Application of three-dimensional composite nanofiber material in preparation of chromatographic monolithic column

Abstract

The invention discloses application of a three-dimensional composite nanofiber material in preparation of a chromatographic monolithic column. The three-dimensional composite nanofiber is prepared by the following steps: homogenizing high-molecular polymer nanofiber prepared by electrostatic spinning into dispersed short fiber in a solution, adding a certain amount of graphene oxide solution and a certain amount of reducing agent into the solution, adding the mixed solution into a columnar glass container, and carrying out hydration reaction, so as to obtain the three-dimensional composite nanofiber. After hydrogel is formed, freeze drying is performed to finally obtain a composite nanofiber block material with a three-dimensional structure, and the composite nanofiber block material is filled into a column with a proper volume and can be used for separating compounds after being combined with a liquid chromatograph.

Description

Technical Field

[0001] The present invention relates to the application of a three-dimensional composite nanofiber material in the preparation of a chromatographic monolithic column, and belongs to the field of chromatographic separation. Background Art

[0002] Chromatographic separation has always occupied an important position in the field of analytical chemistry. As a core component of instrumental analysis, the chromatographic column should have characteristics such as high column efficiency and low column pressure, and its performance plays an important role in the separation effect of compounds. Traditional chromatographic columns generally use particulate fillers as the stationary phase and are prepared by an external column packing technique of filling the fillers into the column tube. Specifically, for fillers with a particle size greater than 20 microns, a dry packing method is often used; for fillers with a particle size less than 20 microns, due to their susceptibility to electrostatic interference, a slurry method is more preferably used for packing. In this process, organic solvents such as n-hexane and methanol are often selected as the driving solvents, while dichloromethane, acetone, carbon tetrachloride, etc. are used as the slurry solvents. With the continuous improvement of the requirements for column efficiency, people have begun to use fillers with smaller and smaller particle sizes. However, small particle fillers will cause a sharp increase in the column pressure of the chromatographic column, and this characteristic limits the further improvement of the performance of ordinary liquid chromatography. Therefore, researchers have to invest a lot of energy and funds in developing ultra-high performance liquid chromatography technology that can withstand high pressure, which undoubtedly increases the cost of sample analysis. At present, many researchers are committed to the research and development of ideal chromatographic fillers. Among them, monolithic columns have been used in various fields such as pharmaceutical analysis and environmental science due to their advantages of good stability, low column pressure, and reusability. Monolithic columns mainly include silica monolithic columns and polymer monolithic columns, and are mainly prepared by mixing organic monomers or alkoxysilanes with cross-linking agents, porogens, initiators and other materials and injecting them into an empty column tube, and then initiating hydrolysis and polycondensation of alkoxysilanes and polymerization of organic monomers in the tube through methods such as thermal polymerization, photoinitiated polymerization, and microwave radiation, omitting the processes of preparing fillers and packing columns. However, although monolithic columns have characteristics such as high mechanical strength, high porosity, low column pressure, and short separation time, the preparation process of monolithic columns is very complex and the processes of hydrolysis, polycondensation, polymerization, catalysis, derivatization, and aging of raw materials are difficult to control. All of the above defects limit the application of monolithic columns. In recent years, electrospun nanofiber materials have been widely used for the enrichment and concentration of trace and ultratrace compounds in various complex samples due to their advantages of high specific surface area, simple preparation, and low preparation cost, and their excellent performance in the field of separation and purification of compounds. However, due to the lack of a support structure in existing two-dimensional nanofibers, the accumulation between nanofibers is serious after the liquid passes through, resulting in an increase in the column pressure. Summary of the Invention

[0003] Object of the Invention: The technical problem to be solved by the present invention is to provide the application of a three-dimensional composite nanofiber material in the preparation of a chromatographic monolithic column, so that the preparation of the monolithic column is simple and easy to control.

[0004] Technical solution: To solve the above technical problems, the present invention provides the application of a three-dimensional composite nanofiber material in the preparation of a chromatographic monolithic column.

[0005] Among them, the three-dimensional composite nanofibers are doped with short nanofibers and graphene oxide, and can be filled into a column with an appropriate shape to make a chromatographic column.

[0006] Among them, the polymer used to form the above short nanofibers is a nanofiber material containing an adsorption functional group.

[0007] Among them, the polymer that composes the short nanofibers is one or a mixture of several of polyethylene oxide, polypyrrole, polyvinyl alcohol, polyethylene naphthalate, polyaniline, nylon, polyphenylene sulfide, cellulose acetate, polystyrene, polyvinylpyrrolidone, polycaprolactam, acrylic resin, and polyacrylonitrile, etc.

[0008] Among them, the short nanofibers are prepared by homogenizing electrospun nanofibers.

[0009] Among them, the electrospun nanofibers are homogenized in a solvent, and the solvent is a polymer that can completely dissolve the electrospun nanofibers.

[0010] Among them, the solvent is one or a mixture of several of ethyl acetate, N, N-dimethylformamide, tetrahydrofuran, formic acid, acetic acid, ethanol, acetone, water, halogenated hydrocarbons or aromatic hydrocarbons.

[0011] Among them, the length of the short fibers is 25 - 200 μm.

[0012] Among them, the mass ratio of the short fibers to graphene oxide is 1:1.

[0013] Among them, the method for preparing the three-dimensional composite nanofiber material includes the following steps:

[0014] Step 1, weigh a certain amount of the above nanofibers, cut them into small pieces, disperse them in an aqueous solution or other solutions, and use a homogenizer to break them into short fibers;

[0015] Step 2, add a certain amount of commercially available graphene oxide dispersion to the above nanofiber dispersion and mix well, then add a certain amount of ascorbic acid and continue to mix well;

[0016] Step 3, transfer the above mixed solution to a glass container with a suitable size, and obtain a three-dimensional composite nanofiber material after a hydration reaction.

[0017] The present invention also provides a chromatographic monolithic column, which contains the three-dimensional composite nanofiber material. The high molecular polymer nanofibers made by electrospinning are homogenized in a solution to be dispersed short fibers, a certain amount of graphene oxide solution and a certain amount of reducing agent are added to the solution, and the mixed solution is then added to containers of different shapes for hydration reaction, and after forming a hydrogel, it is freeze-dried to finally obtain a composite nanofiber block material with a three-dimensional structure. This material is cut into a suitable shape and filled into a column of suitable volume to make a chromatographic separation column, which can be used for compound separation after being used in conjunction with a liquid chromatograph.

[0018] The present invention also provides application of the three-dimensional composite nanofiber material or chromatographic monolithic column in liquid chromatography separation.

[0019] The application includes preparing fillers outside the column, and no organic solvent or vacuum pump is required to apply pressure during the material filling process. The filling can be completed by gently inserting the filler with a size suitable for the column shell into the column. The three-dimensional composite nanofiber material is doped with graphene oxide, which significantly improves the mechanical strength of the nanofiber material. The three-dimensional composite nanofiber material not only has the high specific surface area of ​​the two-dimensional nanofiber, but also because of its internal interconnected and more fluffy structure, the three-dimensional composite nanofiber only needs a small pressure to push the sample solution through the sample solution to penetrate into its structure, so that the target to be tested in the sample is fully contacted with the adsorption sites on the three-dimensional composite nanofiber, and the target compound is separated by the different interaction abilities with the surface of the nanofiber material. The target is eluted isocratically or gradiently with the mobile phase and then enters the detector to perform qualitative and quantitative analysis on the target.

[0020] Among them, it can be used for the separation and detection of two pigments, brilliant blue and rhodamine B.

[0021] Among them, it can be used for the separation and detection of two compounds: cortisol and 3-hydroxyphenanthrene.

[0022] The method for using the nanofiber separation monolithic column of the present invention is as follows: the monolithic column is connected to the liquid chromatography flow path, a certain flow rate is set to clean the separation monolithic column with pure methanol and distilled water respectively, and then the separation monolithic column is balanced with the starting mobile phase. After the injection, an isocratic or gradient mobile phase can be set to elute the target adsorbed on the monolithic column, and the eluted target is sent to the detector for analysis.

[0023] In the patent with the application number 202311373331.5, three-dimensional composite nanofibers are applied to a solid-phase extractor. The three-dimensional composite nanofiber separation monolithic column involved in the present invention is mainly used for chromatographic separation. The main difference between the two is that the three-dimensional composite nanofiber solid-phase extractor in the patent (application number: 202311373331.5) is suitable for selectively enriching target compounds or removing impurities from complex samples. The sample treatment process includes step-by-step operations such as activation, sample loading, washing, and elution to ensure the improvement of the sensitivity and accuracy of subsequent instrumental analysis. In contrast, the three-dimensional composite nanofiber separation monolithic column in the present invention is directly connected to the mobile phase system of a liquid chromatography instrument, and its main function is to separate different components in a mixture for qualitative and quantitative analysis. This column focuses on resolving each component in the mixture, and its separation principle is based on the difference in the distribution coefficients of different compounds in the three-dimensional composite nanofiber separation monolithic column and the mobile phase, which is achieved through a continuous separation process, and the finally separated substances directly enter the detector for qualitative and quantitative analysis.

[0024] Principle of the present invention: The three-dimensional composite nanofiber separation monolithic column can be prepared by selecting materials with specific adsorption functional groups and can effectively separate different types of compounds through various mechanisms such as electrostatic interaction, hydrogen bond, hydrophobic interaction, and π-π interaction. In addition, the porous structure inside the monolithic column can also utilize the size exclusion effect to achieve the separation of molecules according to their size and volume.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The present invention provides a three-dimensional composite nanofiber column that can be integrated into a liquid chromatography instrument for compound separation. This three-dimensional composite nanofiber material has a three-dimensional structure, high porosity, large specific surface area, good mechanical properties. Compared with conventional monolithic columns, the preparation method is simple and controllable, and the preparation cost is low. By connecting the three-dimensional composite nanofibers as separation materials to the liquid chromatography system, the separation of two compounds can be achieved under relatively low column pressure conditions, overcoming the defects such as high column pressure caused by the use of small particle fillers in conventional liquid chromatography columns. Description of the drawings

[0026] Figure 1 It is the preparation flow chart of the PAN / PPy / rGO three-dimensional composite nanofiber separation monolithic column;

[0027] Figure 2 It is the electron microscope image of the PAN / PPy / rGO three-dimensional composite nanofibers;

[0028] Figure 3 It is the schematic diagram of the pressure-induced deformation of the PAN / PPy / rGO three-dimensional composite nanofibers: (A) The non-pressurized morphology of the three-dimensional composite nanofiber material; (B) The morphology of the three-dimensional composite nanofiber material after being pressurized;

[0029] Figure 4 It is the chromatogram of the analysis of two pigments, brilliant blue and rhodamine B, by using the prepared PAN / PPy / rGO three-dimensional composite three-dimensional nanofiber separation monolithic column in combination with liquid chromatography-ultraviolet detector in Example 2;

[0030] Figure 5 It is the chromatogram of the analysis of two biological metabolites, cortisol and 3-hydroxyphenanthrene, by using the prepared PAN / PPy / rGO three-dimensional composite nanofiber separation monolithic column in combination with liquid chromatography-ultraviolet detector in Example 3. Detailed implementation manners

[0031] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings.

[0032] Preparation and performance test of the three-dimensional composite nanofiber separation monolithic column in Example 1

[0033] As Figure 1 shown, the specific steps are as follows:

[0034] Step 1: Weigh 20 mg of the electrospun polyacrylonitrile fiber material modified with polypyrrole (PAN / PPy) respectively. After cutting it into small pieces, disperse the fibers in 10 mL of distilled water and use a handheld homogenizer to break them into short fibers.

[0035] Step 2: Add 4 mL of the commercially available 5 mg / mL graphene oxide (rGO) dispersion to the above PAN / PPy nanofiber dispersion and mix well. After adding 20 mg of ascorbic acid (the ascorbic acid uses a commercially available reagent with a purity above the superior grade), continue to mix well.

[0036] Step 3: Transfer the above mixed solution to a glass container with a suitable size and carry out a hydration reaction at a temperature of 85 °C - 90 °C to finally form a three-dimensional structured PAN / PPy / rGO nanofiber hydrogel.

[0037] Step 4: Carry out freeze-drying on the above PAN / PPy / rGO nanofiber hydrogel.

[0038] Step 5: Take out the freeze-dried three-dimensional structured PAN / PPy / rGO composite nanofiber bulk material.

[0039] As Figure 2 shown, the flaky structure of graphene oxide is successfully doped in the microstructure of the prepared PAN / PPy / rGO three-dimensional composite nanofiber. This lamellar structure plays a supporting role between the nanofibers to prevent the stacking of the fibers.

[0040] Table 1. Comparison of specific surface areas of PAN / PPy nanofibers, rGO and PAN / PPy / rGO three-dimensional composite nanofibers

[0041]

[0042] As shown in Table 1, by using the BET method to investigate the specific surface area of ​​PAN / PPy nanofiber two-dimensional structure, rGO and PAN / PPy / rGO three-dimensional composite nanofiber, it was found that the specific surface area of ​​the three-dimensional composite nanofiber was greatly improved compared with the two-dimensional material. Graphene oxide without nanofiber doping has basically no mechanical strength, but after doping with nanofibers, the fibers are interspersed in the graphene oxide sheets, thereby increasing the mechanical strength of the three-dimensional structure. Figure 3 As shown, a vial weighing 18.4 g and containing an aqueous solution is placed on the block-shaped three-dimensional composite nanofibers, and the degree of its morphological change is small, indicating that the three-dimensional composite nanofiber material has good compression resistance, providing mechanical performance guarantee for the water pressure resistance after being loaded into the C18 pre-column tube.

[0043] The prepared PAN / PPy / rGO three-dimensional composite nanofiber material was cut and carefully loaded into the pre-prepared chromatographic pre-column empty column tube. After installing the sieve plates at both ends of the pre-column, it was connected to the SHIMADZU LC-20AD (including UV detector, automatic sampler and workstation) chromatography system. It was first rinsed with a methanol solution at a flow rate of 1.0mL / min for about 20 minutes, and then changed to an aqueous solution and continued to be rinsed at a flow rate of 1.0mL / min for about 20 minutes. During the flushing, the column pressure of the three-dimensional composite nanofiber monolithic column was observed when pure water and pure methanol were used as the mobile phase. The results are shown in Table 2. After the flushing is completed, the column pressure is balanced with the initial mobile phase, and the detection can be started after the column pressure is balanced.

[0044] Table 2. Column pressure test values ​​of 3D-PAN / PPy / rGO separation column

[0045]

[0046] Example 2 PAN / PPy / rGO three-dimensional composite nanofiber monolith coupled with liquid chromatography-ultraviolet detector for separation and detection of brilliant blue and rhodamine B

[0047] A certain amount of Brilliant Blue and Rhodamine B standard were weighed to prepare a mixed aqueous solution test sample containing Brilliant Blue at a concentration of 1.0 μg / mL and Rhodamine B at a concentration of 10.0 μg / mL. The separation monolithic column prepared according to the process of Example 1 was connected to the liquid chromatography flow path. After the separation monolithic column was cleaned and the column was balanced, the mobile phase flow rate was set to 1.0 mL / min, and the gradient elution program was shown in Table 3.

[0048] Table 3. Mobile phase gradient elution program

[0049]

[0050] The sample injection volume was 20 μL and the column temperature was 30°C. The UV detection wavelength of Brilliant Blue was set to 628 nm and that of Rhodamine B was set to 550 nm. The chromatogram is shown in Figure 4 The separation effect of the three-dimensional composite nanofiber monolithic column was evaluated by calculating the separation degree of brilliant blue and rhodamine B under the chromatographic conditions using formula 1. The results are shown in Table 4.

[0051]

[0052] Where R is the chromatographic peak separation; T RB T is the retention time of the next peak after two adjacent peaks; RA is the retention time of the previous peak between two adjacent peaks; W B W is the peak base width of the peak behind two adjacent peaks; A It is the base width of the peak before two adjacent peaks.

[0053] Table 4. Calculation results of separation of brilliant blue and rhodamine B based on three-dimensional composite nanofiber monolithic column

[0054]

[0055] Through calculation, under the set gradient elution conditions, the three-dimensional composite nanofiber monolithic column can well separate the two pigments, brilliant blue and rhodamine B, with a separation R value greater than 1.5, which meets the requirements of chromatographic separation. Through continuous injection of 10 times, the deviation values ​​of the peak time of the two pigments are less than 2.0%, and the deviation values ​​of the peak area are less than 1.0%, which meets the requirements of chromatographic stability. The above results show that the three-dimensional composite nanofiber separation monolithic column has good separation stability. The calculation results are shown in Table 5.

[0056] Table 5. Calculation of peak time stability of separation of brilliant blue and rhodamine B based on three-dimensional composite nanofiber monolithic column (n=10)

[0057]

[0058] Example 3 PAN / PPy / rGO three-dimensional composite nanofiber monolithic column coupled with liquid chromatography-ultraviolet detector for separation and detection of cortisol and 3-hydroxyphenanthrene

[0059] Weigh a certain amount of cortisol (hydrocortisone) and 3-hydroxyphenanthrene standard products, and prepare a mixed aqueous solution containing 2.0 μg / mL cortisol and 2.0 μg / mL 3-hydroxyphenanthrene as the test sample. Connect the separation monolithic column prepared according to the process of Example 1 to the liquid chromatography flow path. After cleaning and column equilibration of the separation monolithic column, set the mobile phase flow rate to 1.0 mL / min, and the gradient elution program is shown in Table 6.

[0060] Table 6. Mobile phase gradient elution program for two metabolites

[0061]

[0062] The sample injection volume is 20 μL, and the column temperature is 30 °C. Set the ultraviolet detection wavelength of cortisol to 247 nm and that of 3-hydroxyphenanthrene to 254 nm. The chromatogram is shown in Figure 5 . Evaluate the separation effect of the three-dimensional composite nanofiber monolithic column by calculating the resolution of cortisol and 3-hydroxyphenanthrene under this chromatographic condition using Equation 1. The results are shown in Table 7.

[0063] Table 7. Calculation results of the resolution for separating cortisol and 3-hydroxyphenanthrene based on the three-dimensional composite nanofiber monolithic column

[0064]

[0065] By calculation, under the set gradient elution conditions, the three-dimensional composite nanofiber monolithic column can well separate the two metabolites of cortisol and 3-hydroxyphenanthrene, and the resolution R value is greater than 1.5, meeting the requirements of chromatographic separation.

[0066] Table 8. Calculation of the peak elution time stability for separating cortisol and 3-hydroxyphenanthrene based on the three-dimensional composite nanofiber monolithic column (n = 10)

[0067]

[0068] As shown in Table 8, by injecting samples continuously 10 times, the deviation values of the peak elution times of the two metabolites are both less than 2.0%, and the deviation values of the peak areas are both less than 1.0%, meeting the requirements of chromatographic stability. The above shows that the three-dimensional composite nanofiber separation monolithic column has good separation stability.

Claims

1. Application of a three-dimensional composite nanofiber material in the preparation of a monolithic column for chromatography, characterized in that, The three-dimensional composite nanofiber material is doped with short nanofibers and graphene oxide.

2. The application according to claim 1, wherein The polymer that makes up the short nanofibers is a nanofiber material containing adsorption functional groups.

3. The application according to claim 1, wherein The polymer that makes up the short nanofibers includes one or more of polyethylene oxide, polypyrrole, polyvinyl alcohol, polyethylene naphthalate, polyaniline, nylon, polyphenylene sulfide, cellulose acetate, polystyrene, polyvinylpyrrolidone, polycaprolactam, acrylic resin, and polyacrylonitrile.

4. The application according to claim 1, wherein The short nanofibers are prepared by homogenizing electrospun nanofibers.

5. The application according to claim 1, characterized in that The length of the short fibers is 25-200 μm.

6. A monolithic chromatographic column, characterized in that, It contains the three-dimensional composite nanofiber material described in claim 1.

7. Application of the three-dimensional composite nanofiber material or the chromatographic monolith described in claim 6 in liquid chromatography separation.

8. The application according to claim 7, wherein For the separation and detection of two pigments, brilliant blue and rhodamine B.

9. The application according to claim 7, wherein For the separation and detection of two compounds, cortisol and 3-hydroxyphenanthrene.

10. The application according to any one of claims 7 to 9, characterized in that, The application is a combination of liquid chromatography and ultraviolet detector.

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