Application of ionic liquid reverse micelle membrane in extraction of tetracycline antibiotics

Abstract

This invention discloses the application of an ionic liquid reverse micelle membrane in the extraction of tetracycline antibiotics, belonging to the field of membrane extraction technology. The preparation of the ionic liquid reverse micelle membrane includes the following steps: mixing 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt and 1-decyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt, and fully impregnating a PC membrane with the clarified liquid after the reaction to obtain the ionic liquid reverse micelle membrane. The method for extracting tetracycline antibiotics in this application does not require additional separation and purification steps, the preparation process is simple, and when combined with high-performance liquid chromatography (HPLC), it can quantitatively detect the concentration of tetracycline antibiotics, achieving an extraction rate of 77.79%, providing a new and effective pretreatment method for the detection of antibiotic contamination.

Description

Technical Field

[0001] This invention relates to the application of an ionic liquid reverse micelle membrane in the extraction of tetracycline antibiotics, belonging to the field of membrane extraction technology. Background Technology

[0002] Reversed micelles are nanoscale aggregates spontaneously formed by surfactants in organic solvents; they are transparent, thermodynamically stable systems. Reversed micelle extraction is a liquid-liquid extraction method that utilizes reverse micelles formed by various surfactants to separate organic and inorganic substances. This method features high selectivity, low energy consumption, and mild thermal operating conditions. During extraction, the bioactive molecules to be extracted are encapsulated within reverse micelles, thus avoiding denaturation from direct contact with the organic solvent. Therefore, reverse micelle extraction can improve the recovery rate of bioactive molecules. Organic solvents and surfactants can be easily recovered and reused, reducing process costs. It also has the potential for large-scale downstream processing of biomolecules from fermentation broths. Downstream processing in antibiotic production often involves solvent extraction. However, traditional solvent extraction methods suffer from difficulties such as emulsion stability hindering the separation process, limited solvent selection, and long separation times. Reversed micelle extraction is a potential alternative method for antibiotic extraction.

[0003] Tetracyclines (TCs) are a group of antibiotics that inhibit the synthesis of microbial proteins by binding to the 30S subunit of microbial ribosomes. Due to their superior antibacterial activity and minimal side effects, TCs are among the most widely used broad-spectrum antibiotics for treating bacterial infections in humans and animals. However, TC residues are continuously released into the environment, and traditional treatment systems are inadequate in removing them, leading to their widespread presence in soil, surface water, groundwater, and even drinking water, causing water pollution.

[0004] Electrolytic membrane extraction (EME) is a novel, miniaturized extraction technique that has been widely applied in the analysis and removal of environmental pollutants in recent years. It is based on the electrokinetic migration across a supported liquid membrane (SLM) under the influence of an external electric field between two water chambers. Leveraging the characteristics of the SLM and the electric field, EME offers rapid extraction, effective sample purification, and good selectivity, limiting the amount of organic solvent used per sample to just a few microliters. Choosing a suitable solvent is crucial for achieving efficient extraction. Toluene, undecane, 1-octanol, and dihexyl ether are among the most commonly used solvents in EME. However, due to their low viscosity and high volatility, potential solvent loss exists when extraction is performed at high stirring rates, relatively high extraction temperatures, and extended extraction times. The extractability of these solvents for polar compounds is also limited. In recent years, room-temperature ionic liquids have been proposed as alternatives to traditional organic solvents, possessing unique physicochemical properties such as low volatility, low toxicity, high viscosity, tunable polarity, and high extractability for both organic and inorganic compounds, and are widely used in the extraction field. Existing literature has established an ionic liquid electro-membrane extraction (IL-EME) method and compared its performance with that of 2-ethylnitrobenzene (ENB) electro-membrane extraction for the determination of strychnine and brucine in human urine. The results show that IL-EME is more reliable than ENB-based EME and can provide better extraction performance for the determination of strychnine and brucine in human urine. Summary of the Invention

[0005] Objectives of the Invention: The first objective of this invention is to provide an application of ionic liquid reverse micelle membranes in the extraction of tetracycline antibiotics. The second objective of this invention is to provide a method for extracting tetracycline antibiotics.

[0006] Technical solution: Application of an ionic liquid reverse micelle membrane in the extraction of tetracycline antibiotics.

[0007] The preparation of the ionic liquid reverse micelle membrane includes the following steps: mixing 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt and 1-decyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt, and fully impregnating the PC membrane with the clear liquid after the reaction to obtain the ionic liquid reverse micelle membrane.

[0008] The present invention also provides a method for extracting tetracycline antibiotics, comprising the following steps:

[0009] (1) Mix 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt with 1-decyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt, sonicate, and take the lower clear liquid.

[0010] (2) The PC membrane is fully impregnated with the lower clear liquid prepared in step (1), and the excess liquid on the membrane is blown off with nitrogen gas to obtain the ionic liquid reverse micelle membrane for extracting tetracycline antibiotics.

[0011] (3) An ionic liquid reverse micelle membrane is fixed in the middle of the donor and acceptor chambers. The donor phase solution and sample solution are added to the donor chamber and connected to the positive terminal of the power supply. The acceptor phase solution is added to the acceptor chamber and connected to the negative terminal of the power supply to form an electro-membrane extraction device. The device is placed in an ultrasonic instrument for extraction to obtain a solution containing tetracycline antibiotics. The donor and acceptor phase solutions are 0.05 mol·L⁻¹. -1 The solution contains a phosphate buffer solution, and salt is also added to the supply phase solution; the extraction conditions are 20–40 V.

[0012] In step (1), the mixing mass ratio of 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt and 1-decyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt is 50 to 150:1.

[0013] The PC membrane mentioned in step (2) has a pore size of 0.2 to 5 μm.

[0014] The PC film mentioned in step (2) is made of one of the following materials: polycarbonate, polytetrafluoroethylene, polypropylene polyethersulfone, polyvinylidene fluoride, nylon, cellulose acetate, and cellulose nitrate.

[0015] In step (3), the pH of the supply solution is 2 to 2.5, and the pH of the receiving solution is 7.5 to 8.

[0016] In step (3), salt is also added to the phase supply solution.

[0017] In step (3), the salt content is 0.05–0.15 mol·L⁻¹. -1 Li[NTf2].

[0018] The step (3) is followed by a step of detecting the concentration of tetracycline antibiotics using high-performance liquid chromatography.

[0019] (1) Add phosphate buffer solution to standard samples of tetracycline antibiotics of different concentrations, and plot the standard equations by the peak area and absorbance of different concentrations.

[0020] (2) Detect the peak area of ​​the extracted liquid and substitute it into the standard equation to obtain the concentration of tetracycline antibiotics.

[0021] The tetracycline antibiotics include tetracycline or oxytetracycline.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: The method for extracting tetracycline antibiotics in this application does not require additional separation and purification steps, the preparation process is simple, and when combined with high performance liquid chromatography, it can quantitatively detect the concentration of tetracycline antibiotics, with an extraction rate of up to 77.79%, providing a new and effective pretreatment method for the detection of antibiotic drug contamination. Attached Figure Description

[0023] Figure 1 a. [Bmim][NTf2] forms the pre-system of the reverse micelles; b. [Bmim][NTf2] forms the mid-system of the reverse micelles;

[0024] c. [Bmim][NTf2] after forming reverse micelles; d. Methyl orange in the reverse micelles has the highest visible UV absorption A. max ;

[0025] Figure 2 A schematic diagram of a method for extracting tetracycline using a [Bmim][NTf2] reverse micelle electrophoresis membrane;

[0026] Figure 3 The results of reverse micelle electroporation using different [NTf2]-type ionic liquids as liquid membranes;

[0027] Figure 4 [Bmim][NTf2] Reverse micelle electroporation results using PC membranes with different pore sizes;

[0028] Figure 5 [Bmim][NTf2] Reverse micelle electroporation results using different voltages;

[0029] Figure 6 [Bmim][NTf2] reverse micelle electroporation results using different donor phase pH;

[0030] Figure 7 [Bmim][NTf2] reverse micelle electroporation results using different acceptor phase pH;

[0031] Figure 8 [Bmim][NTf2] reverse micelle electroporation results using different salt concentrations;

[0032] Figure 9 Comparison of [Bmim][NTf2] reverse micelle electrophoresis extraction and Tween-80 neutral reverse micelle electrophoresis extraction;

[0033] Figure 10 Standard addition of 2 mg·L -1 High-performance liquid chromatography separation chromatogram of tetracycline and oxytetracycline. Detailed Implementation

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

[0035] Example 1. Preparation of ionic liquid reverse micelles (IL-RM)

[0036] Methyl orange is often used as a solvation colorimetric probe, and its maximum UV-Vis absorption (A) is high. max Sensitive to local environment. Take 1.5 mL (2.16 g) [Bmim][NTf2] (Shanghai Aladdin Biochemical Technology Co., Ltd.), 0.01684 mL (0.0216 g) [C10mim][NTf2] (Shanghai Aladdin Biochemical Technology Co., Ltd.) (mass ratio I = 100) and 5 mL 2.4 × 10 - 4 mol·L -1 Mixed with methyl orange solution, such as Figure 1 As shown in a, methyl orange is insoluble in [Bmim][NTf2], and therefore can be used to confirm the presence of water in the system. In this patent, [C] is used. 10 [mim][NTf2] replaces traditional surfactants and together with [Bmim][NTf2] constructs an antimicelle system.

[0037] The preparation process of ionic liquid reverse micelles is as follows: 1.5 mL (2.16 g) of [Bmim][NTf2] was mixed with different concentrations of [C] 10 Mix [mim][NTf2], then add 5 mL of 2.4 × 10⁻⁶ [NTf2] to it. -4 mol·L -1 A methyl orange solution was subjected to thorough ultrasonic agitation (40 kHz, 120 W) for 15 minutes. Specifically, [Bmim][NTf2] and [C]... 10 The mass ratios I of [mm][NTf2] are 25, 50, 100, 150, 200, and 300, respectively. It can be seen that... Figure 1 The mixed solution in b. After standing for 5 minutes, the solution separates into layers again. After removing the upper layer, the solution is obtained. Figure 1 Clarified ionic liquid reverse micelles (IL-RM) in c.

[0038] Methyl orange was used as a probe to determine the ultraviolet absorption peak of methyl orange in the reverse micelles, thereby determining the water content in the system. The ultraviolet absorption peak A of methyl orange in the reverse micelles was measured at a wavelength of 432 nm using an SV-2100 ultraviolet-visible spectrophotometer (Korea Komei Instruments). max Different I (I = m) were determined [Bmim][NTf2] / m [C10mim][NTf2] The A of methyl orange in the [Bmim][NTf2] aqueous microemulsion (i.e., the clear ionic liquid reverse micelles obtained in the above steps) maxThe result is as follows Figure 1 As shown in d. When I is between 0 and 100, A max As I increases, A also increases, and the rate of increase becomes increasingly gradual, indicating that the water content in the system is gradually increasing; when the I value reaches around 100, A... max It reaches its maximum value, then gradually decreases as I continues to increase, finally ceasing to change significantly after I = 200. This indicates that when [C]... 10 When the content of [Bmim][NTf2] is low enough, its role in the formation of reverse micelles may be negligible. The results show that only [Bmim][NTf2] and [C]... 10 When the mass ratio of mim][NTf2]I is 100, the water content in the reverse micelles is the highest, and the formation of the reverse micelles is the most complete. Therefore, this patent determines that the ratio of I=100 is the optimal condition for constructing ionic liquid reverse micelle films.

[0039] Example 2. Construction of Pre-fabricated Reverse Micellar Membrane and Extraction Apparatus

[0040] Pre-fabricated reverse micelle membrane: First, take 0.01684 mL of [C 10 [Bmim][NTf2] was mixed with 1.5 mL of [Bmim][NTf2] ionic liquid (I = 100) and sonicated at 40 kHz and 120 W for 15 min to obtain a clear mixed ionic liquid (IL-RM). A polycarbonate (PC) membrane (5 μm pore size) was then placed on a glass slide, and the clear mixed ionic liquid solution (IL-RM) was added dropwise until the membrane was fully impregnated. After that, excess ionic liquid on the membrane was blown off with nitrogen gas and placed in a plastic dish for later use.

[0041] The electro-membrane extraction device used is as follows: Figure 2 As shown, the left side is the extraction chamber, with its upper opening connected to the positive terminal of the power supply via foamed titanium to form the anode; the right side is the extraction chamber, with its upper opening connected to the negative terminal of the power supply via platinum wire to form the cathode. Both chambers have the same volume, and the middle section can be fixed with a steel clamp. The side openings of the chambers are connected with rubber rings. The supply phase is 5 mL of 0.05 mol·L⁻¹ solution. -1 60 μmol·L phosphate buffer solution was prepared -1 The tetracycline (TC) sample was adjusted to an initial pH of 2.5 with hydrochloric acid; the receiving phase was 5 mL of 0.05 mol·L⁻¹ hydrochloric acid with an initial pH of 8. -1 A phosphate buffer solution is used to sandwich a pre-formed reverse micelle membrane between the rubber rings at the side openings of the supply and receiving chambers. The two components are then secured with steel clamps to form a single extraction chamber. The extraction chamber is then suspended and connected to an iron frame via hooks, with an ultrasonic machine placed underneath. The height of the iron frame is adjusted so that the ultrasonic liquid level exceeds the extraction chamber by 5 mm, thus providing the necessary conditions for reverse micelle formation through ultrasonic energy transfer.

[0042] Example 3. Comparison of Types of Supported Liquid Films (SLMs)

[0043] The reverse micelle membrane was prepared according to the pre-prepared reverse micelle membrane step in Example 2. [Hmim][NTf2] and [Omim][NTf2] were used to replace [Bmim][NTf2] to explore the effects of these three bis(trifluoromethanesulfonyl)imide ionic liquids with different alkyl chains.

[0044] The extraction conditions for tetracycline (TC) using an ionic liquid reverse micelle electrophoresis membrane were: voltage 30 V, donor phase pH 2.5, acceptor phase pH 8.0, extraction times of 3 and 6 min, without salt addition. The UV absorbance of tetracycline (TC) at 387 nm was measured. The results of tetracycline extraction using different ionic liquid membranes are shown below. Figure 3 As shown. Due to the solubility of ILs in water and their migration in an electric field, the solubility and conductivity of the three ionic liquids decrease with increasing alkyl chain length. Therefore, theoretically, [Omim][NTf2] will provide the most stable extraction effect. However, as... Figure 3 As shown, [Bmim][NTf2] exhibits the best extraction efficiency at both 3 and 6 min, which can be attributed to the "like dissolves like" principle. It possesses polarity similar to tetracycline, enabling better transmembrane transport. Therefore, [Bmim][NTf2] will be used as the SLM for reverse micelle extraction in the future.

[0045] Example 4. Pore size selection of PC membrane carrier

[0046] Reverse micelle membranes were prepared according to the pre-prepared reverse micelle membrane steps in Example 2, wherein PC membranes with pore sizes of 0.2 μm, 0.8 μm, and 5 μm were used, respectively. The effect of different PC membrane pore sizes on tetracycline extraction was investigated.

[0047] The reverse micelle electrophoresis conditions were: voltage 30V, donor phase pH 2.5, acceptor phase pH 8.0, no salt added, and extraction time 18 min. The results are as follows: Figure 4As shown, although there are differences between 0.2-μm and 0.8-μm polycarbonate (PC) membranes, both have much smaller pore sizes than the 5-μm PC membrane, failing to provide sufficient reverse micelle transport phase. The extraction efficiencies of both are similar, and their extraction effect on tetracycline is lower than that of the larger-channel 5-μm PC membrane. This example also explored a 10-μm PC membrane, but due to its excessively large pore size, the SLM could not withstand the solution pressure and was directly destroyed, leading to direct miscibility of the solutions in the donor and acceptor chambers. Therefore, the 10-μm PC membrane cannot be used as a support for ionic liquids. In conclusion, a 5-μm pore size PC membrane is the optimal choice for the support. Furthermore, the absorbance of the extracted tetracycline became almost constant after 15 minutes of extraction; therefore, the extraction time for subsequent experiments was determined to be 15 minutes.

[0048] Example 5. Selection of Applied Voltage

[0049] The reverse micelle membrane was prepared according to the pre-prepared reverse micelle membrane steps in Example 2; the extraction conditions were: sample donor phase pH 2.5, acceptor phase pH 8.0, no salt added, voltages of 5V, 10V, 15V, 20V, 25V, 30V, 35V, 40V and 50V respectively, and extraction time of 15min.

[0050] The results are as follows Figure 5 As shown, this indicates that a lower voltage can provide higher extraction efficiency. This phenomenon can be explained as follows: the entire IL-EME setup is a circuit where the reverse micelle liquid film acts as a resistor, and the charged analyte migrates following the current. This current must be kept at a low value to suppress electrolysis. Given the relatively low resistance of the IL used as the SLM, the potential applied at low voltages should be higher. Therefore, voltages up to 50V can provide an effective electric field strength for the migration of ionized analytes. Figure 5 It can also be seen that the extraction efficiency gradually increases with increasing voltage. Considering the influence of relative measurement deviation, the extraction efficiency remains almost constant within the range of 25 to 30V. However, when the voltage is further increased, the SLM becomes unstable, the current increases, leading to electrolysis and a decrease in extraction efficiency. Under constant voltage, since the total resistance of the EME system is mainly given by the SLM, according to Ohm's law, the current in the EME system varies with the non-uniform extraction unit, i.e., the RMIL membrane. Any small difference in the uniformity of the SLM will cause a measurable change in the resulting current. The total charge through the system (Q in coulombs) will be different, and according to Faraday's law, the total amount of charged substance transferred through the SLM will also be different. Therefore, 30V will be selected as the operating voltage for the EME, and careful operation will be performed to reduce errors and ensure the stability of the liquid film.

[0051] Example 6. Optimization of pH conditions for donor and acceptor solutions

[0052] 1. Reverse micelle membranes were prepared according to the pre-prepared reverse micelle membrane steps in Example 2. The extraction conditions were a voltage of 30V. The sample donor phase used phosphate buffer solutions with pH values ​​of 1.5, 2.0, 2.5, and 3.0, and citric acid buffer solutions with pH values ​​of 3.5, 4.0, 4.5, and 5.0, respectively, with pH adjusted using hydrochloric acid. The acceptor phase used a phosphate buffer solution at pH 7.5. The concentration of all buffer solutions was 0.05 mol·L⁻¹. -1 The extraction efficiency of TC under cationic and zwitterionic conditions was investigated separately.

[0053] The results are as follows Figure 6 As shown, when the pH of the phosphate buffer solution in the donor phase is between 1.5 and 3, total chlorine (TC) is almost completely ionized. Within this range, the donor phase solution at pH 2.5 provides the most stable conditions for tetracycline extraction. Although the figure shows that the pH 2.0 donor phase significantly improves the extraction efficiency after 12 minutes, flocculation and turbidity have already appeared in the acceptor phase solution at this point. This suggests that under low pH and prolonged high current, the slurry lamp (SLM) has been severely damaged, with a large amount of ionic liquid dissolving to the acceptor side, affecting the absorbance measurement. This also explains why the extraction effect is poor when the donor phase pH is less than 2.5 and the degree of TC ionization is greater. When the donor phase solvent was changed to citric acid buffer solution to seek a wider pH range, the extraction efficiency of tetracycline gradually decreased with increasing donor phase pH, and the best extraction effect was provided at pH 3.5. This indicates that a strong acid environment can ensure that the analyte exists in an ionized form, thus allowing charged analytes to migrate effectively in the electric field. As the pH increases, some charged analytes will be converted into molecular form and back-extracted into the SLM. Generally, a strongly alkaline environment can provide better extraction results. However, under alkaline conditions, TC easily forms isomers with lactone structures, and the process is irreversible, which contradicts the original intention of this application to extract and recover TC. Therefore, the pH of the supply phase solution was maintained at 2.5.

[0054] 2. A reverse micelle membrane was prepared according to the pre-prepared reverse micelle membrane steps in Example 2; the extraction conditions were: voltage 30V, and sample donor phase was 60 μmol·L⁻¹ phosphate buffer solution at pH 2.5. -1 Tetracycline (TC) solution; the acceptor phase was prepared using phosphate buffer solutions with pH values ​​of 6.5, 7.0, 7.5, and 8.0, respectively, all with a concentration of 0.05 mol·L⁻¹. -1 The effect of pH value of the neutral acceptor phase on the extraction results was investigated.

[0055] The results are as follows Figure 7As shown, the results indicate that the extraction efficiency at pH 6.5 and 7.0 is superior to that at pH 7.5 and 8.0 in the first 12 min. However, with increasing extraction time, electrolysis occurred in the SLM, rendering the results unreliable. With further increases in extraction time, the amount of TC received by the pH 7.5 and pH 8.0 receiving solutions continued to increase, finally reaching peak values ​​at 24 and 36 min, respectively, at which point the TC absorbance in the pH 7.5 receiving phase was 0.575. Extraction solutions of 10, 20, 40, 50, and 60 μmol·L⁻¹ were prepared. -1 The linear equation for TC standard solution, measured using a UV-Vis spectrophotometer, is: A = 0.11766C + 0.02581(R). 2 =0.9986), C is the concentration of TC (10 μmol·L⁻¹). -1 Let A be the absorbance of TC at a wavelength of 387 nm. Substituting the TC absorbance of 0.575 in the receiving phase into the linear equation A = 0.11766C + 0.02581, we obtain the TC concentration in the receiving phase as 46.676 μmol·L⁻¹. -1 The extraction rate Er was calculated using the formula Er = (Ca / Cd)% , where Cd represents the concentration of the donor phase sample and Ca represents the concentration of the recipient phase sample. The calculated TC extraction rate was 77.79%. Considering all factors, a recipient phase condition of pH 7.5 was selected, as the extraction efficiency steadily increases over time.

[0056] Example 7. Effect of Salt Concentration

[0057] In traditional liquid-liquid extraction (SLEM), the addition of salts typically increases the ionic strength of the aqueous solution and reduces the solubility of analytes in the water sample, thereby increasing their partitioning in the organic phase. However, in ionic liquid reverse micelle extraction (EME), the addition of salts may have different effects. This experiment used lithium bis(trifluoromethanesulfonyl)imide (Li[NTf2]) as the target, which can inhibit the dissolution of ILs in SLMs. Furthermore, the effects of different Li[NTf2] addition amounts (0, 0.05, 0.10, 0.15, 0.20, and 0.25 mol·L⁻¹) were investigated. -1 The effect of the phase extraction rate.

[0058] Reverse micelle membranes were prepared according to the pre-prepared reverse micelle membrane steps in Example 2. Extraction conditions were: voltage 30V, donor phase pH 2.5, acceptor phase pH 7.5. Final concentrations of 0, 0.05, 0.10, 0.15, 0.20, and 0.25 mol·L⁻¹ were added to the acceptor phase, respectively. -1 Li[NTf2] (Shanghai Aladdin Biochemical Technology Co., Ltd.);

[0059] The results are as follows Figure 8 As shown, the extraction efficiency of TC gradually increases with the increase of Li[NTf2] concentration, reaching a peak at 0.1 mol·L⁻¹. -1High extraction efficiency was achieved at a salt concentration of 0.15 mol·L⁻¹. -1 Subsequently, due to the excessively high salt concentration, the system current increased, and the SLM was electrolytically destroyed. Therefore, 0.1 mol·L⁻¹ was chosen. -1 The optimal salt concentration was determined as the extraction condition.

[0060] Example 8. Comparison of [Bmim][NTf2] and Tween-80 neutral reverse micelle SLM

[0061] A reverse micelle membrane was prepared according to the pre-prepared reverse micelle membrane steps in Example 2, except that the [Bmim][NTf2] ionic liquid was replaced with a nonionic surfactant (Tween-80, isopropanol, and n-hexane were mixed in a mass ratio of 1:1:5) to prepare a Tween-80 neutral reverse micelle SLM. The extraction effect of the SLM prepared with ionic and nonionic surfactants was investigated.

[0062] Extraction conditions were: voltage 30V, sample donor phase pH 2.5 (with 0.1 mol·L⁻¹). -1 The Li[NTf2]) was used as the acceptor phase at pH 7.5, and the results were as follows: Figure 9 As shown, the extraction rates of both Tween-80 neutral reverse micelle SLM and IL-SLM increased rapidly over time in the first 10 minutes, but remained almost constant thereafter, indicating that their extraction rates were similar under electric drive in the short term. However, the extraction effect of IL-SLM was far superior to that of neutral reverse micelle SLM in all time periods. This is because, unlike the simple diffusion transport in neutral nonionic reverse micelles, in the electroextraction process of [Bmim][NTf2], the applied electric field not only applies a driving force to the TC in the donor phase, but also provides electrocapillary action to the IL reverse micelle particles in the SLM, promoting the transfer of TC particles from the donor phase to the acceptor phase.

[0063] Example 9. Recovery, determination and separation of environmental water samples

[0064] The environmental water samples were taken from Langyue Lake, located at Yangzhou University in Jiangsu Province.

[0065] 100 mL of lake water was collected and filtered through 0.45 μm and 0.22 μm filters to remove suspended impurities, and stored in a refrigerator at 4 °C. A [Bmim][NTf2] reverse micelle electrophoresis membrane with a PC membrane pore size of 5 μm was prepared according to the conditions in Example 2. The extraction conditions were: operating voltage 30 V, donor phase pH 2.5, acceptor phase pH 7.5, and Li[NTf2] salt concentration 0.1 mol·L⁻¹. -1Extraction time was 15 min. TC (tetracycline) and OTC (oxytetracycline) were separated and determined using LC 1220 liquid chromatography (Agilent Technologies). Conditions were: C18 column, column temperature 30℃, mobile phase A: 0.1% formic acid, mobile phase B: methanol (70:30 v / v), detection at 387 nm for TC and OTC, flow rate 1 mL / min. -1 The injection volume was 10 μL. Before sample analysis, TC and OTC were plotted using a chromatograph at 1, 2, 5, 10, 20, and 25 mg·L⁻¹. -1 The standard curve at a given concentration yielded the linear equation for the absorbance of TC concentration: Y = 19.4946X - 1.5382(R). 2 =0.998), and the linear equation for absorbance of OTC concentration is Y = 16.0551X + 3.5413(R). 2 =0.990), the detection limit is 0.02 mg·L⁻¹ -1 Then, 5 mL of lake water was subjected to electroporation to obtain the initial sample. HPLC analysis of the initial sample showed no TC or OTC levels due to sample selection factors. Subsequently, 2, 4, 8, and 12 mg / L of the solution were added to the initial sample, respectively. -1 The recoveries of the TC-OTC mixed standard were determined. Results are as follows: Figure 10 As shown in Table 1.

[0066] Table 1. Spiked Recovery Determination of Tetracycline and Oxytetracycline Separated by High Performance Liquid Chromatography

[0067]

[0068] It can be seen that by using [Bmim][NTf2]RM-EME coupled with HPLC, good recovery and separation of tetracycline antibiotics can be achieved, providing options for further analysis and quantification.

Claims

1. The application of an ionic liquid reverse micelle membrane in the extraction of tetracycline antibiotics, characterized in that, The preparation of the ionic liquid reverse micelle membrane includes the following steps: mixing 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt and 1-decyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt, and fully impregnating the PC membrane with the clear liquid after the reaction to obtain the ionic liquid reverse micelle membrane; the application is to extract tetracycline antibiotics through electroporation.

2. A method for extracting tetracycline antibiotics, characterized in that, Includes the following steps: (1) Mix 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt and 1-decyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt, sonicate, and take the lower clear liquid; (2) The PC membrane is fully impregnated with the lower clear liquid prepared in step (1), and the excess liquid on the membrane is blown off with nitrogen gas to obtain the ionic liquid reverse micelle membrane for extracting tetracycline antibiotics. (3) Fix the ionic liquid reverse micelle membrane in the middle of the donor and acceptor chambers, add the donor phase solution and sample solution to the donor chamber and connect it to the positive terminal of the power supply; add the acceptor phase solution to the acceptor chamber and connect it to the negative terminal of the power supply to form an electro-membrane extraction device. Place it in an ultrasonic instrument for extraction to obtain a solution containing tetracycline antibiotics; the donor phase solution and acceptor phase solution are 0.05 mol·L −1 The solution contains a phosphate buffer solution, and salt is also added to the supply phase solution; the extraction conditions are 20~40V.

3. The method according to claim 2, characterized in that, The mass ratio of 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt and 1-decyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt in step (1) is 50~150:

1.

4. The method according to claim 2, characterized in that, The pore size of the PC membrane mentioned in step (2) is 0.2~5 μm.

5. The method according to claim 2, characterized in that, The pH of the supply solution in step (3) is 2 to 2.5, and the pH of the receiving solution is 7.5 to 8.

6. The method according to claim 2, characterized in that, The salt mentioned in step (3) is 0.05 ~ 0.15 mol·L. −1 Li[NTf2].

7. The method according to claim 2, characterized in that, Step (3) is followed by a step of detecting the concentration of tetracycline antibiotics using high-performance liquid chromatography: (1) Add phosphate buffer solution to standard samples of tetracycline antibiotics of different concentrations, and plot the standard equations by the peak area and absorbance of different concentrations. (2) Detect the peak area of ​​the extracted liquid and substitute it into the standard equation to obtain the concentration of tetracycline antibiotics.

8. The method according to claim 2, characterized in that, The tetracycline antibiotics include tetracycline or oxytetracycline.

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