Molecular imprinting separation method for gallic acid in tara enzymolysis tannic acid waste liquid

Abstract

The invention discloses a molecular imprinting separation method for gallic acid in tara enzymolysis tannic acid waste liquid. The molecular imprinting separation method comprises the following steps: (1) preparing Fe3O4 nanoparticles; (2) preparing Fe3O4 (at) SiO2 nano particles; (3) preparing Fe3O4 (at) SiO2 (at) NH2 nano particles; (4) preparing a Fe3O4 (at) SiO2 (at) NH2 molecularly imprinted polymer; (5) pretreatment of waste liquid; and (6) gallic acid separation. The method has obvious advantages in the aspects of operation convenience, separation selectivity and repeatability, and an efficient and feasible technical scheme is provided for the field of natural product extraction.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction technology, specifically relating to a molecular imprinting method for separating gallic acid from tannic acid waste liquid obtained by enzymatic hydrolysis of tannins. Background Technology

[0002] Gallic acid, chemically known as 3,4,5-trihydroxybenzoic acid, is an important natural polyphenol compound with a wide range of biological activities. Studies have shown that gallic acid has various beneficial effects on human health, such as antioxidant, anti-inflammatory, antibacterial, and antitumor properties, and therefore has been widely used in the food, pharmaceutical, cosmetic, and chemical industries. The main sources of gallic acid include tannins from plants, obtained through hydrolysis. Among them, *Caesalpinia spinosa* is a plant rich in gallantanine, and its pod powder (tara powder) is an important raw material for the industrial production of gallic acid. Tara tannins can be decomposed to produce gallic acid under the catalysis of acids, alkalis, or enzymes; enzymatic hydrolysis is favored due to its mild conditions and environmental friendliness.

[0003] During enzymatic hydrolysis, taratanine is hydrolyzed by tannase, with gallic acid as the main product. However, a certain amount of gallic acid remains in the waste liquid. Currently, the gallic acid in taratanine enzymatic hydrolysis waste liquid is usually discarded without further extraction, which not only wastes resources but may also have a negative impact on the environment. Existing literature reports that the gallic acid content in taratanine enzymatic hydrolysis waste liquid is high; if not recovered, it will lead to the loss of by-products and reduce the overall industrial output value. Traditional methods for producing gallic acid include acid hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis. For example, acid hydrolysis uses hydrochloric acid or sulfuric acid to treat tannins, but this process generates a large amount of acidic wastewater, which easily causes environmental pollution, and the product purity is low. Although alkaline hydrolysis can avoid some acidity problems, it has harsh operating conditions and high energy consumption. Biological enzymatic hydrolysis utilizes microorganisms such as Aspergillus awamori or Klebsiella pneumoniae to produce tannins for hydrolysis. It is mild and selective, but the enzyme preparations are expensive and the waste liquid treatment is complicated.

[0004] To recover gallic acid from wastewater, existing technologies have explored various separation methods, such as solvent extraction, ion exchange, and adsorption. Solvent extraction can extract gallic acid from wastewater, but it requires large amounts of organic solvents, easily introduces secondary pollution, and suffers from poor selectivity. Ion exchange resins can achieve preliminary purification, but resin regeneration is difficult, and processing capacity is limited. Adsorption methods use materials such as activated carbon or silica gel, but these adsorbents lack specificity and are easily interfered with in complex wastewater systems, leading to low separation efficiency. Furthermore, microbial transformation methods have also been used for wastewater recovery, such as using certain bacteria to directly hydrolyze residual tannins, but this method has a long cycle, unstable yield, and requires strict control of fermentation conditions. Overall, these traditional methods suffer from high energy consumption, complex operation, low separation selectivity, and non-reusability, making them inefficient for treating gallic acid in Tara enzymatic hydrolysis of tannic acid wastewater.

[0005] Molecular imprinting technology is an emerging separation technique that uses target molecules as templates to form polymers (MIPs) with specific recognition sites through polymerization reactions. This technology mimics molecular recognition processes in nature, such as enzyme-substrate or antibody-antigen binding, and offers advantages such as high selectivity, high stability, and reusability. In the field of natural product extraction, molecular imprinting technology has shown broad application prospects. For example, studies have reported the selective extraction of gallic acid from complex matrices such as urine, fruit shell extracts, or olive wastewater using molecularly imprinted polymers. These polymers achieve specific adsorption of target molecules through the pre-assembly of template molecules and functional monomers to form specific cavities. However, existing molecular imprinting applications are mostly limited to simple systems or general extraction, lacking customized designs for Tara enzymatic hydrolysis of tannic acid wastewater. This wastewater has a complex composition, containing residual tannins, gallic acid, and enzymatic hydrolysis byproducts, making efficient separation difficult with traditional MIPs. In addition, although molecular imprinting technology combined with magnetic nanocarriers (such as Fe3O4-based materials) can simplify the separation process, it has not yet been systematically applied in Tara waste liquid recycling. Summary of the Invention

[0006] The purpose of this invention is to overcome the defects of the prior art and provide a molecular imprinting separation method for gallic acid in tannic acid waste liquid from Tara enzymatic hydrolysis.

[0007] The technical solution of the present invention is as follows:

[0008] A molecular imprinted separation method for gallic acid in tannin hydrolysis waste liquid of Tara enzymatic hydrolysis includes the following steps: (1) Preparation of Fe3O4 nanoparticles: FeCl3•6H2O is ultrasonically dispersed in ethylene glycol, followed by the addition of trisodium citrate and PEG-2000, and ultrasonic dispersion is continued until uniform. Then anhydrous sodium acetate is added and stirred until clear and transparent. The reaction is carried out at 195-205 °C for 7-9 h. The product is then separated by magnetic separation and washed with water and anhydrous ethanol. Fe3O4 nanoparticles are obtained by freeze drying.

[0009] (2) Preparation of Fe3O4@SiO2 nanoparticles: Fe3O4 nanoparticles were added to an ethanol-water mixed solution, ultrasonically dispersed, and then 5 mL of ammonia water was added and stirred. TEOS was then added dropwise, and the reaction was mechanically stirred at 38-42 °C and 450-550 r / min for 10-15 h. The product was then separated by magnetic separation and washed alternately with water and anhydrous ethanol. After freeze-drying, Fe3O4@SiO2 nanoparticles were obtained.

[0010] (3) Preparation of Fe3O4@SiO2@NH2 nanoparticles: Fe3O4@SiO2 nanoparticles, ultrapure water and anhydrous ethanol were mixed and ultrasonically dispersed, then APTES were added, and the reaction was mechanically stirred at 55-65 °C and 450-550 r / min for 10-15 h. Then, solid-liquid separation was performed by magnetic separation method, and the nanoparticles were washed alternately with water and anhydrous ethanol and freeze-dried to obtain Fe3O4@SiO2@NH2 nanoparticles.

[0011] (4) Preparation of Fe3O4@SiO2@NH2 molecularly imprinted polymer: Gallic acid and MAA were dissolved in acetonitrile, ultrasonically dispersed, and then allowed to stand at 2-4 °C for 10-15 h to obtain a pre-assembled gallic acid and MAA solution; at the same time, Fe3O4@SiO2@NH2 nanoparticles were ultrasonically dispersed in acetonitrile and allowed to stand at room temperature for 3-5 h. Then EGDMA, AIBN and the pre-assembled gallic acid and MAA solution were added, and the reaction was carried out at 55-65 °C for 20-30 h under nitrogen protection. The product was then separated by a magnet and eluted with a methanol-acetic acid mixed solution until gallic acid could not be detected in the eluent. The product was then washed alternately with water and anhydrous ethanol and freeze-dried to obtain the Fe3O4@SiO2@NH2 molecularly imprinted polymer.

[0012] (5) Waste liquid pretreatment: Take the waste liquid of tannic acid hydrolysis by Tarax and dilute it with acetonitrile for later use;

[0013] (6) Gallic acid separation: The prepared Fe3O4@SiO2@NH2 molecularly imprinted polymer was dispersed in the diluted Tara enzymatic hydrolysis tannic acid waste liquid obtained in step (5) and shaken at 28-32 ℃ for 5-7 h; then the Fe3O4@SiO2@NH2 molecularly imprinted polymer was separated by magnet and eluted by Soxhlet extraction until gallic acid could not be detected in the eluent; the Soxhlet extraction eluent was taken and concentrated by rotary evaporation and freeze-dried in sequence to obtain gallic acid sample.

[0014] In a preferred embodiment of the present invention, in step (1), the mass ratio of FeCl3•6H2O, trisodium citrate, PEG-2000 and anhydrous sodium acetate is 2.1-2.4: 1.2-1.5: 2.2-2.5: 9.1-9.5, and the ratio of FeCl3•6H2O to ethylene glycol is 2.1-2.4 g: 150-180 mL.

[0015] In a preferred embodiment of the present invention, in step (2), the ratio of Fe3O4 nanoparticles, ethanol-water mixed solution, ammonia and TEOS is 0.2-0.4 g: 200 mL: 5 mL: 0.6-0.7 mL.

[0016] More preferably, in step (2), the ethanol-water mixture is composed of anhydrous ethanol and water in a volume ratio of 4:1.

[0017] In a preferred embodiment of the present invention, in step (3), the ratio of Fe3O4@SiO2 nanoparticles to APTES is 0.2-0.3 g: 0.6-0.7 mL.

[0018] More preferably, in step (3), the volume ratio of anhydrous ethanol to ultrapure water is 4:1.

[0019] In a preferred embodiment of the present invention, in step (4), the ratio of gallic acid, MAA, Fe3O4@SiO2@NH2 nanoparticles, EGDMA and AIBN is 0.05-0.08 g: 0.15-0.25 mL: 50-150 mg: 1-3 mL: 0.2-0.6 g.

[0020] More preferably, in step (4), the volume ratio of methanol to acetic acid in the methanol-acetic acid mixed solution is 9:1.

[0021] In a preferred embodiment of the present invention, in step (5), acetonitrile is used to dilute the product by 20-30 times.

[0022] In a preferred embodiment of the present invention, step (7) is further included: Fe3O4@SiO2@NH2 molecularly imprinted polymer regeneration: the solid in the extraction chamber after Soxhlet extraction is taken, washed alternately with water and anhydrous ethanol, and then freeze-dried to obtain the regenerated Fe3O4@SiO2@NH2 molecularly imprinted polymer.

[0023] The beneficial effects of this invention are:

[0024] 1. This invention utilizes the specific recognition characteristics of molecularly imprinted polymers to achieve selective adsorption and separation of gallic acid, thereby simplifying the operation steps and improving the separation efficiency.

[0025] 2. This invention is simple to operate and can be completed with only conventional laboratory equipment, avoiding the complex multi-step extraction or fermentation process in traditional methods, and is easy to scale up for industrial applications.

[0026] 3. The molecularly imprinted polymer in this invention has high stability and can maintain good selectivity in complex waste liquid environments, reduce impurity interference, and improve separation purity.

[0027] 4. This invention introduces a magnetic nanocarrier, facilitating rapid separation of the adsorbent via an external magnetic field, shortening processing time, and enabling polymer regeneration and reuse, thereby reducing material consumption and environmental burden. Compared to existing technologies, this invention fills the gap in gallic acid recovery from Tara enzymatic hydrolysis wastewater, possessing significant resource utilization value, contributing to improved overall economic efficiency in the Tara industry, and promoting sustainable development.

[0028] 5. This invention is highly environmentally friendly, reducing the pollution risks caused by direct discharge of waste liquid and conforming to the principles of green chemistry. Attached Figure Description

[0029] Figure 1 This is a TEM characterization image of the Fe3O4 nanoparticles prepared in Example 1 of the present invention.

[0030] Figure 2 This is a TEM characterization image of the Fe3O4@SiO2 nanoparticles prepared in Example 1 of this invention.

[0031] Figure 3 The hydrophobicity of the Fe3O4@SiO2@NH2 nanoparticles prepared in Example 1 of this invention is characterized.

[0032] Figure 4 This is a SEM characterization image of the Fe3O4@SiO2@NH2 molecularly imprinted polymer prepared in Example 1 of this invention.

[0033] Figure 5 This is a comparison of the high-performance liquid chromatography (HPLC) chromatograms of the gallic acid standard sample and the separated gallic acid in Example 1 of the present invention. Detailed Implementation

[0034] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0035] The compositional analysis of the tannic acid hydrolysate treated in the following examples and comparative examples is as follows: The composition of the tannic acid hydrolysate was measured after spray drying, as shown in Table 1.

[0036] Table 1. Composition of dried powder from Tara enzymatic hydrolysis tannic acid waste liquid

[0037] Inspection items Test results Detection methods Appearance brown powder Visual inspection Moisture content 6.4% Oven drying method Gallic acid content 11% LY-T 1642-2005 Tannic acid content 1.7% LY-T 1644-2005 Quinic acid content Approximately 8% HPLC

[0038] Example 1

[0039] (1) Preparation of Fe3O4 nanoparticles: 2.25 g FeCl3•6H2O was ultrasonically dispersed in 180 mL ethylene glycol. Subsequently, 1.5 g trisodium citrate and 2.5 g PEG-2000 were added to the solution sequentially, and ultrasonic dispersion was continued until uniform. Finally, 9.1 g anhydrous sodium acetate was added. The mixed solution was stirred until clear and transparent, then transferred to a 200 mL reaction vessel and reacted at 200 °C for 8 h. After the reaction was completed, the product was separated by magnetic separation and washed three times alternately with water and anhydrous ethanol. After freeze-drying, dried Fe3O4 nanoparticles (e.g., FeCl3•6H2O) were obtained. Figure 1 (As shown).

[0040] (2) Preparation of Fe3O4@SiO2 nanoparticles: 0.28 g of Fe3O4 nanoparticles were added to an ethanol-water mixture (160 mL anhydrous ethanol and 40 mL water) and ultrasonically dispersed for 30 min. The resulting solution was transferred to a 250 mL three-necked flask, stirred at 500 r / min for 15 min, and then 5 mL of ammonia water was added, and stirring was continued for 10 min. Subsequently, 0.6 mL of TEOS was added dropwise. The mixture was mechanically stirred at 40 °C and 500 r / min for 12 h. After the reaction was completed, the product was separated by magnetic separation and washed three times alternately with water and anhydrous ethanol. After freeze-drying, Fe3O4@SiO2 nanoparticles were obtained (e.g., Fe3O4@SiO2 nanoparticles). Figure 2 (As shown).

[0041] (3) Preparation of Fe3O4@SiO2@NH2 nanoparticles: Weigh 0.25 g of Fe3O4@SiO2 nanoparticles and add them to a 500 mL three-necked round-bottom flask. Then, add 40 mL of ultrapure water and 160 mL of anhydrous ethanol, and ultrasonically disperse for 10 min. Next, add 2.2 mL of APTES and mechanically stir at 60 °C and 500 r / min for 12 h. After the reaction is complete, solid-liquid separation is performed by magnetic separation to obtain black solid Fe3O4@SiO2@NH2, which is washed three times alternately with water and anhydrous ethanol. After freeze-drying, Fe3O4@SiO2@NH2 nanoparticles are obtained (e.g., ...). Figure 3 (As shown).

[0042] (4) Preparation of Fe3O4@SiO2@NH2 molecularly imprinted polymer: 0.05 g gallic acid and 0.15 mL MAA were dissolved in 50 mL acetonitrile, ultrasonically dispersed, and then allowed to stand at 4 °C for 12 h. Simultaneously, 50 mg of Fe3O4@SiO2@NH2 nanoparticles were dispersed in 10 mL acetonitrile, ultrasonically dispersed, and then allowed to stand at room temperature for 4 h. Subsequently, 1 mL EGDMA, 0.2 g AIBN, and the pre-assembled gallic acid and MAA solution were added to the above Fe3O4@SiO2@NH2 solution, and the reaction was carried out at 60 °C for 24 h under nitrogen protection. After the reaction, the product was separated using a magnet and eluted with a methanol-acetic acid (9:1 volume ratio) solution until gallic acid was undetectable in the eluent. Then, the product was washed three times alternately with water and anhydrous ethanol. After freeze-drying, the Fe3O4@SiO2@NH2 molecularly imprinted polymer (e.g., Figure 4 (As shown).

[0043] (5) Waste liquid pretreatment: Take 100 mL of Tara enzymatic hydrolysis tannic acid waste liquid, dilute it 30 times with acetonitrile, and set it aside.

[0044] (6) Gallic acid separation: The prepared Fe3O4@SiO2@NH2 molecularly imprinted polymer was dispersed in the diluted Tara enzymatic hydrolysis tannic acid waste liquid obtained in step (5), and shaken at 30℃ for 6 h. Subsequently, the Fe3O4@SiO2@NH2 molecularly imprinted polymer was separated using a magnet, and eluted using Soxhlet extraction until gallic acid could not be detected in the eluent. The Soxhlet extraction eluent was concentrated by rotary evaporation and then freeze-dried to obtain a gallic acid sample with a mass of 0.42 g, an extraction rate of 70%, and a separation effect as shown in the figure. Figure 5 As shown.

[0045] (7) Regeneration of Fe3O4@SiO2@NH2 molecularly imprinted polymer: Take the solid from the extraction chamber after Soxhlet extraction and wash it three times alternately with water and anhydrous ethanol. After freeze-drying, the regenerated Fe3O4@SiO2@NH2 molecularly imprinted polymer is obtained.

[0046] Example 2

[0047] (1) Preparation of Fe3O4 nanoparticles: 2.13 g FeCl3•6H2O was ultrasonically dispersed in 160 mL ethylene glycol. Subsequently, 1.4 g trisodium citrate and 2.2 g PEG-2000 were added to the solution sequentially, and ultrasonic dispersion was continued until uniform. Finally, 9.1 g anhydrous sodium acetate was added. The mixed solution was stirred until clear and transparent, then transferred to a 200 mL reaction vessel and reacted at 200 °C for 8 h. After the reaction was completed, the product was separated by magnetic separation and washed three times alternately with water and anhydrous ethanol. After freeze-drying, dried Fe3O4 nanoparticles were obtained.

[0048] (2) Preparation of Fe3O4@SiO2 nanoparticles: 0.35 g of Fe3O4 nanoparticles were added to an ethanol-water mixture (160 mL anhydrous ethanol and 40 mL water) and ultrasonically dispersed for 30 min. The resulting solution was transferred to a 250 mL three-necked flask and stirred at 500 r / min for 15 min. Then, 5 mL of ammonia water was added and stirring was continued for 10 min. Subsequently, 0.7 mL of TEOS was added dropwise. The mixture was mechanically stirred at 40 °C and 500 r / min for 12 h. After the reaction was completed, the product was separated by magnetic separation and washed three times alternately with water and anhydrous ethanol. After freeze-drying, Fe3O4@SiO2 nanoparticles were obtained.

[0049] (3) Preparation of Fe3O4@SiO2@NH2 nanoparticles: 0.27 g of Fe3O4@SiO2 nanoparticles were weighed and added to a 500 mL three-necked round-bottom flask. Then, 40 mL of ultrapure water and 160 mL of anhydrous ethanol were added, and the mixture was ultrasonically dispersed for 10 min. Next, 2 mL of APTES was added, and the mixture was mechanically stirred at 60 °C and 500 r / min for 13 h. After the reaction was completed, solid-liquid separation was performed using magnetic separation to obtain black solid Fe3O4@SiO2@NH2, which was washed three times alternately with water and anhydrous ethanol. After freeze-drying, Fe3O4@SiO2@NH2 nanoparticles were obtained.

[0050] (4) Preparation of Fe3O4@SiO2@NH2 molecularly imprinted polymer: 0.08 g gallic acid and 0.2 mL MAA were dissolved in 150 mL acetonitrile, ultrasonically dispersed, and then allowed to stand at 4 °C for 12 h. Simultaneously, 150 mg of Fe3O4@SiO2@NH2 nanoparticles were dispersed in 30 mL acetonitrile, ultrasonically dispersed, and then allowed to stand at room temperature for 6 h. Subsequently, 3 mL of EGDMA, 0.6 g of AIBN, and the pre-assembled gallic acid and MAA solution were added to the above Fe3O4@SiO2@NH2 solution, and the reaction was carried out at 60 °C for 24 h under nitrogen protection. After the reaction, the product was separated using a magnet and eluted with a methanol-acetic acid (9:1 volume ratio) mixture until gallic acid was undetectable in the eluent. Then, the product was washed three times alternately with water and anhydrous ethanol. After freeze-drying, the Fe3O4@SiO2@NH2 molecularly imprinted polymer was obtained.

[0051] (5) Waste liquid pretreatment: Take 100 mL of Tara enzymatic hydrolysis tannic acid waste liquid, dilute it 25 times with acetonitrile, and set it aside.

[0052] (6) Gallic acid separation: The prepared Fe3O4@SiO2@NH2 molecularly imprinted polymer was dispersed in the diluted Tara enzymatic hydrolysis tannic acid waste liquid obtained in step (5), and shaken at 30℃ for 6 h. Subsequently, the Fe3O4@SiO2@NH2 molecularly imprinted polymer was separated using a magnet, and eluted using Soxhlet extraction until gallic acid could not be detected in the eluent. The Soxhlet extraction eluent was concentrated by rotary evaporation and then freeze-dried to obtain a gallic acid sample with a mass of 0.63 g and an extraction rate of 67%.

[0053] (7) Regeneration of Fe3O4@SiO2@NH2 molecularly imprinted polymer: Take the solid from the extraction chamber after Soxhlet extraction and wash it three times alternately with water and anhydrous ethanol. After freeze-drying, the regenerated Fe3O4@SiO2@NH2 molecularly imprinted polymer is obtained.

[0054] Example 3

[0055] (1) Preparation of Fe3O4 nanoparticles: 2.31 g FeCl3•6H2O was ultrasonically dispersed in 150 mL ethylene glycol. Subsequently, 1.2 g trisodium citrate and 2.2 g PEG-2000 were added to the solution sequentially, and ultrasonic dispersion was continued until uniform. Finally, 9.5 g anhydrous sodium acetate was added. The mixed solution was stirred until clear and transparent, then transferred to a 200 mL reaction vessel and reacted at 200 °C for 8 h. After the reaction was completed, the product was separated by magnetic separation and washed three times alternately with water and anhydrous ethanol. After freeze-drying, dried Fe3O4 nanoparticles were obtained.

[0056] (2) Preparation of Fe3O4@SiO2 nanoparticles: 0.22 g of Fe3O4 nanoparticles were added to an ethanol-water mixture (160 mL anhydrous ethanol and 40 mL water) and ultrasonically dispersed for 30 min. The resulting solution was transferred to a 250 mL three-necked flask and stirred at 500 r / min for 15 min. Then, 5 mL of ammonia water was added and stirring was continued for 10 min. Subsequently, 0.7 mL of TEOS was added dropwise. The mixture was mechanically stirred at 40 °C and 500 r / min for 12 h. After the reaction was completed, the product was separated by magnetic separation and washed three times alternately with water and anhydrous ethanol. After freeze-drying, Fe3O4@SiO2 nanoparticles were obtained.

[0057] (3) Preparation of Fe3O4@SiO2@NH2 nanoparticles: 0.2 g of Fe3O4@SiO2 nanoparticles were weighed and added to a 500 mL three-necked round-bottom flask. Then, 40 mL of ultrapure water and 160 mL of anhydrous ethanol were added, and the mixture was ultrasonically dispersed for 10 min. Next, 2 mL of APTES was added, and the mixture was mechanically stirred at 60 °C and 500 r / min for 12 h. After the reaction was completed, solid-liquid separation was performed using magnetic separation to obtain black solid Fe3O4@SiO2@NH2, which was washed three times alternately with water and anhydrous ethanol. After freeze-drying, Fe3O4@SiO2@NH2 nanoparticles were obtained.

[0058] (4) Preparation of Fe3O4@SiO2@NH2 molecularly imprinted polymer: 0.06 g gallic acid and 0.25 mL MAA were dissolved in 140 mL acetonitrile, ultrasonically dispersed, and then allowed to stand at 4 °C for 12 h. Simultaneously, 140 mg of Fe3O4@SiO2@NH2 nanoparticles were dispersed in 30 mL acetonitrile, ultrasonically dispersed, and then allowed to stand at room temperature for 6 h. Subsequently, 3 mL of EGDMA, 0.5 g of AIBN, and the pre-assembled gallic acid and MAA solution were added to the above Fe3O4@SiO2@NH2 solution, and the reaction was carried out at 60 °C for 24 h under nitrogen protection. After the reaction, the product was separated using a magnet and eluted with a methanol-acetic acid (9:1 volume ratio) mixture until gallic acid was undetectable in the eluent. Then, the product was washed three times alternately with water and anhydrous ethanol. After freeze-drying, the Fe3O4@SiO2@NH2 molecularly imprinted polymer was obtained.

[0059] (5) Waste liquid pretreatment: Take 100 mL of Tara enzymatic hydrolysis tannic acid waste liquid, dilute it 20 times with acetonitrile, and set it aside.

[0060] (6) Gallic acid separation: The prepared Fe3O4@SiO2@NH2 molecularly imprinted polymer was dispersed in the diluted Tara enzymatic hydrolysis tannic acid waste liquid obtained in step (5), and shaken at 30℃ for 6 h. Subsequently, the Fe3O4@SiO2@NH2 molecularly imprinted polymer was separated using a magnet, and eluted using Soxhlet extraction until gallic acid could not be detected in the eluent. The Soxhlet extraction eluent was concentrated by rotary evaporation and then freeze-dried to obtain a gallic acid sample with a mass of 0.41 g and an extraction rate of 65%.

[0061] (7) Regeneration of Fe3O4@SiO2@NH2 molecularly imprinted polymer: Take the solid from the extraction chamber after Soxhlet extraction and wash it three times alternately with water and anhydrous ethanol. After freeze-drying, the regenerated Fe3O4@SiO2@NH2 molecularly imprinted polymer is obtained.

[0062] The process parameters of the above embodiments are compared in Table 2 below:

[0063] Table 2

[0064] step Example 1 Example 2 Example 3 （1） FeCl3-6H2O: 2.25 g Ethylene glycol: 180 mL Trisodium citrate: 1.5 g PEG-2000: 2.5 g Sodium acetate anhydrous: 9.1 g Reaction: 200 °C, 8 h FeCl3-6H2O: 2.13 g ethylene glycol: 160 mL trisodium citrate: 1.4 g PEG-2000: 2.2 g anhydrous sodium acetate: 9.1 g reaction: 200 °C, 8 h FeCl3-6H2O: 2.31 g ethylene glycol: 150 mL trisodium citrate: 1.2 g PEG-2000: 2.2 g anhydrous sodium acetate: 9.5 g reaction: 200 °C, 8 h （2） Fe304: 0.28 g TEOS: 0.6 mL Reaction: 40 °C, 500 r / min, 12 h Fe304: 0.35 g TEOS: 0.7 mL Reaction: 40 °C, 500 r / min, 12 h Fe304: 0.22 g TEOS: 0.7 mL Reaction: 40 °C, 500 r / min, 12 h （3） Fe304@Si02: 0.25 g APTES: 2.2 mL Reaction: 60 °C, 500 r / min, 12 h Fe304@Si02: 0.27 g APTES: 2 mL Reaction: 60 °C, 500 r / min, 13 h Fe304@Si02: 0.2 g APTES: 2 mL Reaction: 60 °C, 500 r / min, 12 h （4） Gallic acid: 0.05 g MAA: 0.15 mL Acetonitrile: 50 mL Fe304@Si02@NH2: 50 mg Acetonitrile: 10 mL EGDMA: 1 mL AIBN: 0.2 g Pre-assembly: 4 °C, 12 h Rest: room temperature, 4 h Reaction: 60 °C, 24 h Gallic acid: 0.08 g MAA: 0.2 mL Acetonitrile: 150 mL Fe304@Si02@NH2: 150 mg Acetonitrile: 30 mL EGDMA: 3 mL AIBN: 0.6 g Pre-assembly: 4 °C, 12 h Rest: room temperature, 6 h Reaction: 60 °C, 24 h <![CDATA[Gallic acid: 0.06 g MAA: 0.25 mL Acetonitrile: 140 mL Fe3O4@SiO2@NH2: 140 mg Acetonitrile: 30 mL EG DMA: 3 mL AIBN: 0.5 g Pre-assembly: 4 °C, 12 h Standing: room temperature, 6 h Reaction: 60 °C, 24 h]]> （5） Acetonitrile dilution: 30 times Acetonitrile dilution: 25 times Acetonitrile dilution: 20 times （6） Shaking: 30℃, 6 h; Recovery mass: 0.42 g (other steps are the same) Shaking: 30℃, 6 h; Recovery mass: 0.63 g (other steps are the same) Shaking: 30℃, 6 h; Recovery mass: 0.41 g (other steps are the same) （7） (All embodiments are identical and have no differences) (All embodiments are identical and have no differences) (All embodiments are identical and have no differences)

[0065] Comparative Example 1

[0066] 36 g of wet HP-20 macroporous resin was packed into a chromatography column (20 cm long, 2 cm outer diameter). A certain amount of distilled water was added to wash the column first. After the filtrate became clear and odorless, 240 mL of tannic acid hydrolysis wastewater from Tara was added to the column for gallic acid adsorption, with a flow rate controlled at 30 mL / h. First, distilled water was added for elution. After the filtrate became clear, it was replaced with a 50% ethanol solution for elution, and the filtrate was collected. The filtrate was concentrated by rotary evaporation and freeze-dried to obtain a 50% ethanol-eluted sample. After removing the ethanol solvent by rotary evaporation at 45°C, the aqueous phase was freeze-dried to obtain the gallic acid sample, with a mass of 0.47 g and an extraction rate of 32.6%.

[0067] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A molecular imprinting separation method of gallic acid from enzymatic hydrolysis tannin acid waste liquid, characterized in that: Comprising the following steps: ​ (1) Preparation of Fe3O4 nanoparticles: FeCl3•6H2O is ultrasonically dispersed in ethylene glycol, followed by the addition of trisodium citrate and PEG-2000 in sequence, and then ultrasonic dispersion is continued until uniform, followed by the addition of anhydrous sodium acetate, stirring until clear and transparent, and then reaction at 195-205 ℃ for 7-9 h, followed by separation of the product by magnetic separation, and washing with water and anhydrous ethanol, and then freeze-drying to obtain Fe3O4 nanoparticles; (2) Preparation of Fe3O4@SiO2 nanoparticles: Fe3O4 nanoparticles are added to an ethanol-water mixed solution, ultrasonic dispersion is performed, followed by stirring after the addition of 5 mL of ammonia water, and then dropwise addition of TEOS, followed by mechanical stirring at 38-42 ℃ and 450-550 r / min for 10-15 h, and then separation of the product by magnetic separation, and alternating washing with water and anhydrous ethanol, and then freeze-drying to obtain Fe3O4@SiO2 nanoparticles; (3) Preparation of Fe3O4@SiO2@NH2 nanoparticles: Fe3O4@SiO2 nanoparticles, ultrapure water and anhydrous ethanol are mixed and ultrasonically dispersed, followed by the addition of APTES, and then mechanical stirring at 55-65 ℃ and 450-550 r / min for 10-15 h, and then solid-liquid separation by magnetic separation, and alternating washing with water and anhydrous ethanol, and then freeze-drying to obtain Fe3O4@SiO2@NH2 nanoparticles; (4) Preparation of Fe3O4@SiO2@NH2 molecularly imprinted polymer: gallic acid and MAA are dissolved in acetonitrile, ultrasonic dispersion is performed, and then the solution is placed at 2-4 ℃ for 10-15 h to obtain a pre-assembled gallic acid and MAA solution; simultaneously, Fe3O4@SiO2@NH2 nanoparticles are ultrasonically dispersed in acetonitrile, and then placed at room temperature for 3-5 h, followed by the addition of EGDMA, AIBN and the pre-assembled gallic acid and MAA solution, and then reaction at 55-65 ℃ for 20-30 h under nitrogen protection, and then separation of the product by a magnet, and elution with a methanol-acetic acid mixed solution until gallic acid cannot be detected in the eluent; and then alternating washing with water and anhydrous ethanol, and then freeze-drying to obtain a Fe3O4@SiO2@NH2 molecularly imprinted polymer; (5) Waste liquid pretreatment: tara enzymatic tannin acid waste liquid is taken, diluted with acetonitrile and then reserved; (6) Gallic acid separation: the Fe3O4@SiO2@NH2 molecularly imprinted polymer prepared is dispersed in the diluted tara enzymatic tannin acid waste liquid obtained in step (5), and then oscillation is performed at 28-32 ℃ for 5-7 h; and then the Fe3O4@SiO2@NH2 molecularly imprinted polymer is separated by a magnet, and elution is performed by Soxhlet extraction until gallic acid cannot be detected in the eluent; and then the Soxhlet extraction eluent is taken, concentrated by rotary evaporation and freeze-dried in sequence to obtain a gallic acid sample.

2. The molecular imprinting separation method according to claim 1, wherein: In the step (1), the mass ratio of FeCl3•6H2O, trisodium citrate, PEG-2000 and anhydrous sodium acetate is 2.1-2.4: 1.2-1.5: 2.2-2.5: 9.1-9.5, and the ratio of FeCl3•6H2O to ethylene glycol is 2.1-2.4 g: 150-180 mL.

3. The molecular imprinting separation method of claim 1, wherein: In the step (2), the ratio of Fe3O4 nanoparticles, ethanol-water mixed solution, ammonia and TEOS is 0.2-0.4 g: 200 mL: 5 mL: 0.6-0.7 mL.

4. The molecular imprinting separation method according to claim 3, wherein: In the step (2), the ethanol-water mixed solution is composed of anhydrous ethanol and water in a volume ratio of 4:

1.

5. The molecular imprinting separation method of claim 1 wherein: In the step (3), the ratio of Fe3O4@SiO2 nanoparticles and APTES is 0.2-0.3 g: 0.6-0.7 mL.

6. The molecular imprinting separation method according to claim 5, wherein: In the step (3), the volume ratio of anhydrous ethanol and ultrapure water is 4:

1.

7. The molecular imprinting separation method of claim 1 wherein: In the step (4), the ratio of gallic acid, MAA, Fe3O4@SiO2@NH2 nanoparticles, EGDMA and AIBN is 0.05-0.08 g: 0.15-0.25 mL: 50-150 mg: 1-3 mL: 0.2-0.6 g.

8. The molecular imprinting separation method of claim 5 wherein: In the step (4), the volume ratio of methanol to acetic acid in the methanol-acetic acid mixed solution is 9:

1.

9. The molecular imprinting separation method of claim 1 wherein: In the step (5), acetonitrile is used to dilute 20-30 times.

10. The molecular imprinting separation method according to any one of claims 1 to 9, wherein: It also includes step (7): regeneration of Fe3O4@SiO2@NH2 molecularly imprinted polymer: the solid in the extraction chamber after Soxhlet extraction elution is washed alternately with water and anhydrous ethanol, and then freeze-dried to obtain regenerated Fe3O4@SiO2@NH2 molecularly imprinted polymer.

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