Preparation method of chlorella biochar and method for solid phase extraction of tetracycline antibiotics

Abstract

The present application relates to the preparation of biochar material and its application in the field of solid phase extraction pretreatment, mainly used for the recovery of tetracycline antibiotics, specifically a preparation method of chlorella biochar: including the following steps: S1. Preparation; S2. Roasting; S3. Cooling. The application provides a method for preparing chlorella biochar based on easily available chlorella vulgaris as raw material, which realizes an efficient, environmentally friendly and low-cost preparation process by controlling the vacuum degree in the pyrolysis process. The chlorella biochar is a graphitized non-porous structure of polyvinylpyridine, and the surface has high ion exchange capacity and polarity, which is suitable for the rapid separation of strong polarity and charged compounds.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biochar and tetracycline antibiotics recovery, in particular to a preparation method of chlorella biochar and a method for solid phase extraction of tetracycline antibiotics. BACKGROUND

[0002] With the development of environment, food, biology and pharmaceutical fields, the demand for efficient, reliable and environmentally friendly sample pretreatment and analysis detection technology is increasing. As an advanced separation and enrichment method, solid phase extraction technology has been widely used in sample pretreatment and analysis detection. Traditional solid phase extraction adsorbents such as silica gel, C 18 , C8, C2 and the like have problems such as poor selectivity, sensitivity and specificity, and new adsorbents need to be sought to improve their separation efficiency and selectivity.

[0003] Biochar is a new type of separation material obtained by high-temperature pyrolysis of biomass raw materials, which has high specific surface area, pore volume and pore size, high adsorption capacity and other characteristics. It is a simple, low-cost and environmentally friendly separation material. However, due to the low content of surface functional groups, the separation selectivity of conventional biochar is poor, and chemical modification or compounding with other materials is needed to improve its separation effect. In addition, the conventional biochar adsorbent has problems such as desorption lag, large elution volume and low absolute recovery rate in sample pretreatment application. Better desorption effect often uses high-temperature desorption method of gas chromatography, which seriously limits its application in sample separation and analysis field.

[0004] Therefore, developing new biochar materials to effectively separate antibiotic residues is crucial to improve the pretreatment efficiency of complex samples and further improve the sensitivity and stability of antibiotic detection. Chlorella biochar has high ion exchange capacity, non-porous structure and rich surface nitrogen-containing groups, which can be used as a new type of solid phase extraction adsorbent and has wide application prospect in distribution separation mode. SUMMARY

[0005] To solve the above problems, the present application provides a preparation method of chlorella biochar and a method for solid phase extraction of tetracycline antibiotics.

[0006] The technical solution adopted by the present application to solve the technical problem is: a preparation method of chlorella biochar, comprising the following steps:

[0007] S1. Preparation: weigh the biomass raw material and place it in a crucible, cover the crucible cover, and prepare for use;

[0008] S2. Firing: place the crucible in step S1 in an electric furnace and perform firing under vacuum atmosphere by high-temperature pyrolysis;

[0009] S3. Cooling: After the completion of the firing in step S2, the crucible is taken out and naturally cooled to room temperature.

[0010] As an optimization, the biomass raw material in step S1 is Chlorella vulgaris broken cell protein freeze-dried powder, with a protein content ≥55 g / 100 g, a fat content ≥5 g / 100 g, and a total sugar content (calculated as glucose) ≥5.5 g / 100 g.

[0011] As an optimization, in step S2, the alumina crucible is placed in an atmosphere furnace for high-temperature pyrolysis under a vacuum degree of -0.04 Mpa and at a temperature rise rate of 10°C / min, with a pyrolysis temperature of 300-700°C and a thermal residence time of 30-60 min.

[0012] A method for extracting tetracycline antibiotics by using a Chlorella biochar, comprising the following steps:

[0013] A1. Preparing column packing material by using the Chlorella biochar, and filling the column packing material in a solid-phase extraction empty column to obtain a Chlorella biochar-filled solid-phase extraction column;

[0014] A2. Sequentially using methanol and NaCl aqueous solution to rinse and activate the solid-phase extraction column;

[0015] A3. Slowly passing the sample extract through the solid-phase extraction column activated in step A2, and after the sample extract flows out, using acetonitrile to rinse the solid-phase extraction column, and then vacuum-drying the solid-phase extraction column;

[0016] A4. Eluting the solid-phase extraction column treated in step A3 three times using a methanol solution, and collecting the eluate.

[0017] As an optimization, in step A1, the amount of column packing material is 100 mg, and the specification of the solid-phase extraction empty column is 3 mL;

[0018] The volume of the methanol solution is 5 mL, the concentration of the NaCl aqueous solution is 0.01 mol / L, the pH value is 4.0, and the volume is 5 mL.

[0019] In step A3, the volume of acetonitrile is 3 mL, and the Chlorella biochar solid-phase extraction column is rinsed with acetonitrile to remove non-target substances, and vacuum-drying is performed for 2 min;

[0020] In step A4, the volume of the eluate is 2.0 mL.

[0021] As an optimization, in step A1, the specific steps for preparing column packing material by using Chlorella biochar are as follows:

[0022] (1) The prepared Chlorella vulgaris biochar is ground to obtain Chlorella vulgaris biochar powder, which is stored in a light-proof glass container;

[0023] (2) The Chlorella vulgaris biochar powder obtained in step (1) is weighed and added to a phosphoric acid solution, stirred for 8-12 h, and then filtered to obtain acid-treated Chlorella vulgaris biochar;

[0024] (3) The acid-treated Chlorella vulgaris biochar obtained in step (2) is washed with ethanol for 3-5 times, and then washed with distilled water for 3-5 times until the washing liquid is neutral;

[0025] (4) The washed Chlorella vulgaris biochar in step (3) is dried to obtain a column packing material, which is stored in a light-proof glass container for standby.

[0026] As an optimization, the particle size of the Chlorella vulgaris biochar powder in step (1) is 100-200 mesh.

[0027] As an optimization, in step (2), the concentration of the phosphoric acid solution used is 3 mol / L, and an electric stirrer is used during stirring at a speed of 25-35 rpm.

[0028] The method has the following advantages:

[0029] The method provided by the application is based on readily available Chlorella pyrenoidosa as raw material to prepare Chlorella vulgaris biochar, which realizes an efficient, environmentally friendly and low-cost preparation process by controlling the vacuum degree during pyrolysis, reducing the use of protective gas and the emission of harmful gas.

[0030] The Chlorella vulgaris biochar surface contains rich functional groups, forms a graphite-like non-porous structure with polyvinyl pyridine, and has high ion exchange capacity and polarity on the surface, which is suitable for efficient separation of polar and charged compounds.

[0031] Unlike traditional porous biochar, Chlorella vulgaris biochar is a non-porous layered material, which effectively avoids the desorption hysteresis problem.

[0032] The non-porous Chlorella vulgaris biochar material has the following advantages in solid phase extraction:

[0033] High selectivity: Due to the absence of pores on the surface of non-porous materials, target compounds can only interact with the chemical groups on the surface through adsorption, thus improving the selectivity of solid-phase extraction. This is particularly suitable for the separation and analysis of complex samples. Good regenerability: Because non-porous materials have no pores, they are less likely to interact with other compounds in the sample, allowing for material regeneration through simple elution operations, reducing costs. Low non-specific adsorption: The absence of pores on the surface of non-porous materials means that the interaction between their chemical groups and target compounds is based on chemical affinity rather than physical adsorption, exhibiting lower non-specific adsorption. Faster separation speed: The absence of pores on the surface of non-porous materials allows for faster interaction between target compounds and the material surface, enabling faster separation during solid-phase extraction. This is significant for high-throughput analysis, improving analytical efficiency. In solid-phase extraction, the partition separation mode significantly shortens sample loading and elution time, reduces the use of organic reagents and acidic solutions, and is highly efficient. Sensitive solid-phase extraction of multiple tetracycline residues improves separation efficiency and stability. Attached Figure Description

[0034] Appendix Figure 1 This is a schematic diagram of the surface properties of Chlorella biochar of the present invention.

[0035] Appendix Figure 2 This is a schematic diagram of XPS spectral analysis of Chlorella biochar according to the present invention.

[0036] Appendix Figure 3 This is a schematic scanning electron microscope image of Chlorella biochar and commercial bamboo of the present invention.

[0037] Figure 1 In the figure, a represents the surface C / O element ratio; b represents the surface potential.

[0038] Figure 2 In the diagram, a represents C1s; b represents O1s; and c represents N1s.

[0039] Figure 3 In the diagram, a and b are Chlorella biochar, and c and d are commercial bamboo biochar. Detailed Implementation

[0040] Example 1:

[0041] A method for preparing Chlorella biochar includes the following steps:

[0042] S1. Preparation: Weigh 10.0g of cell wall broken protein Chlorella freeze-dried powder and place it in a crucible. Cover the crucible and set aside.

[0043] The cell wall broken protein Chlorella freeze-dried powder has a protein content of 56g / 100g, a fat content of 5.2g / 100g, and a total sugar content (calculated as glucose) of 5.8g / 100g.

[0044] S2. Firing: Place the crucible in step S1 in an electric furnace under a vacuum of -0.04 Mpa, and use a heating rate of 10°C / min to perform pyrolysis under a vacuum atmosphere. The pyrolysis temperature is 700°C, and the thermal residence time is 60 min.

[0045] S3. Cooling: After the firing in step S2 is completed, remove the crucible and allow it to cool naturally to room temperature.

[0046] The specific steps for preparing a column packing material using the above Chlorella vulgaris biochar are as follows:

[0047] (1) Grind 20.0 g of the fired Chlorella vulgaris biochar to obtain Chlorella vulgaris biochar powder. The Chlorella vulgaris biochar powder is 100 mesh, and is stored in a light-proof glass container.

[0048] (2) Weigh the Chlorella vulgaris biochar powder obtained in step (1) and add it to 100 mL of a 3 mol / L phosphoric acid solution. After stirring for 8 h, filter to obtain acid-treated Chlorella vulgaris biochar.

[0049] An electric stirrer is used during the stirring process, and the speed is 25 rpm.

[0050] (3) Wash the acid-treated Chlorella vulgaris biochar obtained in step (2) with ethanol 3 times, and then wash it with distilled water 3 times until the washing liquid is neutral.

[0051] (4) Dry the Chlorella vulgaris biochar after washing in step (3) to obtain the column packing material. Store the column packing material in a light-proof glass container for later use.

[0052] A method for extracting tetracycline antibiotics using Chlorella vulgaris biochar, which uses any of the above-described Chlorella vulgaris biochars, includes the following steps:

[0053] A1. Prepare a column packing material using Chlorella vulgaris biochar, and fill 100 mg of the column packing material into a solid-phase extraction empty column. The specification of the solid-phase extraction empty column is 3 mL, and a Chlorella vulgaris biochar-filled solid-phase extraction column is obtained.

[0054] A2. Use methanol and a NaCl aqueous solution to sequentially rinse and activate the solid-phase extraction column.

[0055] The volume of the methanol solution is 5 mL, the concentration of the NaCl aqueous solution is 0.01 mol / L, the pH value is 4.0, and the volume is 5 mL.

[0056] A3. Slowly pass the sample extract through the solid-phase extraction column after activation in step A2. After the sample extract flows out completely, use acetonitrile to elute the solid-phase extraction column, and then vacuum dry the solid-phase extraction column.

[0057] A4. Eluting the solid phase extraction column after step A3 with formic acid solution for 3 times, and collecting the eluent.

[0058] Example 2:

[0059] A method for preparing a Chlorella biochar, comprising the following steps:

[0060] S1. Preparing materials: weighing 10.0 g of Chlorella zofingiensis freeze-dried powder and placing it in a crucible, covering the crucible cover, and reserving it;

[0061] The Chlorella zofingiensis freeze-dried powder has a protein content of 56 g / 100 g, a fat content of 5.2 g / 100 g, and a total sugar content (calculated as glucose) of 5.8 g / 100 g;

[0062] S2. Firing: placing the crucible in step S1 in an electric furnace under a vacuum degree of -0.03 Mpa, using a heating rate of 10°C / min, and using a high-temperature pyrolysis method under a vacuum atmosphere to fire at a pyrolysis temperature of 500°C for a heat residence time of 45 min;

[0063] S3. Cooling: after the firing in step S2 is completed, the crucible is taken out and naturally cooled to room temperature.

[0064] The specific steps for preparing a column packing material using the above Chlorella biochar are as follows:

[0065] (1) Grinding 20.0 g of the fired Chlorella biochar to obtain Chlorella biochar powder, the Chlorella biochar powder is 100 mesh, and is stored in a light-proof glass container;

[0066] (2) Weighing the Chlorella biochar powder obtained in step (1) and adding it to 100 mL of a 3 mol / L phosphoric acid solution, stirring for 10 h, and then filtering to obtain acid-treated Chlorella biochar;

[0067] The concentration of the phosphoric acid solution used is 3 mol / L, and an electric stirrer is used during stirring at a speed of 30 pm;

[0068] (3) Washing the acid-treated Chlorella biochar obtained in step (2) with ethanol for 3 times, and then washing it with distilled water for 3 times until the washing liquid is neutral;

[0069] (4) Drying the Chlorella biochar after washing in step (3) to obtain a column packing material, which is stored in a light-proof glass container for standby.

[0070] A method for extracting tetracycline antibiotics using a Chlorella biochar-solid phase extraction, using any one of the above Chlorella biochars, comprising the following steps:

[0071] A1. Using Chlorella biochar to prepare column packing material, 100 mg of column packing material was packed in a solid phase extraction empty column with a size of 3 mL to obtain a Chlorella biochar packed solid phase extraction column;

[0072] A2. The solid phase extraction column was sequentially rinsed and activated with methanol and NaCl aqueous solution;

[0073] The volume of the methanol solution was 5 mL, the concentration of the NaCl aqueous solution was 0.01 mol / L, the pH value was 4.0, and the volume was 5 mL;

[0074] A3. The sample extract was slowly passed through the solid phase extraction column activated in step A2, and after the sample extract flowed out, acetonitrile was used to rinse the solid phase extraction column, and then the solid phase extraction column was vacuum dried;

[0075] A4. The solid phase extraction column treated in step A3 was eluted with formic acid solution for 3 times, and the eluate was collected.

[0076] Example 3:

[0077] A method for preparing Chlorella biochar comprises the following steps:

[0078] S1. Preparation: 10.0 g of broken wall protein Chlorella pyrenoidosa freeze-dried powder was placed in a crucible, the crucible cover was covered, and the crucible was ready for use;

[0079] The protein content of the broken wall protein Chlorella pyrenoidosa freeze-dried powder was 56 g / 100 g, the fat content was 5.2 g / 100 g, and the total sugar content (calculated as glucose) was 5.8 g / 100 g;

[0080] S2. Firing: The crucible in step S1 was placed in an electric furnace under a vacuum degree of -0.02 Mpa, and a high temperature pyrolysis method was used under a vacuum atmosphere with a heating rate of 10°C / min. The pyrolysis temperature was 700°C, and the heat residence time was 30 min;

[0081] S3. Cooling: After the firing in step S2 was completed, the crucible was taken out and naturally cooled to room temperature.

[0082] The specific steps for preparing column packing material using the above Chlorella biochar are as follows:

[0083] (1) The fired Chlorella biochar 20.0 g was ground to obtain Chlorella biochar powder, and the Chlorella biochar powder was 100 mesh and stored in a light-proof glass container;

[0084] (2) The Chlorella biocarbon powder obtained in step (1) is weighed and added to 100 mL of a 3 mol / L phosphoric acid solution. After stirring for 12 h, the acid-treated Chlorella biocarbon is obtained by filtration;

[0085] The concentration of the phosphoric acid solution used is 3 mol / L, and an electric stirrer is used in the stirring process, with a rotation speed of 35 rpm;

[0086] (3) The acid-treated Chlorella biocarbon obtained in step (2) is washed with ethanol for 3 times, and then washed with distilled water for 3 times until the washing liquid is neutral;

[0087] (4) The Chlorella biocarbon washed in step (3) is dried to obtain a column filling material. The column filling material is stored in a light-proof glass container for standby.

[0088] A method for extracting tetracycline antibiotics by using a Chlorella biocarbon, which comprises the following steps:

[0089] A1. A column filling material is prepared by using the Chlorella biocarbon, and 100 mg of the column filling material is filled into a solid-phase extraction empty column with a specification of 3 mL to obtain a Chlorella biocarbon-filled solid-phase extraction column;

[0090] A2. The solid-phase extraction column is sequentially rinsed and activated by using methanol and a NaCl aqueous solution;

[0091] The volume of the methanol solution is 5 mL, the concentration of the NaCl aqueous solution is 0.01 mol / L, the pH value is 4.0, and the volume is 5 mL;

[0092] A3. The sample extraction liquid is slowly passed through the solid-phase extraction column activated in step A2, and after the sample extraction liquid flows out, acetonitrile is used to rinse the solid-phase extraction column, and then the solid-phase extraction column is vacuum-dried;

[0093] A4. The solid-phase extraction column treated in step A3 is eluted 3 times by using a formic acid solution, and the eluate is collected.

[0094] Comparative Example 1:

[0095] A method for extracting tetracycline antibiotics by using a commercial bamboo biocarbon, which comprises the following steps:

[0096] A1. A column filling material is prepared by using the commercial bamboo biocarbon, and 100 mg of the column filling material is filled into a solid-phase extraction empty column with a specification of 3 mL to obtain a bamboo biocarbon-filled solid-phase extraction column;

[0097] Commercialized bamboo charcoal powder was purchased from Zhengzhou Lvhang Water Purification Material Co., Ltd. (https: / / lvhang.1688.com / ), and the raw material was high mountain bamboo with a particle size of 100 mesh.

[0098] A2. sequentially rinse and activate the solid phase extraction column with methanol and NaCl aqueous solution;

[0099] The volume of the methanol solution was 5 mL, the concentration of the NaCl aqueous solution was 0.01 mol / L, the pH value was 4.0, and the volume was 5 mL;

[0100] A3. slowly pass the sample extract through the solid phase extraction column activated in step A2, after the sample extract flows out, use acetonitrile to rinse the solid phase extraction column, and then vacuum dry the solid phase extraction column;

[0101] A4. use the eluent to elute the solid phase extraction column treated in step A3, and collect the eluent.

[0102] The eluent was a methanol / formic acid solution (v / v, 4 / 6), the volume of the eluent was 2.0 mL, and the elution times were 3 times (3x2 mL).

[0103] By filling the Chlorella vulgaris charcoal column packing material obtained in Examples 1-3 and Comparative Example 1 into an empty column, a solid phase extraction column was obtained, and tetracycline antibiotics were extracted by the solid phase extraction column, and the extraction results were as follows:

[0104] Table 1 Extraction efficiency of tetracycline in Examples 1, 2, 3 and Comparative Example 1

[0105]

[0106] (1) To analyze the surface chemical properties and pore structure of Chlorella vulgaris charcoal, element analysis, Zeta potential tester and X-ray photoelectron spectroscopy were used, and the results were as follows:

[0107] Figure 1 In a, the ratio of surface elements C / O of Chlorella vulgaris prepared in Examples 1, 2 and 3 gradually decreased, indicating that the polarity of the three Chlorella vulgaris charcoals gradually increased; Figure 1 In b, the surface potential of the three Chlorella vulgaris charcoals gradually increased. Among them, the Chlorella vulgaris charcoal in Example 3 had the strongest polarity and the largest surface potential, and the extraction efficiency of the four tetracyclines was the best (Table 1).

[0108] Figure 2 In a, the C1s spectrum of Chlorella vulgaris charcoal has three characteristic peaks, which are graphitic structure polycyclic aromatic hydrocarbon at 284.3 ev, polyether imide at 284.7 ev and polyethylene pyridine at 285.5 ev;Figure 2 b In the Ols spectrum of Chlorella biochar, there are two characteristic peaks, one is the oxygen-containing functional group in polyvinylpyridine at 531.8 ev, and the other is the oxygen-containing functional group in polyetherimide at 533.2 ev. Figure 2 c In the Nls spectrum of Chlorella biochar, there are three characteristic peaks, one is polyvinylpyridine at 398.3 and 399.9 ev, and the other is deprotonated nitrogen-containing group ((NH2)3(C3N3)) (PO2(alkyl3)) at 400.08 ev.

[0109] (2) In order to analyze the pore structure of Chlorella biochar and commercial bamboo biochar, N2 adsorption-desorption experiment (BET) and scanning electron microscopy (SEM) method were used. The analysis results are as follows:

[0110] Table 2 Specific surface area, pore size and pore volume of biochar in example 1, 2, 3 and comparative example 1

[0111] Biochar Specific surface area (m2g-1) Average pore size (nm) Average pore volume (mLg-1) 1 0.243 3.859 0.0021 2 23.183 1.124 0.0011 3 18.935 1.937 0.0061 Commercialized bamboo charcoal 322.861 5.965 0.3017

[0112] The specific surface area of Chlorella biochar in example 1, 2, 3 is 0.24~23.18m 2 g -1 , the average pore size is 1.12~3.85nm, and the average pore volume is 0.0021~0.0061mLg -1 . The specific surface area of commercial bamboo biochar in example 4 is 322.86m 2 g -1 , the average pore size is 5.97nm, and the average pore volume is 0.30mLg -1 .

[0113] Figure 3 The surface of Chlorella biochar in a is smooth and dense, forming a layered aggregate. In Figure 3 b, there is almost no pore structure observed on the surface of Chlorella biochar, which confirms that Chlorella biochar is a non-porous layered material. Figure 3 c, the surface of bamboo biochar is rough and shows a large number of large pore structures. Figure 3 d, it can be observed that the surface of bamboo biochar is distributed with different pore structures, and the number of pores with a pore size less than 50nm is large and widely distributed, which shows that bamboo biochar is a porous material.

[0114] Comparing the relationship between pore structure and solid phase extraction efficiency, it is found that biochar with high specific surface area, pore volume and pore size has high adsorption but low desorption efficiency, which is suitable for pollutant removal. The non-porous Chlorella biochar of the present invention can meet the demand of rapid separation and analysis of trace chemical substances.

[0115] The specific operation method of the Chlorella biochar-solid phase extraction tetracycline method of the present application is as follows:

[0116] 1. Determine the key factors affecting the separation efficiency of solid phase extraction by single factor test. In Design-Expert 13 software, the sample volume, column filling volume, column specification, elution volume and elution volume are set as high level and low level respectively. Details are shown in Table 3:

[0117] Table 3 Two-level five-factor test design of Chlorella biochar solid phase extraction

[0118]

[0119] 2. Statistical analysis can be performed using the ANOVA (Analysis of Variance) tool provided by Design-Expert 13 to obtain the significance of each factor and the significance of interaction, thereby determining the main factors. According to the ANOVA results, further response surface analysis and optimization are performed to obtain the optimal solid phase extraction conditions.

[0120] 3. In the statistical results of the test design in Table 4, except for the elution volume, the other factors of solid phase extraction have a significant effect on the recovery rate of tetracycline antibiotics. In the analysis results of multi-factor interaction, it is found that the interaction of sample volume and column specification is the most significant. This indicates that the main reason affecting the recovery rate of Chlorella biochar solid phase extraction may be the distribution separation mechanism dominated by flow rate and column length.

[0121] Table 4 Analysis results of two-level multi-factor test of Chlorella biochar-solid phase extraction

[0122]

[0123] 4. After fitting by DesignExpert 13 software, the optimal conditions for solid phase extraction of tetracycline by Chlorella biochar are obtained, i.e. the sample volume is 12 mL, the loading amount of Chlorella biochar is 100 mg, the eluent is methanol solution, the eluent volume is 2.0 mL, and the elution times are 3 times (3x2 mL). Good recovery effect of tetracycline is obtained, and the recovery rate is 94.30±3.55%.

[0124] The above specific embodiments are only specific cases of the present application, and the patent protection scope of the present application includes but is not limited to the product forms and styles of the above specific embodiments. Any appropriate changes or modifications made by any ordinary skilled person in the corresponding technical field to the preparation method of Chlorella biochar and the method of solid phase extraction of tetracycline antibiotics in accordance with the claims of the present application shall fall within the patent protection scope of the present application.

Claims

1. A method for solid-phase extraction of tetracycline antibiotics using Chlorella biochar, characterized in that: Includes the following steps: A1. A column packing material was prepared using Chlorella biochar, and the column packing material was filled into the empty solid-phase extraction column to obtain a solid-phase extraction column filled with Chlorella biochar. A2. The solid-phase extraction column was rinsed and activated sequentially with methanol and NaCl aqueous solution; A3. Slowly pass the sample extract through the solid-phase extraction column activated in step A2. After all the sample extract has flowed out, wash the solid-phase extraction column with acetonitrile and then vacuum dry the solid-phase extraction column. A4. Elute the solid-phase extraction column treated in step A3 three times with methanol solution and collect the eluent; The method for preparing Chlorella biochar is characterized by comprising the following steps: S1. Preparation: Weigh the biomass raw materials and place them in a crucible. Cover the crucible and set aside. The biomass raw materials are cell wall broken protein-nucleated Chlorella freeze-dried powder with a protein content ≥55g / 100g, a fat content ≥5g / 100g, and a total sugar content (calculated as glucose) ≥5.5g / 100g. S2. Firing: Place the crucible from step S1 in an electric furnace and fire it using a high-temperature pyrolysis method under a vacuum atmosphere. Under a vacuum of -0.02 to -0.04 MPa, with a heating rate of 10℃ / min, place the alumina crucible into an atmosphere furnace for high-temperature pyrolysis. The pyrolysis temperature is 300 to 700℃ and the hot residence time is 30 to 60 minutes. S3. Cooling: After firing in step S2, remove the crucible and allow it to cool naturally to room temperature.

2. The method for Chlorella biochar-solid phase extraction of tetracycline antibiotics according to claim 1, characterized in that: In step A1, the amount of column packing material used is 100 mg, and the specification of the empty solid phase extraction column is 3 mL; In step A2, the volume of the methanol solution is 5 mL, the concentration of the NaCl aqueous solution is 0.01 mol / L, the pH value is 4.0, and the volume is 5 mL. In step A3, the volume of acetonitrile is 3 mL. The Chlorella biochar solid-phase extraction column is washed with acetonitrile to remove non-target substances, and then vacuum dried for 2 min. In step A4, the volume of the elution buffer is 2.0 mL.

3. The method for Chlorella biochar-solid phase extraction of tetracycline antibiotics according to claim 1, characterized in that: In step A1, the specific steps for preparing column packing material using Chlorella biochar are as follows: (1) Grind the calcined Chlorella biochar to obtain Chlorella biochar powder and store it in a light-proof glass container; (2) Weigh the Chlorella biochar powder obtained in step (1) and add it to the phosphoric acid solution. Stir for 8-12 hours and then filter to obtain acid-treated Chlorella biochar. (3) Wash the acid-treated Chlorella biochar obtained in step (2) with ethanol 3 to 5 times, and then wash with distilled water 3 to 5 times until the washing solution is neutral. (4) Dry the Chlorella biochar washed in step (3) to obtain column packing material. Store the column packing material in a light-proof glass container for later use.

4. The method for Chlorella biochar-solid phase extraction of tetracycline antibiotics according to claim 3, characterized in that: The particle size of the Chlorella biochar powder in step (1) is 100~200 mesh.

5. The method for Chlorella biochar-solid phase extraction of tetracycline antibiotics according to claim 3, characterized in that: In step (2), the concentration of the phosphoric acid solution used is 3 mol / L, and an electric stirrer is used during the stirring process at a speed of 25~35 rpm.

Images