample loading: Seawater samples were loaded at a flow rate of 450 mL / min;

[0070] 3) Freeze-drying: Freeze-dry at -60℃ for 24 hours to remove moisture;

[0071] 4) Elution: In-situ ultrasonic desorption. The specific procedure is as follows: 40 mL of methanol is added to the extraction column prepared in Example 3, and in-situ ultrasonic desorption is performed (ultrasonic power ratio 90%, 12 min). This is repeated 3 times to obtain a total of 120 mL of eluent.

[0072] 5) Rotary evaporate to near dryness, transfer to a 15 mL glass tube, concentrate to near dryness by nitrogen blowing, transfer to a 1.5 mL vial, bring the volume to 1 mL with methanol, filter through a 0.22 μm organic syringe filter, and wait for instrumental analysis.

[0073] 2. HPLC-MS / Orbitrap analytical method

[0074] Instrument: Vanquish Flex Ultra HighPerformance Liquid Chromatography coupled with Orbitrap Exploris 120 Mass Spectrometer (Thermo Fisher, CA, USA)

[0075] Chromatographic parameters:

[0076] Column temperature: 25 ℃; injection volume: 5 μL; mobile phase: A: 5 mM ammonium acetate aqueous solution, B: methanol; flow rate: 0.3 mL / min; column: Hypersil GOLD, 100 mm × 2.1 mm, 1.9 μm (Thermo Scientific, USA); mobile phase gradient is shown in Table 1.

[0077] Table 1. Mobile phase gradient

[0078]

[0079] Mass spectrometry parameters:

[0080] Ion source: H-ESI; Spray voltage: Static, positive ion: 3500, negative ion: 3000; Gas mode: Static, sheath gas: 45, auxiliary gas: 10, purge gas: 0; Ion transmission tube temperature: 320℃; Vaporization temperature: 350℃. Scanning mode: Data-dependent acquisition (DDA), full scan, resolution: 120000, scan range (m / z): 100-1000, lens voltage: 80%, positive charge mode. Target chemical formulas, mass-to-charge ratios, and adducts are shown in Table 2.

[0081] Table 2 Chemical formulas, mass-to-charge ratios, and adducts of the target compounds

[0082]

[0083] Table Note: Tris(2-chloropropyl) phosphate-d 18 It is a deuterated derivative of "tris(2-chloropropyl) phosphate", d 18It is an abbreviation for deuterium, indicating that the compound molecule has a total of 18 hydrogen atoms (H) surrounded by deuterium atoms (H). 2 H) substitution.

[0084] Test case

[0085] This study focuses on the "eluent" in large-volume solid-phase extraction. First, the desorption conditions of the large-volume solid-phase extraction process were optimized using single-factor methods to explore the influence of the eluent on the desorption efficiency. The results are as follows: Figure 5 As shown, the effects of methanol, acetonitrile, dichloromethane, and methanol + dichloromethane (1:1, v / v) on the desorption efficiency were explored. Methanol was found to be the most effective, and therefore methanol was selected as the optimal eluent.

[0086] A three-factor, three-level experiment was designed using response surface methodology to investigate the effects of three factors—sample loading rate (A), elution time (B), and elution cycles (C)—on the recovery rate. Each factor was divided into three levels: low, medium, and high. Methanol was used as the desorption solvent. The experimental scheme for the three-factor, three-level experiment is shown in Table 3. The influence of each condition on the response level of tris(2-chloropropyl) phosphate was examined, and the results are shown in Table 4.

[0087] Table 3. Box-Behnken three-factor, three-level experimental design

[0088]

[0089] Table 4. Results of the Box-Behnken three-factor, three-level experiment

[0090]

[0091] Response surface methodology was performed on the experimental results to obtain the response surface plot. Figure 6 ) and the regression model variance table (Table 5). Figure 2 The results show that the interaction of different factors in pairs affects the recovery rate, and the predicted correlation coefficient of the established model (Predicted R-value) is... 2 The adjusted correlation coefficient (R²) was 0.8021. 2 The value of 0.9577 indicates that the model fitting result meets the requirements.

[0092] In Table 5, the independent variables A (flow rate), B (extraction time), C (number of extractions), and A 2 B 2 C 2The p-value was less than 0.05, indicating that the above model had a significant impact on the experimental results. The model results showed that flow rate, extraction time, and number of extractions were important factors affecting the recovery rate. Based on the above results and the prediction conditions of the response surface model, the optimal extraction conditions were finally determined to be: an optimal flow rate of 450 mL / min, an elution time of 12 min, and 3 elutions.

[0093] Table 5. Simulation variance results of the Box-Behnken three-factor, three-level experiment.

[0094]

[0095] This method uses linear range, correlation coefficient, limit of detection, limit of quantitation, precision, and accuracy as evaluation criteria to verify its reliability. Table 6 lists the linear range, limit of detection, and limit of quantitation of the method; Table 7 shows the method precision; and Tables 8 and 9 show the spiked recoveries for tap water and seawater samples, respectively.

[0096] Table 6. Linearity range, limit of detection, and limit of quantitation of the method

[0097]

[0098] Table 7. Precision of the method

[0099]

[0100] Table 8. Spike recovery rate (accuracy) of tap water samples

[0101]

[0102] Table 9. Spike recoveries (accuracy) of seawater samples

[0103]

[0104] In summary, based on the novel molecularly imprinted polymer-chitosan microsphere material, and with optimized large-volume solid-phase extraction conditions, an analytical method based on HPLC-Q / Orbitrap was constructed, establishing a solid-phase extraction and analysis method for tris(2-chloropropyl) phosphate in seawater with selectivity, high enrichment factor, and high sensitivity.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the scope of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing molecularly imprinted polymer-chitosan composite microspheres for large-volume solid-phase extraction, characterized in that, Using tris(2-chloropropyl) phosphate as a template molecule, acrylamide as a functional monomer, ethylene glycol dimethacrylate as a crosslinking agent, azobisisobutyronitrile as an initiator, and anhydrous methanol as a solvent, a prepolymerization reaction was carried out to remove oxygen from the system, thereby preparing tris(2-chloropropyl) phosphate-molecularly imprinted polymer powder. The molecularly imprinted polymer powder reacted with chitosan to form molecularly imprinted polymer-chitosan hydrogel beads, thus obtaining molecularly imprinted polymer-chitosan composite microspheres. The molar ratio of tris(2-chloropropyl) phosphate, acrylamide, and ethylene glycol dimethacrylate is 1:(2.7-3.3):(22-26); the ratio of the total molar amount of tris(2-chloropropyl) phosphate, acrylamide, and ethylene glycol dimethacrylate to the mass of azobisisobutyronitrile is 1 mmol:(24-26) mg; the ratio of the total mass of tris(2-chloropropyl) phosphate and acrylamide to the volume of anhydrous methanol is 1 g:(90-120) mL; and the mass ratio of the molecularly imprinted polymer powder to chitosan is 1:(1.8-2.2). Includes the following steps: (1) Tris(2-chloropropyl) phosphate, acrylamide and anhydrous methanol are mixed and prepolymerized first; then ethylene glycol dimethacrylate and azobisisobutyronitrile are added and prepolymerized again. (2) Deoxygenate the system and react at 55-65℃. After the reaction is completed, wash and Soxhlet extract with methanol-acetic acid solution to remove the template. After washing, vacuum drying and grinding, tri(2-chloropropyl) phosphate-molecularly imprinted polymer powder is obtained. (3) Dissolve chitosan in acetic acid solution, mix it with the tris(2-chloropropyl) phosphate-molecularly imprinted polymer powder, add it dropwise to sodium hydroxide solution to form hydrogel beads, continue stirring, let stand, wash with pure water until neutral, pre-freeze, freeze dry to obtain molecularly imprinted polymer-chitosan composite microspheres.

2. The method for preparing molecularly imprinted polymer-chitosan composite microspheres for large-volume solid-phase extraction according to claim 1, characterized in that, In step (1), the prepolymerization time is 5-7 h, and the prepolymerization time is 2-4 h.

3. The method for preparing molecularly imprinted polymer-chitosan composite microspheres for large-volume solid-phase extraction according to claim 1, characterized in that, In step (2), the reaction time is 12-20 h; the methanol-acetic acid solution is prepared by methanol and acetic acid in a volume ratio of (3.5-4.5):(0.9-1.1); the Soxhlet extraction time is 110-130 h; the vacuum drying temperature is 55-65℃ and the drying time is 20-28 h.

4. The method for preparing molecularly imprinted polymer-chitosan composite microspheres for large-volume solid-phase extraction according to claim 1, characterized in that, In step (3), the volume concentration of the acetic acid solution is 1%-3%; the mass-to-volume ratio of chitosan to acetic acid solution is 2.0g:(90-120)ml; the molar concentration of the sodium hydroxide solution is 2-3mol / L; the ratio of the total mass of the molecularly imprinted polymer powder and chitosan to the volume of the sodium hydroxide solution is 3.0g:(350-450)ml; the mixing is by stirring for 20-28h; the stirring time is 4-6h; the standing time is 0.8-1.2h; the pre-freezing temperature is -24~-18℃, and the pre-freezing time is 1.8-2.2h.

5. A molecularly imprinted polymer-chitosan composite microsphere, characterized in that, It is prepared by the method described in any one of claims 1-4.

6. The application of the molecularly imprinted polymer-chitosan composite microspheres according to claim 5 in large-volume solid-phase extraction.

7. The application according to claim 6, characterized in that, A large-volume solid-phase extraction column was prepared using molecularly imprinted polymer-chitosan microspheres as packing material. The preparation method included the following steps: taking an empty column and connector, cleaning it, and calcining it at 400-500℃ for 3-5 hours; adding the molecularly imprinted polymer-chitosan microspheres while continuously immersing it in water, allowing the packing material to settle uniformly downwards; rinsing the packing material, drying it, placing it in a sleeve, adding methanol, and completely immersing the sampling column in the liquid; discarding the extractant after sonication; and reconnecting the connector after the sampling column has dried, sealing and storing it; the large volume was 10-200 L.

8. The application according to claim 7, characterized in that, Based on the aforementioned large-volume solid-phase extraction column, a method for large-volume solid-phase extraction and high-performance liquid chromatography-high-resolution mass spectrometry analysis of tris(2-chloropropyl) phosphate in seawater was established, including the following steps: (1) Large-volume solid-phase extraction: Seawater samples were spiked with 40-60 ng of tris(2-chloropropyl) phosphate-d 18 Then, the sample was loaded at a flow rate of 400-800 mL / min; the sample was freeze-dried for 20-28 h to remove water; methanol was added to the large-volume solid-phase extraction column, and ultrasonic elution was performed for 10-15 min. The elution was repeated 2-4 times, and the eluent was collected; the eluent was concentrated by rotary evaporation, purged with nitrogen to near dryness, diluted to volume with methanol, and filtered through an organic filter membrane to obtain the sample to be tested. (2) Detection instrument: High performance liquid chromatography-quadrupole / orbital ion trap mass spectrometry, column temperature 24-26℃, injection volume 4-6 μL, flow rate 0.2-0.4 mL / min, mobile phase A is 4-6 mM ammonium acetate aqueous solution, mobile phase B is methanol, gradient elution, gradient elution program is: 0-2 min, 90% v / v mobile phase A; 5 min, 10% v / v mobile phase A; 5.1-10 min, 1% v / v mobile phase A; 10.1-12 min, 90% v / v mobile phase A; Mass spectrometry conditions: H-ESI source, positive ion 3.5kV, negative ion 3.0kV; sheath gas value 45, auxiliary gas value 10; ion transmission tube temperature 320℃, vaporization temperature 350℃; DDA full scan, resolution 120000, m / z 100-1000.

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