A pretreatment process for microcystin in sediments and its application

Abstract

The present application relates to the technical field of organic matter detection, and specifically discloses a pretreatment process and application of microcystin in sediments. A pretreatment process for microcystin in sediments, comprising the following steps: drying and grinding the sediment; dispersing the pretreated sediment into a solvent and then adding a modified adsorbent, ultrasonicating, filtering to obtain an extract A and a precipitate; washing the precipitate with acetone, taking out the modified adsorbent, adding it to the solvent, heating for desorption, and filtering to obtain an extract B; mixing the extract A and the extract B to obtain a crude extract; the preparation method of the modified adsorbent is: adding tannic acid and a porous material to a chitosan solution, stirring and mixing, then adding a cross-linking agent, stirring and mixing, to obtain; the crude extract is passed through a column and eluted. Adding a modified adsorbent to this process can increase the extraction amount of MC, which is beneficial to the detection of trace microcystin in sediments and reduces the influence of impurities such as proteins on the detection.

Description

Technical Field

[0001] The present application relates to the technical field of organic matter detection, and more specifically, to a pretreatment process for microcystin in sediment and its application. Background Art

[0002] Microcystin (hereinafter referred to as MC) is a toxin released by the rupture of algae cells. Since there are many algae genera that produce microcystin, such as Microcystis, Anabaena, Oscillatoria, Nostoc and Aphanizomenon, and with the intensification of eutrophication of water bodies leading to massive reproduction of algae, algal toxin pollution has also become a global environmental problem and has attracted people's attention. People are also increasingly studying the detection method of algal toxins in the environment. At present, this detection method is mainly for the detection of microcystin in water environment.

[0003] Sediment is a type of loose mineral particles, biological debris and organic matter that is deposited on the earth's surface in the form of solid, liquid or gas under water environments such as land or ocean. Microcystins in water bodies are also adsorbed into sediments. The current detection method for microcystins in sediments includes MC pretreatment and HPLC analysis. During the pretreatment process, MC is first extracted with a solution, followed by column purification and elution.

[0004] However, studies have found that when MC is adsorbed on sediments, the hydrophilic groups in MC-RR, such as arginine and glutamic acid, will be firmly bound to the sediments. Simple solvent extraction methods have poor extraction effects on MC in sediments, resulting in a reduced amount of MC extracted, which is not conducive to the detection of trace microcystins. Summary of the Invention

[0005] In order to increase the extraction amount of MC in sediments and facilitate the detection of microcystins or trace amounts of microcystins in sediments, the present application provides a pretreatment process and application of microcystins in sediments.

[0006] In a first aspect, the present application provides a pre-treatment process for microcystin in sediments, which adopts the following technical solution:

[0007] A pretreatment process for microcystin in sediments, characterized by comprising the following steps: (1) pretreatment of sediments: drying and grinding the sediments;

[0008] (2) Extraction of MC: The pretreated sediment is dispersed in a solvent and then a modified adsorbent is added. The mixture is ultrasonicated and filtered to obtain an extract A and a precipitate. The precipitate is washed with acetone, the modified adsorbent is removed and added to a solvent, the temperature is increased for desorption, and the mixture is filtered to obtain an extract B. The extract A and the extract B are mixed to obtain a crude extract.

[0009] The preparation method of the modified adsorbent is as follows: adding tannic acid and porous material to a chitosan solution, stirring and mixing, and then adding a cross-linking agent, stirring and mixing, to obtain the modified adsorbent;

[0010] (3) Passing the crude extract through a column and eluting.

[0011] By adopting the above technical solution: in this application, a crude extract is obtained by pre-treating the sediment and extracting MC. After the crude extract is passed through a column and eluted, the eluted effluent fraction is tested, and the MC-RR content is detected to be 7.19 mg. When the modified adsorbent is not used in the process, the MC-RR content in the effluent fraction is detected to be 4.73 mg. This shows that the addition of the modified adsorbent during the MC extraction process can increase the MC extraction amount, which is beneficial to the detection of microcystins or trace amounts of microcystins in the sediment. Analysis of the possible reasons is:

[0012] The modified adsorbent uses a porous material loaded chitosan membrane as a base material, which has a certain adsorption effect. The addition of tannic acid causes MC bonds to break, allowing MC, which is originally difficult to separate from the sediment, to escape and adhere to the modified adsorbent. Through subsequent desorption at elevated temperatures, MC enters the extract B. Extract B and extract A together serve as the crude extract, thereby increasing the extraction yield of MC.

[0013] At the same time, proteins undergo complexation under the action of tannic acid, thereby precipitating and becoming difficult to adsorb on the surface of the modified adsorbent, and are lost during the subsequent cleaning process. Therefore, adding modified adsorbents can reduce the impact of impurities such as proteins on detection.

[0014] Optionally, the weight ratio of chitosan to porous material is (0.1-0.2):1.

[0015] By adopting the above technical solution: when the weight ratio of chitosan to porous material is within the above range, the modified adsorbent has the best effect in adsorbing impurities. When the amount of chitosan used exceeds the above range, the effect of the modified adsorbent will decrease.

[0016] Optionally, the weight ratio of the tannic acid to the porous material is (0.05-0.1):1.

[0017] By adopting the above technical solution: when the addition amount of tannic acid is within the above range, the modified adsorbent has better adsorption effect on impurities due to the appropriate ratio; when the addition amount of tannic acid is lower than the above range, the synergistic effect of tannic acid on the modified adsorbent needs to be improved; when the addition amount of tannic acid is greater than the above range, the effect of the modified adsorbent is not further significantly improved. Therefore, considering the comprehensive cost and performance, the addition amount of tannic acid should be within the above range.

[0018] Optionally, the porous material is one of activated carbon, activated clay, diatomaceous earth, activated alumina, activated zeolite, and macroporous resin.

[0019] Optionally, the frequency of the ultrasound is 35-45kHz and the intensity is 1.40-1.45W / cm 2 , time 30-45 minutes.

[0020] By adopting the above technical solution: by controlling the parameters of ultrasonic treatment, the temperature rise of the liquid caused by excessive intensity is avoided, the volatilization of MC is reduced, the MC content retained in the crude extract is increased, and the detection accuracy is improved.

[0021] Optionally, the solvent is one or more of water, methanol, and acetic acid.

[0022] By adopting the above technical solution, water, methanol or acetic acid aqueous solution can be used as the solvent for extracting MC, and can be selected according to needs during actual operation.

[0023] Optionally, the solvent is a mixture of a formaldehyde aqueous solution with a volume concentration of 70-80% and an acetic acid aqueous solution with a concentration of 4-6%.

[0024] By adopting the above technical solution: mixing solutions with the above concentrations, the prepared solvent has the best extraction effect on MC, which is conducive to improving the detection accuracy.

[0025] Optionally, the specific steps of the column are:

[0026] The crude extract was rotary evaporated to a volume of 0.5-1 mL and diluted with water to obtain the sample to be passed through the column;

[0027] The sample to be passed through the column was flowed through the SPE (C 18 ) column, and eluted with water, methanol with a volume concentration of 4-6%, and methanol with a volume concentration of 9-11%.

[0028] By adopting the above technical solution: since the loss of MC will be enhanced when eluting with methanol at a volume concentration of 20%, the methanol concentration during elution should not be too high. At the same time, gradient elution can not only remove impurities with different retention strengths, but also reduce the continuous elution time of high-concentration methanol. Therefore, it is possible to reduce the loss of MC while washing away most of the impurities, thereby improving the accuracy of detection and analysis.

[0029] Optionally, the specific steps of eluting are: eluting the algal toxins on the column with a methanol aqueous solution containing TFA, and collecting the effluent fraction;

[0030] In the methanol solution containing TFA, the concentration of TFA is 0.05-0.15%.

[0031] By adopting the above technical solution: during the elution process, it was found that when the methanol concentration was greater than 70%, further increasing the methanol concentration did not improve the elution of MC, but instead increased the amount of impurities washed away. However, reducing the methanol concentration would greatly increase the time for subsequent concentration, so the formaldehyde concentration was controlled within the above range; and the presence of TFA could make MC easier to elute, thereby improving the elution recovery rate of MC.

[0032] In a second aspect, the present application provides an application of a pretreatment process for microcystins in sediments in the detection of microcystins.

[0033] By adopting the above technical solution: in this application, by adding a modified adsorbent, MC is promoted to break bonds and be removed from the sediment, a part of which adheres to the modified adsorbent and enters the extract B during the subsequent temperature rise and analysis process, thereby increasing the MC content in the crude extract and then increasing the MC extraction amount, which not only improves the detection accuracy but also facilitates the detection of trace MC in the sediment.

[0034] In summary, this application has the following beneficial effects:

[0035] 1. In the MC extraction process of the present application, a modified adsorbent is also added. Tannic acid, on the one hand, promotes the removal of MC from the sediment and increases the MC content in the crude extract. On the other hand, it reduces the impact of impurity detection, facilitates more accurate detection of microcystin content in the sediment, and facilitates the detection of trace amounts of Microcystis.

[0036] 2. In this application, by controlling the time, frequency and other parameters of ultrasound, the occurrence of water vapor generated by temperature increase carrying away MC is reduced, which is beneficial to reducing the loss of MC during the detection process and improving the detection accuracy;

[0037] 3. The specific steps of controlling elution and washing in this application can better remove impurities and reduce impurity contamination, and facilitate the elution of MC from the column, thereby improving detection accuracy. DETAILED DESCRIPTION

[0038] The present application is further described in detail below with reference to the embodiments.

[0039] Preparation Example 1

[0040] A modified adsorbent is prepared by the following steps:

[0041] 5 g of chitosan was dissolved in 250 mL of 1% acetic acid aqueous solution to obtain a chitosan solution;

[0042] Add 4g of tannic acid and 100g of porous material to the chitosan solution, stir and mix until uniform, add 40g of cross-linking agent (7% volume concentration of glutaraldehyde solution) at 60°C, heat to 90°C and solidify for 2h, spray dry to obtain;

[0043] Chitosan: Deacetylation degree 93%; porous materials can be selected from activated carbon, activated clay, diatomaceous earth, activated alumina, activated zeolite, and macroporous resin. These options do not significantly affect the test results. In the examples of this application, only activated carbon is used as an example for a brief description.

[0044] Preparation Example 2-7, Comparative Preparation Example 1-2

[0045] A modified adsorbent, which differs from Preparation Example 1 in that the components and their corresponding weights are shown in Table 1.

[0046] Table 1 Components and their weights (kg) in Preparation Examples 1-7 and Comparative Preparation Examples 1-2

[0047]

[0048] Example 1

[0049] A pretreatment process for microcystin in sediments, the specific steps of which are:

[0050] (1) Sediment pretreatment: air-dry the sediment at 25°C, grind it and pass it through an 80-mesh sieve;

[0051] (II) Extraction of MC: 10 g of the pretreated sediment was dispersed in 30 mL of solvent (distilled water), and 10 g of the modified adsorbent (prepared in Preparation Example 1) was added. Ultrasonication (frequency of 35 kHz, intensity of 1.40 W / cm 2 , time 30 min), then centrifuged at 3000 r / min for 10 min, and filtered through a 0.45 μm filter membrane to obtain extract A and a precipitate; the precipitate was washed with 50 mL of acetone, the modified adsorbent in the precipitate was removed and added to 10 mL of solvent (distilled water), the temperature was raised to 80° C., and the mixture was allowed to stand for 20 min for desorption, and the extract B was obtained by filtration; the extract B was added to the extract A, and the mixture was mixed to obtain a crude extract;

[0052] (3) Passing the crude extract through the column and eluting:

[0053] The specific steps of column are: rotary evaporation of the crude extract to a volume of 0.5 mL, adding water to dilute to a methanol content of 20% to obtain the sample to be column-passed; SPE (C 18 ) was activated with 10 mL of methanol, adjusted with 10 mL of redistilled water, and dried to obtain the activated SPE (C 18 );

[0054] Take 500mL of the sample to be passed through the activated SPE (C 18 ) column, and gradient elution was performed using 10 mL of water, 10 mL of 4% methanol aqueous solution, and 10 mL of 9% methanol aqueous solution in sequence;

[0055] The specific steps of elution are: using 10 mL of 70% methanol to elute the algal toxins on the column and collecting the effluent fractions.

[0056] Example 2

[0057] A pretreatment process for microcystin in sediments, the specific steps of which are:

[0058] 1. Sediment pretreatment: air dry the sediment at 25°C, grind it and pass it through an 80-mesh sieve;

[0059] (II) Extraction of MC: 10 g of pretreated sediment was dispersed in 30 mL of solvent (distilled water) and ultrasonicated once (frequency 40 kHz, intensity 1.42 W / cm 2 , time 40 min), then centrifuged at 3000 r / min for 10 min, and filtered through a 0.45 μm filter membrane to obtain extract A and a precipitate; the precipitate was washed with 50 mL of acetone, the modified adsorbent in the precipitate was removed and added to 10 mL of solvent (distilled water), the temperature was raised to 80° C., and the mixture was allowed to stand for 20 min for desorption, and filtered to obtain extract B; extract B was added to extract A, mixed, and a crude extract was obtained;

[0060] (3) Passing the crude extract through the column and eluting:

[0061] The specific steps of column are: rotary evaporation of the crude extract to a volume of 0.8 mL, adding water to dilute to a methanol content of 20% to obtain the sample to be column-passed; SPE (C 18 ) was first activated with 10 mL of methanol, adjusted with 10 mL of reforming water, and dried to obtain the activated SPE (C 18 );

[0062] Take 500mL of the sample to be passed through the activated SPE (C 18 ) column, and gradient elution was performed using 10 mL of water, 10 mL of 5% methanol aqueous solution, and 10 mL of 10% methanol aqueous solution in sequence; the specific steps of elution were: 10 mL of 70% methanol was used to elute the algal toxins on the column, and the effluent components were collected.

[0063] Example 3

[0064] A pretreatment process for microcystin in sediments, the specific steps of which are:

[0065] (1) Sediment pretreatment: The surface sediments of Dianchi Lake were dried naturally at 25°C, ground and pulverized to pass through an 80-mesh sieve;

[0066] (II) Extraction of MC: 10 g of pretreated sediment was dispersed in 20 mL of solvent (distilled water) and ultrasonicated once (frequency 45 kHz, intensity 1.45 W / cm 2 , time 45 minutes), then centrifuged at a speed of 3000 r / min for 10 minutes, and filtered through a 0.45 μm filter membrane to obtain extract A and a precipitate; the precipitate was washed with 50 mL of acetone, and the modified adsorbent in the precipitate was removed and added to 10 mL of solvent (distilled water), heated to 80° C., allowed to stand for 20 minutes for desorption, and filtered to obtain extract B; extract B was added to extract A, mixed, and a crude extract was obtained;

[0067] (3) Passing the crude extract through the column and eluting:

[0068] The specific steps of column are: rotary evaporation of the crude extract to a volume of 1 mL, dilution with water to a methanol content of 20%, and obtaining the sample to be column-passed; 18 ) was first activated with 10 mL of methanol, adjusted with 10 mL of reforming water, and dried to obtain the activated SPE (C 18 );

[0069] Take 500mL of the sample to be passed through the activated SPE (C 18 ) column, and gradient elution was performed using 10 mL of water, 10 mL of 6% methanol aqueous solution, and 10 mL of 11% methanol aqueous solution in sequence; the specific steps of elution were: 10 mL of 70% methanol was used to elute the algal toxins on the column, and the effluent components were collected.

[0070] Comparative Example 1

[0071] A pretreatment process for microcystin in sediment differs from Example 1 in that no modified adsorbent is added in step (2). Step (2) is specifically as follows:

[0072] (II) Extraction of MC: 10 g of pretreated sediment was dispersed in 20 mL of solvent (distilled water) and ultrasonicated (frequency 40 kHz, intensity 1.42 W / cm 2, time 40 min), then centrifuged at a speed of 3000 r / min for 10 min, and filtered through a 0.45 μm filter membrane to obtain extract A and a precipitate; after washing the precipitate with 50 mL of acetone, 10 mL of solvent (distilled water) was added, the temperature was raised to 60° C., allowed to stand for 20 min, and filtered to obtain extract B; extract B was added to extract A, mixed, and a crude extract was obtained.

[0073] Comparative Example 2

[0074] A pretreatment process for microcystin in sediments differs from Example 1 in that an equal amount of activated carbon is used instead of the modified adsorbent.

[0075] Examples 4-9, Comparative Examples 3-4

[0076] The usage of the modified adsorbent is shown in Table 2. The modified adsorbent prepared in Preparation Example 1 is replaced with the following substances in equal amounts.

[0077] Table 2 Usage of modified adsorbents in Examples 1, 4-9, and Comparative Examples 3-4

[0078] Example 1 4 5 6 7 8 9 Preparation example of modified adsorbent 1 2 3 4 5 6 7 Comparative Example 3 4 \ \ \ \ \ Comparative preparation example of modified adsorbent 1 2 \ \ \ \ \

[0079] Examples 10-13

[0080] A pretreatment process for microcystin in sediment differs from Example 8 in that the solvent is used differently, as follows:

[0081] Example 10: An equal amount of a methanol aqueous solution with a volume concentration of 75% is used instead of water.

[0082] Example 11: A mixture of equal amounts of a methanol aqueous solution with a volume concentration of 70% and an ethanol aqueous solution with a volume concentration of 4% is used instead of water; wherein the volume ratio of the methanol aqueous solution to the ethanol aqueous solution is 8:2.

[0083] Example 12: A mixture of equal amounts of a methanol aqueous solution with a volume concentration of 75% and an ethanol aqueous solution with a volume concentration of 5% is used instead of water; wherein the volume ratio of the methanol aqueous solution to the ethanol aqueous solution is 8:2.

[0084] Example 13: A mixture of equal amounts of an 80% by volume methanol aqueous solution and a 6% by volume ethanol aqueous solution is used instead of water; wherein the volume ratio of the methanol aqueous solution to the ethanol aqueous solution is 8:2.

[0085] Examples 14-16

[0086] A pretreatment process for microcystin in sediments differs from Example 8 in that an equal amount of methanol-water solution containing TFA is used in place of methanol to elute the microcystin on the column in step (iii), specifically as follows:

[0087] [Methanol aqueous solution containing TFA: obtained by mixing methanol aqueous solution with TFA at a volume concentration of 70% until uniformly mixed.]

[0088] Example 14: In the methanol aqueous solution containing TFA, the mass concentration of TFA is 0.05%.

[0089] Example 15: In the methanol aqueous solution containing TFA, the mass concentration of TFA is 0.10%.

[0090] Example 16: In the methanol aqueous solution containing TFA, the mass concentration of TFA is 0.15%.

[0091] MC-RR content detection

[0092] The surface sediments of Dianchi Lake were pretreated using the pretreatment processes described in the Examples and Comparative Examples to obtain an outflow fraction. The outflow fraction was rotary evaporated and then diluted to 300 μL with a 75% methanol-water solution to obtain a sample solution. The sample was analyzed using a Waters 600 chromatograph, and the MC-RR content in the sample solution was determined using an external standard method. Chromatographic parameters included: analytical column: BDS Hypersil C18 (4.6×250 mm), column temperature: 25°C, mobile phase: methanol:water (containing 0.05% TFA) = 62:38, mobile phase flow rate: 1 ml / min; injection volume: 20 μL.

[0093] Table 3 Performance test results

[0094]

[0095] As can be seen from Table 3, when the modified adsorbent prepared in Preparation Example 1 was added to Example 1, the MC-RR content in the effluent component was detected to be 7.19 mg, while in Comparative Example 1, no modified adsorbent was used, and the MC-RR content in the effluent component was detected to be 4.73 mg. In Comparative Example 2, an equal amount of activated carbon was used instead of the modified adsorbent, and the MC-RR content was detected to be 3.16 mg. This shows that the use of the modified adsorbent in the MC extraction process can increase the extraction amount of MC-RR in the pretreatment process, while the use of adsorbents such as activated carbon reduces the extraction amount of MC-RR.

[0096] Analysis suggests that the reason for this may be that the tannic acid in the modified adsorbent, on the one hand, breaks the bonds of some MC in the sediment that is difficult to release, allowing it to escape from the sediment and attach to the activated carbon and chitosan. As the temperature rises and the pressure decreases, it enters Extract B and merges into the crude extract, thereby increasing the MC content in the crude extract. On the other hand, it reduces the adhesion of proteins to the surface of the modified adsorbent, reducing the interference of impurities in the analysis. When only activated carbon is used, not only does the lack of tannic acid fail to promote the detachment of MC from the sediment, but it also reduces the extraction efficiency of the solvent, resulting in a decrease in the MC extraction amount. At the same time, the adsorption of activated carbon increases the amount of impurities that enter the crude extract.

[0097] The difference between Examples 4-6 and Example 1 is that the amount of chitosan used in the modified adsorbent is different. When the amount of chitosan used is within the range of Examples 4-6, the content of MC in the outflow component is higher and the effect of the modified adsorbent is better. The difference between Comparative Example 3 and Example 1 is that chitosan is not used in the modified adsorbent, and the MC content in the outflow component is reduced.

[0098] The difference between Examples 7-9 and Example 1 is that the amount of tannic acid used in the modified adsorbent is different. When the amount of tannic acid used is within the range of Examples 7-9, the content of MC in the effluent component is higher and the effect of the modified adsorbent is better. The difference between Comparative Example 4 and Example 1 is that tannic acid is not used in the modified adsorbent, and the content of MC in the effluent component decreases.

[0099] The difference between Examples 14-16 and Example 8 is that the elution liquid contains TFA at different concentrations. The presence of TFA can make MC easier to elute and improve the elution recovery rate of MC.

[0100] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A pretreatment process for microcystin in sediments, characterized in that: The following steps are involved: (1) Sediment pretreatment: drying, grinding and crushing the sediment; (II) Extraction of MC: The pretreated sediment is dispersed in a solvent and then a modified adsorbent is added. Ultrasonic treatment is performed and the precipitate is filtered to obtain an extract A. The precipitate is washed with acetone, the modified adsorbent is removed and added to a solvent, the temperature is increased to desorb, and the extract B is filtered to obtain an extract. The extract A and the extract B are mixed to obtain a crude extract. The preparation method of the modified adsorbent is as follows: adding tannic acid and porous material to a chitosan solution, stirring and mixing, and then adding a cross-linking agent, stirring and mixing, to obtain the modified adsorbent; (3) Passing the crude extract through a column and eluting.

2. The pretreatment process for microcystins in sediments according to claim 1, characterized in that: The weight ratio of the chitosan to the porous material is (0.1-0.2):

1.

3. The pretreatment process for microcystins in sediments according to claim 1, characterized in that: The weight ratio of the tannic acid to the porous material is (0.05-0.1):

1.

4. The pretreatment process for microcystins in sediments according to claim 1, characterized in that: The porous material is one of activated carbon, activated clay, diatomaceous earth, activated alumina, activated zeolite and macroporous resin.

5. The pretreatment process for microcystin in sediment according to claim 1, characterized in that: The ultrasound has a frequency of 35-45 kHz and an intensity of 1.40-1.45 W / cm 2 , time 30-45 minutes.

6. The pretreatment process for microcystins in sediments according to claim 1, characterized in that: The solvent is water, methanol, or a mixture of methanol and acetic acid.

7. The pretreatment process for microcystins in sediments according to claim 6, characterized in that: The solvent is a mixture of a methanol aqueous solution with a volume concentration of 70-80% and an acetic acid aqueous solution with a concentration of 4-6%.

8. The pretreatment process for microcystins in sediments according to claim 1, characterized in that: The specific steps of the column are: The crude extract was rotary evaporated to a volume of 0.5-1 mL and diluted with water to obtain the sample to be passed through the column; The sample to be passed through the SPE column was flowed at a rate of 4-5 mL / min, and eluted with water, methanol with a volume concentration of 4-6%, and methanol with a volume concentration of 9-11%.

9. The pretreatment process for microcystins in sediments according to claim 1, characterized in that: The specific steps of the elution are: using a methanol aqueous solution containing TFA to elute the microcystin on the column, and collecting the effluent fraction; In the methanol solution containing TFA, the concentration of TFA is 0.05-0.15%.

10. Use of the pretreatment process for microcystins in sediments according to any one of claims 1 to 9 in the detection of microcystins.

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