Preparation of silver nanocluster functionalized monolithic column and selective solid-phase microextraction application thereof

Abstract

The invention discloses preparation of a silver nano-cluster functionalized monolithic column and selective solid-phase microextraction application of the silver nano-cluster functionalized monolithic column. According to the invention, urea and formaldehyde are taken as matrix monomers, glutathione or sulfanilic acid is taken as a functionalization-reaction catalysis bifunctional agent, and a glutathione functionalized monolithic column or sulfanilic acid functionalized monolithic column is prepared by a one-step polycondensation method; and by utilizing multiple interaction between glutathione or p-aminobenzene sulfonic acid and silver ions, in-situ reduction is carried out on the surface of the monolithic column to generate and fix the silver nano-cluster, and the silver nano-cluster functionalized monolithic column is prepared. The invention provides a new way of combining a cluster material and the monolithic column, the prepared monolithic column can be used as an in-tube solid-phase microextraction stationary phase, and online selective enrichment and high-sensitivity analysis of trace trans-fatty acid in a complex sample are realized. The method is ingenious in process, simple and convenient to operate, free of expensive instruments, easy to popularize and high in analysis object specificity.

Description

Technical Field

[0001] The invention belongs to the field of monolithic column preparation, and in particular relates to the preparation of a silver nanocluster functionalized monolithic column and its selective solid phase microextraction application. Background Art

[0002] Monolithic columns, also known as continuous beds, are a type of continuous column bed with a porous structure prepared by in-situ polymerization in a tube. Monolithic columns have many advantages, including good permeability, fast mass transfer, strong mechanical stability, easy chemical modification, and good biocompatibility, and are widely used in the fields of chromatographic separation and sample pretreatment.

[0003] Silver nanoclusters are ultra-small clusters composed of several to hundreds of silver atoms, and their size is generally less than 5nm. Due to the quantum size effect, silver nanoclusters not only exhibit non-metallic properties and photophysical properties similar to molecules, but also retain some affinity activities similar to silver ions. It has broad application prospects in biology, optoelectronics and other fields, but its research is still in its infancy and various properties are still being explored. However, silver nanoclusters, due to their small scale effect, will cause agglomeration, which reduces their stability and surface activity. How to improve their stability has attracted much attention.

[0004] Small-sized silver nanoclusters usually need to rely on certain carriers to exist stably. To this end, the present invention uses a monolithic column as a carrier and silver nanoclusters as functionalized substances to prepare a silver nanocluster functionalized monolithic column, and proposes a new way to combine silver nanoclusters with monolithic columns, in order to solve the stability problem of silver nanoclusters while utilizing the silver ion-like affinity between silver nanoclusters and unsaturated carbon-carbon double bonds to achieve selective enrichment and extraction of trans fatty acids with unsaturated carbon-carbon double bonds. The monolithic column prepared by the present invention can be used as a selective in-tube solid phase microextraction stationary phase, and then used in conjunction with relevant analytical methods to achieve selective and efficient enrichment and online high-sensitivity analysis of trace trans fatty acids in complex actual samples. Summary of the invention

[0005] The purpose of the present invention is to provide a silver nanocluster functionalized monolithic column and use it as a selective in-tube solid phase microextraction stationary phase to achieve selective solid phase microextraction and highly sensitive analysis of trace trans fatty acids in complex samples.

[0006] To achieve the above object, the present invention adopts the following technical solution: A silver nanocluster functionalized monolithic column, which uses urea and formaldehyde as matrix monomers, glutathione or p-aminobenzenesulfonic acid as functionalization-reaction catalytic bifunctional agents, and prepares the glutathione functionalized monolithic column or p-aminobenzenesulfonic acid functionalized monolithic column by a one-step polycondensation method; and then utilizes the multiple interactions between glutathione or p-aminobenzenesulfonic acid and silver ions to in-situ reduce and generate and fix silver nanoclusters on the surface of the monolithic column to prepare the silver nanocluster functionalized monolithic column; wherein the amount of glutathione used is 110-220 mg / g urea, and the amount of p-aminobenzenesulfonic acid used is 27-55 mg / g urea; Wherein, the silver ions are derived from a silver nitrate solution; the concentration of the silver nitrate solution is 4 to 10 mmol / L.

[0007] The method for preparing the above-mentioned silver nanocluster functionalized monolithic column comprises the following steps: 1) Preparation of glutathione functionalized monolithic column: 550 μL of 1 g / mL urea solution, 450 μL of 33 wt% to 37 wt% formaldehyde solution, 100 μL of water and 60 to 120 mg of glutathione were mixed and vortexed for 30 seconds to prepare a monolithic column polycondensation reaction solution; the monolithic column polycondensation reaction solution was sucked up with a disposable sterile syringe and quickly injected into a clean and dried polytetrafluoroethylene tube, and after sealing both ends, it was placed in a 65° C. water bath for reaction for 2 to 12 hours; the obtained monolithic column was connected to a μHPLC pump and rinsed with ultrapure water for 30 minutes to remove unreacted polycondensation precursors, oligomers and other impurities in the monolithic column to obtain a glutathione functionalized monolithic column; 2) Preparation of silver nanocluster functionalized monolithic column: prepare silver nitrate solution with a concentration of 4-10 mmol / L, inject 1.0 mL of silver nitrate solution into the glutathione functionalized monolithic column using a microinjection pump, then cap it, and then place the glutathione functionalized monolithic column in a 65°C water bath to react for 30 minutes, repeat the above steps three times; connect the obtained monolithic column to a μHPLC pump, and rinse with methanol, isopropanol, and n-hexane as mobile phases for 30 minutes respectively to obtain a silver nanocluster functionalized monolithic column.

[0008] The method for preparing the above-mentioned silver nanocluster functionalized monolithic column comprises the following steps: 1) Preparation of p-aminobenzenesulfonic acid functionalized monolithic column: 550 μL 1g / mL urea solution, 450 μL 33wt% to 37wt% formaldehyde solution, 100 μL water and 15 to 30 mg p-aminobenzenesulfonic acid were mixed and vortexed for 30 seconds to prepare a monolithic column polycondensation reaction solution; the monolithic column polycondensation reaction solution was drawn up with a disposable sterile syringe and quickly injected into a clean and dried polytetrafluoroethylene tube, and after sealing both ends, it was placed in a 50°C water bath for reaction for 2 to 12 hours; the obtained monolithic column was connected to a μHPLC pump and rinsed with ultrapure water for 30 minutes to remove unreacted polycondensation precursors, oligomers and other impurities in the monolithic column to obtain a p-aminobenzenesulfonic acid functionalized monolithic column; 2) Preparation of silver nanocluster functionalized monolithic column: prepare a silver nitrate solution with a concentration of 4-10 mmol / L, inject 1.0 mL of the silver nitrate solution into the aminobenzenesulfonic acid functionalized monolithic column using a microinjection pump, then cap it, and then place the aminobenzenesulfonic acid functionalized monolithic column in a 65°C water bath to react for 30 minutes, and repeat the above steps three times; connect the obtained monolithic column to a μHPLC pump, and rinse with methanol, isopropanol, and n-hexane as the mobile phase for 30 minutes respectively to obtain a silver nanocluster functionalized monolithic column.

[0009] Among them, in the first step of preparing the silver nanocluster functionalized monolithic column, glutathione (or p-aminobenzenesulfonic acid) simultaneously plays the role of a reaction catalyst and a functionalizing agent; specifically, glutathione can ionize hydrogen ions to catalyze the urea-formaldehyde polycondensation reaction, and glutathione can also directly participate in the polycondensation reaction of urea-formaldehyde resin, thereby realizing the glutathione functionalization of urea-formaldehyde resin; p-aminobenzenesulfonic acid can also ionize hydrogen ions to catalyze the urea-formaldehyde polycondensation reaction, and utilize the substitution reaction between the aromatic ring hydrogen at the adjacent position of the amino group and formaldehyde to connect p-aminobenzenesulfonic acid to the polymer backbone of the urea-formaldehyde resin, thereby realizing the p-aminobenzenesulfonic acid functionalization of the urea-formaldehyde resin.

[0010] Among them, in the second step of the preparation of the silver nanocluster functionalized monolithic column, an in-situ reduction and immobilization strategy is adopted to prepare the silver nanocluster functionalized monolithic column; specifically, for the glutathione functionalized monolithic column, the silver ions are in-situ reduced to silver nanoclusters under the reducing action of glutathione, and the silver nanoclusters are fixed on the surface of the monolithic column through the metal-thiol interaction between the residual thiol groups of glutathione on the surface of the monolithic column and the silver nanoclusters; for the p-aminobenzenesulfonic acid functionalized monolithic column, the silver ions are reduced to silver nanoclusters in situ by utilizing the reducing property of the amino group conjugated to the benzene ring and the strengthening effect of the para-sulfonic acid group on the reducing property, and the silver nanoclusters are fixed on the surface of the monolithic column through the electrostatic interaction between the sulfonic acid groups of p-aminobenzenesulfonic acid and the silver nanoclusters.

[0011] The present invention further provides the use of the above-mentioned silver nanocluster functionalized monolithic column in the selective solid phase microextraction and online combined analysis of trace trans fatty acids in complex samples. Since there is a unique silver ion-like affinity between the silver nanocluster on the surface of the monolithic column and the unsaturated carbon-carbon double bonds, trans fatty acids with unsaturated carbon-carbon double bonds can be selectively enriched and extracted on the silver nanocluster functionalized monolithic column, and then combined with relevant analytical methods to achieve selective and efficient enrichment and online high-sensitivity analysis of trace trans fatty acids in complex actual samples.

[0012] The significant advantages of the present invention are: 1) The monolithic column preparation scheme of the present invention is ingeniously and concisely designed. In the process of preparing the monolithic column, glutathione or p-aminobenzenesulfonic acid is used as a functionalizing agent and a reaction catalyst, and a glutathione-functionalized monolithic column or p-aminobenzenesulfonic acid-functionalized monolithic column is prepared by a one-step polycondensation method, which provides a large number of active sites for the subsequent in-situ reduction and surface immobilization of silver nanoclusters.

[0013] 2) The present invention proposes for the first time a strategy for in situ reduction and immobilization of silver nanoclusters, utilizing the multiple interactions between glutathione or p-aminobenzenesulfonic acid and silver ions to in situ reduce and generate and immobilize silver nanoclusters on the surface of the monolithic column to prepare a silver nanocluster functionalized monolithic column; the preparation process is simple, which greatly improves the preparation efficiency and success rate of the silver nanocluster functionalized monolithic column.

[0014] 3) The present invention provides a new way to combine cluster materials with integral columns, which has ingenious process, simple operation, does not require expensive instruments, and is easy to promote.

[0015] 4) The monolithic column prepared by the present invention can be used as an in-tube solid phase microextraction stationary phase, has clear specificity for the analysis object, and can realize the online selective enrichment and highly sensitive analysis of trace trans fatty acids in complex samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The following are the energy dispersive X-ray spectroscopy (EDS) diagrams corresponding to the three monolithic column materials. Among them, a is a urea-formaldehyde resin matrix monolithic column material; b is a glutathione functionalized monolithic column material; and c is a silver nanocluster functionalized monolithic column material.

[0017] Figure 2 The solid UV-visible spectra of two monolithic column materials are shown in Figure 1. Among them, a is a glutathione functionalized monolithic column material; b is a silver nanocluster functionalized monolithic column material.

[0018] Figure 3is a chromatogram of high performance liquid chromatography. a is a chromatogram of the detection of 9t-C18:1 and 9t,12t-C18:2 standard solutions by in-tube solid phase microextraction-silver ion liquid chromatography with silver nanocluster functionalized monolithic column as solid phase microextraction stationary phase; b is a chromatogram of the detection of spiked bread samples by the same method; c is a chromatogram of the detection of blank bread samples by the same method; d is a chromatogram of the detection of spiked bread samples by direct silver ion liquid chromatography. Among them, the spiked concentration of the analyte is 10μg / g; peak attributes: (1) trans-oleic acid methyl ester (9t-C18:1); (2) trans-linoleic acid methyl ester (9t,12t-C18:2).

[0019] Figure 4 The following are the EDS spectra of three monolithic column materials. Among them, a is a urea-formaldehyde resin matrix monolithic column material; b is a p-aminobenzenesulfonic acid functionalized monolithic column material; and c is a silver nanocluster functionalized monolithic column material.

[0020] Figure 5 Solid UV-visible spectra of two monolithic column materials. A is the monolithic column material functionalized with p-aminobenzenesulfonic acid; B is the monolithic column material functionalized with silver nanoclusters.

[0021] Figure 6 The following are HPLC chromatograms. a is a chromatogram of the detection of 9t-C18:1 and 9t,12t-C18:2 standard solutions by in-tube solid phase microextraction-silver ion liquid chromatography, using silver nanocluster functionalized monolithic column material as solid phase microextraction stationary phase; b is a chromatogram of the detection of spiked bread samples by the same method; c is a chromatogram of the detection of blank bread samples by the same method; d is a chromatogram of the detection of spiked bread samples by direct silver ion liquid chromatography. The spiked concentration of the analyte is 10 μg / g; peak attributes: (1) trans-oleic acid methyl ester (9t-C18:1); (2) trans-linoleic acid methyl ester (9t,12t-C18:2). DETAILED DESCRIPTION

[0022] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0023] The preparation method of the urea-formaldehyde resin matrix monolithic column involved in the following embodiment is as follows: 550 μL urea solution (1 g / mL), 450 μL commercial formaldehyde solution (Sinopharm Group Chemical Reagent Co., Ltd.) with a mass concentration of 33% to 37%, and 100 μL ultrapure water are mixed and vortexed for 30 seconds to prepare a monolithic column polycondensation reaction liquid; the monolithic column polycondensation reaction liquid is sucked with a disposable sterile syringe, and quickly injected into a clean and dried polytetrafluoroethylene (PTFE) tube (with an inner diameter of 750 μm and a column length of 25 cm), and after sealing both ends, the PTFE tube containing the monolithic column polycondensation reaction liquid is placed in a 65°C water bath for 6 hours. After the reaction is completed, the obtained monolithic column is connected to a μHPLC pump, and ultrapure water is used to rinse for 30 minutes at a flow rate of 0.10 mL / min to remove unreacted polycondensation precursors, oligomers and other impurities in the monolithic column to obtain a urea-formaldehyde resin matrix monolithic column.

[0024] Example 1 This embodiment provides the preparation of a silver nanocluster functionalized monolithic column and its selective solid phase microextraction application, which specifically includes the following steps: 1) Preparation of glutathione functionalized monolithic column: 550 μL urea solution (1 g / mL), 450 μL commercially available formaldehyde solution (Sinopharm Chemical Reagent Co., Ltd.) with a mass concentration of 33% to 37%, 100 μL ultrapure water and 100 mg glutathione were mixed and vortexed for 30 seconds to prepare the monolithic column polycondensation reaction solution; the monolithic column polycondensation reaction solution was aspirated with a disposable sterile syringe and quickly injected into a clean and dried polytetrafluoroethylene (PTFE) tube (with an inner diameter of 750 μm and a column length of 25 cm), and after sealing both ends, the PTFE tube containing the monolithic column polycondensation reaction solution was placed in a 65°C water bath for 6 hours. After the reaction was completed, the obtained monolithic column was connected to a μHPLC pump and flushed with ultrapure water at a flow rate of 0.10 mL / min for 30 minutes to remove unreacted polycondensation precursors, oligomers and other impurities in the monolithic column to obtain a glutathione functionalized monolithic column; 2) Preparation of silver nanocluster functionalized monolithic column: prepare a silver nitrate solution with a concentration of 10 mmol / L, inject 1.0 mL of the silver nitrate solution into the glutathione functionalized monolithic column using a microinjection pump at a flow rate of 0.05 mL / min, then cap it, and then place the glutathione functionalized monolithic column in a 65°C water bath to react for 30 minutes, and repeat the above steps three times; connect the obtained monolithic column to a μHPLC pump, and use methanol, isopropanol, and n-hexane as the mobile phases, and flush them at a flow rate of 0.10 mL / min for 30 minutes respectively to obtain a silver nanocluster functionalized monolithic column.

[0025] Figure 1The elemental compositions of urea-formaldehyde resin matrix monolithic column materials, glutathione functionalized monolithic column materials and silver nanocluster functionalized monolithic column materials were analyzed by energy dispersive X-ray spectroscopy (EDS). Figure 1 -a) only showed signals of three elements: C, N, and O (the appearance of Au was caused by gold spraying); while the glutathione functionalized monolithic column material ( Figure 1 -b) also added the signal of S element, proving that glutathione was successfully bound to urea-formaldehyde resin material; in the silver nanocluster functionalized monolithic column material ( Figure 1 -c), a silver signal further appeared, which indicates that the silver element has achieved the functionalization of the overall column through its strong interaction with the thiol group.

[0026] Figure 2 Solid UV-visible spectroscopy was used to characterize the glutathione functionalized monolithic column material and the silver nanocluster functionalized monolithic column material. Figure 2 -a) compared to the silver nanocluster functionalized monolithic pillar material ( Figure 2 -b) An obvious absorption peak appeared at 355nm, which was caused by the nanoconfinement effect of silver nanoclusters, further confirming the successful preparation of silver nanocluster functionalized monolithic columns.

[0027] The silver nanocluster functionalized monolith prepared according to the above specific implementation method is used as the solid phase microextraction stationary phase, combined with the in-tube solid phase microextraction-silver ion liquid chromatography coupling system (the construction of the coupling system refers to the utility model patent 2018207623454), to achieve the selective enrichment and efficient analysis of trace trans fatty acid methyl esters (9t-C18:1, 9t,12t-C18:2) in complex actual samples, and with Ag + -HPLC direct detection (Microchemical Journal, 2022, 179, 107540) analysis results were compared. The optimal detection conditions of this combined method are as follows: sample loading liquid composition: n-hexane; injection flow rate: 0.06mL / min; eluent composition: n-hexane / acetonitrile / dichloromethane = 99.5% / 0.4% / 0.1%; elution flow rate: 0.06mL / min; injection volume: 500μL; elution volume: 250μL; column oven temperature: 40℃; detection wavelength: 203nm.

[0028] Figure 3 Chromatograms of spiked and blank bread samples analyzed by different detection methods are shown. + -HPLC testing of spiked bread samples ( Figure 3-d), the matrix in the sample produced strong signal interference, many unknown signal peaks overlapped with the target 9t-C18:1 signal peak, and the signal peaks of 9t, 12t-C18:2 could not be detected, so the target could not be accurately identified and quantitatively analyzed. When the method proposed in this embodiment was used to detect the blank sample ( Figure 3 -c), the matrix interference of the sample was significantly reduced, the interference signal peak of the unknown substance was greatly weakened, and no other interference signal appeared near the peak position of the analyzed object, indicating that the prepared silver nanocluster functionalized monolithic column can significantly reduce the matrix interference in complex samples and has excellent matrix purification ability; when the spiked bread sample was analyzed by the method of the present invention ( Figure 3 -b), the spiked trace amounts of two trans fatty acid methyl esters can be efficiently enriched and accurately identified, indicating that the silver nanocluster functionalized monolithic column prepared in this work has excellent selective enrichment and extraction performance for the target trans fatty acid methyl esters.

[0029] Example 2 This embodiment provides the preparation of a silver nanocluster functionalized monolithic column and its selective solid phase microextraction application, which specifically includes the following steps: 1) Preparation of p-aminobenzenesulfonic acid functionalized monolithic column: 550 μL urea solution (1 g / mL), 450 μL commercially available formaldehyde solution (Sinopharm Chemical Reagent Co., Ltd.) with a mass concentration of 33% to 37%, 100 μL ultrapure water and 25 mg p-aminobenzenesulfonic acid were mixed and vortexed for 30 seconds to prepare the monolithic column polycondensation reaction solution; the monolithic column polycondensation reaction solution was aspirated with a disposable sterile syringe and quickly injected into the cleaned and dried PTFE tube, and after sealing both ends, it was placed in a 50°C water bath for reaction for 10 hours. After the reaction was completed, the obtained monolithic column was connected to a μHPLC pump and rinsed with ultrapure water at a flow rate of 0.10 mL / min for 30 minutes to remove the unreacted polycondensation precursors, oligomers and other impurities in the monolithic column to obtain a p-aminobenzenesulfonic acid functionalized monolithic column; 2) Preparation of silver nanocluster functionalized monolithic column: prepare a silver nitrate solution with a concentration of 5 mmol / L, inject 1.0 mL of silver nitrate solution into the p-aminobenzenesulfonic acid functionalized monolithic column using a microinjection pump at a flow rate of 0.05 mL / min, then cap it, and then place the p-aminobenzenesulfonic acid functionalized monolithic column in a 60°C water bath to react for 30 minutes, and repeat the above steps three times; connect the obtained monolithic column to a μHPLC pump, and use methanol, isopropanol, and n-hexane as the mobile phase, and rinse at a flow rate of 0.10 mL / min for 30 minutes respectively to obtain a silver nanocluster functionalized monolithic column.

[0030] Figure 4 The elemental compositions of urea-formaldehyde resin matrix monolithic column materials, p-aminobenzenesulfonic acid functionalized monolithic column materials and silver nanocluster functionalized monolithic column materials were analyzed by energy dispersive X-ray spectroscopy (EDS). Figure 4 It can be seen that the urea-formaldehyde resin matrix monolithic column material ( Figure 4 -a) mainly contains three elements: C, N, and O; p-aminobenzenesulfonic acid functionalized monolithic column material ( Figure 4 -b) contains not only C, N, and O, but also a new S element, which is mainly derived from the functionalization reagent p-aminobenzenesulfonic acid, indicating that p-aminobenzenesulfonic acid has achieved the functionalization of urea-formaldehyde resin by participating in the polycondensation reaction of urea-formaldehyde resin; in addition, it can be found that the N element signal peak of the p-aminobenzenesulfonic acid functionalized monolithic column material has increased significantly, indicating that p-aminobenzenesulfonic acid also participates in the polymerization reaction of the monolithic column, providing a large amount of N element for the monolithic material. Compared with the p-aminobenzenesulfonic acid functionalized monolithic column material, the silver nanocluster functionalized monolithic column material ( Figure 4 -c) further increased the signal of the Ag element, indicating that silver elements were attached to the surface of the aminobenzenesulfonic acid functionalized monolithic column, which means that the silver element has achieved the functionalization of the monolithic column through its strong interaction with the sulfonate group.

[0031] Figure 5 Solid-state UV-visible spectroscopy was used to characterize the p-aminobenzenesulfonic acid functionalized monolithic column material and the silver nanocluster functionalized monolithic column material. Figure 5 -a) compared to the silver nanocluster functionalized monolithic pillar material ( Figure 5 -b) A broad absorption peak appeared near 420nm, which is the embodiment of the nanoconfinement effect of silver nanoclusters, further confirming the successful preparation of silver nanocluster functionalized monolithic columns.

[0032] The silver nanocluster functionalized p-aminobenzenesulfonic acid urea-formaldehyde resin monolith prepared according to the above specific implementation method was used as the solid phase microextraction stationary phase, combined with the in-tube solid phase microextraction-silver ion liquid chromatography coupling system (the construction of the coupling system refers to the utility model patent 2018207623454), to achieve the selective enrichment and efficient analysis of trace trans fatty acid methyl esters (9t-C18:1, 9t,12t-C18:2) in complex actual samples, and compared with conventional Ag + -HPLC method direct detection (Microchemical Journal, 2022, 179, 107540) analysis results were compared. The optimal detection conditions of this combined method are as follows: sample loading liquid composition: n-hexane; injection flow rate: 0.06mL / min; eluent composition: n-hexane / acetonitrile / dichloromethane = (95.8% / 2.2% / 2%); elution flow rate: 0.06mL / min; injection volume: 500μL; elution volume: 250μL; column oven temperature: 40℃; detection wavelength: 203nm.

[0033] Figure 6Chromatograms of spiked and blank bread samples analyzed using different detection methods are shown. + -HPLC detection ( Figure 6 -d), the chromatographic peaks of the two trans fatty acid methyl esters in the spiked bread sample were not obvious, and there were many unknown signal peaks nearby, which made it impossible to accurately analyze the target qualitatively and quantitatively. When the method proposed in this embodiment was used to detect the spiked sample ( Figure 6 -b), the signal of the analysis object is significantly enhanced, the peak shape is clearly visible, and there is no obvious impurity peak interference near the peak position of the target object, which shows that the silver nanocluster functionalized monolithic column prepared in this example has excellent selective enrichment and extraction ability for trace trans fatty acids in complex samples. When the blank bread sample is detected by the method of the present invention ( Figure 6 -c), the interference of the sample matrix was greatly reduced, indicating that the silver nanocluster functionalized monolithic column has good matrix purification ability. The above results show that the silver nanocluster functionalized monolithic column prepared in this work has excellent selective enrichment and extraction performance for the target trans fatty acid methyl ester, and can realize the efficient selective enrichment analysis and accurate identification of trace trans fatty acid methyl ester in complex samples.

[0034] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Silver nanocluster functionalized monolithic column, characterized in that: The monolithic column uses urea and formaldehyde as matrix monomers and glutathione or p-aminobenzenesulfonic acid as a functionalization-reaction catalytic bifunctional agent to prepare a glutathione-functionalized monolithic column or a p-aminobenzenesulfonic acid-functionalized monolithic column through a one-step polycondensation method; and then utilizes the multiple interactions between glutathione or p-aminobenzenesulfonic acid and silver ions to in-situ reduce and generate and fix silver nanoclusters on the surface of the monolithic column to prepare a silver nanocluster-functionalized monolithic column.

2. The silver nanocluster functionalized monolithic column according to claim 1, characterized in that: The dosage of the glutathione is 110-220 mg / g urea, and the dosage of the p-aminobenzenesulfonic acid is 27-55 mg / g urea.

3. The silver nanocluster functionalized monolithic column according to claim 1, characterized in that: The silver ions come from a silver nitrate solution; the concentration of the silver nitrate solution is 4-10 mmol / L.

4. The method for preparing the silver nanocluster functionalized monolithic column according to claim 1, characterized in that: The following steps are involved: 1) Preparation of glutathione functionalized monolithic column: 550 μL 1 g / mL urea solution, 450 μL 33wt%~37wt% formaldehyde solution, 100 μL water and 60~120 mg glutathione were mixed and vortexed for 30 seconds to prepare the monolithic column polycondensation reaction solution; the monolithic column polycondensation reaction solution was aspirated with a disposable sterile syringe and quickly injected into a clean and dried polytetrafluoroethylene tube, and after sealing both ends, it was placed in a 65°C water bath for reaction for 2~12 hours; the obtained monolithic column was connected to a μHPLC pump and rinsed with ultrapure water for 30 minutes to remove unreacted polycondensation precursors, oligomers and other impurities in the monolithic column to obtain a glutathione functionalized monolithic column; 2) Preparation of silver nanocluster functionalized monolithic column: prepare silver nitrate solution with a concentration of 4-10 mmol / L, inject 1.0 mL of silver nitrate solution into the glutathione functionalized monolithic column using a microinjection pump, then cap it, and then place the glutathione functionalized monolithic column in a 65°C water bath to react for 30 minutes. Repeat the above steps three times; connect the obtained monolithic column to a μHPLC pump, and rinse with methanol, isopropanol, and n-hexane as the mobile phase for 30 minutes each to obtain a silver nanocluster functionalized monolithic column.

5. The method for preparing the silver nanocluster functionalized monolithic column according to claim 1, characterized in that: The following steps are involved: 1) Preparation of p-aminobenzenesulfonic acid functionalized monolithic column: 550 μL 1 g / mL urea solution, 450 μL 33wt%~37wt% formaldehyde solution, 100 μL water and 15~30 mg p-aminobenzenesulfonic acid were mixed and vortexed for 30 seconds to prepare a monolithic column polycondensation reaction solution; the monolithic column polycondensation reaction solution was aspirated with a disposable sterile syringe and quickly injected into a clean and dried polytetrafluoroethylene tube, and after sealing both ends, it was placed in a 50°C water bath for reaction for 2~12 hours; the obtained monolithic column was connected to a μHPLC pump and rinsed with ultrapure water for 30 minutes to remove unreacted polycondensation precursors, oligomers and other impurities in the monolithic column to obtain a p-aminobenzenesulfonic acid functionalized monolithic column; 2) Preparation of silver nanocluster functionalized monolithic column: prepare silver nitrate solution with a concentration of 4-10 mmol / L, inject 1.0 mL of silver nitrate solution into the p-aminobenzenesulfonic acid functionalized monolithic column using a microinjection pump, then cap it, and then place the p-aminobenzenesulfonic acid functionalized monolithic column in a 65°C water bath to react for 30 minutes. Repeat the above steps three times; connect the obtained monolithic column to a μHPLC pump, and rinse with methanol, isopropanol, and n-hexane as the mobile phase for 30 minutes each to obtain a silver nanocluster functionalized monolithic column.

6. The use of the silver nanocluster functionalized monolithic column as claimed in claim 1, characterized in that: The silver nanocluster functionalized monolithic column can be used as a selective in-tube solid phase microextraction stationary phase to achieve selective solid phase microextraction and highly sensitive analysis of trace trans fatty acids in complex samples.

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