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

Abstract

The application discloses preparation of silver nanocluster functionalized monolithic column and selective solid phase microextraction application thereof. The application takes urea and formaldehyde as matrix monomers, takes glutathione or p-aminobenzenesulfonic acid as functionalization-reaction catalysis bifunctional agent, and prepares glutathione functionalized monolithic column or p-aminobenzenesulfonic acid functionalized monolithic column through one-step polycondensation. Then, the multiple interactions between glutathione or p-aminobenzenesulfonic acid and silver ions are utilized to reduce and fix silver nanoclusters in situ on the surface of the monolithic column, so that the silver nanocluster functionalized monolithic column is prepared. The application provides a new way of combining cluster materials with monolithic columns, and the prepared monolithic column can be used as an in-tube solid phase microextraction stationary phase to realize online selective enrichment and high-sensitivity analysis of trace trans fatty acids in complex samples. The application has the advantages of ingenious process, simple operation, no need of expensive instruments, easiness in popularization and strong specificity of analysis objects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of monolithic column preparation, and particularly relates to preparation of silver nanocluster functionalized monolithic column and selective solid phase microextraction application thereof. BACKGROUND

[0002] Monolithic column, also known as continuous bed, is a kind of continuous column bed with porous structure prepared by in-situ polymerization in a tube. Due to the unique network space structure, monolithic column has good permeability, fast mass transfer speed, strong mechanical stability, easy chemical modification and good biocompatibility, and is widely used in the fields of chromatographic separation and sample pretreatment.

[0003] Silver nanocluster is a super small size cluster body composed of several to hundreds of silver atoms, and the size is generally less than 5 nm. Due to the quantum size effect, silver nanocluster exhibits non-metallic characteristics and similar molecular optical physical properties, and still retains part of the affinity activity similar to silver ions. It has broad application prospects in the fields of biology, optoelectronics and the like, but at present its research is still in the initial stage, and each performance is still in exploration. However, silver nanocluster will cause agglomeration due to its small size effect, so that its stability and surface activity are reduced, and how to improve its stability is concerned.

[0004] Small size silver nanocluster usually needs to rely on a certain carrier to exist stably. Therefore, the present application takes monolithic column as the carrier, takes silver nanocluster as the functionalized substance, prepares silver nanocluster functionalized monolithic column, and proposes a new way of combination of silver nanocluster and monolithic column, so as to solve the problem of stability of silver nanocluster, and realize selective enrichment and extraction of trans fatty acids with unsaturated carbon-carbon double bond by using the silver ion type affinity between silver nanocluster and unsaturated carbon-carbon double bond. The monolithic column prepared in the present application can be used as a selective in-tube solid phase microextraction stationary phase, and then combined with related analysis means to realize selective and efficient enrichment and on-line high-sensitivity analysis of trace trans fatty acids in complex actual samples. SUMMARY

[0005] The present application aims to provide silver nanocluster functionalized monolithic column, and use it as a selective in-tube solid phase microextraction stationary phase to realize selective solid phase microextraction and high-sensitivity analysis of trace trans fatty acids in complex samples.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] A silver nanocluster functionalized monolithic column is prepared by a one-step condensation method using urea and formaldehyde as matrix monomers, glutathione or p-aminobenzenesulfonic acid as functionalized-reaction catalysis bifunctional agent to prepare glutathione functionalized monolithic column or p-aminobenzenesulfonic acid functionalized monolithic column; and then using the multiple interactions between glutathione or p-aminobenzenesulfonic acid and silver ions, silver nanoclusters are generated and fixed in situ on the surface of the monolithic column to obtain the silver nanocluster functionalized monolithic column; wherein the amount of glutathione is 110-220 mg / g of urea, and the amount of p-aminobenzenesulfonic acid is 27-55 mg / g of urea.

[0008] The silver ions are derived from a silver nitrate solution; and the concentration of the silver nitrate solution is 4-10 mmol / L.

[0009] The preparation method of the above-mentioned silver nanocluster functionalized monolithic column comprises the following steps:

[0010] 1) Preparation of glutathione functionalized monolithic column: 550 μL of 1 g / mL urea solution, 450 μL of 33wt%-37wt% formaldehyde solution, 100 μL of water and 60-120 mg of glutathione are mixed and vortexed for 30 seconds to prepare a monolithic column condensation reaction solution; the monolithic column condensation reaction solution is taken with a disposable sterile syringe, quickly injected into a washed and dried polytetrafluoroethylene tube, both ends are sealed, and then placed in a 65°C water bath for 2-12 hours; the obtained monolithic column is connected to a μHPLC pump and washed with ultrapure water for 30 minutes to remove unreacted condensation precursors, oligomers and other impurities in the monolithic column, thereby obtaining a glutathione functionalized monolithic column.

[0011] 2) Preparation of silver nanocluster functionalized monolithic column: a silver nitrate solution with a concentration of 4-10 mmol / L is prepared, 1.0 mL of the silver nitrate solution is injected into the glutathione functionalized monolithic column with a microsyringe, then the column is sealed, and the glutathione functionalized monolithic column is placed in a 65°C water bath for 30 minutes, and the above steps are repeated three times; the obtained monolithic column is connected to a μHPLC pump, and then washed with methanol, isopropanol and n-hexane as mobile phases for 30 minutes, respectively, to obtain a silver nanocluster functionalized monolithic column.

[0012] The preparation method of the above-mentioned silver nanocluster functionalized monolithic column comprises the following steps:

[0013] 1) Preparation of p-aminobenzenesulfonic acid functionalized monolithic column: 550 μL of 1 g / mL urea solution, 450 μL of 33wt%-37wt% formaldehyde solution, 100 μL of water and 15-30 mg of 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 taken up with a sterile disposable syringe and quickly injected into a washed and dried polytetrafluoroethylene tube, after the two ends were sealed, it was placed in a 50°C water bath for 2-12 hours; the obtained monolithic column was connected to a μHPLC pump and washed with ultrapure water for 30 minutes to remove unreacted polycondensation precursors, oligomers and other impurities in the monolithic column, thereby obtaining a p-aminobenzenesulfonic acid functionalized monolithic column;

[0014] 2) Preparation of silver nanocluster functionalized monolithic column: a silver nitrate solution with a concentration of 4-10 mmol / L was prepared, 1.0 mL of the silver nitrate solution was injected into the p-aminobenzenesulfonic acid functionalized monolithic column with a microsyringe, then the column was sealed, and the p-aminobenzenesulfonic acid functionalized monolithic column was placed in a 65°C water bath for 30 minutes, the above steps were repeated three times; the obtained monolithic column was connected to a μHPLC pump and washed with methanol, isopropanol and n-hexane as mobile phases for 30 minutes, respectively, thereby obtaining a silver nanocluster functionalized monolithic column.

[0015] In the first step of the preparation of the silver nanocluster functionalized monolithic column, glutathione (or p-aminobenzenesulfonic acid) simultaneously functions as a reaction catalyst and a functionalization reagent; 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 the substitution reaction between the amino ortho ring hydrogen of p-aminobenzenesulfonic acid and formaldehyde can be used to connect p-aminobenzenesulfonic acid to the polymer backbone of urea-formaldehyde resin, thereby realizing the p-aminobenzenesulfonic acid functionalization of urea-formaldehyde resin.

[0016] In the second step of the preparation of the silver nanocluster functionalized monolithic column, an in-situ reduction immobilization strategy is adopted to prepare the silver nanocluster functionalized monolithic column; specifically, for the glutathione functionalized monolithic column, silver ions are reduced in-situ to silver nanoclusters under the reduction of glutathione, and the metal-sulfhydryl interaction between the thiol groups of the glutathione residues on the surface of the monolithic column and the silver nanoclusters is used to realize the immobilization of the silver nanoclusters on the surface of the monolithic column; for the p-aminobenzenesulfonic acid functionalized monolithic column, the reduction of the amino group conjugated with the benzene ring and the strengthening effect of the para-sulfonic acid group on the reduction are used to reduce silver ions in-situ to silver nanoclusters, and the electrostatic interaction between the sulfonic acid groups of p-aminobenzenesulfonic acid and the silver nanoclusters is used to realize the immobilization of the silver nanoclusters on the surface of the monolithic column.

[0017] The application further provides application of the silver nanocluster functionalized monolithic column in selective solid phase microextraction and on-line analysis of trace trans fatty acids in complex samples.

[0018] The application has the following advantages:

[0019] 1) The monolithic column preparation scheme of the application is ingenious and concise. In the preparation process of the monolithic column, glutathione or p-aminobenzenesulfonic acid is used as a functionalization reagent and a reaction catalyst, and a glutathione functionalized monolithic column or a p-aminobenzenesulfonic acid functionalized monolithic column is prepared by one-step polycondensation, thereby providing a large number of active sites for subsequent in-situ reduction and surface immobilization of silver nanoclusters.

[0020] 2) The application first proposes an in-situ reduction and immobilization strategy of silver nanoclusters, and uses the multiple interactions between glutathione or p-aminobenzenesulfonic acid and silver ions to generate and immobilize silver nanoclusters in-situ on the surface of the monolithic column, thereby preparing a silver nanocluster functionalized monolithic column. The preparation process is simple, and the preparation efficiency and success rate of the silver nanocluster functionalized monolithic column are greatly improved.

[0021] 3) The application provides a new way of combining cluster materials with monolithic columns, and has the advantages of ingenious process, simple operation, no need for expensive instruments and easy popularization.

[0022] 4) The monolithic column prepared by the application can be used as an in-tube solid phase microextraction stationary phase, has clear specificity for analysis objects, and can realize on-line selective enrichment and high-sensitivity analysis of trace trans fatty acids in complex samples. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Fig. 1 is an energy dispersive X-ray spectroscopy (EDS) diagram of three kinds of monolithic column materials. Wherein, 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.

[0024] Figure 2 Fig. 2 is a solid ultraviolet-visible spectrogram of two kinds of monolithic column materials. Wherein, a is a glutathione functionalized monolithic column material; and b is a silver nanocluster functionalized monolithic column material.

[0025] Figure 3are high performance liquid chromatograms. a is the chromatogram of the detection of 9t-C18:1 and 9t,12t-C18:2 standard solution by in-tube solid phase microextraction-silver ion liquid chromatography using silver nanocluster functionalized monolithic column as solid phase microextraction stationary phase; b is the chromatogram of the detection of spiked bread sample by the same method; c is the chromatogram of the detection of blank bread sample by the same method; d is the chromatogram of the detection of spiked bread sample by silver ion liquid chromatography directly. The spiked concentration of analyte is 10 μg / g; peak assignment: (1) trans-oleic acid methyl ester (9t-C18:1); (2) trans-linoleic acid methyl ester (9t,12t-C18:2).

[0026] Figure 4 are EDS spectra of three kinds of monolithic column materials. a is urea-formaldehyde resin matrix monolithic column material; b is p-aminobenzenesulfonic acid functionalized monolithic column material; c is silver nanocluster functionalized monolithic column material.

[0027] Figure 5 are solid UV-Vis spectra of two kinds of monolithic column materials. a is p-aminobenzenesulfonic acid functionalized monolithic column material; b is silver nanocluster functionalized monolithic column material.

[0028] Figure 6 are high performance liquid chromatograms. a is the chromatogram of the detection of 9t-C18:1 and 9t,12t-C18:2 standard solution 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 the chromatogram of the detection of spiked bread sample by the same method; c is the chromatogram of the detection of blank bread sample by the same method; d is the chromatogram of the detection of spiked bread sample by silver ion liquid chromatography directly. The spiked concentration of analyte is 10 μg / g; peak assignment: (1) trans-oleic acid methyl ester (9t-C18:1); (2) trans-linoleic acid methyl ester (9t,12t-C18:2). DETAILED DESCRIPTION

[0029] In order to make the content of the present application more convenient to understand, the technical solutions of the present application will be further described below in combination with specific embodiments, but the present application is not limited to this.

[0030] The preparation method of the urea-formaldehyde resin matrix monolithic column involved in the following examples is as follows: 550 μL of urea solution (1 g / mL), 450 μL of commercially available formaldehyde solution (mass concentration of 33%-37%) (National Pharmaceutical Group Chemical Reagent Co., Ltd.), 100 μL of ultrapure water are mixed and vortexed for 30 seconds to prepare a monolithic column polycondensation reaction solution; the monolithic column polycondensation reaction solution is taken with a disposable sterile syringe and quickly injected into a polytetrafluoroethylene (PTFE) tube (inner diameter of 750 μm, column length of 25 cm) which has been washed and dried, both ends are sealed, and then the PTFE tube containing the monolithic column polycondensation reaction solution is placed in a 65°C water bath for reaction for 6 hours. After the reaction is completed, the prepared monolithic column is connected to a μHPLC pump, and ultrapure water is used to flush 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, thereby obtaining a urea-formaldehyde resin matrix monolithic column.

[0031] Example 1

[0032] The present example provides the preparation of a silver nanocluster functionalized monolithic column and its selective solid phase microextraction application, which specifically comprises the following steps:

[0033] 1) Preparation of glutathione functionalized monolithic column: 550 μL of urea solution (1 g / mL), 450 μL of commercially available formaldehyde solution (mass concentration of 33%-37%) (National Pharmaceutical Group Chemical Reagent Co., Ltd.), 100 μL of ultrapure water and 100 mg of glutathione are mixed and vortexed for 30 seconds to prepare a monolithic column polycondensation reaction solution; the monolithic column polycondensation reaction solution is taken with a disposable sterile syringe and quickly injected into a polytetrafluoroethylene (PTFE) tube (inner diameter of 750 μm, column length of 25 cm) which has been washed and dried, both ends are sealed, and then the PTFE tube containing the monolithic column polycondensation reaction solution is placed in a 65°C water bath for reaction for 6 hours. After the reaction is completed, the prepared monolithic column is connected to a μHPLC pump, and ultrapure water is used to flush 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, thereby obtaining a glutathione functionalized monolithic column;

[0034] 2) Preparation of silver nanocluster functionalized monolithic column: a silver nitrate solution with a concentration of 10 mmol / L is prepared, 1.0 mL of the silver nitrate solution is injected into the glutathione functionalized monolithic column at a flow rate of 0.05 mL / min with a microsyringe pump, and then the column is capped; the glutathione functionalized monolithic column is then placed in a 65°C water bath for reaction for 30 minutes, and the above steps are repeated three times; the obtained monolithic column is connected to a μHPLC pump, and is sequentially flushed with methanol, isopropanol and n-hexane as mobile phases at a flow rate of 0.10 mL / min for 30 minutes, thereby obtaining a silver nanocluster functionalized monolithic column.

[0035] Figure 1The element composition of the urea-formaldehyde resin matrix monolithic column material, the glutathione functionalized monolithic column material and the silver nanocluster functionalized monolithic column material was analyzed by an energy dispersive X-ray spectrometer (EDS). As can be seen from the figure, the urea-formaldehyde resin matrix monolithic column material ( Figure 1 -a) only signals of C, N and O (the appearance of Au element is caused by gold spraying) three elements appeared; while the glutathione functionalized monolithic column material ( Figure 1 -b) also added a signal of S element, which proved that glutathione was successfully combined into the urea-formaldehyde resin material; and in the silver nanocluster functionalized monolithic column material ( Figure 1 -c) silver element signal further appeared, which indicated that silver element had realized the functionalization of the monolithic column through its strong interaction with the mercapto group.

[0036] Figure 2 The glutathione functionalized monolithic column material and the silver nanocluster functionalized monolithic column material were characterized by solid UV-Vis spectroscopy. As can be seen from the figure, compared with the glutathione functionalized monolithic column material ( Figure 2 -a), the silver nanocluster functionalized monolithic column material ( Figure 2 -b) appeared a clear absorption peak at 355 nm, which was caused by the nanometer confinement effect of silver nanoclusters, further proving the successful preparation of the silver nanocluster functionalized monolithic column.

[0037] The silver nanocluster functionalized monolithic column prepared according to the above specific embodiment was used as a solid phase microextraction stationary phase, combined with an in-tube solid phase microextraction-silver ion liquid chromatography coupled system (the coupled system was constructed according to the utility model patent 2018207623454), to realize the selective enrichment and high-efficiency analysis of trace amounts of trans fatty acid methyl esters (9t-C18:1, 9t,12t-C18:2) in complex actual samples, and compared with the analysis results of Ag + -HPLC direct detection (Microchemical Journal, 2022, 179, 107540). The optimal detection conditions of the coupled method are as follows: composition of sample loading liquid: n-hexane; sample injection flow rate: 0.06 mL / min; composition of eluent: n-hexane / acetonitrile / dichloromethane = 99.5% / 0.4% / 0.1%; elution flow rate: 0.06 mL / min; sample injection volume: 500 μL; elution volume: 250 μL; column oven temperature: 40℃; detection wavelength: 203 nm.

[0038] Figure 3 The chromatograms of the spiked and blank bread samples analyzed by different detection methods are shown. When the Ag + -HPLC detection spiked bread sample ( 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 peak of 9t,12t-C18:2 could not be detected, so that the target could not be accurately identified and quantitatively analyzed. When the blank sample was detected by the method provided in the embodiment, Figure 3 -c), the matrix interference of the sample was significantly reduced, and the interference signal peak of the unknown was greatly weakened, and no other interference signal appeared near the peak position of the analysis object, which indicated that the prepared silver nanocluster functionalized monolithic column could significantly reduce the matrix interference in the complex sample and had excellent matrix purification capacity; when the spiked bread sample was analyzed by the method of the application, Figure 3 -b), the trace amount of two trans fatty acid methyl esters added could be efficiently enriched and accurately identified, which indicated that the silver nanocluster functionalized monolithic column prepared in the work had excellent selective enrichment and extraction performance for the target trans fatty acid methyl ester.

[0039] Example 2

[0040] The embodiment provides preparation of a silver nanocluster functionalized monolithic column and selective solid phase microextraction application thereof, and specifically comprises the following steps:

[0041] 1) Preparation of p-aminobenzenesulfonic acid functionalized monolithic column: 550 μL of urea solution (1 g / mL), 450 μL of commercially available formaldehyde solution (mass concentration of 33%-37%) (National Pharmaceutical Group Chemical Reagent Co., Ltd.), 100 μL of ultrapure water and 25 mg of 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 taken by a disposable sterile syringe and quickly injected into a washed and dried PTFE tube, both ends were sealed, and then placed in a 50°C water bath for reaction for 10 hours. After the reaction was completed, the prepared monolithic column was connected to a μHPLC pump, and ultrapure water was used at a flow rate of 0.10 mL / min for flushing for 30 minutes to remove unreacted polycondensation precursors, oligomers and other impurities in the monolithic column, thereby obtaining a p-aminobenzenesulfonic acid functionalized monolithic column;

[0042] 2) Preparation of silver nanocluster functionalized monolithic column: a silver nitrate solution with a concentration of 5 mmol / L was prepared, 1.0 mL of the silver nitrate solution was injected into the p-aminobenzenesulfonic acid functionalized monolithic column by using a micro-injection pump at a flow rate of 0.05 mL / min, then the column was sealed, and the p-aminobenzenesulfonic acid functionalized monolithic column was placed in a 60°C water bath for reaction for 30 minutes, and the above steps were repeated three times; the obtained monolithic column was connected to a μHPLC pump, and methanol, isopropanol and n-hexane were used as mobile phases at a flow rate of 0.10 mL / min for flushing for 30 minutes, respectively, thereby obtaining a silver nanocluster functionalized monolithic column.

[0043] Figure 4The element composition of the urea-formaldehyde resin matrix monolithic column material, the p-aminobenzenesulfonic acid functionalized monolithic column material and the silver nanocluster functionalized monolithic column material was analyzed by an energy dispersive X-ray spectrometer (EDS), respectively. Figure 4 It can be seen that the urea-formaldehyde resin matrix monolithic column material ( Figure 4 -a) mainly contains C, N and O elements; the p-aminobenzenesulfonic acid functionalized monolithic column material ( Figure 4 -b) contains not only C, N and O elements, but also S element, which is mainly derived from the functionalization reagent p-aminobenzenesulfonic acid, indicating that the p-aminobenzenesulfonic acid realizes the functionalization of the urea-formaldehyde resin by participating in the polycondensation reaction of the urea-formaldehyde resin; in addition, it can be found that the N element signal peak of the p-aminobenzenesulfonic acid functionalized monolithic column material increases significantly, indicating that the p-aminobenzenesulfonic acid also participates in the polymerization reaction of the monolithic column and provides 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 increases the signal of Ag element, indicating that the silver element is attached to the surface of the p-aminobenzenesulfonic acid functionalized monolithic column, which indicates that the silver element realizes the functionalization of the monolithic column through the strong interaction between the silver element and the sulfonate group.

[0044] Figure 5 The p-aminobenzenesulfonic acid functionalized monolithic column material and the silver nanocluster functionalized monolithic column material were characterized by solid UV-Vis spectroscopy. Compared with the p-aminobenzenesulfonic acid functionalized monolithic column material ( Figure 5 -a), the silver nanocluster functionalized monolithic column material ( Figure 5 -b) has an absorption wide peak near 420 nm, which is the embodiment of the nanometer confinement effect of the silver nanocluster, further confirming the successful preparation of the silver nanocluster functionalized monolithic column.

[0045] The silver nanocluster functionalized p-aminobenzenesulfonic acid urea-formaldehyde resin monolithic column prepared according to the above specific embodiment was used as a solid phase microextraction stationary phase, and combined with a tube-in-syringe solid phase microextraction-silver ion liquid chromatography coupled system (the coupled system was constructed according to the utility model patent 2018207623454) to realize the selective enrichment and high-efficiency analysis of trace trans fatty acid methyl esters (9t-C18:1, 9t, 12t-C18:2) in complex actual samples, and compared with the conventional Ag +The analysis results of the HPLC method direct detection (Microchemical Journal, 2022, 179, 107540) were compared. The optimal detection conditions of the combined method are as follows: composition of sample loading liquid: n-hexane; sample flow rate: 0.06 mL / min; composition of eluent: n-hexane / acetonitrile / dichloromethane = (95.8% / 2.2% / 2%); elution flow rate: 0.06 mL / min; sample injection volume: 500 μL; elution volume: 250 μL; column oven temperature: 40 °C; detection wavelength: 203 nm.

[0046] Figure 6 The chromatograms of the spiked and blank bread samples analyzed by different detection methods are shown. The Ag + In the case of HPLC detection, Figure 6 In the case of HPLC detection, Figure 6 In the case of HPLC detection, Figure 6 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 efficient selective enrichment analysis and accurate identification of trace trans fatty acid methyl ester in complex samples.

[0047] The above only describes the preferred embodiments of the present application, and any equivalent changes and modifications made within the scope of the patent application of the present application shall be included in the scope of the present application.

Claims

1. A silver nanocluster functionalized monolith, characterized in that: The monolithic column is prepared by one-step condensation polymerization method using urea and formaldehyde as matrix monomers, glutathione or p-aminobenzenesulfonic acid as functionalized-reaction catalysis bifunctional agent to prepare glutathione functionalized monolithic column or p-aminobenzenesulfonic acid functionalized monolithic column; and then using the multiple interactions between glutathione or p-aminobenzenesulfonic acid and silver ions, silver nanocluster functionalized monolithic column is prepared by in-situ reduction and fixation of silver nanoclusters on the surface of the monolithic column.

2. The silver nanocluster functionalized monolithic column of claim 1, wherein: The amount of glutathione is 110-220 mg / g urea, and the amount of p-aminobenzenesulfonic acid is 27-55 mg / g urea.

3. The silver nanocluster functionalized monolithic column of claim 1, wherein: The silver ions are derived from silver nitrate solution, and the concentration of the silver nitrate solution is 4-10 mmol / L.

4. The method for preparing the silver nanocluster functionalized monolithic column as described in claim 1, characterized in that: The method comprises the following steps: 1) Preparation of glutathione functionalized monolithic column: 550 μL of 1 g / mL urea solution, 450 μL of 33wt%-37wt% formaldehyde solution, 100 μL of water and 60-120 mg of glutathione are mixed and vortexed for 30 seconds to prepare a monolithic column condensation polymerization reaction solution; the monolithic column condensation polymerization reaction solution is taken with a disposable sterile syringe and quickly injected into a washed and dried polytetrafluoroethylene tube, the two ends are sealed, and then placed in a 65°C water bath for 2-12 hours; the obtained monolithic column is connected to a μHPLC pump and washed with ultrapure water for 30 minutes to remove unreacted condensation polymerization precursors, oligomers and other impurities in the monolithic column, thereby obtaining a glutathione functionalized monolithic column; 2) Preparation of silver nanocluster functionalized monolithic column: a silver nitrate solution with a concentration of 4-10 mmol / L is prepared, 1.0 mL of the silver nitrate solution is injected into the glutathione functionalized monolithic column with a micro-injection pump, then the column is sealed, and the glutathione functionalized monolithic column is placed in a 65°C water bath for 30 minutes, and the above steps are repeated three times; the obtained monolithic column is connected to a μHPLC pump, and then washed with methanol, isopropanol and n-hexane as mobile phases for 30 minutes, respectively, to obtain a silver nanocluster functionalized monolithic column.

5. The method for preparing the functionalized monolithic column of silver nanoclusters as described in claim 1, characterized in that: 1) Preparation of p-aminobenzenesulfonic acid functionalized monolithic column: 550 μL of 1 g / mL urea solution, 450 μL of 33wt%-37wt% formaldehyde solution, 100 μL of water and 15-30 mg of p-aminobenzenesulfonic acid are mixed and vortexed for 30 seconds to prepare a monolithic column condensation polymerization reaction solution; the monolithic column condensation polymerization reaction solution is taken with a disposable sterile syringe and quickly injected into a washed and dried polytetrafluoroethylene tube, the two ends are sealed, and then placed in a 50°C water bath for 2-12 hours; the obtained monolithic column is connected to a μHPLC pump and washed with ultrapure water for 30 minutes to remove unreacted condensation polymerization precursors, oligomers and other impurities in the monolithic column, thereby obtaining a p-aminobenzenesulfonic acid functionalized monolithic column; ​ 2) Preparation of silver nanoclusters functionalized monolithic column: prepare silver nitrate solution with concentration of 4-10 mmol / L, inject 1.0 mL silver nitrate solution into p-aminobenzenesulfonic acid functionalized monolithic column by micro-injection pump, then seal the end, and then place the p-aminobenzenesulfonic acid functionalized monolithic column in 65℃ water bath for reaction for 30 minutes, repeat the above steps for three times; connect the obtained monolithic column to μHPLC pump, flush with methanol, isopropanol and n-hexane as mobile phase respectively for 30 minutes, and obtain silver nanoclusters functionalized monolithic column.

6. The use of silver nanocluster functionalized monolithic column according to claim 1, characterized in that: The silver nanoclusters functionalized monolithic column can be used as selective in-tube solid phase microextraction stationary phase, so as to realize selective solid phase microextraction of trace trans-fatty acids in complex sample and high-sensitivity analysis.

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