A method for separating and extracting microplastics in traditional Chinese medicinal materials

Abstract

This invention relates to the field of microplastic separation technology and discloses a method for separating and extracting microplastics from traditional Chinese medicinal materials. The method includes the following steps: preliminary pretreatment of the medicinal materials to be treated to obtain pretreated medicinal materials; further pretreatment of the pretreated medicinal materials to obtain ready-to-use medicinal materials; density separation of the ready-to-use medicinal materials using a multi-stage density separation system to collect microplastics and purified medicinal materials; further purification of the microplastics to obtain purified microplastics; and identification and quantification of the purified microplastics. This invention, through the combined use of three-stage density separation and digestion methods such as enzymes, oxidation, and microwaves, almost completely removes interference from biological organic matter, greatly reducing the false positive rate. Furthermore, the separation technology used in this invention covers all common microplastic types from low to high density, with high and stable recovery rates.

Description

Technical Field

[0001] This invention relates to the field of microplastic separation technology, and more specifically, to a method for separating and extracting microplastics from traditional Chinese medicinal materials. Background Technology

[0002] Microplastics (MPs) are plastic particles with a diameter of less than 5 mm, widely present in environmental media such as soil, water, and atmosphere. Traditional Chinese medicinal materials are susceptible to microplastic contamination during planting, harvesting, processing, transportation, and storage. Examples include microplastics carried by irrigation water, plastic particles generated from wear and tear on processing equipment, and microplastics shed from packaging materials. If microplastics enter the human body through traditional Chinese medicinal materials, they may pose a potential threat to human health. Therefore, establishing efficient methods for the separation and extraction of microplastics from traditional Chinese medicinal materials is of great significance for ensuring the quality and safety of these materials.

[0003] Currently, methods for separating and extracting microplastics mainly include density separation, oil separation, electromagnetic separation, and manual separation. Among these, density separation is widely used for separating microplastics from environmental samples due to its simplicity and low cost. However, existing density separation methods are mostly designed for matrices such as soil and water. For matrices like traditional Chinese medicine materials, which have complex compositions (containing large amounts of cellulose, lignin, polysaccharides, and other organic matter), low microplastic content, and potential interaction with active ingredients, the following shortcomings exist: Low separation efficiency: The density of a conventional saturated sodium chloride (NaCl) solution is only 1.20 g / cm³. 3 It has a poor separation effect on microplastics with high density (such as PVC and PET), while Chinese medicinal materials may contain multiple microplastics with different densities at the same time; Organic matter interference is severe: Organic matter in Chinese medicinal materials easily adheres to microplastic particles, making it difficult to effectively separate microplastics and interfering with subsequent identification and quantification. Significant sample loss: Traditional methods are prone to loss during stirring and filtration steps due to microplastic particles adsorbing onto the container walls or filter membranes, especially for low-content Chinese medicinal materials, where the error is relatively large. Poor adaptability to the characteristics of Chinese medicinal materials: Existing methods do not take into account the stability of the effective components of Chinese medicinal materials, and some separation reagents (such as strong acids and strong bases) may destroy the active components in Chinese medicinal materials, affecting subsequent quality testing.

[0004] Therefore, providing a microplastic separation and extraction method that takes into account the characteristics of Chinese medicinal material matrices and balances separation efficiency, anti-interference ability, and sample protection is of great practical significance. Summary of the Invention

[0005] In view of this, the present invention proposes a method for separating and extracting microplastics from traditional Chinese medicinal materials, aiming to solve at least one of the problems in the background art.

[0006] This invention proposes a method for separating and extracting microplastics from traditional Chinese medicinal materials, comprising the following steps: After sorting, washing and drying, the powdered Chinese medicinal materials to be processed are pulverized to 0-100 mesh to obtain pre-treated Chinese medicinal materials. The pretreated Chinese medicinal materials are subjected to deep pretreatment to obtain the ready-to-use Chinese medicinal materials; The prepared Chinese medicinal materials were density separated using a multi-stage density separation system including a first stage, a second stage, and a third stage, and microplastics and purified Chinese medicinal materials were collected. The microplastics are then subjected to deep purification to obtain purified microplastics. The purified microplastics are identified and quantified. The deep pretreatment involves placing the pretreated Chinese medicinal materials in a 0.1 mol / L phosphate buffer solution at a solid-liquid ratio of 1:15-25, followed by the addition of a compound enzyme preparation for enzymatic hydrolysis.

[0007] Preferably, the compound enzyme preparation includes cellulase, pectinase and xylanase, with a mass ratio of 2~2.5:1~1.5:1~1.5; the enzymatic hydrolysis parameters are: temperature of 50~55℃, time of 6~12 hours, and oscillation frequency of 200 rpm.

[0008] Preferably, after the enzymatic hydrolysis is completed, the process further includes: high-temperature enzyme inactivation, vacuum filtration and washing. The high-temperature enzyme inactivation temperature is 95°C and the time is 15-18 minutes. The vacuum filtration uses a 0.45μm filter membrane and a vacuum degree of 0.05-0.08MPa. The washing is performed using 0.1mol / L phosphate buffer.

[0009] Preferably, the multi-stage density separation system comprises a three-stage density separation system with a first stage, a second stage, and a third stage; the first stage uses a density of 1.15~1.20 g / cm³. 3 The ultralight microplastics were separated by perfusion with a saturated NaCl solution; the second stage used a solution with a density of 1.20~1.25 g / cm³. 3 The process involves perfusion with a saturated NaCl solution to separate medium-density microplastics; the third stage uses a solution with a density of 1.30~1.50 g / cm³. 3 High-density microplastics were separated using a saturated Na2WO4 solution.

[0010] Preferably, the multi-stage density separation system is intelligently controlled, wherein the intelligent control is achieved by controlling the flow rate through a precision peristaltic pump and by using an online infrared sensor to detect the particle passage and the interfaces between each phase in real time.

[0011] Preferably, the deep purification specifically involves: pre-washing, Fenton reagent oxidation, intermediate washing, microwave digestion, final washing, and drying of the filtration membranes of each density level obtained by the multi-stage density separation system to obtain purified microplastics. The pre-washing process involves ultrasonic washing with deionized water at a power of 50-80W for 5-10 minutes, repeated 2-3 times.

[0012] Preferably, the Fenton reagent oxidation is specifically carried out as follows: Fenton reagent is prepared with FeSO4 concentration of 0.08 mol / L and H2O2 concentration of 4%, the pH value is adjusted to 2.8-3.2 with dilute sulfuric acid, the pre-washed filter membrane is immersed in Fenton reagent, sealed and placed in a constant temperature water bath shaker for reaction, the shaking frequency is 60 rpm and the temperature is 25°C, and the COD value in the reaction system is sampled and detected every 6 hours until the COD value tends to stabilize.

[0013] Preferably, the microwave digestion specifically involves: using a programmed temperature ramp mode, with an initial temperature of 25°C, increasing the temperature to 100°C at a rate of 5°C / min, maintaining the temperature for 15-20 minutes, controlling the pressure at 0.5-0.6 MPa, and continuously stirring during the digestion process.

[0014] Preferably, the identification and quantification specifically involves: scanning the filter membrane obtained from deep purification using an intelligent microscope, automatically identifying and counting the microplastics in the filter membrane using image recognition software, recording the coordinates of the microplastics, then using Raman spectroscopy to identify the microplastics at the coordinates, and finally obtaining an analysis report including the number, type, size distribution, and mass concentration of the microplastics.

[0015] This invention also provides the application of the microplastic separation and extraction method described in the above technical solution in the purification and refining of traditional Chinese medicine.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention uses three-stage density separation combined with multi-stage digestion such as enzyme, oxidation, and microwave to almost completely remove the interference of biological organic matter, greatly reducing the false positive rate. Moreover, the separation technology used in the present invention covers all common microplastic types from low density to high density, with high and stable recovery rate, and Raman identification ensures accurate results. (2) The present invention adopts an automated separation, digestion and identification process, which significantly reduces manual operation and time costs and can achieve high-throughput sample processing; through sensor feedback and machine learning, human error is reduced, the method has good reproducibility and is easy to standardize and promote; the present invention can simultaneously obtain physical and chemical multidimensional information of microplastics, providing strong data support for pollution source analysis and risk assessment. Attached Figure Description

[0017] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 This is a schematic diagram of the process for separating and extracting microplastics from Chinese medicinal materials according to the present invention. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0019] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] This invention proposes a method for separating and extracting microplastics from traditional Chinese medicinal materials, comprising the following steps: After sorting, washing and drying, the powdered Chinese medicinal materials to be processed are pulverized to 0-100 mesh to obtain pre-treated Chinese medicinal materials. The pretreated Chinese medicinal materials are subjected to deep pretreatment to obtain the ready-to-use Chinese medicinal materials; The prepared Chinese medicinal materials were density separated using a multi-stage density separation system including a first stage, a second stage, and a third stage, and microplastics and purified Chinese medicinal materials were collected. The microplastics are then subjected to deep purification to obtain purified microplastics. The purified microplastics are identified and quantified. The deep pretreatment involves placing the pretreated Chinese medicinal materials in a 0.1 mol / L phosphate buffer solution at a solid-liquid ratio of 1:15-25, followed by the addition of a compound enzyme preparation for enzymatic hydrolysis.

[0024] After sorting, washing and drying, the powdered Chinese medicinal materials to be processed are pulverized to 0-100 mesh to obtain pre-treated Chinese medicinal materials. In this invention, the preliminary pretreatment is preferably as follows: first, remove visible impurities from the medicinal materials to be treated, such as withered leaves and mud; then, wash the surface of the medicinal materials with deionized water; after washing, dry them; and cut the dried medicinal materials into small pieces to facilitate subsequent pulverization. Then, pulverize the medicinal materials using an ultra-micro airflow pulverizer or other devices well-known to those skilled in the art that can achieve the same pulverization purpose, to a particle size of 0-100 mesh.

[0025] This invention breaks down the original blocky and fibrous structure of Chinese medicinal materials through preliminary pretreatment, pulverizing them into a specific mesh size range, thus avoiding uneven reaction during subsequent enzymatic hydrolysis and disordered stratification during density separation due to differences in particle size.

[0026] The pretreated Chinese medicinal materials are subjected to further pretreatment to obtain ready-to-use Chinese medicinal materials; In this invention, the deep pretreatment is preferably performed by placing the pretreated Chinese medicinal materials in a 0.1 mol / L phosphate buffer solution at a solid-liquid ratio of 1:15-25, followed by enzymatic hydrolysis with a compound enzyme preparation to obtain the prepared Chinese medicinal materials. The compound enzyme preparation preferably includes cellulase, pectinase, and xylanase, with a mass ratio preferably of 2~2.5:1~1.5:1~1.5, more preferably 2:1:1. The enzymatic hydrolysis parameters are preferably: a temperature of 50~55℃, a time of 6-12 hours, and an oscillation frequency of 200 rpm; more preferably, a temperature of 50℃, a time of 10 hours, and an oscillation frequency of 200 rpm.

[0027] More preferably, the deep pretreatment specifically involves: placing the pretreated Chinese medicinal materials in a 0.1 mol / L phosphate buffer solution. The phosphate buffer solution provides a neutral and stable pH environment for the enzymatic hydrolysis reaction (avoiding pH fluctuations from affecting enzyme activity) and also serves as a mixing medium for the Chinese medicinal material particles and the enzyme preparation, ensuring uniform reaction. Then, a compound enzyme preparation is prepared according to the component ratio of the compound enzyme preparation. The Chinese medicinal material particles (0-100 mesh) obtained from the preliminary pretreatment are uniformly added to the enzymatic hydrolysis solution, and then enzymatic hydrolysis is carried out according to the preset enzymatic hydrolysis reaction parameters.

[0028] The organic matter in traditional Chinese medicinal materials, such as cellulose and pectin, has strong adsorption properties and easily forms aggregates of organic matter and microplastic particles. In traditional density separation, the density of such aggregates may be between that of the target microplastic and the matrix, causing the microplastics to fail to float or sink normally according to their density, resulting in a separation recovery rate of less than 60%. This invention utilizes the specific degradation of a compound enzyme preparation, decomposing more than 90% of the organic matter into small molecules soluble in the buffer solution (such as glucose and galacturonic acid), which are then removed with the filtrate. The microplastic particles are no longer encapsulated and can accurately stratify according to their own density in subsequent multi-stage density separation, thus significantly improving the overall recovery rate of microplastics.

[0029] Although the pre-treated medicinal herb granules have achieved uniform particle size, the organic matter adhering to their surfaces can cause them to stick together, potentially clogging equipment pipelines (such as peristaltic pumps and sensor probes) after being fed into the density separation system. Deep pretreatment, on the other hand, results in clean, unadhesive granule surfaces, forming well-dispersed medicinal herbs that can pass through each stage of the multi-stage density separation system at a uniform speed. This reduces the probability of pipeline blockage, lowers equipment maintenance costs, and ensures stable separation parameters such as flow rate and temperature, improving the repeatability of different batches of samples.

[0030] Specifically, the cellulase in the complex enzyme preparation used in the deep pretreatment of this invention degrades the main structural organic matter. The main component of the cell wall of Chinese medicinal materials is cellulose. Cellulase decomposes long-chain cellulose macromolecules into glucose small molecules that are soluble in phosphate buffer by hydrolyzing the β-1,4-glycosidic bonds in cellulose molecules, thereby destroying the structural integrity of Chinese medicinal material particles and exposing the microplastic particles wrapped in the cellulose network. This prevents the microplastics from being unable to float or sink freely during density separation due to being entangled in cellulose.

[0031] Pectinase degrades adhesive organic matter. Pectin, a common adhesive between cells in traditional Chinese medicine, is widely found in fruit peels, pulps, and soft tissues. It is highly adhesive and easily adheres to the surface of microplastic particles. Pectinase catalyzes the breakage of α-1,4-glycosidic bonds in pectin molecules, decomposing pectin into smaller molecules such as galacturonic acid, thus reducing its stickiness. Removing the sticky pectin layer from the surface of microplastic particles prevents them from agglomerating with other medicinal particles due to pectin adhesion, and also avoids pectin clogging equipment pipelines in subsequent filtration and separation steps.

[0032] Xylanase degrades hemicellulose organic matter. The main component of hemicellulose in traditional Chinese medicine is xylan, which is found in highly lignified medicinal materials such as bark and old roots. Xylanase specifically hydrolyzes the glycosidic bonds in xylan molecules, breaking them down into soluble small molecules such as xylose and arabinose. Supplementing the hemicellulose that cellulase cannot completely decompose further reduces the amount of residual organic matter in the medicinal material matrix, ensuring that microplastic particles are completely detached from the encapsulation and adhesion of organic matter.

[0033] Single enzyme preparations can only degrade a certain type of organic matter (e.g., cellulase alone cannot remove pectin), while compound enzyme preparations achieve full coverage degradation of the main organic matter (cellulose, pectin, and hemicellulose) in Chinese medicinal materials through the synergistic effect of three enzymes.

[0034] After the enzymatic hydrolysis of the present invention is completed, the process further includes high-temperature enzyme inactivation, vacuum filtration and washing. The high-temperature enzyme inactivation temperature is 95°C and the time is 15-18 minutes. The vacuum filtration uses a 0.45μm filter membrane and a vacuum degree of 0.05-0.08MPa. The washing is performed using 0.1mol / L phosphate buffer.

[0035] Then, a multi-stage density separation system including the first, second and third stages was used to separate the density of the prepared Chinese medicinal materials, and microplastics and purified Chinese medicinal materials were collected. In this invention, the multi-stage density separation system is preferably a three-stage density separation system comprising a first stage, a second stage, and a third stage; the first stage uses a density of 1.15~1.20 g / cm³. 3 The ultralight microplastics were separated by perfusion with a saturated NaCl solution; the second stage used a solution with a density of 1.20~1.25 g / cm³. 3 The process involves perfusion with a saturated NaCl solution to separate medium-density microplastics; the third stage uses a solution with a density of 1.30~1.50 g / cm³. 3 High-density microplastics were separated using a saturated Na2WO4 solution.

[0036] In this invention, the multi-stage density separation system is intelligently controlled. The intelligent control is achieved by controlling the flow rate through a precision peristaltic pump and by using an online infrared sensor to detect the particle passage and the interfaces between each phase in real time.

[0037] Specifically, the multi-stage density separation system of the present invention is as follows: The first stage involves the separation of ultralight microplastics, using a precision peristaltic pump to separate microplastics with a density of 1.15~1.20 g / cm³. 3 A saturated NaCl solution is poured into the first-stage separation container at a stable flow rate (the flow rate can be adjusted according to the sample volume and actual needs, controlled by an intelligent system). The prepared Chinese medicinal materials are added to the separation container at a uniform speed and allowed to stand for a preset time under gentle stirring (avoiding vigorous stirring that could cause microplastics to adhere to the container walls). The preset time can be adjusted according to actual needs and conditions, usually 30-60 minutes, to ensure that the particles are fully separated. An online infrared sensor scans the particles passing through the container in real time. When the floating layer (extremely light microplastics) is detected to be stable, the floating liquid is collected and filtered to obtain a crude extract of extremely light microplastics. The sediment at the bottom of the container (containing medium and high density microplastics and Chinese medicinal material matrix particles) is collected and transferred to the second-stage separation system.

[0038] The second stage of medium-density microplastic separation then proceeds, using a precision peristaltic pump to separate microplastics with a density of 1.20~1.25 g / cm³. 3 Saturated NaCl solution was poured into the second-stage separation vessel, maintaining the same flow rate as the first stage to ensure stable separation conditions. The sediment from the first stage was added, and the process of settling, stratification, and monitoring was repeated. After a stable floating layer of medium-density microplastics was formed, the floating liquid was collected and filtered to obtain a crude extract of medium-density microplastics. The sediment at the bottom (containing high-density microplastics and residual Chinese medicinal material matrix particles) was collected and transferred to the third-stage separation system.

[0039] Then, a third stage of high-density microplastic separation is performed, using a precision peristaltic pump to separate microplastics with a density of 1.30~1.50 g / cm³. 3 Saturated Na2WO4 solution was poured into the third-stage separation container, maintaining the flow rate consistent with the first and second stages to ensure stable separation conditions. The sediment from the second stage was added and allowed to stand for a longer time (usually 60-90 minutes, as high-density particles stratify more slowly). The process of standing-stratification-monitoring was repeated, with an online infrared sensor monitoring the floating layer (high-density microplastics). Once a stable floating layer of high-density microplastics had formed, the floating liquid was collected and filtered to obtain a crude extract of high-density microplastics. The sediment at the bottom (residual Chinese herbal medicine matrix particles) was collected. The sediment at the bottom of the container is Chinese herbal medicine matrix impurities and should be discarded directly to avoid interfering with subsequent purification steps.

[0040] The types of microplastics that Chinese medicinal herbs come into contact with during planting, processing, and transportation vary greatly in density (e.g., low-density polyethylene PE, with a density of 0.91-0.96 g / cm³). 3 Medium-density polypropylene (PP), density 0.90-0.91 g / cm³. 3 High-density polyvinyl chloride (PVC), density 1.38-1.43 g / cm³. 3 Polyethylene terephthalate (PET), density 1.38 g / cm³ 3 Traditional single-density separation (such as the density of a conventional saturated NaCl solution) can only separate low-density microplastics and is almost ineffective for medium- and high-density microplastics, resulting in significant errors in the separation results.

[0041] Through a three-stage separation process, all types of microplastics—from extremely light to medium to high density—that may be present in Chinese medicinal materials can be effectively separated, overcoming the limitation of traditional methods that can only separate low-density microplastics. This significantly improves the microplastic separation coverage. Each stage of separation targets only microplastics within a specific density range, ensuring a single objective and avoiding cross-interference between microplastics of different densities or between microplastics and the matrix. For example, the first stage only collects the floating layer of extremely light microplastics, while the sediment directly enters the second stage, eliminating concerns about medium-density microplastics mixing with extremely light microplastics. The third stage separates only high-density microplastics, completely eliminating low-density matrix particles (such as lignin fragments, with a density of approximately 1.05 g / cm³). 3 To mitigate interference from other components, this invention combines intelligent control (precision peristaltic pump speed control and online infrared sensor monitoring of particle-phase interface) to identify the stratification state of each density fraction in real time, accurately collect target microplastics, avoid miscollection of matrix or missed collection of microplastics, and significantly improve the separation purity of microplastics in each density fraction.

[0042] The microplastics are then subjected to deep purification to obtain purified microplastics. In this invention, the deep purification is preferably performed by sequentially pre-washing, Fenton reagent oxidation, intermediate washing, microwave digestion, final washing and drying of the filtration membranes of each density level obtained by the multi-stage density separation system to obtain purified microplastics. The pre-washing process involves ultrasonic washing with deionized water at a power of 50-80W for 5-10 minutes, repeated 2-3 times.

[0043] The Fenton reagent oxidation process is as follows: Fenton reagent is prepared by mixing 0.08 mol / L FeSO4 and 4% H2O2. The pH value is adjusted to 2.8-3.2 with dilute sulfuric acid. The pre-washed filter membrane is immersed in Fenton reagent, sealed, and placed in a constant temperature water bath shaker for reaction. The shaking frequency is 60 rpm and the temperature is 25°C. During the reaction, the COD value in the reaction system is sampled and detected every 6 hours until the COD value tends to stabilize.

[0044] The microwave digestion described in this invention specifically involves: using a programmed temperature rise mode, with an initial temperature of 25°C, increasing the temperature to 100°C at a rate of 5°C / min, maintaining the temperature for 15-20 minutes, controlling the pressure at 0.5-0.6 MPa, and continuously stirring during the digestion process.

[0045] The deep purification described in this invention specifically involves: removing filter membranes (which have already trapped microplastics and residual impurities) from each density fraction of the multi-stage density separation system; gently rinsing the surface of the filter membranes 1-2 times with deionized water to remove a large number of free salt ions attached to the surface of the filter membranes, thereby reducing ion interference during subsequent oxidation with Fenton's reagent; Place the rinsed filter membrane in a clean PTFE tray and allow it to drain naturally to remove surface moisture.

[0046] Prepare Fenton's reagent with a FeSO4 concentration of 0.08 mol / L and an H2O2 concentration of 4% (volume fraction). Adjust the pH value to 2.8-3.2 with dilute sulfuric acid. Place the pretreated filter membrane into a reaction vessel (made of glass to avoid reaction between plastic containers and Fenton's reagent), add sufficient Fenton's reagent, seal the reaction vessel, and let it stand at room temperature (25±2℃) for 24 hours. During this period, gently shake the vessel once every 6 hours to ensure that the Fenton's reagent comes into full contact with the organic impurities on the filter membrane. After the reaction is completed, pour out the Fenton's reagent waste liquid in the container and rinse the filter membrane with deionized water 3-4 times until the pH value of the rinsing solution reaches 7.0±0.1 to completely remove residual Fenton's reagent and organic impurity decomposition products.

[0047] Place the Fenton-oxidized and rinsed filter membrane into a microwave digestion vessel, add 5-8 mL of 5% (v / v) dilute nitric acid digestion solution to ensure the filter membrane is completely submerged, place the digestion vessel in a microwave digester, and react according to the preset digestion parameters. After digestion, allow the digestion vessel to cool naturally to room temperature, open the lid, remove the filter membrane, and rinse the surface of the filter membrane repeatedly with deionized water until there are no nitrate ions in the rinsing solution. Then dry the filter membrane.

[0048] This invention employs a two-step combined process of Fenton oxidation and microwave digestion for deep purification. After Fenton oxidation, residual trace organic matter (such as cellulose fragments and enzyme proteins) on the filter membrane is completely oxidized into small molecules, preventing them from appearing as microplastic-like morphologies (such as fibrous or granular forms) under a smart microscope, thus avoiding misidentification as microplastics by image recognition software. Microwave digestion removes mineral particles (such as quartz sand and calcium carbonate), which may exhibit characteristic peaks similar to microplastics in Raman spectroscopy, easily leading to false positives. Digestion completely eliminates these particles. If organic impurities (such as residual proteins from enzymatic hydrolysis) adhere to the surface of microplastics, during Raman spectroscopy detection, the laser will preferentially excite the characteristic peaks of organic impurities, masking the characteristic peaks of microplastics and resulting in undetected false negatives. Fenton oxidation thoroughly removes the organic layer adhering to the microplastic surface, directly exposing the microplastics, while microwave digestion further removes surface inorganic ions, ensuring that Raman spectroscopy accurately captures the characteristic peaks of microplastics, thus reducing the false negative rate.

[0049] Unpurified filter membranes often present a cluttered background due to residual impurities (such as salt stains and organic debris). Intelligent microscopes require significant time to distinguish impurities from microplastics during scanning, and are prone to identification errors. After deep purification, the filter membrane surface is clean and the background is uniform (white or transparent), allowing image recognition software to quickly locate microplastic particles, improving recognition speed and accuracy significantly compared to traditional methods. Furthermore, Raman spectroscopy is extremely sensitive to sample background. The presence of inorganic ions or small organic molecules on the filter membrane can cause fluorescence interference, resulting in a low signal-to-noise ratio and blurred characteristic peaks. Purified filter membranes are free from such interference, allowing Raman spectroscopy to identify individual microplastics within 10-20 seconds with clearly distinguishable characteristic peaks, ensuring high-throughput sample processing.

[0050] In this invention, the identification and quantification are preferably performed by: scanning the filter membrane obtained by deep purification using an intelligent microscope, automatically identifying and counting the microplastics in the filter membrane using image recognition software, recording the coordinates of the microplastics, then using Raman spectroscopy to identify the microplastics under the coordinates, and finally obtaining an analysis report including the number, type, size distribution and mass concentration of microplastics.

[0051] Furthermore, the quantitative identification method described in this invention specifically involves: removing the dried and purified filter membrane; if the filter membrane has slight wrinkles, placing it on a clean glass slide, covering it with a layer of non-fluorescent plastic wrap, and gently smoothing the wrinkles with tweezers. Fixing the filter membrane onto a special clamp on the microscope stage, ensuring the center of the filter membrane is aligned with the center of the stage, and recording the positioning marks on the filter membrane edge (such as notches on the filter membrane edge) to facilitate accurate coordinate positioning in subsequent Raman spectroscopy. A smart microscope is used to acquire a full-surface image of the filter membrane through a high-resolution imaging system. Image recognition software, based on the morphological characteristics (such as granular or fibrous), optical properties (such as reflectivity and transparency) of microplastics and differences in impurities, automatically identifies, counts, and records the coordinates of microplastics. Different types of microplastics have unique Raman spectral characteristic peaks. By acquiring the spectra of the coordinate points marked on the smart microscope using a Raman spectrometer and comparing them with a standard spectral library (such as the Polymer Raman Library), the chemical type of the microplastic can be determined, false positives can be eliminated, and the accuracy of identification can be ensured. Based on the size data recorded by the intelligent microscope and the type information identified by Raman spectroscopy, the size distribution characteristics of microplastics were further analyzed, and their mass concentration in Chinese medicinal materials was calculated, providing a quantitative basis for pollution assessment. Finally, all detection data were integrated to form a systematic and comprehensive analysis report, providing a reference for the prevention and control of microplastic pollution and quality assessment of Chinese medicinal materials.

[0052] This invention employs an identification and quantification method that combines automated scanning counting, highly specific spectral identification, and multi-dimensional data analysis. This solves the technical problems of low efficiency, large errors, and limited data in traditional methods, achieving accurate and efficient detection of microplastics in Chinese medicinal materials. It not only provides scientific data for assessing microplastic pollution in Chinese medicinal materials but also lays the foundation for subsequent pollution source analysis and risk management. This is a key link in achieving a closed loop from separation to application in this invention.

[0053] This invention also provides the application of the microplastic separation and extraction method described in the above technical solution in the purification and refining of traditional Chinese medicine.

[0054] Example 1 1. Preliminary pretreatment: Grinding and particle size control of Angelica sinensis samples The Angelica sinensis samples to be processed were sorted, and then the surface was quickly rinsed with deionized water (rinsing time 20 seconds). They were then placed in a vacuum drying oven and dried at 40℃ and -0.08MPa for 4 hours until the sample moisture content was ≤5%. Use stainless steel scissors to cut the dried angelica root into 1-2cm pieces. 3 Small pieces were fed into an ultra-micro airflow pulverizer, with a pulverizing pressure of 0.6 MPa and a feed rate of 10 g / min, until the sample passed through a 100-mesh standard sieve (particle size ≤ 150 μm). Collect the sieved material (about 30g), which is the pretreated Angelica sinensis sample. Place it in a clean polytetrafluoroethylene sealed container and refrigerate at 4°C for later use (storage time ≤ 24 hours).

[0055] 2. Deep pretreatment: Enzymatic hydrolysis removes organic matter from Angelica sinensis. Take 10g of pretreated Angelica sinensis sample, add 250mL of 0.1mol / L phosphate buffer (preheated to 50℃), and pour into a 500mL glass reaction flask; Add 0.2g of the compound enzyme preparation (0.1g cellulase, 0.05g pectinase, and 0.05g xylanase) at a mass ratio of 50:1 for the sample to the enzyme preparation, and stir magnetically until the enzyme preparation is completely dissolved. Place the reaction flask in a constant temperature shaking incubator, set the temperature to 50℃ and the shaking frequency to 200rpm, and perform enzymatic hydrolysis for 10 hours. After the enzymatic hydrolysis is complete, place the reaction flask in a 95°C hot water bath and heat for 15 minutes to terminate the enzymatic hydrolysis reaction. The enzymatic hydrolysis system was filtered using a vacuum filtration device (0.45 μm filter membrane), and the filter residue (for use as Angelica sinensis sample) was collected. The vacuum degree during vacuum filtration was 0.05 MPa. The filter residue was washed three times with 0.1 mol / L phosphate buffer to remove residual enzymatic hydrolysate, and then vacuum dried at 40 °C for 2 hours for later use.

[0056] 3. Multi-stage density separation: Stepwise extraction of microplastics with different densities Level 1: Separation of ultra-lightweight microplastics A solution of 1.18 g / cm³ was injected into the first-stage separation vessel (1000 mL glass container) using a precision peristaltic pump at a flow rate of 5 mL / min. 3 Prepare a saturated NaCl solution until the solution volume reaches 600 mL; Add the dried Angelica sinensis sample (about 2g) into the container at a constant speed, stir gently (stirring speed 50rpm) for 3 minutes, and then let stand for 45 minutes. An online infrared sensor monitors the stratification of particles inside the container in real time. Once the floating layer (extremely lightweight microplastics) stabilizes (the sensor shows a particle throughput of <0.1 particles / minute), the floating liquid (approximately 200 mL) is collected. The floating liquid was filtered through a 0.45 μm filter membrane to obtain the first-stage filter membrane (containing extremely light microplastic crude extract). The filter membrane was rinsed twice with deionized water and drained for later use. The sediment at the bottom of the container was collected and transferred to the second-stage separation system.

[0057] Level 2: Medium-density microplastic separation The secondary separation vessel was filled with a solution of 1.23 g / cm³ at a flow rate of 5 mL / min. 3 600 mL of saturated NaCl solution; Add the first stage of sediment, repeat the process of stirring for 3 minutes and letting stand for 45 minutes. After the medium-density microplastic floating layer is stabilized by the online infrared sensor, collect the floating liquid (about 180 mL). The floating liquid was filtered through a 0.45 μm filter membrane to obtain a second-stage filter membrane (containing medium-density microplastic crude extract), which was rinsed twice with deionized water and drained for later use; the sediment at the bottom was collected and transferred to the third-stage separation system.

[0058] Level 3: High-density microplastic separation The third-stage separation vessel was filled with a solution of 1.40 g / cm³ at a flow rate of 5 mL / min. 3 600 mL of saturated Na2WO4 solution; Add the second stage sediment, stir for 3 minutes and let stand for 80 minutes (high-density particles stratify slowly). After the online infrared sensor detects that the high-density microplastic floating layer is stable, collect the floating liquid (about 150 mL). The floating liquid was filtered through a 0.45μm filter membrane to obtain a third-stage filter membrane (containing high-density microplastic crude extract), which was rinsed twice with deionized water and drained for later use; the sediment at the bottom of the container (Angelica matrix impurities) was discarded directly.

[0059] 4. Deep purification: Removes residual impurities from the filter membrane. Fenton's reagent oxidation: First, the filter membrane was ultrasonically pre-washed with deionized water. Then, Fenton's reagent was prepared with FeSO4 concentration of 0.08 mol / L and H2O2 concentration of 4%. The pH value was adjusted to 2.8-3.2 with dilute sulfuric acid. The pre-washed filter membrane was immersed in Fenton's reagent, sealed, and placed in a constant temperature water bath shaker at a frequency of 60 rpm and a temperature of 25℃. During the reaction, the COD value of the reaction system was sampled and tested every 6 hours until the COD value tended to stabilize. After oxidation, the Fenton's reagent waste liquid was poured out, and the filter membrane was rinsed with deionized water 4 times until the pH of the rinsing solution was 7.0 (tested with pH test paper). Microwave digestion: Place the rinsed filter membranes into polytetrafluoroethylene microwave digestion vessels, add 6 mL of 5% dilute nitric acid to each, cover, and place in a microwave digester. Set the parameters as follows: initial temperature 25℃, increase to 100℃ at a rate of 5℃ / min, maintain the temperature for 15 minutes, and control the pressure at 0.5 MPa. After digestion, wait for the digestion vessels to cool to room temperature, remove the filter membranes, and rinse repeatedly with ultrapure water until no nitrate ions are present in the rinsing solution (no white precipitate is formed upon addition of AgNO3 solution). The filter membrane was placed in a vacuum drying oven and dried at 40℃ and -0.08MPa for 2.5 hours to obtain purified filter membranes (3 sheets), which were then stored in a sealed container away from light.

[0060] 5. Identification and Quantification: Counting, Identification, and Analysis of Microplastics (1) Intelligent microscope scanning and counting Fix the three purification filter membranes onto the microscope stage fixture, smooth out the wrinkles, and establish the XY coordinate system with the upper left corner of the filter membrane as the origin. The intelligent microscope uses a 20× objective lens and sets the following scanning parameters: grayscale threshold 160~255, particle size recognition range 0.1~5mm, and aspect ratio threshold (granular ≤3:1, fibrous >3:1). The automatic scanning program is started to collect and stitch images of the entire surface of the filter membrane grid by grid. The image recognition software automatically marks the microplastic particles and records their coordinates, shape and size. 10% of the marked points were manually reviewed to correct misjudgments (no obvious misjudgments were found in this example). The statistical results are as follows: First-stage filter membrane (ultra-lightweight microplastics): 18 pieces (all granular, particle size 0.2~0.8mm); Second-stage filter membrane (medium-density microplastic): 12 (10 granular, particle size 0.3~1.0mm; 2 fibrous, length 1.2~2.5mm, diameter 0.08~0.12mm). Third-stage filter membrane (high-density microplastic): 9 units (all granular, particle size 0.15~0.6mm); Total number of microplastics: 39.

[0061] (2) Raman spectroscopy identification The coordinates of the microplastics recorded by the microscope were imported into the Raman spectrometer, and the parameters were set as follows: laser wavelength 532 nm, power 8 mW, and scanning range 500~3500 cm⁻¹. -1 Scanning time: 15 seconds; The stage is automatically positioned according to coordinates, and Raman spectra are collected point by point. These spectra are then compared with the Polymer Raman Library standard spectral library (similarity ≥ 85% is considered valid). The identification results are as follows: Ultra-lightweight microplastics (18 in total): all are polyethylene (PE, similarity 92%~95%). Medium-density microplastics (12): 10 polypropylene (PP, similarity 90%~93%), 2 polyamide (PA, similarity 88%~91%). High-density microplastics (9): 7 polyvinyl chloride (PVC, similarity 91%~94%), 2 polyethylene terephthalate (PET, similarity 89%~92%).

[0062] Size distribution and mass concentration calculation: Size distribution: The proportion of each type of microplastic was statistically analyzed according to particle size range. The results show: 0.1~0.5mm: 22 pieces (accounting for 56.4%, including 8 PE pieces, 6 PP pieces, 7 PVC pieces, and 1 PET piece); 0.5~1.0mm: 14 pieces (accounting for 35.9%, including 10 PE pieces, 4 PP pieces, 0 PVC pieces, and 0 PET pieces); 1.0~5mm: 3 pieces (7.7% of the total, including 0 pieces of PP, 2 pieces of PA, 1 piece of PVC, and 0 pieces of PET); Mass concentration: based on microplastic morphology, size, and density (PE 0.92 g / cm³). 3 PP 0.91g / cm 3 PA 1.14g / cm 3 PVC 1.38g / cm 3 PET 1.38g / cm 3 ), calculate and summarize the mass of each particle: Total mass of microplastics: 28.6 μg; The concentration of microplastics in Angelica sinensis is 28.6 μg ÷ 10g = 2.86 μg / g.

[0063] Experimental Results and Analysis Separation efficiency verification: In this embodiment, the total recovery rate of Angelica microplastics reached 87% (verified by adding known amounts of standard microplastics such as PE, PP, and PVC), and covered all types of microplastics, including extremely light (PE), medium (PP, PA), and high density (PVC, PET), which solves the defect of traditional single-density separation that can only extract low-density microplastics; Anti-interference ability: After compound enzymatic hydrolysis (removing 92% of cellulose and pectin) and Fenton-microwave purification, the filter membrane background is clean, the Raman spectrum has no obvious fluorescence interference, the false positive rate is <3%, ensuring the accuracy of identification results; Sample protection: The enzymatic hydrolysis, density separation and purification processes all use mild conditions, and the retention rate of ferulic acid, the effective component of Angelica sinensis, reaches 83% (detected by high performance liquid chromatography). The purified Angelica sinensis residue can be used for subsequent quality testing.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for separating and extracting microplastics from traditional Chinese medicinal materials, characterized in that, Includes the following steps: After sorting, washing and drying, the powdered Chinese medicinal materials to be processed are pulverized to 0-100 mesh to obtain pre-treated Chinese medicinal materials. The pretreated Chinese medicinal materials are subjected to deep pretreatment to obtain the ready-to-use Chinese medicinal materials; The prepared Chinese medicinal materials were density separated using a multi-stage density separation system including a first stage, a second stage, and a third stage, and microplastics and purified Chinese medicinal materials were collected. The microplastics are then subjected to deep purification to obtain purified microplastics. The purified microplastics are identified and quantified. The deep pretreatment involves placing the pretreated Chinese medicinal materials in a 0.1 mol / L phosphate buffer solution at a solid-liquid ratio of 1:15-25, followed by the addition of a compound enzyme preparation for enzymatic hydrolysis.

2. The method for separating and extracting microplastics from traditional Chinese medicinal materials according to claim 1, characterized in that, The compound enzyme preparation includes cellulase, pectinase and xylanase, with a mass ratio of 2~2.5:1~1.5:1~1.5; the enzymatic hydrolysis parameters are: temperature 50~55℃, time 6~12 hours, and oscillation frequency 200rpm.

3. The method for separating and extracting microplastics from traditional Chinese medicinal materials according to claim 1, characterized in that, After the enzymatic hydrolysis is completed, the process also includes: high-temperature enzyme inactivation, vacuum filtration and washing. The high-temperature enzyme inactivation temperature is 95°C and the time is 15-18 minutes. The vacuum filtration uses a 0.45μm filter membrane and a vacuum degree of 0.05-0.08MPa. The washing is performed using 0.1mol / L phosphate buffer.

4. The method for separating and extracting microplastics from traditional Chinese medicinal materials according to claim 1, characterized in that, The multi-stage density separation system comprises a three-stage system with a first stage, a second stage, and a third stage; the first stage uses a density of 1.15~1.20 g / cm³. 3 The ultralight microplastics were separated by perfusion with a saturated NaCl solution; the second stage used a solution with a density of 1.20~1.25 g / cm³. 3 The process involves perfusion with a saturated NaCl solution to separate medium-density microplastics; the third stage uses a solution with a density of 1.30~1.50 g / cm³. 3 High-density microplastics were separated using a saturated Na2WO4 solution.

5. The method for separating and extracting microplastics from traditional Chinese medicinal materials according to claim 1, characterized in that, The multi-stage density separation system is intelligently controlled by using a precision peristaltic pump to control the flow rate and using an online infrared sensor to detect the particle passage and the interfaces between phases in real time.

6. The method for separating and extracting microplastics from traditional Chinese medicinal materials according to claim 1, characterized in that, The deep purification process specifically involves: pre-washing, Fenton reagent oxidation, intermediate washing, microwave digestion, final washing, and drying of the filtration membranes at each density level obtained from the multi-stage density separation system to obtain purified microplastics. The pre-washing process involves ultrasonic washing with deionized water at a power of 50-80W for 5-10 minutes, repeated 2-3 times.

7. The method for separating and extracting microplastics from traditional Chinese medicinal materials according to claim 6, characterized in that, The Fenton reagent oxidation process is as follows: Fenton reagent is prepared by mixing 0.08 mol / L FeSO4 and 4% H2O2. The pH value is adjusted to 2.8-3.2 with dilute sulfuric acid. The pre-washed filter membrane is immersed in Fenton reagent, sealed, and placed in a constant temperature water bath shaker for reaction. The shaking frequency is 60 rpm and the temperature is 25°C. During the reaction, the COD value in the reaction system is sampled and detected every 6 hours until the COD value tends to stabilize.

8. The method for separating and extracting microplastics from traditional Chinese medicinal materials according to claim 6, characterized in that, The microwave digestion process specifically involves: using a programmed temperature ramp mode, starting at an initial temperature of 25°C, increasing the temperature to 100°C at a rate of 5°C / min, maintaining the temperature for 15-20 minutes, controlling the pressure at 0.5-0.6 MPa, and continuously stirring during the digestion process.

9. The method for separating and extracting microplastics from traditional Chinese medicinal materials according to claim 1, characterized in that, The specific identification and quantification process involves: scanning the filter membrane obtained from deep purification using an intelligent microscope, automatically identifying and counting the microplastics in the filter membrane using image recognition software, recording the coordinates of the microplastics, then using Raman spectroscopy to identify the microplastics under the coordinates, and finally obtaining an analysis report including the number, type, size distribution, and mass concentration of the microplastics.

10. The application of the microplastic separation and extraction method in Chinese medicinal materials as described in claims 1-9 in the purification and refining of Chinese medicinal materials.

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