A method for simultaneously detecting five trace herbicides in water

Abstract

A method for simultaneously detecting five trace herbicides in water belongs to the field of environmental analysis and detection technology. It uses thymol as a hydrogen bond acceptor, and an organic acid selected from decanoic acid, n-hexanoic acid, n-butyric acid, and undecylenic acid as a hydrogen bond donor to synthesize a low eutectic solvent as an extractant, and performs dispersed liquid-liquid microextraction on the water sample; then, high performance liquid chromatography is performed to determine five trace herbicides in the water, wherein the herbicides are bentazon, pyrazosulfuron-methyl, cypermethrin, fenoxaprop-butyl, and pyrimidine oxadiazine. The present invention uses a green solvent low eutectic solvent as a pre-concentrator and extractant, which has the advantages of low cost, safety, and non-toxicity. At the same time, the hydrophobic DES has the characteristics of a low freezing point, so it can be solidified, which greatly reduces the difficulty of enriching trace herbicides in water, makes the experimental operation simple, and greatly reduces the amount of extractant used.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental analysis and detection, and particularly relates to a method for simultaneously detecting five trace herbicides in water. Background Art

[0002] With the development of modern agriculture, herbicides have been widely used in agricultural production. Although the commonly used herbicides in agricultural production have the characteristics of low toxicity and short half-life, their large amount and high frequency of use lead to their "persistent" existence in the environment. Therefore, herbicide pollutants have become one of the most common organic pollutants in the world. Although herbicides are usually sprayed in the air, aquatic ecosystems can be exposed to herbicides through runoff or spray drift, accidental leakage, etc. Secondly, rain washing can also cause herbicides to enter the groundwater environment through soil faults. Most importantly, most herbicides exist in the form of ions and are easy to transfer. Herbicides that enter the water area are accumulated in the water environment through a series of transformations, and finally enter the organism through bioaccumulation under the influence of the food chain. Considering the above facts, the detection and quantitative analysis of herbicide residues in water areas are of great significance.

[0003] So far, a variety of sample preparation methods have been used for pesticide analysis. Among them, SPE, SPME and liquid-liquid extraction are the most commonly used pesticide pretreatment methods and technologies. In recent years, separation and enrichment technologies have developed towards simplification, miniaturization, and minimization of organic reagent consumption. Among them, dispersed liquid-liquid microextraction (DLLME) has shown high sample extraction ability and good enrichment effect, and is simple and fast. However, the selection of extraction solvent in this method is one of the most critical factors. Deep eutectic solvents (DES) are green solvents with the advantages of low cost, good thermal and electrochemical stability, non-toxicity, high purity, and no chemical reaction with water. They have received widespread attention; they have been used as extraction solvents for various analytes. Pre-concentration of sample extracts by DES-based DLLME can achieve higher sensitivity and reduce potential matrix interference. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for simultaneously detecting five trace herbicides in water. The present invention designs a new type of hydrophobic DES for the first time for the pre-concentration of five herbicides in water samples. By adding a dispersant, the DES forms an insoluble self-aggregate of nanometer or molecular size, and adding salt as an electrolyte to produce a salting-out or salt-dissolving effect, thereby reducing the solubility of the herbicide in the aqueous solution and promoting its distribution into the extractant phase, that is, the hydrophobic DES phase in the present invention. Compared with other detection methods, the amount of organic solvent used in the present invention is greatly reduced, and the detection is more sensitive.

[0005] The present invention specifically adopts the following technical solutions:

[0006] A method for simultaneously detecting five trace herbicides in water, comprising: using thymol as a hydrogen bond acceptor and an organic acid selected from decanoic acid, n-hexanoic acid, n-butyric acid and undecylenic acid as a hydrogen bond donor to synthesize a deep eutectic solvent (DES) as an extractant; performing dispersed liquid-liquid microextraction on a water sample; and then performing high performance liquid chromatography to determine the five trace herbicides in water, wherein the herbicides are bentazon (BTZ), pyrazosulfuron-methyl (PSE), alfaphos (ALF), fenoxaprop-butyl (FPE) and pyrifos-butyl (PBZ).

[0007] Furthermore, the method specifically comprises the following steps:

[0008] 1) Synthesis of hydrophobic DES: DES was synthesized using thymol as a hydrogen bond acceptor and an organic acid as a hydrogen bond donor as an extractant;

[0009] 2) Collect water samples, remove impurities in the water samples by vacuum filtration, filter them with a 0.45 μm aqueous phase filter membrane, and store them in a refrigerator at 4°C for future use;

[0010] 3) adding the extractant obtained in step 1) to the water sample obtained in step 2), and adding dispersant and salt at the same time, adjusting the pH to 6-7, placing on a vortex mixer for vortexing, centrifuging to accelerate phase separation after reaching extraction equilibrium, taking the upper DES phase after centrifugation, diluting to 1 mL with methanol, and performing high performance liquid chromatography analysis to obtain a sample spectrum;

[0011] 4) Establishment of standard curve: weigh the standard substances of BTZ, PSE, ALF, FPE and PBZ, prepare single standard stock solutions with methanol or acetonitrile as solvent, and then prepare mixed standard working solutions as needed, and test the obtained mixed standard solutions under the same high performance liquid chromatography analysis conditions as in step 3) to obtain standard substance chromatograms, and draw standard curves of BTZ, PSE, ALF, FPE and PBZ respectively with the injection concentration of each standard substance in the mixed standard solution as the abscissa x and the peak area of ​​the corresponding standard substance in the standard substance spectrum as the ordinate y;

[0012] 5) Analysis of test results: The peak area values ​​of each herbicide component in the sample spectrum obtained in step 3) are respectively substituted into the corresponding standard curve obtained in step 4), and the content of the herbicide component in the sample is obtained by calculation.

[0013] Furthermore, in the step 1), the molar ratio of thymol to the organic acid hydrogen bond donor is 1:3 to 3:2.

[0014] Furthermore, in the step 1), the organic acid is preferably n-butyric acid, and the molar ratio of thymol to n-butyric acid is 1:1.

[0015] Furthermore, in the step 3), the amount of water sample is 5-10 mL, preferably 8 mL, and the amount of extractant is 100-500 μL, preferably 150 μL.

[0016] Furthermore, in step 3), the salt is sodium sulfate or sodium chloride with a concentration of 0-0.6 mol / L, preferably sodium chloride with a concentration of 0.4 mol / L.

[0017] Furthermore, in step 3), the dispersant is methanol, ethanol, acetone and acetonitrile.

[0018] Furthermore, in step 3), the vortex time is 2-5 min, preferably 3 min.

[0019] Furthermore, in step 3), the centrifugal speed is 4000 rpm and the time is 10 min.

[0020] Furthermore, the conditions for the high performance liquid chromatography analysis in step 3) and step 4) are as follows: chromatographic column: C18 column, 250×4.6 mm, 5 μm; mobile phase is acetonitrile and 0.1% phosphoric acid aqueous solution, mobile phase flow rate is 0.8-1.2 mL / min, preferably 1 mL / min; column temperature is 20-40° C., preferably 35° C., detection wavelength is 220-260 nm, preferably 250 nm.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention adopts the technology of combining dispersed liquid-liquid microextraction with vortex to pre-concentrate trace herbicides in water samples, and invents, designs and synthesizes a green new hydrophobic DES solution to replace traditional organic solvents as extractants, and uses vortex to replace toxic organic dispersants, integrating concentration and extraction, and has the advantages of simple and convenient operation, short extraction time, high extraction efficiency, safety and environmental protection, and small amount of extractant and sample.

[0023] At present, herbicide pollution has become an important research topic in environmental protection, and its detection and removal are the primary goals. The present invention uses the green solvent DES as a pre-concentrator and extractant, which has the advantages of low cost, safety and non-toxicity. At the same time, hydrophobic DES has the characteristics of a low freezing point, so it can be solidified, which greatly reduces the difficulty of removing and collecting trace herbicides in water, makes experimental operations simple, and greatly reduces the amount of extractant used. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Liquid phase diagram of five herbicide standards;

[0025] Figure 2 Liquid phase diagram of herbicide samples;

[0026] Figure 3 Effects of different extractants on the extraction of five herbicides;

[0027] Figure 4 Effect of extraction agent dosage on extraction effect;

[0028] Figure 5 The effect of sample amount on extraction effect;

[0029] Figure 6 Effects of different dispersants on extraction performance;

[0030] Figure 7 Effect of different salts on extraction performance;

[0031] Figure 8 Effect of NaCl dosage on extraction effect;

[0032] Fig. 9 This method is used for actual water sample extraction. DETAILED DESCRIPTION

[0033] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0034] Example 1: A method for simultaneously detecting five trace herbicides in water

[0035] 1) Synthesis of hydrophobic DES: Using thymol as a hydrogen bond acceptor and an organic acid as a hydrogen bond donor to synthesize DES as an extractant, wherein the molar ratio of thymol to the hydrogen bond donor is 1:3 to 3:2, wherein the organic acid comprises one of decanoic acid, n-hexanoic acid, n-butyric acid and undecylenic acid, preferably the hydrogen bond donor is n-butyric acid, and the molar ratio of thymol to n-butyric acid is 1:1;

[0036] 2) Collect water samples, remove impurities in the water samples by vacuum filtration, filter them with a 0.45 μm aqueous phase filter membrane, and store them in a refrigerator at 4°C for future use;

[0037] 3) adding the extractant obtained in step 1) to the water sample obtained in step 2), the water sample volume is 5-10 mL, preferably 8 mL, the extractant dosage is 100-500 μL, preferably 150 μL, and dispersant and salt are added at the same time, the salt is sodium sulfate or sodium chloride, the concentration is 0-0.6 mol / L, preferably sodium chloride, the concentration is 0.4 mol / L, the dispersant is methanol, ethanol, acetone and acetonitrile, the pH is adjusted to 6-7, and vortexed on a vortex mixer for assistance, the vortex time is 2-5 min, preferably 3 min, after reaching extraction equilibrium, centrifuge to accelerate phase separation, the centrifugal speed is 4000 rpm, the time is 10 min, after centrifugation, the upper DES phase is aspirated and fixed to 1 mL with methanol, and analyzed by high performance liquid chromatography to obtain a sample spectrum;

[0038] 4) Establishment of standard curve: weigh the standard substances of BTZ, PSE, ALF, FPE and PBZ, prepare single standard stock solutions with methanol or acetonitrile as solvent, and then prepare mixed standard working solutions as needed, and test the obtained mixed standard solutions under the same high performance liquid chromatography analysis conditions as in step 3) to obtain standard substance chromatograms, and draw standard curves of BTZ, PSE, ALF, FPE and PBZ respectively with the injection concentration of each standard substance in the mixed standard solution as the abscissa x and the peak area of ​​the corresponding standard substance in the standard substance spectrum as the ordinate y;

[0039] 5) Analysis of test results: The peak area values ​​of each herbicide component in the sample spectrum obtained in step 3) are respectively substituted into the corresponding standard curve obtained in step 4) to calculate the content of the herbicide component in the sample.

[0040] The conditions for the HPLC analysis in the above steps 3) and 4) are as follows: chromatographic column: C18 column, 250×4.6 mm, 5 μm; mobile phase is acetonitrile and 0.1% phosphoric acid aqueous solution, mobile phase flow rate is 0.8-1.2 mL / min, preferably 1 mL / min; column temperature is 20-40° C., preferably 35° C., detection wavelength is 220-260 nm, preferably 250 nm.

[0041] Example 2: Optimization and verification of method conditions for simultaneous detection of five trace herbicides in water 1.1 Main instruments and reagents

[0042] The herbicide content after DES pre-enrichment was determined using a Shimadzu high performance liquid chromatography analysis system (Shimadzu), a separation chromatography system (LC-20AD), an automatic sampling system (SIL-20A), a column chamber (CTO-20A) and a UV / visible detector (SPD-20A). A vortex mixer (LMS, Japan) was used as an auxiliary device to extract the herbicides from the sample phase to the extraction phase. The extraction phase in the sample was separated using a 80-2 centrifuge (China OEM, China).

[0043] 1.2 Preparation of hydrophobic deep eutectic solvent

[0044] According to a certain molar ratio, the mass of HBA and HBD were accurately weighed, mixed, and placed in a dry beaker. Heat and stir in a 90°C water bath until a uniform and clear liquid is formed. Let stand at room temperature overnight. Table 1 lists the HBA, HBD, and molar ratio of the DES used in this experiment.

[0045] Table 1

[0046]

[0047] 1.3 Sample processing

[0048] 50 mL of the collected water sample was centrifuged at 4000 rpm for 10 min. The supernatant was filtered through a 0.45 μm filter membrane to obtain a water sample, which was then stored in a refrigerator at 4°C for use.

[0049] 1.4 Extraction process

[0050] In a 15.0mL centrifuge tube, add 8mL of the water sample in 1.3, 150μL DESs, and 0.187g NaCl respectively. After vortexing for 3min, centrifuge the tube at 4000r / min for 10min. After stratification, draw the upper DES layer and dilute it to 1.0mL with methanol to obtain the test solution; for HPLC testing.

[0051] 1.5 HPLC determination

[0052] The peak areas of the five herbicides to be tested were determined using the following chromatographic conditions, and the contents of BTZ, PSE, ALF, FPE, and PBZ were obtained by substituting the standard working curve equation (Table 2). Chromatographic conditions: chromatographic column: WondaCract ODS-2 column (5μm, 250×4.6mm); acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution program: 0-10min, 50-54%A; 10-20min, 54%A; 20-35min, 54-80%A; 35-42min, 80%A, 42-49min, 50%A. The detection wavelength was 250nm, the column temperature was 35°C, and the injection volume was 10μL.

[0053] 2 Results and analysis

[0054] 2.1 Optimization of extraction conditions

[0055] In order to obtain a higher extraction efficiency, DES was used to extract water samples containing herbicides, including BTZ, PSE, ALF, FPE, and PBZ, and the effects of different extraction parameters on the amount of herbicides extracted in water samples were studied. Therefore, in order to establish a liquid-liquid microextraction method for the determination of herbicides by DESs, the parameters controlling the performance, such as the selection of extraction phase type, sampling volume, dispersant, DESs volume, and optimization of ionic strength, were investigated.

[0056] 2.1.1 DESs types and volumes

[0057] The selection of extractants is crucial in the process of liquid phase microextraction. A good extractant can often effectively extract the analyte from a large amount of matrix with a small amount, achieving the purpose of separation and enrichment at the same time. The composition of DES will affect the properties of the DES solvent, and then affect the extraction rate of the compound. In this study, thymol was used as HBA, and four organic acids, hexanoic acid, butyric acid, caprylic acid and undecylenic acid, were used as HBD to prepare six DESs. Figure 3 As shown, under the same other conditions, the DES formed by thymol and n-butyric acid (1:1) has higher extraction efficiency than other DES.

[0058] It is determined that DES prepared by thymol and n-butyric acid in a ratio of 1:1 is used as the extractant. The volume of DES has a certain influence on the extraction efficiency. This study conducted experiments on the extraction efficiency of 100, 150, 200, 250 μL and 130 μL volumes. Figure 4 It can be seen that when the DES volume is 150 μL, the extraction peak area reaches the maximum, so it is selected for the next step of the experiment.

[0059] 2.1.2 Extraction sample volume

[0060] The volume of the sample has a certain influence on the extraction efficiency. This study conducted experiments on the extraction efficiency of 2, 4, 6, 8 and 10 mL volumes. Figure 5 It can be seen that when the water sample volume is 8 mL, the extraction peak area reaches the maximum, so the water sample volume of 8 mL and the DES volume of 150 μL were selected for the next experiment.

[0061] 2.1.3 Types of dispersants

[0062] In microextraction studies, the addition of a dispersant can cause the extractant to form insoluble self-aggregates of nanometer or molecular size, which are separated from the sample solution after centrifugation. In this way, the interaction between the analyte and the extraction solvent is increased. Therefore, the effects of different dispersant solvents (methanol, ethanol, acetone and acetonitrile) on the extraction effect were studied. Figure 6It can be seen that for PBZ, adding methanol can improve its extraction efficiency; for BTZ, PSE, ALF and FPE, adding extractants has no effect on their extraction effects. Therefore, according to the results, it is possible to choose not to add dispersants in subsequent studies. This may be related to the properties of DESs. The density of DES is less than that of water. In the extraction process, the use of external forces (such as temperature control, boiling, etc.) to assist the extraction can achieve the goal of not using dispersants. In this study, the vortex method was used, which allowed the components of different polarities in the sample solution to be adsorbed on different parts of the DESs or enter the hydrophobic core and be extracted by the DESs.

[0063] 2.1.3 Solution ionic strength

[0064] Electrolytes have different effects in different systems, so it is necessary to investigate the effects of different electrolytes in the system. Adding soluble salts to water samples makes organic compounds more dispersed in the medium and easier to combine with the extractant. In fact, adding electrolytes to aqueous solutions may produce different effects, including salting out, salting out, or no effect on the extraction efficiency. Generally, the salting out effect can reduce the solubility of the analyte in aqueous solution and promote its distribution into the extractant phase, that is, the hydrophobic DES phase in this study. In order to study the effect of the addition of electrolytes on the extraction effect, different types of salts and their dosages were explored. First, sodium acetate (CH40), the most commonly used salt in DLLME, was studied. 3 COONa), magnesium sulfate (MgSO 4 ), sodium carbonate (Na 2 CO 3 ), sodium sulfate (Na 2 SO 4 ) and sodium chloride (NaCl) at the same molar concentration on the extraction effect, Figure 7 The results in the results show that at the same molar concentration, the addition of NaCl has a stronger promoting effect on the extraction, so NaCl electrolyte was chosen for extraction research.

[0065] At the same time, the effect of NaCl dosage on the extraction effect was further studied. The NaCl addition amount was optimized within the range of 0-0.6 mol / L. Figure 8 The results show that the extraction effect is improved when the NaCl content is in the range of 0-0.4mol / L, while further increase in the amount of salt has no significant effect on the extraction effect. From the results, it can be seen that there is a salting-out effect in the system in this study, and it is believed that the salting-out effect increases the extraction rate. Therefore, 0.4mol / L NaCl is the most appropriate choice.

[0066] 2.2 Validation of analytical methods

[0067] After the above experimental optimization, the analytical methodology of the five herbicides was further verified, including linear range, limit of detection (LOD), limit of quantification (LOQ), sample recovery rate, etc. (Table 2). The peak area and herbicide concentration were plotted into a linear regression curve to obtain a linear regression equation. The linear range of BTZ was 800-25000μg / L, and the linear range of PSE, ALF, FPE and PBZ was 800-10000μg / L. All linear regression equations showed good linear relationships (r 2 ≥0.998). LODs and LOQs were calculated when the signal-to-noise ratio was 3 and 10, respectively, and the LODs were 20-400μg / L and the LOQs were 70-1400μg / L. Under the optimal conditions, three concentration levels of standard solutions were added to the sample, namely 1000, 2000, and 3000μg / L. Each sample spike solution with different concentrations was subjected to three DES-DLLME processes. The extract phase obtained in each process was analyzed by HPLC. The RSD (n=3) of the five herbicides was calculated to be between 0.997-9.669%; the spike recovery rate was between 70-125% (Table 2). This shows that the method has good accuracy.

[0068] Table 2 Standard curve equation, linear correlation coefficient, linear range, limit of detection (LOD), limit of quantification (LOQ), recovery rate and enrichment factor (EF) of each component

[0069]

[0070] In addition, the intra-day and inter-day precision were tested and analyzed with mixed standard solutions of 3 different concentration gradients. The results showed that the RSD range of intra-day and inter-day precision was 0.04%-7.11%, indicating that the method has good precision.

[0071] 2.3 Analysis of real samples

[0072] The above DES-DLLME combined with HPLC analysis method was used to analyze 11 different environmental water samples (using the optimal method of Example 1) to further evaluate the applicability and reliability of the method. Fig. 9 As shown in the data, alfaphos (ALF) was not detected in any of the 11 water samples, which may be related to the degradation cycle of ALF and the sampling time; pyrazosulfuron-methyl (PSE) had the highest detection amount, followed by bentazon (BTZ), fenoxaprop-butyl (FPE) and pyrifos-butyl (PBZ).

[0073] 3. Conclusion

[0074] The present invention proposes an analytical method for simultaneously detecting the content of five herbicides, BTZ, PSE, ALF, FPE, and PBZ, in water samples by combining a vortex-assisted hydrophobic low eutectic solvent dispersed liquid-liquid microextraction method (DES-DLLME) with HPLC. Under the action of the vortex, the enrichment multiple can reach 19-98 times or more without the use of a dispersant, thereby reducing the use of organic solvents. The experimental results of the accuracy, precision, linear range, detection limit, etc. of the method show that the method can be used for the analysis of actual samples such as water, which can provide technical support for the research and analysis of environmental pesticide residues.

Claims

1. A method for simultaneously detecting five trace herbicides in water, characterized in that A low eutectic solvent was synthesized using thymol as a hydrogen bond acceptor and an organic acid selected from decanoic acid, hexanoic acid, butyric acid and undecylenic acid as a hydrogen bond donor as an extractant; a water sample was subjected to dispersed liquid-liquid microextraction; and then high performance liquid chromatography was used to determine five trace herbicides in the water, the herbicides being bentazon, pyrazosulfuron-methyl, thiabendazim, fenoxaprop-butyl and pyrimidinesulfuron-butyl; The specific steps include: 1) Synthesis of hydrophobic DES: Using thymol as a hydrogen bond acceptor and an organic acid as a hydrogen bond donor to synthesize DES as an extractant; the molar ratio of thymol to the organic acid hydrogen bond donor is 1:3 to 3:2; 2) Collect water samples, remove impurities in the water samples by vacuum filtration, filter them with a 0.45 μm aqueous phase filter membrane, and store them in a refrigerator at 4 ℃ for later use; 3) Add the extractant obtained in step 1) to the water sample obtained in step 2), and add dispersant and salt at the same time, adjust the pH to 6-7, place on a vortex mixer for vortexing, centrifuge to accelerate phase separation after reaching extraction equilibrium, draw the upper DES phase after centrifugation, dilute to 1 mL with methanol, perform high performance liquid chromatography analysis, and obtain a sample spectrum; 4) Establishment of standard curve: weigh the standard substances of BTZ, PSE, ALF, FPE, and PBZ, prepare single standard stock solutions with methanol or acetonitrile as solvent, and then prepare mixed standard working solutions as needed. Detect the obtained mixed standard working solutions under the same HPLC analysis conditions as in step 3) to obtain standard chromatograms. Draw standard curves of BTZ, PSE, ALF, FPE, and PBZ respectively, with the injection concentration of each standard substance in the mixed standard working solution as the abscissa x and the peak area of ​​the corresponding standard substance in the standard chromatogram as the ordinate y; 5) Analysis of test results: The peak area values ​​of each herbicide component in the sample spectrum obtained in step 3) are respectively substituted into the corresponding standard curve obtained in step 4), and the content of the herbicide component in the sample is obtained by calculation; The conditions for the high performance liquid chromatography analysis in step 3) and step 4) are as follows: chromatographic column: C18 column, 250 × 4.6 mm, 5 μm; mobile phase is acetonitrile and 0.1% phosphoric acid aqueous solution, mobile phase flow rate is 0.8-1.2 mL / min; column temperature is 20-40° C., and detection wavelength is 220-260 nm.

2. A method for simultaneously detecting five trace herbicides in water as claimed in claim 1, characterized in that In the step 1), the organic acid is n-butyric acid, and the molar ratio of thymol to n-butyric acid is 1:

1.

3. A method for simultaneously detecting five trace herbicides in water as claimed in claim 1, characterized in that In step 3), the amount of water sample is 8 mL, and the amount of extractant used is 150 μL.

4. A method for simultaneously detecting five trace herbicides in water as claimed in claim 1, characterized in that The salt in step 3) is sodium chloride with a concentration of 0.4 mol / L.

5. A method for simultaneously detecting five trace herbicides in water as claimed in claim 1, characterized in that In the step 3), the dispersant is methanol, ethanol, acetone or acetonitrile.

6. A method for simultaneously detecting five trace herbicides in water as claimed in claim 1, characterized in that The vortex time in step 3) is 3 minutes.

7. A method for simultaneously detecting five trace herbicides in water as claimed in claim 1, characterized in that The centrifugal speed in step 3) is 4000 rpm and the time is 10 min.

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